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.     

Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mm butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1.     

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.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

Zinc salicylate reduces airway smooth muscle cells remodelling by blocking mTOR and activating p21(Waf1/Cip1)

Asthma is characterized by chronic inflammation and tissue remodeling of the airways. Remodeling is resistant to pharmaceutical therapies. This study investigated the effect of zinc salicylate-methylsulfonylmethane (Zn-Sal-MSM) compared to zinc salicylate (Zn-Sal), or sodium salicylate (Na-Sal), or zinc chloride (ZnCl₂) on remodeling parameters of human airway smooth muscle cells (ASMC). Human ASMC obtained from asthma patients (n=7) and non-asthma controls (n=7) were treated with one of the reagents. Cell proliferation and viability was determined by direct cell counts and MTT assay. The expression of and phosphorylation proteins was determined by Western-blotting, ELISA, immunofluorescence, and mass spectrometry. Extracellular matrix deposition by ELISA. Zn-Sal-MSM, Zn-Sal and Na-Sal (0.1–100 µg/mL) significantly reduced PDGF-BB-induced proliferation in a concentration dependent manner, while ZnCl₂ was toxic. The reduced proliferation correlated with increased expression of the cell cycle inhibitor p21^(Waf1/Cip1), and reduced activity of Akt, p70S6K, and Erk1/2. Zn-Sal-MSM, Zn-Sal, but not Na-Sal reduced the deposition of fibronectin and collagen type-I. Furthermore, Zn-Sal-MSM reduced the mitochondria specific COX4 expression. Mass spectrometry indicated that Zn-Sal-MSM modified the expression of several signaling proteins and zinc-dependent enzymes. In conclusion, Zn-Sal-MSM and Zn-Sal potentially prevent airway wall remodeling in asthma by inhibition of both the Erk1/2 and mTOR signaling pathways.     

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.     

Susceptibility to killer T cells of gastric cancer cells enhanced by Mitomycin-C involves induction of ATBF1 and activation of p21 (Waf1/Cip1) promoter

Alpha-fetoprotein (AFP) expression is observed in embryonic tissues and, the expression of this protein is absent in normal adult tissues. The re-elevation of serum AFP strongly suggests generation of a malignant tumor in an adult. We demonstrated here that AFP-producing gastric cancer (AFP-gastric cancer) could be treated by a combination therapy with a low dose of Mitomycin-C (MMC) and lymphokineactivated killer T (LAK-T) cells. Treatment with MMC of AFP-gastric cancer cells enhanced their susceptibility to LAK-T cells and induced ATBF1 gene expression. We revealed here a novel signal pathway for regulation of the cell cycle of AFP-gastric cancer cells through ATBF1, which enhances the promoter activity of the p21 (Waf1/Cip1) gene. Immunoprecipitation revealed the direct interaction between ATBF1 and p53. Overexpressed ATBF1 stimulated p21 (Waf1/Cip1) promoter activity up to 4-fold compared with basal activity. The expression level of ATBF1 mRNA was doubled by MMC (0.05 microg/ml) treatment. The MMC treatment and ATBF1 overexpression synergistically activated the p21 (Waf1/Cip1) promoter activity in a dose-dependent manner up to 7-fold compared with basal activity.     

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.     

Age-dependent changes of p57(Kip2) and p21(Cip1/Waf1) expression in skeletal muscle and lung of mice

p57(Kip2) and p21(Cip1/Waf1) are members of cyclin-dependent kinase (Cdk) inhibitors which play critical roles in the terminal differentiation of skeletal muscle and lung. We investigated mRNA levels of p57(Kip2) and p21(Cip1/Waf1) in skeletal muscle and lung of mice during maturation and aging using Northern hybridization. The mRNA levels of p57(Kip2) and p21(Cip1/Waf1) decreased in skeletal muscle and lung of mice during maturation and aging except that the level of p21(Cip1/Waf1) mRNA in skeletal muscle of mice showed an increase only during maturation. The decrease of the p57(Kip2) mRNA level involved neither a change of DNA methylation at the promoter region nor an alteration of the imprinting status in aged mice. The decreases of p57(Kip2) and p21(Cip1/Waf1) mRNA levels during aging suggest that the process of tissue-specific terminal differentiation may be gradually downregulated with senescence in tissues where p57(Kip2) and p21(Cip1/Waf1) play key roles in differentiation. The downregulation of p57(Kip2) and p21(Cip1/Waf1) during aging is contrary to the upregulation of Cdk inhibitors during cellular replicative senescence, indicating that aging in an organismal level is mediated by mechanisms different from replicative senescence of cultured cells.     

T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21(CIP1/WAF1) expression

Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53(-/-) mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21(CIP1/WAF1), but not p27(Kip1), expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21(CIP1/WAF1) expression and was eliminated in p21(CIP1/WAF1)-deficient fibroblasts. Collectively, these observations suggest a novel mechanism of growth regulation for T-cadherin involving p21(CIP1/WAF1) expression and G2 arrest.