Differential expression and role of p21<Superscript>cip/waf1</Superscript> and p27<Superscript>kip1</Superscript> in TNF-α-induced inhibition of proliferation in human glioma cells

Background  

The role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) – p21^cip/waf1 and p27^kip1 in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) as in vitro models.     

Paradoxical correlations of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1 in metastatic colorectal carcinoma.

The cyclin-dependent kinase inhibitors (CDIs) p27kip1 and p21waf1/cip1 are key cell cycle-negative regulatory enzymes. The objective of this study was to correlate expression of p27kip1 and p21waf1/cip1 with survival, chemotherapy responsiveness, and expression of the proliferation marker Ki-67 in patients with advanced colorectal cancer. Immunohistochemistry was performed with antibodies to p27kip1, p21waf1/cip1, and Ki-67 on samples from 66 patients with metastatic colorectal carcinoma. Interpretation was performed by visual inspection and automated image analysis. Patients who obtained a response to chemotherapy had greater p21waf1/cip1 tumor staining with a mean of 10.0 positive cells/high-powered field, compared with 4.5 positive cells/high-powered field for nonresponders (P = 0.03). A positive Spearman correlation was seen between Ki-67 and p27kip1 (r = 0.48; P = 0.0001), as well as between Ki-67 and p21waf1/cip1 (r = 0.48; P = 0.0001). A trend toward shorter survival was seen in patients with positive specimens (median survival of 10 months for patients with both p27kip1- and p21waf1/cip1-positive specimens, compared with 22 months for patients with neither p27kip1- nor p21waf1/cip1-positive specimens). In contrast to that previously reported in normal colonic mucosa or early-stage colorectal cancer, we observed positive correlations of Ki-67 with both p27kip1 and p21waf1/cip1, a trend toward greater CDI staining indicating worse prognosis, and greater p21waf1/cip1 staining in tumors that were chemosensitive. These findings suggest that in the metastatic setting, CDIs may show altered function, compared with their role in the normal cell cycle.     

Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.     

Expressions of cyclin D1 and p27kip1 in carcinogenesis of stomach mucosa

Objective  To evaluate the relationship between the expressions of cyclin D1 and p27^kip1 in the canceration course of the stomach. Methods  The immunohistochemical staining technique (SP method) was used to detect the expressions of cyclin D1, p27^kip1 in chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), intestinal metaplasia (IM), dysplasia (DYS), gastric carcinoma (GCA) biopsy specimens. Results  The positive cyclin D1 expression rates increased with the progressing from CAG→IM→DYS→GCA respectively, and those in IM, DYS and GCA were different from those in CSG, P < 0.05, while DYS group was indifferent from GCA group, P > 0. 05. The positive p27^kip1 expression rates decreased with the mucosa progressing from CAG→IM→DYS→GCA. There was a negative correlation between the expression cyclin D1 and p27^kip1 (Y = −0.53, P = 0.000). Conclusion  Expression rates of cyclin D1 in the canceration course of the stomach mucosa trend were increased and those of p27^kip1 were decreased; the abnormal expressions of them were found in the early term of the canceration course of the stomach mucosa, and the inverse expression suggests there may be a negative feedback regulatory loop between cyclin D1 and p27^kip1.     

CD15<Superscript>+</Superscript>/CD16<Superscript>low</Superscript> human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function

Myeloid-derived suppressor cells (MDSCs) are a subpopulation of myeloid cells with immunosuppressive function whose numbers are increased in conditions such as chronic infection, trauma, and cancer. Unlike murine MDSCs defined as CD11b⁺/Gr-1⁺, there are no specific markers for human MDSCs. The goal of this study was to delineate a specific human MDSCs subpopulation in granulocytes from terminal cancer patients and investigate its clinical implications. Here, we show that the CD15⁺/CD16^low subset was increased in terminal cancer patients compared with healthy donors (P = 0.009). Phorbol 12-myristate 13-acetate-activated granulocytes (CD16^low/CD66b⁺⁺/CD15⁺) that have a phenotype similar to MDSCs from cancer patients, effectively suppressed both proliferation and cytotoxicity of normal T cells. Among cancer patients, T-cell proliferation was highly suppressed by granulocytes isolated from terminal cancer patients with a high proportion of CD15⁺/CD16^low cells. Patients with low peripheral blood levels of CD15⁺/CD16^low cells had significantly longer survival than those with high levels (P = 0.0011). Patients with higher levels of CD15⁺/CD16^low also tended to have poor performance status (P = 0.05). These data suggest that CD15⁺/CD16^low granulocytes found in terminal cancer patients may play a role in the progression of cancer by inhibiting tumor immunity.     

Expression of cyclins A and D and p21(waf1/cip1) proteins in renal cell cancer and their relation to clinicopathological variables and patient survival.

We have studied 118 renal cell carcinomas to analyse the expressions of cyclins A and D1 and p21(waf1/cip1), and their relationship to clinical and histopathological parameters as well as to clinical outcome. Cyclins A and D1 and cyclin-dependent kinase inhibitor p21 (waf1/cip1) were not expressed in normal renal tissue. Staining signals of cyclin D1 and p21(waf1/cip1) were always nuclear but cyclin A was also expressed in the cytoplasm of the tumour cells. The mean (range) fractions of cyclin A, cyclin D1 and p21(waf1/cip1)-positive tumour cells were 2.2% (range 0-20%), 23.3% (range 0-90%) and 6.8% (range 0-70%) respectively. The expression of cyclin A was related to venous invasion, high nuclear grade, high mitotic rate, high Ki-67 and high PCNA expressions (P < or = 0.006 for all). The expression of cyclin D1 was linked with age over 65 years, low nuclear grade and high p53 expression (P < or = 0.05 for all). An inverse correlation was present between p21(waf1/cip1) and cyclin D1 (P = 0.011). Cyclin A predicted survival in the entire study group (P = 0.0014), in T1-4/N0-2/M0 (P = 0.0007) and in T1-2/N0/M0 tumours (P = 0.0007). Cyclin A was also a powerful predictor of disease-free survival in T1-4/N0/M0 (P = 0.0027) tumours (P = 0.0007). Cyclin D1 and p21(waf1/cip1) were not significantly related to survival or disease-free survival in any of the groups. In the entire material the independent prognostic factors were the presence of distant metastases (relative risk (RR) 5.16, P < 0.001), T category (RR 2.68, P < 0.001), Ki-67 expression (RR 1.02, P = 0.026) and cyclin A expression (RR 1.12, P = 0.001). The independent predictors in T1-4/N0/M0 tumours were T-category (RR 2.67, P = 0.001) and cyclin A (RR 1.21, P < 0.001), and in T1-2/N0/M0 tumours the only significant predictor was cyclin A (RR 1.19, P = 0.0002). In renal cell carcinoma, cyclin A is a powerful and independent prognostic factor in all clinical stages of the disease, whereas cyclin D1 and p21(waf1/cip1) have no prognostic value.     

Research on cell cycle retardation,apoptosis and the expression of antioncogene p57<Superscript>Kip2</Superscript> by radioactive rays in nasopharyngeal carcinoma cell line in vitro

Objective To study cell cycle retardation, apoptosis and the expression of antioncogene p57^kip2 by radioactive rays in nasopharyngeal carcinoma cells. Methods Cell cycle retardation, apoptosis and cell survival rate induced by radioactive rays were tested by the methods of flow cytometry and MTT method. The expression of antioncogene p57^kip2 was detected by immunohistochemistry and Western blot. Results After irradiation, G1 phase had no obvious retardation, S phase showed transient delay. There was a positive correlation between irradiation dosage and retardation strength in G2/M phase (P < 0.01). Peak value appeared at 24 h after 12 Gy irradiation, then decreased. There was a positive correlation between apoptosis incidence and irradiation dosage or after-irradiation time extention (P < 0.01). There was a negative correlation between cell survival rate and irradiation dosage or apoptosis incidence (P < 0.01). The expression of p57^kip2 protein was up-regulated along with the prolongation of time and dosage after irradiation (P < 0.01). Conclusion G2/M phase arrest, apoptosis and the up-regulation of the expression of p57^kip2 protein all can reflect predict the radiosensitivity of nasopharyngeal carcinoma cells.     

p21WAF1/CIPI基因DNA序列分析及其与骨肉瘤表型的关系

To investigate the p21^WAF1/CIP1 gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. p21^WAF1/CIP1 gene DNA of 36 osteosarcoma specimens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. The PCR products were sequenced directly. In p21^WAF1/CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of CT in the p21^WAF1/CIP1 gene CDNA sequence of position 609^th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of CT in the p21^WAF1/CIP1 gene CDNA sequence of position 609^th occurred in 8 cases with the incidence being 80%. Being the novel location of p21^WAF1/CIP1 gene polymorphism of osteosarcoma, but not mutation was defined, and this location maybe might provide the meaningful reference for the further research of p21^WAF1/CIP1 gene.     

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21waf 1/cip 1

The LEC rat is an inbred mutant strain which spontaneously developsliver injury and subsequent liver cancer. Liver injury in LECrats has recently been shown to be closely related to abnormalcopper accumulation in the liver. Previously, we reported thatLEC rat hepatocytes lose their growth potential, probably allowingselective growth of preneoplastic cells. In this study, to elucidatethe effects of copper accumulation on the growth activity ofLEC rat hepatocytes, we examined the growth activity and theexpression of p53 and p21^{waf 1/cip 1} in the livers of LEC ratsfed on either a control or a low-copper diet. Potential forcell proliferation of hepatocytes obtained from normal dietfed LEC rats was almost comparable to that of the cells fromage-matched Sprague-Dawley (SD) rats. Northern blot analysisshowed that the expression of p53 and p21^{waf 1/cip 1} was significantlyhigh in the livers of LEC rats fed a control diet, while theexpression of p53 and p21^{waf 1/cip 1} in the LEC rats fed a low-copperdiet was as low as that of SD rat livers. Western blot analysisconsistently showed that the amount of p21^{waf 1/cip 1} boundto the nuclear matrix scaffold of the LEC rat liver was reducedby feeding a low-copper diet. These findings suggest that abnormalaccumulation of copper induced the expression of p53 and p21^{waf1/cip 1}, resulting in the inhibition of cell proliferation ofLEC rat hepatocytes.     

槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性抑制口腔鳞状细胞癌细胞增殖

目的:探究槲皮素通过调控p21/cip1和p27/kip1蛋白的稳定性对口腔鳞状细胞癌细胞增殖的影响.方法:CCK-8试剂盒检测口腔鳞状细胞癌细胞(SCC-15)增殖情况;流式细胞术检测SCC-15细胞周期分布;Western blot检测CyclinD1/CDK复合体、p21/cip1、p27/kip1、p-GSK3...     

p21cip1和p27kip1基因遗传多态与食管癌及贲门癌发病风险的关联

背景与目的：有研究表明p21^cip1和p27^kip1的基因多态性与乳腺癌、肺癌、前列腺癌等肿瘤易感性有关。本研究分析中国北方高发区人群食管鳞状细胞癌（ESCC）和贲门腺癌（GCA）与勺p21^cip1和p27^kip1基因多态性之间的关系。方法：应用聚合酶链反应-限制性片段长度多态性（PCR—RFLP）方法检测299例ESCC患者、256例GCA患者及437名健康对照人群p21^cip1 3’非翻译区和p27^kip1第109位密码子基因多态性分布情况。结果：ESCC患者组p21^cip1 T等位基因型频率（42．8％）显著高于健康对照组（36．7％）（P=0．02）,ESCC和GCA患者组p27^kip1等位基因型频率（分别为96．8％和96．1％）均显著高于健康对照组（92．9％）（P值分别为0．00和0．02）。ESCC患者组p21^cip1基因型频率分布与健康对照组相比有显著性差异（P=0．04）,与C／C和C／T基因型相比,T／T基因型可显著增加ESCC的发病风险（校正OR=1．93,95％CI=1．12～3．94）ESCC和GCA患者细p27^kip1基因型频率分布与健康对照组相比均有显著性差异（P分别为0．00和0．01）,与V／G和G／G基因型相比,V／V基因型可显著增加ESCC和GCA的发病风险（校正OR分别为2．44和2．01,95％CI分别为1．2l～4．02和1.12～3．68）。当按吸烟和上消化道肿瘤家族史状况进行分层分析时发现,与V／G和G／G基因型相比,V／V基因型可显著增加吸烟人群患ESCC和GCA（校正OR分别为2．24和2．61,95％C1分别为1.14～4．03和1．25～3．82）以及有家族史人群患ESCC的发病风险（校正OR=2．04,95%CI=1．04～3．43）．两基因联合分析显示,携带p21^cip1T／T和p27^kip1V／V基因型可显著增加患食管癌和贲门癌的发病风险（校正OR分别为3．78和2．56,95?分别为1．46～5．89和1．06～4．78）。结论：在中国北方人群中,p21^cip1基因多态性可能与食管癌的易感性有关,p27^kip1基因多态性可能与食管癌和贲门癌的易感性有关．而且这两个基因的多念性可能存食管癌和贲门癌发病中起联合作用．     

Cyclooxygenase-2 promotes human cholangiocarcinoma growth: evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest

The expression of cyclooxygenase-2 (COX-2) is increased in human cholangiocarcinoma. However, the biologic function and molecular mechanisms of COX-2 in the control of cholangiocarcinoma cell growth have not been well established. This study was designed to examine the direct effect of COX-2 and its inhibitor celecoxib on the growth of human intrahepatic cholangiocarcinoma cells. Overexpression of COX-2 or treatment with prostaglandin E(2) (PGE(2)) enhanced human cholangiocarcinoma cell growth, whereas antisense depletion of COX-2 in these cells decreased PGE(2) production and inhibited growth. These findings demonstrate a direct role of COX-2-mediated PGE(2) in the growth regulation of human cholangiocarcinoma cells. Furthermore, the COX-2 inhibitor celecoxib induced a dose-dependent inhibition of cell growth, cell cycle arrest at the G(1)-S checkpoint, and induction of cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). However, the high concentration of celecoxib (50 micro M) required for inhibition of growth, the incomplete protection of celecoxib-induced inhibition of cell growth by PGE(2) or COX-2 overexpression, and the fact that overexpression or antisense depletion of COX-2 failed to alter the level of p21(waf1/cip1) and p27(kip1) indicate the existence of a COX-2-independent mechanism in celecoxib-induced inhibition of cholangiocarcinoma cell growth.     

p27<Superscript>Kip1</Superscript>deficiency promotes prostate carcinogenesis but does not affect the efficacy of retinoids in suppressing the neoplastic process

Background  

p27 is a cell cycle suppressor gene, whose protein is a negative regulator of cyclin/cdk complexes. p27 is also a potential target of retinoids in cancer prevention studies. In benign prostate hyperplasia (BPH), and in most carcinomas, p27^Kip1 is down-regulated, suggesting its potential resistance to retinoids. To test this hypothesis, we examined the efficacy of 9-cis retinoic acid (9cRA) to suppress prostate cell proliferation (PECP) and carcinogenesis in p27^Kip1 deficient mice.     

Effect of <Emphasis Type="Italic">cis</Emphasis>-9, <Emphasis Type="Italic">trans</Emphasis>-11-conjugated linoleic acid on cell cycle of mammary adenocarcinoma cells(MCF-7)

Objective: To determine the effect of cis-9, trans-1 1-conjugated linoleic acid on the cell cycle of mammary cancer cells (MCF-7) and the possible mechanism of the inhibitory effect of c9,t11-CLA. Methods: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B₁, D₁, p16^ink4a and p21^cip/waf1 of MCF-7 cells at various c9,t11-CLA concentrations (25μM, 50μM, 100μM and 200μM), at 24h and 48h. 96% ethand was used as negative control. Results: The cell growth and DNA synthesis of MCF-7 cells were inhibited by c9,t11-CLA. After treatment with various doses of c9,t11-CLA mentioned above for 8 days, the inhibition frequency was 27.18%, 35.43%, 91.05%, and 92.86%, respectively. Inhibitory effect of c9,t11-CLA on DNA synthesis (except for 25μM, 24h) was demonstrated by significantly less incorporation of ³H-TdR than the negative control (P<0.05 and P<0.01). To further investigate the influence of the cell cycle progression, we found that c9,t11-CLA may arrest the cell cycle of MCF-7 cells. Immunocytochemical staining demonstrated that incubation with different concentration of c9,t11-CLA at various times significantly decreased the expression of PCNA, Cyclin A, B₁, D₁ in MCF-7 cells compared to the negative control (P<0.01), whereas the expression of p16^ink4a and p21^cip/waf1, cyclin-dependent kinases inhibitors (CDKI), were increased. Conclusions: The cell growth and proliferation of MCF-7 cells is inhibited by c9,t11-CLA via blocking cell cycle, accompanying reduced expression of cyclin A, B₁, D₁ and enhanced expression of CDKI (p16^ink4a and p21^cip/waf1). Foundation item: This work was supported by the National Natural Science Foundation of China(No.39870661). Biography: Liu Jia-ren(1968–), PhD, assistant professor, Public Health College, Harbin Medical University, majors in health toxicology.     

Prognostic value of CD57<Superscript>+</Superscript> T lymphocytes in the peripheral blood of patients with advanced gastric cancer

Background  Natural killer (NK)-like T cells comprising CD56⁺ T cells and CD57⁺ T cells belong to a subset of CD1d-independent NKT cells playing an important role in regulating immune responses. Although NK-like T cells are reported to increase in patients with advanced gastric carcinomas, it remains unknown how NK-like T cells are involved in disease progression in gastric cancer patients. Methods  The proportions of Th1 cells (interferon [IFN]-γ-producing CD4⁺ T cells), Th2 cells (IL-4-producing CD4⁺ T cells), and NK-like T cells (CD56⁺ T cells and CD57⁺ T cells) in the peripheral blood of 48 gastric cancer patients and 20 healthy controls were measured by two-color flow cytometry analysis and by intracellular cytokine analysis to investigate an association of these immune cells with the survival rate of gastric cancer patients. Results  Univariate analysis showed that Th1 cells and CD57⁺ T cells, as well as some clinicopathological factors, significantly influenced the survival rate. CD57-high (≧18%) patients survived for a significantly shorter period after surgery compared to CD57-low patients (P = 0.046; Kaplan-Meier, log-rank test) in the stage III–IV patients, but not in the stage I–II patients. Further, multivariate analysis showed that lymphatic invasion was a statistically significant independent risk factor in all the gastric cancer patients, but the proportion of CD57⁺ T cells as well as depth of tumor were statistically significant independent risk factors in patients with advanced carcinomas (stage III–IV). Conclusion  An increased proportion (≧18%) of CD57⁺ T cells in the peripheral blood of patients with advanced gastric carcinomas could indicate a poor prognosis.     

Terbinafine inhibits oral squamous cell carcinoma growth through anti-cancer cell proliferation and anti-angiogenesis

Terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, has been reported to exert an anti‐tumor effect in various cancer cells. However, the effect of TB on oral cancer has not been evaluated. Herein we demonstrate that TB (0–60 µM) concentration‐dependently decreased cell number in cultured human oral squamous cell carcinoma (OSCC), KB cells. The anti‐proliferation effect of TB was also observed in two other OSCC cell lines, SAS and SCC 15. TB (60 µM) was not cytotoxic and its inhibition on KB cell growth was reversible. [³H]thymidine incorporation and flow cytometric analyses revealed that TB‐inhibited DNA synthesis and induced the G0/G1 cell‐cycle arrest. The TB‐induced cell‐cycle arrest occurred when the cyclin‐dependent kinase 2 activity was inhibited just as the protein levels of p21^cip1 and p27^kip1 were increased. The TB‐induced G0/G1 cell‐cycle arrest was completely blocked when the expressions of p21^cip1 and p27^kip1 were knocked‐down together. Taken together, these results suggest that the p21^cip1‐ and p27^kip1‐associated signaling pathways might be involved in the TB‐induced anti‐proliferation in KB cells. In vivo, TB (50 mg/kg, i.p.) significantly inhibited the KB tumor size. In these TB‐treated tumors, increases in the levels of p21^cip1 and p27^kip1 protein and decreases in the number of proliferating cell nuclear antigen‐positive cells and the microvessel density were observed. These findings demonstrate for the first time that TB might have potential to serve as a therapeutic tool in the treatment of oral cancer. Mol. Carcinog. © 2011 Wiley Periodicals, Inc.