Martina Anton

Requirement for Both Shc and Phosphatidylinositol 3′ Kinase Signaling Pathways in Polyomavirus Middle T-Mediated Mammary Tumorigenesis

ABSTRACT Transgenic mice expressing the polyomavirus (PyV) middle T antigen (MT) develop multifocal mammary tumors which frequently metastasize to the lung. The potent transforming activity of PyV MT is correlated with its capacity to activate and associate with a number of signaling molecules, including the Src family tyrosine kinases, the 85-kDa Src homology 2 subunit of the phosphatidylinositol 3′ (PI-3′) kinase, and the Shc adapter protein. To uncover the role of these signaling proteins in MT-mediated mammary tumorigenesis, we have generated transgenic mice that express mutant PyV MT antigens decoupled from either the Shc or the PI-3′ kinase signaling pathway. In contrast to the rapid induction of metastatic mammary tumors observed in the strains expressing wild-type PyV MT, mammary epithelial cell-specific expression of either mutant PyV MT resulted in the induction of extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias expressing the mutant PyV MT defective in recruiting the PI-3′ kinase were highly apoptotic, suggesting that recruitment of PI-3′ kinase by MT affects cell survival. Whereas the initial phenotypes observed in both strains were global mammary epithelial hyperplasias, focal mammary tumors eventually arose in all female transgenic mice. Genetic and biochemical analyses of tumorigenesis in the transgenic strains expressing the PyV MT mutant lacking the Shc binding site revealed that a proportion of the metastatic tumors arising in these mice displayed evidence of reversion of the mutant Shc binding site. In contrast, no evidence of reversion of the PI-3′ kinase binding site was noted in tumors derived from the strains expressing the PI-3′ kinase binding site MT mutant. Tumor progression in both mutant strains was further correlated with upregulation of the epidermal growth factor receptor family members which are known to couple to the PI-3′ kinase and Shc signaling pathways. Taken together, these observations suggest that PyV MT-mediated tumorigenesis requires activation of both Shc and PI-3′ kinase, which appear to be required for stimulation of cell proliferation and survival signaling pathways, respectively.

Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre.

We have constructed 293 cell lines expressing the site-specific Cre recombinase from bacteriophage P1, that acts on a 34 bp target sequence calledloxP. Stably transformed cells were obtained by transfection with a plasmid containing Cre and a selectable marker under the control of viral promoters. The resulting 293 Cre cell lines could be used to induce expression from adenovirus vectors containing reporter genes under the control of a Cre responsive “molecular switch” High efficiency recombination was observed for Ad viral DNA containingloxP sites. The Cre expressing cell lines described here are likely to be useful for several purposes: For expression of toxic gene products from Cre inducible viral vectors, to induce recombination betweenloxP sites in transfected plasmids, and to induce deletions or rearrangements of genes defined byloxP sites in viral genomes.

Reprogramming of Telomerase by Expression of Mutant Telomerase RNA Template in Human Cells Leads to Altered Telomeres That Correlate with Reduced Cell Viability

Telomerase synthesizes telomeric DNA by copying the template sequence of its own RNA component. In Tetrahymena thermophila and yeast (G. Yu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature 344:126-131, 1990; M. McEachern and E. H. Blackburn, Nature 376:403-409, 1995), mutations in the template domain of this RNA result in synthesis of mutant telomeres and in impaired cell growth and survival. We have investigated whether mutant telomerase affects the proliferative potential and viability of immortal human cells. Plasmids encoding mutant or wild-type template RNAs (hTRs) of human telomerase and the neomycin resistance gene were transfected into human cells to generate stable transformants. Expression of mutant hTR resulted in the appearance of mutant telomerase activity and in the synthesis of mutant telomeres. Transformed cells were not visibly affected in their growth and viability when grown as mass populations. However, a reduction in plating efficiency and growth rate and an increase in the number of senescent cells were detected in populations with mutant telomeres by colony-forming assays. These results suggest that the presence of mutant telomerase, even if coexpressed with the wild-type enzyme, can be deleterious to cells, likely as a result of the impaired function of hybrid telomeres.

High frequency recombination betweenloxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination betweenloxP sites in human chromosomes. G418 resistant cells with oneloxP site, generated by transfection with a plasmid containingloxP between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containingloxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked byloxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.