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

Abstract

p16 ink4a and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb Dcdk mutant both blocked G1 in shortterm experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb Dcdk entered S phase after 2 days, followed by endoreduplication between days 4 ± 6. The distinct phenotypes evoked by p16 vs pRb Dcdk appear mediated by cyclin E/CDK2 which, while active in the pRb Dcdkexpressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.

E2F activity is essential for survival of Myc-overexpressing human cancer cells

“…Altogether, these data indicate that deregulated expression of Myc in the presence of constitutively active Rb induces apoptosis in U2OS cells. It is noteworthy that decreased survival was not observed in U2OS-RbDcdk or U2OS-RbDcdk/CycE cell lines, which express RbDcdk alone or together with another cell cycle stimulator, cyclin E, respectively (Lukas et al, 1999b;Santoni-Rugiu et al, 2000 and data not shown). This implies that the induction of apoptosis is a specific response to the coexpression of Myc and RbDcdk.…”

“…We have recently shown in a specifically engineered U2OS-derived cell line, U2OS-RbDcdk/Myc, that Myc can stimulate G1/S transition and persistent DNA replication in cells long-term deprived of E2F activity (Santoni-Rugiu et al, 2000) by a constitutively active pRb mutant, pRbDcdk (Lukas et al, 1997(Lukas et al, , 1999b. However, both E2F and Myc activities are required to ensure timely and proper levels of DNA synthesis and orderly completion of cell cycles (Lukas et al, 1999a;Santoni-Rugiu et al, 2000).…”

“…Inappropriate, sustained expression of the constitutively active RbDcdk mutant alone in U2OS cells results in temporary G1 arrest followed by gradual entry into S phase due to residual cyclin E-associated kinase activity stimulated by endogenous Myc activity (Lukas et al, 1999b;Santoni-Rugiu et al, 2000). Instead, cells with forced co-expression of Myc and RbDcdk, rapidly enter S phase and persistently replicate DNA, owing to the high cyclin E-associated kinase activity induced by ectopic Myc, without, however, being able to productively divide (SantoniRugiu et al, 2000).…”

“…The hemagglutinin (HA)-tagged pBI-HA-RbDcdk and pECE-HA-RbDcdk vectors, expressing the phosphorylation-deficient murine pRb mutant RbDcdk in a tetracycline (TET)-repressible or constitutive manner, respectively, the PECEpRbDB/X expressing wild-type pRb, and the pBI-Myc or pCMV-Myc vectors for TET-repressed or constitutive c-Myc expression, as well as pCMV-CD20, pCMV-HA-dnDP-1 (D103-126), and pX-p16, were described before (Lukas et al, 1997(Lukas et al, , 1999bSantoni-Rugiu et al, 2000). The pCMV-HA-ARF plasmid for expression of human ARF was generated as previously published (Della Valle et al, 1997).…”

“…These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (10 U/ml), streptomycin (10 U/ml), geneticin (G418; 400 mg/ ml), puromycin (1 mg/ml) and TET (2 mg/ml). Transgene derepression was performed by removal of TET according to procedures previously published (Lukas et al, 1999b). SAOS-2 and BJ cells were cultured in DMEM containing 10% FCS, penicillin and streptomycin.…”