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Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with À7/del7q and 11 with normal karyotype) with 23 CD34 þ preparations from healthy bone marrow donors. SKI, a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with À7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RARa signaling in AML. Ski was co-immunoprecipitated and colocalized with RARa. We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RARa signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RARa signaling.

Glucocorticoids exert antiproliferative effects on a number of cell types, including the HeLa cervical carcinoma cell line. However, the mechanism responsible for the antiproliferative effect is poorly understood. In this report we have investigated the role of the recently identified cyclin-dependent kinase inhibitor (CDI) p57Kip2 in the antiproliferative effect conferred by glucocorticoids. When HeLa cells were treated with the synthetic glucocorticoid dexamethasone (DEX), the doubling time of exponentially growing cells increased 2-fold. Within 11 h of DEX treatment, this was accompanied by an accumulation of cells in the G1 phase of the cell cycle with a corresponding decreased proportion of cells in the S phase and decreased CDK2 activity. DEX treatment of the HeLa cells dramatically induced the protein and mRNA expression of the CDI p57Kip2. This induction was seen within 4 h of DEX treatment, preceding a major DEX-induced accumulation of cells in the G1 phase. DEX-induced mRNA expression of p57Kip2 did not require de novo protein synthesis, and the transcription of the p57Kip2 gene was increased as determined by a run-on transcription assay. Furthermore, DEX induction of p57Kip2 was not a consequence of the cell cycle arrest, since other growth inhibition signals did not result in strong p57Kip2 induction. Overexpression of p57Kip2 using HeLa cells stably transfected with a tetracycline-inducible vector showed that p57Kip2 is sufficient to reconstitute an antiproliferative effect similar to that seen in DEX-treated cells. Selective p57Kip2 expression by the tetracycline analog doxycycline to levels comparable to those observed on DEX induction resulted in a 1.7-fold increase in the doubling time and a shift of HeLa cells to the G1 phase as well as a decrease in CDK2 activity. Taken together, these results suggest that glucocorticoid treatment directly induces transcription of the p57Kip2 gene and that the p57Kip2 protein is involved in the glucocorticoid-induced antiproliferative effect.

No abstract

Glucocorticoids are known regulators of the cell cycle, normally exerting an anti-proliferative effect. We have previously shown that glucocorticoids stimulate expression of p57 Kip2 , a member of the Cip/Kip family of cyclin-dependent kinase inhibitors which, in some cell types, may account for the anti-proliferative responses seen after glucocorticoid treatment. The induction of p57Kip2 involves primary transcriptional effects where no de novo protein synthesis is necessary, suggesting a direct interaction of the glucocorticoid receptor with the p57 Kip2 gene. In this study we have identified a functional glucocorticoid response element (GRE), located 5 kilo bases (kb) upstream of the transcription start site in the human p57Kip2 promoter. This GRE was functional also when isolated, suggesting a direct transcriptional effect of the glucocorticoid receptor. Furthermore, mutation of this GRE abolished glucocorticoid induction of the reporter gene, whereas mutation of a nearby Sp1 site did not. Using electrophoretic mobility shift assays, we have shown that the -5 kb p57Kip2 promoter GRE was able to compete with a well-known GRE for glucocorticoid receptor binding. Sequence comparisons with the mouse genome showed that this GRE is highly conserved, further strengthening the biological importance of this site. All these data emphasize the involvement of this GRE in the glucocorticoid-mediated induction of p57 Kip2 expression.