p27Kip1, a cyclin-dependent kinase inhibitor, might be a marker of choice for preclinical in vitro screening of nutritional and chemopreventive anti-breast cancer agents.

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Background: A marker for preclinical in vitro screening of potential nutritional and chemopreventive anti-breast cancer agents must be efficient and reliable. p27(Kip1) is a cyclin-dependent kinase inhibitor and, when up-regulated, p27 inhibits G1-to-S phase transition of the cell cycle, thereby inhibiting DNA replication and cell division. In this study, we investigated whether p27 could be a useful marker for preclinical in vitro screening of nutritional and chemopreventive anti-breast cancer agents. Methods: Human breast cancer (MCF7, MDA-MB-321, and AU565) and mouse epidermal (JB6) cells were transfected with luciferase reporter plasmid containing proximal 5'-upstream region of p27 gene and then exposed to various nutritional and chemopreventive anti-breast cancer agents. Results: The results presented in the first half of this report show that various nutritional and chemopreventive anti-breast cancer agents fairly faithfully up-regulated synthesis of p27 in human breast cancer (MCF7, MDA-MB-321, AU565) and mouse epidermal (JB6) cells. Up-regulation appeared to be specific to p27 because synthesis of cyclin D1, E, and A, and p21Cip1/Waf1 was not modulated by these agents. Up-regulation of p27 is likely due to activation of translation rather than transcription of p27 because (a) up-regulation was mediated by the 5'-untranslated region (-575) of the p27 gene and (b) the antibiotic actinomycin D, an inhibitor of transcription, did not attenuate the up-regulation of the synthesis of p27. The results, presented in the second half of this report, was obtained using the 5'-untranslated region (-575) of p27 gene. The evidence suggested that cancer preventive agents up-regulate the synthesis of p27 by at least four different molecular signaling pathways: (a) Caloric restriction is likely to up-regulate the synthesis of p27 via 5'-AMP-activated protein kinase (AMPK; a metabolic energy sensor or cellular fuel gauge), tuberous sclerosis complex (TSC), and mammalian target of rapamycin (mTOR). Amino acid deficiencies also up-regulate the synthesis of p27 using some components of this pathway. (b) 4-Hydroxytamoxifen (but not tamoxifen), genistein (but not genistin), daidzein, and probably other nutritional and chemopreventive anti-cancer agents up-regulate the synthesis of p27 via receptor protein tyrosine kinases (RPTKs), phosphoinositide 3-kinase (PI3K), phosphoinosite-dependent kinase (PDK), Akt/PKB and mTOR. (c) The synthesis of p27 is also up-regulated via RPTKs followed by MAPKs - MEK, ERK and p38MAPK - and probably MNK. Finally, (d) global hypomethylation of 5'-m⁷G cap of mRNAs also up-regulates the synthesis of p27. Conclusion: Based on these findings, we conclude that p27 might be a marker of choice for preclinical in vitro screening of potential nutritional and chemopreventive anti-breast cancer agents.