In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-lspg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-lspg cell line. Additionally, after transfection of GC-lspg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-lspg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-lspg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin a6, L6 antigen, annexin VIII, BVL- 1 retrotransposon, protective protein, replacement variant histone 3.3, α-catenin and LPS-binding protein, are over-expressed in invasive GC-lspg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-lspg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.

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