Cyclin D1 and D3 associate with the SCF complex and are coordinately elevated in breast cancer

Abstract

D-type cyclins are important cell cycle regulators that promote cellular proliferation in response to growth factors by inactivation of the retinoblastoma protein (Rb). Cyclin D1 has been shown to be overexpressed in several cancer types and to act as an oncogene in breast cancers. As D-type cyclins are rate limiting for progression into S phase, the level at which they accumulate must be carefully regulated. Several mechanisms leading to overexpression of cyclin D1 have been reported including ampli®cation, translocation and stabilization of the mRNA. Here, we present data showing elevated cyclin D1 protein in breast cancer samples in the absence of elevated mRNA level. Further, we found that in these cases, cyclin D3 protein also accumulates and that the coordinate increase in cyclin D1 and D3 occurs in 15% (7/47) of breast cancers. In addition we show that blocking the activity of the 26S proteosome results in the accumulation of cyclin D1 and D3, that both D-type cyclins are ubiquitinated and associate with Cul-1, a component of the SCF ubiquitin ligase complex. Finally, we show that the coordinated elevation of cyclin D1 and D3 is also observed in the breast cell line MCF-7 and demonstrate that the degradation of cyclin D1 and D3 is de®cient in this cell line. These results indicate that cyclin D1 and cyclin D3 share a common mechanism of degradation and we propose that the coordinate increase of D-type cyclins observed in primary breast cancers re¯ects a defect in their proteolysis.

Inhibitory effect of p21 in MCF-7 cells is overcome by its coordinated stabilization with D-type cyclins

“…We previously showed that the half-life of cyclins D1 and D3 was increased in this cell line (Russell et al, 1999). In agreement with the data obtained in primary breast cancers, the level of p21 is coordinately elevated with cyclins D1 and D3 in this cell line (Figure 3a).…”

“…We have previously shown that human cyclins D1 and D3 can both associate with a SCF complex containing the cullin Cul-1 (Russell et al, 1999). Recently, cyclin D1 has been shown to associate not only with Cul-1, con®rming our previous observation, but also with the F-box protein Skp2 (Zhong-Kang et al, 1998).…”

“…p21 is elevated in cyclin D1 + D3 + breast cancers Cyclins D1 and D3 are coordinately elevated in *15% of breast cancers and we have proposed that this simultaneous accumulation re¯ect a defect in common elements of their proteolysis (Russell et al, 1999). We reasoned that if D-type cyclins proteolysis is defective in these patients and p21 is degraded by the same proteolytic pathway, p21 should also be elevated.…”

“…Protein extraction and immunoprecipitation were performed as described elsewhere (Russell et al, 1999). For inhibition of the 26S proteosome, cells were treated with the peptide aldehyde leucinyl-leucinyl-norleucinyl (LLnL) solubilized in DMSO at a ®nal concentration of 100 mM for 4 h. Following immunoprecipitation, proteins were resolved on 12% polyacrylamide gels, transferred onto nitrocellulose membranes, subjected to standard Western blotting analysis and visualised using ECL (Amersham Life Science).…”

“…We have previously identi®ed a subclass of breast cancers, which are potentially defective in the proteolysis of D-type cyclins (Russell et al, 1999). Considering that p21 and D-type cyclins may be degraded by the same pathway (Zhong-Kang et al, 1998), we investigated whether the level of p21 protein is altered in this subset of breast cancers.…”