B

Figure 3. JCV T-antigen transformed cells form tumors when injected subcutaneously into the flanks of Nude mice. (A) HJC-15 cells injected subcutaneously form large palpable masses within 2-3 weeks. (B) Histological evaluation of the excised tumors reveals poorly differentiated neural-origin tumors. Original magnification, x400.

helicase

DNA host pol NLS binding ATPase range

Figure 4. Structural and functional domains of the JC viral regulatory oncoprotein, T-antigen. Domains represented include the nuclear localization signal (NLS), and regions responsible for DNA binding, polymerase (pol), helicase, ATPase activity and host range specificity. The black boxes depict the regions of the protein which are responsible for binding to the cellular factors, p53 and pRb.

for complex formation with the cellular tumor suppressor protein, p53. Despite sequence conservation in the regions encompassing p53 binding sites, the interaction between p53 and JCV T-antigen has not been completely established. In earlier studies, analysis of proteins from owl monkey brain tumors induced by intracranial inoculation of JCV, or proteins from JCV-infected human fetal glial cells, revealed nuclear expression of JCV T-antigen which was not associated with the host cell p53 protein [24, 42]. However, later studies have demonstrated complex formation between p53 and the early protein of JCV in owl monkey glioblastoma. These tumors were generated by intracranial inoculation of a juvenile owl monkey with a cell suspension of an explanted JCV-induced owl monkey glioblastoma [25]. The T-antigen protein synthesized by this virus reacted with several monoclonal antibodies which differentially detect T-antigens of SV40 and the other strains of JCV (Mad-1). Im-munoprecipitation of T-antigen from JC V-transformed primary hamster brain cells showed an association of cellular proteins (50 to 56 kDa), which may represent cellular p53 protein. These discrepancies may stem from heterogeneities within the regions of T-antigen important for its interaction with p53. Additionally, JCV T-antigen potentially interacts with the cellular tumor suppressor proteins pRb and its related family, including pi07, pi30 and p300 and perhaps, upon in-activation of these proteins, mediates its transforming and tumorigenic activities.

It is postulated that JCV T-antigen may mediate its transforming potential through its interaction with the cellular tumor suppressor proteins, p53, pl07, pl30/Rb2, p300 and the retinoblastoma gene product, pRb. In support of this hypothesis, results from co-immunoprecipitation assays have revealed the association of JCV T-antigen with p53 [43, 44], pi07

[45] and pRb [46], The critical question is whether, or not, the expression of T-antigen functionally blocks the inhibitory effects of these proteins in normal cells, leading to the oncogenic transformation of glial cells. As well, the question remains as to whether expression and/or biological activities of cell-cycle regulators, including cyclins and their inhibitors, which are important for activation/inactivation of the pRb family, alter transformation and the course of initiation and progression of brain tumors in an in vivo whole animal system.

A large number of studies during the past 5 years have shown that several oncoproteins from DNA tumor viruses, by sequestering pRb, disrupt the normal interaction of this protein with its cellular partners, including the transcription factor, E2F [47-50]. E2F was initially identified as a cellular protein whose ability to bind DNA was stimulated upon adenovirus infection [51]. Functional E2F binding sites, with the consensus sequence, 5'TTTSSCGC-3'(S = C or G), have been identified in promoters of several cellular genes, including E2F-1 itself, dihydrofolate reductase (DHFR), c-myc, cdc2, thymidine kinase (tk), cyclin D1, c-myb, ribonucleotide reductase and DNA polymerase-« (for reviews, see [52, 53]). Furthermore, E2F binding sites have been shown to be necessary and sufficient for proper temporal expression [54, 55]. In the JCV hamster glioma cell line, HJC-15, T-antigen expression leads to high levels of p53 and E2F-1, possibly through similar mechanisms (Fig. 5).

E2F itself appears to be a multicomponent transcription factor whose ability to bind DNA increases upon association with DP proteins [56]. A number of cDNA clones have recently been isolated that encode distinct members of the E2F family [57-60]. These various proteins bind to the same consensus E2F sites, but differ in their interaction with various cellular proteins, and in their cell-cycle profile.

An interesting aspect of E2F function is that its expression in nontransformed cells in culture is cell-cycle regulated, as it rises from undetectable levels during quiescence to very high levels prior to the onset of DNA synthesis in cell culture systems [61, 62]. Of interest, it has been reported that E2F1 expression is very high in adult brain [63] in which there typically are low levels of proliferating cells. It appears that other members of the E2F family (E2F2, E2F3 and DPI) are not expressed in brain [57,59, 64]. One pos-

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