Isolation and characterization of differentially expressed genes in invasive and non-invasive immortalized murine male germ cells in vitro

In an attempt to elucidate the potential of premeiotic male germ cells to m alignant transformation both the invasiveness and the differential gene exp ression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg 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 tumo rs demonstrated that the expression of the endogenous mouse embryonic alkal ine phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally , after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT con struct, an increased promoter activity of the human germ cell alkaline phos phatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg . Furthermore, an in vitro Matrigel invasion assay revealed a significant h igher invasive potential of GC-1spg cells as compared to GC-4spc cells. Fin ally, a suppression subtractive hybridization on RNA of invasive GC-1spg ce lls and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequenc es 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 alpha6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement varia nt histone 3.3, alpha -catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activ ity of GC-1spg cells and the upregulated expression of genes involved in th e process of tumor progression suggest that the immortalized spermatogonia- derived cell line GC-1spg does have a higher potential to malignant transfo rmation than the immortalized spermatocyte-derived cell line GC-4spc.