The expression of retinoblastoma and Sp1 is increased by low concentrations of cyclin‐dependent kinase inhibitors

Abstract

We examined the effect of suboptimal concentrations of cyclin-dependent kinase inhibitors, which do not interfere with cell proliferation, on retinoblastoma expression in hamster (Chinese hamster ovary K1) and human (K562 and HeLa) cells. To achieve this, we used the chemical inhibitors roscovitine and olomoucine (which inhibit CDK2 preferentially), UCN-01 (which also inhibits CDK4/6) and p21 (as an intrinsic inhibitor). All chemical inhibitors and overexpression of p21 strongly induced retinoblastoma protein expression. UCN-01-mediated retinoblastoma expression was caused by an increase in both the levels of retinoblastoma mRNA and the stability of the protein. The expression of the transcription factor Sp1, a retinoblastomainteracting protein, was also enhanced by all the cyclin-dependent kinase inhibitors tested. However, Sp1 expression was caused by an increase in the levels of Sp1 mRNA without modification in the stability of the protein. By using luciferase experiments, the transcriptional activation of both retinoblastoma and Sp1 promoters by UCN-01 was confirmed. Bisindolylmaleimide I, at concentrations causing a similar or higher inhibition of protein kinase C than UCN-01, provoked a lower activation of retinoblastoma and Sp1 expression. Finally, the effects of cyclin-dependent kinase inhibitors on dihydrofolate reductase gene expression were evaluated. Treatment with UCN-01 increased cellular dihydrofolate reductase mRNA levels, and dihydrofolate reductase enzymatic activity was enhanced by UCN-01, roscovitine, olomoucine and p21, in transient transfection experiments. These results support a mechanism for the self-regulation of retinoblastoma expression, and point to the need to establish the appropriate dose of cyclin-dependent kinase inhibitors as antiproliferative agents in anticancer treatments.Keywords: retinoblastoma gene product; Sp1; UCN-01; roscovitine; dihydrofolate reductase.Cyclin-dependent kinases (CDKs) are key regulators of cell cycle progression. They constitute the catalytic subunits of holoenzymes formed in combination with regulatory subunits named cyclins. Thirteen CDKs [1,2] and at least 25 cyclins [2] have been reported to date. Cyclin expression varies during the cell cycle and the cyclin/CDK holoenzyme is activated by phosphorylation of specific residues in the CDK catalytic subunit by the cdk-activating kinase [3,4]. CDKs are involved in transcriptional control [5], mitotic progression [6], DNA repair (CDK7) [7], differentiation of brain neurons (CDK5) [8] and play a crucial role in the progression of cells from G1 to S phase by regulating the phosphorylation state of the retinoblastoma gene product (Rb). The tumor suppressor Rb is a nuclear protein of 928 amino acids [9] that is present in distinct phosphorylation states depending on the phase of the cell cycle [10,11]: it is nonphosphorylated when newly synthesized; hypophosphorylated in early G1; and hyperphosphorylated in late G1, S, and G2/M phases. In mitosis, a protein phosphatase 1-like protein removes all phosphates f...

Regulation of Sp1 by cell cycle related proteins

“…We confirmed that p21 was able to activate transcription form the Sp1 promoter and to increase Sp1 mRNA expression in this system, as previously observed in HeLa cells transiently transfected with p21. The activation of Sp1 at the promoter and mRNA levels after p21 induction is in accordance with previous observations using CDK inhibitors 45. This effect could be caused by dephosphorylation of Sp1 at concrete sites that could increase its transcriptional activity directly or increase the degradation of its inhibitory domain 8.…”

Sp1 is involved in the transcriptional activation of p16INK4 by p21Waf1 in HeLa cells

“…This is the classic pathway. In addition, CDK inhibitors, for example p21 Waf1 , can also affect certain transcription factors directly to activate or inactivate target genes [17,18]. There is much evidence to support the fact that p21 Waf1 is an effector of p16 INK4 , which is associated with a posttranscriptional induction of p21 Waf1 [19].…”

E2F1 stability is regulated by a novel-PKC/p38β MAP kinase signaling pathway during keratinocyte differentiation

“…We examined pRb levels as a function of time and found a moderate increase after 10 h of incubation in the presence of CHX. Incubation of cells in the presence of both CHX and TPA decreased pRb levels by about 50% after 10 h. pRB has been shown to be a substrate for PKC (Suzuma et al, 2002), and increased pRb phosphorylation can decrease the stability of this protein (Pen˜uelas et al, 2003). Whether PKC-induced phosphorylation of pRb caused the observed decreases in pRb levels remains to be established.…”