p21Waf1/Cip1 Deficiency Stimulates Centriole Overduplication

Inactivation of the cyclin-dependent kinase (CDK) inhibitor p21Waf1/Cip1 (CDKN1; hereafter p21) has previously been implicated in the induction of numerical centrosome alterations. It is unclear, however, whether p21 deficiency deregulates the centrosome duplication cycle itself or causes an accumulation of centrosomes due to cell division failure and/or polyploidization. Using a novel marker for maternal centrioles, Cep170, we show here that knock-down of p21 protein expression in murine myeloblasts can stimulate excessive centriole numbers in the presence of only one or two mature centrioles. These results indicate that p21 deficiency can trigger a bona fide overduplication of centrioles and that aberrant centrosome numbers cannot solely be explained by polyploidization as suggested by previous studies. Our findings underscore that impaired p21 expression may function as a driving force for chromosomal instability and highlight the importance of markers for maternal centrioles such as Cep170 to elucidate the pathogenesis of numerical centriole aberrations in tumor cells.     

BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21Cip1/Waf1

Bone-morphogenetic proteins (BMP)-2 and -7, multifunctional members of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, cause cell cycle arrest in a variety of transformed cell lines by activating signaling cascades that involve several cyclin-dependent kinase inhibitors (CDKIs). CDKIs in the cip/kip family, p21(Cip1/Waf1) and p27(Kip1), have been shown to negatively regulate the G1 cyclins and their partner cyclin-dependent kinase proteins, resulting in BMP-mediated growth arrest. Bone morphogens have also been associated with antiproliferative effects in vascular tissue by unknown mechanisms. We now show that BMP-2-mediated inhibition of platelet-derived growth factor (PDGF)-stimulated human aortic smooth muscle cell (HASMC) proliferation is accompanied by increased levels of p21 protein. Antisense oligodeoxynucleotides specific for p21 attenuate BMP-2-induced inhibition of proliferation when transfected into HASMCs, demonstrating that BMP-2 inhibits PDGF-stimulated proliferation of HASMCs through induction of p21. Whether p21-mediated induction of cell cycle arrest by BMP-2 sets the stage for osteogenic differentiation of vascular smooth muscle cells, ultimately leading to vascular mineralization, remains to be investigated.     

Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1

Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.     

Cytosolic p21Waf1/Cip1 increases cell cycle transit in vascular smooth muscle cells

The intracellular localization of signaling proteins is critical in directing their interactions with both upstream and downstream signaling cascade components. While initially described as a cyclin kinase inhibitor, p21Waf1/Cip1 has since been shown to have bimodal effects on cell cycle progression and cell proliferation, and evidence is emerging that intracellular localization of this protein plays a role in directing its signaling properties by dictating its interactions with downstream molecules. Since we have previously demonstrated a pro-apoptotic and cell cycle inhibitory effect of p21 attenuation after transfection of antisense p21 oligodeoxynucleotides (ODN) in several cell lines, we asked whether cytosolic p21 mediates a positive effect on vascular smooth muscle (VSM) cell cycle transit. We now show that transfection of a nuclear-localization signal deficient (DeltaNLS) p21 construct into VSM cells results in increased cytosolic levels of p21 and causes increased cell cycle transit as measured by [3H]thymidine incorporation. Thus, at least in VSM cells, cytosolic localization of p21 is a means by which this signaling protein transmits pro-mitogenic signals to the proteins responsible for G1/S transition. Furthermore, compartmentalization of p21 may help explain the biphasic nature of p21 in a variety of cell types and may lead to therapeutic advances directed at modulating pathologic cell growth in vascular diseases and cancer.     

1,25-Dihydroxycholecalciferol enhances butyrate-induced p21(Waf1/Cip1) expression

Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.     

p21(Waf1/Cip1) is an assembly factor required for platelet-derived growth factor-induced vascular smooth muscle cell proliferation

The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet-derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation.     

The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21^Waf1/Cip1. We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53^S18P) and targets it for degradation in low-dose-UV-irradiated cells. DDB2^−/− mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53^S18P. Accumulation of p53^S18P in DDB2^−/− MEFs causes higher expression p21^Waf1/Cip1. We show that the increased expression of p21^Waf1/Cip1 is the cause NER deficiency in DDB2^−/− cells because deletion or knockdown of p21^Waf1/Cip1 reverses their NER-deficient phenotype. p21^Waf1/Cip1 was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21^Waf1/Cip1 was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21^Waf1/Cip1 to the NER activity of DDB2.     

RASSF6 promotes p21Cip1/Waf1-dependent cell cycle arrest and apoptosis through activation of the JNK/SAPK pathway in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common pathological subtype of renal cancer. This cancer is characterized by biallelic inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene, which leads to the accumulation of hypoxia-inducible factors (HIFs). Although therapies targeted at HIFs can significantly improve survival, nearly all patients with advanced ccRCC eventually succumb to the disease. Thus, additional oncogenic events are thought to be involved in the development of ccRCC tumors. In this study, we investigated the role of RASSF6 in ccRCC. Downregulation of RASSF6 was commonly observed in primary tumors relative to matched adjacent normal tissues. Moreover, functional studies established that ectopic re-expression of RASSF6 in ccRCC cells inhibited cell proliferation, clonogenicity, and tumor growth in mice, whereas silencing of RASSF6 dramatically enhanced cell proliferation in vitro and in vivo. Mechanistic investigation suggested that RASSF6 triggers p21^Cip1/Waf1 accumulation to induce G₁ cell cycle arrest and promote apoptosis upon exposure to pro-apoptotic agents, and both of these mechanisms appear to be mediated by activated JNK signaling. Together, these findings suggest that RASSF6 may play a tumor suppressor role in the progression of ccRCC.     

Involvement of p21(Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression

Protein kinase C (PKC) plays an important role in the regulation of glioma growth; however, the identity of the specific isoform and mechanism by which PKC fulfills this function remain unknown. In this study, we demonstrate that PKC activation in glioma cells increased their progression through the cell cycle. Of the six PKC isoforms that were present in glioma cells, PKC alpha was both necessary and sufficient to promote cell cycle progression when stimulated with phorbol 12-myristate 13-acetate. Also, decreased PKC alpha expression resulted in a marked decrease in cell proliferation. The only cell cycle-regulatory molecule whose expression was rapidly altered and increased by PKC alpha activity was the cyclin-cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). Coimmunoprecipitation studies revealed that p21(Waf1/Cip1) upregulation was accompanied by an incorporation of p21(Waf1/Cip1) into various cyclin-CDK complexes and that the kinase activity of these complexes was increased, thus resulting in cell cycle progression. Furthermore, depletion of p21(Waf1/Cip1) by antisense strategy attenuated the PKC-induced cell cycle progression. These results suggest that PKC alpha activity controls glioma cell cycle progression through the upregulation of p21(Waf1/Cip1), which facilitates active cyclin-CDK complex formation.     

HDAC inhibitors act with 5-aza-2'-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells

5-Aza-2'-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [(3)H]-5-aza-CdR incorporated in DNA. However, incorporated [(3)H]-5-aza-CdR gradually decreased when cells were incubated in [(3)H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [(3)H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as gamma-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.