Biochemical and molecular properties of cisplatin-resistant A2780 cells grown in folinic acid

In this study, A2780 human ovarian carcinoma cells were grown in folinic acid in contrast to folic acid, and the molecular and biochemical properties of cisplatin-resistant A2780 cells were analyzed for changes in the dTMP synthase cycle. At concentrations of folinic acid that were optimal for cell growth (10(-8) M), the ED50 for cisplatin was 2.5 and 43 microM in the A2780S and A2780DDP cells, respectively. Resistance to cisplatin was associated with a 2-fold cross-resistance to 5-fluorodeoxyuridine and 5-fluorouracil as well as a 3-fold increase in both dTMP synthase activity and mRNA. The ED50 for methotrexate was similar in both A2780S and A2780DDP cells (1.2 microM). When both the A2780S and A2780DDP cells were grown in folinic acid, there was no significant difference in the level of dihydrofolate reductase activity. This data would suggest that cisplatin resistance is associated with changes in folate metabolism.     

MiR-130a and MiR-374a Function as Novel Regulators of Cisplatin Resistance in Human Ovarian Cancer A2780 Cells

Chemoresistance remains a major obstacle to effective treatment in patients with ovarian cancer, and recently increasing evidences suggest that miRNAs are involved in drug-resistance. In this study, we investigated the role of miRNAs in regulating cisplatin resistance in ovarian cancer cell line and analyzed their possible mechanisms. We profiled miRNAs differentially expressed in cisplatin-resistant human ovarian cancer cell line A2780/DDP compared with parental A2780 cells using microarray. Four abnormally expressed miRNAs were selected (miR-146a,-130a, -374a and miR-182) for further studies. Their expression were verified by qRT-PCR. MiRNA mimics or inhibitor were transfected into A2780 and A2780/DDP cells and then drug sensitivity was analyzed by MTS array. RT-PCR and Western blot were carried out to examine the alteration of MDR1, PTEN gene expression. A total of 32 miRNAs were found to be differentially expressed in A2780/DDP cells. Among them, miR-146a was down-regulated and miR-130a,-374a,-182 were upregulated in A2780/DDP cells, which was verified by RT-PCR. MiR-130a and miR-374a mimics decreased the sensitivity of A2780 cells to cisplatin, reversely, their inhibitors could resensitize A2780/DDP cells. Furthermore, overexpression of miR-130a could increase the MDR1 mRNA and P-gp levels in A2780 and A2780/DDP cells, whereas knockdown of miR-130a could inhibit MDR1 gene expression and upregulate the PTEN protein expression .In a conclusion, the deregulation of miR-374a and miR-130a may be involved in the development and regulation of cisplatin resistance in ovarian cancer cells. This role of miR-130a may be achieved by regulating the MDR1 and PTEN gene expression.     

Cellular proto-oncogene expression following exposure of mice to gamma rays.

Many studies have shown the importance of altered cellular proto-oncogene expression in contributing to changes in cell survival, cell transformation, and cell cycle progression. In these experiments we examined the effects of total-body exposure of BCF1 mice to gamma rays (3 Gy) in modulating expression of cellular oncogenes in both gut and liver tissues. We selected specific cellular oncogenes (c-fos, c-myc, c-src, and c-H-ras), based on their normal expression in liver and gut tissues from untreated mice. As early as 5 min following whole-body exposure of BCF1 mice to gamma rays we detected induction of mRNA specific for c-src and c-H-ras in both liver and gut tissues. Accumulation of c-fos-RNA was slightly decreased in gut but was unaffected in liver tissue from irradiated mice relative to untreated controls. Accumulation of c-myc mRNA was unaffected in all tissues examined. These experiments document that modulation of cellular proto-oncogene expression can occur as an early event in tissues following irradiation and suggest that this modulation may play a role in radiation-induced cellular changes.     

外源性神经节苷脂GM_3影响人肝癌细胞生长的可能机制的初步研究

通过苔酚蓝染色细胞发现,外源性GM3（10μg/ml）能明显抑制人肝癌细胞株SMMC-7721细胞生长,在GM3处理3d时,出现明显差异．通过NorthernBlot分析发现,外源性GM3可明显影响人肝癌细胞株SMMC-7721细胞中c-fos、c-jun、c-myc和N-ras这四种癌基因的mRAN表达．未经GM3处理的细胞中没有检测到c-fosmRNA,但c-jun微量表达,并有c-myc和N-rasmRNA的高水平表达而GM3可在短时间内快速大量地诱导c-fos、c-junmRNA的生成．GM3处理的细胞,c-myc和N-rasmRNA的表达均明显减少．GM3处理45min时,c-myc基因表达只为对照组的39．55％;GM3处理24h时,N-ras基因表达为对照组的30.48％．以上结果提示：GM3抑制SMMC-7721细胞生长很可能是通过改变癌基因表达来实现的．     

Augmentation of the expression of c-myc and c-fos oncogenes as a function of the mechanical activity of the isolated adult rat heart

c-myc and c-fos oncogenes encode nuclear DNA binding proteins, and are involved in both growth regulation and differentiation. Using the molecular hybridization technique and DNA probes complementary to c-myc and c-fos mRNA, we report an increase in c-myc and c-fos expression level in the isolated beating adult rat heart with reference to the arrested isolated heart. This suggests a causal relationship between mechanical activity of the heart and c-myc and c-fos expression. It evidences for the first time a messenger between mechanical factor and adaptational changes in the phenotype which occurs at the beginning of cardiac hypertrophy.     

The effects of hydrocortisone on c-fos, c-myc and c-ras oncogene expression in IMR-90 fibroblasts

The effects of hydrocortisone on oncogene expression in human IMR-90 fibroblasts was analyzed by Northern blotting of total RNA. In synchronized fibroblasts stimulated with serum alone, there were two time periods of increased c-fos expression during the G1 phase of the cell cycle. There was no significant difference between cells treated with serum plus hydrocortisone, and cells treated with serum alone with respect to c-fos expression. Quiescent cells showed no change in c-fos expression during the G1 phase of the cell cycle. Three peaks of c-fos expression occur when cells are treated with hydrocortisone alone, but hydrocortisone in the absence of serum is insufficient to initiate DNA synthesis. Hydrocortisone has no effect on c-myc or c-Ha-ras expression in the presence or absence of serum in synchronized fibroblasts. Therefore, the control of mRNA production of the nuclear oncogenes c-fos and c-myc, and the cytoplasmic oncogene c-ras are independent and hydrocortisone may enhance DNA synthesis by increasing c-fos expression.     

Transforming growth factor alpha (TGF alpha) induction of C-FOS and C-MYC expression in C3H 10T1/2 cells

We have investigated the effects of transforming growth factor alpha (TGF alpha) in C3H10T1/2 cells, on S phase entry and early gene activation events associated with cell cycle progression. We find that EGF and TGF alpha, which both utilize the EGF receptor for signal generation, are able to stimulate DNA synthesis in these cells with nearly superimposable kinetics; however, the stimulation by TGF alpha was slightly greater at nearly all time points assayed. This report is the first showing that TGF alpha, like EGF, vigorously induces c-myc and c-fos gene expression in these cells. A significant stimulation of c-myc and c-fos mRNA levels is observed with both TGF alpha and EGF; c-myc mRNA levels show an 8-fold induction with both mitogens, while c-fos inductions were on the order of 12 to 14-fold at maximum. However, the induction of c-myc mRNA by TGF alpha has slower kinetics than by EGF.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Interference with SMO increases chemotherapy drug sensitivity of A2780/DDP cells by inhibiting the Hh/Gli signaling pathway

Aberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus-infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin-resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin-resistant A2780/DDP cells were decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP-2, MMP-9, VEGF, and Snail in cisplatin-resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus–infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin-resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.     

人膀胱癌中某些癌基因的研究

本实验应用Northern和斑点印渍杂交技术探测了人膀胱癌细胞株中c-myc、c-fos、erbB等癌基因的表达,以及TPA对这些癌基因表达的调控,发现BIU-87细胞有这些癌基因的表达,并能被TPA所增强,同时也发现人膀胱癌组织有c-myc、c-fos、erbB、N-ras基因的高表达。提示蛋白激酶C的激活可以诱导某些癌基因的表达。多种癌基因的表达异常可能在膀胱癌中起重要作用。     

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide alters expression of c-fos and c-myc oncogenes

Expression of the c-fos, c-myc, and c-fms proto-oncogenes has been studied in thioglycollate-elicited murine peritoneal macrophages after exposure to lipopolysaccharide (LPS). After incubation with LPS (20 ng/ml), a transient and rapid induction of the expression of c-fos and c-myc oncogenes could be observed, whereas the RNA levels for c-fms were not affected. Treatment with lipid A, the active moiety of the LPS molecule, increased the c-fos and c-myc expression to a comparable degree. Similar induction of c-fos and c-myc was observed after treatment with phorbol myristate acetate, suggesting that this effect of LPS on murine macrophages might be mediated through stimulation of protein kinase C. Under similar experimental conditions, LPS treatment of macrophages did not trigger DNA synthesis. Treatment with LPS blocked DNA synthesis in macrophages treated with L cell-conditioned medium containing colony-stimulating factor. Thus changes in c-fos and c-myc expression may be elements in the complex series of biochemical events that contribute to macrophage activation and are not necessarily related to induction or priming for cellular proliferation.