Induction of anti-proliferative mechanisms in hepatitis B virus producing cells

BACKGROUND/AIMS: Hepatitis B virus (HBV) preferentially replicates in quiescent cells. It was analyzed whether HBV affects cell cycle control. METHODS: The amount of EGF-receptor (EGFR) and the binding capacity for 125I-EGF was determined. Expression of mdm2 and p21 and relevance of p53 for it were analyzed by reporter gene assays and western blotting. Cyclin A/E associated cdk2 activities were determined by immunocomplex assays. Cell proliferation was quantified by measurement of BrdU incorporation. RESULTS: In HBV producing cells a significant reduction of EGFR expression, diminished 125I-EGF-binding capacity and insensitivity to EGF-stimulation were observed as compared to the control. Moreover, c-Raf-1-dependent induction of mdm2-P2 and p21cip1/waf1-promoter and elevated amounts of the respective proteins were observed in HBV producing cells. Whereas activation of mdm2-P2-promoter requires p53, activation of p21cip1/waf1-promoter is mediated partially by a p53-independent process. Induction of p21cip1/waf1 is reflected by a reduction of cyclin A associated cdk2 activity and an increase of cyclin E associated cdk2 activity. In accordance with this proliferation rate of HBV-producing hepatocytes is reduced as compared to control cells. CONCLUSIONS: These results describe novel cell-cycle inhibitory functions of HBV that correlate well with the general concept of enhanced HBV replication in quiescent cells.     

Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21(waf1/cip1) in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like alpha-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 microM, and DFMO elicited effects at concentrations of 100 microM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21(waf1/cip1). This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21(waf1/cip1) depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21(waf1/cip1) were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21(waf1/cip1) were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21(waf1/cip1) and consequent inhibition of cell proliferation.     

姜黄素对血管平滑肌细胞增殖及cyclinD1和p21waf1/cip1蛋白表达的影响

目的 观察姜黄素（curcumin）抑制大鼠血管平滑肌细胞(VSMC)增殖和诱导细胞周期停滞的作用,以及对细胞周期蛋白cyclinD1,p21waf1/cip1表达的影响。方法 采用组织贴块法体外培养大鼠VSMC,MTT法测定姜黄素对VSMC增殖的抑制作用,流式细胞仪分析细胞周期分布,Western印迹法检测cyclinD1,p21waf1/cip1蛋白的变化。结果 MTT示不同浓度姜黄素(7.5～120 μmol/L)在24～72 h范围内,浓度和时间依赖性抑制VSMC增殖;30 μmol/L以上浓度姜黄素显著抑制增殖的VSMC细胞周期进程,使S及G2/M期减少(P<0.05),G0/G1期增加(P<0.05);30 μmol/L的姜黄素可抑制cyclinD1表达,促进p21waf1/cip1蛋白表达。结论 姜黄素具有明确的抑制VSMC增殖和细胞周期停滞的作用,其与cyclinD1,p21waf1/cip1蛋白变化有关。     

Overexpression of protein kinase C-beta1 isoenzyme suppresses indomethacin-induced apoptosis in gastric epithelial cells

BACKGROUND & AIMS: We have previously reported that nonsteroidal anti-inflammatory drugs (NSAIDs) could induce apoptosis of gastric epithelial cells both in vivo and in vitro. This study investigated the role of protein kinase C (PKC) isoforms in the regulation of NSAID-induced apoptosis. METHODS: Protein levels of 12 PKC isoforms in AGS cells, in the presence or absence of indomethacin, were determined by Western blot. The effect of PKC-beta1 overexpression by transfection with its complementary DNA (cDNA) on indomethacin-induced apoptosis and apoptosis-related genes, including p53, p21(waf1/cip1), and c-myc, was further investigated. RESULTS: Treatment with indomethacin decreased the abundance of PKC-beta1 and increased that of PKC-beta2, eta, and epsilon, but did not alter the expression of PKC alpha, gamma, zeta, delta, iota, and micro. Overexpression of PKC-beta1 attenuated the apoptotic response of AGS cells to indomethacin, associated with overexpression of p21(waf1/cip1) in both messenger RNA and protein levels. Inhibition of PKC-beta1-mediated overexpression of p21(waf1/cip1) by its antisense cDNA partially reduced the antiapoptotic effect of PKC-beta1. CONCLUSIONS: Indomethacin-induced apoptosis in gastric cancer cells is partly mediated by differential regulation of PKC isoform expression. Enhanced expression of exogenous PKC-beta1 protects against indomethacin-induced apoptosis through up-regulation of p21(waf1/cip1).     

Pioglitazone inhibits growth of carcinoid cells and promotes TRAIL-induced apoptosis by induction of p21waf1/cip1.

BACKGROUND/AIMS: We investigated the effect of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone on growth and TRAIL-induced apoptosis in carcinoid cells. METHODS: Carcinoid cells were incubated without and with pioglitazone. Effects on growth were examined by cell count and cell cycle analysis. p21waf1/cip1 expression was determined by Western blotting. Cytotoxicity assay was performed by FACS analysis. RESULTS: Pioglitazone suppressed the growth and induced apoptosis of carcinoid cells. Additionally, pioglitazone significantly enhanced carcinoid cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The enhancement of TRAIL-induced apoptosis was associated with an upregulation of cyclin-dependent kinase inhibitor p21waf1/cip1 in pioglitazone-treated carcinoid cells. Importantly, overexpression of p21waf1/cip1 in carcinoid cells by adenoviral gene transfer of p21 sensitized them to TRAIL-induced apoptosis. CONCLUSIONS: These results suggest that pioglitazone inhibits cell growth and sensitizes cells to TRAIL-induced apoptosis by induction of p21waf1/cip1. Therefore, pioglitazone can be an effective therapeutic adjuvant for the treatment of carcinoid tumors.     

p53 functional impairment and high p21waf1/cip1 expression in human T- cell lymphotropic/leukemia virus type I-transformed T cells

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53- regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I- infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I- transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T- cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.     

Blocking effects of genistein on cell proliferation and possible mechanism in human gastric carcinoma

AIM: To study the blocking effects of genistein on cell proliferation cycle in human gastric carcinoma cells (SGC-7901) and the possible mechanism. METHODS: MTT assay was applied in the detection of the inhibitory effects of genistein on cell proliferation. Flow cytometry was used to analyze the cell cycle distribution. Immunocytochemical technique and Western blotting were performed to detect the protein expression of cyclin D_1, cyclin B_1 and p21~(waf1/cip1). RESULTS: Genistein significantly inhibited the growth and proliferation of human gastric carcinoma cells (SGC-7901). Seven days after treatment with different concentrations of genistein (2.5, 5.0, 10.0, 20.0 μg/mL), the growth inhibitory rates were 11.2%, 28.8%, 55.3%, 84.7% respectively and cell cycles were arrested at the G(2)/ M phase. Genistein decreased cyclin D_1 protein expression and enhanced cyclin B_1 and p21~(waf1/cip1) protein expression in a concentration-dependent manner. CONCLUSION: The growth and proliferation of SGC-7901 cells can be inhibited by genistein via blocking the cell cycle, with reduced expression of cyclin D_1 and enhanced expression of cyclin B_1 and p21~(waf1/cip1) protein in the concentration range of 0-20 μg/mL.     

The role of XPB in cell apoptosis and viability and its relationship with p53, p21 and c-myc in hepatoma cells

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.     

p27kip1 Regulates cdk2 activity in the proliferating zone of the mouse intestinal epithelium: potential role in neoplasia

BACKGROUND & AIMS: Reduced p27(kip1) expression is a marker of poor prognosis in colorectal neoplasia, and inactivation of p27 in mice (p27(Delta51/Delta51)) causes increased intestinal epithelial cell proliferation and small and large intestinal neoplasia in a diet-dependent manner. Here, we addressed the role of p27 in untransformed intestinal epithelial cells in vivo and the consequence of its targeted inactivation. METHODS: A sequential fractionation procedure was used to isolate murine intestinal epithelial cells relative to their position along the crypt-villus axis, and the levels of cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors and of the complexes formed among them was determined by immunoprecipitation-immunoblotting and kinase assays. RESULTS: As cells exited the proliferative crypt compartment, expression and activity of both cdk2 and cdk4 decreased, in parallel with reduced expression of cyclin A and proliferating cell nuclear antigen (PCNA); expression of cyclin D1, D2, and cyclin E showed little change. As expected, expression of the cdk inhibitors p21, p57, and p16 was highest in differentiated villus cells. Unexpectedly, p27 protein expression was highest in cells of the proliferative crypt compartment where it bound both cdk2 and cdk4. Cdk2 activity was increased in crypt cells from p27(Delta51/Delta51) mice, although cyclin D-associated kinase activity was unchanged (indeed, cyclin D1/2-cdk4 complex levels were reduced). Importantly, cdk2 activity was unchanged in crypt cells from p21(-/-) mice, which do not develop intestinal tumors. CONCLUSIONS: We propose that p27 contributes to intestinal epithelial homeostasis by regulating cdk2 activity in proliferating cells, thus gating cell cycle progression and suppressing intestinal neoplasia.     

Immunohistochemical expression of Cyclin D1, Cyclin E,p21/waf1 and p27/kip1 in inflammatory bowel disease: correlation with other cell-cycle-related proteins (Rb,p53, ki-67 and PCNA) and clinicopathological features

Background and aims The expression patterns of cyclins D1 and E as well as cyclin-dependent kinase inhibitors p21/waf1 and p27/kip1 and their correlation with clinical parameters and other cell cycle regulators was investigated in inflammatory bowel disease (IBD).Patients and methods These molecular markers were localized immunohistochemically using the monoclonal antibodies anti-cyclin D1 (DCS-6), anti-cyclin E (13A3), anti-p21 (4D10) and anti-p27 (1B4) in 70 patients with IBD, 30 patients with colorectal cancer and eight healthy subjects. Data were analyzed statistically using the software program.Results Cyclin D1 expression was higher in both UC and CD compared with the healthy control group. In addition, CD cyclin D1 expression was higher compared with UC cases and colorectal carcinomas. Cyclin D1 expression was correlated with disease activity and cell proliferation in UC cases. A positive relationship of cyclin D1 with p27/kip1 in both UC and CD was detected. Cyclin E expression was higher in UC, CD and carcinomas compared with healthy control group and its expression correlated with proliferative activity in both UC and CD cases. p21/waf1 expression was higher in IBD cases compared with that of the control group, while a decreased p21/waf1 expression in the group of carcinomas was noted. This expression was correlated with disease activity in UC and the proliferative activity in both UC and CD. The expression of cyclins D1 and E as well as p21/waf1 was also correlated with the existence of dysplastic lesions. A lower p27/kip1 expression in the group of carcinomas compared with IBD cases and healthy controls was found.Conclusions The expression patterns of cyclin D1, cyclin E, p21/waf1 and p27/kip1 in IBD may indicate their contribution in epithelial cell turnover and their possible implication in IBD-related dysplasia-carcinoma.     

Exogenous nitric oxide upregulates p21(waf1/cip1) in pulmonary microvascular smooth muscle cells

The histopathology of chronic pulmonary hypertension includes microvascular proliferation and neointimal formation. Nitric oxide (NO) has been implicated in the regulation of these mechanisms, but how NO controls microvascular proliferation and its effect on pulmonary microvascular cells is still unclear. In this study, we characterized the in vitro effects of NO on rat pulmonary microvascular smooth muscle cell (PMVSMC) proliferation and investigated the contribution of the p42/44 mitogen-activated protein kinase (MAPK) pathway and p21(waf1/cip1) induction to this response. NO donors inhibited PMVSMC proliferation in a dose-dependent manner. In the presence of hypoxia, the degree of inhibition was significantly enhanced. This inhibition was reversible and independent of apoptosis. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) had no impact on proliferation rates, suggesting a cyclic guanosine monophosphate-independent process. Administration of MEK1/2 inhibitors failed to abrogate the antimitotic effect of NO. There was a two- fold induction of the cyclin-dependent kinase inhibitor p21 in PMVSMC treated with NO donors. Under hypoxic conditions, NO caused a three-fold increase in p21 levels. These results demonstrate that NO inhibits PMVSMC proliferation and that this inhibition is not the result of p42/44 MAPK activation. The ability of NO to induce p21 upregulation may be a mechanism by which it exerts antiproliferative effects in PMVSMC.     

Effect of cis—9,trans—11—conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line（SGC—7901）

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P<0.05 and P<0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P<0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).     

Role of epidermal growth factor in pathogenesis of uterine leiomyomas

Objective:To investigate the role of epidermal growth factor(EGF) in the pathogenesis of uterine leiomyomas.Methods:Human myometrial smooth muscle cells(HM-SMCs) and smooth muscle cells of human uterine leiomyomas(HL-SMCs) were separated from patients' specimens and cultured.After processed by EGF or PD98059(inhibitor of MKK/MEK) +EGF,the proliferation rate of both SMCs was detected by BrdU method and the phosphorylation level of p44/42 mitogen-activated protein kinase(MAPK) was determined by Western-blot.After different processing time by EGF,the phosphorylation levels of p44/42 MAPK and AKT and p27 expression level in both SMCs were detected by Western-blot.Results:EGF could significantly promote HL-SMCs proliferation and PD98059 could inhibit this effect(P0.05);besides,PD98059 could inhibit the increase of the phosphorylation level of p44/42 MAPK in both SMCs induced by EGF.When the processing time by EGF was over 15 min,the phosphorylation levels of p44/42 MAPK and AKT in both SMCs decreased sharply and were close to zero:p27 expression in HM-SMCs raised significantly while the upregulation in HL-SMCs was little.Conclusions:EGF could not cause activation of EGFR because of the dephosphorylation of p44/42 MAPK and AKT in HL-SMCs,which caused p27 expression insufficiently and cell cycle dysregulation.     

Epidermal growth factor induces cyclin D1 in human pancreatic carcinoma: evidence for a cyclin D1-dependent cell cycle progression

INTRODUCTION: We recently showed that cyclin D1 is overexpressed in human pancreatic carcinoma cells, and that this overexpression correlates significantly with a poor prognosis. AIMS: To assess the interrelations of epidermal growth factor (EGF), EGF receptor (EGFR), and cyclin D1 in human pancreatic carcinoma. METHODOLOGY AND RESULTS: In pancreatic carcinoma cell lines (BxPC-3, AsPC-1), cell cycle analysis revealed an increase in cells in the S/G1 phase between 18 and 30 hours after stimulation with 50 ng/mL EGF. Cyclin D1 mRNA increased after 2 hours, corresponding to an increase in cyclin D1 protein, with the maximum level between 7.5 and 10 hours after stimulation, as demonstrated by Western blot analysis. We performed immunohistochemical analysis on 61 adenocarcinoma tissues for the expression of EGF, EGFR, and cyclin D1 and demonstrated an overexpression in the tumor cells in 51%, 54%, and 62.3%, respectively, whereas normal human pancreas stained negative for all of the three factors. Interestingly, EGF and EGFR expression correlated significantly with the cyclin D1 expression in human pancreatic tumor cells (p < 0.001 and p < 0.01, respectively). CONCLUSION: These results demonstrate that cyclin D1 overexpression in the tumor cells of pancreatic carcinoma tissue is at least partly dependent on the mitogenic effects of EGF signaling through the EGFR.     

表皮生长因子受体在胶质瘤中的表达变化及其在细胞信号传导中的作用

目的观察胶质瘤组织及细胞U251中表皮生长因子受体（EGFR）的表达变化,探讨其在细胞信号传导通路中的作用。方法采用免疫组化SP法检测78例脑胶质瘤组织和U251中的EGFR。用EGF作用U251,MTT法检测U251细胞增殖情况,Western blot法检测U251中磷酸化EGFR（p-EGFR）。结果 78例胶质瘤组织中EGFR阳性表达率为66.67%（52/78）,且与胶质瘤的病理分级呈正相关（r=0.441,P〈0.05）。EGF作用后,U251细胞增殖显著、U251中p-EGFR水平明显提高（P均〈0.05）。结论胶质瘤组织、细胞中EGFR均呈过度表达。EGFR通过其介导的细胞信号传导通路促进细胞增殖,EGFR在细胞信号传导通路中发挥重要作用。     

Pharmacological Inhibition of Protein Kinase C Activity Could Induce Apoptosis in Gastric Cancer Cells by Differential Regulation of Apoptosis-Related Genes

The protein kinase C (PKC) signaling pathwayplays a key role in tumor cell proliferation,differentiation, and apoptosis. Gastric cancer usuallypossesses a higher level of PKC activity than normaltissue. We evaluated inhibition of PKC activity inapoptosis induction of gastric cancer cells and theexpression profile of apoptosis-related genes. Gastriccancer cells (AGS) were incubated with two highlyspecific PKC inhibitors (RO-31-8220 and chelerythrine).Cell viability and cell cycle were determined bymethyl-tetrazolium (MTT) assay and flow cytometry,respectively. Apoptosis was characterized by acridineorange staining, DNA gel electrophoresis, and flowcytometry. The expression of p53,p21^waf/cip1, c-myc, bcl-2, and bax wasdetermined by western blot. The results showed that bothPKC inhibitors hindered cell growth, arrested cells atG₀/G₁ phase and induced apoptosis.The protein level of p53, p21^waf/cip1, c-myc,and bax was elevated while bcl-2 kept unchangedfollowing drug exposure. In conclusion, PKC inhibitorssuppress growth of gastric cancer cells throughapoptosis induction and cell cycle quiescence, which maybe regulated by differential expression ofapoptosis-related genes.     

p21(Waf1/Cip1) regulates proliferation and apoptosis in airway epithelial cells and alternative forms have altered binding activities

p21(Waf1/Cip1) plays central roles in proliferation, differentiation, and apoptosis. Alterations in the expression and subcellular localisation of p21 occur during several lung diseases but the roles of p21 in the lung epithelium are unknown. The effects of p21 on proliferation and apoptosis in mouse airway epithelial cells (AECs) were examined using p21-null mice. AECs isolated from p21-null mice had increased proliferation and apoptotic rates compared to AECs from wild-type mice. Alterations in the subcellular localization of the cell cycle regulatory proteins p27, PCNA, and p53 were also evident in p21(-/-) cells. The nuclear and cytoplasmic forms of p21 present in AECs were also examined. Full-length p21 (20 kDa) was detected in nuclear fractions but a C-terminal truncated form (17 kDa) of p21 was present in cytoplasmic fractions. The binding activities of truncated p21 were altered compared to full-length p21. Although the latter was complexed with PCNA, Cdk2, Cdk4, Cdk6, cyclin D3, and cyclin E, truncated p21 was bound only to Cdk4 and cyclin D3. In conclusion, p21 regulates proliferation and protects against apoptosis in AECs. In addition, different forms of p21 are present in AECs and the subcellular localization of these forms reflects differences in p21 activity.     

JTT-705 blocks cell proliferation and angiogenesis through p38 kinase/p27(kip1) and Ras/p21(waf1) pathways

The excessive proliferation and migration of vascular smooth muscle cells (SMCs) participate in the growth and instability of atherosclerotic plaque. We examined the direct role of a newly developed chemical inhibitor of cholesteryl ester transfer protein, JTT-705, on SMC proliferation and angiogenesis in endothelial cells (ECs). JTT-705 inhibited human coronary artery SMC proliferation. JTT-705 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular-signal-regulated kinases (ERK) in SMCs. In addition, the anti-proliferative effects of JTT-705 in SMCs were blocked by p38 MAPK inhibitor. JTT-705 induced the upregulation of p-p21(waf1), and this effect was blocked by dominant-negative Ras (N17), but not by inhibitors of p38 MAPK or ERK. In addition, JTT-705 also induced the upregulation of p27(kip1), and this effect was blocked by p38 MAPK inhibitor. Interestingly, culture medium from JTT-705-treated SMCs blocked human coronary artery EC tube formation in an in vitro model of angiogenesis indirectly via a decrease in vascular endothelial growth factor (VEGF) from SMCs and directly via an anti-proliferative effect in ECs. JTT-705 blocked the proliferation of SMCs through the activation of p38 kinase/p27(kip1) and Ras/p21(waf1) pathways, and simultaneously blocked EC tube formation associated with a decrease in VEGF production from SMCs and an anti-proliferative effect in ECs. Our results indicate that JTT-705 may induce a direct anti-atherogenic effect in addition to its inhibitory effect of CETP activity.     

牛磺酸通过调控细胞周期蛋白抑制肝星状细胞增殖

目的进一步研究牛磺酸对肝星状细胞(HSC)增殖抑制作用的机制。方法用四甲基偶氮唑盐法检测细胞增殖;流式细胞仪测定细胞周期;免疫细胞化学和实时荧光定量PCR测定细胞周期调控蛋白Cyclin D1和P21waf1表达。结果牛磺酸对HSC增殖具有抑制作用,在浓度为5、10、20,30、40、50 mmol/L 作用48h时的抑制率分别为6.7%、14.4%、23.3%、32.2%、36.7%和45.6%,t值为2.939～6.369,P<0.05～0.01。流式细胞仪检测发现牛磺酸可阻滞HSC由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少。G0/G1期、S期细胞,牛磺酸浓度为40 mmol/L时,分别为(68.2±1.4)%和(26.2±1.3)%,与对照组分别为(56.2±1.7)%和(38.5±0.8)%,差异有统计学意义,t≥5.422,P<0.01。牛磺酸可抑制Cyclin D1表达、促进P21waf1表达,用免疫细胞化学染色结合数码图像分析系统软件分析发现牛磺酸浓度在40 mmol/L时HSC的Cyclin D1表达的平均吸光度为0.13±0.02,P21waf1为0.19±0.02,对照组分别为0.18±0.02和0.14±0.01,差异有统计学意义,t=6.689和t=6.528,P<0.01。实时荧光定量PCR检测也发现经40 mmol/L牛磺酸处理的HSC的Cyclin D1 mRNA表达量(拷贝数与106磷酸甘油醛脱氢酶比值)降低为5776.7±3345.0,对照组为18 400.6±1374.8,而P21waf1 mRNA表达量(拷贝/106磷酸甘油醛脱氢酶)增多为44 866.7±3910.7,对照组为16 933.3±960.9。结论牛磺酸通过抑制Cyclin D1表达、促进P21waf1表达,使HSC阻滞于G0/G1期,而抑制HSC增殖。