Cell cross-talk mediates PPARalpha null hepatocyte proliferation after peroxisome proliferator exposure

Peroxisome proliferator activated receptor(alpha) (PPARalpha) mediates the liver's responses to peroxisome proliferator compounds. These responses include induction of specific hepatic enzymes, peroxisome proliferation and hepatocyte proliferation. PPARalpha null mice, which lack receptor in all cells of the body, do not respond to peroxisome proliferators, indicating that hepatocellular proliferation and other responses require the presence of this receptor in at least some cells. To determine if PPARalpha is required specifically in hepatocytes for each response, we used hepatocyte transplantation to generate chimeric livers composed of PPARalpha null and positive hepatocytes in PPARalpha null or positive hosts. Upon exposure to a peroxisome proliferator, peroxisome proliferation and enzyme induction were restricted to receptor positive hepatocytes, indicating that these responses are cell autonomous with respect to hepatocyte receptor status. However, both PPARalpha null and positive hepatocytes in chimeric livers displayed elevated DNA synthesis regardless of host receptor status, as long as at least some hepatocytes contained receptor. These findings indicate that the mitogenic response to peroxisome proliferators does not require PPARalpha in all hepatocytes.     

Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells

Stable re-expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy-induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control-transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43-mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl-2. Over-expression of bcl-2 in cx43-transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43-mediated apoptosis to chemotherapeutic agents is regulated in part through the down-regulation of bcl-2 expression. Furthermore, the cx43-mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43-transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy.     

Expression of the human Bcl-2 increases resistance of Ewing's sarcoma cells to apoptosis and inhibits poly(ADP-ribose) polymerase cleavage induced by radiation

We reported previously that Ewing's sarcoma (ES) cells respond to ionizing radiation exposure by arrest in G(2)/M phase and induction of apoptosis which occurs in conjunction with poly(ADP-ribose) polymerase (PARP) proteolytic cleavage. ES cells (A4573 cell line) do not express immunodetectable levels of Bcl-2. To determine if expression of Bcl-2 could modulate radiation-induced ES cell death, we have stably transfected A4573 cells with a full-length human bcl-2 cDNA. Expression of Bcl-2 protein rendered ES cells relatively resistant to radiation-induced apoptosis. Moreover, the anti-apoptotic activity of Bcl-2 was directly related to levels of its expression in different ES clones. Cell cycle characteristics were similar for both parental and Bcl-2 expressing ES cells following radiation treatment, although bcl-2 transfectants exhibited a more protracted G(2)/M phase arrest and lower rate of apoptosis after release from the block. Constitutive expression of Bcl-2 resulted in about two-fold inhibition of PARR cleavage in ES cells dying after ionizing radiation exposure. These data support a role for Bcl-2 protein as a negative regulatory element of PARP proteolysis at the early stages of radiation-induced apoptosis in ES cells.     

The peroxisome proliferators are hepatocyte mitogens in chemically- defined media: glucocorticoid-induced PPAR alpha is linked to peroxisome proliferator mitogenesis

Peroxisome proliferator-induced mitogenesis is believed to play a role in hepatocarcinogenesis, but it has not been possible to demonstrate high level induction of DNA synthesis by peroxisome proliferators in cultured hepatocytes. We now show that four structurally dissimilar peroxisome proliferators (methylclofenapate, Wy-14 643, tetradecyl-3- thia acetic acid and clofibrate) cause high level induction of DNA synthesis in primary cultures of rat hepatocytes, routinely 7-9 fold above control, with up to 29% of cells undergoing S-phase. Peroxisome proliferators induce DNA synthesis rapidly, with maximal response 24 h after dosing [compared with 48 h for epidermal growth factor (EGF)]; indeed, peroxisome proliferators were mitogenic in a chemically defined medium, i.e. with no added exogenous growth factors. EGF-treated hepatocytes that had undergone DNA synthesis comprised 23% binucleated cells, whereas hepatocytes induced into S-phase by peroxisome proliferators contained only 3% binucleated cells, demonstrating a distinct response of hepatocytes to peroxisome proliferators and EGF. The presence of a glucocorticoid was essential for peroxisome proliferator-induced DNA synthesis, but not for EGF-induced DNA synthesis, demonstrating that the requirement for glucocorticoids is selective for peroxisome proliferators. Hydrocortisone was shown to induce the expression of peroxisome proliferator activated receptor- alpha (PPAR alpha), and we propose that it is the glucocorticoid- induced expression of PPAR alpha that is essential for peroxisome proliferator mitogenesis. This in vitro system provides a powerful tool for investigating the mechanism and role of peroxisome proliferator- induced mitogenesis in liver growth and carcinogenesis.      

不同途径免疫的AFP基因修饰DC瘤苗体内抗肿瘤效应的研究

目的:评价bcl-2反义寡核苷酸对肾细胞癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用.方法:合成与已知人类基因无同源性的bcl-2反义寡核苷酸(AS1与翻译起始端互补,AS2与编码区互补),以阳离子脂质体Lipofectin为转染载体,MTS比色实验测定细胞活率,反转录-聚合酶链反应(RT-PCR)测定bcl-2 mRNA的表达,Western杂交法测定Bcl-2蛋白表达,碘化丙啶(PI)染色流式细胞术测定凋亡细胞.结果:所测定的5个肾癌细胞株均有稳定的bcl-2 mRNA表达;转染的反义寡核苷酸对ACHN细胞Bcl-2蛋白的表达有明显抑制作用,而正义寡核苷酸无明显影响,AS2的抑制作用大于AS1;反义寡核苷酸可诱导ACHN细胞的凋亡,AS1和AS2的诱导率分别为32.1%和43.2%.结论:bcl-2反义寡核苷酸可抑制肾癌细胞Bcl-2蛋白的表达,并进而诱导细胞的调亡.     

Bcl-2基因家族相关蛋白在人卵巢癌耐药细胞凋亡中的作用

[目的]探讨bcl-2基因家族相关蛋白在卵巢癌耐药细胞SKOV3／ADM发生凋亡中的作用。[方法]用免疫细胞化学法测定SKOV3和SKOV3／ADM细胞的bcl-2蛋白；以不同浓度的阿霉素作用于SKOV3/ADM细胞，用流式细胞仪动态分析细胞周期和凋亡，并在不同时间点观察其bcl-2、bax、bad和p21蛋白变化。[结果]SKOV3/ADM细胞的bcl-2蛋白显著高于SKOV3（P〈0．01）；大剂量阿霉素（10μg／ml和40μg/ml）作用3h后，能明显下调SKOV3／ADM的bcl-2蛋白和增加bad蛋白；小剂量（0．5μg／ml）阿霉素作用（24、48、72h）后，SKOV3/ADM细胞的凋亡发生率（10．08％、30．53％和33．7％），显著低于SKOV3（40．01％、65.38％和77．65％）（P〈0．01）；SKOV3／ADM细胞p21、bad蛋白增加，bcl-2蛋白下降。[结论]SKOV3／ADM细胞bcl-2高表达能拮抗化疗剂诱导凋亡的作用，bcl-2基因家族相关蛋白与卵巢癌耐药细胞凋亡密切相关。     

5-Fluorouracil induces apoptosis in human colon cancer cell lines with modulation of Bcl-2 family proteins.

Recently, apoptosis has been implicated as one of the end points of cells exposed to chemotherapeutic agents. The p53 and Bcl-2 family of proteins are involved in chemotherapy-induced apoptosis, but in a cell type-dependent manner. We sought to determine the roles played by the p53 and Bcl-2 family of proteins in 5-fluorouracil (5-FU)-induced apoptosis of human colon cancer cell lines. We first studied the p53 genetic and functional status, and then 5-FU, at inhibitory concentration of 50% (IC50) doses, was used to induce apoptosis, which was confirmed by morphological analysis and enzyme-linked immunosorbent assay (ELISA). Bcl-2, Bcl-X(L), Bax, Bad, Bak and p53 protein expression was analysed by Western blotting. Using five human colon cancer cell lines, we found that equitoxic (IC50) doses of 5-FU induced apoptosis in both wild-type p53 and mutant p53 cells. Analysis of the steady-state levels of Bcl-2 family proteins showed high expression of Bcl-X(L) in all of the cell lines except Colo320. Bcl-2 was expressed in two of them. Bax presented with the lowest basal expression and Bad showed homogeneous expression. On the other hand, Bak expression varied more than fivefold among these cells. In cells containing wild-type p53 (e.g. LoVo), 5-FU-induced apoptosis was accompanied by increased expression of Bax and Bak without consistent modulation of other bcl-2 family proteins. In contrast in cells containing mutant p53 (e.g. DLD1), Bak expression was remarkably increased. There was a significant correlation between chemosensitivity and Bcl-X(L) to Bax ratio, rather than Bcl-2 to Bax. In conclusion, these results suggest that some members of the Bcl-2 family of proteins, in human colon cancer cell lines, are modulated by 5-FU and that the ratio of Bcl-X(L) to Bax may be related to chemosensitivity to 5-FU.     

PPARγ配体Ciglitazone对人胃癌MGC803细胞增殖的影响及其机制

目的探讨PPARγ配体Ciglitazone对人胃癌MGC803细胞增殖的影响及其机制。方法RTPCR检测MGC803细胞PPARγ mRNA。MTT及流式细胞术检测Ciglitazone对MGC803细胞增殖及凋亡的影响。相差倒置显微镜和Hoechst33342染色观察凋亡的形态学变化。免疫组化和流式细胞术检测PPARγ、Bcl-2、Bag-1及Bax蛋白表达和变化。结果PPARγ配体Ciglitazone（5、10、20和50μmol／L）可抑制MGC803细胞增殖和诱导其凋亡,增殖的抑制和凋亡的诱导有平行性,且有浓度和时间依赖性。随20μmol／L Ciglitazone作用时间的延长,MGC803细胞的PPARγ mRNA及蛋白表达、Bax蛋白表达及Bax／Bcl-2蛋白的比值升高,Bcl-2和Bag-1蛋白表达降低。结论Ciglitazone可能主要通过激活PPARγ抑制人胃癌MGC803细胞增殖和诱导其凋亡。Bax蛋白表达和Bax／Bcl-2蛋白表达比值升高,Bcl-2和Bag-1蛋白表达降低在Ciglitazone诱导人胃癌MGC803细胞凋亡中起重要作用。提示Ciglitazone可能对治疗胃癌有效。     

Apoptosis in the absence of caspase 3

MCF-7 human breast cancer cells do not express caspase 3, thought by some to be a critical component of the apoptosis cascade. Nonetheless, both mock- and bcl-2-transfected MCF-7 cells undergo apoptosis after treatment with a variety of stimuli, including the DNA-cleaving antimitotic agent, neocarzinostatin (NCS). Transfection with bcl-2 shifts the concentration-response curve to NCS but does not change the phenomenology of apoptosis when it occurs. In both cases, NCS treatment results in condensation and fragmentation of MCF-7 cell nuclei and release of cytochrome c from the mitochondria to the cytosol. This apoptosis is accompanied by decreased levels of Bcl-2 and increased levels of Bax. Using a series of caspase inhibitors with overlapping specificities, enzyme-specific chromogenic substrates, and an antibody specific for activated caspase 7, we have determined that apoptosis in MCF-7 cells proceeds via sequential activation of caspases 9, 7 and 6. P21 is detected only after activation of caspase 7, and P53 is neither expressed at baseline nor up-regulated with apoptosis induction. This pathway bypasses the need for activated caspase 3 in these cells.     

TRAIL (APO-2L) induces apoptosis in human prostate cancer cells that is inhibitable by Bcl-2.

To determine if TRAIL-induced apoptosis in human prostate tumor cells was suppressed by bcl-2, we compared the levels of apoptosis induced by recombinant human TRAIL in pairs of isogenic cell lines that do or do not express bcl-2. Three human prostate tumor cell lines (PC3, DU145 and LNCaP) and their bcl-2-expressing counterparts were tested for their susceptibility to TRAIL. Cells were exposed to TRAIL in the presence of cycloheximide which acted as a sensitizer. Apoptosis was induced rapidly in PC3 and DU145 neo-control transfected cells, whereas induction in LNCaP required 24 h. All three cell line variants expressing bcl-2 were resistant to the apoptotic effects of TRAIL. Caspase 3 and 8 activation was also detected in the neo control cells after treatment with TRAIL, demonstrating the rapid activation of the caspase cascade similar to that seen with other death receptors. Bcl-2 overexpression in these cells blocked activation of these caspases, suggesting that bcl-2 expression of human cancer cells may be a critical factor in the therapeutic efficacy of TRAIL.