The Expression Of Cyclin D1 And Cyclin E In Female Non-small Cell Lung Cancer

IntroductionNon - small cell lung cancer is one of the leading causes of cancer - related death in china for females. Despite major advances in cancer treatment in past two decades, the overall survival rate of patients with lung cancer has improved only minimally, and the 5 - year survival rate remains less than 15%. To improve the prognosis of lung cancer patients, attempts have been made to develop tests facilitate the early diagnosis and treatment. Recent progress in the study of the molecular biology of cancer has contributed to a better understanding of its molecular pathogenesis. Under such situations, it may be possible to identify patients with a good or poor prognosis using molecular biological alterations as clinical biomarkers, including alterations of cell cycle regulators.Abnormalities in cell cycle regulators and subsequent deregulation of the Cl/S transition may be one of the most important biological events in malignant cell transformation. The cell cycle is governed by cdks, the activity of which is regulated by the binding of positive effectors, the cyclins, and by negative regulators , the cdk inhibitors. The cdks integrate mitogenic and growth - inhibitory signals and coordinate cell cycle transitions . Progression through the Gl phase of the cell cycle is dependent upon the activity of Gl cyclins, which include the D - type cyclins and cyclin E. A few studies have reported that overexpression of cyclin D1 is implicated in the tumorigenesis of NSCLCs. However, there has been controversy and limited available data for the significance of cyclin Dl overexpression on the prognosis of NSCLCs patients. In addition, the clinico-pathological implications of cyclin E overexpression have been reported to varywith the type of tumor; cyclin E overexpression was initially established as a negative prognostic factor in carcinoma of the colon, ovary, and breast, but other studies have reported that cyclin E up - regulation is a positive prognostic factor in carcinoma of the stomach, bladder and skin。 The inconsistency may be due to the heterogeneity of the tumors examined and the diversity in the mechanism of cyclin E expression. Our present study was designed to evaluate expressions of cyclin D1 in relation to cyclin E in female non - small cell lung cancers (NSCLCs).Materials and methodsPatients and samplesThe study was performed using materials from primary female non - small lung cancers obtained from Iiaoning Tumor Hospital between 2003 and 2004. All the specimens (tumor tissue 66 cases, neighbor tissue 66 cases) were obtained from surgical resections from patients. All cases were classified in advance according to the last World Health Organization (WHO) histological classification. Histopatological classification and pTNM stages were in accordance with the standard of International Anti - cancer Alliance.AntibodiesAnti - cyclin E ( clone C - 19, dilution 1: 300 ) and Anti - cyclin Dl (clone A -12,dilution 1:200) , a Mo IgG( H + L) / AP( dilution 1:500) , a Rb IgG(H +L) / AP (dilution 1:500) and NBT/BCIP Reagent Kit were produced by Beijing Zhongshan Biotechnology in China。ApparatusSpectrophotometer and centrifugal machine and electrophoresis apparatus were used as major apparatusWestern blotThe tissue samples were homogenized in lyses buffer (50mM N - 2 -hydroxyethylpiperazine - N'-2 - ethanesulfonic acid ( HEPES;PH7.0) ,400mM sodium chloride(Nacl) ,0.1% Nonidet P -40,100mM NaF, 200μM Sodium or-thovanadate, 0. 5mM phenylmethylsulphonyl fluoride(PMSF) , and 10mg ml-1aprotinin, 5mg ml-1 leupeptin, 5mg ml-1Antipain) and lysates were centrifuged at 14,000 g for 15 min at 4℃. Protein concentration was measured by bicincho-ninic acid protein assay. Equal amounts of protein per sample (50ug/lane) were electrophoresed on a 12% SDS - polyacrlamide gel and were then electro-phoretically transferred from the gel to a nitrocellulose membrane. Blot were washed once with Tris - buffered saline saline (TBS )/Tween and blocked with (TBS)/0. 1%Tween containing 3% BSA for 2 hours at room temperature. The blocking solution was removed and replaced with fresh solution containing primary antibodies. After incubation overnight at 4℃, blots were washed by gently shaking three times (for 10 minute each time) in(TBS)/0.1l%Tween and then incubated for 2 hours in (PBS)/0.1%Tween containing Alkaline Phosphatase -linked secondary antibodies . After three washes in (TBS)/0.1%Tween, immu-noreactive proteins were visualized by incubation with NBT/BCIP reagent kit.Statistical analysisStatistical associations between cyclin D1 and cyclin E immunoreactivity and various clinicopathological factors were determined using the chi square test for categorical variables. A p value less than 0. 05 was considered statistically significant.ResultsThirty cases of female non - small lung cancer (30/66 45.5% ) showed the overexpression of cyclin D1, and thirty - nine cases (39/66 59.1% ) were positive for cyclin E. Expression levels of cyclin E and cyclin Dl were significantly higher in the tumor samples than in normal controls (p <0.05). Cyclin D1 expression levels were higher in poorly differentiated NSCLCs and in patients with tumor T2-4 ( p < 0.05 ). No significant relationships between cyclin E expression and tumor differentiation or tumor size were observed. High - level cyclin D1 and cyclin E expression were more frequently found in patients with lymph node metastasis (p <0.05) , but were not associated with different age or histological type or tumor stage or tumor position.DiscussionCyclin D1 and cyclin E are G1 cyclins that have been shown to be the key regulators of the G1 - S transition and could consequently be deregulated molecules in tumors. In the present study, we have characterized cyclin D1 and cyclin E expression by western blot in 66 resected female non - small lung cancer. To our knowledge, this is the first study to evaluate the expressions of cyclin D1 as well as cyclin E in female non - small lung cancer. Our data demonstrates that cyclin D1 expression was closely related to T factor. High - level cyclin D1 and cyclin E expression were more frequently found in patients with lymph node metastasis.The clinical significances of cyclin Dl expression were previously evaluated in NSCLCs, where a frequency of cyclin Dl expression between 11.7% and 58% was reported, in accordance with the present result (45.5% ). However, there was conflict about the role of overexpression of cyclin Dl in relation to pathological findings and prognosis. Betticher et al could not demonstrate the relationship between cyclin Dl overexpression and stage of disease, histology in a-greement with our present study. Keum et al demonstrated that cyclin Dl immu-noreactivity was well correlated with lymph node metastasis concurred with us. They suggested that cyclin D1 overexpression is involved in tumorigenesis of NSCLCs and could be a predictive molecular marker for poor prognosis in resectable NSCLC patients. Mario et al draw the same conclusions. However, Bram-billa et al indicated that the Kb pathway of G1 arrest is initially disrupted in the vast majority of NSCLCs (83 percent) , but could not confirm an unfavorable role for each individual event (pl6INK4A loss or cyclin D1 up -regulation) in prognosis. Therefore, divergent reports have been produced in the literature, further studies are necessary to evaluate the relationships between cyclin Dl and prognosis.Overexpression of cyclin E has been observed in several malignancies and is associated with high proliferation, aberrant expression of other cell cycle regulators and chromosomal instability in vitro. There are few reports about the asso-...