Purpose: To investigate the biological correlation between vascular endothelial growth factor (VEGF)-C expression and invasive phenotype in ovarian carcinomas. Experimental Design: Gene and protein expression levels of VEGF-C in 10 ovarian carcinoma cell lines were correlated with invasive activity of the cells. The correlation between immunohistochemical expression of VEGF-C and tumor aggressiveness in 73 ovarian carcinomas was also examined with respect to clinicopathologic features and patient outcome. Results:VEGF-C gene and protein expression differed remarkably among the cell lines, and there was a statistical correlation among VEGF-C expression, in vitro invasive activity, and matrix metalloproteinase-2 (MMP-2) gene expression and its activity. Anti-VEGF-C and anti-MMP-2 antibodies inhibited the invasive activity of tumor cells. VEGF-C expression in clinical tissue samples was well correlated with clinical stages, retroperitoneal lymph node metastasis, MMP-2 expression, angiogenesis, lymphangiogenesis, and low apoptotic index (AI). The patients whose tumors had strong VEGF-C expression and low AI underwent a poorer prognosis than did those with weak VEGF-C expression and high AI. Conclusion: VEGF-C expression is closely related to invasive phenotype and affects the patient's survival in ovarian carcinomas.

A total of 63 pigmented or non-pigmented endometriotic, 26 endometrial carcinoma, and 12 normal eutopic endometrial tissues were examined for mRNA expression of survivin, matrix metalloproteinase (MMP)-2, MMP-9, and membrane-type 1 (MT1)-MMP. The expression levels of the survivin and MMPs genes in clinically aggressive pigmented lesions were significantly higher than those in normal eutopic endometrium, and survivin gene expression in pigmented lesions was also higher than that in non-pigmented lesions (P < 0.05). There was a close correlation between the expression levels of the survivin and MMPs genes in 63 endometriotic tissues examined (P < 0.01). Apoptotic cells detected by dUTP nick-end labeling were rare in I I ovarian endometriotic tissues, which showed positive immunohistochemical expression for survivin and MMPs. Expression levels of the survivin and MMPs genes in endometrial carcinomas were higher than those in normal eutopic endometrium and were well correlated with the depth of myometrial invasion (P < 0.05). There was a close correlation between the expression levels of the survivin and MMPs genes in 26 endometrial carcinoma tissues examined (P < 0.01). These findings suggest that upregulation of survivin and MMPs may contribute cooperatively to survival and invasion of endometriosis and endometrial carcinomas.

An association between deregulation of homeobox (HOX) gene expression and oncogenic transformation has been recently reported in human tumors. In this study, we investigated HOX gene expression and mutation in cervical carcinoma cells. Using reverse transcription‐PCR, 11 human cervical carcinoma cell lines and 14 normal cervical tissue samples were examined for mRNA expression of the 39 class I HOX genes. DNA samples from 11 cell lines were tested for mutations in exons 1 and 2 of the HOXA10 and A13 genes using overlapping primer pairs which also cover intron 1 of these genes. HOXA1, B2, B4, C5, C10 and D13 genes were expressed in 8, 7, 9, 9, 9 and 11 of 11 cervical carcinoma cell lines, respectively, but not in any of the normal cervical tissues. HOXA9, A11, A13, B5, C4, D3 and D9 genes were expressed in all cell lines and normal tissues. In contrast, 13 of 39 HOX genes were silent in all materials examined. Single‐strand conforma‐tional polymorphism and sequence analysis revealed a C insertion after base 1042 and/or a G to C substitution at base 1113 in intron 1 of the HOXA13 gene in 4 of 11 cell lines, however, neither deletions nor mutations were detected in exons 1 and 2 of the HOXA10 and A13 genes. Our data suggest that the expression of HOXA1, B2, B4, C5, C10 and D13 genes might be involved in the process leading to the transformation of normal cervical cells. (Cancer Sci 2003; 94: 437–441)

The genes of the glutathione S-transferase (GST) family encode enzymes that appear to be critical in cellular protection against the cytotoxic effects, whereas p53 is a tumor suppressor gene. Despite a large number of studies on germline polymorphisms of GSTM1, GSTT1 and p53 genes, there have been very few reports on genotyping of these genes in human malignant tumor cells. In this study, we investigated GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human tumor cell lines originating from different organs to clarify tissuespedic polymorphic frequency of these genes in human solid tumors. The GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex PCR techniques and PCR-RFLP analysis was conducted to identify p53 codon 72 genotypes. Gene expression of GSTM1 or GSTT1 was detected by RT-PCR in the cells with respective present genotype for each. Polymorphisms of p53 codon 72 detected by PCR-RFLP were also confirmed using SSCP and sequence analyses. GSTM1 and GSTT1 genotypes were various in 104 cell lines examined. Null GSTM1 genotype was dominant in small cell lung, kidney and ovarian carcinoma cells, whereas null GSTT1 genotype was dominant in cervical and endometrial Carcinoma cells. GSTM1 and GSTT1 genotypes in ovarian carcinoma cells were quite similar to those in small cell lung carcinoma cells. Polymorphic frequency of p53 codon 72 was also various among the cells, however, the Pro allele was found in only 1 of 6 kidney, 14 cervical and 4 endometrial carcinoma cell lines. There was a significant difference in GSTM1 and p53 genotypes between 34 small cell and 24 non small cell lung carcinoma cells (P < 0.01). Combined study on the distribution of GSTM1, GSTT1 and p53 genotypes revealed that null GSTM1 genotype was associated with the Arg allele of p53 codon 72 in 58 lung carcinoma cells and null GSTT1 genotype was associated with the Pro/Pro homozygote in 104 tumor cell lines examined. This is the first study examining GSTM1, GSTT1 and p53 codon 72 polymorphisms in a variety of human solid tumor cells and suggesting that polymorphic frequency of these genes may be tissue- and organ-specific. The molecular interaction between GST gene defects and p53 codon 72 genotype in the development of human malignant tumors should be further investigated.

The correlation between vascular endothelial growth factor (VEGF)‐C gene expression and in vitro invasive activity and matrix metalloproteinase (MMP)‐2 or 9 gene expression and proteolytic activity in 11 cervical carcinoma cell lines, was investigated. Immunohistochemical expression of VEGF‐C in 52 cervical carcinoma tissues was also correlated with tumor aggressiveness with respect to clinicopathologic features, tumor vascularity, MMP‐2 expression and patient outcome. Expression of VEGF‐C mRNA differed remarkably among the cell lines and there was a statistical correlation between VEGF‐C gene expression and the number of invaded tumor cells (p = 0.0009) and MMP‐2 gene expression and activity (p < 0.05). Anti‐VEGF‐C antibody inhibited the invasive and proteolytic activity of tumor cells in a concentration‐dependent manner. VEGF‐C or MMP‐2 expression in clinical tissue samples was well correlated with depth of myometrial invasion, endometrial invasion, pelvic lymphnode metastasis and tumor vascularity (p < 0.05) and there was a close relation between VEGF‐C and MMP‐2 expression (p < 0.0001) in cervical carcinomas. Overall survival rates for 14 patients with strong VEGF‐C staining tumors were lower than those for 38 patients with weak VEGF‐C staining tumors (p = 0.0132) and VEGF‐C tissue status emerged as an independent prognostic parameter (p = 0.0232). These results suggest that VEGF‐C expression is closely related to invasion phenotype and affects the patient's survival in cervical carcinomas. © 2002 Wiley‐Liss, Inc.

Survivin is a novel inhibitor of apoptosis and is expressed during fetal development and in cancer tissues, but its expression has not been reported in normal adult tissues or benign diseases. We investigated survivin gene and protein expression in a tumor-like benign disease, endometriosis, and correlated them with apoptosis and invasive phenotype of endometriotic tissues. Gene expression levels of survivin, matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1 (MT1)-MMP in 63 pigmented or nonpigmented endometriotic tissues surgically obtained from 35 women with endometriosis were compared with those in normal eutopic endometrium obtained from 12 women without endometriosis. Survivin, MMP-2, MMP-9, and MT1-MMP mRNA expression levels in clinically aggressive pigmented lesions were significantly higher than those in normal eutopic endometrium, and survivin gene expression in pigmented lesions was also higher than that in nonpigmented lesions (P < 0.05). There was a close correlation between survivin and MMP-2, MMP-9, or MT1-MMP gene expression levels in 63 endometriotic tissues examined (P < 0.01). Apoptotic cells detected by the dUTP nick-end labeling were rare in 11 ovarian endometriotic tissues, which showed positive immunohistochemical expression for survivin and MMPs. Our findings suggest that up-regulation of survivin and MMPs may cooperatively contribute to survival and invasion of endometriosis.

OBJECTIVE:The correlation between the gene expression of various angiogenic factors and in vitro invasive activity in 16 human gynecological cancer cell lines was investigated.METHODS:Semiquantitative reverse transcription polymerase chain reaction analysis was performed to investigate the mRNA expression levels of vascular endothelial growth factors (VEGF-A, -B, -C, and -D), basic fibroblast growth factor (bFGF), and matrix metalloproteinase (MMP)-2 with beta-actin coamplified as an internal standard. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were evaluated by haptotactic migration and invasion assay.RESULTS:Expression of VEGF-A mRNA was detected in all 16 cell lines, whereas the relative expression levels of other VEGF family members and bFGF, differed markedly among the cell lines. There was a statistical correlation between VEGF-C gene expression and the number of cells that migrated and invaded (P < 0.01). However, expression of mRNAs of other angiogenic factors did not correlate with motility and invasive activity of the cells. Moreover, there was a close correlation between VEGF-C and MMP-2 gene expression levels (P < 0.05).CONCLUSION:Tumor cells that produce VEGF-C may have a higher invasive and metastatic potential because of their capacity to pass through tissue barriers.