Method for determining tissue of origin and presence and extent of cellular abnormalities

A biochemical procedure for identification and characterization of cells in a biopsy or sample of a body fluid. The method can be used to determine cell type, i.e. epidermal, neuronal; tissue of origin, i.e. breast tissue, liver tissue; and degree of abnormality. The procedure can also be used to make antibodies and hybridization probes to detect cell or tissue specific antigens and nuclear matrix associated nucleic acids in cellular material and body fluids.The procedure is based on the isolation and analysis of the components of a specific subcellular protein fraction referred to here as the "nuclear matrix". The nuclear matrix includes proteins and nuclear matrix associated DNA specific to different cell types. These proteins and nucleic acids are altered or new ones expressed as a result of viral infection, genetic defects or malignancy.The method has a number of important clinical applications in determining tissue type, tissue of origin, degree of malignancy and extent of metastasis in cancer patients; in detecting and analyzing chromosomal deficiencies or genetic defects, especially in cells obtained by amniocentesis; in identifying viral or other infections; and in measuring the extent and location of cell damage, particularly in patients with localized cell damage or autoimmune disease. The isolated nuclear matrix proteins are also useful in screening for drugs binding to and affecting the nuclear matrix.

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Claims

1. A method for characterizing the origin and malignancy of cellular materials comprising:

(a) isolating interior nuclear matrix proteins from at least one type of cell of known origin;
(b) separating the isolated interior nuclear matrix proteins;
(c) comparing isolated and separated interior nuclear matrix proteins from cells of unknown origin with the separated interior nuclear matrix proteins of known origin; and
(d) characterizing interior nuclear matrix proteins from cells of unknown origin based upon the comparison with interior nuclear matrix proteins from cells of known origin.

2. The method of claim 1 further comprising

(a) isolating interior nuclear matrix proteins from cells of unknown origin; and
(b) separating the isolated nuclear matrix proteins from cells of unknown origin;

3. The method of claim 1.Iadd.or 2.Iaddend.wherein the interior nuclear matrix proteins are isolated by:

(a) extracting eucaryotic cells with a non-ionic detergent solution at physiological pH and ionic strength to extract the proteins in the nucleus and cytoskeleton which are soluble in the physiological detergent solution;
(b) separating the nuclear matrix proteins from the cytoskeleton proteins remaining in the extracted cells of step.Iadd.(.Iaddend.a.Iadd.).Iaddend.by solubilizing the cytoskeleton proteins in a solution which does not dissolve the nuclear proteins;
(c) separating the chromatin proteins from the nuclear matrix by digesting the insoluble material from step.Iadd.(.Iaddend.b.Iadd.).Iaddend.with DNAase and RNAase and dissolving the chromatin proteins with a buffered ammonium sulfate solution;
(d) separating the interior and exterior proteins of the nuclear matrix by first dissolving the insoluble nuclear matrix proteins from step.Iadd.(.Iaddend.c.Iadd.).Iaddend.in a solubilizing agent and then dialyzing the dissolved proteins back into a physiological buffer, wherein the interior nuclear matrix proteins are soluble in the physiological buffer and the exterior nuclear matrix proteins are insoluble.

5. The method of claim 1 further comprising purifying the exterior nuclear matrix proteins from cells of known origin and degree of malignancy.

10. The method of claim 7 further comprising digesting the nuclear matrix associated nucleic acids with restriction enzymes.

11. The method of claim 7 wherein the nuclear matrix associated nucleic acids are isolated by:

(a) extracting eucaryotic cells with a non-ionic detergent solution at physiological pH and ionic strength to extract the proteins in the nucleus and cytoskeleton which are soluble in the physiological detergent solution;
(b) digesting the non-extracted material from step.Iadd.(.Iaddend.a.Iadd.).Iaddend.with DNAase and then dissolving the digested extracted material into a buffered ammonium sulfate solution; and
(c) removing any remaining protein from the material in step.Iadd.(.Iaddend.b.Iadd.).Iaddend.which does not dissolve in the buffered ammonium sulfate solution using a method which does not denature or remove nucleic acids.

12. The method of claim 1 further comprising selecting the cells of known origin of step a from the group of cells consisting of normal cells, cells containing nucleotide sequences of viral origin or proteins derived thereof, malignant cells, and cells having chromosomal deficiencies or defects for the production of specific proteins.

Referenced Cited
U.S. Patent Documents
4358535 November 9, 1982 Falkow
4569916 February 11, 1986 Penman
4882268 November 21, 1989 Penman et al.
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Patent History
Patent number: RE35747
Type: Grant
Filed: Nov 19, 1991
Date of Patent: Mar 17, 1998
Assignee: Massachusetts Institute of Technology (Cambridge, MA)
Inventors: Sheldon Penman (Brookline, MA), Edward G. Fey (Boston, MA)
Primary Examiner: Toni R. Scheiner
Law Firm: Testa, Hurwitz & Thibeault
Application Number: 7/794,586