Posttranslational regulation of cyclin D1 by retinoic acid: A chemoprevention mechanism

Abstract

The retinoids are reported to reduce incidence of second primary aerodigestive cancers. Mechanisms for this chemoprevention are previously linked to all-trans retinoic acid (RA) signaling growth inhibition at G 1 in carcinogen-exposed immortalized human bronchial epithelial cells. This study investigated how RA suppresses human bronchial epithelial cell growth at the G 1 -S cell cycle transition. RA signaled growth suppression of human bronchial epithelial cells and a decline in cyclin D1 protein but not mRNA expression. Exogenous cyclin D1 protein also declined after RA treatment of transfected, immortalized human bronchial epithelial cells, suggesting that posttranslational mechanisms were active in this regulation of cyclin D1 expression. Findings were extended by showing treatment with ubiquitindependent proteasome inhibitors: calpain inhibitor I and lactacystin each prevented this decreased cyclin D1 protein expression, despite RA treatment. Treatment with the cysteine proteinase inhibitor, E-64, did not prevent this cyclin D1 decline. High molecular weight cyclin D1 protein species appeared after proteasome inhibitor treatments, suggesting that ubiquitinated species were present. To learn whether RA directly promoted degradation of cyclin D1 protein, studies using human bronchial epithelial cell protein extracts and in vitro-translated cyclin D1 were performed. In vitro-translated cyclin D1 degraded more rapidly when incubated with extracts from RA treated vs. untreated cells. Notably, this RA-signaled cyclin D1 proteolysis depended on the C-terminal PEST sequence, a region rich in proline (P), glutamate (E), serine (S), and threonine (T). Taken together, these data highlight RA-induced cyclin D1 proteolysis as a mechanism signaling growth inhibition at G 1 active in the prevention of human bronchial epithelial cell transformation.

pRb and Cdk regulation by N-(4-hydroxyphenyl)retinamide

“…Since the levels of the Cdk4-regulating protein Cyclin D1 decline in all cell lines, the drop in Cdk-4 activity may, at least in part, be explained by this event. Based on the fact that RA and other receptor-non-selective and growth-suppressive retinoids enhance proteasomedependent Cyclin D1 degradation, it has been hypothesized that proteolysis of this protein is a common chemoprevention signal, at least in normal and transformed human bronchial epithelial cells (Langenfeld et al, 1997;Boyle et al, 1999). Although we cannot exclude that Cyclin D1 downregulation may equally represent a chemoprevention signal in response to HPR, nevertheless our results point out that unlike RA, HPR suppresses Cyclin D1 expression by a transcriptional rather than posttranslational mechanism, emphasizing further the di erent mode of action of these two retinoids.…”

“…Signi®cantly, however, Cyclin D1 protein levels markedly decreased in all three cell lines after retinoid treatment (Figure 6), a ®nding that may explain the drop in Cdk4 function given that its activity depends on Cyclin D1. Since RA and other retinoids induce proteasome-mediated Cyclin D1 degradation, and this may represent an e ective growth suppressing and cancer chemoprevention signal (Langenfeld et al, 1997;Boyle et al, 1999), we determined whether a similar mechanism accounted for the downregulation of Cyclin D1 by HPR. For this purpose, cells were pre-treated with a non-toxic dose of calpain inhibitor I (the inhibitor of the 26S proteasome pathway) prior to treatment with HPR.…”

“…Cell suspensions from adherent cultures were obtained by incubation with Trypsin-Versene (BioWhittaker), and viable cells counted on a hemocytometer after trypan blue staining. The calpain inhibitor I (Calbiochem, CA, USA) was used as described (Langenfeld et al, 1997). Brie¯y, cells were pre-treated for 1.5 h with non-toxic doses of calpain inhibitor I (100 mg for T47D and 25 mg for MCF7) before addition or not of 3 mM HPR, and after 12 h were harvested and used for Western analysis.…”

Cotargeting Cyclin D1 Starts a New Chapter in Lung Cancer Prevention and Therapy

“…Downregulation of cyclin D1 with the GG genotype in these lesions with a regimen based on the bexarotene relative 13-cis-retinoic acid (13cRA) was associated with cancer prevention (14, 15); the regimen did not downregulate overexpression of cyclin D1 with the AG or AA genotype [the A allele preferentially produces an altered transcript that resists retinoic acid degradation; ref. (16)] or prevent cancer in patients with such lesions. The prevalence of the GG genotype in cyclin D1 overexpression seems to be about 30%.…”

Expression profile and molecular genetic regulation of cyclin D1 expression in epithelioid sarcoma

“…It has been suggested that mTOR/PI3 kinase regulated p70S6 kinase-dependent control and/or PI3 kinase/eIF4E-mediated control may participate in cyclin D1 translational control. [32][33][34][35][36] In addition, a study from human sarcoma cell lines has suggested that overexpression of cyclin D1 protein may result from enhanced protein stability due to an altered ubiquitin/proteasome pathway involved in the degradation of cyclin D1 protein. 37 Therefore, it is possible that translational/post-translational regulation of cyclin D1 protein may provide a timing fashion for control of rapid cell cycle progression.…”