Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol

Flavopiridol is a novel flavonoid that induces cell cycle arrest at differe nt stages of the cell cycle because of the inhibition of cyclin-dependent k inases (cdks). In previous studies from our laboratory, (B. A. Carlson et a l., Cancer Res., 56: 2973-2978, 1996), we observed that exposure of the MCF -7 breast carcinoma cell line to flavopiridol resulted in G(1)-S arrest, wh ich was associated with the loss of cdk4 and cdk2 activity by 24 h of expos ure. Along with this inhibition, flavopiridol decreased total cyclin-D prot ein levels in this cell line. In this work, we demonstrate that using isofo rm-specific antibodies, flavopiridol induces an early (by 6 h) decrease in cyclin D1 protein levels. This decline is followed by a decline in cyclin D 3 with no effect on cyclin D2 or cyclin E levels by 10 h. Furthermore, at e arly time points (up to 8 h), the activity of cdk4 and the expression of en dogenous phosphorylated retinoblastoma species from intact cells exposed to flavopiridol are unchanged. Thus, the decline in cdk4 activity and the ind uction of retinoblastoma hypophosphorylation follows cyclin D1 decline. Tur nover studies demonstrate that the half life of cyclin D1 (similar to 30 mi n) is not shortened in flavopiridol-exposed cells, and that the turnover of cdk4-bound cyclin D1 is unaltered. However, steady-state levels of cyclin D1 mRNA display a significant decrease by 24 h of flavopiridol treatment, w ith total disappearance by 8 h. This mRNA decline is not abrogated by the p resence of cycloheximide. Furthermore, we have found that flavopiridol spec ifically represses the activity of the full-length cyclin D1 promoter linke d to a luciferase reporter gene. In summary, we have found that the flavopi ridol-induced decline in cyclin D1 is an early event, specific and, at leas t in part, due to the transcriptional repression of the cyclin D1 promoter. These results extend our understanding of flavopiridol's action to include regulation of cyclin D1 transcription.