The Cyclin-Dependent Kinase Inhibitor Butyrolactone Is a Potent Inhibitor of p21WAF1/CIP1 Expression

Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damaging agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21-dependent checkpoints.

Key Words

p21, Butyrolactone, Proteasome, Cell Cycle, Checkpoint     

Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1

We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well-understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.     

The Cyclin-Dependent Kinase Inhibitor p21CIP1/WAF1 Blocks Paclitaxel-Induced G2M Arrest and Attenuates Mitochondrial Injury and Apoptosis in p53-Null Human Leukemia Cells

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21Cip1/WAF1 in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21Cip1/Waf1 expression. Nevertheless, stable expression of U937 cells with a p21Cip1/WAF1 antisense construct blocked paclitaxel-induced G2M arrest and significantly, albeit modestly, increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. These protective effects were less than those observed in cells exposed to the antimetabolite ara-C. Consistent with these results, enforced expression of p21Cip1/WAF1 in Jurkat cells transfected with a construct driven by a doxycycline-responsive promoter increased the percentage of cells arrested in G2M, but attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21Cip1/WAF1 diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that the CDKI p21Cip1/WAF1 modestly but significantly protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21Cip1/WAF1-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.     

Not Just a CDK Inhibitor: Regulation of Transcription by p21WAF1/CIP1/SDI1

No Abstract Available

Key Words

D-type Cyclins, Cyclin D1, Ras     

Cyclin-Dependent Kinase Inhibitor p21 Modulates the DNA Primer-Template Recognition Complex

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5′-O-(3-thiotriphosphate) (ATPγS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPγS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.     

The immunosuppressive agent tacrolimus induces p21WAF/CIP1WAF1/CIP1 via TGF-beta secretion

Tacrolimus (Tac) is more immunosuppressive drug compared to cyclosporine (CsA). Our previous studies have demonstrated that CsA induces the expression of p21WAF/CIP1 expression. In this study we explored if like CsA, Tac also induces expression of p21WAF/CIP1. We also determined if induction of p21WAF/CIP1 by Tac is dependent on TGF-beta. Using RT-PCR and Western blot analysis, we studied the induction of p21WAF/CIP1 mRNA and protein in human T cells and A-549 cells (human lung adenocarcinoma cells) by Tac. The stimulation of p21WAF/CIP1 promoter activity was studied by luciferase assay using p21WAF/CIP1-luc, chimeric plasmid DNA containing a p21WAF/CIP1 promoter segment and luciferase reporter gene. Using anti-TGF-beta antibody, we studied if induction of p21WAF/CIP1 by tacrolimus is dependent on TGF-beta. The results demonstrate that Tac induced p21WAF/CIP1 mRNA and protein expression as well as stimulated its promoter activity in T cells and A-549 cells. The induction of p21WAF/CIP1 expression by tacrolimus was dependent on TGF-beta since a neutralizing anti-TGF-beta antibody inhibited induction of p21WAF/CIP1in A-549 cells. These data support the hypothesis that cyclin inhibitor p21WAF/CIP1 might represent a unified mediator of the anti-proliferative effects of Tac and other immunosuppressive agents. Strategies involving p21WAF/CIP1 induction should be considered a viable alternative strategy to achieve immunosuppression possibly with reduced toxicity associated with current immunosuppression.     

A mimic of p21WAF1/CIP1 ameliorates murine lupus

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease characterized by the production of high levels of affinity-matured IgG autoantibodies to dsDNA and, possibly, visceral involvement. Pathogenic autoantibodies result from the activation and proliferation of autoreactive T and B lymphocytes stimulated by epitopes borne by nucleosomal histones. To inhibit the proliferation of autoreactive cells and abrogate the development of SLE, a novel tool, cell cycle inhibiting peptide therapy, was used. Thus, a peptidyl mimic of p21WAF1/CIP1 that inhibits the interaction between cyclin-dependent kinase 4 and type D cyclins abrogated the in vitro proliferative response of T cells to histones and T-independent and T-dependent proliferative responses of B cells. The WAF1/CIP1 mimic also abrogated the in vitro production of total and anti-dsDNA IgG Abs by B cells. Similarly, the p21WAF1/CIP1 construct inhibited the ex vivo T and B cell proliferative responses to histones and decreased the numbers of activated/memory B and T spleen cells. The alterations in the balance of spleen cell subsets resulted from proapoptotic effects of the p21WAF1/CIP1)construct on activated splenocytes. Finally, in vivo, four i.v. injections of the p21WAF1/CIP1 mimic were sufficient to inhibit the progression of the lupus-like syndrome in (NZB x NZW)F1 mice. The levels of anti-dsDNA IgG autoantibodies and the incidence and severity of renal involvement were lower in treated mice than in nontreated mice. Those observations open new avenues for the treatment of SLE and prompt us to evaluate the potential interest of peptidic therapy in human SLE.     

p21WAF1/CIP1基因与肿瘤

肿瘤的发生发展是多基因参与的复杂而多步骤的过程.p21WAF1/CIP1基因作为一种重要的细胞周期抑制基因与肿瘤的发生发展密切相关,近年来的研究表明它在不同肿瘤发生发展中有着不同的作用.     

The p21(WAF1/CIP1) promoter is methylated in Rat-1 cells: stable restoration of p53-dependent p21(WAF1/CIP1) expression after transfection of a genomic clone containing the p21(WAF1/CIP1) gene

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.     

Regulation of the Cyclin-Dependent Kinase Inhibitor p57Kip2 Expression by p63

The cyclin-dependent kinase (CDK) inhibitor p57^Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57^Kip2 gene was recognized as a target gene for p73β, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knock out mice do not resemble each other while there is a phenotypic overlap betweeen the p57^Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57^Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the δNp63α protein is associated to three different regions of the p57Kip2 gene. δNp63 can activate both the endogenous p57^Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57^Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57^Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57^Kip2 expression and that this regulation is subverted in AEC p63 mutants.     

Chemical DNA damage activates p21 WAF1/CIP1-dependent intra-S checkpoint

When cells progressing in G₁ phase are irradiated with UV light, two damage checkpoint pathways are activated: CHK1-Cdc25A and p53-p21^WAF1/CIP1, both targeting Cdk2 but the latter inducing long lasting inactivation. In similarly irradiated S phase cells, however, p21^WAF1/CIP1-dependent checkpoint is largely inactive. We report here that p21-dependent checkpoint can effectively be activated and induce a prolonged S phase arrest with similarly extended inactivation of Cdk2 by association of p21 if mid-S phase cells are damaged with a base-modifying agent instead of UV light, indicating that the poor utilization of p21-dependent checkpoint is not an innate property of S phase cells.     

Control of Cyclin-Dependent Kinase Inhibitor p27 Expression by Cap-Independent Translation

p27 is a key regulator of cell proliferation through inhibition of G(1) cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5' cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5' untranslated region (5'-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5'-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.