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Definitions

• the genes corresponding to these clones are expressed by all normal mammary epithelial cells, but not by any primary mammary tumors or mammary tumor cell lines.
• One such gene encodes keratin 5, which is said to be a valuable marker to distinguish normal and primary tumor cells in culture.
• keratin 5 is said to be a valuable marker to distinguish normal and primary tumor cells in culture.
• fibronectin is also identified.
• Tumor suppressor genes are proposed to play a key role in cancer protection, and it is suggested that tumor suppressor genes provide a vast untapped resource for anti-cancer therapy.
• DNA methylation is a consistent feature of tumorigenesis (Jones et al.. Adv. Cancer Res . 54.:1- 23, 1990) but local sites of hypermethylation have also been found in tumor cells (Jones et al.. Adv. Cancer Res . 54.U-23, 1990; Baylin et al.. Blood 10 : 412-411 , 1987). Elevated expression of the DNA methyltransferase gene has recently been described in progressive stages of colon cancer (El-Deiry et al., Proc. Natl . Acad. Sci . USA 88.:3470-3474, 1991), suggesting a general mechanism for hypermethylation, but not explaining the specificity seen on particular genes.
• This invention features novel methods for identifying cancerous cells present in a human, particularly in solid tumors.
• the invention also features methods for identifying drugs useful for treatment of such cancer cells, and for treatment of the cancerous condition.
• the invention provides a means for identifying cancer cells at an early stage of development, such that premalignant cells can be identified prior to their spreading throughout the human body. This allows early detection of potentially cancerous conditions, and treatment of those cancerous conditions prior to spread of the cancerous cells throughout the body, or prior to development of an irreversible cancerous condition.
• Tumor suppressor genes have been divided into two general types, termed class I and class II.
• Class I tumor suppressor genes are said to be those in which a genetic alteration (e.g., the deletion, addition or substitution of one or more nucleotides) in the coding sequence of the gene has been found to contribute to tumor cell development.
• class II tumor suppressor genes are identified as those which have a lower level of expression in a cancer or precancer cell compared to a normal cell, which decreased level of expression is due to alteration in the regulation of expression of that gene, rather than to the loss of genetic information in the coding sequence of the gene.
• a diagnostic test based upon levels of expression of either a class I or a class II gene, or of another marker gene that is identified as a candidate tumor suppressor gene by one of the differential screening methods described below, but which does not turn out to have tumor suppressor activity, is useful for detecting the presence of cancerous or pre-cancerous cells in a tissue sample from a patient.
• a patient with a cancer characterized by a lower-than-normal level of expression of one or more tumor suppressor genes can be treated (e.g., with a drug or radiation, or by transforming one or more of the tumor suppressor genes into the cancerous cells) to induce a higher level of expression of such gene(s) in the cancerous cells, thus halting or reversing the growth of the cancer.
• the invention features a method for identifying a cancer cell in a human by providing nucleic acid from a candidate tumor suppressor gene which specifically hybridizes to RNA expressed from such a gene in a cancer cell at a level less than one third the level of hybridization with the equivalent RNA expressed from that gene in a normal cell.
• the method involves providing an antibody to the gene product of such a candidate tumor suppressor gene, which antibody specifically reacts (in the sense of an antibody-antigen reaction to form an immune complex) with the polypeptide expressed from the candidate tumor suppressor gene in a cancer cell, at a level less than one-third the level of reaction (i.e., binding) with the equivalent gene product expressed from that gene in a normal cell.
• the method further features obtaining from the human a tissue sample which potentially includes the cancer cell to be detected, and contacting this sample with either (1) the nucleic acid probe, under conditions which would permit hybridization with the mRNA transcribed from the gene, or (2) the antibody, under conditions appropriate for immune complex formation between the antibody and its antigen.
• the method involves determining the amount of hybridization of the nucleic acid or the amount of binding of the antibody with the tissue sample, compared to the amount of hybridization of that nucleic acid or binding of that antibody with a normal tissue sample which includes only normal cells.
• An amount of hybridization or immune complex formation with the tissue sample less than one third the amount of hybridization or immune complex formation with the normal tissue sample is indicative of the presence of cancerous or pre-cancerous cells in the tissue sample.
• the method of using a nucleic acid probe to determine the presence of cancerous cells in a tissue from a patient includes the steps of: providing a nucleic acid probe (i.e., a single- stranded nucleic acid such as DNA, or a double stranded nucleic acid which is made single-stranded prior to doing the hybridization step) comprising a nucleotide sequence at least 8 nucleotides in length (preferably at least 15 nucleotides, and more preferably at least 40 nucleotides, and up to all or nearly all of the coding sequence) which is identical to a portion of either strand of the coding sequence of a candidate tumor suppressor gene; obtaining from a patient a first tissue sample potentially comprising cancerous cells; providing a second tissue sample containing cells substantially all of which are non-cancerous; contacting the nucleic acid probe under high- stringency hybridizing conditions with RNA of each of said first and second tissue samples (e.g., in a northern blot or in situ
• the diagnostic assay may be carried out with antibodies to the candidate tumor suppressor gene product, instead of a nucleic acid probe.
• Such an assay would include the following steps: providing an antibody specific for the gene product of a candidate tumor suppressor gene, the gene product being present in cancerous tissue of a given tissue type (e.g., mammary, ovarian, bladder or prostate epithelium) at a level less than one third the level of the gene product in noncancerous tissue of the same tissue type; obtaining from a patient a first sample of tissue of the given tissue type, which sample potentially includes cancerous cells; providing a second sample of tissue of the same tissue type (which may be from the same patient or from a normal control, e.g.
• a given tissue type e.g., mammary, ovarian, bladder or prostate epithelium
• this second sample containing normal cells and essentially no cancerous cells; contacting the antibody with protein (which may be partially purified, in lysed but unfractionated cells, or in situ) of the first and second samples under conditions permitting immunocomplex formation between the antibody and any tumor suppressor gene product present in the samples; and comparing (a) the amount of immunocomplex formation in the first sample, with (b) the amount of immunocomplex formation in the second sample, wherein an amount of immunocomplex formation in the first sample less than one third (preferably less than one fourth, and more preferably less than one tenth) the amount of immunocomplex formation in the second sample indicates the presence of cancerous cells in the first sample of tissue.
• protein which may be partially purified, in lysed but unfractionated cells, or in situ
• the level of a candidate tumor suppressor gene product in a biological fluid (e.g., blood or urine) of a person may be determined as a way of monitoring the level of expression of the gene in cells of that person.
• a biological fluid e.g., blood or urine
• Such a method would include the steps of obtaining a sample of a biological fluid from the person, contacting the sample (or proteins from the sample) with an antibody specific for a candidate tumor suppressor gene product, and determining the amount of immune complex formation by the antibody, with the amount of immune complex formation being indicative of the level of the gene product in the sample.
• This determination is particularly instructive when compared to the amount of immune complex formation by the same antibody in a control sample taken from a normal individual or cancer patient, or in one or more samples previously or subsequently obtained from the same person.
• a candidate tumor suppressor gene By a candidate tumor suppressor gene is meant those genes which are found to be expressed to a significantly higher degree in normal cells than in cancerous or precancerous cells, as generally discussed above.
• Such a candidate tumor suppressor gene is generally identified by northern analysis or its equivalent (for example, by in situ hybridization) as a gene whose expression is lower in a cancer cell compared to a normal cell. If the gene bears a disabling mutation in its coding sequence, then it is termed a "candidate class I tumor suppressor gene".
• cancer cell If the coding sequence of the gene is intact, inasmuch as the DNA forming the exons of that gene is not significantly altered, a southern analysis of such a gene in a cancer cell does not reveal any significant difference in the tumor suppressor coding sequence in a cancer cell compared to a normal cell. In such genes, termed “candidate class II tumor suppressor genes", it is the regulatory mechanism of the gene that is altered in a cancerous cell compared to a normal cell.
• class I tumor suppressor gene or class II tumor suppressor gene
• class II tumor suppressor gene or class II tumor suppressor gene
• hybridizing conditions conditions under which the nucleic acid used as a probe in the method is able to specifically hybridize with RNA expressed from a candidate tumor suppressor gene without significantly hybridizing to any other RNA expressed from either normal or cancerous human cells (e.g., conditions of high stringency, as described, for example, in Sambrook et al.. Molecular Cloning, a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratories, Cold Spring
• RNA specifically indicates the presence or absence of a candidate tumor suppressor gene transcript (usually mRNA) .
• reaction of the antibody with the candidate tumor suppressor gene product (protein) is performed under normal antibody-antigen reaction conditions which allow specific recognition of the candidate tumor suppressor gene product by antibody, with little or no cross-reaction of the antibody with other proteins normally present in the cancerous or normal cells.
• measurement of the amount of antigen-antibody immune complex formed in the sample is indicative of the amount of candidate tumor suppressor gene product present in that sample.
• the candidate tumor suppressor gene is a gene encoding keratin 5, NB-1 gene product, fibronectin, connexin 26, glutathione-S- transferase pi, CaN19 protein (formerly called clone 19 gene product) , small proline-rich (spr-1) protein, amphireguiin, thymosin beta-4, gamma actin, calpactin light chain (pll) , HBpl7, myosin regulatory light chain, V-Fos transformation effector protein, or one of the following mitochondrial genome-encoded proteins: URF4, Co III, and ATPase ⁇ .
• the candidate tumor suppressor gene may alternatively be one of the newly-identified genes herein referred to as U1-U10, partial sequences of which are given as SEQ ID NOs: 3-12. These genes are specifically described in detail below.
• the amount of hybridization of the nucleic acid with RNA from precancerous or cancerous cells in the human is less than one third the level detected with a normal cell, more preferably less than one tenth the level, or even more preferably is undetectable.
• the invention features a method for identifying a drug useful for treatment of a cancer cell.
• the method includes the steps of identifying a candidate class II tumor suppressor gene, expression of which is suppressed [i.e., significantly diminished (e.g., by two thirds or more)] in a given type of cancerous cell from a given type of tissue, compared to a normal cell in the same type of tissue; providing a first and a second sample of that given type of cancerous cell; treating the second sample with a candidate drug; and determining the level of expression of the gene in the second sample after treatment with the candidate drug, wherein a drug which increases the level of expression of the gene in the second sample, compared to the level of expression of the gene in the untreated first sample, is potentially useful for treatment of the given type of cancer cell, and perhaps for other types of cancer cells, as well.
• the candidate class II tumor suppressor gene and the level of expression of that gene are identified or determined as discussed herein.
• Potentially useful drugs may be chosen from, for example, those which alter signal transduction pathways, or which facilitate demethylation of methylated residues on DNA. Such drugs may increase tumor suppressor gene expression by, for example, increasing tumor suppressor gene messenger RNA synthesis, or mRNA processing, or protein synthesis, or by decreasing RNA degradation or protein degradation.
• the invention features methods for treating a patient who has cancer.
• One such method involves the steps of identifying, in a human, a cell having a low level of expression of a candidate tumor suppressor gene compared to a normal cell, and treating that cell with a drug identified as one which raises the level of expression of that candidate tumor suppressor gene in the cell.
• the method of treatment includes the steps of identifying a patient with a cancer cell characterized by a low level of expression of a candidate tumor suppressor gene, compared to the level of expression of such gene in a normal cell of the same tissue type as the cancer cell; and either treating the cancer cell with a compound which raises the level of expression of the gene in the cancer cell, or introducing into the cancer cell a nucleic acid encoding the gene.
• the nucleic acid would include an expression control element permitting expression of the gene in the cancer cell. Treating patients with such drugs or gene therapy provides a means to control or eliminate their cancers.
• the invention also includes an isolated DNA which hybridizes under high-stringency conditions to any one of the sequences shown as SEQ ID NOs: 3-12, including but not limited to an isolated DNA which has a sequence identical to any one of SEQ ID NOs: 3-12.
• isolated DNA denotes a DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the candidate tumor suppressor gene that hybridizes to the sequence shown in the applicable SEQ ID NO.
• the term therefore includes, for example, a cDNA encoding the applicable candidate tumor suppressor gene product; a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or a genomic DNA fragment produced by PCR or restriction endonuclease treatment. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
• the Figure illustrates the DNA sequence of a cDNA encoding human connexin 26 (Cx26) , and the amino acid sequence deduced therefrom.
• Candidate class I and class II tumor suppressor genes are generally described above. These candidate tumor suppressor genes can be identified as described by
• the subtractive hybridization method described below may be used.
• the subtractive hybridization method is particularly advantageous in screening for candidate tumor suppressor genes since it provides a positive selection procedure.
• the medium, DFCI-1, described by Band and Sager, 86 Proc. Natl . Acad. Sci . , USA 1249, 1989 was used because of its ability to support similar growth of both normal and tumor-derived human mammary epithelial cells.
• cDNA rather than genomic DNA was used for screening since the cDNAs are smaller and easier to manipulate than their genomic counterparts, and are present in multiple copies. Recovery of such cDNAs allows their use as probes to isolate the equivalent genomic DNA.
• the cDNA can be expressed in a expression vector to produce the tumor suppressor gene product, and thus allow production of antibodies to that product for use in the methods described herein.
• the cDNA may be genetically manipulated so that it encodes only a chosen portion of the full-length gene product, resulting in expression of a defined oligopeptide fragment of the tumor suppressor gene product that may be used to generate antibodies useful for detecting the full-length gene product.
• the oligopeptides may be chemically synthesized. Design and production of such defined fragments may be accomplished by standard methods.
• the normal cells used in the methods described herein were derived from a strain 76N established from discarded reduction mammoplasty tissue as described by Band and Sager, supra. These cells are diploid and senesce after 15-20 passages.
• the tumor cells were derived from an aneuploid cell line established from a pleural effusion as described by Band et al.
• any cells used for subtractive hybridization can be derived from any individuals, and substituted as described below.
• Primary .tumor cells or metastatic cells can be used.
• both parental cell populations were grown in DFCI-1 medium at similar population doubling times of about 30 hours. These cells were harvested at 70% confluency directly into 4M guanidium isothiocyanate. 0.5M sodium citrate, and 0.1M ⁇ -mercaptoethanol for RNA preparation.
• the cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase from Bethesda Research Laboratories with an oligodeoxynucleotide oligo(dT) 12 _ 18 as a primer.
• the 32 P pre-labeled SS cDNA from 76N cells was hybridized with a 10-fold excess of tumor poly(A) + mRNA from 21MT-2 cells (Band et al.. Cancer Research 1990).
• FN fibronectin
• 500 ng fibronectin (FN) mRNA, prepared by in vitro transcription was added to subtract out FN cDNA, which is present at high abundance in the mRNA of the normal cells.
• the hybridization reaction mixture was loaded onto a hydroxylapatite column maintained at 60°C and eluted with 0.1M phosphate buffer (pH 6.8). After rerunning the effluent through the column three times, the effluent was collected and rehybridized as above (2nd subtraction) without added FN mRNA. The final effluent was concentrated to 100 ⁇ l, a sample was removed for quantitation, and the rest frozen for subsequent screening.
• cDNA from 76N poly(A) + RNA was used to produce a recombinant library in the phagemid lambda Zap II (Stratagene Corp., La Jolla, CA) by procedures recommended by the vender.
• the 76N library was screened by differential hybridization using the 32 P random-primer labelled subtracted cDNA probe against the tumor specific cDNA. After a secondary screening the differentially expressed clones were isolated, and the inserts were amplified by PCR from phage using T3 and T7 sequences as primers. After gel electrophoresis, the PCR products were purified by phenol/chloroform extraction from agarose and 32 P random-primer labelled for RNA northern analysis.
• RNA (20ug) was heat denatured at 68°C for 15-20 min. followed by electrophoresis in 1.2% agarose- formaldehyde gels and transferred to nylon membranes
• clones were recovered. After two rounds of screening, seven different clones showed unique or highly preferential expression in normal cells compared to tumor cells. The clones were identified by northern hybridization using standard techniques. The size range of mRNAs varied from 0.6 kb to almost 5 kb. These clones include genes expressed at rare to high abundance in mRNAs.
• TTCCAG consensus splice accepter signal
• This signal sequence is not present in the 5' region of the rat Cx26 sequence, which does not produce two transcripts.
• the 3' untranslated regions contains a possible polyadenylation signal sequence AATAAA positioned 87 nucleotides upstream from the poly(A) + tail.
• ATTTA putative instability sequence
• the overall nucleotide homology between human and rat Cx26 is 86.2% within the open reading frame.
• the amino acid sequence deduced from the human cDNA is 92.5% identical to rat Cx26.
• the 5' and 3• untranslated regions show no significant similarity between human and rat.
• Connexins are structural proteins that surround the channels of which gap junctions are composed; the channels in turn provide direct communication between adjacent cells.
• Gap junctions have been postulated to play a growth regulatory role, on the basis of numerous correlations between growth control and junctional communication. Of these, one of the earliest and still the most striking is Stoker's experiment in which polyoma-transformed BHK cells were inhibited from colony formation by contact (later shown to be junctional communication) with a monolayer of normal BHK cells. Recent experiments by Loewenstein and coworkers and others have correlated post-translational modulation of junctional communication with growth inhibition. Our results, in contrast, suggest transcriptional regulation. This opens the possibility for experimental and clinical modulation at the level of transcription as described below.
• a clone termed clone 2-3 encodes glutathione-S- transferase __i , identified by sequence comparison with known genes in GENBANK.
• the DNA sequence for glutathione-S-transferase pi is provided by Moscow et al., 49 Cancer Research 1422, 1989.
• This protein is a well-characterized enzyme, present in many cell types, that has detoxifying activity against many lipophilic toxic agents including carcinogens. We have found that it is down-regulated in a number of mammary tumor-derived cell lines, both primary and metastatic, but strongly expressed in normal and immortalized mammary epithelial cells grown in culture.
• a clone originally termed clone 19, and now referred to as CaN19 represents a gene expressed in normal mammary epithelial cell strains but not in tumor- derived cell lines.
• the DNA sequence (and corresponding amino acid sequence, or "gene product") of CaN19 is shown as SEQ ID NO: 1 below. Sequence comparisons have shown that CaN19 is a member of the S100 gene family, encoding small Ca " * " * " binding proteins (about 10 kD) with diverse functions. These proteins have two "EF hands", domains where Ca 2+ is bound, in contrast to calmodulin proteins which have four.
• the S100 beta protein is a major constituent of glial cells, whereas related proteins are expressed in differentiated but not in undifferentiated PC 12 (rat pheochromocytoma) cells.
• CaN19 is also related in structure to the small regulatory subunit of calpactin, pll.
• MRP8 and MRP14 are also related and are S100 proteins expressed by macrophages during chronic inflammation. Calabretta et al., 261 J. Biol . Chem. 12628, 1986.
• Another related protein, calcyclin has been found in serum-induced cycling cells, but not in quiescent cells, and in leukocytes from CML patients.
• a related mouse protein is also cell cycle induced.
• calcyclin expression might be cancer related is particularly interesting in view of our evidence that CaN19 is not expressed in breast tumor cells. CaN19 appears to be negatively regulated in tumors, in contrast to calcyclin. Other related proteins are described by Kligman and Hilt 13 TIBS 437, 1988.
• genes which are useful in the present invention include NB-1 described by Yaswen et al., supra: keratin 5 as described by Trask et al. , supra. the DNA sequence of which is published in 8 Molecular Cell Biology 486, 1988; and small proline-rich protein (spr- 1) , the sequence of which is published in 18 Nucl . Acid Res. 4401-4407, 1990.
• the latter gene is known to be expressed at higher levels following treatment with ultraviolet radiation, suggesting that the protein may have a DNA repair function.
• spr-1 is a very promising gene for further investigation.
• a single strand phagemid cDNA library from normal cell polyA + mRNA is hybridized with excess biotinylated tumor polyA + mRNA, and the resulting double stranded sequences are removed by binding to streptavidin.
• the remaining single-stranded phagemid cDNAs are converted to double-stranded form and used to transform bacterial host cells.
• the resulting subtracted cDNA library is differentially screened with total cDNA from normal and tumor cells. This method produced some 20 additional cloned cDNAs, including some which, upon partial sequencing, proved to have been previously identified by others, and some which appear to be novel.
• genes which were found by this method to be candidate tumor suppressor genes potentially useful in the methods of the invention include genes encoding human amphiregulin (the full sequence of which can be found in GENBANK at locus HUMARXC, Accession #M30704) ; thymosin beta-4 (locus HUMTHYB4, Accession #M17733) ; gamma actin (locus HUMACTCGR, Accession ##X04098, K00791, M24241) ; calpactin light chain (pll) (locus HUMCALPAIL, Accession #M81457) ; HBpl7 (locus HUMHEPBP, Accession #M60047) , myosin regulatory light chain (locus HUMMRLCM, Accession #X54304) ; v-fos transformation effector protein (locus HUMFTE1A, Accession #M84711) ; and the mitochondrial genome-encoded proteins URF4 (locus HUMMTHS)
• genes which, on the basis of the partial DNA sequences set forth as SEQ ID NOs: 3-12, respectively, appear to be novel sequences not previously entered into GENBANK.
• the portion of the cDNAs so sequenced represents part of the coding region and/or part of the 3• untranslated region of each cDNA.
• Still other genes can be identified as described above using northern analysis of isolated clones and determining whether tumor cell expression of the gene is reduced by at least 2/3 compared to normal cells.
• genes described herein have been found to be expressed at a low but detectible level in at least some tumor cells (which is taken to be an indication that the coding sequence is intact in these cells) , and thus appear to be candidate class II rather than class I tumor suppressor genes in these tumors.
• Another indication that a particular candidate tumor suppressor gene falls within class II in a particular tumor is a normal-appearing Southern blot of the tumor's genomic DNA when probed with the tumor suppressor gene cDNA.
• Candidate class I tumor suppressor genes in which the coding sequence of the gene is altered in a way to yield no biologically active gene product or an altered gene product, could also be detected by the differential hybridization screening method of the invention if the genetic alterations are such that (1) no detectable mRNA is transcribed from the mutant gene, or (2) the mRNA transcribed from the gene is sufficiently different from wild type that it cannot hybridize to the hybridization probe utilized, or (3) the mRNA has an altered sequence resulting in a different location on a Northern gel than the normal mRNA, or (4) the mRNA is hydrolyzed by the cell rapidly after transcription.
• the alternative method of detection disclosed herein in which an antibody to the wild type candidate tumor suppressor gene product is used to detect gene expression in cell samples, would also be useful for identifying candidate Class I tumor suppressor genes, and for detecting their expression in a given cell sample, if the mutations in the coding sequence of the gene are such that (1) no stable gene product is expressed by the mutant gene, or (2) the gene product that is expressed is so altered that the antibody utilized cannot bind to it.
• Class II genes are of particular interest because the suppressor gene has not been lost, and may therefore be available for up-regulation by drugs or other treatment. Restoration of suppressor gene function by regulatory intervention offers new opportunities in the design of novel drugs for cancer therapy. Both Class I and Class II genes are immediately valuable for early diagnosis and prognosis, which are especially pressing needs in breast cancer where the course of the disease is so unpredictable. Some genes expressed preferentially in normal cells may not have tumor suppressor functions. They are nonetheless useful as diagnostic markers.
• the candidate suppressor genes described herein represent just the "tip of the iceberg" with respect to loss-of-function genes that may be useful in diagnosis, prognosis, and therapy.
• Genes with numerous and diverse functions are anticipated to participate in protecting the long-lived human species from cancer. They include DNA repair genes that maintain genomic integrity and stability, genes that promote irreversible steps in differentiation, and genes that regulate proliferation. Cancer starts at the cellular level, but becomes a systemic disease, and at that point, systemic mechanisms of protection play important roles. These include cell- cell communication by gap junctions, paracrine regulation by growth factors and cytokines, protection by the immune system, control of angiogenesis, and the regulation of tumor invasion. For each of these, specific genes encode key proteins whose loss may facilitate neoplasia.
• the experimental system described herein allows early recognition of aberrant tumor suppressor and diagnostic genes.
• both candidate class I and candidate class II tumor suppressor genes can be used for diagnosis of cancer. All of those genes described above, and other genes identified in a similar manner, are potentially useful for diagnosis of cancerous conditions. For example, they are particularly useful for identification of cancerous cells in solid tumors, such as in breast cancer.
• a portion of that lump may be removed and analyzed by northern analysis or by in situ hybridization using the cloned gene (or antibodies to the gene product produced by standard techniques) to determine whether the level of expression of the candidate tumor suppressor gene is normal or at a reduced level. If it is at a reduced level, this will be indicative that the cells in that lump are cancerous or pre-cancerous and appropriate steps may be taken to either remove or treat those cells in vivo.
• routine diagnosis can be obtained in a manner similar to a papsmear in which cells are taken from a human and tested by hybridization with any one or more of the above candidate tumor suppressor genes or by immune complex formation with antibodies to the gene products. Such testing will allow earlier diagnosis of cancerous conditions than has previously been possible.
• the northern analysis and in situ hybridization or immune complex formation can be carried out by any of a number of standard techniques.
• the DNA of a candidate tumor suppressor gene or its equivalent cDNA may be used as a probe for RNA transcribed from those genes in cells to be tested.
• DNA which hybridizes to the RNA produced by such genes can also be used.
• the cDNA or its equivalent may be placed in expression vectors to cause production of candidate tumor suppressor gene products which may be purified and used to isolate polyclonal or monoclonal antibodies to those candidate tumor suppressor gene products.
• candidate tumor suppressor gene products which may be purified and used to isolate polyclonal or monoclonal antibodies to those candidate tumor suppressor gene products.
• Those particular antibodies which are specific for (i.e., form readily detectible immune complexes with) the candidate tumor suppressor gene product can be identified by standard procedures. Generally, it is preferred that a specific monoclonal antibody be identified so that a large amount of that antibody can be readily produced and used in diagnostic procedures.
• Immunoprecipitation by antibodies of candidate tumor suppressor gene products is performed by standard methodology such as western blotting.
• diagnostic methods can be adapted for use as a way to monitor changes in the level of expression of a given candidate tumor suppressor gene in a given patient over time. This would be useful, for example, as a routine measure for monitoring for the presence of cancer in apparently healthy subjects, much as pap smears and mammograms are used.
• This technique relies upon the normal expression of a given candidate tumor suppressor gene product in a readily obtainable biological fluid such as blood, urine, or saliva.
• a baseline normal level of expression of the gene product would be established by analyzing samples taken from the subject over the years, or by comparison with standards obtained from other, disease-free individuals. A drop in the amount of the gene product present in a given sample would be an indication of the presence of tumor cells in the subject.
• the method could be adapted to serve as a means for following the clinical progression of a tumor. wherein increases or decreases in the level of the gene product in the analyzed sample would be indicative of decreasing or increasing tumor load, respectively.
• the above-described method for assaying a biological fluid will work reliably only if the candidate tumor suppressor gene product is normally a secreted protein. Whether or not a given gene product is secreted can be determined empirically (e.g., by using an antibody specific for the gene product) , or may be predicted by the presence of a secretion signal sequence in the cDNA (e.g., as taught by Von Heijne, 133 Eur. J. Biochem. 17- 21, 1983) in accordance with standard methods.
• candidate class II tumor suppressor genes can be used to identify useful drugs for treatment of cancers. This may be accomplished by standard procedures by culturing cells which include tumor suppressor genes (which are either expressed at normal or subnormal levels) and treating those cells with a variety of drugs to determine which drugs increase the level of expression of the candidate tumor suppressor gene product within those cells. It is preferred that a cancerous cell be used in such a procedure since the increased level of expression of the candidate tumor suppressor gene product will be more readily detected in such a cell, and the drug may work only on genes the expression of which is lower than normal. Identification of the increase in tumor suppressor gene expression can be analyzed by standard northern or in situ analysis or by antibody testing.
• the concomitant increase in a function of that gene may be detected by standard techniques. Two examples illustrating such a procedure are given below. In these examples, phorbol myristate acetate (PMA) is found to increase expression of the Cx26 tumor suppressor gene in tumor cells but not in normal cells, while azadeoxycytidine increases the level of expression of the CaN19 candidate tumor suppressor gene in tumor cells but not in normal cells.
• PMA phorbol myristate acetate
• the appropriate drug may be administered to humans who are identified as containing cells having a reduced level of the tumor suppressor gene product. This may be accomplished either by direct administration of the drug at the tumor site or by systemic treatment with the drug.
• actinomycin D, cyclohexi ide, A23187, or okadaic acid were without noticeable effect on the level of expression of CaN19 mRNA in tumor cells.
• exposure of mammary tumor cells to azadeoxycytidine induced the expression of CaN19-specific RNA.
• the level of expression of CaN19 in normal cells was not affected by azadeoxycytidine treatment.
• tumor suppressor genes of both class I and class II may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cancer cells.
• gene therapy is particularly appropriate for use in cells, both cancerous and precancerous, in which the level of a particular tumor suppressor gene product is diminished compared to normal cells, it may also be useful to increase the level of expression of a given tumor suppressor gene even in those tumor cells in which the gene is expressed at a "normal" but perhaps not optimal level.
• 21MT-2 cells a line of cultured breast tumor cells (developed in this laboratory) in which the level of Cx26 mRNA is undetectible, were transfected with a plasmid construct containing the full-length cDNA corresponding to Cx26 linked to appropriate expression control elements. Unlike the untransfected cells, the transfectants expressed significant amounts of Cx26 protein. Furthermore, the transfected cells were found to assemble the Cx26 protein into gap junctions that functioned in cell-cell communication in the same manner as described for normal mammary epithelial cells.
• a virus or plasmid containing a copy of such a tumor suppressor gene linked to expression control elements and capable of replicating inside the tumor cells would then be injected into the patient, either locally at the site of the tumor or systemically (in order to reach any tumor cells that may have metastasized to other sites) . If the transfected gene is not permanently incorporated into the genome of each of the targeted tumor cells, the treatment may have to be repeated periodically.
• ACGGTCATCC TTAACCAAGG CACTTCTTAA GCAGAAAATA TTGTTGAGGT TACCTTTGCT 180
• GCTAAAGATC CAATCTTCTA ACGCCACAAC AGCATAGCAA ATCCTAGGAT AATTCACCTC 240
• GGATTCTCAC AATAGCCGAC ATCAGAATTT GTGTTGAAGG AACTTGTCTC TTCATCTAAT 120
• TTCGAATCCA TATTTCAAGC CTGGTAGAAT TGGCTTTTCT AGCAGAACCT TTCCAAAAGT 240 TTTATATTGA GATTCATAAC AACACCAAGA ATTGATTTTG TAGCCAACAT TCATTCAATC 300
• GAGAGACTCC GCTGGACATT GCCAAGCGCC TCAAGCACGA GCACTGTGAG GAGCTGCTGA 960
• CTTCGCGTGA CATCTACCAA TCCCCTGACC CCCACGCCGC CCCCACCCGT TGCCAAGACG 1500
• CTCTCTGCAA CGGAAGCTCT GGGTCCTCTG TCCAATGCTA TGGTCCTGCA GCCCCCTGCA 1740

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