The inhibition of cultured myoblast differentiation by the simian virus 40 large T antigen occurs after myogenin expression and Rb up-regulation and is not exerted by transformation-competent cytoplasmic mutants

Abstract

We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of mouse C2 myoblasts, but this arrest occurred only after the synthesis of myogenin, an initial step in biochemical differentiation. Neither the synthesis nor some basic activities of MyoD appeared to be affected by wt SVLT. In these transformants, mitogen depletion elicited an increase in the Rb level comparable to that in normal C2 cells; wt SVLT, however, promoted the phosphorylation of a large part of the induced Rb. Mutations affecting nuclear transport were far more critical for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expression was fully compatible with the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transforming competence. The remaining SVLT mutants presented different degrees of ability to inhibit differentiation (as shown by the expression of tissue-specific markers in transformants). The inhibiting mutants, including the Rb binding site mutant, were able to promote a higher state of Rb phosphorylation than that observed in either normal cells or cytoplasmic-SVLT transformants.

p300 is required for MyoD-dependent cell cycle arrest and muscle-specific gene transcription

“…However, some differences do exist between the two oncoproteins in their ability to interfere with discrete steps of myogenic differentiation. In fact, SV40 TAg inhibits muscle cell differentiation without affecting the induction of myogenin expression which occurs early in the process (Tedesco et al ., 1995). It will be interesting to investigate whether these differences between E1A and TAg can be explained by their different ability to bind various forms of p300.…”

“…The viral oncoproteins E1A of adenovirus and large T antigens (TAg) of both SV40 and polyomavirus share the ability to inhibit myogenic differentiation (Fogel and Defendi, 1967; Webster et al ., 1988; Braun et al ., 1992; Maione et al ., 1992; Tedesco et al ., 1995). This property has been attributed to their binding to a common region of pRb, p107 and pRb2/p130, called the ‘pocket domain’.…”

Induction of cyclins E and A in response to mitogen removal: a basic alteration associated with the arrest of differentiation of C2 myoblasts transformed by simian virus 40 large T antigen

“…We have shown that this arrest occurs only after the MyoD-dependent induction of myogenin and high Rb levels; an important difference from normal C2 cells, however, is that a large part of this increased Rb is hyperphosphorylated (76). Among SVLT mutants, the ability to inhibit myogenesis correlates with capability of promoting Rb hyperphosphorylation but not with that of physically complexing Rb (76).…”

“…The simian virus 40 large T antigen (SVLT), like many other oncogene-encoded proteins (see references 2 and 44 for reviews), when expressed in C2 myoblasts causes the arrest of differentiation (18,23,25,50,76). We have shown that this arrest occurs only after the MyoD-dependent induction of myogenin and high Rb levels; an important difference from normal C2 cells, however, is that a large part of this increased Rb is hyperphosphorylated (76). Among SVLT mutants, the ability to inhibit myogenesis correlates with capability of promoting Rb hyperphosphorylation but not with that of physically complexing Rb (76).…”

“…We thus analyzed the cell levels and activities of the cyclins and cdks known to be active in the G 1 and S phases of the cell cycle and compared them in three cell types: normal C2 cells, C2 cells transformed by wildtype (wt) SVLT (WTSV cells), and C2 cells transformed by SVLT mutant NKT1 (defective for transport to the nucleus), which does not arrest terminal differentiation (NKT1 cells). Both transformants, unlike normal C2 cells, are able to form colonies when plated in soft agar (76).…”