Stepwise neoplastic transformation of a telomerase immortalized fibroblast cell line

We have described recently a human fibroblast cell line immortalized through ectopic telomerase expression (cen3tel), in which the extension of the life span was associated with the appearance of chromosomal aberrations and with the ability to grow in the absence of solid support. As reported in this article, on further propagation in culture, cen3tel cells became neoplastically transformed, being able to form tumors in nude mice. The analysis of the cells, during the gradual transition toward the tumorigenic phenotype, allowed us to trace cellular and molecular changes associated with different phases of transformation. At the stage in which they were able to grow in agar, cen3tel cells had lost contact growth inhibition but still retained the requirement of serum to proliferate and were not tumorigenic in immunocompromised mice. Moreover, they showed a down-regulation of the INK4A locus and were resistant to oncogenic Ras-induced senescence but still retained a functional p53. Subsequently, cen3tel cells became tumorigenic, lost p53 function because of a mutation in the DNA-binding motif, and overexpressed c-myc. Interestingly, tumorigenic cells did not carry activating mutations either in the ras proto-oncogenes (H-ras, N-ras, and K-ras) or in B-raf. Cen3tel cells gradually became hyperdiploid but did not display centrosome abnormalities. To our knowledge, cen3tel is the first telomerase immortalized fibroblast line, which became neoplastically transformed. In this system, we could associate a down-regulation of the INK4A locus with anchorage-independent growth and with resistance to Ras-induced senescence and link p53 mutations and c-myc overexpression with tumorigenicity

3T3 NIH murine fibroblasts and B78 murine melanoma cells expressing the "E. coli" N3-methyladenine-DNA glycosylase I do not become resistant to alkylating agents"

The role of alkylation of the N3 position of adenine in the cytotoxicity of alkylating agents in mammalian cells is still undefined. By co-transfecting NIH3T3 murine fibroblast and murine B78 H1 melanoma cells with pSG5tag and pSV2neo, we obtained clones expressing the mRNA of the bacterial tag gene coding for N3-methyladenine-DNA glycosylase I (Gly I), which specifically repairs N3-methyladenine. The levels of Gly I were 400 times higher in NIH3T3 pSG5tag (clone 3.9.4.) and 12-33 times higher in B78 H1 tag clones (2A4, 2A6, 2C3 and 2D1) than in the respective control cells. The sensitivity to alkylating agents was evaluated in tag-expressing cells in comparison with pSG5, pSV2neo cotransfected control cells. As regards the cytotoxic activity of methylating agents (N-methylnitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, dimethylsulfate and temozolomide) and other alkylators with different structure and different interactions with DNA such as CC-1065 and FCE-24517 (minor groove binders known to bind to N3 of adenine), 4-[bis(2-chloroethyl)amino]L-phenylalanine and cis-diamino-dichloroplatinum II, cytotoxicity was the same for tag-expressing and non-expressing cells. These results suggest that the increased expression of N3-methyladenine-DNA glycosylase is not necessarily a crucial mechanism for the resistance of cells to alkylating agents