Our current data strongly point to Myc transcriptional activity regulating a cyclin E-targeting mechanism capable of promoting the G1
/S transition in parallel with the pRb/E2F pathway in mammalian cells (Fig. ). By employing novel U2OS-inducible cell lines, we show that ectopic expression of Myc, like that of cyclin E, can rapidly induce S-phase entry and persistent DNA replication in cells deprived of E2F function long term by constitutively active pRb. This effect was not connected with restoration of E2F activity, but was associated with increased Myc activity and abolished by antagonizing the latter with the dnMM mutant. Hence Myc, like the putative downstream target cyclin E, can perform G1
/S-promoting functions independent of E2F activity. This property was associated with induction of cyclin E-dependent kinase and Cdc25A phosphatase activities, implying that control of cyclin E and Cdc25A by Myc is not necessarily consequential to increased E2F activity (3
). Cyclin E- and cyclin A-CDK2 complexes are likely targets for Cdc25A activity in vivo (6
; Mailand et al., submitted). However cyclin A is not expressed, and cyclin A-associated kinase is silenced in cells deprived of E2F activity (43
), even when Myc is induced (Santoni-Rugiu et al., unpublished data). Thus, the similar temporal induction patterns of Cdc25A phosphatase and cyclin E-CDK2 activities in derepressed U2OS-RbΔcdk/Myc cells suggest that, in this context, Cdc25A may contribute to cyclin E-CDK2 activation.
FIG. 8 Schematic model of cyclin E-associated kinase activity as an integrative convergence point for the regulation of G1/S transition by the “classic” pRb/E2F and the “parallel” Myc-dependent pathways in mammalian cells (thick (more ...)
Although the impact of the G1
/S “parallel” pathway with respect to other stages of cell cycle remains to be further explored, we found that coinduction of either Myc or cyclin E with pRbΔcdk was unable to restore productive cell division over an extended period of time. Thus, even in presence of activated cyclin E-associated kinase, the intactness of E2F activity appears essential for the orderly completion of mammalian cell cycle, in accord with a role for the pRb/E2F pathway extending beyond the control of G1
/S transition (see, for instance, references 11
, and 51
To elucidate in more detail the existence of parallel Myc and E2F pathways for S-phase entry, we developed a system to block endogenous E2F and/or Myc activity and assess the impact on G1/S transition in synchronized cells. By this approach, we have shown in different cell types that blocking either of these activities significantly delays and lowers, but does not prevent, DNA replication, while the concurrent blocking of both activities virtually abolishes it. These findings support the concept of an independent role of Myc and E2F activities in controlling the G1/S transition and suggest that they are both necessary to cooperatively allow timely and proper initiation of DNA synthesis in mammalian cells (Fig. ). This independent but apparently synergistic control of S-phase entry depends on the capacity to activate cyclin E-CDK2 kinase. Indeed, the data from U2OS-RbΔcdk/CycE (Fig. ) and U2OS-RbΔcdk/Myc (Fig. ) cells and from transiently transfected cells (Fig. B and C) indicate that both cyclin E and Myc can strongly induce DNA replication in cells deprived of E2F activity. This property of Myc and cyclin E is nullified by the expression of a dnK2 allele. Moreover, while ectopic E2F-1 potently rescues S phase in cells deprived of Myc activity, cyclin E and Cdc25A are able to dramatically overcome the abrogation of DNA synthesis due to a concomitant block of both Myc- and E2F-dependent pathways. Again, the rescue capabilities of E2F-1 or Cdc25A are completely neutralized by dnK2. Collectively, these results indicate that cyclin E-CDK2 activity is a common rate-limiting target of both transcriptional pathways for entry into S phase.
Our findings are supported by the previous observation that activation of cyclin E-dependent kinase by Myc requires transcription and depends on dimerization and DNA binding sites of Myc (70
). They are also consistent with Myc's ability to abrogate a G1
arrest induced by wild-type pRb (23
) and with the fact that serum growth factors, potent activators of the pRb/E2F pathway, synergize with ectopic Myc in activating cyclin E-CDK2 kinase in arrested fibroblasts (70
). By the same token, cyclin D1 and Myc collaborate in lymphomagenesis of transgenic mice (7
). Finally, ectopic Ras and Myc reportedly cooperate in inducing cyclin E-CDK2 activity and S-phase entry in quiescent cells (40
). This was ascribed to a combined effect of Rb pathway control by Ras, Myc-induced E2F-1 gene activation, and modulation of p27 and Cdc25A levels by Myc and Ras signaling (3
). The present work argues, though, that E2F activity is not strictly necessary for Myc-induced cyclin E activation and S-phase entry.
Previous experiments suggest that ectopic Myc may stimulate cyclin E-CDK2 and S-phase entry via direct induction of cyclin E or Cdc25A gene transcription or via functional inactivation of p27 (see the introduction). These mechanisms might also be operating in cells deprived of E2F activity. Indeed, our results indicate that the independent and synergistic control of S-phase entry by Myc and E2F activities relies, at least in part, on the transcriptional regulation of cyclin E and Cdc25A gene expression. In particular, repression of either E2F or Myc activity approximately halved cyclin E and Cdc25A transcription levels in synchronized cells. The effect of MM was not caused indirectly by reduced E2F activity, since MM expression did not significantly affect the activity of E2F-responsive reporters in these cells. Furthermore, ectopic Myc increased cyclin E and Cdc25A RNA levels by more than twofold (Fig. A, Myc) and approximately tripled the levels present in cells deprived of E2F activity (Fig. A, RbΔcdk+Myc). Thus, Myc and E2F activities play, at least in part, independent roles in regulating cyclin E and Cdc25A expression at the G1
/S transition. In addition, suppression of both activities by RbΔcdk+MM caused a further, marked decrease of cyclin E transcription, indicating that such a regulation is synergistic. All together, our data therefore argue against the possibility that Myc may induce cyclin E gene expression only indirectly by increasing E2F activity (3
). Cdc25A RNA levels, instead, do not further decline upon coexpression of MM and pRbΔcdk, possibly because other transcriptional factors may contribute to Cdc25A transcription. Supporting this hypothesis, binding sites for multiple transcription factors have been recently identified in a human Cdc25A promoter region (72
). Observations in Myc-null cells have suggested that Myc makes a contribution of approximately twofold to Cdc25A gene activation and only during early phases of growth factor stimulation (10
). However, the contribution and possible compensation by E2F or other transcription factors to Cdc25A expression in Myc-null cells have not been examined, although compensatory increases in critical Myc targets are considered possible in this experimental model (14
). In any case, our data indicate that both Myc and E2F activities are required for proper cyclin E and Cdc25A expression during G1
/S transition and suggest that this could have a strong impact on critical G1
/S-promoting activities associated with cyclin E and Cdc25A. Indeed, we show that these two proteins can effectively cooperate in reestablishing DNA synthesis in cells devoid of both E2F and Myc activities, even when expressed in amounts unable to do it on their own. This is presumably due to a positive feedback loop between cyclin E-CDK2 and its activator Cdc25A (28
). Thus, even relatively small adjustments of cyclin E and Cdc25A gene expression induced by changes in E2F or Myc activity may be critical for the decision to enter the S phase.
Further work will elucidate alternative mechanisms of cyclin E-CDK2 control by E2F-dependent and -independent pathways. However, our current findings may have important implications for cancer treatment, because they suggest that limiting E2F activity in Myc-overexpressing cells may not suffice to halt DNA replication and therefore the risk of genomic instability. In this regard, a potent growth suppressor like transforming growth factor β (TGF-β) inhibits transcription of both E2F and Myc (2
), and the down-regulation of Myc also occurs in cells lacking pRb function and yet leads to significant cell cycle arrest (2
). On the other hand, despite the plethora of TGF-β inhibitory effects on cell cycle progression (2
), overexpression of Myc, like that of E2F (66
), renders cells in culture or in transgenic animals resistant to this cytokine, even when the expression of CKIs and pRb is preserved (2
). Depending on the cellular context, this may be achieved through functional inactivation of p27 (3
), prevention of p15 induction (73
), or transcriptional control of CDK2 activators (references 20
and this work). This is relevant for those human cancers in which increased expression of TGF-β and increased expression of Myc are frequent and early events (see references 14
and references herein). In these cases, Myc activation may allow neoplastic cells to initially counteract TGF-β and induce unscheduled DNA replication, increasing the risk of additional genetic defects necessary for tumor progression.