Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1

The core protein (Core) of hepatitis C virus (HCV) has been known to play an important role in hepatocarcinogenesis. By using glutathione S-transferase (GST) pull-down assay, we show here that Core formed a complex with p21Waf1/Cip1/Sdi1 (p21) cell cycle regulator. The deletion-mapping analysis revealed that a portion near the N-terminus of Core (amino acids 24-52) and a C-terminal portion of p21 (amino acids 139-164) were involved in the complex formation. The complex formation was not impaired by point mutations of p21 at residues 147, 149, and 150, which have been reported to abrogate interaction of p21 with proliferating cell nuclear antigen (PCNA), discriminating the Core-binding sequence from the PCNA-binding sequence. Due to the close vicinity of the binding sites, however, Core and PCNA competed with each other when interacting with p21. The distinct interaction between Core and p21 may provide a new aspect to the studies of HCV pathogenesis.     

Solution structure of p21(Waf1/Cip1/Sdi1) C-terminal domain bound to Cdk4.

Cyclin-dependent kinase (Cdk) inhibitor p21(Waf1/Cip1/Sdi1), a multifunctional protein, has a major role as tumor suppressor, mediating G1/S arrest through inhibition of Cdks. Recent biological studies of Cyclin D1/Cdk4 have proposed that p21 C-terminal domain (p21(CT)) plays a key role as a potent Cdk4 inhibitor. We report here solution structures of p21(CT) for both the free and Cdk4-bound forms using 2D transferred NOE spectroscopy and dynamical simulated annealing calculations. Even though p21(CT) peptide is very flexible in the free state, when it bound to Cdk4, the structure becomes well structured in the binding domain. Therefore we propose that p21(CT) experiences an extensive conformational change upon Cdk4 binding. This structural change of p21(CT) may suggest the molecular mechanism of p21 for specificity and inhibition mode to assemble different cyclin-Cdk complexes. Especially, our data suggests that the D(149)FYHSKRR(156) region of p21 is critical for Cdk4 binding, indicating that the major driving force for complex originates from hydrophobic interaction between p21 and Cdk4.     

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.     

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.     

Extension of replicative lifespan in WI-38 human fibroblasts by dexamethasone treatment is accompanied by suppression of p21 Waf1/Cip1/Sdi1 levels

Numerous studies have shown that supplementation of the growth medium of human fibroblasts with dexamethasone at physiologic concentrations extends replicative lifespan up to 30%. While this extension of lifespan has been used to probe various aspects of the senescent phenotype, no mechanism for the increased lifespan of human fibroblasts grown in the presence of dexamethasone has ever been identified. In the present study we present evidence that the extended lifespan of human lung fibroblasts (WI-38 cells) that occurs when these cells are maintained in culture medium supplemented with dexamethasone is accompanied by a suppression of p21(Waf1/Cip1/Sdi1) levels, which normally increase as these cells enter senescence, while p16(INK4a) levels are unaffected. These results suggest that the delay of senescence in cultures grown in the presence of dexamethasone is due to a suppression of the senescence related increase in p21(Waf1/Cip1/Sdi1). These results are consistent with models of replicative senescence in which p53 and p21(Waf1/Cip1/Sdi1) play a role in the establishment of the senescent arrest.     

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene

A PstI polymorphism in the 3 flanking region of the p21^CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a CT base substitution in the 3 flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.     

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/Sdi1) prevents apoptosis as well as stimulates growth in cells transformed or immortalized by human T-cell leukemia virus type 1-encoded tax

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21(Waf1/Cip1/Sdi1), resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-kappaB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms.     

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.