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Recently, it has been suggested that chemokine/receptor interactions determine the destination of the invasive tumor cells in several types of cancer. It has also been proposed that the stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system might be involved lymph node metastasis in oral squamous cell carcinoma (SCC). In order to further clarify the role of the SDF-1/CXCR4 system in oral SCC, we generated CXCR4 stable transfectants (IH-CXCR4) using oral SCC cells, and compared them to IH, which did not express CXCR4 and which did not have lymph node metastatic potentials in vivo. We introduced enhanced green fluorescent protein (GFP) fused-CXCR4 into IH cells, and detected the GFP fluorescence in the cytoplasm and cell membrane in approximately 60% of the G418-resistant cells. This bulk-transfectant expressed a high level of CXCR4 mRNA and protein, and exhibited the characteristic calcium fluxes and chemotactic activity observed in treatment with SDF-1. SDF-1 biphasically activated extracellular signal-regulated kinase (ERK)1/2, but continuously activated Akt/protein kinase B (PKB) in IH-CXCR4 cells. Most importantly, IH-CXCR4 cells frequently metastasized to the cervical lymph node, but not to the distant organs in the orthotopic inoculation of nude mice. Furthermore, these lymph node metastases were inhibited by the treatment of a mitogen-activated protein kinase/ERK kinase inhibitor, U0126, or a phosphatidylinositol 3 kinase inhibitor, wortmannin. These results indicate that SDF-1/CXCR4 signaling mediates the establishment of lymph node metastasis in oral SCC via ERK1/2 or Akt/PKB pathway.

We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.