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

Abstract

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.

Description

BACKGROUND OF THE INVENTION

The U.S. Government has certain rights in this invention by virtue of National Institute of Health Grant Numbers 5R01 CA08416-20 and 1R01CA37330-01 and National Science Foundation Grant Number PCM 8309334.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of our copending U.S. patent application Ser. No. 812,955, filed Dec. 24, 1985, 2a2b 2C and 2b' 2D these include human colon, lung, adrenal cortex and bladder cell lines. Although the electropherogram patterns are markedly different in different cell types, each cell type containing unique as well as common proteins, the pattern for each cell type is specific and reproducible. Similar results can be obtained with animal tissue, having been demonstrated using mouse tissue.

In addition to identifying the tissue of origin of the nuclear matrix proteins, the matrix protein patterns reflect cell transformation, viral infection, and genetic defects. This has been demonstrated in spontaneous transformation of rodent primary fibroblasts, viral transformation of established rodent fibroblasts, transformation of a kidney cell line by transaction with the ras oncogene, and transformation of a kidney cell line by the ultimate carcinogen, BAP diole epoxide, using a number of cloned cell lines in each case. Each showed different and marked variation in their matrix protein pattern, but retained sufficient information to determine the cell of origin.

The type and degree of transformation or infection are also closely correlated with changes in matrix protein composition. Of particular interest are the qualitative differences between transformation by the complete carcinogen and by ras gene transfection. Six cloned isolates of cells transformed by ras transfection and 10 isolates of the carcinogen-transformed cells were analyzed. A brief summary of the results is:

a. Transformation by chemical carcinogen leads to the appearance in the matrix of 12 to 15 new or previously undetected proteins.

b. Transfection by the ras gene results in the loss of about 6 proteins and, in some cases, the appearance of 2 or 3 new proteins. There appears to be a correlation between the degree of morphological aberrance and the number of matrix protein changes.

The data from transformed cells reveals different types of transformation events with characteristic signatures.

Analysis of the nuclear matrix associated DNA provides further information for determining the cell type, tissue of origin, and degree of abnormality of cells. "Actively" transcribed DNA in a cell is usually associated with the nuclear matrix proteins. This DNA represents approximately 6% of the total cell DNA. Approximately one-third (2% of total DNA) is directly bound to the protein portion of the matrix and approximately two-thirds (4% of the total DNA) is bound to the RNA component of the nuclear matrix. Different quantities of DNA as well as specific sequences of DNA will be associated with the nuclear matrix and nuclear matrix proteins, depending on cell type and whether the cell is abnormal and to what degree the cell is abnormal. The isolated DNA can be analyzed and identified using gel electrophoresis and blot hybridization with probes specific for a particular unique sequence or repetitive sequence. Probes may be made by inserting the sequence of interest, either a synthetic sequence or a portion of the gene of interest, into a recombinant plasmid using methods known to those skilled in the art. The probes can be used to screen commercially available libraries, such as the λGT11 library, for cDNA encoding nuclear matrix proteins. The library could also be screened using antibodies to the proteins. The probes can also be conjugated with a radiolabel, biotinylated, or crosslinked with psoralin and derivaties thereof for use as a double stranded probe, and used in assays, imaging, isolation and identification procedures. 5

The isolated DNA can be inserted into a vector for expression of nuclear matrix proteins or expressed directly in a system such as one of the frog oocytes direct expression systems.

Analysis of either the proteins or the DNA provides a means to determine the presence of chromosomal defects or genetic deficiencies which might otherwise be undetectable. One application is in the analysis of cells obtained by amniocentesis. Another is in the identification and assessment of autoimmune diseases. Both the antibodies and hybridization probes can be used to analyze cellular materials and body fluids, both in vivo (tissue imaging) and in vitro, for nuclear matrix proteins. However, steps to concentrate or enhance the antigen levels in the body fluids may be required to insure adequate levels for detection. Once probes or antibodies are developed, for example, to carcinoma specific proteins, they can be labeled and used to rapidly screen either histological sections such as a pap smear, or the body fluid.

The extremely rapid, simple extraction and analytical procedure of the present invention provides a means for an objective determination of the tissue of origin of normal and abnormal cells and their degradation products, thereby indicating whether transformation or infection of normal cells has occurred, whether there has been metastasis and to what degree, and whether the individual has any autoimmune disease.

Although this invention has been described with reference to specific embodiments, variations and modifications of the method for isolating and diagnosing nuclear matrix proteins and associated DNA from cells of unknown tissue type or state of malignancy, infection or abnormality will be obvious to those skilled in the art. It is intended that such modifications and variations fall within the scope of the appended claims.

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;

wherein the isolated and separated nuclear matrix proteins from cells of unknown origin are then compared and characterized according to steps (c) and (d) of claim 1.

3. The method of claim 1 or 2 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 (a) 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 (b) 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 (c) 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.

4. The method of claim 1 3 wherein the solubilizing agent in step (d) is urea.

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

6. The method of claim 2 3 wherein cytoskeleton proteins and chromotin proteins in the extract of step a are removed together from the nuclear matrix proteins by digesting the non-extracted material from step (a) with DNAase and RNAase.

7. The method of claim 1 or 2 further comprising isolating nucleic acids associated with the interior nuclear matrix proteins isolated in step (a) from at least one cell of known origin.

8. The method of claim 7 further comprising making antibodies or hybridization probes to the isolated, separated interior nuclear matrix proteins of step (b) and said nucleic acids associated with the interior nuclear matrix proteins of step (a).

9. The method of claim 8 further comprising

(a) labeling the antibodies to the interior nuclear matrix proteins or said hybridization probes to nucleic acids associated with the interior nuclear matrix proteins and

comparing the interior nuclear matrix proteins and associated nucleic acids from cells of unknown origin by reacting the proteins and nucleic acids with the labeled antibodies and hybridization probes to interior nuclear matrix proteins and;

(b) detecting interior nuclear matrix protein associated nucleic acids with said labeled hybridization probes; and

(c) comparing said detected nucleic acids with interior nuclear matrix associated nucleic acids of known origin.

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 (a) 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 (b) 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.

13. A method for detecting cell type-specific interior and exterior nuclear matrix proteins and associated nucleic acids in samples a sample of cellular material and or body fluids fluid, said method comprising:

(a) reacting the sample to be analyzed with labeled antibodies to isolated cell type-specific interior and exterior nuclear matrix proteins of known origin with the samples to be analyzes; and

(b) detecting the presence in the sample of proteins recognized by said labeled antibodies to the interior and exterior nuclear matrix proteins from cells of known origin in the samples of cellular material or body fluid, said isolated cell type-specific interior nuclear matrix proteins being characterized as non-cytoskeletal, non-lamin, non-chromatin protein isolated from mammalian nuclei, resistant to solubiliztation from said nuclei in 0.25M ammonium sulfate, remaining in solution following dialysis into physiological buffer from 8M urea, and segregating into a reproducible pattern unique to a given cell type on a two-dimensional electrophoresis gel.

14. A method for detecting cell type-specific interior nuclear matrix protein associated nucleic acids in samples a sample of cellular material and or a body fluids fluid, said method comprising:

(a) reacting the sample to be analyzed with labeled hybridization probes to isolated, cell type-specific interior and exterior nuclear matrix protein associated nucleic acids of known origin with the sample to be analyzed; and

(b) detecting the presence in the sample of nucleic acids recognized by said labeled hybridization probes to nucleic acids associated with either interior or exterior nuclear matrix proteins from cells of known origin in the samples of cellular material or body fluid, said isolated cell type-specific interior nuclear matrix protein associated nucleic acids being characterized as co-purifying with non-cytoskeletal, non-lamin, non-chromatin protein isolated from mammalian nuclei, being resistant to nuclease digestion during isolation from said nucleic, and segregating into a reproducible pattern unique to a given cell type when digested with restriction endonuclease and separated by gel electrophoresis, said co-purifying protein being resistant to solubilization from said nuclei in 0.25M ammonium sulfate, remaining in solution following dialysis into physiological buffer from 8M urea, and segregating into a reproducible pattern unique to a given cell type on a two-dimensional electrophoresis gel.

15. A method for detecting chemical compounds which bind cell type-specific interior nuclear matrix proteins or nuclear matrix associated nucleic acids comprising

(a) reacting selected compounds with isolated, cell type-specific interior and exterior nuclear matrix proteins or nucleic acids associated with either interior or exterior nuclear matrix protein, said isolated cell type-specific interior nuclear matrix proteins being characterized as non-cytoskeletal, non-lamin, non-chromatin protein isolated from mammalian nuclei, resistant to solubilization from said nuclei in 0.25M ammonium sulfate, remaining in solution following dialysis into physiological buffer from 8M urea, and segregating into a reproducible pattern unique to a given cell type on a two-dimensional electrophoresis gel;

(b) detecting the presence of binding between the compound and the a said isolated cell type-specific interior or exterior nuclear matrix protein or associated nucleic acid; and

(c) determining the extent of binding.

16. Interior A cell type-specific nuclear matrix proteins protein isolated from eucaryotic cells transformed mammalian cell nucleic of known origin selected from the group consisting of lung, adrenal cortex, colon, bladder and kidney cells by:

(a) extracting eucaryotic cells with a non-ionic detergent-salt 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 remaining in the extracted cells of step (a) by solubilizing the cytoskeleton proteins in a solution which does not dissolve the nuclear matrix proteins;

(c) separating the chromatin proteins from the nuclear matrix proteins by digesting the insoluble material from step (b) with DNAase and RNAase and dissolving the chromatin in a buffered ammonium sulfate solution; and

(d) separating the interior and exterior proteins of the nuclear matrix by dissolving the insoluble nuclear matrix proteins from step (c) in a solubilizing agent and 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 and removing the insoluble exterior matrix proteins from the solution, said isolated, cell type-specific interior nuclear matrix protein being characterized as non-cytoskeletal, non-lamin, non-chromatin protein present in the nuclei of said transformed cells, having an isoelectric point of at least 4.0 and not greater than 8.0 and being detectable on a two dimensional electrophoresis gel, said protein not being detectable on said two-dimensional electrophoresis gel from normal cells of said known origin.

17. Antibodies Isolated antibodies to the isolated cell type-specific interior nuclear matrix proteins of claim 16. 18. cDNA and derivatives thereof encoding the isolated interior nuclear matrix proteins of claim 16.19. Interior nuclear matrix protein associated nucleic acids isolated from eucaryotic cells by

(a) separating the soluble proteins from the nucleus and cytoskeleton by extracting eucaryotic cells with a non-ionic detergent solution at physiological pH and ionic strength;

(b) digesting the insoluble protein and nucleic acid from step a with DNAase and then eluting with a buffered ammonium sulfate solution to yield nucleic acids associated with nuclear matrix proteins;

(c) separating the nucleic acids associated with interior nuclear matrix proteins from the nucleic acids associated with the exterior nuclear matrix proteins; and

(d) removing any remaining protein from the separated insoluble interior nuclear matrix protein associated nucleic acids and exterior nuclear

matrix protein associated nucleic acids from step c.20. Hybridization probes derived from the interior nuclear matrix protein associated nucleic acids and exterior nuclear matrix protein associated nucleic acids of step c of claim 19.21. Restriction fragments of nuclear matrix associated nucleic acid prepared by

(a) separating the soluble proteins from the nucleus and cytoskeleton by extracting eucaryotic cells with a non-ionic detergent solution at physiological pH and ionic strength;

(b) digesting the insoluble protein and nucleic acid from step a with one or more restriction enzymes in the appropriate buffer and then eluting with a buffered ammonium sulfate solution to yield restriction fragments of nucleic acids associated with nuclear matrix proteins;

(c) separating the nucleic acids associated with interior nuclear matrix proteins from the nucleic acids associated with the exterior nuclear matrix proteins; and

(d) removing any remaining protein from the separated insoluble interior nuclear matrix protein associated nucleic acids and exterior nuclear matrix protein associated nucleic acids from step c.22. The method of claim 1 or 2 further comprising making antibodies to the isolated interior nuclear matrix proteins of step (a) or (b). 23. The method of claim 22 further comprising

(a) labelling said antibodies

(b) detecting interior nuclear matrix proteins from cells of unknown origin with said labeled antibodies; and

(c) comparing said detected proteins with interior nuclear matrix proteins of known origin. 24. The method of claim 13 wherein said proteins detected are released from cells as degradation products.

25. The method of claim 14 wherein said nucleic acids detected are released from cells as degradation products. 26. An isolated cell type-specific interior nuclear matrix protein, said protein being characterized as non-cytoskeletal, non-lamin, non-chromatin protein present in the nuclei of transformed mammalian cells of known origin and selected from the group consisting of lung, colon, adrenal cortex, bladder and kidney cells, resistant to solubilization from said nuclei in 0.25M ammonium sulfate, remaining in solution following dialysis into physiological buffer from 8M urea, having an isoelectric point of at least 4.0 and not greater than 8.0 and being detectable on a two dimensional electrophoresis gel, said protein not being detectable on said two dimensional eletrophoresis gel from normal cells of said known origin. 27. Isolated antibodies to the isolated cell type-specific interior nuclear matrix proteins of claim 26.