A METHOD FOR DETECTING A DIFFERENTIATION MARKER IN NORMAL AND CARCINOMA CELLS AND IN BIOLOGICAL SAMPLES AND BODY FLUID

Abstract: The present invention provides a novel and improved method for the diagnosis, and continuing assessment, of squamous cell carcinoma (SCC). The protein encoded by a rabbit cDNA termed SQ10 is shown to be a molecular marker of squamous cell differentiation. By immunoblotting with antibodies raised against a 22 residue SQ10 synthetic peptide, SQ10 is shown not to be present in undifferentiated cells. SQ10 expression is up-regulated during the differentiation process and inhibited by retinoids. DNA and amino acid homologies between SQ10 and various preprorelaxin proteins allowed SQ10 to be identified as a preprorelaxin-like molecule. The SQ10 relaxin-like protein is secreted from squamous epithelial cells, and as such represents the first molecular marker of squamous cell differentiation that can be detected in body fluids. The rabbit SQ10-derived peptide was found to have a counterpart in human preprorelaxin, with the sequence MSTWSKRSLSQEDAPQTPRPV. Antibodies raised to a peptide of this sequence are also specific differentiated squamous cells. Such antibodies can be used to detect the presence of preprorelaxin-related molecules in normal and malignant squamous cells and in human biological samples and, importantly, in tissue fluids, and thus can be used in a diagnostic test for SCC.

Description: A METHOD FOR DETECTING A DIFFERENTIATION MARKER IN NORMAL AND CARCINOMA CELLS AND IN BIOLOGICAL SAMPLES AND BODY FLUID. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to compositions and methods for the diagnosis of carcinomas, particularly squamous cell carcinoma, by the identification of intracellular and secreted molecular markers of squamous cell differentiation. In particular, the present invention relates to the use of antibodies directed against squamous cell carcinoma markers to detect the presence of such markers in normal and malignant squamous cells and in human biological samples and tissue fluids. 2. Description of the Related Art. The normal tracheobronchial epithelium is a columnar pseudostratified epithelium that is continuously renewing itself (1,2). This epithelium is replaced by a stratified squamous epithelium under a variety of adverse conditions, such as mechanical injury, exposure to carcinogens or tumour promoters and vitamin A-deficiency (2) . This programme of squamous differentiation in tracheobronchial epithelial cells shares many biochemical and molecular characteristics with squamous differentiation in other epithelia such as the skin (3,4). Recently, a multi-stage model has been proposed for the program of squamous differentiation (2,4) . In the first stage, cells become irreversibly growth-arrested. In normal cells irreversible growth arrest appears to be a prerequisite for the second stage of the differentiation program, namely the induction of the squamous differentiated phenotype. Squamous differentiation is influenced by several factors, including retinoids, transforming growth bl and phorbol esters (5,11,12). Retinoic acid, a vitamin A analogue, has been shown to act at a very specific stage in this differentiation program. It effectively suppresses the induction of squamous cell markers without blocking irreversible growth arrest (4-10) . Understanding the mechanism of aberrant squamous cell differentiation is of great interest due to the association between squamous metaplasia and of squamous cell carcinoma (SCC) in humans. This has led to the search for biochemical and molecular markers of differentiated squamous cells, which would be of use in cancer screening or monitoring. Several potential markers have been identified, the expression of which is believed to correlate with squamous cell differentiation. These include, transglutaminase type I mRNA and protein (5,6), cholesterol sulfotransf rase activity and consequent cholesterol sulphate levels (7-9) , keratin (10) , involucrin, loricrin and filaggrin. However, all the above differentiation markers are known to be intracellular. This severely limits their clinical usefulness, as such substances cannot be detected in body fluids. A secreted protein, termed TA-4, has been proposed to be a marker of squamous ovarian carcinoma of the uterine cervix. However, TA-4 also proved to be inappropriate for clinical use as it was found to be present in the sera of only 50% of patients with squamous ovarian cancer and not to be present in any patients with other types of squamous cell carcinoma. Recently, several cDNAs encoding mRNAs abundantly expressed in squamous-differentiated rabbit tracheal epithelial (RbTE) cells have been cloned (13) . However, prior to the current invention, there was no sequence information available relating to such cDNA clones, and their molecular identity remained unknown. Thus, there clearly exists a demand for further squamous cell differentiation markers to be identified and characterized. Such markers would be of considerable use both in basic research and in the diagnosis of carcinomas, and in particular, in the diagnosis of SCC. SUMMARY OF THE INVENTION The present invention relates generally to compositions and methods for the diagnosis, and continuing assessment, of squamous cell carcinoma (SCC) . The present invention provides materials and methods for SCC diagnosis by the identification of intracellular and secreted molecular markers of squamous cell differentiation. In particular, this invention relates to antibodies directed against antigens having cross- reactivity with preprorelaxin, and to the use of such antibodies to detect the presence of preprorelaxin- related molecules in normal and malignant squamous cells and in human biological samples and tissue fluids. In preferred embodiments, the present invention relates to methods for testing for the presence of a squamous cell differentiation marker in a biological sample comprising 1) obtaining a sample from a patient suspected of having squamous cell carcinoma; and 2) testing for the presence of a protein that is antigenically cross-reactive with preprorelaxin by contacting the sample with an antibody directed against human preprorelaxin-related proteins, or antigenic peptides derived therefrom, under conditions effective to allow the antibody to form an immunocomplex with the antigen that may be present in the sample; and 3) detecting the presence of the antigen in the sample by detecting the formation of such an immunocomplex. A particular advantage of the present invention is that preprorelaxin-like molecules, herein disclosed to be squamous cell differentiation markers, are secreted into the extracellular milieu. This allows, for the first time, body fluids such as sera, saliva and urine to be analysed in the diagnosis of squamous cell carcinoma. Various immunoassay methods are proposed to be of use in the practice of the present invention, such as enzyme- linked immunosorbent assays (ELISAs) ; radioimmunoassays (RIAs) ; fluorescence-based immunoassays (FIAs) ; immunoblotting and dotblotting techniques, and the like. The inventors propose that the analysis body fluids by different forms of ELISAs, RIAs and FIAs, will be found to be of most use in SCC diagnosis. However, it will be understood that this invention is not limited to the analysis of body fluids by these methods. Tissue sample analysis by immunocytological methods, for example indirect immunofluorescence, is also within the scope of the invention. Immunocytological methods are known to comprise contacting the sample with specific primary and secondary antibodies and subsequent analysis by microscopic examination. In preferred embodiments, as stated above, a tissue fluid sample suspected of containing a squamous cell differentiation marker, i.e. a preprorelaxin-like molecule, would be analysed by immunoassay. The antibodies known to recognize proteins that are immunologically cross-reactive with preprorelaxin could themselves be conjugated to an enzyme, a radioactive compound, or fluorophore, to allow direct detection following immunocomplex formation. However, in preferred embodiments, an amplification cascade would be employed. This would involve detection of the immunocomplex by the use of a secondary antibody that is specific for the primary antibody. In that the primary will typically be of rabbit, goat or mouse origin, the secondary antibody will preferably be an antibody having specificity in general for rabbit, goat or mouse IgG, or for a particular mouse Ig subclass in the case of a monoclonal primary antibody. To provide means of detection, the second antibody will preferably be conjugated to a radioactive compound or fluorophore to allow detection by autoradiography or fluorography, respectively; or to an enzyme that will generate a coloured product upon incubation with an appropriate chromogenic substrate. Enzymes such as urease, hydrogen peroxidase or alkaline phosphatase are known in the art to be of use in these ELISAs methods. Of course, one may find additional advantages through the use of a secondary binding ligand such as a biotin/avidin ligand binding arrangement, as is known to those of skill in the art. In preferred embodiments the antibodies employed in such immunoassays will be directed against a peptide derived from the human preprorelaxin protein, and in particular a peptide with the sequence MSTWSKRSLSQEDAPQTPRPV. However, it will be appreciated that there is no requirement for antibodies to be generated against peptides with this exact sequence. All that is required is to obtain an antibody that exhibits cross-reactivity with human preprorelaxin. This can be elicited by immunization of experimental animals with peptide fragments that are "antigenic functional equivalents" of the above peptide. For the purposes of the present invention, polypeptides which are variations of the above peptide sequence, but which polypeptides nevertheless elicit the production of antibodies that are cross-reactive with preprorelaxin, are considered to be antigenic functional equivalents of the above peptide and are thus within the scope of the present invention. As described above, certain changes may be made in peptides sequences without necessarily reducing or negating their antigenic/immunogenic identity. Furthermore, it is known in the art that certain amino acid substitutions may actually improve the antigenic or immunogenic activity of a peptide as a result of the small conformational changes which they induce. Changes in the primary sequence can be made through considerations based on similarity in charge (e.g., acidity or basicity charges of the amino acid side group) , hydropathic index, or amphipathic score. Exemplary amino acids that can be substituted by conserved equivalents are known in the art, and include: serine and threonine; valine, leucine and isoleucine; and arginine and lysine. The techniques for oligopeptides generation, by automated peptide synthesis, and purification are generally known to those of skill in the art. In certain embodiments, the invention relates to the generation of antibodies that are cross-reactive with preprorelaxin. Particular techniques for the preparation of such antibodies in accordance with the invention are disclosed hereinbelow. The preparation of polyclonal antibodies is preferably achieved by immunization of experimental animals such as rabbits or goats. However, it is proposed by the inventors that any of the current techniques known in the art for the preparation of antibodies in general may be employed, through the application of either monoclonal or polyclonal technology. It is generally known in the art that the generation of high-titre polyclonal antibodies against peptide antigens can be achieved by coupling the small peptide to a larger carrier molecule prior to experimental immunization. Therefore, in certain embodiments, the present invention relates to the coupling of a peptide with a sequence homologous to that of a preprorelaxin- like protein to a carrier molecule. Suitable carrier molecules include, but are not limited to, bovine serum albumin (BSA) and keyhole lympet hemocyanin (KLH) . Techniques for monoclonal antibody generation in accordance with the present invention will be known to those of skill in the art, in light of the present disclosure. Such techniques generally include the immunization of an experimental animal, such as a mouse, from which a population of spleen or lymph cells are subsequently obtained. The spleen or lymph cells can then be fused with immortalized cells, such as human or mouse myeloma cell lines, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody which is cross-reactive with human preprorelaxin. In further embodiments, the present invention concerns a kit for the immunodetection of a squamous cell differentiation marker, i.e. a preprorelaxin-like molecule, for use in the diagnosis and continuing assessment of squamous cell carcinoma. It is proposed that antibodies prepared in accordance with the present invention, may be employed to detect preprorelaxin-like antigens in tissue samples or body fluids employing the immunoassay methods described above. For diagnostic purposes, it is proposed that virtually any sample suspected of containing the preprorelaxin-like antigen to be detected may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, semen samples, saliva samples, urine samples and lavage samples, or even biopsy materials from various tissues such as lung, breast, prostate and oral tissues. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. Diagnostic kits for use in accordance with the present invention will include a suitable antibody directed against antigens having cross-reactivity with preprorelaxin, together with an immunodetection reagent and a means for containing the antibody and reagent. The immunodetection reagent will typically comprise a label associated with the antibody. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The container means will generally include a vial into which the antibody or detection reagent may be placed, and preferably suitably aliquoted. The kits of the present invention will also typically include a means for containing the antibody and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. In still further embodiments, the present invention contemplates the use of specific antibodies in basic research. For example, in an analysis of the processes occurring in squamous cell differentiation. Further knowledge of the squamous cell differentiation mechanisms will be of interest, not only in relation to the development of human cancers, but also in extending our knowledge of molecular events underlying several cellular processes such as hormonal signalling and gene regulation. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. DNA sequence of SQIO. Codons corresponding to the initiation (ATG) and termination (TAG) of translation and the polyadenylation signal (AATAAA) are shown in boldface. DNA sequence is compared with the sequence of human preprorelaxin 2 (Hrelx) . Gaps are indicated by hyphens and identical nucleotides by periods. Figure 2. Comparison of the amino acid sequence of SQIO with that of human preprorelaxin (Hrelx) . Underlined are the positions of the A and B chains of human preprorelaxin. Arrows indicate conserved cysteine residues. Vertical lines indicate identical amino acids; colons and periods indicate highly and moderately conserved changes in amino acids, respectively. Figure 3. Separation and microsequencing of SQ16 tryptic peptides. The SQ16 blotted on nitrocellulose membrane was trypsinized and the resulting peptides were separated by reverse phase high pressure liquid chromatography. The elution profile of the peptides is shown. The amino acid sequences of the indicated peptides were determined as described. Figure 4. The antiserum pepIIIb-Ab reacts specifically with a 20 and 18 kD cellular protein. Proteins from squamous differentiated RbTE cells were analyzed by immunoblotting using preimmune serum, pepIIIb-Ab and anti-Rb-relaxin. The pepIIIb-Ab was used in the absence or presence of 1, 10, 100 mg peplllb. Numbers on the right indicate the size of the molecular markers. Figure 5. Immunoblot analysis of SQ20 expression in cell lysates (cells) and conditioned medium (CM) of RbTE cells. Proteins from cells and conditioned media from logarithmic, undifferentiated cultures of RbTE cells (Undiff.), confluent, squamous-differentiated cultures (Diff.) and confluent, retinoic acid-treated cultures (RA) were analysed. The numbers on the right represent the molecular weight of marker proteins. Figure 6. Immunohistochemical staining of RbTE cells by pepIV-Ab. RbTE cells were grown in Lab-Tek culture chambers, fixed and stained with the antibody pepIIIb-Ab or preimmune serum. A: Confluent, squamous-differentiated RbTE cells stained with preimmune serum; B: Squamous-differentiated RbTE cells stained with pepIIIb-Ab; C: Confluent retinoic acid-treated RbTE cells stained with pepIIIb-Ab. Fig. 7 Suppression of the expression of SQ20 proteins by retinoids. RbTE cells were grown until just confluent and were then treated with retinoic acid (A) or the analog Ch55 (B) for 4 days at the concentrations indicated. The expression of SQ20 was examined by immunoblot analysis using pepIIIb-Ab. Fig. 8 Northern blot analysis of the expression of SQ10 RNA in RbTE cells. Confluent cultures of RbTE cells were treated for 5 days with retinoic acid and Ch55 at the concentrations indicated. Total RNAs (30 mg) isolated from RbTE cells were fractionated, blotted to Nytran, and hybridized to [32P]-labeled probes for SQ10 and glyceraldehyde-3-phosphate dehydrogenase (GPDH; pGAD-28) . Blots were exposed with enhancing screens at -70°C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Squamous cell differentiation in tracheobronchial epithelial cells is accompanied by many biochemical and molecular changes. One of the molecular changes in rabbit tracheal epithelial (RbTE) cells is the differential expression of a squamous cell-specific mRNA encoded by the cDNA SQIO. The DNA sequence of SQIO is herein shown to correspond to a secreted protein that is related to preprorelaxin. The DNA sequence of the coding region of SQIO has 63% homology with the human preprorelaxin mRNA whereas the deduced amino acid sequence exhibits 45% homology with human preprorelaxin. Antibodies, termed pepIIIb-Ab, raised against a 22 amino acid oligopeptide of the protein encoded by SQIO reacted with 20 and 22kD proteins in immunoblots of squamous differentiated RbTE cells. These proteins are highly likely to be prepro- and prorelaxin, respectively. PepIIIb-Ab stained specifically the cytosol of squamous-differentiated RbTE cells but not that of undifferentiated cells. The expression of SQIO was up-regulated during differentiation of RbTE cells. PepIIIb-Ab recognized 20 and 16kD polypeptides present in medium conditioned by squamous differentiated RbTE cells, believed to be prorelaxin and a further proteolytically processed form. The amino acid sequence of three tryptic peptides from the 16kD protein were identical to sequences found in SQ10. The induction of SQ10 protein as well as mRNA was suppressed by retinoids in a concentration dependent manner. The EC 50 for retinoic acid inhibition was lOOnM. The analog Ch55 was approximately 100-fold more effective than retinoic acid, with an EC 50 of approximately InM. Retinoic acid treatment caused a decrease in the stability of the mRNA encoded by SQIO. The 22 residue rabbit-derived peptide was found to have a counterpart in the sequence of human preprorelaxin, the human peptide having the sequence MSTWSKRSLSQEDAPQTPRPV. Antibodies raised to this peptide were also specific for a marker of squamous cell differentiation and can be used in the diagnosis of patients with squamous cell carcinoma. Example 1: Sequencing of the Rabbit cDNA clone SQIO. A cDNA library had previously been constructed from poly(A)+ RNA isolated from squamous differentiated RbTE cells (13). The cDNA clone, SQIO, isolated from this library encoded an mRNA which was abundantly expressed in squamous RbTE cells (13) . The insert of the plas id SQIO was sequenced in both directions by the partial dideoxynucleotide chain-termination sequencing method (19) , using a Sequenase kit (United States Biochemical) . The DNA and deduced amino acid sequences were analyzed by the GCG sequence analysis software package (20) . The obtained DNA sequence is shown in Fig. 1. An initiation codon is evident 91 bases from the beginning of the cDNA at the 5'-end of the sequence. A translation stop codon is present at 625 bases downstream from the beginning followed 150 bases further by an AATAAA polyadenylation signal at the 3'-end of the sequence. Comparison of this DNA sequence with DNA sequences in Genbank revealed that SQ10 was homologous to human and porcine relaxin, the degree of homology with the human sequence was determined to be 63%. Based on the deduced amino acid sequence SQ10 encodes a 20 kD protein (Fig. 2) . Its amino acid sequence exhibited 45% homology with human relaxin and was also homologous to porcine relaxin. There are six conserved cysteine residues within the SQIO sequence (arrows in Fig. 2) which are known to be conserved among human and porcine relaxins. Example 2: Isolation of Rabbit rSQ16 Protein. Rabbit tracheal epithelial (RbTE) cells were isolated and maintained in culture. Tracheas were excised from 8-10 week old male New Zealand White rabbits (Hazelton Inc., Denver, PA). The tracheal lumen was filled with a solution of 0.1% protease type XIV in minimal essential medium (MEM) and incubated for 18 hours at 4°C. The epithelial cells were collected by flushing the trachea with MEM, and the cells washed with MEM. The RbTE cells were resuspended in Ham's F-12 medium containing 34ng/ml epidermal growth factor (EGF) , lOmg/ml insulin, 5mg/ml transferrin and ImM hydrocortisone (5) and grown in primary culture in this buffer. 150mm dishes containing confluent, differentiated cultures of RbTE cells were washed with Ham's F12 medium without growth factors and re-fed with the same medium. After an additional 48 hours, the conditioned medium was collected and concentrated 100-fold using an Amicon pressure concentrator with a 10 kD MW cut-off filter. The proteins present in the concentrated medium were analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS/PAGE) . Aliquots of the conditioned medium were added to an equal volume of 2X SDS-sample buffer (60mM Tris, pH 6.8, 2% SDS, 10% glycerol, lOOmM DTT) , heated for 5 min 95°C, subjected to SDS/PAGE on 14% polyacrylamide slab gels (1.5 x 13 x 15 cm) and then transferred electrophoretically to a nitrocellulose membrane (Micron Separations) using the Moos buffer system (21) . After the transfer, the nitrocellulose sheet was stained with Ponceau red in 0.1% acetic acid and destained briefly in distilled H 2 0. By using the methods described immediately above, an abundant secreted protein of 20kD, designated rSQ20, was identified in medium conditioned by squamous- differentiated RbTE cells (18) . This protein was found to absent in conditioned medium from undifferentiated cells. Prior labelling of cells with radioactive precursors and subsequent protein analysis revealed that the protein did not incorporate radioactive label from [ 3H ]glucosamine or [ 32 ]P0 4 , which demonstrated that it was not a glyco- or phosphoprotein. It was determined that the 20kD protein was converted to a 16kD protein (rSQ16) by the action of proteases present in the medium. The band corresponding to SQ16 was excised, portioned into 0.3 x 1cm pieces and frozen at -70°C. Example 3: Protein Sequencing of Rabbit SQ16. A sample of the nitrocellulose-excised SQ16 polypeptide was processed for microsequencing, as follows. Approximately lOpmol of SQ16 was digested with trypsin and the resulting peptides separated on a reverse phase Vydac C-18 high pressure liquid chromatography (HPLC) column (2.1x150 mm). The resultant peptide profile (Figure 3) was found to exhibit three prominent peaks, termed I, II and III, in order of elution time. The material in these three most abundant peaks was subjected to peptide microsequencing using a gas phase sequencer (Applied Biosystems instrument with a 120A Online PTH-amino acid analyzer) . Peptide separation and microsequencing were performed by Harvard MicroChem (Harvard University, Cambridge, MA) . It was found that peaks I and II contained a single purified peptide which yielded a clean amino acid sequence. However, peak three yielded two distinct sequences, indicating that peak III contained two unresolved peptides, Ilia and Illb. The amino acid sequences of the major peptides were determined to be: peptide I: SNVSFEEFK peptide II: ESPSPENPFLSSGPAAETVPSSIK peptide Ilia: RVTYEWMMENVK peptide Illb: ESPSPENPFLSSGPAAETVPSS The sequence of these peptides could be identified within the amino acid sequence of SQIO (Fig. 2) at the following regions: Using computer analysis, the termini of these peptides were found to exactly correspond to the points in the deduced amino acid sequence where trypsin would be expected to cleave. These results provide evidenced that the previously identified secreted protein pSQ20 is the product of the SQIO gene. Furthermore, the first 21 amino acid residues of SQIO could serve as a signal peptide for this secretory protein. Example 4: Anti-rabbit SQ10 Antibody Generation. A synthetic Illb peptide (ESPSPENPFLSSGPAAETVPSS) was synthesized on a Vega 250C automated peptide synthesizer (22) , in the Synthetic Antigen Laboratory (M.D. Anderson Cancer Center, Houston, TX) . As the generation of antibodies against peptide antigens is known to be greatly increased by coupling the small peptide to a larger carrier molecule prior to experimental immunization, the synthetic Illb peptide was crosslinked to keyhole lympet hemocyanin (KLH) . The peptide was dissolved in phosphate-buffered saline, pH 7.4 at lmg/ml and mixed with an equal volume of lmg/ml KLH solution (Calbiochem, La Jolla, CA) . Crosslinking was achieved by incubating the mixture for 1 hour at 23°C with glutaraldehyde (0.04% v/v, final concentration). The KLH-peptide conjugate (200mg peptide) , in complete Freund's adjuvant, was subcutaneously injected at multiple sites on the back of a New Zealand White rabbit. Immunization boosts were repeated every 3 weeks and serum samples were collected 7 days after each injection. All immunizations and serum isolations were performed by Bethyl Laboratories (Montgomery, TX) . The antibody generated in this manner is termed pepIIIb-Ab. A guinea pig antiserum raised against rabbit placental relaxin (23) was a gift from Dr. P.A. Fields, University of South Alabama, Mobile, AL. Example 5. Immunoblotting and Immunofluorescent staining Analyses. Cell extracts and conditioned media for immunoblotting analyses were prepared from 60mm culture dishes of RbTE cells. Conditioned medium was collected just prior to cell harvest and combined 1:1 with 2X SDS-sample buffer (60mM Tris, pH 6.8, 2% SDS, 10% glycerol, lOOmM DTT) . Cells were rinsed with cold PBS containing ImM PMSF, and scraped directly into 250ml SDS-sample buffer. All samples were heated for 5 min 95°C and stored at -70°C. Proteins were separated on precast, 10-20% polyacrylamide gradient gels (Integrated Separation Systems, city, state) , and transferred to NitroPlus 2000 membrane (Micron Separations) using a Polyblot (American Bionetics, city, state) semidry electrophoretic transfer system (24) . Prior to antibody binding, blots were incubated overnight at 4°C in blocking buffer (15% w/v nonfat dry milk powder in PBS) . Blocking buffer was used for all washes and dilutions. The blot was incubated in pepIV-Ab (1:500) for 4 hours at room temperature, with agitation, then washed three times for 10 minutes each. The blot was incubated for 1 hour in 0.1 mCi/ml [ 125 I]-protein A (Amersham Corp., Arlington Heights, IL) followed by four 10 minute washes, with 0.1% Tween-20 included in the final two washes. Autoradiography was performed at -70°C using XAR-5 X-ray film (Kodak, Rochester, NY) and intensifying screens (Dupont, Boston, MA) . Immunoblotting of whole cell extracts demonstrated that the peplllb antibodies recognized two polypeptides of approximately 22 and 20kD (termed rSQ22 and rSQ20) , present in squamous-differentiated RbTE cells, but not in undifferentiated cells (Fig. 4) . To determine whether this protein was secreted by the cells, the medium conditioned by squamous-differentiated and undifferentiated RbTE cultures was analysed by immunoblotting against pepIIIb-Ab. A positive reaction was observed against a 20kD polypeptide in the squamous- differentiated conditioned medium (Fig. 4) . Sometimes the antibody reacted with an additional 16kD protein band, possibly a product generated by proteolytic cleavage of rSQ20. The peplllb antibodies did not react with any protein in conditioned medium prepared from undifferentiated RbTE cells (Fig. 4) . The specificity of the antibody was demonstrated by the ability of the immunizing peptide (Illb) to block antibody binding to rSQ22 and rSQ20 (Fig. 5, lanes 5-7). In addition, preimmune serum did not react with these proteins (Fig. 5, lane 4) . A guinea pig antibody raised against relaxin isolated from the placenta of rabbits (23) also reacted specifically with two proteins of 22 and 20kD (lanes 8-11) . The pepIIIb-Ab immunofluorescent staining pattern of differentiated and undifferentiated cells was analysed. RbTE cells grown in Lab-Tek culture chambers were washed twice in phosphate buffered saline (PBS) and then fixed for 2 minutes in methanol at room temperature. Cells were washed twice again in PBS and incubated for 1 hour in pepIIIb-Ab or preimmune rabbit serum diluted 1:100 in PBS containing 3% BSA. Cells were washed three times in PBS containing 1% Triton X-100, and then incubated for 30 minutes in a 1:100 dilution of FITC-conjugated donkey anti-rabbit lgG (Jackson Immunoresearch) . Cells were washed as above, followed by one wash in PBS without Triton X-100. Cells were mounted in 70% glycerol with 5% w/v n-propyl gallate to retard fading of the fluorescence (25). Squamous-differentiated RbTE cells showed a diffuse Illb-positive staining throughout the cytoplasm (Fig. 6B) , whilst undifferentiated cells were negative. Preimmune serum also failed to stain the cytoplasm of differentiated cells (Fig. 6A) . These observations indicate that the presence of this protein in the cytoplasm is restricted to differentiated RbTE cells. Example 6: Action of Retinoids. Since various retinoids have been shown to suppress squamous cell differentiation in RbTE cells (2), the effect of retinoic acid (RA) on the expression of rSQ20 was examined. In contrast to squamous differentiated cells, the pepIIIb-Ab did not recognize any protein in RA-treated cells grown under differentiating conditions or in their conditioned media (Fig. 4, lanes 3 & 6) . RA-treated cells also did not stain strongly with the pepIIIb-Ab (Fig. 6C) . The suppression of rSQ20 was found to be dependent on the concentration of RA (Fig. 7) , with an EC 50 value of lOOnM. Several synthetic retinoids, including Ch55, Ro 40-6055, Ro 18-6622, Ro 13-7410 and SRI 6754-84, were also found to be effective suppressors; for example, Ch55 had an IC 50 value of InM. In contrast, Ch20, a poor inhibitor of squamous cell differentiation had no effect on the levels of these proteins. The influence of RA on the expression of SQ10 mRNA was analysed using Northern Blot Analysis. RbTE cells were solubilized in guanidinium isothiocyanate and total RNA was isolated by centrifugation of the extract through a CsCl cushion (26). RNA was separated on a 1.2% agarose-formaldehyde gel and transferred to Nytran nitrocellulose membrane (Schleicher and Schuell) with a Vacublot vacuum transfer system (American Bionetics) . Following transfer, RNA was cross-linked to the blot by UV irradiation. Blots were prehybridized for 4-24 hours at 42°C in buffer containing 50% formamide, 5x SSPE 2x Denhardt's solution, 1.0% SDS, and lOOmg/ml sheared salmon sperm DNA. Northern blots were probed with either the 0.8kb BamEl insert of SQ10 (13) or with a 1.12kb, Pstl fragment of the recombinant cDNA clone pGAD-28 (27) , which contained an insert encoding chicken glyceraldehyde-3-phosphate dehydrogenase. Probes were labeled with [α- 32 P]dCTP (-3000 Ci/mmol; Amersham Corp.) using a random priming kit (Boehringer Mannheim) . Following hybridization for 4-24 hours at 42°C, the blots were washed (26) and the final wash was carried out at 55°C in 0.1 x SSC, 0.1% SDS. Densitometric analysis was performed by scanning the autoradiogra s with an LKB Ultra Scan Laser Densitometer. The expression of SQIO mRNA was determined to be upregulated during squamous differentiation of RbTE cells and suppressed by retinoic acid (Fig. 8) . The degree of RA-induced suppression of SQIO expression was found to be concentration-dependent (Fig. 8) , with an IC 50 value of 5nM. The RA analog Ch55 was an even more effective SQIO suppressor, with an EC 50 value of 50pM. The suppression of SQIO mRNA by these two retinoids correlates with their ability to reduce SQ20/18 protein levels. Possibly different rates in metabolism of the two retinoids may account for their differential effectiveness. Specific nuclear retinoic acid (RARs) have been implicated in the regulation of gene expression by retinoids (34-37) . The RARs which belong to the superfamily of steroid/thyroid hormone receptors can influence gene transcription through binding to specific retinoic acid response elements in the promoter region of target genes (37) and may be involved in regulating the stability of certain mRNAs. Recently, expression of RARs has been demonstrated in tracheobronchial epithelial cells (38) . To examine the effect of RA on the stability of SQIO mRNA, squamous differentiated RbTE cells were treated with RA in the presence of actinomycin D, and the relative levels of SQIO mRNA were determined at various time intervals. The half-life of the SQIO mRNA was calculated to be 27 hours. There was no statistically significant difference between the half-lives calculated for control and retinoic acid-treated cells. These results indicate that retinoic acid does not significantly alter the stability of SQ10 mRNA and probably regulates SQ10 at a transcriptional level. Example 7: Generation of Antibodies against Related Human peptides. In immunoblotting analyses, the antibodies raised against the rabbit-derived peptide Illb, ESPSPENPFLSSGPAAETVPSS, showed no reactivity against any human protein. However, as described above (Example 3) , the rabbit DNA and deduced amino acid sequence was found to be homologous to human preprorelaxin. A detailed analysis of the human relaxin sequence and organization enabled the identification of a human peptide with a sequence that resembled that of the rabbit peptide Illb. This peptide has the following sequence: MSTWSKRSLSQEDAPQTPRPV A synthetic peptide with a sequence corresponding exactly to this was generated by automated peptide sequencing, as described above. This peptide was conjugated to a carrier protein and antibodies were raised in rabbits, as follows. KLH-peptide conjugate (200μg) in complete Freund's adjuvant, was injected subcutaneously at multiple sites on the back of a New Zealand White rabbit. Immunization boosts were repeated every 3 weeks and serum samples were collected 7 days after each injection. All immunizations and serum isolations were performed by Bethyl Laboratories (Montgomery, TX) . Immunoblotting studies employing these antibodies demonstrated that, in a similar manner to their rabbit counterparts, they recognize a squamous cell differentiation marker of 16-2OkD. The human target protein was determined to be expressed only in differentiated squamous cells, and not in their undifferentiated counterparts. DISCUSSION The isolation of several cDNA clones that encode mRNAs abundantly expressed in squamous differentiating RbTE cells has been reported (13) . Based on DNA and protein sequence homology with preprorelaxins, the cDNA clone SQIO is herein shown to encode a preprorelaxin-like protein. Although the coding sequence of rabbit relaxin is not known, the amino acid sequences of relaxin from a number of species have been reported including the sequence of the human relaxin (14-16,28-30). The amino acid sequence of the protein encoded by SQIO exhibited 45% homology with human preprorelaxin (28,29). Relaxin is synthesized as a single polypeptide precursor, preprorelaxin, that contains a signal peptide and three polypeptide chains, designated A, B and C (15,16,28-30). The C chain is proteolytically removed, leaving the sulfhydryl-bonded A and B chains to form the mature heterodimer. Although the primary structure of different relaxins exhibit variation, the six cysteine residues in the A and B chains are 100% conserved among the relaxins of different species as well among insulin and insulin-like growth I (15,16). In the protein encoded by SQ10 these six cysteine residues are also conserved. Further evidence that suggests that SQ10 encodes preprorelaxin is provided by the reactivity of rSQ22, rSQ20 and rSQ16 with an antiserum raised against placental relaxin, and conversely, the recognition of rabbit placental prorelaxin by anti-SQIO peptide antibodies. Relaxin is a member of the insulin family and is known to exert its biological effects on various parts of the mammalian reproductive system (14,15). In females, preprorelaxin is produced by the corpus luteum of the ovary during pregnancy, by trophoblasts (16,32) and by mammary cells (32,33). In males, preprorelaxin is synthesized in the prostate (17) . The expression of preprorelaxin mRNA and protein is herein shown to be differentially regulated during squamous cell differentiation, and to be suppressed by retinoids. These observations suggest a role for this hormone outside the reproductive system. Since the induction of preprorelaxin occurs at a specific stage in the differentiation of tracheobronchial epithelial cells, the prorelaxin or relaxin may act by an autocrine mechanism and signal the cell to proceed to the next stage in the differentiation process. Alternatively, relaxin may act by a paracrine mechanism. For example, prorelaxin released by the epithelial cells may affect gene expression in neighboring fibroblast cells. Relaxin has been shown to increase the synthesis and secretion of procollagenase and to decrease the synthesis of collagen in dermal fibroblasts (41) . Moreover, in these cells the induction of collagen I and III synthesis by TGF-b is antagonized by relaxin. Relaxin has been shown to stimulate the secretion of collagenase and plas inogen activator in human amnion and chorion cells (42) . The identification of such receptors on tracheobronchial epithelial or fibroblast cells may provide evidence whether relaxin acts via a paracrine or autocrine mechanism. Antibodies against a peptide from a human relaxin- like molecule will prove useful in the diagnosis and continuing analysis of squamous cell carcinoma (SCC) . SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: BOARD OF REGENTS, THE UNIVERISTY OF TEXAS SYSTEM (ϋ) INVENTORS: LOTAN, REUBEN JETTEN, ANTON M. (iii) TITLE OF INVENTION: A METHOD FOR DETECTING A DIFFERENTIATION MARKER IN NORMAL AND MALGINANT CARCINOMA CELLS AND IN BIOLOGICAL SAMPLES AND BODY FLUIDS (iv) NUMBER OF SEQUENCES: 14 (v) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: ARNOLD, WHITE & DURKEE (B) STREET: P.O. BOX 4433 (C) CITY: HOUSTON (D) STATE: TEXAS (E) COUNTRY: USA (F) ZIP: 77210 (vi) COMPUTER READABLE FORM: (A) MEDIUM TYPE: FLOPPY DISK (B) COMPUTER: IBM PC COMPATIBLE (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: WORDPERFECT 5.1 (vii) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: UNKNOWN (B) FILING DATE: UNKNOWN (C) CLASSIFICATION: UNKNOWN (viii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: 07/783,046 (B) FILING DATE: 10/28/91 (C) CLASSIFICATION: 424 (ix) ATTORNEY/AGENT INFORMATION: (A) NAME: PARKER, DAVID L. (B) REGISTRATION NUMBER: 32,165 (C) REFERENCE/DOCKET NUMBER: UTFC247PCT (X) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 512-320-7200 (B) TELEFAX: 713-789-2679 (C) TELEX: 79-0924 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 100 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GGTAAGCACC CAGGCCCACT TCAGTCGGCG AAACGACAGG CGGCTGCCAA AGACAGCAGA 60 15 GAAACCCGGA GCAGAAGAGA CCAGCCGAGG ATGCCTGCCC 100 (3) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 425 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 a. TCTTGTTCTA CCTTCTAGGA TTCTGTCTCC TCCAGGGCCA AGTTACCGGA AGAGTCACGT 60 15 ATGAATGGAT GATGGAGAAT GTTAAGATAT GCCGTAATGA TTTTGTCAGA ACCGCGATCG 120 AAGTCTGTGG CCATGTCCAC TTGGAAAGAG AGTCTCCGAG TCCAGAAAAC CCCTTTCTGA 180 GTTCTGGACC AGCTGCAGAA ACTGTGCCAT CATCCATCAA GAAAGATGCA GAAAATGCAA 240 20 ATACTATGTT GGAATCCATT CCTAACTTGC CACAGGAGCT GACGGCAACA CTATTTGAGA 300 AGCAGCCATC AAAGCTGTAT CTACAATACC TACCTACATT GAAGAAGTCA AATGTTAGTT 360 TTGAAGAATT TAAGAAAATT ATTCAGAATA TACAAAGAGG AGTGCAAGGT AGCAGTGCTT 420 CAGAA 425 (4) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 280 base pairs 10 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear « (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 15 TCGAATACTT TCTCCCGAAA AAAGAGACAG TTCTCTGAGA GTCTGCCTGA AGAATGTTGC 60 AAATATGGTT GTCCCAGATA TTATCTTCTT ATGTATTGCT GAGATGGAGA TAACTGTATA 120 20 GATTTCACAT AATTTTCACG TGTATTTCAC ATGTATTCTT GATATTTTCA CTTATAATTC 180 TGTCAGGATG CTACTGATTA CTAGAATATA AGACCTCCAC AGCTGAAGGT TTGTGCATTG 240 TTCATTTTAT AATAAATGGT ATGTCTGTGC AAAAAAAAA 280 (5) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TTAAAATACT TAGGC 15 i 7 15 (6) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acid residues 20 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Pro Ala Leu Leu Phe Tyr 5 1 5 (7) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: 10 (A) LENGTH: 104 amino acid residues (B) TYPE: amino acid \ (C) STRANDEDNESS: single 0-. o (D) TOPOLOGY: linear 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Leu Leu Gly Phe Cys Leu Leu Gin Gly Gin Val Thr Gly Arg Val Thr 1 5 10 15 20 Tyr Glu Trp Met Met Glu Asn Val Lys lie Cys Arg Asn Asp Phe Val 20 25 30 Arg Thr Ala lie Glu Val Cys Gly His Val His Leu Glu Arg Glu Ser Pro Ser Pro Glu Asn Pro Phe Leu Ser Ser Gly Pro Ala Ala Glu Thr 5 50 55 60 Val Pro Ser Ser lie Lys Lys Asp Ala Glu Asn Ala Asn Thr Met Leu 65 70 75 80 10 Glu Ser lie Pro Asn Leu Pro Gin Glu Leu Thr Ala Thr Leu Phe Glu 85 90 95 * > Lys Gin Pro Ser Lys Leu Tyr Leu 100 15 (8) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: 20 (A) LENGTH: 34 amino acid residues (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Gln Tyr Leu Pro Thr Leu Lys Lys Ser Asn Val Ser Phe Glu Glu Phe 1 5 10 15 5 Lys Lys He He Gin Asn He Gin Arg Gly Val Gin Gly Ser Ser Ala 20 25 30 Ser Glu 10 (9) INFORMATION FOR SEQ ID NO: 9: f. (i) SEQUENCE CHARACTERISTICS: 15 (A) LENGTH: 33 amino acid residues (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Ser Asn Thr Phe Ser Arg Lys Lys Arg Gin Phe Ser Glu Ser Leu Pro 1 5 10 15 Glu Glu Cys Cys Lys Tyr Gly Cys Pro Arg Tyr Tyr Leu Leu Met Tyr 20 25 30 Cys 10 (10) INFORMATION FOR SEQ ID NO: 10: CM (i) SEQUENCE CHARACTERISTICS: 15 (A) LENGTH: 185 amino acid residues (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Pro Arg Leu Phe Phe Phe H s Leu Leu Gly Val Cys Leu Leu Leu 1 5 10 15 5 Asn Gin Phe Ser Arg Ala Val Ala Asp Ser Trp Met Glu Glu Val He 20 25 30 Lys Leu Cys Gly Arg Glu Leu Val Arg Ala Gin He Ala He Cys Gly 10 35 40 45 Met Ser Thr Trp Ser Lys Arg Ser Leu Ser Gin Glu Asp Ala Pro Gin (^J. 50 55 60 V 15 Thr Pro Arg Pro Val Ala Glu He Val Pro Ser Phe He Asn Lys Asp 65 70 75 80 Thr Glu Thr He Asn Met Met Ser Glu Phe Val Ala Asn Leu Pro Gin 85 90 95 Glu Leu Lys Leu Thr Leu Ser Glu Met Gin Pro Ala Leu Pro Gin Leu 100 105 110 Gin Gin His Val Pro Val Leu Lys Asp Ser Ser Leu Leu Phe Glu Glu 115 120 125 Phe Lys Lys Leu He Arg Asn Arg Gin Ser Glu Ala Ala Asp Ser Ser 5 130 135 140 Pro Ser Glu Leu Lys Tyr Leu Gly Leu Asp Thr His Ser Arg Lys Lys 145 150 155 160 10 Arg Gin Leu Tyr Ser Ala Leu Ala Asn Lys Cys Cys His Val Gly Cys 165 170 175 - . Thr Lys Arg Ser Leu Ala Arg Phe Cys 180 185 15 (11) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: 20 (A) LENGTH: 9 amino acid residues (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11! Ser Asn Val Ser Phe Glu Glu Phe Lys 1 5 (12) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: 10 (A) LENGTH: 24 amino acid residues (B) TYPE: amino acid 4 (C) STRANDEDNESS: single \ (D) TOPOLOGY: linear 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Glu Ser Pro Ser Pro Glu Asn Pro Phe Leu Ser Ser Gly Pro Ala Ala 1 5 10 15 20 Glu Thr Val Pro Ser Ser He Lys 20 ( 13 ) INFORMATION FOR SEQ ID NO : 13 : (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 12 amino acid residues (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Arg Val Thr Tyr Glu Trp Met Met Glu Asn Val Lys ( 1 5 10 ^ l 15 (14) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acid residues 20 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Glu Ser Pro Ser Pro Glu Asn Pro Phe Leu Ser Ser Gly Pro Ala Ala 1 5 10 15 5 Glu Thr Val Pro Ser Ser 20 10 4 REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Evans, M.J. and Shami, S.G. 1989. Lung cell kinetics, In Lung Cell Biology (Massaro, D., ed.) Vol. 41, pp. 341-347, Dekker, New York. 2. Jetten, A.M. 1991. Growth and differentiation factors in tracheobronchial epithelium. Am. J. Physiol. (Lung Cell. Mol. Biol.) 260:L361-L373. 3. Holbrook, K.A. , and Hennings, H. 1983. Phenotypic expression of epidermal cells in vitro. J. Invest. Dermatol. 81:125-130. 4. Jetten, A.M. 1989. Multi-step process of squamous differentiation of tracheobronchial epithelial cells in vitro. Analogy with epidermal differentiation. Environ. Health Perspect. 80:149-160. 5. Jetten, A.M., and Shirley, J.E. 1986. Characterization of transglutaminase activity in rabbit tracheal epithelial cells. Regulation by retinoids. J. Biol. Chem. 261:15097-15101. 6. Floyd, E.E., and Jetten, A.M. 1989. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down-regulation by retinoids. Mol. Cell Biol. 9:4846-4851. 7. Rearick, J.I., and Jetten, A.M. 1986. Accumulation of cholesterol sulfate during in vitro squamous differentiation of rabbit tracheal epithelial cells and its regulation by retinoids. J. Biol. Chem. 261:13898-13904. 8. Rearick, J.I., Albro, P.W. , and Jetten, A.M. 1987. Cholesterol sulfotransferase activity is induced upon squamous differentiation of RbTE cells in vitro. J. Biol. Chem. 262:13069-13074. 9. Jetten, A.M., George, M.D., Smits, H.L., and Voliberg, T.M. 1989. Keratin 13 expression is linked to squamous cell differentiation in rabbit tracheal epithelial cells and down-regulated by retinoic acid. Exp. Cell Res. 182:622-634. 10. Rearick, J.I., Hesterberg, T.W. , and Jetten, A.M. 1987. Human bronchial epithelial cells synthesize cholesterol sulfate during squamous differentiation in vitro. J. Cell. Physiol. 133:573-578. 11. Jetten, A.M., Shirley, J.E., and Stoner, G. 1986. Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFb. Exp. Cell Res. 167:539-549. 12. Jetten, A.M., George, M.D., Pettit, G.R., and Rearick, J.I. 1989. Effects of bryostatins and retinoic acid on phorbol ester- and diacylglycerol-induced squamous differentiation in human tracheobronchial epithelial cells. Cancer Res. 49:3990-3995. 13. Smits, H.L., Floyd, E.e., and Jetten, A.M. 1987. Molecular cloning of gene sequences regulated during squamous differentiation of tracheal epithelial cells and controlled by retinoic acid. Mol. Cell. Biol. 7:4017-4023. 14. Schwabe, C. , and Bullesbach, E.E. 1990. Relaxin. Comp. Biochem. Physiol. 96B:15-21. 15. Schwabe, C . , Goan, L.K. , and Reinig, J.W. 1982. Evolution, relaxin and insulin: a new perspective. Ann N.Y. Acad. Sci. 380:6-12. 16. Bagnell, CA. , Tashima, L. , Tsark, W. , Ali, S.M., and McMurtry, J.P. 1990. Relaxin gene expression in the sow corpus luteum during cycle, pregnancy, and lactation. Endocrinology 126:2514-2520. 17. Weiss, G. 1989. Relaxins in the male. Biol. Reprod. 40:17-200. 18. Lotan, R. , Pieniazek, J. , George, M.D., and Jetten, A.M. Identification of a new squamous cell differentiation marker and its suppression by retinoids, J. Cell Physiol. Submitted. 19. Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 20. Devereux, J., Haeberli, P., and Smithies, 0. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. 21. Moos, M. , Nguyen, N.Y., and Liu, T-Y. 1988. J. Biol. Chem. 263:6005-6008. 22. Merrifield, R.B. 1963. J. Am. Chem. Soc. 85:2149-2154. 23. Eldridge, R.K., and Fields, P.A. 1985. Rabbit placental relaxin: purification and immunohistochemical localization. Endocrinology 117:2512-251. 24. Harlow, E., and Lane, D. 1988. Antibodies, A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 25. Giloh, H. , and Sedat, J.W. 1982. Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217:1252-1255. 26. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1979. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 27. Dugaiczyk, A.D., Haron, J.A., Stone, E.M., Dennison, O.E., Rothblum, K.N., and Schwartz, R.J. 1983. Cloning and sequencing of a ribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger acid isolated from chicken muscle. Biochemistry 22:1605-1613. 28. Haley, J. , Crawford, R., Hudson, P., Scanlon, D., Tregear, G. , Shine, J. , and Niall, H. Porcine relaxin. 1987. Gene structure and expression. J. Biol. Chem. 262:11940-11946. 29. Hudson, P., Haley, J. , John, M. , Cronk, M. , Crawford, R. , Haralambidis, J. , Tregear, G., Shine, J. , and Niall, H. 1983. Structure of a genomic clone encoding biologically active human relaxin. Nature 301:628-631. 30. Schwabe, C. , Bullesbach, E.E., Heyn, H. , and Yoshioka, M. 1989. Cetacean relaxin. Isolation and sequence of relaxins from Balaenoptera acutorostrata and Balaenoptera edini. J. Biol. Chem. 264:940-943. 31. Rawitch, A.B., and Moore, W.V. 1980. Relaxin-insulin homology: predictions of secondary structure and lack of competitive binding. Int. J. Biochem. 11:357-362. 32. Crish, J.F., Soloff, M.S., and Shaw, A.R. 1986. Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy. J. Biol. Chem. 261:1909-1913. 33. Bryant-Greenwood, G.D. 1991. At the cutting edge. The human relaxins: consensus and dissent. Mol. Cell. Endocrinol. 79:C125-C132. 34. Mazoujian, G. , and Bryant-Greenwood, G.D. 1990. Lancet 335:298-299. 35. Petkovich, M. , Brand, N. , Krust, and Chambon, P. 1987. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444-450. 36. Giguere, V., Ong, E.S., Segui, P., and Evans, R.M. 1987. Identification of a receptor for the morphogen retinoic acid. Nature 330:624-629. 37. Jetten, A.M. 1990. Induction of differentiation of embryonal carcinoma cells by retinoids. In: Mechanisms of Differentiation. Editor: Fisher, P. CRC Press, Boca Raton, FL. Vol. I, pp 49-74. 38. Nervi, C. , Vollberg, T.M. , George, M.D., Zelent, A., Chambon, P., and Jetten, A.M. 1991. Expression of nuclear retinoic acid receptors in normal tracheobronchial cells and in lung carcinoma cells. Exp. Cell Res. 195:163-170. 39. Bullesbach, E.E., and Schwabe, C. 1988. On the receptor binding site of relaxins. J. Peptide Protein Res. 32:361-367. 40. Osheroff, P.L. , Ling, V.T., Vandlen, R.L., Cronin, M.J., and Lofgren, J.A. 1990. Preparation of biologically active 32P-labeled human relaxin. J. Biol. Chem. 265:9396-9401. 41. Unemori, E.N. , and Amento, E.P. 1990. Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts. J. Biol. Chem. 265:10681-10685. 42. Koay, E.S.C., Too, C.K.L., Greenwood, F.C., and Bryant-Greenwood, G.D. 1983. Relaxin stimulates collagenase and plasminogen activator secretion by dispersed human amnion and chorion cells in vitro J. Clinic Endocrinol. Metab. 56:1332-1334.

Claims: CLAIMS 1. A method for detecting carcinoma in humans, comprising the steps of: obtaining a sample from a patient suspected of having carcinoma; testing for the presence of a protein that is antigenically cross-reactive with preprorelaxin. The method of claim 1, comprising the steps of: contacting the sample with an antibody directed against a protein that is antigenically cross- reactive with preprorelaxin under conditions effective to allow the antibody to form an immunocomplex with the antigen that may be present in the sample; detecting the presence of the antigen in the sample by detecting the formation of such an immunocomplex. 3. The method of claim 2, wherein the detection of the immunocomplex employs a secondary enzyme-linked antibody, 4. The method of claim 2, wherein the detection of the immunocomplex employs a secondary radiolabeled antibody. 5. The method of claim 2, wherein the detection of the immunocomplex employs a secondary fluorescent-tagged antibody. 6. A protem-conjugated peptide, wherein the peptide has the sequence MSTWSKRSLSQEDAPQTPRPV. 7. The protein-conjugated peptide of claim 6, wherein the protein is bovine serum albumin. 8. The protein-conjugated peptide of claim 6, wherein the protein is keyhole lympet hemocyanin. 9. An antibody directed against a peptide with the sequence MSTWSKRSLSQEDAPQTPRPV. 10. An antibody directed against a protein-conjugated peptide, wherein the peptide has the sequence MSTWSKRSLSQEDAPQTPRPV. 11. The method of claim 2, wherein the antibody is directed against a peptide with the sequence MSTWSKRSLSQEDAPQTPRPV. 12. The method of claim 2, wherein the antibody is directed against a protein-conjugated peptide, the peptide having the sequence MSTWSKRSLSQEDAPQTPRPV. 13. The method of claim 2, wherein the antibody is a monoclonal antibody. 14. The method of claim 2, wherein the sample is a human tissue biopsy. 15. The method of claim 2, wherein the sample is human sera. 16. The method of claim 2, wherein the sample is human saliva. 17. The method of claim 2, wherein the sample is human urine. 18. The method of claim 2, wherein the sample is human semen. 19. The method of claim 2, wherein the sample is a human lavage sample. 20. A kit for use in detecting the presence of an antigen in a sample, the kit comprising: an antibody directed against a protein that is antigenically cross-reactive with preprorelaxin; an immunodetection reagent; means for containing said antibody and reagent.

Inventors: JETTEN ANTON M, LOTAN REUBEN, JETTEN, ANTON, M., LOTAN, REUBEN
Applicants: UNIV TEXAS, US HEALTH, BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, THE GOVERNMENT OF THE UNITED STATES AS REPRESENTED
Application year: 1992
Main classification: C07K15
IPC codes: C07K14, C07K16, G01N33