Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists

Abstract

Recent studies identified a short peptide motif that serves as a docking site for cyclin͞cyclin-dependent kinase (cdk) 2 complexes . Peptides containing this motif block the phosphorylation of substrates by cyclin A͞cdk2 or cyclin E͞cdk2. Here we report that cell membrane-permeable forms of such peptides preferentially induced transformed cells to undergo apoptosis relative to nontransformed cells. Deregulation of E2F family transcription factors is a common event during transformation and was sufficient to sensitize cells to the cyclin͞cdk2 inhibitory peptides. These results suggest that deregulation of E2F and inhibition of cdk2 are synthetically lethal and provide a rationale for the development of cdk2 antagonists as antineoplastic agents.Certain molecular pathways frequently are altered during human carcinogenesis. Therefore one approach to treating cancer, while minimizing host toxicity, would be to develop drugs that preferentially kill cells in which such pathways are altered.An example of a molecular pathway that is recurrently altered in cancer involves the retinoblastoma tumor suppressor protein (pRB) (1-3). Many tumors lack a wild-type RB-1 allele, thus depriving them of pRB. Furthermore, pRB is negatively regulated by cyclin-dependent kinases (cdks). These cdks are, in turn, negatively regulated by certain cdk inhibitors and positively regulated by certain cyclins. These upstream pRB regulators frequently are altered in tumors that retain a wild-type RB-1 allele. Thus, functional inactivation of pRB, a known inhibitor of cell growth, may be a necessary step in human carcinogenesis.Members of the E2F cell-cycle regulatory transcription factor family are critical downstream targets of pRB. Binding to pRB converts E2F from a transcriptional activator to a potent transcriptional repressor. Consequently, E2F-responsive genes are activated in cancer cells because of loss of pRB͞E2F repressor complexes and liberation of free, transcriptionally active E2F. Of note, at least some E2F family members, including E2F1, are themselves transcribed from E2F-responsive promoters (4-7). Thus, pathological activation of E2F responsive genes can establish a positive feedback loop. Paradoxically, forced activation of E2F-responsive genes, such as through the overproduction of E2F1, can induce both cellular proliferation and cell death (apoptosis) (8-11).E2F family members bind to DNA as heterodimers with members of the DP family. The DNA-binding capability of some of these heterodimers is negatively regulated by cyclin A͞cdk2 (12-15). For example, E2F1, E2F2, and E2F3 each contain a short, collinear cyclin A͞cdk2 binding motif that is required for the timely neutralization of E2F DNA-binding capability as cells traverse and prepare to exit S phase (13,16,17). Mutation of this motif in E2F1 enhances its ability to induce apoptosis (13). In summary, E2F is negatively regulated by both pRB and cyclin A͞cdk2. We therefore reasoned that pharmacologic inhibition of cyclin A͞cdk2 might preferentially kill cells in whic...

Mutant Bik expression mediated by the enhanced minimal topoisomerase IIα promoter selectively suppressed breast tumors in an animal model

“…44 Furthermore, interrupting the association between cyclin A and cdk2 can specifically suppress breast cancer cell growth. 45,46 Together with our results, these data indicate that cyclin A/Cdk2 is a specific upstream activator of topoIIa promoter in breast cancer cells. Interestingly, among the deletion mutants of topoIIa promoter, only the minimal À90 promoter was potentiated successfully by the CMV enhancer, resulting in activity comparable to that of the CMV promoter while retaining specificity for breast cancer.…”

In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R‐roscovitine)

“…Ltd,, Dundee, UK or recombinant active ERK-2 or protein kinase C␣ (Upstate Biotechnology). CDK2, CDK4, CDK7, SAPK2a (p38) and ERK-2 assays were performed in assay buffer (25 mM ␤-glycerophosphate, 20 mM MOPS, 5 mM EGTA, 1 mM DTT and 1 mM NaVO 3 , pH 7.4), into which were added 2-4 g of active enzyme with appropriate substrates (purified histone H1 for CDK2/cyclin E, GST-retinoblastoma(773-928) for CDK2/cyclin A and CDK4/cyclin D1, biotinyl-Ahx-(YSPTSPS) 4 for CDK7 or myelin basic protein for SAPK2a (p38) and ERK-2). The reaction was initiated by addition of Mg/ATP mix (15 mM MgCl 2 ϩ 100 M ATP with 30 -50 kBq per well of [␥-32 P]-ATP) and mixtures incubated for 10 min (CDK2/cyclin E, ERK-2, SAPK2a) or 45 min (CDK4/cyclin D1, CDK7/cyclin H) as required, at 30°C.…”

“…Questions remain, however, regarding the importance of CDK selectivity for this type of agent. Both CDK2 and CDK4 have been targeted for small molecule inhibitor development, and recent results suggest that CDK2 antagonists may induce apoptosis selectively in transformed cells regardless of p53 status, 4 while the function of CDK4 has now been linked to modulation of the rate of cellular growth and has been suggested to be (in Drosophila at least) dispensable for cell-cycle progression. 5 CDK7 has an interesting dual role.…”

Bcl-2 activates a programme of premature senescence in human carcinoma cells

“…Accordingly, the cell-cycle inhibitory activity of Bcl-2 is more severe in tumour than in normal cells [7,12]. In this context, it is interesting to note that transformed cells are much more sensitive to Cdk2 inhibition relative to normal cells [48]. Therefore we propose that the capability of Bcl-2 to inhibit Cdk2 only affects the G 1 -to-S transition in normal cells [5,11], whereas Bcl-2 expression inhibits proliferation and results in a senescent-like phenotype in carcinoma cells.…”