p53 promoter-based reporter gene in vitro assays for quick assessment of agents with genotoxic potential

The p53 promoter-based green fluorescent protein(GFP)and luciferase reporter gene assayshave been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cellsexposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of thecells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reportergene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assess-ment or screening of agents with genotoxic potential.     

Activation of p53 in gastric cancer cells by tripchlorolide specifically induces apoptosis

There are two possible outcomes when DNA damage occurs in normal mammalian cells: either induction of cell-cycle checkpoint which inhibits the progress of the cell cycles as well as activates DNA repair pathways, or activation of apoptosis to eliminate damaged cells. The p53 tumour-suppressor gene plays a key role in selecting these pathways. In our present works, the human gastric cancer cell line AGS was treated with tripchlorolide, a potent antitumor compound purified from a Chinese herb Tripterygium Wilfordii Hook. Single cell gel electrophoresis (Comet assay) showed that the treatment of tripchlorolide resulted in DNA damage in AGS cells. The damaged AGS cells went through apoptosis, which was time- and dose- dependent.     

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell lines

This study aimed to investigate the effects of arsenic trioxide(As2O3) on the mitochondrial DNA(mtDNA) of acute promyelocytic leukemia(APL) cells.The NB4 cell line was treated with 2.0 μmol/L As2O3 in vitro,and the primary APL cells were treated with 2.0 μmol/L As2O3 in vitro and 0.16 mg kg-1 d-1 As2O3 in vivo.The mitochondrial DNA of all the cells above was amplified by PCR,directly sequenced and analyzed by Sequence Navigatore and Factura software.The apoptosis rates were assayed by flow cytometry.Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As2O3 use,but the mutation spots were remarkably increased after As2O3 treatment,which was positively correlated to the rates of cellular apoptosis,the correlation coefficient:rNB4-As2O3=0.973818,and rAPL-As2O3=0.934703.The mutation types include transition,transversion,codon insertion or deletion,and the mutation spots in all samples were not constant and regular.It is revealed that As2O3 aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo.Mitochondrial DNA might be one of the targets of As2O3 in APL treatment.     

AKT2基因的过表达抑制H2O2诱导的乳腺癌细胞凋亡

To study the effect of Akt2 gene on the apoptosis of breast cancer cells induced by H2O2. The full length cDNA of Akt2 gene was amplified by RT-PCR, and then cloned into pcDNA3.1 /myc-His(-)A vector (Wild type, WT-Akt2). Dominant negative mutant of AKT2 (DN-Ak2) were made by QuikChange site-directed mutagenesis. The eukaryotic expression vector of WT-Akt2 and DN-Akt2 were constructed, and were then transfected into MCF-7 breast cancer cells, respectively. Clones stably expressing Akt2 or DN-Akt2 were obtained by neomycin screening; Two different siRNA fragments targeted Akt2 gene were designed and synthesized, and were then transfected into the same cells. Cell apoptosis pre or post-H2O2 treatment was determined by TUNEL 和DNA Laddering assays. The sequencing result confirmed WT-Akt2 and DN-Akt2 were successfully constructed, and the results of Western Blot show They had good expression in MCF-7 cells, and Akt2 siRNA could effectively silence Akt2 expression. The resistance for apoptosis-induced by H2O2 in MCF-7 cells with WT-Akt2 over-expression was significantly increased (DN-Akt2 showed opposite function). The apoptotic cell number induced by H2O2 was significantly lower in stable transfectants with the WT-Akt2 vector than in those with empty vector or in untransfected cells (P ＜0.05), whereas no significant difference was found between the latter two groups (P ＞0.05). The function of inhibition of apoptosis by Akt2 was blocked by Akt2 siRNA and PI3K/Akt inhibitor, wortmannin. Thus, Akt2’s effect was further confirmed by these endogenous results. Overall, our study suggests that Akt2 can increase the resistance of human breast cancer cells to the apoptosis induced by H2O2, and it may be used as a therapeutic target for breast cancer, providing a foundation for investigation the molecular mechanism of breast cancer cells resistant to the apoptosis induced by reactive oxygen.     

Modulation of the effect of camptothecin in x-irradiated L5178Y-R and L5178Y-S cells by benzamide

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 μM CPT enhanced the radiosensitivity of LY-S cells (D₀ decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly(ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D₀ decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e. g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases α and δ) applied concomitantly with CPT in cells not pre-treated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication. Received: 28 November 1995 / Accepted in revised form: 20 April 1996     

Downregulation of wild-type p53 protein by HER-2／neu mediated PI3K pathway activation in human breast cancer cells： its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy.Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.     

野生型P53、P16基因协同对肺腺癌细胞系生长抑制作用的研究

为了探讨野生型P53基因及P16基因在恶性肿瘤基因治疗中的作用,用腺病毒为载体将野生型P53基因转入高、低转移的肺腺癌细胞系Anip973、AGZY83-a和经野生型P16基因质粒转染的高、低转移肺腺癌细胞系Anip973（Anip973P16）、AGZY83-a（AGZY83-aP16）。对各组转染细胞进行生长曲线、MTT生长抑制率、原位末端标记、Western-blotting等技术检测分析。结果发现（1）野生型P53蛋白的过表达对上述肺腺癌细胞系均呈现出较强的生长抑制作用。（2）野生型P53蛋白的过表达对高转移肺癌细胞系Anip973的抑制作用明显高于低转移细胞系AGZY83-a。（3）野生型p53蛋白的过表达对经野生型P16基因转染的高、低转移的肺癌细胞Anip973、AGZY83-a抑制作用明显高于未经P16基因转染的细胞。野生型P53基因可以作为肺腺癌基因治疗的候选基因。肿瘤抑制基因P53、P16的联合转染可能是对肺腺癌进行基因治疗的有效手段。 Abstract：To investigate the suppression effect of tumor suppressor genes in lung adenocarcinoma cell lines,we transferred a pair of lung adenocarcinoma cell lines with different metastasis potential,Anip973(High-metastasis potential cell line) and AGZY83-a (Low-metastasis potential cell line)and this pair of cell lines transfected with P16 gene:AGZY83-a P16 and Anip973 P16 with wild type P53 gene with adenovirus vector.The suppression effects of P53 gene were evaluated by cell growth curve,MTT,western-blotting analysis and TUNEL technique.Overexpression of wild-type P53 gene in AGZY83-a,Anip973,Anip973 P16 and AGZY83-a P16 inhibited the growth of these four kinds of lung cancer cells and induced apoptosis of the cells.The suppression effect of P53 gene in Anip973 and Anip973 P16 was higher than AGZY83-a and AGZY83-a P16 while co-expression of P53 and　P16 in this pair of cell lines inhibited the cells more efficiently comparing with the expression of P53 alone.Wild-type P53 gene might act as a candidate gene in lung adenocarcinoma gene therapy while co-transfection of P53 and P16 genes was a more effective method.     

离子注入对微生物细胞的刻蚀与对DNA的损伤及修复

以耐辐射异常球菌为试材,以E. coli 为对照,用显微扫描电镜和3H-TdR标记,研究了离子注入对微生物细胞的刻蚀与对DNA的损伤及其修复。结果表明,注入离子对细胞存在着刻蚀损伤;中性蔗糖梯度密度离心沉降分析证明, 大剂量下离子注入可直接导致DNA损伤,并观察到在对应的存活率峰值注入剂量下,D. radiodurans修复损伤DNA的能力比E. coli 强,还证明了细胞经不同时间温育后,损伤的DNA分子得到了部分修复。 Abstract：　The direct action of N+implantationin on D. radioduransand E. coliwas investigated by SEM, and their cells were labeled with 3H-TdR, which were implanted by 20keV N+after incubation 18hours, then the DNA of lysed cells was subjected to the neutral sucrose gradient(5%～20%) ultra-centrifugation sedimentation analysis. The results showed that N+implantation exerted direct action on two kinds of microorganisms; the momentum transfer and energy deposition of implantation ions produced the direct etching damage on cells, and repair DNA efficiency of D.radiodurans was higher than that of E. coli. Meanwhile, the damaged DNA incomplete repairing was observed. When incubation was continued up to 6 hours, the rejoined DNA molecules broke again. The repair of damaged DNA could be inhibited by 200μg/ml chloramphenicol. This suggested that DNA damage was serious by ion implantation and damaged DNA repair of cells need continuously synthesizing repair enzyme.