| BACKGROUDPro-oncogene can promote cell growth and proliferation in normal cells, It codes for gene sequences involved in cell signal transduction, gene expression and regulation. Oncogene is mutated form of proto-oncogenes, which may lead to malignant transformation of cells. Ras is an oncogene, including K-ras, H-ras, N-ras, the main mutant forms of which are main point mutations and gene amplification. The main point mutation occurs in codons 12,13, and 61, among which the codon 12 is most common. Oncogene Ras activation alone induces senescence and inhibits proliferation. It needs some tumor suppressor gene inactivation or other conditions to transform cells. It is reported that K-Ras activation and tumor suppressor gene p16 inactivation can be found frequently in human pancreatic epithelial, and actived K-Ras and p16 inactivation can bypass sensescence. We also found p16 inativation increases tumorigenicity in mice. Many literatures have reported that oncogene Ras induced senescence, during which it can also alter the autophagic activity of cells.Autophagy, a lysosome-independent degradation pathway, regulates cells fate by selectively degrading long-lives proteins and damaged organelles, in such maintais cellular homeostasis and genenomic integrity. Appropriate autophagy can remove unfolded proteins or damaged organelles, which is critical for cell survival. Autophagy has opposing roles in cancer inititation and in established tumors. On the one hand autophagy can degrade unfolded and aggregation-prone proteins, as well as damaged organelles to maintain cellular homeostasis, which may be important for tumor suppression. On the other hand, autophagy may provide metabolic substates during periods of nutrient deprivation and preserve organelle function required for cell growth. It seems that autophagy plays a different role at different stages of tumor progression.OBJECTIVEThe activation of Ras genes alone in primary normal cells does not promote oncogenic transformation, but rather induces growth arrest known as premature senescence or oncogene-induced senescence (OIS). Other than OIS, there are also other cellular processes involved in preventing oncogenic transformation. Up-regulated autophagy has been demonstrated both in untransformed and transformed cells after overexpression/activation of oncogenic Ras, which subsequently lead to negative control of cell growth and tumor suppression. However, how can cancer progenitor cell overcome these defensive mechanisms to undergo transformation? Recently, opposing results also appeared in the literature, in which Ras overexpression may inhibit autophagy, or inhibition of Ras may induce autophagy, in untransformed mouse cell lines.Methods1. SA-p-gal staining to detect cell senescenceRemove the cell culture medium, wash cells once with PBS, then add 1 ml β-gal dye fixative solution (0.5% glutaraldehyde in PBS) and fixed at room temperature for 15 min. Remove fixative solution and wash cells with 2 mM MgCl2 three times. Cells were incubated at in 37℃for 2-4 h or up to 12-16 h. with fresh senescence-associated P-Gal stain solution 100 cells were counted in three separate fields and the results are expressed as percentage of SA-β-gal-positive cells.2. Growth curve detect cell senescenceCells infected with Ras or control viruses were selected with proper drugs for 4 days. Cells were culture in chloroquine or rapamycin and analyzed for senescence. The day when drug selection was completed (6 days after infection) was defined as day 0. For growth curves, cells were plated in 12-well plates at 104/well in triplicates. every 4 days, cells were harvested and counted. At each split,104 cells were reseeded to fresh plates, and allowed to grow until next split. Population doublings (PD) were calculated with the formula PD=log(N2/N1)/log2, where N1 is the number of cells seeded and N2 is that of recovered Cells with senescence-associated β-galactosidase activity were detected as previously described. At least 200 cells were counted in randomly chosen fields for each sample.3. Western blot detect proteinCells were lysed in immunoprecipitation assay buffer. Samples of protein were subjected to SDS-PAGE gel and then transferred to nitrocellulose membranes. The primary antibodies used for p62, LC3, p16, Phospho-p53 (Ser15), Reactive proteins were visualized using enhanced chemiluminescence.4. Transduced siRNAUse HiPerFect transfection reagent to transduce Atg7, Atg5, Beclinl siRNA into cells. Cells were cultured for 48 h. Western blot was used to determine the expression levels of p16, LC3, p62.5. Apoptotic assays.Cells infected with Ras or control viruses were selected with proper drugs for 4 days. Cells were culture in chloroquine or rapamycin. Cells were collected by trypsinization, washed with cold PBS twice and resuspended 300 1×Binding buffer containing 5μl AnnexinVand 5μl PI for 30 min at room temperature. After incubated, using the FACScan flow cytometer to quantify the apoptotic rate.6. Hoechst 33342 and propidium iodide stainingCells were seeded in a six-well plate, and after 48 hours were stained with 1 μg/mL Hoechst 33342 and 1μg/mL propidium iodide for 10-15 mins. Percentage of apoptotic cells, identified by condensed, bright blue chromatins or pink chromatins, was determined by counting>200 cells under an UV microscope in triplicates.RESULTS1. Oncogene Ras-induced premature senescenceCells expressed high level of Ras displayed significant increase in SA-β-GAL activity. Cells growth curve also shows growth arrest. We detect cells protein, and found p16 accumulated in H-Ras cells. The amount of p53 protein phosphorylated on Ser 15 (p-p53ser15) also increased as part of the senescence response. In contrast, BJ cells transfected with control vector continued to divide with low p16 and pp53.2. Oncogene Ras inhibites cell autophagyTo determine whether autophagic activity is enhanced or inhibited in H-Ras cells, we evaluated the amount of autophagosome-associated lipidated form of LC3 (LC3II) and the level of p62 by Western blot. Western blot analysis displayed marked accumulations of LC3II and p62 in H-Ras cells as compared with those in the control cells, indicating that the autophagy is actually inhibited in H-Ras cells. After treatment with rapamycin, protein levels of LC3II and p62 in H-Ras cells were significantly decreased.3. Chloroquine inhibites autophagy and promotes senescence in Ras-overexpressing cellsWhen chloroquine (CQ) was added to the culture media, The cells displayed increased number of SA-P-GAL-positive cells and more depressed growth curve, Western blot analysis displayed significant and continuous increase in the levels of p16 and p-p53serl5. The cells transfected with control vector only showed some decrease in the levels of p16 and p-p53ser15, and did not show apparent change in terms of SA-β-GAL activity and PD.4. Rapamycin can induce autophagy and attenuate senescence in Ras overexpressing cellsLess SA-β-GAL activity mild increase in PD, and apparent decrease in the levels of p16 and p-p53ser15 were demonstrated in Ras-overexpressing cell. On the contrary, cell transfected with control vector only show some decrease in the levels of p16 and p-p53serl5, and did not show apparent change in terms of SA-β-GAL activity and PD.5. Knock down of ATG7, ATG5 can promote senescence in Ras-overexpressing cellsUsing siRNA to knock down ATG7, ATG5 and BECN1, western blot showed significant p62 accumulated, together with marked increase of p16 and pp53, indicating inhibition of autophagy and increase of senescence. In control cells, this parameters didn’t change.6. Chloroquine and rapamycin can alter the apoptosis rate in Ras-overexpressing cellsResults from cell cycle indicated that after chloroquine treatment, the proportion of sub-G1 population rapidly increased in Ras-overexpressing cells. In contrast, the proportion of sub-Gl population first increased, but then rapidly decreased to a level comparable to Ras-overexpressing cells without rapamycin treatment. Hochest and PI staining revealed that PI-positive population in Ras-overexpressing cells significantly increased after chloroquine treatment, whereas decreased after rapamycin treatment. These phenomenon were not seen in control cells.Conclusion1. H-Ras oncogene overexpression induce senescence in human fibroblasts;2. H-Ras oncogene overexpression inhibits autophagy in human fibroblasts;3. Down-regulating the autophagy activity using chloroquine or knock-down of the key genes of autophagy can promote senescence in hunman fibroblast overexpressing Ras oncogene.4. Rapamycin can induce autophagy and attenuate senescence in human fibroblast overexpressing Ras oncogene.The above results suggest that inhibition of autophagy to a just right point might be the mechanism by which Ras overexpression may induce cancer formation through the bottleneck effect. |
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