Method for identifying hormonally modulated genes
This invention provides methods of identifying hormone-regulated traits in a cell. The methods involve cultivating the cell as a graft in two different hormonal environments and determining whether expression of the trait differs in the two grafts.
[0001] This application claims the benefit of the priority date of U.S. provisional patent application No. 60/041,246, filed Mar. 7, 1997, and provisional patent application No. 60/047,811, filed May 15, 1997, incorporated herein by reference in their entirety.
FIELD OF THE INVENTION[0002] The present invention is directed to animal model systems for identifying hormonally regulated traits, such as gene expression and, especially, hormonally regulated, disease-associated traits.
BACKGROUND OF THE INVENTION[0003] The structure and function of a cell, tissue or organ depends, in part, upon environmental signals it receives. For example, external signals induce the transformation of normal cells into cancer cells. There are three broadly defined events in tumor formation: initiation, promotion and progression. Yuspa, S. H. and Poirier, M. C. (1988) Adv. Cancer Res., 50:25-70. During initiation, carcinogenic signals cause mutations in oncogenes and/or tumor suppressor genes when carcinogens form adducts with DNA. These mutations then become fixed upon mitosis. Promotion occurs when the initiated cells are stimulated to divide by mechanical, hormonal or genetic signals resulting in pre-neoplasias and benign tumors. This stage can occur long after the cells have been initiated and can sometimes be reversed if the promotional factors are removed. The final stage of tumorigenesis, progression to a malignant phenotype, occurs with the acquisition of metastatic capabilities. This stage is believed to be brought on by the increasing genetic instability of the tumor, leading to mutations at key genetic sites. Metastasis of human cancer is a complex process in itself, involving cell migration, invasion, seeding, adhesion, and all the processes involved in cell growth. Hoffman, R. M. (1994) Journal of Cellular Biochemistry 56:1-3.
[0004] Many mediators of tumorigenesis operate at the initiation stage and are also functional at the promotional stage of tumorigenesis. In hormonal carcinogenesis, the endocrine system plays a role as the intermediator between the outer environment and the target tissue in the course of tumorigenesis. Kodama, M. and Kodama, T (1994) Anticancer Research 14:2653-2666. In this case, the host endocrine system plays a cardinal role as the medium that “senses” the carcinogenic signal and converts it to a corresponding hormonal signal, which in urn is conveyed to the target tissue to trigger the progression of carcinogenesis within the recipient tissue.
[0005] For example, during carcinogenesis of the mammary gland, the promotional stage of tumorigenesis relies on the hormonal state of the individual and the proliferative ability of the initiated cells. Sukumar, S, McKenzie, K., Chen, Y. (1995) Mutation Research 333:37-44. Initiated cells require the presence of estrogen for tumor formation. The generally accepted function of estrogen is to expand the population of activated cells. However, there is also a hormone-mediated differentiation aspect. In this case, estrogen-induced differentiation of mammary epithelial cells is necessary prior to neoplastic development.
[0006] Endocrine-responsive cancers include cancers of the prostate (androgens), larynx (androgen), endometrium (estradiol, progesterone), breast (estrogen), ovary (estrogen) and lymphoid tissues (i.e., leukemias and lymphomas) (glucocorticoids).
[0007] Xenograft propagation of human tumors in athymic nude mice is a well established technique for growing neoplasms. Giovanella, B. C. and Fogh, J. (1985) Advances in Cancer Research 44:70-120. Human tumor tissue or cultured cell suspensions are xeno-transplanted into this congenitally athymic and thus immunologically hypo-responsive mouse strain. Because the nude mouse is immunologically incompetent, the human xenografts are readily accepted without requiring additional immunosuppression. The tumor can grow and become vascularized from the periphery. Human connective tissue and other benign stromal elements, transplanted together with the malignant tumor cells, will not grow in the nude mouse; only the malignant portion of the human tumor will grow. Welander, C. E. and Lewis, J. L. Jr. (1983) Gynecologic Oncology, Griffiths, C. T. and Fuller, A. F. (eds), Martinus Nijhoff Publishers, Boston, pp. 314-342. Cell lines of human tumors in tissue culture also can be readily grown in the nude mouse.
[0008] In order to understand and prevent tumorigenesis and metastasis, models that reflect the human processes are required.
SUMMARY OF THE INVENTION[0009] This invention provides methods for identifying hormonally modulated traits, that is, traits whose expression is influenced by the hormonal environment of a cell, tissue or organ. The methods of this invention involve exposing biological material to different hormonal environments, and detecting traits whose expression differs in the two environments. Many cell types are known to be hormonally responsive. Many diseases and, in particular, certain cancers, are known to be hormonally dependent: their occurrence, progression or severity is regulated by hormonal environment. The identification of hormone-dependent traits, and in particular, hormone-dependent genes, in these cells would be a contribution to field. Such genes may, themselves, be regulators of malignant transformation or may be signs of transformation. In one embodiment, the methods involve exposing hormone-dependent human neoplasms, either benign or malignant, as xenografts to hormonal environments in female and male host animals, and identifying genes whose expression is up- or down-regulated in one environment compared with the other.
[0010] The methods of this invention use diseased cells or tissue propagated under different hormonal backgrounds. Prior models have used only normal versus diseased cells or tissue propagated or compared under a single background. In contrast, the methods of this invention provide both pathologically relevant and pharmacologically relevant gene sequences: Genes whose expression is modulated in disease states compared with healthy states are pathologically relevant because they mark the disease and can provide a target for therapeutic intervention. Disease-specific genes that also are hormonally regulated are pharmacologically relevant: Classes of drugs or molecules that modulate the activity of the hormone will automatically be relevant candidates for modulating the expression of these hormonally modulated genes. This includes, for example, steroid chemistry products (synthetic, natural, analogs, metabolites).
[0011] In summary, this model inherently provides gene sequences with pathologic and pharmacologic relevance. The genes are isolated from cells or tissues in a diseased state. The hormonal dependence of these genes is inherent in the model (differential expression under varied hormonal growth conditions). Because the genes are hormonally dependent there is an inherent pharmacologic relevance.
[0012] In one aspect this invention provides a method for identifying a hormone-regulated trait. The method comprises the steps of a) exposing grafts of a biological material to different hormonal environments in first and second non-human animals of different reproductive states, and b) detecting a difference in expression of a trait in the biological material between the first and second animals. Detecting a difference identifies the trait as a hormone-regulated trait. In one embodiment the hormonal environments are natural hormonal environments and the non-human animals are same species, non-human mammals. In another embodiment the first non-human mammal is a sexually mature female and second non-human mammal is a sexually mature male. In another embodiment the first and second non-human mammals differ in a state of sexual maturity. In another embodiment the first and second non-human mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant. In another embodiment the biological material comprises human benign or malignant neoplastic cells from a laryngeal cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, an endometrial cancer, a vaginal cancer, a testicular cancer, a prostate cancer, a leiomyoma or a benign prostate hyperplasia. In another embodiment the biological material comprises human endocrine cells or human exocrine cells from gall bladder, adrenal gland, pancreas, thyroid, salivary gland, pituitary gland, hypothalamus, ovary or testis. In another embodiment the biological material comprises hormonally responsive human cells selected from adipocytes, osteoblasts, osteoclasts, chondrocytes, hematopoietic cells, cells from lymphoid or myeloid cell lines, lymphoreticular cells, cells from neural tissue, cells from hair follicles, cells from sebaceous glands, cells from bladder tissue, cells from male or female reproductive tract tissue or cells from mammary gland tissue. In another embodiment the biological material comprises hormonally responsive human cells selected from endometrial tissue, endometriotic tissue, pelvic floor tissue or vaginal tissue. In another embodiment the trait is expression of a gene.
[0013] In another aspect this invention provides a model system for identifying hormone-regulated traits comprising at least two non-human, same species animals of different reproductive states, each animal hosting a craft of a biological material, whereby the grafts are exposed to different hormonal environments.
[0014] In one embodiment the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals. In another embodiment at least one non-human mammal is a sexually mature female and at least one non-human mammal is a sexually mature male. In another embodiment at least two non-human mammals differ in a state of sexual maturity. In another embodiment at least two non-human mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.
[0015] In another aspect this invention provides a method for identifying a trait whose expression is regulated by a hormone-regulated gene. The method involves the steps of: a) exposing a graft of biological material comprising the hormone-regulated gene to a natural hormonal environment in a first non-human animal, the environment comprising a hormone that up-regulates or down-regulates expression of the gene; b) exposing a graft of the biological material to the hormonal environment in a second non-human animal, wherein cells of the material are provided with means for down-regulating expression of the gene if the hormone up-regulates its expression, or means for up-regulating expression of the gene if the hormone down-regulates its expression; and c) detecting whether a trait is differently expressed in the graft from the first animal compared with the second animal. Detecting a difference identifies the trait as regulated by the hormone-regulated gene.
[0016] In one embodiment the means for up-regulating comprises an expression vector comprises an expression control sequence operative in the cells and operatively linked with a nucleotide sequence encoding the gene, and the means for down-regulating comprises an antisense polynucleotide that specifically hybridizes with a nucleotide sequence of the gene or its promoter.
[0017] In another aspect this invention provides a method for determining whether a first gene regulates expression of a second, hormone-regulated gene. The method comprises the steps of: a) exposing a graft of biological material comprising the first and second genes to a natural hormonal environment in a first non-human animal, wherein the hormonal environment comprises a hormone that regulates expression of the second hormone-regulated gene; b) exposing a graft of the biological material to the same hormonal environment in a second non-human animal, wherein the cells are provided with means for up-regulating or down-regulating expression of the first gene; and c) detecting whether the second gene is differently expressed in the first animal and the second animal. Detecting a difference provides a determination that the-first gene regulates expression of the second, hormone-regulated gene.
[0018] On another aspect this invention provides a method for detecting a differential effect of a drug on animals of at least two different reproductive states. The method comprises the steps of: a) providing at least two immunocompromised, non-human, same species animals of different reproductive states serving as hosts for grafts of hormonally responsive biological material; b) administering to the animals equivalent dosage regimens of the drug and c) recording a statistically significant (p<0.05) difference in the effect of the dosage regimen on a trait of the biological material in the animals.
[0019] In another aspect this invention provides a method for determining reproductive-state-specific dosages of a pharmaceutical. The method comprises the steps of: a) providing at least two immunocompromised, non-human, same species animals of different reproductive states serving as hosts for grafts of hormonally regulated, diseased, human tissue; b) administering to the animals equivalent dosage regimens of the pharmaceutical; and c) determining the efficacy of the pharmaceutical in treating the diseased tissue in each of the animals of the set.
[0020] In another aspect this invention provides a method for determining whether an agent modulates expression of a disease-associated, hormone regulated trait, wherein the trait is expression of a gene. The method comprises the steps of: (a) exposing diseased biological material comprising a disease-associated, hormone regulated gene to a hormonal environment in an animal that regulates expression of the gene; (b) contacting the biological material with the agent; (c) measuring the amount of expression of the gene; and (d) determining whether the measured amount is different than an amount of expression of the gene in the cell under control conditions in which the biological material is not exposed to the agent. A difference between the measured amount and the amount under control conditions indicates that the agent modulates expression of the gene.
[0021] In one embodiment the step of exposing comprises exposing the cell to the hormonal environment in an animal in vivo. In another embodiment the agent is an agent that modulates the action of a hormone that regulates expression of the gene. In another embodiment the agent modulates the action of estrogen or testosterone. In another embodiment the agent is an estrogen or testosterone analog.
[0022] Another embodiment further comprises the steps of: identifying a second hormone regulated trait: measuring the amount of expression of the second trait in the animal; and determining whether the measured amount of expression of the second trait is different than an amount of expression of the second trait in the animal under control conditions in which the biological material is not exposed to the agent. A further embodiment comprises the steps of: determining the degree of difference between the measured amounts of gene expression to provide a degree of gene expression modulation; determining the degree of difference between the measured amounts of expression of the trait to provide a degree of trait expression modulation; and determining whether the degree of gene expression modulation is different than the degree of trait expression modulation. A difference indicates whether the agent preferentially modulates expression of the gene.
BRIEF DESCRIPTION OF THE DRAWINGS[0023] FIGS. 1A-1B is a photograph of LNCaP cells grown in male (1A) and female (1B) mice.
[0024] FIGS. 2A-2B is a photograph of a Northern analysis. FIG. 2A shows RNA from LNCaP cells and hybridized with Repro-PC-1.0-specific probe. FIG. 2B shows rehybridization of the same blot with probes for tubulin and actin. Male =male-grown tumors. Female =female-grown tumors. C=LNCaP cells. P=PC3 cells.
[0025] FIG. 3 shows the alignment of overlapping clones which, together, produce the Repro-PC-1.0 cDNA of SEQ ID NO:1. FIG. 4 presents a comparison of the organization of PKC, Repro-PC-1.0 and synaptotagmin polypeptides.
[0026] FIG. 5 shows the alignment of the amino acid sequences of Repro-PC-1.0 (“PC-20”) and rat synaptotagmin 4.
[0027] FIG. 6 shows the alignment of the internal repeats of PKC-C2, Repro-PC-1.0 (PC-20) “B” repeat, synaptotagmin “B” repeat, synaptotagmin “A” repeat and Repro-PC-1.0 (PC-20) “A” repeat.
DETAILED DESCRIPTION OF THE INVENTION[0028] I. Definitions
[0029] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
[0030] “Polynucleotide” refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (“PNAs”), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. “Nucleic acid” typically refers to large polynucleotides. “Oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”
[0031] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5′-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5′-direction. The direction of 5′ to 3′ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand”; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5′ to the 5′-end of the RNA transcript are referred to as “upstream sequences”; sequences on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the coding RNA transcript are referred to as “downstream sequences.”
[0032] “Recombinant polvnucleotide” refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, e.g., a “recombinant polypeptide.” A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well. Appropriate unicellular hosts include any of those routinely used in expressing eukaryotic or mammalian polynucleotides, including, for example, prokaryotes, such as E. coli; and eukaryotes, including for example, fungi, such as yeast; and mammalian cells, including insect cells (e.g., Sf9) and animal cells such as CHO, R1.1, B-W, L-M, African Green Monkey Kidney cells (e.g. COS 1, COS 7, BSC 1, BSC 40 and BMT 10) and cultured human cells.
[0033] “Expression control sequence” refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and/or translation) of a nucleotide sequence operatively linked to it. “Operatively linked” refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence). Expression control sequences can include, for example and without limitation, sequences of promoters (e.g., inducible, repressible or constitutive), enhancers, transcription terminators, a start codon (i.e., ATG), splicing signals for introns, and stop codons.
[0034] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
[0035] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
[0036] “Allelic variant” refers to any of two or more polymorphic forms of a gene occupying the same genetic locus. Allelic variations arise naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. “Allelic variant” also refers to polymorphisms in non-coding sequences at a genetic locus and cDNAs derived from mRNA transcripts of genetic allelic variants, as well as the proteins encoded by them.
[0037] “Hybridizing specifically to” or “specific hybridization” or “selectively hybridize to,” refers to the binding, duplexing, or hybridizing of a polynucleotide preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
[0038] “Stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. “Stringent hybridization” and “stringent hybridization wash conditions” in the context of polynucleotide hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of polynucleotides is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
[0039] An example of stringent hybridization conditions for hybridization of complementary polynucleotides which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2× SSC wash at 65° C. for 15 minutes (see Sambrook et al. for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6× SSC at 40° C. for 15 minutes. In general, a signal-to-noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
[0040] A first sequence is an “antisense sequence” with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically hybridizes with a polynucleotide whose sequence is the second sequence.
[0041] “Primer” refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
[0042] “Probe” refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.
[0043] “Detectable moiety” or a “label” refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or polynucleotides with a sequence complementary to a target. The detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantitate the amount of bound detectable moiety in a sample. The detectable moiety can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin. The detectable moiety may be directly or indirectly detectable. Indirect detection can involve the binding of a second directly or indirectly detectable moiety to the detectable moiety. For example, the detectable moiety can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a polynucleotide having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled polynucleotides. Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
[0044] “Linker” refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a polynucleotide that hybridizes to one complementary sequence at the 5′ end and to another complementary sequence at the 3′ end, thus joining two non-complementary sequences.
[0045] “Amplification” refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotides, e.g., by reverse transcription, polymerase chain reaction, and ligase chain reaction.
[0046] “Polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides. The term “peptide” typically refers to short polypeptides.
[0047] Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
[0048] “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Thus, the polynucleotide whose sequence 5′-TATAC-3′ is complementary to a polynucleotide whose sequence is 5′-GTATA-3′.
[0049] “Conservative substitution” refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:
[0050] 1) Alanine (A), Serine (S), Threonine (T);
[0051] 2) Aspartic acid (D), Glutamic acid (E);
[0052] 3) Asparagine (N), Glutamine (Q);
[0053] 4) Arginine (R), Lysine (K);
[0054] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
[0055] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0056] “Antibody” refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa and lambda light chain constant region genes and the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, as well as the myriad immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab′ and F(ab)′2 fragments. The term “antibody,” as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies, as well as humanized antibodies.
[0057] An antibody “specifically binds to” or “is specifically immunoreactive with” a protein when the antibody functions in a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
[0058] “Immunoassay” refers to an assay that utilizes an antibody to specifically bind an analyte. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the analyte.
[0059] “Agent” refers to a chemical compound, a mixture of chemical compounds, a sample of undetermined composition, a combinatorial small molecule array, a biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward et al. (1989) Nature 341: 544-546. The protocol described by Huse is rendered more efficient in combination with phage display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.
[0060] “Pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a mammal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. “Pharmacologically effective amount” refers to that amount of an agent effective to produce the intended pharmacological result. “Pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, or intravenous intraperitoneal injection; or topical, transdermal, or transmucosal administration).
[0061] “Biological material” refers to cells, tissues or organs.
[0062] Biological material is “hormonally responsive” if a trait of the biological material differs when the biological material is exposed to two different hormonal environments.
[0063] “Trait” refers to any distinguishable characteristic of a phenotype of a cell, tissue, organ or organism. Traits include, for example, the level of expression of one (or more) genes, as evidenced by, e.g., RNA transcripts or expression of a protein), presence of products resulting from protein interaction with an analyte (e.g., the product of enzymatic activity), biological activity resulting from expression of a gene (e.g., increased or decreased activity of an enzyme or binding protein regulated by the expressed protein), cellular activity resulting from expression a gene (e.g., entry into the mitotic phases of the cell cycle), or changes in physical appearance (e.g., malignant appearing cells, tissues or organs).
[0064] A “hormonal environment” is a set of hormonal signals, including the concentration of hormones and their fluctuation over time, to which a biological material is exposed. Hormonal environments refers to both in vivo and in vitro hormonal environments.
[0065] A “hormone-regulated” gene or trait of a hormonally responsive biological material is a gene or trait whose expression is differentially expressed (e.g., increased (“up-regulated”) or decreased (“down-regulated”)) based on the hormonal environment to which the biological material is exposed.
[0066] A “disease-associated” gene or trait is a gene or trait of a biological material that is differently expressed (e.g., up-regulated or down-regulated) in diseased biological material compared with healthy biological material.
[0067] A hormonal environment regulates expression of a disease-associated, hormone-regulated gene or trait if the hormonal environment modulates expression of the gene or trait in diseased biological material compared with normal biological material.
[0068] In certain embodiments, “differential expression” or “modulation” of a gene or trait in a biological material exposed to a hormonal environment can be compared with reference to healthy biological material in a hormonal environment of a healthy animal.
[0069] “Graft” refers to any free (unattached) cells, tissue or organ for transplantation.
[0070] “Reproductive state” refers to one of several stages of an animal's life characterized by a distinct hormonal environment. Reproductive states depend on sex (male or female), sexual maturity (pre-pubescent, sexually mature, sexually senescent) and reproductive status (cycling, pregnant or pseudo-pregnant).
[0071] “Allograft” refers to a transplanted cell, tissue or organ derived from a different animal of the same species.
[0072] “Xenograft” refers to a transplanted cell, tissue or organ derived from an animal of a different species.
[0073] “Detecting” refers to determining the presence, absence, or amount of a trait in a biological material, and can include quantifying the amount of a compound in a sample or per cell in a sample.
[0074] “Expression of a trait” refers to the occurrence or non-occurrence of the trait, as well as the degree to which a trait occurs. Thus, the occurrence or non-occurrence of metastasis is a trait, as is the rate of metastasis. The expression of a trait differs between grafted biological material in two animals if there is a difference in either the occurrence or non-occurrence or the degree of occurrence of the trait.
[0075] Traits are “differentially expressed” in two environments if there is a qualitative or statistically significant (p<0.05) quantitative difference between the traits in the two environments.
[0076] A “natural hormonal environment” is a hormonal milieu in an animal that exists as a result of endogenously produced hormones. This includes both healthy and unhealthy animals, as well as animals at different reproductive states. Unnatural hormonal environments are created by altering the natural hormonal environment by exogenous administration of hormones source or antagonism of endogenous hormones. For example, the hormonal environment of an animal can be unnaturally reconstituted by, e.g., surgical manipulation of hormone producing tissues (e.g., removal or ovary or testicle) and administration of replacement hormones. Another method of creating an unnatural hormonal environment is by transgenic manipulation of an organism so that it increases or decreases expression of one or more hormones.
[0077] “Functional pathway” refers to a chain of events in a biological system from expression of a gene to expression of a trait linked so that each event in the chain is caused by a preceding event in the chain and, turn, causes a subsequent event in the chain.
[0078] II. Experimental System for Identifying Hormone-Regulated Traits
[0079] A. General
[0080] This invention provides methods for identifying hormone-regulated traits. The methods involve exposing biological material to different hormonal environments and determining whether a trait is differently expressed in the two environments. If so, the trait is hormonally regulated. For example, certain cancer cells, such as prostate cancer cells, express different mRNA and exhibit different tumor morphology depending upon whether they are exposed to a male or a female hormonal environment. Accordingly, practicing the methods of this invention involves choosing different hormonal environments to which to expose biological material. Traits that are preferentially expressed in male environments are referred to as “male-environment-associated traits” and traits that are preferentially expressed in female environments are referred to as “female-environment-associated traits.”
[0081] B. Hormonal Environment
[0082] The hormonal environment that an animal presents to a graft reflects several factors such as the animal's species, reproductive state, and emotional state.
[0083] To detect hormone-regulated traits, biological material is exposed to different non-human animals whose hormonal environment differs at least in reproductive state. Accordingly, the animal hosts differ according to at least one of the following states: sex, sexual maturity or reproductive status. Thus, in one embodiment, the animals are differently sexed, sexually mature animals. However, this invention also contemplates comparing a trait in same sexed animals at different levels of sexual maturity, or of different reproductive status.
[0084] In a preferred embodiment, the biological material is exposed to a natural hormonal environment. However, the hormonal environment of an animal can be primed before grafting. For example, gonadotrophins can be administered to female animals.
[0085] The hormonal signals produced by an animal can change depending on the emotional state of the animal. For example, animals that are chronically stressed produce larger amounts of steroids than animals that are not so stressed.
[0086] An animal's taxonomic classification dictates what sorts of hormones the animal produces and whether these hormones are likely to have an effect on a particular xenograft. Generally, the closer the taxonomic connection between the host species and the source species of the graft, the more likely the hormones, particularly the peptide hormones, will regulate events in the cells of the xenograft. Accordingly, same-species transplants are useful when the transplant tissue is not human. Insofar as the biological material of interest derives from human sources, the animals useful as hosts for xenografts are mammals and, more particularly, rodents (e.g., mice or rats).
[0087] When the graft is a xenograft or allograft, there is the risk that the host animal will reject the grafted biological material. Therefore, immunocompromised hosts that do not reject allografts or xenografts are preferred. Animals can be immunocompromised genetically, surgically or by exposure to an agent that injures or destroys the immune system. Animals whose immune systems are compromised genetically are known in the art. For example, athymic, nude mice are immunocompromised. Nude rats also can be used. (Taconic, Germantown, N.Y.) Another genetic immunodeficiency is severe combined immune deficiency (“SCID”). Animals can be rendered immunocompromised by surgical removal of the thymus or the bursa. Irradiation of an animal renders it immunodeficient by destroying hematopoietic cells in the bone marrow. Hyperacute rejection can be suppressed by inhibiting complement activation. Chemical means of immunocompromising an animal include immunosuppressants such as cyclosporin and prednisone, among others.
[0088] Further factors in choosing animal hosts include the expense to keep the animals and the familiarity of science with the particular animals used.
[0089] C. Biological Material
[0090] The biological material that is to be exposed to different hormonal environments can be selected from any source, including cultured cell lines (e.g. transformed or immortalized), cells from primary cultures, animal tissue or animal organs. The biological material can be from any species. Biological material from human tissue or cells are particularly interesting from a medical perspective. Generally, two samples from the same biological material are each exposed to a different hormonal environment.
[0091] The biological material generally will include hormonally responsive cells. This can include healthy or diseased tissue (e.g., from a hormone-regulated disease), senescent or pre-senescent. Thus, the cells can be hormonally responsive human cells such as, e.g., adipocytes, osteoblasts, osteoclasts, chondrocytes, hematopoietic cells (e.g., stem cells), cells of myeloid or lymphoid cell lines (e.g., B-lymphocytes, T-lymphocytes, red blood cells, platelets, monocytes, neutrophils, eosinophils, basophils, macrophages), lymphoreticular cells, cells from neural tissue, cells from hair follicles, cells from sebaceous glands, cells from bladder tissue (e.g., from a subject with hormonally regulated bladder disfunction), cells from male or female reproductive tract tissue (e.g., endometrial tissue, endometriotic tissue, pelvic floor tissue or vaginal tissue) or cells from mammary gland tissue.
[0092] The biological material also can comprise human neoplasms, either benign or malignant, e.g., cells from a laryngeal cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, an endometrial cancer, a vaginal cancer, a testicular cancer, a prostate cancer, a leiomyoma or benign prostate hyperplastic tissue. LNCaP is a useful prostate cancer cell line.
[0093] The cells also can be human endocrine cells or human exocrine cells from, e.g., gall bladder, adrenal gland, pancreas, thyroid, salivary gland, pituitary gland, hypothalamus, ovary or testis.
[0094] D. Exposing the Biological Material
[0095] After having selected the biological material and the hormonal environments, one then exposes the biological material to different hormonal environments.
[0096] A first step in exposure is administering the graft to the animal. If the graft is a cell line or cell culture, the material can be injected into the host. Tissue can be prepared by mincing it and passing the minced material serially through a needle before injection. Injection can be, e.g., intraperitoneal, intramuscular, or subcutaneous. If the material for transplant is a tissue or organ, it can be inserted surgically. However, regardless of the method, one aim is to administer a sufficient amount of material to an appropriate location so that material can be retrieved for analysis after cultivation. In order to perform parallel experiments, the transplants usually will be made contemporaneously.
[0097] The biological material should be cultured in the animal for a sufficient amount of time for the hormone-regulated trait to become detectable in the different hormonal environments. Also, the material should be cultured for sufficient time to provide sufficient material for subsequent analysis. Depending upon the nature of the hormone-dependent trait to be examined, differences can be seen within a few hours after exposure. However, genetic expression of other traits, such as morphological features, may take weeks to develop. This includes, for example, sufficient time for a grafted tumor to become vascularized or to metastasize. A graft can be cultured through the point of death of the animal.
[0098] If the graft is a cancer cell line, it is useful to expose it until the cells have colonized the host or when a tumor becomes palpable; this provides enough material for analysis. For example, injection of about 1×106 to 1×107 LNCaP cells to nude mice required about two to three months to become palpable.
[0099] After the graft has been exposed for a sufficient period of time, it can be removed from the animals so that differentially expressed traits can be identified and examined.
[0100] III. Methods for Identifying Hormone-Regulated Traits
[0101] A. Introduction
[0102] In general, the methods of this invention for identifying hormone-regulated traits involve exposing biological material to two different hormonal environments and identifying traits that are expressed differently in the environments by comparing their expression in each of the environments. In this method, the traits to be analyzed must be selected and means for their identification provided. Once a hormone-regulated trait has been identified, one can make the trait the subject of further analysis.
[0103] B. Selecting Traits for Identification and Analysis
[0104] Any detectable trait can be the subject of identification and further analysis. This includes structural or functional traits. This invention also contemplates identifying traits that are up- or down-regulated in response to the hormonal environment. “Up-regulation” and “down-regulation” connote statistically significant )p <0.05) increases or decreases in expression of a measurable trait, such as transcription of a particular mRNA.
[0105] Functional traits include biochemical or physiological traits of the cell, tissue or organ. The distinction between biochemical and physiological traits is not particularly distinct, and is used merely for purposes of demonstration. In general, biochemical traits are traits expressed at a cellular level, while physiological traits are expressed at a tissue or organ level.
[0106] Biochemical traits include, most particularly, gene expression (i.e, transcription of DNA into RNA), and also, for example, post-transcriptional modifications, protein expression, post-translational protein modification or protein secretion. Post-transcriptional modifications include, for example, splicing mRNAs or trimethylguanosine (“TMG”) capping. Post-translational modifications include, for example, glycosylation and ADP ribosylation. Differences in gene expression can be identified as absolute induction or repression of a gene, or as up- or down-regulation of a gene expressed in both environments.
[0107] Functional differences also can manifest at the physiological level, especially if the biological material is a tissue or organ transplant. Physiological traits include, for example, secretion of various chemicals (for example, insulin by pancreatic tissue), urine production by a kidney, metabolism by liver tissue or anchorage dependence of cancer cells.
[0108] Structural traits can include cell or tissue architecture, morphology or histology, cell differentiation, tumor shape, tumor staging, angiogenesis, metastasis (e.g., appearance at locations distant to the primary tumor site) and tissue degradation.
[0109] After exposing the biological material to different hormonal environments for a sufficient period of time, the material is retrieved and a trait is compared between the two samples. Differences in mRNA expression can be identified by comparative northern hybridization or any of the more powerful subtractive or differential analytic techniques described below. Differences in protein expression can be determined by comparative quantitative and qualitative techniques such as 2D gel electrophoresis, western hybridization, HPLC, mass spectrometry or immunoassays, among other methods.
[0110] C. Means for Identifying Hormone-Regulated Genes
[0111] In one embodiment of the invention, the trait is expression of a gene. One useful method of identifying differentially expressed genes involves comparing the amount of mRNA expression between the samples. Methods to compare mRNA expression between two tissues or cell types are well known in the art. They include, for example, subtractive hybridization, differential display; representational differential analysis and suppression subtractive hybridization. These methods can yield probes enriched for genes preferentially expressed in one hormonal environment versus the other.
[0112] 1. Subtractive Hybridization
[0113] Subtractive hybridization is a useful method of identifying genes that are differentially expressed in different hormonal environments. For example, one can subtract female-derived mRNA populations from male-derived mRNA population, and vice-versa.
[0114] RNA prepared by conventional methods from a first cell population (e.g. cells from the graft exposed to the female hormonal environment) and RNA from a second cell population (e.g., cells from the graft exposed to the male hormonal environment) are separately reverse-transcribed and second-strand synthesized to form two pools of double-stranded cDNA. a tester pool comprising sequences of the mRNA species for which enrichment is desired, and a driver pool comprising the sequences to be subtracted from the tester pool. The two pools may be fragmented by endonuclease digestion (restriction endonuclease or non-specific endonuclease) if desired to degrade cDNA consisting of tandem repeated sequences and to enhance hybridization efficiency.
[0115] The driver pool is labeled, such as by photobiotinylation or attachment of another suitable recoverable label. The driver pool and tester pool are denatured and mixed together in a reaction mixture under hybridization conditions and incubated for a suitable hybridization period. The reaction mixture is contacted with a ligand which binds the recoverable label on the driver cDNA and which can be readily recovered from the reaction mixture (e.g., using avidin attached to magnetic beads), such that a substantial fraction of the driver cDNA and any tester cDNA hybridized thereto is selectively removed from the reaction mixture. The remaining reaction mixture is enriched for tester cDNA species that are preferentially expressed in the first cell population as compared to the second cell population.
[0116] The enriched (subtracted) tester cDNA pool may be subjected to one or more additional rounds of subtraction hybridization with a pool of labeled driver cDNA. The driver cDNA can be substantially identical to the initial pool of driver cDNA or which may represent a different cell population having mRNA species which are desired to be subtracted from the subtracted tester cDNA pool.
[0117] A variety of means for accomplishing the subtraction hybridization and suitable methodological guidance are available to the artisan. See, e.g., Lee et al., 1991, Proc. Natl. Acad. Sci. (U.S.A.) 88:2825; Milner et al., 1995, Nucleic Acids Res. 23:176; Luqmani et al., 1994, Anal. Biochem. 222: 102; Zebrowski et al., 1994, Anal. Biochem. 222:285; Robertson et al., 1994, Genomics 23:42; Rosenberg et al., 1994, Proc. Natl. Acad. Sci. (U.S.A.) 91:6113; Li et al., 1994, Biotechniques 16:722; Hakvoort et al., 1994, Nucleic Acids Res. 22:878; Satoh et al., 1994, Mutat. Res. 316:25; Austruy et al. (1993) Cancer Res. 53:2888; Marechal et al., 1993, Anal. Biochem. 208:330; El-Deiry et al., 1993, Cell 75:817; Hara et al., 1991, Nucleic Acids Res. 19:7097; and Herfort and Garber, 1991, Biotechniques 11:598.
[0118] 2. Differential Display of Amplified Products
[0119] In differential display, cDNA is subjected to selective amplification. One can amplify the DNA by means known in the art. The general strategy involves amplification of cDNAs from species in the subtracted tester cDNA pool by PCR using one or a set of arbitrary sequence primers. Arbitrary primers are selected according to various criteria at the discretion of the practitioner so that each will amplify only a fraction of the cDNAs in the subtracted cDNA pool so that the amplification products can be resolved and individually recovered on a separation system, such as a polyacrylamide gel. The selection of arbitrary primers and their sequence is determined by the practitioner with reference to the literature. See, e.g., U.S. patent application Ser. No. 08/235,180, filed Apr. 29, 1994; Linskens et al., 1995, Nucleic Acids Res. 23 (16): 3244-3251; Liang et al., 1993, Nucleic Acids Res. 21:3269; Utans et al., 1994, Proc. Natl. Acad. Sci. (U.S.A.) 91:6463; Zimmermann et al., 1994, Proc. Natl. Acad. Sci. (U.S.A.) 91:5456; Fischer et al., 1995, Proc. Natl. Acad. Sci. (U.S.A.) 92:5331; Lohmann et al., 1995, Biotechniques 18:200; Reeves et al., 1995, Biotechniques 18:18; and Maser et al., 1995, Semin Nephrol 15:29.
[0120] Selection of the arbitrary primers is conducted using known theoretical and empirical parameters. In brief, the shorter a 5′ arbitrary primer with a selected sequence of nucleotides, the more frequently it will anneal near the end of a cDNA strand. However, if primers are too short, they will not efficiently serve as a specific site for primer extension by a DNA polymerase such as taq. The theoretical calculations for determining the frequency with which a given primer will specifically prime a PCR or other polymerase-mediated primer extension are known, and depend, inter alia, on the frequency with which a primer binds the template, the upper limit of the desired products, the number of cDNA species in the template mixture, and the like. The frequency with which a primer will prime a reaction typically depends largely upon the 3′ terminal nucleotides of the primer. For example, if the 3′ primer is a poly-dT primer which binds to the common poly-A tail of mRNA (or corresponding cDNA), an anchor 3′ primer having about two (or more) additional bases at the 3′ end of the 3′ primer is preferably used. By standard probability, any two 3′ bases (excluding dT at the penultimate base) will bind one-twelfth of the time to a perfectly complementary mRNA. Thus, any arbitrary 3′ primer with the sequence (dT)ZMN, wherein Z is about 18-24, M is dA dC or dG and N is dA. dT. dC or dG will prime about one twelfth of all mammalian mRNAs.
[0121] Enhanced Differential Display (EDD), a method that improves on the reproducibility and efficiency of the differential display technique, is described in Linskens et al. (1995) Nucleic Acids Res. 23(16):3244-51, and Villeponteau et al., U.S. Pat. No. 5,580,726. EDD is characterized by a two-stage amplification process. Both stages involve long primers of at least 21 nucleotides. The first stage is a low stringency amplification involving about 2-4 cycles of amplification by, e.g., PCR, at low temperature, e.g., 35° to 45°, 39° to 41°, or about 41°. The second stage is a high stringency amplification involving about 10 to 25 cycles, typically 18 cycles, at high temperature, e.g., 55° to 70° or about 60°.
[0122] As a result of this amplification process, the cDNA pool is normalized. That is, the amount of more abundant cDNA species (e.g., present in the original cDNA population at greater than about 0.5%) is decreased relative to other species in the pool and the amount of rare species is increased relative to the other species.
[0123] The amplified products are optionally labeled and are typically resolved by electrophoresis on a polyacrylamide gel; the locations where label is present are excised and the labeled product species are recovered from the gel portion, typically by elution. The resultant recovered product species (typically an expressed sequence tag or EST cDNA) can be subcloned into a replicable vector with or without attachment of linkers, amplified further, and/or sequenced directly. Once an EST is recovered, it can be used to obtain a substantially full-length cDNA from a cDNA library. The EST(s) can be sequenced and the sequence information used to generate a primer for primer extension (5′-RACE) or the EST can be labeled and used as a hybridization probe to identify larger cDNA clones from a cDNA library. Genomic or full-length cDNA clones corresponding to ESTs can be isolated from clone libraries (e.g., available from Clontech, Palo Alto, Calif.) using the labeled EST (e.g., by nick-translation, random prime labelling or end-labeling) or other hybridization probes with nucleotide sequences corresponding to those identified in the EST in conventional hybridization screening methods.
[0124] Once an EST is recovered, it may be used to obtain a substantially full-length cDNA from a cDNA library. The EST can be sequenced and the sequence information used to generate a primer for primer extension (5′-RACE technique) or the EST can be labeled and used as a hybridization probe to identify larger cDNA clones from a cDNA library.
[0125] Genomic or cDNA clones of the subtracted cDNA species are isolated from clone libraries (e.g., available from Clontech, Palo Alto, Calif.) using the labeled EST (e.g., by nick-translation or end-labeling) or using hybridization probes designed on the basis of the nucleotide sequences identified and using conventional hybridization screening methods (e.g., Bentori W D and Davis R W (1977) Science 196:180; Goodspeed et al. (1989) Gene 76:1). Where a low abundance protein cDNA clone is desired, clone libraries containing cDNA derived from somatic cell mRNA or other expressing cell mRNA are preferred. Additionally, polymerase chain reaction (PCR) using primers based on predetermined sequence data are used to amplify DNA fragments from genomic DNA, mRNA pools, or from cDNA clone libraries. U.S. Pat. Nos. 4,683,195 and 4,683,202 describe the PCR method. Additionally, PCR methods employing one primer that is based on predetermined or predicted sequence data and a second primer that is not based on that sequence data is used.
[0126] 3. Suppression Subtractive Hybridization (SSH)
[0127] Suppression subtractive hybridization (SSH) techniques can be used to generate subtracted probes and cDNA libraries for the identification of differentially expressed transcripts. This technique includes a “normalization” step that optimizes the identification of rare transcripts that are differentially expressed. The process involves “normalizing” the levels of high and low abundance sequences by generating two differently prepared “tester” cDNA (contains differentially expressed sequences) fractions with different 5′-end primer annealing sites and hybridizing each “tester” fraction to “driver” cDNA (contains non-target molecules), which is present in excess. Due to second-order kinetics, rare transcripts accumulate as single-stranded cDNA (ss-cDNA) molecules that are not capable of serving as template during PCR amplification using adaptor primers. To resolve this issue and allow for the preferential amplification of rare sequences, a second hybridization step is performed that includes mixing each “tester”“driver” reaction, which contains ss-cDNA with different 5′-end adaptor primer sites. The second hybridization reaction results in an increase in the apparent concentration of ss-cDNA, which facilitates the formation of double-stranded cDNA (ds-cDNA) with different 5′-end primer sites. The ds-cDNA populations with different 5′-end primer sites is competent template for the preferential amplification of differentially expressed sequences that represent rare sequences.
[0128] Briefly, the “tester” cDNAs are prepared by splitting the cDNA into two equal aliquots and ligating different oligonucleotide adapters, which function as PCR primer annealing sites, to the cDNA ends in each fraction. Following adaptor ligation, each aliquot is hybridized to excess amounts of “driver” cDNA, which forms complexes with non-target cDNA that do not contain adaptor-primer sites. Following the first hybridization step, the highly abundant target cDNAs form ds-cDNA that contain the same 5′-end adaptor-primer sites on each end, the rare transcripts remain as single-stranded cDNA (ss-cDNA) molecules and the non-targeted cDNAs anneal to the “driver” cDNA. The ss-cDNAs are capable of forming ds-cDNA using the ss-cDNA targets that contain different 5′-adaptor-primer sites once the two “tester” aliquots are mixed during the second hybridization step. The second hybridization reaction is used as template for the amplification of cDNAs using the two different adaptor-primers. Due to the preferential amplification of the ds-cDNAs with different 5′-end adaptor-primer sites, the low abundance sequences are amplified during the PCR step, while the amplification of the highly abundant sequences (ds-cDNAs with same 5′-end adaptor-primer site) is suppressed. The suppressed amplification of highly abundant sequences is due to pan-like formation caused by the annealing of complementary ends, which results in reduced representation following PCR amplification. In addition to “normalizing” the representation of highly abundant sequences, SSH also allows for the abated amplification of sequences present in the “driver” population. This is accomplished by hybridizing “driver” cDNA, which does not contain ligated adapters, to the “tester” cDNA. The resulting ds-cDNAs will not contain adaptor-primer sites on both 5′-ends, thus, rendering the molecules incompetent for exponential PCR amplification. See, e.g., L. Diatchenko et al. (1996), Proc. Natl. Acad. Sci. USA 93:6025-6030.
[0129] 4. Representational Difference Analysis (RDA)
[0130] RDA provides a method that enriches for sequences present in one complex sample that are absent or substantially depleted from another. Restriction digested cDNA fragments from each complex sample (“tester” and “driver”) are ligated to adaptor oligonucleotides and amplified (“amplicons”). Differential amplification, principally because of preferential amplification of smaller restriction fragments in the initial complex pool, results in a reduced-complexity “representation” of the original sample. Ligation of a second adaptor olizonucleotide to the 3′ ends of the fragments in the “tester” amplicon provides a means for amplifying “tester” sequences. After denaturation and hybridization in the presence of an excess of “driver”, “tester” sequences are preferentially recovered in a form that can be amplified by subsequent PCR. Additional rounds of selection allow further subtractive hybridization by amplifying the initial enrichment factor that results from the dependence of the annealing rate of each fragment on its relative abundance in the pool (kinetic enrichment).
[0131] In brief, double-stranded cDNAs are synthesized from different poly A+RNA populations, restriction digested and adapters are ligated. This material is used to generate representations by PCR amplification. The oligonucleotide adapters are removed from each representation by restriction digestion. “Driver” is the adaptor-less preparation. “Tester” is prepared by ligating new oligonucleotide adapters. Representations are then used as both a “tester” and as a “driver” in reciprocal subtractive hybridizations. First difference products are then generated by PCR amplification. The adapters are changed by digestion, removal, and ligation of new adapters. Second difference products are generated by PCR amplification using the new adapters. This process is repeated to generate third difference products which are cloned into an appropriate vector (e.g., pBluescript). The difference products are confirmed as differential by probing against blots of the original amplicons.
[0132] See, e.g., N. Lisitsyn et al. (1993) Science 259:946-951 and M. Hubank and D. Schatz (1994) Nucleic Acids Research 22:5640-5648.
[0133] D. Uses of Identified Hormone-Regulated Traits
[0134] Once a hormone-regulated trait has been identified or a polynucleotide encoding the hormone-regulated gene isolated, one can make the trait the subject of further analysis.
[0135] For example, a hormone-regulated gene can be used to produce various reagents useful in the model system. These include, for example, (1) polynucleotide probes and primers, useful for detecting and amplifying sequences of the hormone-regulated gene; (2) inhibitory polynucleotides, useful to inhibit expression of a hormone-regulated gene; (3) recombinant expression constructs for expressing polypeptides encoded by a hormone-regulated gene; (4) polypeptides encoded by a hormone-regulated gene, useful in, e.g., activity assays and in the preparation of antibodies; (5) antibodies, useful in detecting polypeptides in, e.g., immunoassays; and (6) ligands for polypeptide receptors, useful, e.g., as agonists or antagonists for up- or down-regulating protein activity.
[0136] These reagents are useful in further methods of the invention including, for example, (1) detecting expression of hormone-regulated traits (e.g., gene or protein expression); (2) identifying up- or down-stream traits in a functional pathway that includes the hormone-regulated trait, and determining expression of the trait in cells of various tissues; (3) screening compounds for their ability to modulate expression of hormone-regulated traits; (4) genotyping individuals for mutations in hormone-regulated genes (e.g., mutations, amplifications, deletions or translocations) and (5) karyotyping for chromosomal aberrations in the region of the hormone-regulated gene.
[0137] IV. Polynucleotides Encoding Hormone-Regulated Genes
[0138] Upon identifying a hormone-regulated gene, one can clone the gene to produce polypeptides encoding the gene, polynucleotide probes for identifying the gene and inhibitory polynucleotides for inhibiting expression of the gene.
[0139] Hormone-regulated genes can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Q&bgr; replicase amplification system (QB). A wide variety of cloning and in vitro amplification methodologies are well-known to persons of skill. PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the gene under stringent hybridization conditions.
[0140] Mutant versions of the proteins can be made by site-specific mutagenesis of other polynucleotides encoding the proteins, or by random mutagenesis caused by increasing the error rate of PCR of the original polynucleotide with 0.1 mM MnCl2 and unbalanced nucleotide concentrations.
[0141] This invention also provides expression vectors, e.g., recombinant polynucleotide molecules comprising expression control sequences operatively linked to a nucleotide sequence encoding the target polypeptide. Expression vectors can be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, markers, etc. for transcription and translation of mRNA. The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques also well known in the art. Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.) Useful promoters for such purposes include a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter, a MRP polIII promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), and a constitutive CMV promoter. A plasmid useful for gene therapy can comprise other functional elements, such as selectable markers, identification regions, and other genes.
[0142] Expression vectors useful in this invention depend on their intended use. Such expression vectors must, of course, contain expression and replication signals compatible with the host cell. Expression vectors useful for expressing the protein of this invention include viral vectors such as retroviruses, adenoviruses and adeno-associated viruses, plasmid vectors, cosmids, and the like. Viral and plasmid vectors are preferred for transfecting mammalian cells. The expression vector pcDNA1 (Invitrogen, San Diego, Calif.), in which the expression control sequence comprises the CMV promoter, provides good rates of transfection and expression. Adeno-associated viral vectors are useful in the gene therapy methods of this invention.
[0143] A variety of means are available for delivering polynucleotides to cells including, for example, direct uptake of the molecule by a cell from solution, facilitated uptake through lipofection (e.g., liposomes or immunoliposomes), particle-mediated transfection, and intracellular expression from an expression cassette having an expression control sequence operably linked to a nucleotide sequence that encodes the inhibitory polynucleotide. See also Inouye et al., U.S. Pat. No. 5,272,065; Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego, Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger, Gene Transfer and Expression—A Laboratory Manual, Stockton Press, New York, N.Y., (1990). Recombinant DNA expression plasmids can also be used to prepare the polynucleotides of the invention for delivery by means other than by gene therapy, although it may be more economical to make short oligonucleotides by in vitro chemical synthesis.
[0144] The construct can also contain a tag to simplify isolation of the protein. For example, a polyhistidine tag of, e.g., six histidine residues, can be incorporated at the amino terminal end of the protein. The polyhistidine tag allows convenient isolation of the protein in a single step by nickel-chelate chromatography.
[0145] In another embodiment, endogenous genes are transcribed by operatively linking them to expression control sequences supplied endogenously that recombine with genomic DNA. In one method, one provides the cell with a recombinant polynucleotide containing a targeting sequence, which permits homologous recombination into the genome upstream of the transcriptional start site of target gene; the expression control sequences; an exon of the target gene; and an unpaired splice-donor site which pairs with a splice acceptor in the target gene. Such methods are discussed in Treco et al., WO 94/12650; Treco et al., WO 95/31560 and Treco et al., WO 96/29411.
[0146] The invention also provides recombinant cells comprising a recombinant polynucleotide having expression control sequences operatively linked with a nucleotide sequence encoding a polypeptide of this invention. Host cells can be selected for high levels of expression in order to purify the protein. Mammalian cells are preferred for this purpose, but prokaryotic cells, such as E. coli, also are useful. The cell can be, e.g., a recombinant cell in culture or a cell in vivo.
[0147] V. Polynucleotide Probes and Primers
[0148] The nucleotide sequence of a hormone-regulated gene is useful for preparing polynucleotide probes and primers that specifically hybridize to a polynucleotide encoding a hormone-regulated gene or cDNA under stringent hybridization conditions. The probes and primers of this invention are polynucleotides of at least 7 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides or at least 25 nucleotides. Any suitable region of the hormone-regulated gene may be chosen as a target for polynucleotide hybridization. Nucleotide substitutions, deletions, and additions may be incorporated into the polynucleotides as long as the characteristic ability to specifically hybridize to the target sequence or its complement is retained. Nucleotide sequence % variation may result from sequence polymorphisms of various alleles, minor sequencing errors, and the like.
[0149] The probes and primers of the invention are useful as probes in hybridization assays, such as Southern and Northern blots, for identifying polynucleotides having a nucleotide sequence encoding a hormone-regulated polypeptide, and as primers for amplification procedures. The probes and primers of the invention are also useful in detecting the presence, absence or amount of a hormone-regulated gene in tissue biopsies and histological sections where the detection method is carried out in situ, typically after amplification of a hormone-regulated gene sequences using a primer set.
[0150] The probes and primers of this invention also are useful for identifying allelic forms of a hormone-regulated gene and animal cognate genes. Probes and primers can be used to screen human or animal genomic DNA or cDNA libraries under, e.g., stringent conditions. DNA molecules that specifically hybridize to the probe are then further examined to determine whether they are allelic variants or animal cognates.
[0151] The probes also are useful in oligonucleotide arrays. Such arrays are used in hybridization assays to check the identity of bases in a target polynucleotide. In essence, when a target hybridizes perfectly to a probe on the array, the target contains the nucleotide sequence of the probe. When the target hybridizes less well, or does not hybridize at all, then the target and probe differ in sequence by one or more nucleotide. By proper selection of probes, one can check bases on a target molecule. See, e.g., Chee et al., WO 95/11995. The use the hormone-regulated gene sequence in genomics is described further below.
[0152] In one embodiment, the polynucleotide further comprises a label. A detectable moiety bound to either an oligonucleotide primer or a probe is subsequently used to detect hybridization of an oligonucleotide primer to the RNA component. Detection of labeled material bound to a hormone-regulated polynucleotide in a sample provides a means of determining a diagnostic or prognostic value.
[0153] Although primers and probes can differ in sequence and length, the primary differentiating factor is one of function: primers serve as an initiation point for DNA synthesis of a target polynucleotide, as in RT and PCR reactions, while probes are typically used for hybridization to and detection of a target polynucleotide. Typical lengths of primers or probes can range from 7-50 nucleotides, preferably from 10-40 nucleotides, and most preferably from 15-35 nucleotides. A primer or probe can also be labeled with a detectable moiety for detection of hybridization of the primer or probe to the target polynucleotide.
[0154] In general, those of skill in the art recognize that the polynucleotides used in the invention include both DNA and RNA molecules and naturally occurring modifications thereof, as well as synthetic, non-naturally occurring analogs of the same, and heteropolymers, of deoxyribonucleotides, ribonucleotides, and/or analogues of either. The particular composition of a polynucleotide or polynucleotide analog will depend upon the purpose for which the material will be used and the environment in which the material will be placed. Modified or synthetic, non-naturally occurring nucleotides have been designed to serve a variety of purposes and to remain stable in a variety of environments, such as those in which nucleases are present.
[0155] Oligonucleotides preferably are synthesized, e.g., on an Applied Biosystems or other commercially available oligonucleotide synthesizer according to specifications provided by the manufacturer. Oligonucleotides may be prepared using any suitable method, such as the phosphotriester and phosphodiester methods, or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidates are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters 22: 1859 (1981), and U.S. Pat. No. 4,458,066.
[0156] Polynucleotides, e.g., probes, also can be recombinantly produced through the use of plasmids or other vectors.
[0157] VI. Methods for Detecting Polynucleotides Encoded by Hormone-Regulated Genes
[0158] The probes and primers of this invention are useful, among other things, in detecting in a sample polynucleotides having sequences of hormone-regulated gene. A method for detecting the presence, absence or amount of such polynucleotide in a sample involves two steps: (1) specifically hybridizing a polynucleotide probe or primer to a hormone-regulated polynucleotide, and (2) detecting the specific hybridization.
[0159] For the first step of the method, the polynucleotide used for specific hybridization is chosen to hybridize to any suitable region of a hormone-regulated gene. The polynucleotide can be a DNA or RNA molecule, as well as a synthetic, non-naturally occurring analog of the same. The polynucleotides in this step are polynucleotide primers and polynucleotide probes disclosed herein.
[0160] For the second step of the reaction, any suitable method for detecting specific hybridization of a polynucleotide to a hormone-regulated gene may be used. Such methods include, e.g., amplification by extension of a hybridized primer using reverse transcriptase (RT); extension of a hybridized primer using RT-PCR or other methods of amplification; and in situ detection of a hybridized primer. In in situ hybridization, a sample of tissue or cells is fixed onto a glass slide and permeablized sufficiently for use with in situ hybridization techniques. Detectable moieties used in these methods include, e.g., labeled polynucleotide probes; direct incorporation of label in amplification or RT reactions, and labeled polynucleotide primers.
[0161] Often, cell extracts or tissue samples used in methods for determining the amount of a polynucleotide in a sample will contain variable amounts of cells or extraneous extracellular matrix materials. Thus, a method for determining the cell number in a sample is important for determining the relative amount per cell of a test polynucleotide. A control for cell number and amplification efficiency is useful for determining diagnostic values for a sample of a potential cancer, and a control is particularly useful for comparing the amount of test polynucleotide in sample to a prognostic value for the disease. A preferred embodiment of the control RNA is endogenously expressed 28S rRNA. (See, e.g., Khan et al., Neurosci. Lett. 147: 114-117 (1992) which used 28S rRNA as a control, by diluting reverse transcribed 28S rRNA and adding it to the amplification reaction.)
[0162] VII. Inhibitory Polynucleotides for Inhibiting Hormone-Regulated Gene Expression
[0163] A. General
[0164] This invention also provides inhibitory polynucleotides directed against polynucleotides encoding hormone-regulated genes that inhibit hormone-regulated gene expression and, therefore inhibit its activity in a cell. Inhibitory polynucleotides can inhibit gene activity in a number of ways. According to one mechanism, the polynucleotide prevents transcription of the hormone-regulated gene (for instance, by triple helix formation). In another mechanism, the polynucleotide destabilizes the hormone-regulated gene and reduces its half-life.
[0165] An inhibitory polynucleotide is a polynucleotide that is capable of specifically hybridizing with a target polynucleotide and that interferes with the transcription, processing, translation or other activity the target polynucleotide. Thus, the polynucleotide can be targeted at the promoter, the site of ribosome binding to the mRNA, etc. Inhibitory polynucleotides generally are single-stranded and have a sequence of at least 7, 8, 9, 10, or 11 nucleotides capable of specifically hybridizing to the target sequence. RNA sequences generally require a sequence of at least 10 nucleotides for specific hybridization. Inhibitory polynucleotides include, without limitation, antisense molecules, ribozymes, sense molecules and triplex-forming molecules. In one embodiment, the inhibitory polynucleotide is no more than about 50 nucleotides long.
[0166] While not wishing to be limited by theory, it is believed that inhibitory polynucleotides inhibit the function of a target, in part, by binding to the appropriate target sequence. An inhibitory polynucleotide can inhibit DNA transcription by, for example, interfering with the attachment of RNA polymerase to the promoter by binding to a transcriptional initiation site or a template. It can interfere with processing of mRNA, poly(A) addition to mRNA or translation of mRNA by, for example, binding to regions of the RNA transcript such as the 5′ capping site, the ribosome binding site, splice junctions or poly A tail. It can promote inhibitory mechanisms of the cells, such as promoting RNA degradation via RNase action. The inhibitory polynucleotide can bind to the major groove of the duplex DNA to form a triple helical or “triplex” structure. Methods of inhibition using inhibitory polynucleotides therefore encompass a number of different approaches to altering expression of specific genes that operate by different mechanisms. These different types of inhibitory polynucleotide technology are described in C. Helene and J. Toulme, (1990) Biochim. Biophys. Acta., 1049:99-125. Properties of the polynucleotide can be engineered to impart stability (e.g., nuclease resistance), tighter binding or the desired Tm. See, e.g., International patent publication No. 94/12633.
[0167] The general approach to constructing various polynucleotides useful in inhibitory polynucleotide therapy has been reviewed by A. R. Vander Krol et al. (1988), Biotechniques 6:958-976, and by C.A. Stein et al., (1988) Cancer Res. (1988) 48:2659-2668. See also Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, Cohen, J. S., editor, MacMillan Press, London, pages 79-196 (1989), and Antisense RNA and DNA, (1988), D. A. Melton, Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. In certain embodiments inhibitory polynucleotides comprise a derivatized substituent which is substantially non-interfering with respect to hybridization of the inhibitory polynucleotide to the target polynucleotide.
[0168] For general methods relating to antisense polynucleotides, see Antisense RNA and DNA, (1988), D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). For a review of antisense therapy, see, e.g., Uhlmann et al., Chem. Reviews, 90:543-584 (1990).
[0169] Cleavage of a hormone-regulated gene can be induced by the use of ribozymes or catalytic RNA. In this approach, the ribozyme would contain either naturally occurring RNA (ribozymes) or synthetic nucleic acids with catalytic activity. Bratty et al., (1992) Biochim. Biophys. Acta., 1216:345-59 (1993) and Denhardt, (1992) Ann. N.Y. Acad. Sci., 660:70-76 describe methods for making ribozymes. Optimum target sites for ribozyme-mediated inhibition of activity can be determined as described by Sullivan et al., PCT patent publication No. 94/02595 and Draper et al., PCT patent publication No. 93/23569.
[0170] B. Methods for Making Inhibitory Polynucleotides
[0171] Inhibitory polynucleotides can be made chemically or recombinantly.
[0172] 1. Chemical Synthesis
[0173] Small inhibitory polynucleotides for direct delivery can be made by chemical synthesis. Chemically synthesized polynucleotides can be DNA or RNA, or can include nucleotide analogs or backbones that are not limited to phosphodiester linkages.
[0174] 2. Recombinant Production
[0175] For delivery into cells or for gene therapy methods, recombinant production of inhibitory polynucleotides through the use of expression vectors is particularly useful. Accordingly, this invention also provides expression vectors, e.g., recombinant polynucleotide molecules comprising expression control sequences operatively linked to the nucleotide sequence encoding the inhibitory polynucleotide.
[0176] VIII. Polypeptides Encoded by Hormone-Regulated Genes
[0177] Polynucleotides encoding hormone-regulated genes also are useful for producing the polypeptides they encode (“hormone-regulated polypeptides”). This invention also provides purified, recombinant hormone-regulated polypeptides and analogs. Hormone-regulated analogs include active analogs, inactive analogs and immunogenic analogs.
[0178] Hormone-regulated polypeptides are native polypeptides, including allelic variants. Polynucleotide molecules that encode allelic variants are isolatable from cDNA or genomic DNA and typically hybridize under stringent conditions to the nucleotide sequence encoding a hormone-regulated gene.
[0179] Hormone-regulated polypeptides are useful as immunogens to elicit the production of anti-bodies against hormone-regulated polypeptides, as affinity capture molecules to isolate such antibodies from a mixture, and as controls in diagnostic methods aimed at detecting a hormone-regulated gene in a sample.
[0180] A polypeptide analog is a polypeptide whose sequence is not naturally occurring but is substantially identical over its sequence to a sequence of a native gene. Analogs include active analogs, inactive analogs and immunogenic analogs. Analogs also include fusion proteins, i.e., polypeptides having a hormone-regulated polypeptide or analog moiety fused with another polypeptide moiety at its amino- or carboxy-terminal end.
[0181] Active analogs have the biological activity of a hormone-regulated gene. Active a hormone-regulated gene analogs can be produced by, for example, introducing conservative amino acid substitutions into the sequence of native a hormone-regulated gene. Active fragments of a hormone-regulated gene can be identified empirically by truncating the protein from either the amino-terminus or the carboxy-terminus to generate fragments, and testing the resulting fragments for a hormone-regulated gene activity.
[0182] Inactive analogs are polypeptides of at least 5 amino acids whose amino acid sequence over its length is substantially identical to native a hormone-regulated polypeptide. Inactive analogs include, for example, polypeptides encoding fragments of a native polypeptide. Inactive analogs are useful as inhibitory polypeptide mimics or decoys. When expressed in a cell they can exhibit dominant/negative effects by competing with native polypeptides for interaction with molecules that naturally interact with the polypeptide.
[0183] Immunogenic analogs are polypeptides having a sequence of at least 5 amino acids selected from a native hormone-regulated polypeptide and which, when presented to an animal as an immunogen, elicit a humoral or cell-mediated immune response. This includes polypeptides comprising an amino acid sequence which is an epitope from a hormone-regulated polypeptide, such as immunogenic fragments of a hormone-regulated polypeptides.
[0184] Hormone-regulated polypeptides and analogs are most easily produced recombinantly, as described herein. Recombinant a hormone-regulated gene can be purified by affinity purification. In one method, recombinant hormone-regulated polypeptides or analogs comprise a polyhistidine tag. The protein is purified on a nickel-chelate affinity matrix. In another method, the recombinant protein is purified using an affinity matrix carrying antibody that specifically binds the polypeptide.
[0185] IX. Antibodies and Hybridomas
[0186] Hormone-regulated polypeptides are useful in the preparation of compositions comprising an antibody that specifically binds the hormone-regulated polypeptide. Antibodies preferably have affinity of at least 106 M−1, 107 M−1, 108 M−1, or 109 M−1. This invention contemplates both polyclonal and monoclonal antibody compositions.
[0187] The antibodies of the invention have many uses. For example, such antibodies are useful for detecting hormone-regulated polypeptides in immunoassays. The antibodies also can be used to screen expression libraries for particular expression products of a hormone-regulated gene. They are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens. Usually the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding. Antibodies raised against a hormone-regulated polypeptide can also be used to raise anti-idiotypic antibodies. The antibodies of this invention are also used for affinity chromatography in isolating a hormone-regulated proteins. Antibodies also are useful in the preparation of immunotoxins. Immunotoxins bind to cells that express a hormone-regulated polypeptide and kill them.
[0188] A full-length hormone-regulated polypeptide or an immunogenic fragment of it is a suitable immunogen for producing antibodies. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide. Methods for producing polyclonal antibodies are known to those of skill in the art. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY.
[0189] Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified polypeptides, or screened for agonistic or antagonistic activity, e.g., activity mediated through a hormone-regulated gene.
[0190] Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546.
[0191] Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.
[0192] An alternative approach is the generation of humanized immunoglobulins by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., U.S. Pat. No. 5,585,089.
[0193] A further approach for isolating DNA sequences which encode a human monoclonal antibody or a binding fragment thereof is by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989) and then cloning and amplifying the sequences which encode the antibody (or binding fragment) of the desired specificity. The protocol described by Huse is rendered more efficient in combination with phage display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. Phage display technology can also be used to mutagenize CDR regions of antibodies previously shown to have affinity for a hormone-regulated gene protein receptors or their ligands. Antibodies having improved binding affinity are selected.
[0194] In another embodiment of the invention, fragments of antibodies against a hormone-regulated gene protein or protein analogs are provided. Typically, these fragments exhibit specific binding to the hormone-regulated gene protein receptor similar to that of a complete immunoglobulin. Antibody fragments include separate heavy chains, light chains Fab, Fab′ F(ab′)2 and Fv. Fragments are produced by recombinant DNA techniques, or by enzymic or chemical separation of intact immunoglobulins.
[0195] X. Methods for Detecting Polypeptides
[0196] A hormone-regulated polypeptide can be identified by any methods known in the art. In one embodiment, the methods involve detecting the polypeptide with a ligand that specifically recognizes the polypeptide. The antibodies of the invention are particularly useful for specific detection of a hormone-regulated polypeptide. A variety of antibody-based detection methods are known in the art. These include, for example, radioimmunoassay, sandwich immunoassays (including ELISA), western hybridization (e.g., blot), isolation on antibodies bound to a solid phase and in situ detection with labeled antibodies. Another method for detecting a hormone-regulated polypeptide involves identifying the polypeptide according to its mass through, for example, gel electrophoresis, mass spectrometry or HPLC. Subject samples can be taken from any number of appropriate sources, such as blood, urine, tissue biopsy (e.g., lymph node tissue), etc.
[0197] XI. Polypeptide and Polynucleotide Vaccines Against Hormone-Regulated Diseases
[0198] This invention also provides vaccines against cells expressing a hormone-regulated gene and methods for using them.
[0199] In one aspect, this invention provides methods for eliciting a humoral immune response against a hormone-regulated polypeptide. The method involves immunizing a subject with a vaccine comprising an immunogenic amount of a hormone-regulated polypeptide or an immunogenic analog. Such vaccines elicit antibodies against the hormone-regulated polypeptide.
[0200] In another aspect, this invention provides methods for eliciting an MHC Class II-dependent immune response against cells expressing a hormone-regulated gene. MHC Class II molecules bind peptides having particular amino acid motifs well known in the art. The MHC Class II-dependent response involves the uptake of an antigen by antigen-presenting cells (APC's), its processing, and presentation on the cell surface as pan of an MHC Class II/antigenic peptide complex. Alternatively, MHC Class II molecules on the cell surface can bind peptides having the proper motif.
[0201] Antigen presenting cells interact with CD4-positive T-helper cells, thereby activating the T-helper cells. Activated T-helper cells stimulate B-lymphocytes to produce antibodies against the antigen. Antibodies mark cells bearing the antigen on their surface. The marked cells are subject to antibody-dependent cell-mediated cytotoxicity, in which NK cells or macrophages, which bear Fc receptors, attack the marked cells.
[0202] Methods for eliciting an MHC Class II-dependent immune response involve administering to a subject a vaccine including an immunogenic amount of a hormone-regulated polypeptide or an immunogenic analog that includes an amino acid motif recognized by MHC Class II molecules of the subject. Alternatively, antigen presenting cells can be cultured with such peptides to allow binding, and the cells can be administered to the subject. Preferably, the cells are syngeneic with the subject.
[0203] In another aspect, this invention provides methods for eliciting an MHC Class I-dependent cell-mediated immune response against cells expressing a hormone-regulated gene in a subject. MHC Class I molecules also bind peptides having particular amino acid motifs well known in the art. Proteins expressed in a cell are digested into peptides, become associated with MHC Class I molecules and are presented on the cell surface. There, they are recognized by CD8-positive lymphocytes, generating a cytotoxic T-lymphocyte response against cells expressing the epitopes in association with MHC Class I molecules.
[0204] Another method involves transfecting cells ex vivo with such expression vectors, and administering the cells to the subject. The cells preferably are syngeneic to the subject. Another method of delivering DNA vaccines involves rubbing a solution comprising the polynucleotide into the skin.
[0205] Methods for eliciting an immune response against a disease-associated hormone-regulated polypeptide in a subject are useful in prophylactic methods for preventing a hormone-regulated disease when the vaccine is administered to a subject who does not already suffer from the disease.
[0206] XII. Transgenic Non-Human Animals
[0207] Hormone-regulated genes identified by the methods of this invention can be introduced into animals to produce non-human mammals transgenic for the hormone-regulated gene. As used herein. “animal transgenic for a hormone-regulated gene” refers to an animal, in particular a mammal, whose germ cells (i.e., oocytes or sperm), at least, comprise a recombinant nucleic acid molecule comprising expression control sequences operatively linked to a nucleic acid sequence encoding the hormone-regulated gene. The expression control sequences can be constitutive, or they can be inducible or repressible, so that expression of the gene can be regulated. Such animals are useful, for example, as models in the study of hormone-regulated diseases.
[0208] The transgenic animals of this invention are produced, for example, by introducing the recombinant nucleic acid molecule into a fertilized egg or embryonic stem (ES) cell, typically by microinjection, electroporation, lipofection, particle-mediated gene transfer. The transgenic animals express the heterologous nucleotide sequence in tissues depending upon whether the promoter is inducible by a signal to the cell, or is constitutive. Transgenic animals can be bred with non-transgenic animals to produce transgenic animals with mixed characteristics.
[0209] XIII. Methods for Identifying Traits in Functional Pathways that Include a Hormone-Regulated Gene
[0210] A hormonal signal usually initiates a cascade of cellular events in a hormone-regulated functional pathway that results in expression of a trait or traits. It can include regulation of hormone receptors, receptor number, signal transduction, receptor phosphorylation, or production of transcriptional, translational or cell cycle regulators. A hormone-regulated gene can exist at any point of a hormone-regulated functional pathway. Its identification by the methods of this invention can be the springboard for identifying other genes, biochemical events or traits in the pathway. This invention provides methods for identifying traits located either up-stream or down-stream of the hormone-regulated gene in the functional pathway.
[0211] A. Down-Stream Traits
[0212] A trait is considered to be located down-stream of a hormone-regulated gene in a functional pathway if hormonal regulation of the gene (e.g., hormonally induced induction or repression of the gene) in turn causes expression of the trait. A method for identifying a down-stream trait in a hormone-regulated functional pathway involves determining whether reversing hormone-regulation of the gene in a cell in the hormonal milieu alters expression of the trait.
[0213] In one embodiment, the method involves providing two samples. One sample comprises hormonally responsive biological material. A second sample comprises hormonally responsive biological material in which the cells have been provided with means for down-regulating genes that are up-regulated in response to a hormonal stimulus, or means for up-regulating expression of genes that are down-regulated in response to a hormonal stimulus. The means can be any known in the art or described herein. For example, gene expression can be up-regulated by providing the cells with expression vectors for expressing the gene. The expression control sequences can be inducible or repressible, so that the gene can be activated at will by supplying a transcriptional activator or repressor. Gene expression can be down-regulated by providing inhibitory polynucleotides, such as antisense polynucleotides, that interfere with transcription, processing or translation. Alternatively, gene expression can be knocked-out by, for example, sit-directed mutagenesis of the gene or homologous recombination that inactiviates the gene. Then, the two samples are exposed to a hormonal environment to which the hormone-responsive gene is responsive. The samples are exposed for a time sufficient to induce hormone-regulated expression of the gene. Then, the samples are compared to identify traits whose expression is different. Because these samples have been exposed to the same hormonal environment, any differences in expression are likely due to interference with hormonal regulation of the gene. Such traits are identified as down-stream traits in the functional pathway.
[0214] B. Up-Stream Traits
[0215] A trait is considered to be located up-stream of a hormone-regulated gene in a functional pathway if hormonally induced expression of the trait in turn causes hormonal regulation of the gene. A method for identifying an up-stream trait in a hormone-regulated functional pathway involves determining whether reversing hormone-regulation of the trait in a cell in the hormonal milieu alters hormone regulation of the gene.
[0216] In one embodiment, the method involves providing two samples. One sample comprises hormonally responsive biological material. A second sample comprises hormonally responsive material in which the cells have been provided with means for up- or down-regulating a gene that is a candidate as an up-stream gene. The means can be any known in the art or described herein. Then, the two samples are exposed to a hormonal environment to which the hormone-responsive gene is responsive. The samples are exposed for a time sufficient to induce hormone-regulated expression of the gene. Then, the samples are compared to determine whether expression of the hormone-responsive trait is different. Because these samples have been exposed to the same hormonal environment, any differences in expression in the hormone-responsive gene are likely due to up- or down-regulation of the candidate gene. Such candidate genes are identified as up-stream regulators in the functional pathway of the hormone-regulated gene.
[0217] The altered cells generally are exposed to the same hormonally coded environment. This can be achieved by administering the altered cells and control cells as grafts to the same host or different hosts that provide the hormonal environment that regulates the gene. In another embodiment, altered cells can be administered to male and female hosts. In both cases, after sufficient time for the hormonal code to be processed by the cells, and to retrieve sufficient graft tissue for analysis, traits between the samples are compared.
[0218] XIV. Drug Function and Efficacy Studies
[0219] One aspect of drug testing is determining whether a drug has similar function and efficacy in females and males. The model system of this invention is useful making such determinations and, more generally, in determining drug efficacy in animals of different reproductive states. The methods involve exposing a graft of hormonally responsive biological material in animals providing different hormonal environments (e.g., different reproductive states), contacting the material with the drug, and determining whether the biological material is differently effected in each of the hosts. For example, one could test the effect of a drug on cancer growth or remission in males and females. Such testing would be a much less expensive preliminary test than first performing the test on humans.
[0220] XV. Methods for Screening for Compounds that Regulate Expression or Activity of a Disease-Associated, Hormone-Regulated Gene
[0221] The treatment of certain diseases known to be hormone regulated involves modulating the hormonal environment. For example, estrogen antagonists are used in the treatment of estrogen-regulated diseases, such as ovarian cancer. However, such modulation can have other actions besides its impact on the target diseased material. For example, antagonizing estrogen in the treatment of ovarian cancer also decreases the level of heart-protecting low density lipoproteins in the blood. Therefore, it is useful to screen for agents that inhibit the action of hormones on diseased tissue but do not effect, or have a proportionally smaller effect, on desired biological functions.
[0222] Accordingly, this invention provides screening methods for identifying agents that modulate expression of disease-associated, hormone-regulated genes. The method is particularly useful for screening those agents already known to modulate the activity of a hormone. The methods involve exposing diseased biological material to a hormonal environment that regulates expression of the gene. The biological material is contacted with the agent. Then, one determines whether expression of the hormone-regulated gene is altered compared to biological material under control conditions (e.g., without contacting the biological material with the agent).
[0223] In one embodiment, the methods involve transplanting an animal host that provides a hormonal environment that regulates the gene with a graft of cells responsive to the environment. The compound is administered to the animal and the amount of the hormone-regulated polynucleotide or polypeptide in a sample from the graft is measured. Then one determines whether the measured amount is different than the amount expected in a sample to which no agent has been administered. A difference between the measured amount and the expected amount indicates that the compound alters the amount of expression of the hormone-regulated gene. The cells also can be cultured ex vivo and the compound administered to the cells in culture.
[0224] The agent to be tested can be selected from a number of sources. For example, combinatorial libraries of molecules are available for screening experiments. Using such libraries, thousands of molecules can be screened for regulatory activity. In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. In one embodiment, the agent is a compound known to regulate the action of a hormone.
[0225] Furthermore, this invention provides screening methods for agents that modulate expression of disease-associated, hormone-regulated genes, but that do not modulate (or modulate to a lesser extent) desired biological activities of the hormone. This includes, for example, traits of an organism that one does not desire to modulate. For example, as discussed above, one may wish to screen agents for those that inhibit the inducing effect of testosterone on Repro-PC-1.0 in prostate cancer, but have little or no effect on desired activities of testosterone, such as fertility, urological function or sexual function. Thus, cancerous prostate tissue can be grafted into a male mouse, which provides a hormonal environment that up-regulates Repro-PC-1.0. Then, a testosterone inhibitor can be administered to the mouse. Its effect on both Repro-PC-1.0 and sexual function can be determined in the usual ways. In one embodiment, one can determine whether the extent to which the antagonist inhibits one function is the same, greater or less than the extent to which it inhibits the other function. Drugs that preferentially modulate expression of the disease-specific, hormone-regulated gene are better drug candidates.
[0226] XVI. Diagnostic, Monitoring and Prognostic Methods
[0227] Disease-associated hormone-regulated genes are highly selective and highly specific markers for the disease. Accordingly, the methods described herein for detecting a disease-associated hormone-regulated gene or polypeptide in a sample are useful in methods for diagnosing the disease, monitoring its progress or treatment, and determining patient prognosis. The methods of the present invention allow disease conditions to be detected with increased confidence and at an earlier stage, for example, before cells are detected as cancerous based on pathological characteristics. It is, of course, understood by diagnosticians that diagnostic tests are measured by their degree of specificity and sensitivity. Tests which are not perfectly specific or sensitive are, nevertheless, useful in diagnosis because they provide useful information which, in combination with other evidence, can provide a definitive diagnosis or indicate a course of treatment.
[0228] Methods for diagnosis involve determining a diagnostic amount of a hormone-regulated trait (e.g., mRNA. cDNA or polypeptide) in a patient sample and comparing that amount with a normal range expected to be found in the sample. The samples used to determine the normal range of expression can be normal samples from the individual to be tested, or normal samples from other individuals not suffering from the disease condition.
[0229] XVII. Karyotyping
[0230] A translocation in a chromosome at or near locus of a gene can result in alteration of the gene's activity, such as activated transcription or changed function. Chromosomal translocations in the vicinity of a hormone-regulated gene can be detected by hybridizing a labeled probe of this invention to a chromosome spread. A translocation, duplication or deletion can be identified by aberrant hybridization patterns compared to normal. Such tests are useful in detecting genetic abnormalities such as familial disposition to prostate cancer, or early onset of the disease. A method for fluorescent in situ hybridization of chromosomes is provided in the Examples.
[0231] XVIII. Genomics
[0232] The identification of cognate or polymorphic forms of hormone-regulated genes and the tracking of those polymorphisms in individuals and families is important in genetic screening. Accordingly, this invention provides methods useful in detecting polymorphic forms of hormone-regulated genes. The methods involve comparing the identity of a nucleotide or amino acid at a selected position from the sequence of a test gene with the nucleotide or amino acid at the corresponding position from the sequence of the native hormone-regulated gene. The comparison can be carried out by any methods known in the art, including direct sequence comparison by nucleotide sequencing, sequence comparison or determination by hybridization or identification of RFLPs.
[0233] In one embodiment, the method involves sequencing the entire test polynucleotide or polypeptide, or a subsequence from it, and comparing that sequence with the sequence of a native hormone-regulated gene. In another embodiment, the method involves identifying restriction fragments produced upon restriction enzyme digestion of the test polynucleotide and comparing those fragments with fragments produced by restriction enzyme digestion of a native hormone-regulated gene. Restriction fragments from the native gene can be identified by analysis of the sequence to identify restriction sites. Another embodiment involves the use of oligonucleotide arrays. (See, e.g., Fodor et al., U.S. Pat. No. 5,445,934.) The method involves providing an oligonucleotide array comprising a set of oligonucleotide probes that define sequences selected from the native hormone-regulated gene sequence, generating hybridization data by performing a hybridization reaction between the target polynucleotide molecules and the probes in the set and detecting hybridization between the target molecules and each of the probes in the set and processing the hybridization data to determine nucleotide positions at which the identity of the target molecule differs from that of a native hormone-regulated gene. The comparison can be done manually, but is more conveniently done by a programmable, digital computer.
EXAMPLE[0234] Using the model system of this invention, a hormone-regulated gene from human prostate cancer cells, called Repro-PC-1.0, was identified.
[0235] I. LNCaP Tumor Xenografts Display Morphological and Metastatic Characteristics that Are Dependent upon the Environment in which They Are Generated Using a model system of LNCaP human prostate tumor cells, tumors were raised in both male and female athymic mice. The tumors, which typically took at least 2-3 months to develop into palpable masses, showed remarkably different features depending on whether they were propagated in a male or a female environment. Tumors that developed in male mice (male-LNCaP) showed extensive vascularization and morphological destruction compared to the highly regular and homogeneous composition of the tumors raised in females (female-LNCaP) (FIG. 1). Additionally, the tumors raised in male hosts had gained metastatic potential, whereas no metastases were ever detected in female mice. The differential morphologies of these tumors suggested the possibility that specific sequences involved in metastatic progression and/or angiogenesis had been induced in the male environment.
[0236] The aberrant expression of a number of oncogenes and growth factors (up-regulation) as well as tumor suppressor genes (down-regulation) has been implicated in a number of human tumors and in cancer progression in general. Therefore the expression of a representative panel of these sequences was examined in order to investigate whether the differential appearance of male derived LNCaP tumors was the result of an androgen driven up- or down-regulation of a previously characterized factor. RNA isolated from male-LNCaPs, female-LNCaPs, LNCaP cells, PC-3 cells (a non-androgen responsive human prostate cell line), normal human spleen tissue and normal human liver tissue was subjected to Northern hybridization using a variety of probes for previously characterized oncogenes, growth factors and tumor suppressors. As can be seen in Table 1, there was no difference in the pattern of expression detected in male-LNCaP tumors compared to female-LNCaP tumors or to LNCaP cells grown in vitro indicating that the differentiated state of male-LNCaP tumors was not due to a differential expression of any of these more common factors. 1 TABLE 1 ONCOGENE EXPRESSION TO LNCaP TUMORS Summaxy of Northern hybridization results for a variety of oncogene probes to 4 &mgr;g total RNA from different tissues, cell lines and tissues. a-f, The presence of detectable RNA was scored on a relative basis. +, detectable levels; −, undetectable levels. LNCaPa LNCaPb Probe (Male) (Female) LNCaPc PC-3d Spleene Liverf FGF, acidic + + + + − − FGF, basic + + + + − − PDGF, A-chain + + + + − + PDGF, B-chain + + + + + + PDGF-receptor + + + + − − TGF-alpha + + + + − − TGF-B + + + + − − c-myc + + + + − − EGF-receptor + + + + − − Ha-ras + + + + + + Ki-ras + + + + − − NM23 + + + + − − c-myc (oligo) + + + − − − p53 (oligo) + + + + + + androgen re- + + + + − − ceptor (oligo) TNF-alpha + + + + − + (oligo) TNF-B (oligo) + + + + − − int-2 (oligo) + + + + + + actin + + + + + + alpha-tubulin + + + + + + aRNA isolated from LNCaP prostate adenocarcinoma tumors propagated in male athymic mice. bRNA isolated from LNCaP prostate adenocarcinoma tumors propagated in female athymic mice. cRNA isolated from LNCaP prostate adenocarcinoma cells. dRNA isolated from PC-3 (non-androgen responsive) cells. eRNA isolated from human spleen tissue. fRNA isolated from human liver tissue.
[0237] II. Isolation of Male-LNCaP-Specific Sequences
[0238] In order to isolate sequences that are over-expressed in male-LNCaP tumors and that might elucidate the mechanism(s) responsible for the dramatic morphological differences, a male-LNCaP-specific probe was generated by three rounds of subtractive hybridization with female-LNCaP tumor cDNA. This male-LNCaP specific probe was then used to perform a primary screen of a lambda-ZAP-male-LNCaP tumor cDNA library. Positive plaques were subjected to a dual secondary screen, using the male-LNCaP specific probe and total female-LNCaP tumor cDNA. Clones were considered positive if they hybridized strongly to the “male-specific” probe and weakly to the female probe.
[0239] The resulting positives were subjected to a tertiary screen in which the clones were “rescued” and their plasmid DNA was subjected to duplicate Southern hybridizations using total male-LNCaP or total female-LNCaP cDNA. The DNA from clones hybridizing more strongly to male-LNCaP sequences was then subjected to Northern analyses. The DNA from one clone, Repro-PC-1.0, when hybridized to equivalent amounts of RNA from male-LNCaP tumors, female-LNCaP tumors or LNCaP cells showed an ˜10× amplification of a single 4.4 kb mRNA in male-LNCaP tumors (FIG. 2a). Rehybridization of the same blot with probes for actin and tubulin showed that the amplified signal detected by Repro-PC-1.0 in male-LNCaP tumors was not due to an increased level of RNA in that lane (FIG. 2b).
[0240] III. Sequence Analysis of Repro-PC-1.0
[0241] Initial sequence analysis of clone Repro-PC-1.0 did not reveal any significant open reading frames (ORFs) in either direction. Subsequently, an overlapping clone (PS5-1), was isolated from the male-LNCaP tumor library by hybridization with an oligonucleotide probe encoding 5′ sequences contained within the Repro-PC-1.0 insert. Directionality of the Repro-PC-1.0 clone was inferred from the presence of a putative poly-A tail. The complete coding region was determined by sequencing PS5-1 and an overlapping RACE-PCR derived 5′ end cDNA clone. The alignment of these clones is shown in FIG. 3. Northern analyses of the 2 overlapping clones to panels of RNA isolated from male-LNCaP tumors, female-LNCaP tumors, LNCaP cells and PC-3 cells revealed the same pattern of hybridization as originally observed with Repro-PC-1.0.
[0242] Sequence analysis of the overlapping clones revealed a single 1275 bp ORF encoding 425 amino acids, followed by a 2466 bp 3′ untranslated region to which a polyadenylate tail was added (SEQ ID NO: 1). The sequence obtained for the 5′ untranslated region of Repro-PC-1.0 was much shorter than the approximately 750 bp of sequence predicted, based on the size of the 4.4 kb message that was observed by RNA blot hybridizations, and is therefore probably incomplete. The Mr and pI were calculated to be 48,070 and 8.83, respectively.
[0243] Sequence identity and similarity were determined using the FASTDB program, Bionet suite, Oxford Molecular Group, Campbell, Calif. or BLASTX, NCBI.
[0244] The predicted amino acid sequence of the C-terminal region of Repro-PC-1.0 contained two copies of a 116 amino acid direct repeat that had 34% identity (41% similarity) with each other. These repeats are located from amino acid 150 to 252 and amino acid 276 to 396. The repeats were found to be homologous to the C2 regulatory domain of calcium-dependent isoforms of protein kinase C (PKC), and to isoforms of synaptotagmin (FIG. 4).
[0245] Synaptotagmin is actually a family of highly conserved, abundant synaptic vesicle proteins that has been proposed to play a role in synaptic vesicle translocation to the presynaptic release site of the plasma membrane (docking) and/or fusion of these two membranes. Structurally, synaptotagmin isoforms can be divided into several domains which include: an intravesicular, amino-terminal domain; a single transmembrane domain; and a cytoplasmic, carboxyl-terminal domain that consists of two repeats homologous to the C2 regulatory domain of PKC termed A and B (FIG. 4). Homologues of synaptotagmin have shown greater conservation of sequence identity and similarity in the cytoplasmic domain containing the two PKC C2-homologous repeats than in the N-terminal intravesicular or transmembrane domains.
[0246] FIG. 5 shows the alignment of the amino acid sequences for Repro-PC-1.0 and rat synaptotagmin IV. Repro-PC-1.0 shows 90% overall identity with rat synaptotagmin IV. Like the other synaptotagmin isoforms, Repro-PC-1.0 was most similar to these sequences in the PKC C2 repeat C-terminal region (91% identity). The two internal repeats of Repro-PC-1.0 are approximately as homologous to each other (34% identity) as to the corresponding region of PKC (identity between 35% and 43% depending on the isoform). As in the other forms of synaptotagmin, the amino acid residues that are identical between the two internal repeats of Repro-PC-1.0 are also conserved between Repro-PC-1.0 and PKC, revealing a core consensus sequence of SDPYV/IK followed by a stretch of basic residues (FIG. 6).
[0247] Hydrophobicity plots of the amino acid sequence of Repro-PC-1.0 revealed a single segment, from residues 15-37, of sufficient length and hydrophobicity to constitute a transmembrane domain. Although this domain does not align colinearly with the corresponding domain in the other synaptotagmins, it also displays the unusual transmembrane boundaries reported for other synaptotagmins. The N-terminal border of the putative transmembrane domain is flanked by a proline, the C-terminus of the domain is flanked by cysteine residues followed by a highly positively charged region.
[0248] IV. Chromosomal Location
[0249] Southern blot analyses of Repro-PC-1.0 hybridization to human genomic DNA revealed a non-complex pattern indicating a single copy sequence. Analysis of Repro-PC-1.0 hybridization to genomic DNAs from a panel of phylogenetically distinct species showed that the sequences encoding Repro-PC-1.0 were highly conserved, hybridizing to yeast DNA even under high stringency conditions. A single, evolutionarily conserved Repro-PC-1.0 gene localizes to chromosome 18.
[0250] V. Expression of Repro-PC-1.0 in Prostate Carcinoma and in Other Tissues
[0251] In order to compare the expression of Repro-PC-1.0 in normal prostate to prostate adenocarcinomas and benign hyperplasias, Repro-PC-1.0 sequences were specifically amplified from RNA isolated from a number of well characterized tissue sources by RT-PCR. These products were fractionated, transferred and hybridized with a Repro-PC-1.0 probe and the level of expression was graded by relative signal intensity of the Repro-PC-1.0 specific bands (Table 2).
[0252] Repro-PC-1.0 sequences were only present in the RNAs isolated from prostatic adenocarcinoma samples and were not detectable in samples representing benign hyperplasias. The marginal signal detected in normal prostate tissue may reflect the beginning of undiagnosed prostate cancer, as the source of the tissue was an elderly man. To ensure that the amplified sequences were not due to genomic DNA contaminants, control reactions were performed, adding RNase or DNase prior to the 1st strand synthesis step. As shown in Table 2, the addition of DNase had no effect of the level of signal detected in prostate adenocarcinoma samples, whereas RNase treatment eradicated the signal, indicating that the Repro-PC-1.0 hybridizing signal detected in the adenocarcinomas samples was due to Repro-PC-1.0 RNA sequences and not due to contaminating genomic sequences.
[0253] Repro-PC-1.0 expression in additional carcinomas and tissues was investigated by r110i RNA blot analysis. Table 3 lists the different carcinoma cell lines and human tissues that were screened for Repro-PC-1.0 expression. Repro-PC-1.0 expression was not detected in any other prostate or non-prostate carcinoma cell line besides the LNCaP line, nor was it detected in any other normal tissue except for brain. Interestingly, this is precisely the tissue where the synaptotagmin family is expressed, suggesting the LNCaP cells are aberrantly expressing a gene that encodes a protein involved in the regulated secretory pathway. 2 TABLE 2 EXPRESSION OF REPRO-PC-1.0 IN PROSTATE CARCINOMA Repro-PC-1.0 PCR Tissue Source Hybridization LNCaP Tumor (male)* +++ +DNase +++ +RNase − Normal Prostate** − +DNase − +RNase − Prostate Adenocarcinoma*** ++ +DNase + +RNase − Prostatic Benign Hyperplasia − Uterus − The level of expression was graded by relative signal intensity of the Repro-PC-1.0 specific bands: +++ very strong hybridization ++ strong hybridization + moderate hybridization − no hybridization *LNCaP Tumor tissue grown in male athymic mice. **The prostate was obtained from a 65 yr. old male, who may have had the beginning of undiagnosed prostate cancer. ***Compilation of analysis from 2 different prostate adenocarcinomas.
[0254] 3 TABLE 3 REPRO-PC-1.0 mRNA IN HUMAN CELL LINES AND MAMMALIAN TISSUES CELL LINE EXPRESSION TISSUE SOURCE Repro-PC-1.0 LNCaP prostate adenocarcinoma + LNCaP male tumors* + LNCaP female tumors** + PC-3 prostate carcinoma − DU-145 prostate carcinoma − BT-20 breast carcinoma − T-47D breast ductal carcinoma − BT-474 breast ductal carcinoma − MCF-7 breast adenocarcinoma − SK-BR-3 breast adenocarcinoma − MDA-MB-231 breast adenocarcinoma − LS174T colon adenocarcinoma − NIH:OVCAR-3 ovary adenocarcinoma − 3a5 hybridoma-SP2/0 fusion myeloma − placenta, human − pancreas, human − pituitary, human − liver, human − spleen, human − brain, human + heart, human − skeletal muscle, human − kidney, human − pancreas, canine − pituitary, canine − spleen, mouse − rubella (strain M33) − Summary of Northern hybridization results for different human cell lines and mammalian tissues. + indicates detectable expression − absence of detectable expression *LNCaP xenograft tumors propagated in male athymic mice. **LNCaP xenograft tumors propagated in female athymic mice.
[0255] VI. Differential Expression of Repro-PC-1.0 and Human Synaptotagmin
[0256] In order to address whether or not Repro-PC-1.0 might represent a human synaptotagmin isoform that was differentially expressed in LNCaP tumor cells, the expression of Repro-PC-1.0 and human synaptotagmin in LNCaP cells, brain tissue, PC12 cells (rat adrenal medullary cell line) and in a non-Repro-PC-1.0 or synaptotagmin expressing human carcinoma cell line, RL95-2 was compared. RNA isolated from each of these sources was hybridized to either Repro-PC-1.0-specific or human synaptotagmin-specific probes. Both forms are detectable in RNA isolated from normal human brain, but only Repro-PC-1.0, and not human synaptotagmin is expressed in LNCaP cells. Interestingly, only Repro-PC-1.0 sequences are detectable in PC12 cells which normally express rat brain synaptotagmin I. Rat brain synaptotagmin I would not have been detectable with either the human synaptotagmin-specific or Repro-PC-1.0-specific probes. The cross hybridization of Repro-PC-1.0 to PC12 RNA suggests that a rat homologue of Repro-PC-1.0 might also be expressed in PC12 cells and may participate in regulated secretion. Thus Repro-PC-1.0 expression is specifically and differentially up-regulated in LNCaP tumor cells, representing a novel human brain synaptotagmin isoform that cross hybridizes with a rat homologue, distinct from rat brain synaptotagmin 1, normally expressed in PC12 cells.
[0257] VII. Discussion
[0258] Tumor-specific proteins permit insight into the mechanisms that contribute to the progression to malignancy. The LNCaP cell line provides a useful model of prostate cancer that has proven valuable in understanding prostate cancer. These cells were used to raise human prostate tumor xenografts in both male and female athymic mice. Initial observations of host-sex-dependent histological and morphological differences in tumor production prompted investigation of these two forms of tumor as a potential source of RNA to develop a subtractive screen for identifying uniquely expressed proteins in a tumor that has progressed through vascularization (angiogenesis) and has gained metastatic character.
[0259] Tumorigenesis is often associated with either an activation of an oncogene or the inactivation of a tumor-suppressor gene (anti-oncogene). We compared the level of expression of a number of oncogenes, tumor suppressor genes, growth factors and the androgen receptor between the irregular LNCaP tumors grown in a male environment, to LNCaP tumors grown in a female environment, to LNCaP cells grown in vitro, to another non-androgen responsive human prostate cancer cell line and to normal human tissue. Because tumors grown in male hosts showed a profile that was similar to tumors grown in females and to LNCaP cells grown in vitro, the two morphologically distinct tumors was considered as a suitable source of potential message.
[0260] VIII. Fluorescent in situ Hybridization (FISH) of Metaphase Chromosomes
[0261] Cultured lymphoblast cells are incubated with BrdU and Nacadozole for 3 to 5 hours prior to harvesting with trypsin and collection by centrifugation. The cells are resuspended in KCl and fixed in suspension with methanol/acetic acid on ice for 30 min. The cell suspension is placed on microscope slides and slowly dried at 70° C., 80% humidity. A biotinylated cDNA probe, complementary to Repro-PC-1.0, is generated using standard protocols (Labeling and colorimetric detection of non-isotopic probes. In “Current Protocols in Molecular Biology,” F. M. Ausubel, et al., Eds., pp. 3, 18, 1-3,18.7, John Wiley and Sons, Inc., New York). The biotinylated probe is diluted to 3 ng/&mgr;l in a hybridization buffer containing 50% formamide, 10% dextran, 2× SSC, 50 &mgr;g/ml Cot-1 DNA and incubated overnight at 37° C. with the fixed cell spread which has been preincubated with hybridization buffer. The slides are washed twice with 0.5× SSC at 70° C., once with 4× SSC at room temperature, once with antibody diluent (1% BSA, 4× SSC) at room temperature for 5 min. and incubated for 20 min. at 37° C. with FITC-conjugated avidin diluted (10 &mgr;g/ml) in antibody diluent. The slides are washed three times with a buffer containing 4× SSC, then with a buffer containing 4× SSC, 0.05% Tween20, then with a buffer containing 4× SSC followed by counter staining for 2 min with DAPI (200 ng/ml in 4× SSC) and washing with 4× SSC. The excess fluid is blotted off and the slides are mounted with a FITC stabilizing reagent containing p-phenylenediamine dihydrochloride in 90% glycerol. The hybridization profile is determined using a fluorescent microscope.
[0262] The present invention provides novel methods for detecting hormone-regulated traits. While specific examples have been provided, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.
[0263] All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted.
Claims
1. A method for identifying a hormone-regulated trait comprising the steps of:
- a) exposing grafts of a biological material to different hormonal environments in first and second non-human animals of different reproductive states, and
- b) detecting a difference in expression of a trait in the biological material between the first and second animals;
- whereby detecting a difference identifies the trait as a hormone-regulated trait.
2. The method of claim 1 wherein the hormonal environments are natural hormonal environments and the non-human animals are same species, non-human mammals.
3. The method of claim 2 wherein the first non-human mammal is a sexually mature female and second non-human mammal is a sexually mature male.
4. The method of claim 2 wherein the first and second non-human mammals differ in a state of sexual maturity.
5. The method of claim 2 wherein the first and second non-human mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.
6. The method of claim 3 wherein the biological material comprises human benign or malignant neoplastic cells from a laryngeal cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, an endometrial cancer, a vaginal cancer, a testicular cancer, a prostate cancer, a leiomyoma or a benign prostate hyperplasia.
7. The method of claim 3 wherein the biological material comprises human endocrine cells or human exocrine cells from gall bladder, adrenal gland, pancreas, thyroid, salivary gland, pituitary gland, hypothalamus, ovary or testis.
8. The method of claim 3 wherein the biological material comprises hormonally responsive human cells selected from adipocytes, osteoblasts, osteoclasts, chondrocytes, hematopoietic cells, cells from lymphoid or myeloid cell lines, lymphoreticular cells, cells from neural tissue, cells from hair follicles, cells from sebaceous glands, cells from bladder tissue, cells from male or female reproductive tract tissue or cells from mammary gland tissue.
9. The method of claim 3 wherein the biological material comprises hormonally responsive human cells selected from endometrial tissue, endometriotic tissue, pelvic floor tissue or vaginal tissue.
10. The method of claim 3 wherein the trait is expression of a gene.
11. The method of claim 10 wherein the step of detecting a difference in expression of a gene comprises performing subtractive hybridization on cDNA isolated from the biological material from the first and second animals, and identifying a cDNA species that is either more abundant or less abundant in the biological material from one animal compared with the other.
12. The method of claim 11 further comprising the step of isolating the cDNA species.
13. The method of claim 3 wherein the trait is protein expression, post-translational protein modification or protein secretion.
14. The method of claim 3 wherein the trait is expression of a structural or functional trait of a cell or a tissue.
15. The method of claim 14 wherein the trait is rate of cell cycling, anchorage dependence of cells, tumor staging or histochemical staining.
16. The method of claim 3 wherein the non-human mammals are genetically immunocompromised.
17. The method of claim 3 wherein the non-human mammals are chemically immunocompromised or immunocompromised from exposure to radiation.
18. The method of claim 3 wherein the non-human animals are rodents.
19. The method of claim 3 wherein the non-human animals are nude, athymic mice.
20. The method of claim 3 wherein the step of exposing comprises exposing human tumor tissue in the non-human mammal for a time sufficient for the tumor to become palpable.
21. A model system for identifying hormone-regulated traits comprising at least two non-human, same species animals of different reproductive states, each animal hosting a graft of a biological material, whereby the grafts are exposed to different hormonal environments.
22. The model system of claim 21 wherein the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals.
23. The model system of claim 22 at least one non-human mammal is a sexually mature female and at least one non-human mammal is a sexually mature male.
24. The model system of claim 22 wherein at least two non-human mammals differ in a state of sexual maturity.
25. The model system of claim 22 wherein at least two non-human mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.
26. The model system of claim 22 wherein the biological material comprises human benign or malignant neoplastic cells from a laryngeal cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, an endometrial cancer, a vaginal cancer, a testicular cancer, a prostate cancer, a leiomyoma or a benign prostate hyperplasia.
27. The model system of claim 22 wherein the biological material comprises human endocrine cells or human exocrine cells from gall bladder, adrenal gland, pancreas, thyroid, salivary gland, pituitary gland, hypothalamus, ovary or testis.
28. The model system of claim 22 wherein the biological material comprises hormonally responsive human cells selected from adipocytes, osteoblasts, osteoclasts, chondrocytes, hematopoietic cells, cells from lymphoid or myeloid cell lines, lymphoreticular cells, cells from neural tissue, cells from hair follicles, cells from sebaceous glands, cells from bladder tissue, cells from male or female reproductive tract tissue or cells from mammary gland tissue.
29. A method for identifying a trait whose expression is regulated by a hormone-regulated gene, the method comprising the steps of:
- a) exposing a graft of biological material comprising the hormone-regulated gene to a natural hormonal environment in a first non-human animal, the environment comprising a hormone that up-regulates or down-regulates expression of the gene;
- b) exposing a graft of the biological material to the hormonal environment in a second non-human animal, wherein cells of the material are provided with means for down-regulating expression of the gene if the hormone up-regulates its expression, or means for up-regulating expression of the gene if the hormone down-regulates its expression; and
- c) detecting whether a trait is differently expressed in the graft from the first animal compared with the second animal;
- whereby detecting a difference identifies the trait as regulated by the hormone-regulated gene.
30. The method of claim 29 wherein the means for up-regulating comprises an expression vector comprising an expression control sequence operative in the cells and operatively linked with a nucleotide sequence encoding the gene, and the means for down-regulating comprises an antisense polynucleotide that specifically hybridizes with a nucleotide sequence of the gene or its promoter.
31. The method of claim 30 wherein the expression control sequence is inducible or repressible.
32. A method for determining whether a first gene regulates expression of a second, hormone-regulated gene, the method comprising the steps of:
- a) exposing a graft of biological material comprising the first and second genes to a natural hormonal environment in a first non-human animal, wherein the hormonal environment comprises a hormone that regulates expression of the second hormone-regulated gene;
- b) exposing a graft of the biological material to the same hormonal environment in a second non-human animal, wherein the cells are provided with means for up-regulating or down-regulating expression of the first gene; and
- c) detecting whether the second gene is differently expressed in the first animal and the second animal;
- whereby detecting a difference provides a determination that the first gene regulates expression of the second, hormone-regulated gene.
33. The method of claim 32 wherein the means for up-regulating comprises an expression vector comprising an expression control sequence operative in the cells and operatively linked with a nucleotide sequence encoding the gene, and the means for down-regulating comprises an antisense polynucleotide that specifically hybridizes with a nucleotide sequence of the gene or its promoter.
34. The method of claim 33 wherein the expression control sequence is inducible or repressible.
35. A method for detecting a differential effect of a drug on animals of at least two different reproductive states, the method comprising the steps of:
- a) providing at least two immunocompromised, non-human, same species animals of different reproductive states serving as hosts for grafts of hormonally responsive biological material;
- b) administering to the animals equivalent dosage regimens of the drug and
- c) recording a statistically significant (p<0.05) difference in the effect of the dosage regimen on a trait of the biological material in the animals.
36. The method of claim 35 wherein the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals.
37. The method of claim 36 wherein at least one mammal is a sexually mature female and at least one animal is a sexually mature male.
38. The method of claim 36 wherein at least two mammals differ in a state of sexual maturity.
39. The method of claim 36 wherein at least two mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.
40. A method for determining reproductive-state-specific dosages of a pharmaceutical comprising the steps of:
- a) providing at least two immunocompromised, non-human, same species animals of different reproductive states serving as hosts for grafts of hormonally regulated, diseased, human tissue;
- b) administering to the animals equivalent dosage regimens of the pharmaceutical; and
- c) determining the efficacy of the pharmaceutical in treating the diseased tissue in each of the animals of the set.
41. The method of claim 40 wherein the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals.
42. The method of claim 41 wherein at least one mammal is a sexually mature female and at least one mammal is a sexually mature male.
43. The method of claim 41 wherein at least two mammals differ in a state of sexual maturity.
44. The method of claim 43 wherein at least two mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.
45. A method for determining whether an agent modulates expression of a disease-associated, hormone regulated trait, wherein the trait is expression of a gene, comprising the steps of:
- (a) exposing diseased biological material comprising a disease-associated, hormone regulated gene to a hormonal environment in an animal that regulates expression of the gene;
- (b) contacting the biological material with the agent;
- (c) measuring the amount of expression of the gene; and
- (d) determining whether the measured amount is different than an amount of expression of the gene in the cell under control conditions in which the biological material is not exposed to the agent;
- whereby a difference between the measured amount and the amount under control conditions indicates that the agent modulates expression of the gene.
46. The method of claim 45 wherein the step of exposing comprises exposing the cell to the hormonal environment in an animal in vivo.
47. The method of claim 46 wherein the hormonal environment is a natural hormonal environment.
48. The method of claim 45 wherein the agent is an agent that modulates the action of a hormone that regulates expression of the gene.
49. The method of claim 48 wherein the agent modulates the action of estrogen or testosterone.
50. The method of claim 49 wherein the agent is an estrogen analog.
51. The method of claim 45 wherein the step of measuring comprises measuring an amount of mRNA transcribed from the gene.
52. The method of claim 51 wherein the step of measuring comprises an amount of protein encoded by the gene.
53. The method of claim 46 further comprising the steps of:
- identifying a second hormone regulated trait;
- measuring the amount of expression of the second trait in the animal; and
- determining whether the measured amount of expression of the second trait is different than an amount of expression of the second trait in the animal under control conditions in which the biological material is not exposed to the agent.
54. The method of claim 53 further comprising the steps of:
- determining the degree of difference between the measured amounts of gene expression to provide a degree of gene expression modulation;
- determining the degree of difference between the measured amounts of expression of the trait to provide a degree of trait expression modulation; and
- determining whether the degree of gene expression modulation is different than the degree of trait expression modulation;
- whereby a difference indicates whether the agent preferentially modulates expression of the gene.
Type: Application
Filed: Sep 17, 2001
Publication Date: Feb 13, 2003
Inventors: Cynthia K. French (Irvine, CA), Karen K. Yamamoto (San Clemente, CA)
Application Number: 09954570
International Classification: A01K067/00;
