Figure 5. Immunocytochemical staining of HJC-15 cells for viral T-antigen and the cellular factors, p53 and E2F-1. Immunostaining of the JCV T-antigen transformed cell line, HJC-15. for the viral T-antigen (Panel A), and cellular proteins p53 and E2F-1 (panels B and C, respectively) demonstrate prominent nuclear localization. Original magnification, x400 (A and C) or x200 (B).

sibility is that E2F1, as opposed to other characterized members of the E2F family, is responsible for regulating expression of brain specific genes. This might occur through dimerization with brain specific helix-loop-helix proteins, including neuro-D [65]. In this regard, earlier studies have indicated that E2F1 regulates expression of genes needed for control of contact inhibition of growth as well as for control of cell shape and adherence to the substratum [66]. Also, it has been found that E2F1 regulates movement through the G0/G1 phase of the cell cycle when overexpressed in NIH3T3 fibroblasts [66]. Constitutive expression of E2F1 in these nontransformed fibroblasts causes them to move prematurely into S phase from GO/G1 during serum starvation [66, 67], Thus, while E2F1 regulates transcription of genes needed for exit from GO/G 1 and entry into S phase, it may also be involved in the regulation of transcription of tissue-specific genes. Taken together, these data indicate that E2F1 is likely to be a multifunctional transcription factor, regulating expression of different genes depending on the specific tissue or cell-type analyzed.

As mentioned above, the state of phosphorylation of pRb, presumably by various G1-associated cyclins, cyclin D and cyclin E, and their associated kinases, cdk4,6 and cdk2, respectively, affects its ability to bind to and regulate E2F activity. Thus, the control of E2F activity may be an event that takes place downstream from the action of cdk's in the G1 phase. It has been demonstrated that transforming growth factor /6 [TGF/6], a family of proteins with complex and sometimes opposing activities in various cell types [68, 69] inhibits cell-cycle progression in mid- to late-Gl phase. It appears that TGF/3 exerts its activity by inactivating cyclin D:cdk4,6 and cyclin E:cdk2 complexes which are believed to be important for phosphorylation of pRb. Phosphorylation of pRb liberates E2F from the pRb:E2F complex, allowing E2F to activate genes important for progressing cells into S phase. Therefore, TGF/6, by maintaining pRb in the underphosphorylated form, encourages formation of pRb:E2F complexes. To exert its activity, TGF/J generates signals which induce the production/activation of pl5, a cdk inhibitor, which in turn displaces and replaces another cdk inhibitor, p27, from its complexes with cyclin D:cdk4 and cyclin D:cdk6. The free p27, by forming complexes with cyclinE:cdk2 inhibits its action. Moreover, recent observations have demonstrated that treatment of epithelial cells with

TGF/S leads to a dramatic decrease in the level of E2F1 transcription in the cells, and overexpression of E2F1 in the cells can overcome TGF/S-mediated growth suppression [70]. Conversely, in mouse fibroblasts, addition of TGF/6 to the culture media induced E2F transcriptional activity [71]. These two observations suggest that TGF/6 possesses a unique ability to differentially regulate expression of E2F in various cell types.

Results from our laboratory have demonstrated that in T-antigen producing oligodendrocytic cells, the TGF/3 promoter is more responsive to E2F1 activity, and that overexpression of E2F1 under these conditions elevates the endogenous level of TGF/6 RNA. Of particular interest is the notion that overexpression of E2F1 in oligodendrocytic cells reproducibly results in the appearance of a novel TGF/6-related transcript of 1.8 kb in size in Northern blot assay. From these studies, it appears that under certain conditions, TGF/6 and E2F regulate each others' expression, as well as expression of other related genes, which may play an important roles in determining the rate of cell proliferation in normal and malignant states.

In this respect one may envision a scenario whereby the interplay between the JCV early protein, and p53 and pRb perturbs the regulatory function of these two tumor suppressors in brain cells. For example, the association of T-antigen with p53 may inhibit the ability of p53 to enhance expression of cellular genes including p21WAF-l. p21 WAF-1 is an inhibitor of the cyclin and cyclin-dependent kinase complexes. These complexes can prevent cell progression from G1 into S phase by phosphorylating pRb. The result of pRb phosphorylation, as mentioned earlier, is freedom of E2F-1, a transcription factor which can stimulate S-phase specific genes. Indeed, liberation of E2F-1 from pRb:E2F-l can also be accomplished upon direct association of T-antigen with pRb. Thus, JCV T-antigen directly by associating with pRb, and indirectly through p21 WAF-1, enhance G1 to S phase entry of the cells and causes brain tumor formation.

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