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can be upregulated by the myc or ras oncogenes, we also chose to study putative relationships between cyclin D I protein levels and intrinsic cellular sensitivity to CDDP and y-irradiation. We report that in the 16 human cell lines which we have studied, high cyclin DI expression is related to CDDP resistance but has no relationship with radiation responsiveness, whereas high c-raf-I expression, although related to radiosensitivity has no relationship with CDDP responsiveness.

BackgroundCyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor.ResultsA search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy.ConclusionThese findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic biomarker and a putative functional site for kinase-unrelated activities of Cdk4.

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We have previously noted that high endogenous expression of the protein product of the full-length RAF1 proto-oncogene is related to relative intrinsic cellular radiosensitivity in 19 human cells lines in vitro. This appeared to be unrelated to the parameters of cell kinetics. In rodent and human cell lines transfected with dominant oncogenes, including Myc and MYC, Hras and HRAS and SV40, increased radioresistance has been accompanied by increased delay in progress through the G2 phase of the cell cycle after irradiation. We have thus examined the putative relationship between RAF1 expression and postirradiation perturbation of G2 phase in six of the human cell lines for which data have been reported previously. These lines exhibit a wide range of both radiosensitivity and Raf1 protein levels as measured previously by Western blotting. We report here that the cell lines whose cells appear to exit more rapidly from G2 phase are more radiosensitive (r = 0.91, P = 0.01) and express high levels of Raf1 protein (r = -0.93, P = 0.006).

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