Yoshinori Suminami

Squamous cell carcinoma antigen is a new member of the serine protease inhibitors

We have cloned cDNA of squamous cell carcinoma antigen. Sequence analysis of the complete 1711 basepairs SCC antigen cDNA revealed that it coded 390 amino acids and no typical signal sequence in the NH2-terminus. Northern blot analysis of human squamous cell poly(A)+ RNA using this cDNA insert as the probe showed a single RNA species of about 1.7 kilobases. The cDNA was expressed in Escherichia coli and the product was detected by immunological methods using antibodies against SCC antigen, indicating that this cDNA encodes the entire SCC antigen sequence. The amino acid homology search revealed that SCC antigen was a member of the serine protease inhibitors family.

Inhibition of apoptosis in human tumour cells by the tumour-associated serpin, SCC antigen-1

The squamous cell carcinoma antigen (SCC Ag) is a tumour-associated protein and a member of theserineproteaseinhibitor (serpin) family. The SCC Ag has been used as a serologic tumour marker for SCC progression, and its elevated serum levels are a risk factor for disease relapse. However, the biologic significance of this intracytoplasmic protein in cancer cells remains unknown. In this report, we demonstrated that apoptosis induced by 7-ethyl-10-hydroxycamptothecin, tumour necrosis factor-α (TNF-α) or interleukin (IL)-2-activated natural killer (NK) cells was significantly inhibited in tumour cells transduced with the SCC Ag-1 cDNA, as compared to control cells in vitro. Also, inhibition of the SCC Ag-1 expression in tumour cells by transfection of antisense SCC Ag-1 cDNA was accompanied by significantly increased sensitivity of these cells to apoptosis induced by etoposide or TNF-α. The mechanism of protection of tumour cells from apoptosis involved inhibition of caspase-3 activity and/or upstream proteases. In vivo, tumour cells overexpressing the SCC Ag-1 formed significantly larger tumours in nude mice than the SCC Ag-1-negative controls. Thus, overexpression of the SCC Ag-1, a member of the serpin family, in human cancer cells contributed to their survival by mediating protection from drug-, cytokine- or effector cell-induced apoptosis.

Comparative genomic hybridization detects genetic alterations during early stages of cervical cancer progression.

Invasive cervical carcinoma is thought to arise from cervical intraepithelial neoplasm (CIN). Genetic changes that occur during progression of CIN to cervical carcinoma are poorly understood, although they appear to be directly involved in this process. We used comparative genomic hybridization (CGH) with precise microdissection and degenerate oligonucleotide primed‐polymerase chain reaction (DOP‐PCR) to detect genetic alterations in normal epithelial, CIN, and invasive carcinoma tissues colocalized in tumors from 18 patients with squamous cell carcinoma of the uterine cervix. Gains on chromosome 1 and on 3q and losses on 2q, 3p, 4, 6p, 11q, and 17p were frequent alterations found in CIN and invasive carcinoma lesions. Interestingly, several of these genetic changes were observed in preinvasive carcinoma lesions. The frequency and average number of genetic alterations corresponded directly to the extent to which the cervical carcinoma had progressed. Frequent alterations were found in more than 90% of CIN III lesions. Gains on 3q and losses on 11q were the most prevalent genetic alterations found in association with uterine cervix carcinogenesis. The common regions of alteration were 3q26.1‐q28 and 11q23‐qter. The majority of tumor samples showed variability in genetic alterations across lesion types within a single specimen.

Squamous cell carcinoma antigen suppresses radiation-induced cell death

Previous study has demonstrated that squamous cell carcinoma antigen (SCCA) 1 attenuates apoptosis induced by TNFα, NK cell or anticancer drug. In this study, we have examined the effect of SCCA2, which is highly homologous to SCCA1, but has different target specificity, against radiation-induced apoptosis, together with that of SCCA1. We demonstrated that cell death induced by radiation treatment was remarkably suppressed not only in SCCA1 cDNA-transfected cells, but also in SCCA2 cDNA-transfected cells. In these transfectants, caspase 3 activity and the expression of activated caspase 9 after radiation treatment were suppressed. Furthermore, the expression level of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was suppressed compared to that of the control cells. The expression level of upstream stimulator of p38 MAPK, phosphorylated MKK3/MKK6, was also suppressed in the radiation-treated cells. Thus, both SCCA1 and SCCA2 may contribute to survival of the squamous cells from radiation-induced apoptosis by regulating p38 MAPK pathway.

Biological Role of SCC Antigen

SCC antigen is a tumor-associated protein of squamous cell carcinoma of various organs. So far, two genes (SCC Ag-1 and SCC Ag-2) have been identified, and their products are highly homologous and classified as serine protease inhibitors (serpin). Recombinant SCC antigen-1 inhibits chymotrypsin and cathepsin L in vitro, indicating that it is inhibitory type serpin. Transduction of tumor cells with SCC antigen-1 reveals that SCC antigen-1 inhibits apoptosis of tumor cells induced by anticancer drug, TNFα or NK cells. Therefore SCC antigen-1 may work in cancer cells for tumor growth, and in normal squamous epithelium for differentiation by means of the inhibition of apoptosis. Recombinant SCC antigen-2 inhibits cathepsin G and mast cell chymase, suggesting that it protects epithelial cells from the inflammation induced by these proteases.

Expression of mRNA of SCC Antigen in Squamous Cells

The expression of SCC antigen mRNA was studied by Northern blot analysis and in situ hybridization in human gynecologic tissues. Northern blot analysis revealed that mRNA of SCC antigen was expressed strongly in normal squamous epithelium and columnar epithelium of the uterine cervix, but not in the endometrium, fallopian tube or ovarian tissue. Among gynecologic malignancies, squamous cell carcinoma of the uterine cervix expressed mRNA of SCC antigen, whereas endometrial and ovarian adenocarcinoma were negative. With in situ hybridization, mRNA of SCC antigen is located in the basal and parabasal layers of the normal squamous epithelium, in dysplasia, and also in carcinoma in situ and invasive squamous cell carcinoma. These results confirmed the previous findings by immunohistochemical studies that SCC antigen is closely related to squamous cells, but also raised a new puzzle regarding the different localization of mRNA and its product, SCC antigen, in the normal squamous epithelium.

Altered Proliferative and Metastatic Potential Associated with Increased Expression of Syndecan-1

The process of metastasis involves a series of sequential steps in which malignant cells are released from the primary tumor and metastasize to distant sites. Syndecan-1 is a cell surface proteoglycan that mediates cell adhesion and undergoes changes upon cell transformation of some cells that may contribute to the process of metastasis. To investigate the possible role of syndecan-1 in cell proliferation and metastatic potential, we employed a highly metastatic cell line (KLN 205) derived from mouse lung squamous cell carcinoma that expressed moderate amounts of syndecan-1. At first, endogenous syndecan-1 production was inhibited by an antisense oligodeoxynucleotides (ODNs). Since antisense ODNs of syndecan-1 inhibited cell growth, we established stable transfectants of syndecan-1 in this cell line to examine a proliferative advantage with the level of syndecan-1 expression. Overexpresser cells grew at a significantly faster rate than the vector-transfected control and showed greater incidence of tumor formation when injected subcutaneously into nude mice. Surprisingly, overexpresser cells enhanced pulmonary metastasis when injected intravenously. These results indicate that syndecan-1 expression plays a role in the control of cell proliferation and suggest that syndecan-1 expression may be involved in facilitating distant metastasis of tumor cells once they managed to enter the bloodstream (after intravasation steps).

Specific Detection and Quantitation of SCC Antigen 1 and SCC Antigen 2 mRNAs by Fluorescence-Based Asymmetric Semi-Nested Reverse Transcription PCR

Squamous cell carcinoma antigen (SCCA) is expressed in normal squamous epithelia and malignant squamous cell tissues. The serum level of SCCA has been used to evaluate treatment efficacy, clinical course of disease, and recurrence. SCCA is produced by at least two genes (SCCA1 and SCCA2); both of them have been located on chromosome 18q21.3. It has been difficult to examine the expression levels of SCCA1 and SCCA2 mRNAs separately because of their high homology at nucleotide level. In the present study, asymmetric semi-nested reverse transcription PCR, based on the principle of fluorescence energy transfer, enabled to quantitate the copy numbers of both SCCA1 and SCCA2 mRNAs. Using this method, the expression levels of these mRNAs were evaluated in normal and malignant squamous tissues. The copy number of SCCA2 mRNA was higher in malignant tissues than in normal tissues, while those of SCCA1 mRNA did not significantly differ between normal and malignant tissues. These data indicate that specific quantitation of the expression level of SCCA2 mRNA may be useful for the diagnosis and management of patients with squamous cell carcinoma.

Tumor Necrosis Factor-α Stimulates the Production of Squamous Cell Carcinoma Antigen in Normal Squamous Cells

Squamous cell carcinoma (SCC) antigen, a tumor marker of squamous cell carcinoma, is also increased in several nonmalignant skin lesions, e.g. pemphigus. The aim of the present investigation was to determine if tumor necrosis factor-alpha (TNF-alpha), one of the important environmental factors, stimulated the production of SCC antigen in the normal squamous cells. The exposure of normal human epidermal keratinocytes to TNF-alpha (100 IU/ml) for 72 h greatly increased the SCC antigen production. The stimulatory effect of TNF-alpha (1,000 IU/ml) on the production of SCC antigen was also observed in the normal squamous epithelium tissue. These results would be helpful for understanding the increase of SCC antigen in several nonmalignant skin disorders.

Serum Anti-p53 Antibodies in Uterine and Ovarian Cancer: Association with DNA Sequence Copy Number Abnormalities

The aim of the present study was to evaluate the clinical significance of the serum anti-p53 antibody in patients with uterine and ovarian cancer. Some of the ovarian patients were also evaluated for overexpression of p53 by immunohistochemistry and for cytogenetic alterations by comparative genomic hybridization (CGH). Serum anti-p53 antibodies were determined by an enzyme immunoassay kit. The antibody was detected in 8/30 (27%) of ovarian cancers, in 12/86 (14%) cancers of the uterine cervix, in 5/41 (12%) cancers of the uterine body, and 0/9 (0%) healthy women. The overall survival rate in patients with ovarian cancer was significantly worse in patients with anti-p53 antibody positivity than that in patients with anti-p53-antibody-negative cancers using the log rank test (p = 0.017). There was a significant correlation between the presence of anti-p53 antibody and tissue overexpression of p53 in ovarian cancers. CGH analysis showed that the aberrations in DNA sequence copy number in ovarian cancers were significantly increased in anti-p53-antibody-positive cases compared to antip53-antibody-negative cases including increased copy number on 20q and reduced copy number on 5q and 13q. Although the exact relationship between the presence of serum anti-p53 antibody (specific humoral response) and cytogenetic alterations is still unknown, these findings suggest that the measurement of serum anti-p53 antibody may be useful for the assessment of genetic instability and tumor biological aggressiveness.