Cyclin E and c-Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins

Abstract

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. We suggest that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. Our data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis.

p16INK4a, but not constitutively active pRb, can impose a sustained G1 arrest: molecular mechanisms and implications for oncogenesis

“…Although none of these changes that evolve during multistep oncogenesis is likely to prevent the ability of p16 to directly act on cyclin D-dependent complexes, we expect that they would considerably decrease or eliminate the indirect e ects of the INK4 family of CDK inhibitors (Sherr and Roberts, 1995;Jiang et al, 1998;Reynisdo ttir et al, 1995;Planas-Silva and Weinberg, 1997) on cyclin E/CDK2. The recent demonstration that ectopic expression of cyclin E can rescue the p16-mediated G1 arrest Alevizopoulos et al, 1997) is also consistent with such interpretation. Finally, we believe that our present ®ndings may assist in designing future gene therapy strategies, in that at least Rb-positive malignancies might respond more decisively to p16 than to active pRb, with an additional bene®t of avoiding the potentially hazardous occurrence of aneuploid cells.…”

Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1

“…All mutations were veri®ed by sequencing. Retroviruses encoding human p16 (pBP-p16), p27 (pBP-p27) and wild type 12S E1A (pBH2-E1A) were described previously (Alevizopoulos et al, 1997(Alevizopoulos et al, , 1998Vlach et al, 1996). High-titer retroviral supernatants were generated as described (Vlach et al, 1996).…”

“…Serial infections of cell pools, preparation of cell lysates and biochemical analysis (immunoblots, immunoprecipitations) were as previously described (Vlach et al, 1996). For cell cycle analysis, cells were labeled with 33 mM Bromodeoxyuridine (BrdU) for 30 min and analysed by immunocytochemical detection of BrdU, as previously described (Alevizopoulos et al, 1997). Northern blot analysis were performed as previously described (Alevizopoulos et al, 1998).…”

“…The second family includes the INK4 proteins p16 INK4a , p15 INK4b , p18 INK4c and p19 INK4d , which bind to CDK4 and CDK6. Ectopic expression of both classes of CKIs causes activation of pRb-family proteins, sequestration of E2F and subsequent cell cycle arrest in G1 (Alevizopoulos et al, 1997(Alevizopoulos et al, , 1998Guan et al, 1994;Hirai et al, 1995;Polyak et al, 1994;Quelle et al, 1995;Toyoshima and Hunter, 1994;Vlach et al, 1996).…”

“…For example, pRb-negative cells become insensitive to expression of p16 or microinjection of antibodies neutralizing cyclin D/CDK4 (Koh et al, 1995;Lukas et al, 1994Lukas et al, , 1995aMedema et al, 1995) and ectopic expression of E2F-1 prevents G1 arrest by p16 (Alevizopoulos et al, 1997;DeGregori et al, 1995;Lukas et al, 1996;Mann and Jones, 1996). However, E2F is not su cient to prevent growth inhibition by p27, implying that cyclin E/CDK2 complexes have an additional role(s) at the G1-S transition, distinct from pRb phosphorylation (Alevizopoulos et al, 1997;Mann and Jones, 1996). Similar conclusions were reached by other groups (Chew et al, 1998;Hofmann and Livingston, 1996;Knudsen et al, 1998;Leng et al, 1997;Lukas et al, 1997;Ohtsubo et al, 1995;Resnitzky and Reed, 1995).…”