Coordinated trans activation of DNA synthesis- and precursor-producing enzymes by polyomavirus large T antigen through interaction with the retinoblastoma protein.

Abstract

Previously constructed Swiss mouse 3T3 fibroblasts producing polyomavirus large T antigen after addition of dexamethasone were used to study the transcriptional activation by the viral protein of five genes coding for enzymes involved in DNA synthesis and precursor production, namely, dihydrofolate reductase, thymidine kinase, thymidylate synthase, DNA polymerase a, and proliferating-cell nuclear antigen. It was found that all these genes, whose expression is stimulated at the G1/S boundary of the cell cycle after growth stimulation by serum addition, are coordinately trans activated when T antigen is induced in cells previously growth arrested by serum withdrawal. Cell lines carrying the information for a mutant form of large T antigen, in which a glutamic acid residue in the binding site for the retinoblastoma protein was changed into aspartic acid, were constructed to test the involvement of an interaction of T antigen with the retinoblastoma protein in this reaction. It was found that the mutated T protein is incapable of stimulating transcription of any one of the genes. The promoter of three of the genes (dihydrofolate reductase, thymidine kinase, and DNA polymerase ax) unequivocally carries binding sites for transcription factor E2F, suggesting that complexes forming with this growth-and cell cycle-regulating transcription factor are the targets for T antigen. Although there is so far no evidence that thymidylate synthase and proliferating cell nuclear antigen are regulated via E2F, our data indicate that the retinoblastoma protein still is involved in the control of these genes. mRNA for E2F itself increases in amount at the G,/S border in serum-stimulated cells but not during polyomavirus T antigeninduced transcriptional activation of DNA synthesis enzymes in arrested cells.Enzymes involved in DNA synthesis and precursor production are regulated during growth stimulation and during the cell cycle. Their synthesis is induced at the beginning of the S phase, and in many cases controls at different levels, transcriptional as well as posttranscriptional, are crucial (for reviews, see references 32 and 42). Transcriptional regulation plays the major role when cells are stimulated to grow from an arrested state. Members of a family of transcription factors called E2F were recently found to play a key role in transcriptional regulation of many important genes in the GI phase and at the G,/S boundary of the cell cycle (reviewed in references 4, 15, and 27). E2F was originally identified as a cellular protein binding to the adenovirus E2 promoter, but binding sites for E2F were later also found in cellular promoters and were shown to be important for the transcription of cell cycleregulated genes such as c-myc, dihydrofolate reductase, and thymidine kinase (2,17,27,30,38). E2F is a target for the retinoblastoma tumor suppressor protein (pRB); a related protein, p107 (reviewed in references 4, 15, and 27); and possibly other so-called pocket proteins; complexes formed on binding sites for E2F were also found to c...

Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents

“…This suggested that 5-FU might be more efficiently activated to FdUMP in RB-deficient cells than in RB-proficient cells. Consistent with this concept, the promoters of two genes encoding enzymes involved in 5-FU metabolism to FdUMP, thymidine kinase and RNR (Longley et al, 2003), are regulated by the RB/E2F axis (Mudrak et al, 1994;Angus et al, 2002). Indeed, expression of the RNR small subunit (RNR2) was significantly elevated following acute RB loss in primary MAFs (Figure 7e).…”

“…Interestingly, we detected increased formation of the inactive ternary complex in RBdeficient cells exposed to 5-FU. We speculate that this was due to the RB/E2F-targets RNR and thymidylate kinase (TK) that are elevated in RB-deficient cells (Ogris et al, 1993;Mudrak et al, 1994;DeGregori et al, 1995;Angus et al, 2002). Analysis of upstream DNA damage checkpoint signaling revealed similar kinetics of activation of p53 by 5-FU in both RB-proficient and RB-deficient cells.…”

Polyomavirus large T antigen-dependent DNA amplification

“…Stable REF52 lines, inducibly expressing wild type PyLT (REF52-LT8) and mutated PyLT (REF52-LTRB 7 12) were generated by calcium phosphate cotransfection of the plasmid pSV2neo and pMMTV-PyLT or pMMTV-PyLTmut respectively (Ogris et al, 1992;Mudrak et al, 1994), and subsequent selection in the presence of Geneticin (600 mg/ml of medium). Time determination of population doubling (PDL) was done as described previously (Otto et al, 1989).…”

Polyomavirus large T antigen overcomes p53 dependent growth arrest

“…LT is a nuclear phosphoprotein that induces cellular DNA synthesis and cell division, is responsible for immortalizing cells, and is necessary for viral DNA synthesis (Francke and Eckhart, 1973;Schlegel and Benjamin, 1978;Rassoulzadegan et al, 1983). LT associates with pRB and this association is necessary for the induction of cellular DNA synthesis and immortalization of primary cells (Dyson et al, 1990;Larose et al, 1991;Freund et al, 1992Freund et al, , 1994Mudrak et al, 1994). Several other DNA tumor viruses, including SV40, adenovirus, and human papilloma virus, encode proteins that associate with and inactivate both p53 and pRB (Sarnow et al, 1982;DeCaprio et al, 1988;Whyte et al, 1988;Werness et al, 1990).…”