Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression

Oncology Research, Wyeth Research, Pearl River, NY 10965, USA.
Breast Cancer Research and Treatment (Impact Factor: 4.2). 02/2006; 95(2):185-94. DOI: 10.1007/s10549-005-9066-y
Source: PubMed

ABSTRACT We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1-S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.

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    • "Mitogenic stimuli increase the intracellular levels of cyclin D1, which then form an assembly with CDK4 or CDK6, and ultimately leading to the hyperphosphorylation of Rb protein (Sherr 1993; Matsushime et al. 1994). Elevations in cyclin D1 result in a corresponding increase in cyclin D1/CDK4 activity and hyperphosphorylation of Rb protein, whereas blockade of cyclin D1 activation blocks the hyperphosphorylation of Rb protein and results in cell cycle arrest in G1 (Grillo et al. 2006). Overexpression of cyclin D1 has been observed in approximately 50% of all breast cancers (Hall and Peters 1996; Arnold and Papanikolaou 2005; Caldon et al. 2006), whereas suppression of cyclin D1 activity significantly inhibits mammary tumorigenesis (Bartkova et al. 1994; Arber et al. 1997). "
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    • "In the absence of ectopically expressed cyclin D1, silencing RSK2 decreased the S-phase population to ~50% of the control (Fig. 5F). As expected, over-expression of cyclin D1 increased the number of control cells in S-phase by ~ 1.7X (Grillo et al., 2006). Importantly, the forced expression of cyclin D1 after RSK2 knockdown not only prevented the decrease in S-phase but even increased the number of cells in S-phase. "
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    • "Accordingly, another study showed that the major antiproliferative effect of the antiestrogen tamoxifen could be related to its inhibition of CCND1 expression (Hodges et al. 2003). CCND1 silencing experiments in BCC further confirmed the correlation between CCND1 expression levels and cellular proliferation, emphasizing CCND1 as a potential therapeutic target for breast cancer (Arnold and Papanikolaou 2005; Grillo et al. 2006). "
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