Common and reversible regulation of wild-type p53 function and of ribosomal biogenesis by protein kinases in human cells

Abstract

Two speci®c inhibitors of cyclin-dependent kinase 2 (Cdk2), roscovitine and olomoucine, have been shown recently to induce nuclear accumulation of wt p53 and nucleolar unravelling in interphase human untransformed IMR-90 and breast tumor-derived MCF-7 cells. Here, we show that the early response of MCF-7 cells to roscovitine is fully reversible since a rapid restoration of nucleolar organization followed by an induction of p21 WAF1/CIP1 , a downregulation of nuclear wt p53 and normal cell cycle resumption occurs if the compound is removed after 4 h. Interestingly, similar reversible e ects are also induced by the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole. Upon short-term treatment also, both compounds signi®cantly, but reversibly, reduce the level of 45S precursor ribosomal RNA. Cells exposed to the two types of protein kinase inhibitors for longer times keep exhibiting altered nucleolar and wt p53 features, yet they strikingly di erentiate in that most roscovitine-treated cells fail to ever accumulate high levels of p21 WAF1/CIP1 in contrast with DRB-treated ones. In both cases, however, the cells eventually fall into an irreversible state and die. Moreover, we found that constitutive overexpression of p21 WAF1/CIP1 alters the nucleolar unravelling process in the presence of DRB, but not of roscovitine, suggesting a role for this physiological Cdk inhibitor in the regulation of nucleolar function. Our data also support the notion that both roscovitine-and DRB-sensitive protein kinases, probably including Cdk2 and CKII, via their dual implication in the p53-Rb pathway and in ribosomal biogenesis, would participate in coupling cell growth with cell division. Oncogene (2001) 20, 5951 ± 5963.

p53 activation results in rapid dephosphorylation of the eIF4E-binding protein 4E-BP1, inhibition of ribosomal protein S6 kinase and inhibition of translation initiation

“…Although p53 can associate with ribosomes (Fontoura et al, 1997) and is able to translationally repress the synthesis of proteins such as cdk4 and FGF-2 directly (Miller et al, 2000;Galy et al, 2001), it is also possible that mTOR activity, 4E-BP1 phosphorylation and/or overall protein synthesis are regulated by factors that are transcriptionally induced by p53. We have been unable to test this directly since transcriptional inhibitors such as DRB and actinomycin D themselves affect p53 function and/or the state of phosphorylation of 4E-BP1 (Loreni et al, 2000;David-Pfeuty et al, 2001). Further studies, for example using mutant forms of p53 which are deficient in transcriptional transactivation or selectively defective in the ability to induce different sets of genes (Haupt et al, 1995;Ding et al, 2000;Kokontis et al, 2001), will therefore be required to establish the mechanism by which p53 controls translation as part of its function as a key factor in growth regulation and tumorigenesis.…”

Targeting the cell cycle and the PI3K pathway: A possible universal strategy to reactivate innate tumor suppressor programmes in cancer cells

“…Thus, roscovitine is relatively efficient at inducing apoptosis in (Rb + /p53 + ) tumor cells (10,11) and its cytotoxic effects are enhanced by either mTOR or PI3K inhibitors (which are ineffective on their own). However, it is virtually inactive against p53-deficient, Rb-proficient or -deficient tumor cells, even in the presence of rapamycin.…”

“…It also delayed tumor outgrowth in two xenograft models (25). We investigated whether roscovitine in cotreatment with either LY294002 (a direct PI3K inhibitor; 13) or rapamycin (a specific inhibitor of a downtream PI3K target, mTOR; 13) would affect the longterm irreversible cytotoxic effects of roscovitine (10,11). Three human tumor cell lines differing in the Rb/p53 status were used: MCF-7(Rb + /p53 + ) breast carcinoma cells; MCF-7:E6 cells infected with HPV18-E6 oncoprotein (which facilitates wild-type p53 degradation) and Saos-2(Rb -/p53 -) osteosarcoma cells.…”

“…A further key argument derives from the observation that roscovitine (9) induces both a nuclear p53 accumulation and p53-independent nucleolar disassembly in human cultured cells (10). The roscovitine effects were fully reversible upon short-term treatment, transiently braking cell cycle progression, but became irreversible in longer term, ending in apoptosis, especially in tumor cells (10,11). This suggests that roscovitine-sensitive Cdks not only control cell division, but also facilitate cell growth and mitigate p53 function in cell cycle arrest and apoptosis.…”

Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling of Anti-Neoplastic Agents

“…This is known to occur through mutation in genes that produce regulatory proteins which modulate cell cycle, such as the Cyclin Dependent Kinases (Cdks) [14][15][16]. Cdks are highly conserved serine/threonine kinases whose activation depends on formation of a heterodimeric complex with cyclins [17][18][19].…”