Retinoblastoma protein in human renal cell carcinoma in relation to alterations in G1/S regulatory proteins

The retinoblastoma gene product (pRb) is the main substrate for cyclin-dependent kinases (CDKs) during the G1/S transition. Aberrations in cell cycle regulatory proteins, which have been observed in many malignancies, can theoretically cause increased phosphorylation of pRb due to unbalanced CDK activities. The expression and phosphorylation of pRb and potential associations to cell cycle aberrations in renal cell carcinomas (RCC) has only partly been clarified. We therefore evaluated the presence of pRb and the level of pRb-phosphorylation in 216 RCCs arranged in tissue microarrays by using different pRb-antibodies, including pRb-phosphospecific antibodies. Most RCCs (95%) expressed pRb, while cases with the low pRb levels, potentially indicative for pRb-inactivation, were few. In order to detect secondary alterations to a potential pRb-inactivation, the p16 expression was also monitored. None of the tumors exhibited increased p16 levels, confirming that pRb-inactivation is rare in RCC. Phosphorylated pRb was detected in approximately 50% of the RCCs, using Western blotting or immunohistochemistry. The immunohistochemical ppRb(ser807/811) levels were associated with high proliferation, cyclin D1, cyclin E and p27 protein content. Surprisingly, there was no association between pRb-phosphorylation and clinicopathological data. In summary, pRb seemed to be functional and aberrations in G1/S-regulatory proteins were associated with increased phosphorylation of pRb and proliferation. The data supports that pRb might be one of the main cell cycle regulators in RCC.     

Differential expression of cell cycle regulatory molecules and evidence for a "cyclin switch" during progression of prostate cancer

BACKGROUND: Deregulation of the cell cycle can be viewed as both cause and consequence of cancer. Cyclin expression regulates progression through the cell cycle and although some cyclins have been examined in prostate cancer, the spatial and temporal changes in expression of these molecules during progression of autochthonous disease has not been fully explored. METHODS: Expression patterns of cyclins and cyclin dependent kinases during the different stages of progression in the spontaneous autochthonous TRAMP model were examined by RNAse protection assay, Western blot analysis, and immunohistochemistry. RESULTS: Differential expression of cell cycle regulatory molecules was observed during prostate cancer progression. Levels of the D-type cyclins decreased during progression while expression of cyclin E increased both at the mRNA and protein levels. The level of cyclin A and cyclin B expression increased beginning in early stage tumors and continued to increase throughout progression. The levels of cyclin dependent kinases did not change substantially during progression of the TRAMP model. CONCLUSIONS: The spatial and temporal pattern of mitotic cyclin expression during prostate cancer progression suggests that these molecules represent potential therapeutic targets. The differential expression of D-type cyclins may have implications with respect to androgen receptor mediated gene expression.     

Peplomycin induces G1-phase specific apoptosis in liver carcinoma cell line Bel-7402 involving G2-phase arrest

AIM: To investigate the mechanism of peplomycin (PEP)-induced apoptosis in liver carcinoma cell line (Bel-7402).METHODS: Growth inhibition by PEP was analyzed using 3- 4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide (MTT) assay. Apoptotic cells were detected using Hoechest 33258 staining, and confirmed by flowcytometric analysis and DNA fragmentation analysis. The expression of cyclin A and B 1 were determined by flowcytometry and Western blot. Annexin V assay was measured by flow cytometric analysis. RESULTS: PEPinduced apoptosis and then inhibited cell proliferation in liver carcinoma cell line Bel-7402. Cells treated with PEP50 μmol/L for 15 h were arrested in G2-phase with dramatical expression of cyclin A and a little change in cyclin B 1.Almost all the apoptosis occurred in cells undergoing the G1-phase after treatment for 24 h. CONCLUSION:Peplomycin induced G1-phase specific apoptosis in Bel-7402 involving G2-phase arrest.     

Piroxicam selectively inhibits the growth of premalignant and malignant human oral cell lines by limiting their progression through the S phase and reducing the levels of cyclins and AP-1

Studies have shown that nonsteroidal antiinflammatory drugs (NSAIDs) reduce the risk of and mortality from a variety of cancers. Although cyclooxygenase (COX)-dependent and -independent pathways may be involved, the mechanisms responsible for these effects remain unknown. In our study, we found that piroxicam inhibited cell growth in premalignant and malignant, but not normal, human oral epithelial cell lines in a concentration- and time-dependent manner. After 6 days of exposure, the concentration that inhibited growth by 50% was 181 and 211 microM for premalignant and malignant cells, respectively. Piroxicam did not induce apoptosis. The growth inhibitory effect was COX and PGE2 independent. Adding PGE2 or infecting cells with a COX-1 transgene did not abrogate piroxicam-induced growth inhibition. After treatment of the premalignant and malignant cell lines with piroxicam, cells accumulated in the S phase of the cell cycle. Upon removal of piroxicam, cells entered the G2 phase. The S phase block was accompanied by a reduction in the protein levels of cyclin A, cyclin B1, cyclin D1, cdc2, PCNA and the c-jun AP-1 component. Therefore, piroxicam may exert its growth inhibitory effects selectively on the premalignant and malignant human oral epithelial cells lines via signaling pathways regulating the progression of cells through the S phase of the cell cycle.     

Diversity in expression and prognostic significance of G1/S cyclins in human primary lung carcinomas

Expression of cyclin A, cyclin E and cdk2 was examined immunohistochemically in 144 cases of primary non-small cell lung carcinoma to evaluate their prognostic value. Cyclin A was co-expressed with cdk2 in the proliferating cells, ie those showing positive Ki-67 staining. The labelling index (LI) of cyclin A revealed a positive correlation with the S-phase fraction and an inverse correlation with histological differentiation. Furthermore, high cyclin A LIs indicated a poor prognosis in all histological types. Cyclin E exhibited a characteristic staining pattern: in squamous cell carcinoma (SCC), differentiated cells without Ki-67 staining revealed cyclin E positivity with expression of cdk2. Conversely, in adenocarcinoma (AC), proliferating cells revealed cyclin E positivity. Cases of large cell carcinoma showed heterogeneous cyclin E staining patterns, unlike those of SCC or AC. Statistical analyses also revealed a marked contrast between SCC and AC. In AC, the LI of cyclin E was inversely correlated with histological differentiation and a high LI predicted a worse prognosis. In contrast, in SCC, the LI of cyclin E correlated positively with histological differentiation and better prognosis. However, the expression levels of cyclin E mRNA evaluated by quantitative RT-PCR were higher in poorly differentiated SCC and AC, suggesting that protein turnover plays a large role in determining cyclin E protein levels. Although the expression of cyclins was demonstrated to be diversely regulated depending on the histological type, the combined immunohistochemical analyses performed in this study on these proteins could be useful tools for evaluating patient prognosis in lung carcinomas.     

Cyclins D1 and D3 and topoisomerase II alpha in inactive pituitary adenomas

The oncogenes cyclin D1 and D3 are overexpressed in many tumors. Topoisomerase IIα is found in proliferating cells. The immunohistological expression of cyclin D1, cyclin D3, and Topoisomerase IIα was studied in a collection of 60 clinically inactive surgically removed pituitary adenomas of the follicle-stimulating hormone/luteinizing hormone (FSH/LH) cell complex (20 null cell adenomas, 20 oncocytomas, and 20 FSH/LH cell adenomas) for correlation with other proliferation markers (Ki-67, PCNA) and with clinical data. Whereas cyclin D1 was positive only in one invasive null cell adenoma (1.7%) with some p53-positive nuclei, cyclin D3 was overexpressed in the nuclei of 41 tumors (68%). Topoisomerase IIα was demonstrated in the nuclei of 42 adenomas (70%) with no significant differences discernible between the three adenoma subtypes. There was no significant correlation to the time of development of tumor symptoms, but a correlation of Topoisomerase IIα with cyclin D3 and the proliferation marker Ki-67 (Mib1). From these data we conclude that cyclin D3 and Toposomerase IIα appear to be additional markers for proliferation which can be used for prognosis index in surgical pathology of the pituitary.     

cyclin D1和CDK4在口腔正常上皮、炎症及鳞癌中表达的定量分析

目的 通过检测口腔粘膜的正常上皮、非特异性炎症上皮及鳞癌中cyclin D1与CDK4的表达,探讨其在上皮组织不同状态中的变化及意义。方法 用临床病理标本,设立对照,选择cyclin D1和CDK4抗体免疫组化染色。结果 cyclin D1和CDK4在正常口腔上皮与口腔鳞癌上皮、非特异性炎症与口腔鳞癌上皮中的表达间有统计学差别（P＜0．05）,正常组与炎症组间无差别,cyclin D1及CDK4在口腔鳞癌上皮中过表达（P＜0．05）。结论 cyclin D1与CDK4过表达的机制可能由于基因扩增或其他因素使其蓄积,口腔鳞癌组分级间的差异性提示其可作为肿瘤分级的评价指标之一。     

舌鳞癌组织中cyclin A和cyclin D1的基因转录

目的 :探讨不同级别舌鳞癌组织中细胞周期相关蛋白cyclinA和cyclinD1基因转录情况。方法 :DIG标记及检测试剂盒进行组织原位杂交。结果 :舌鳞状细胞癌组织Ⅰ、Ⅱ、Ⅲ级中cyclinA基因转录阳性率分别为 47 6%、76 9%和 87 5 % ;cyclinD1基因转录阳性率分别为 62 0 %、 69 2 %和75 0 %。在有淋巴结转移的情况下 ,cyclinAmRNA和cyclinD1mRNA的表达较高。cy clinA和cyclinD1表达与舌鳞癌的恶性程度及淋巴结的转移均呈正相关 ,P <0 0 5。结论 :cyclinA和cyclinD1表达与舌鳞癌分化程度及淋巴结转移有关 ,可能是舌鳞癌预后不良的指标     

Effect of Cell Cycle Inhibitor Olomoucine on Astroglial Proliferation and Scar Formation after Focal Cerebral Infarction in Rats

Background:Astrocytes become reactive following many types of CNS injuries.Excessive astrogliosis is detrimental and contributes to neuronal damage.We sought to determine whether inhibition of cell cycle could decrease the proliferation of astroglial cells and therefore reduce excessive gliosis and glial scar formation after focal ischemia.Methods:Cerebral infarction model was induced by photothrombosis method.Rats were examined using MRI,and lesion volumes were estimated on day 3 post-infarction.The expression of glial fibrillary acidic protein(GFAP) and proliferating cell nuclear antigen(PCNA) was observed by immunofluorescence staining.Protein levels for GFAP,PCNA,Cyclin A and Cyclin B1 were determined by Western blot analysis from the ischemic and sham animals sacrificed at 3,7,30 days after operation.Results:Cell cycle inhibitor olomoucine significantly suppressed GFAP and PCNA expression and reduced lesion volume after cerebral ischemia.In parallel studies,we found dense astroglial scar in boundary zone of vehicle-treated rats at 7 and 30 days.Olomoucine can markedly attenuate astroglial scar formation.Western blot analysis showed increased protein levels of GFAP,PCNA,Cyclin A and Cyclin B1 after ischemia,which was reduced by olomoucine treatment.Conclusion: Our results suggested that astroglial activation,proliferation and subsequently astroglial scar formation could be partially inhibited by regulation of cell cycle.Cell cycle modulation thereby provides a potential promising strategy to treat cerebral ischemia.     

Cell proliferation and cell cycle control: a mini review

Tumourigenesis is the result of cell cycle disorganisation, leading to an uncontrolled cellular proliferation. Specific cellular processes-mechanisms that control cell cycle progression and checkpoint traversation through the intermitotic phases are deregulated. Normally, these events are highly conserved due to the existence of conservatory mechanisms and molecules such as cell cycle genes and their products: cyclins, cyclin dependent kinases (Cdks), Cdk inhibitors (CKI) and extra cellular factors (i.e. growth factors). Revolutionary techniques using laser cytometry and commercial software are available to quantify and evaluate cell cycle processes and cellular growth. S-phase fraction measurements, including ploidy values, using histograms and estimation of indices such as the mitotic index and tumour-doubling time indices, provide adequate information to the clinician to evaluate tumour aggressiveness, prognosis and the strategies for radiotherapy and chemotherapy in experimental researches.