| ObjectiveLung cancer is a malignant tumor which high mortality and morbidity.Although tremendous amount of financial and material resources have been treated for the lung cancer control and treatment.November 2008 in the eight a "Lung Cancer Awareness Month",yearbook of the World Health Organization showed that in recent years,lung cancer in Europe and the United States is high accurence.The statistical data gathered from 50 countries and regions lung cancer is the munber one killer among the malignant tumor in 36 countries and regions.The epidemic data published by the Department of Human Health Board that lung cancer in " the leading cause of death in urban and rural residents in China in 2006 "with a morbidity of 61.4/100000.More worriedly,the accurenceof lung cancer is still climbing up,and it was predicted that by 2015,China will have the largest population of lung cancer patient in the world.Lung cancer can be pathologically divided into squamous cell carcinoma, adenocarcinoma,large cell lung cancer and small cell lung cancer.Small cell lung cancer accounts for 12-15%of all type of lung cacer,small cell lung cancer is a highly malignant tumor of growing rapidly and spreading to the whole body early,and small cell lung cancer is the most difficult to be treated.Nowadays,people become more concerned about their health,and have tried their best to conque cancer.With the social development and the technology advancement and human have made many achievement in many fundamental study,such as tumor cell differentiation,cell cycle and cancer,oncogenes,tumor suppressor genes and signal transduction,tumor angiogenesis and tumor metastasis.Uncontrolled cell proliferation is a characteristic of malignant tumors.From biological point of view malignant proliferation of tumor has two notable features:on one hand,tumor cells are immortal, that is apoptosis barriers;on the other hand,cells division is out-of-control,that is cell cycle regulation and control system network obstacles.These two are the hot spots in current cancer research.Very often,oncogene and oncoprotein participate in the cell apoptosis.bc1-2 (B-cell lymphoma-2) gene found in t(14;18) chromosomal translocation in B-cell lymphoma.Being different from other oncogenes,bcl-2's function is to inhibit cell apoptosis rather than promoting cell proliferation.Bcl-2 protein inhibits cell apoptosis by repressing the activity of Bax and Bak which are pro-apoptotic proteins.Due to the weaken of apoptosis should not clear the cells which are cell cycle disorder and mutant timely,the anti-apoptotic protein over-expression is closely related to the occurrence of tumor.Many studies show that Bcl-2 protein is overexpressed in many kinds of tumor cells.Abnormal regulation of cell cycle is not only an important prerequisite for tumorigenesis,but also the important factor of promoting the proliferation and metastasis of malignant tumor.Cell cycle has two important checking points:Gl/S and G2/M,and controlly these two points can regulate cell cycle.At these two points out-of-control,the cell which should stop proliferation or physiological apoptosis entry of the cell cycle continuously leading to malignant cells proliferation.Tumorigenesis is a complex process in which chromosome undergoes multiple demages and is characterized as a disorder cell cycle regulation,and unrestricted and autonomous proliferation and division.Abnormal cell cycle has a common feature of deviant expression of regulatory factors,such as cyclins,CDKs,as well as CKIs.CyclinE is the cell cycle regulation factor of G1 phase and mainly participates in cell cycle G1/S phase transition control.Its interaction with other regulatory factors cyclinD, retinoblastoma protein(Rb),transcription factor E2F,p53,Kip family,CDK2,4,6 will decide cell survival and apoptosis.CyclinE is expressed in late Gl and S phase and its expression reached to peak at G1/S phase.The premature and excessive expression of CyclinE will speed up the cell into the S phase,promoting cell proliferation.The CyclinE-CDK2 complex was considered to be the important factor to initiate DNA replication and enter into S phase,and its function is closely related to the expression level of cyclinE and CDK2 kinase activity.Shortened G1 phase can decrease the dependence of cells on these growth factors,benefiting the occurrence of tumor. Previous studiesshowed that CyclinE over-expression often occurred in majority of tumors,such as liver cancer,nasopharyngeal cancer,ovarian cancer,breast cancer and pancreatic cancer and is closely related with the occurrence of ovarian cancer,breast cancer,pancreatic cancer.CyclinE has been considered as biomarker for tumor diagnosis.In addition,CyclinE expression has also demonstated a close relationship with tumor angiogenesis and tumor metastasis.Studis show that CyclinE participates in the transference most likely through the following two mechanisms:The first one is the cyclinE-CDK2 complexes' target proteins involved in the loss of cell adhesion,which is charaterised as the cell migration which regulates mitosis increase;the second one is, that cyclinE gene over-expression reduced the stability of chromosomes.High level of expression of cyclinE protein affected the S phase process and could produce aneuploid cells.Studis on renal cell carcinoma,pancreatic cancer,B-cell lymphoma and other tumor showed that the expression of cyclinE protein in the cases which had occurred metastasis was significantly higher than the case which did not,which are consistent with the result of the tumor pathology and clinical classification.,it was also observed that the over-expression of CyclinE has a direct impact on the prognosis of patients.Currend studys on Bcl-2 protein are more focused on tumors other than small-cell lung cancer.Particularly,small-cell lung cancer lines are used as the model,the research about CyclinE protein focus on the digestive tract tumors and gynecological tumors,rarely involves in lung cancer.Therefore,in this study,we use large cell and small cell lung cancer cell line as our subjects and investigate the roles of Bcl-2 protein and CyclinE protein in the development of lung cancer as well as the relationship between their expression changes and biological behavior.Methods1.Culture MRC-5 cells in MEM culture medium of 10%calf serum,H460 and H446 cells in RPMI1640 culture medium with 10%calf serum,at saturated humidity,and at 37℃,in 5%CO2 incubator.2.Determine the expression of Bcl-2 protein in MRC-5,H460 and H446 with Western-blot.3.Use H460 cell line as a standard,estimated cell growth and proliferation with MTT assay,and draw growth curve;the dose of UV irradiation and the follow-up cell culture time.4.Estimated the expression of Bcl-2 protein in the UV irradiated MRC-5,H460 and H446 cells,respectively,with immunohistochemical.5.Use a double thymidine(TdR) to perform the cell synchronization treatment.6.Determine thymidine(TdR)-induced cell synchronization using flow cytometry.7.Assay the expresstion of CyclinE protein in MRC-5,H460 and H446 with Western-blot.Results1.Bcl-2 protein expressionThe expression of Bcl-2 protein in MRC-5,H460 and H446 cells is significant different.The expression of Bcl-2 protein in lung tumor cell lines H460 and H446 measured in terms of the gray-scale was increased by 6.10 and 4.60 fold in corparison with that of MRC-5 cell lines.2.Effect of the exposure time and dose of UV radiation(1) When Fixed culture time,UV radiation dose is chosen 20s,40s,60s and 80s. As the is irradiation dose increased,cell proliferation become decreased and 80s, exposure could cause more cells death.The UV irradiation will inhibit the cell proliferation in a dose-dependent manner,and over-dose could cause cell death. (2) When the UV exposure is fixed at 60s,a follow-up time of 10.5 hours demonstrated a maximal inhibition of cell proliferation.3.Bcl-2 protein in the UV-irradiated cells by immunohistochemistry after UV irradiationAfter UV irradiation,the expression level of Bcl-2 protein in MRC-5,H460 and H446 cell lines was significantly higher than the cells without UV irradiation.When elongating the incubation time after irradiation,the percentage of positive cells increased and the positive cells become darkened.Particularly,the cells in the post-10.5h cultivate,the cells who have lost their adhesion to the surrounding cells in dark brown color,suggesting that the expression of Bcl-2 protein is possiblely related with the incubation time which after irradiation.4.Cell synchronizationThymidine-induced cell,was examined with flow cytometry,showing that more than 80%of cells are arressed in Gl phase,10%in S phase,and only a tiny fraction of cells in G2 and M phase in each group,indicating that cells in each group have synchronized in the G1/S phase.5.Western blot testThe expression levels of CyclinE protein was significant different from MRC-5, H460 and H446 cells(P＜0.01).The Cyclin E protein in lung tumor cell line H460 and H446 arel0.59 and 5.72 fold,respectively higher than normal lung cells MRC-5.Conclusion1.Over-expression of Bcl-2 protein has a relationship with the development of lung cancer.2.The UV-irradiation tumor cells retains the normal capacity of the stress response, which may be related to the immortality of tumor cells at certain degrees.3.Over-expression of Cyclin E protein is closely related to the origination of lung cancer.4.CyclinE protein might associate with the metastasis of lung cancer.
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