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.     

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.     

肿瘤抑制因子p21/waf1与肝炎病毒复制与表达的调节研究

0 引言作为一种肿瘤抑制基因,p21/wafl在细胞周期、细胞凋亡、信号转导以及癌变中具有十分重要的调节作用。肝炎病毒,特别是乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)的感染,不仅引起急性和慢性病毒性肝炎,而且与肝纤维化、肝细胞癌(HCC)的发生、发展密切相关。这些肝炎病毒感染靶细胞之后,在细胞内对于细胞正常的调节机制产生严重干扰,这是肝炎病毒致病的分子生物学机制的重要组成部分。其中,肝炎病毒蛋白对于p21/wafl的异常调节具有十分重要的生物学和医学意义。     

p21/waf1/cip1 in gastric cancer: associations with histopathological subtypes, lymphonodal metastasis, prognosis and p53 status

BACKGROUND: Cyclins and cyclin-dependent kinases are determining factors of the cell cycle. In the present study, we investigated the role of p21 and p53 in the biology of gastric cancer, focusing on its influence on progression and prognosis (n = 195). METHODS: P21 and p53 immunoreactivity was analysed immunohistochemically, applying monoclonal antibodies. The p53 status was comparatively evaluated by PCR-SSCP analysis of p53 mutations in selected tumours. RESULTS: Fifty-eight percent of the carcinomas were p21+ in more than 5% of the cancer cell nuclei, whereas 19% exhibited a p21 immunoreactivity in more than 20% of the nuclei. On the other hand, p53 was over-expressed (in more than 50% of the nuclei) in about 45% of the specimens. P21 immunoreactivity in more than 5% of the nuclei was inversely related to the pN as well as pTNM cancer stage, whereas only a strong p21 expression (in >20% of the nuclei) was correlated with a better survival probability in a univariate analysis. The p53 status was associated with lymphonodal metastasis, but not with prognostic data. In multivariate survival analyses, neither p21 nor p53 emerged as independent prognostic factors. Compared with the results of p53 mutation analysis by PCR-SSCP. p21 immunoreactivity was reduced in p53-mutated cases. CONCLUSIONS: These features show an association of p21 over-expression with certain clinico-pathological parameters of gastric cancer. In this context, our data suggest that p21 immunoreactivity in more than 5% of the tumour cells has a predictive value for the course of adenocarcinoma of the stomach.     

p21/waf1/cip1 in Gastric Cancer: Associations with Histopathological Subtypes,Lymphonodal Metastasis,Prognosis and p53 Status

Background: Cyclins and cyclin-dependent kinases are determining factors of the cell cycle. In the present study, we investigated the role of p21 and p53 in the biology of gastric cancer, focusing on its influence on progression and prognosis (n = 195). Methods: P21 and p53 immunoreactivity was analysed immunohistochemically, applying monoclonal antibodies. The p53 status was comparatively evaluated by PCR-SSCP analysis of p53 mutations in selected tumours. Results: Fifty-eight percent of the carcinomas were p21⁺ in more than 5% of the cancer cell nuclei, whereas 19% exhibited a p21 immunoreactivity in more than 20% of the nuclei. On the other hand, p53 was over-expressed (in more than 50% of the nuclei) in about 45% of the specimens. P21 immunoreactivity in more than 5% of the nuclei was inversely related to the pN as well as pTNM cancer stage, whereas only a strong p21 expression (in >20% of the nuclei) was correlated with a better survival probability in a univariate analysis. The p53 status was associated with lymphonodal metastasis, but not with prognostic data. In multivariate survival analyses, neither p21 nor p53 emerged as independent prognostic factors. Compared with the results of p53 mutation analysis by PCR-SSCP, p21 immunoreactivity was reduced in p53- mutated cases. Conclusions: These features show an association of p21 over-expression with certain clinico-pathological parameters of gastric cancer. In this context, our data suggest that p21 immunoreactivity in more than 5% of the tumour cells has a predictive value for the course of adenocarcinoma of the stomach.     

Recruitment of ataxia-telangiectasia mutated to the p21(waf1) promoter by ZBP-89 plays a role in mucosal protection

BACKGROUND & AIMS: Histone deacetylase inhibitors (HDACi) induce growth arrest, apoptosis, and differentiation, particularly in colon cancer cells where they are potential chemopreventive agents. HDACi induction of the cyclin-dependent kinase inhibitor p21(waf1) has been shown to require ataxia-telangiectasia mutated (ATM). Nevertheless, how ATM participates in p21(waf1) gene expression has not been defined. METHODS: In vivo protein complexes forming in response to butyrate were studied using co-immunoprecipitation and mass spectroscopy. DNA elements in the p21(waf1) promoter were analyzed in vivo by chromatin immunoprecipitation and in vitro DNA affinity precipitation assays. The expression of p21(waf1) was analyzed by immunoblots and reporter assays. RESULTS: Reduction of ZBP-89 or ATM with small interfering RNAs blocked HDACi-induced p21(waf1) expression. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that both ZBP-89 and ATM are recruited to the GC-rich DNA elements of the p21(waf1) promoter with HDACi treatment. Co-immunoprecipitation revealed that ATM associates with ZBP-89 in an HDACi-dependent manner. Serial deletions revealed that ATM interacts with both the N-terminal and DNA binding domains of ZBP-89. Moreover, we found that immunodepletion of ZBP-89 prevented recruitment of ATM to the p21(waf1) promoter in vitro. Silencing of ZBP-89 expression blocked HDACi-induced phosphorylation of ATM(Ser1981) and p53(Ser15). ATM(Ser1981) phosphorylation in the colons of mutant mice expressing an N-terminally truncated form of ZBP-89 was not observed after ingestion of dextran sodium sulfate and correlated with exacerbation of the mucosal injury. CONCLUSIONS: ZBP-89 interacts with ATM in a butyrate-dependent manner and is essential for colonic homeostasis in the setting of acute mucosal injury.     

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.     

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.     

Tranilast inhibits vascular smooth muscle cell growth and intimal hyperplasia by induction of p21(waf1/cip1/sdi1) and p53

Tranilast, which is an antiallergic drug, has a potent effect on preventing postangioplasty restenosis. To elucidate this mechanism, we studied the effect of tranilast on the proliferation of vascular smooth muscle cells (SMCs) in vitro and in vivo. Tranilast decreased the growth rate of SMCs stimulated by either 10% FBS or platelet-derived growth factor. The IC50 value, evaluated as cell number, was 100 micromol/L. These inhibitory effects were associated with inhibition of the retinoblastoma gene product (pRb) phosphorylation. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDK), we investigated CDK2 and CDK4 activities and the expression of CDK inhibitor p21(waf1/cip1/sdi1) (p21). When SMCs were stimulated by 10% FBS or platelet-derived growth factor, CDK2 and CDK4 activities reached a maximum near the G1/S transition. Tranilast suppressed their activities by >80% without reduction of CDK2/cyclin E and CDK4/cyclin D1 protein levels. These inhibitory effects were associated with enhanced expression of p21 and elevated complexing of p21 with CDK2/CDK4. Next, rat balloon-injured carotid artery was analyzed for intimal thickening and p21 expression. Tranilast-treated rats had a 70% (P<0.001) smaller neointima/media area ratio at 14 days after balloon injury compared with the controls. Immunohistochemical staining demonstrated that, in tranilast-treated rats, p21 was already present in the neointima at day 7 and strongly expressed throughout the neointima at day 14. In control rats, p21 was not observed in the neointima at day 7 but was sparsely expressed at day 14. These data demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 may be one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis.     

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.     

Tranilast inhibits the proliferation of human coronary smooth muscle cell through the activation of p21waf1

Restenosis after percutaneous transluminal coronary angioplasty (PTCA) occurs due to vascular smooth muscle cell proliferation and migration. Recently, tranilast, an anti-allergic drug, has been used for the prevention of restenosis after PTCA. To determine the molecular mechanism involved, the effect of tranilast on the proliferation of human coronary smooth muscle cells (SMCs) was investigated. Tranilast arrested the proliferation of human coronary SMCs at the G0/G1 phase of the cell cycle. In association with this inhibitory effect, tranilast increased p21waf1 and p53 tumor suppressor factor, and decreased cyclin-dependent kinase 2 (CDK2) activity. These results suggest that tranilast inhibits the proliferation of human coronary SMCs during restenosis after PTCA via an induction of p21waf1 and p53. Tranilast may thus allow us to prevent restenosis after PTCA by interfering with this mechanism.     

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。     

Tranilast inhibits the proliferation of uterine leiomyoma cells in vitro through G1 arrest associated with the induction of p21(waf1) and p53

Uterine leiomyoma is a mesenchymal tumor composed of smooth muscle cells with fibrous tissues and many mast cells. Tranilast is known to suppress fibrosis or to work as a mast cell stabilizer and is reported to inhibit proliferation of vascular smooth muscle cells. In this study, we examined the effects of tranilast on cultured human leiomyoma cells in vitro to evaluate whether this agent has the potential to inhibit the growth of uterine leiomyomas. Tranilast inhibited the proliferation of cultured leiomyoma cells in a dose-dependent manner without any cytotoxic effect or induction of apoptosis. In association with the inhibitory effect, tranilast induced the cyclin-dependent kinase (CDK) inhibitor p21(waf1) and tumor suppressor gene p53 and decreased CDK2 activity. These results suggest that tranilast arrests the proliferation of uterine leiomyoma cells at the G0/G1 phase, through the suppression of CDK2 activity via an induction of p21(waf1) and p53. Tranilast was concluded to be a potent agent to inhibit proliferative activity of uterine leiomyoma cells.     

Role of interleukin 1 and interleukin 1 receptor antagonist in the mediation of rheumatoid arthritis

Chronic arthritis is characterised by chronic joint inflammation and concurrent joint erosion and destruction. The inflammatory cytokine interleukin 1 (IL1) has been shown to be a key mediator in the autoimmune disease rheumatoid arthritis (RA). Interleukin 1 mediates bone resorption and cartilage destruction, but may not play as dominant a part in joint swelling and inflammation. Interleukin 1 receptor antagonist (IL1Ra) selectively inhibits the effects of IL1 by competing for the IL1 receptor on all surfaces of the synovium. In a randomised controlled trial in 472 patients with active disease, IL1Ra 30 mg/day, 75 mg/day or 150 mg/day given by subcutaneous injection significantly reduced the signs and symptoms of RA at 24 weeks. An American College of Rheumatology (ACR) 20% response was seen in 43% of the patients treated with 150 mg/day at 24 weeks. IL1Ra was well tolerated; injection site reactions were the most common adverse event. In another trial, in 419 patients with active RA treated concomitantly with methotrexate, there were ACR 20% responses after 24 weeks in 42% of the patients treated with 1 mg/kg/day by subcutaneous injection and in 35% of those treated with 2 mg/kg/day. I1Ra offers a unique selective, targeted mechanism of action to block the IL1 mediated effects of RA.     

Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1)

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras^61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21^Cip1/Waf1. Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-α for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2 % vs. 4.3 %, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58 % of Ras-infected cells stained positive for senescence-associated β-galactosidase activity as opposed to 2 % in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21^Cip1/Waf1 in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-α induced-apoptosis. Surprisingly, senescence-associated β-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21^Cip1/Waf1. Future studies have to investigate a potential role of Ras in human vascular biology. Received: 5 June 2001, Returned for revision: 28 June 2001, Revision received: 6 July 2001, Accepted: 31 July 2001     

A20 protects mice from lethal radical hepatectomy by promoting hepatocyte proliferation via a p21waf1-dependent mechanism

The liver has a remarkable regenerative capacity, allowing recovery following injury. Regeneration after injury is contingent on maintenance of healthy residual liver mass, otherwise fulminant hepatic failure (FHF) may arise. Understanding the protective mechanisms safeguarding hepatocytes and promoting their proliferation is critical for devising therapeutic strategies for FHF. We demonstrate that A20 is part of the physiological response of hepatocytes to injury. In particular, A20 is significantly upregulated in the liver following partial hepatectomy. A20 protects hepatocytes from apoptosis and ongoing inflammation by inhibiting NF-kappaB. Hepatic expression of A20 in BALB/c mice dramatically improves survival following extended and radical lethal hepatectomy. A20 expression in the liver limits hepatocellular damage hence maintains bilirubin clearance and the liver synthetic function. In addition, A20 confers a proliferative advantage to hepatocytes via decreased expression of the cyclin-dependent kinase inhibitor p21(waf1). In conclusion, A20 provides a proliferative advantage to hepatocytes. By combining anti-inflammatory, antiapoptotic and pro-proliferative functions, A20-based therapies could be beneficial in prevention and treatment of FHF.     

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.     

Oxygen sensing by primary cardiac fibroblasts: a key role of p21(Waf1/Cip1/Sdi1)

In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to approximately 1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the PO2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the PO2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-beta1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-beta1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.