Suppression of metastasis by tissue inhibitor of metalloproteinase-1 in a newly established human oral squamous cell carcinoma cell line.

We have established a highly-metastatic cell line (designated as HNOS) and a non-metastatic cell line (designated as SAT) derived from human oral cavity squamous cell carcinoma (SCC). Both lines were transplantable in nude mice. The invasive activity through Matrigel-coated membrane of HNOS cells was also higher than that of SAT cells. mRNA of TIMP-1 was expressed in SAT cells but not in HNOS cells. Metastatic and invasive abilities were suppressed by the overexpression of TIMP-1 in HNOS cells. These results suggest that TIMP-1 may have an important role in inhibiting invasion and metastasis of human oral cavity SCC.

Down-regulated expression of CD44 variant 6 in oral squamous cell carcinomas and its relationship to regional lymph node metastasis.

The expression of the adhesion molecule CD44 variant 6 (CD44v6) was studied immunohistochemically on 38 oral squamous cell carcinomas (SCCs) and 10 biopsies of healthy oral mucosa. The relationship between the expression of CD44v6 and regional lymph node metastasis was also investigated. The expression of CD44v6 was apparently down-regulated in oral SCC, but not in normal oral mucosa. Carcinomas expressing lower levels of CD44v6 exhibited more frequent regional lymph node metastasis. The expression of CD44v6 showed no statistically significant relationship to the degree of differentiation, but tended to be down-regulated in poorly differentiated carcinoma. No significant relation was found between the expression of CD44v6 in primary and metastatic lesions.

CD44 variant 6 (CD44v6) expression as a progression marker in benign, premalignant and malignant oral epithelial tissues.

The change in the expression pattern of CD44 variant 6 (CD44v6) protein in benign, premalignant, and malignant (SCC) oral epithelial lesions was studied immunohistochemically and compared with the pattern in normal mucosa in order to examine whether this gene can serve as a progression marker in patients with SCC. The principal findings is that CD44v6 expression was clearly downregulated in most cases of severe premalignant lesions as well as in most of the SCCs. The staining pattern and intensity varied according to the degree of dysplasia and to the degree of differentiation of the SCCs. Premalignant severe epithelial dysplasia cases with early features of invasion, not yet developed into SCC, showed distinctly downregulated expression of CD44v6 protein whereas hyperplastic and benign epithelial lesions (papilloma) expressed positive staining patterns comparable to those of the normal counterparts. The authors conclude that alteration in CD44v6 may occur as an early event in primary oral SCC development, as well as in premalignant severe epithelial dysplasia. It can thus, be used as a molecular progression marker when screening for oral cancer.

[Expression of CD44 variant 6 and its metastatic potential in squamous cell carcinoma of the oral cavity].

Using antihuman CD44 variant 6 monoclonal antibody (2F10), immunohistochemical screenings were performed for 38 oral squamous cell carcinoma cases and 10 oral mucosa in healthy cases as normal counterpart. Normal epithelium in the oral surface was stained intensely by the antibody. The reactivity was particularly strong in the spinous layers of stratified squamous epithelium. Cells in the basal layers exhibited moderate staining. In contrast, expression of CD44v6 tended to be downregulated in 38 oral squamous cell carcinoma materials. Interestingly, more faint or no staining by the anti-CD44v6 monoclonal antibody was found in the primary squamous cell carcinomas involving regional lymphnode metastasis. Downregulation of CD44v6 isoform was suggested to occur during regional lymphnode metastasis on oral squamous, cell carcinomas.

Expression of type IV collagen-degrading metalloproteinases and tissue inhibitors of metalloproteinases in newly established human oral malignant tumor lines.

We have established 11 human oral tumor lines maintained as in subcutaneous xenografts in BALB/c athymic nude mice, and examined their metastatic and invasive characteristics, and expression of type IV collagen-degrading metalloproteinases and their intrinsic inhibitors (TIMPs). These tumor lines have approximately maintained the histological appearance of the parental tumors. Generally, human tumors maintained subcutaneously in nude mice metastasize only very rarely. However, one mesenchymal tumor line, a malignant melanoma designated MTN, was found to metastasize spontaneously to the lung and a lysate of the MTN cells had a high level of type IV collagenolytic activity. Among epithelial tumor lines, SKH, derived from squamous cell carcinoma, showed high expression of TIMP-1 in Northern blotting and had low type IV collagenolytic activity. SN, derived from squamous cell carcinoma, also showed low type IV collagenolytic activity. These two squamous cell carcinoma lines showed a non-invasive growth pattern when they were implanted orthotopically into the tongues of athymic nude mice. By contrast, tumor lines which showed higher type IV collagenolytic activity had a tendency to grow invasively in mouse tongue. These findings suggest that our 11 newly established tumor lines may provide useful systems for studies of tumor biology and therapy.

A biochemical evaluation of oral squamous cell carcinoma growth by measurement of specific activity of succinate dehydrogenase in the subrenal capsule assay.

An auxiliary method for determination of chemosensitivity with the subrenal capsule assay (SRCA) was developed in which the specific activity of succinate dehydrogenase (SD) of tumor implanted beneath the renal capsule is measured. The appropriate conditions for measuring the specific activity of SD were determined. The chemosensitivity of tumors, derived from six xenograft lines originating from oral squamous cell carcinomas, to peplomycin (PEP), cisplatin (CDDP), and 5-fluorouracil (5-FU) were evaluated by the SRCA and the nude mouse assay (NMA). The chemosensitivity evaluated by NMA displayed a higher degree of correlation with that determined by the improved SRCA than with that determined by the conventional SRCA. The correlations between overall accuracy of prediction with the NMA and those with the conventional SRCA and the improved SRCA were 72.2% and 88.9%, respectively. These findings suggest that our new assay may be useful for evaluation of chemosensitivity in the SRCA.

[Fundamental study of subrenal capsule assay by measuring specific activity of succinate dehydrogenase].

It may not show accurate results if subrenal capsule assay (SRCA) is made only by measuring tumor size, because of infiltration of host inflammation cells resulted from host immune reaction. We developed a new method which make possible an accurate determination of chemosensitivity by measuring specific activity of succinate dehydrogenase (SD) of the tumor cells implanted in the subrenal capsular space. With reference to SDI test, the assay condition for measuring specific activity of SD was determined. A comparative study was carried out in which malignant tumors of the oral cavity serially transplanted in nude mice were tested with SRCA and subcutaneous transplantation assay in nude mice. Chemosensitivity to peplomycin (PEP), CDDP and 5-fluorouracil (5-FU) evaluated SSDI method and nude mouse assay showed a high correlation than those evaluated by TGIR method and nude mouse assay. The overall predictive accuracy compared with nude mouse assay was 72.2% by TGIR method and 88.9% by SSDI method. SSDI method seemed to be a useful method to evaluate the chemosensitivity in SRCA.

Vascular endothelial cell growth factor (VEGF) produced by A-431 human epidermoid carcinoma cells and identification of VEGF membrane binding sites.

A distinct family of endothelial cell mitogens that are homologous to platelet-derived growth factor has recently been identified. Unlike other known endothelial cell mitogens, these vascular endothelial cell growth factors (VEGFs) are secreted and appear to act specifically on endothelial cells. We have purified VEGF 2083-fold to apparent homogeneity from protein-free culture medium conditioned by A-431 human epidermoid carcinoma cells. This A-431-derived VEGF was characterized as a homodimer composed of 22-kDa subunits with an N-terminal sequence that was similar to VEGFs produced by human HL-60 leukemic and U-937 histiocytic lymphoma cells. A-431 VEGF was used to identify specific and saturable binding sites for VEGF on human umbilical vein endothelial cells (HUVEC). By affinity cross-linking, VEGF-binding site complexes of 230, 170, and 125 kDa were detected on HUVEC. VEGF specifically induced the tyrosine phosphorylation of a 190-kDa polypeptide, which was similar in mass to the largest binding site detected by affinity cross-linking.

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium.

The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [3H]lathosterol to [3H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesterone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells.