Multiple mechanisms of N-(phosphonoacetyl)-L-aspartate drug resistance in SV40-infected precrisis human fibroblasts.

Normal and SV40-infected human fibroblasts were grown in the presence of the drug N-(phosphonoacetyl)-L-aspartate (PALA) and examined for evidence of genetic instability. Both cell populations were precrisis and showed a normal, diploid karyotype at early passage. In contrast to the normal IMR-90 cells, which showed growth arrest and did not form colonies in PALA, the SV40-infected IMR-90 cells formed colonies at a very high frequency and continued to cycle in the drug. The drug-resistant colonies senesced after continued growth in culture, indicating that this change in ability to amplify preceded immortalization. This is the first observation of mortal human cells overcoming the drug-induced growth arrest. Although all previously isolated PALA-resistant colonies demonstrated CAD gene amplification as the mechanism of the drug-resistant phenotype, these SV40-infected human cells also showed alternative mechanisms, including increases in gene copy number by aneuploidy and formation of an isochromosome 2p.

Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.

Genomic integrity is maintained by a network of cellular activities that assess the status of the genome at a given point in time, provide signals to proceed with or halt cell cycle progression, and provide for repair of damaged DNA. Mutations in any part of these pathways can have the ultimate effect of disturbing chromosomal integrity. Recent work suggests that p53 performs this integrator function in mammalian cells. Our present study demonstrates that in mortal cells, the expression of E6 and E7 viral oncoproteins of type 16 human papillomavirus each disrupts the integration of these signals by diverged pathways. Cells expressing E6 protein, which binds and degrades the p53 protein, exhibited alterations in cell cycle control when placed in drug and displayed the ability to amplify the CAD gene. The expression of E7, which binds different cellular proteins important for transformation, including Rb, led to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. These results demonstrate that diverse perturbations of molecular pathways can have different effects on chromosomal integrity.

Alteration of cell cycle kinase complexes in human papillomavirus E6- and E7-expressing fibroblasts precedes neoplastic transformation.

Expression of viral oncoproteins results in the loss of cell cycle checkpoint control and the accumulation of chromosomal abnormalities. Expression of both human papillomavirus type 16 oncoproteins, E6 and E7, in normal human fibroblasts completely dissociates p21 and proliferating cell nuclear antigen from the quarternary cyclin-cyclin-dependent kinase (CDK) complexes present in normal cells, causes disruption of the cyclin D-CDK4 complex and replacement with a CDK4-p16 complex, and leaves binary complexes of cyclin B1-CDC2 and cyclin A-CDK2 intact. These results are identical to those observed in fully transformed cells. The expression of the individual oncoproteins dramatically affects the association of proliferating cell nuclear antigen into the complexes while leaving the total cellular levels unaltered. Expression of low-risk human papillomavirus has no effect on cyclin complexes. These findings provide evidence for the gross alteration of cyclin-CDK complexes in preneoplastic cells and links this alteration to the loss of genomic stability.