Effects of p73 gene overexpression on apoptosis and chemosensitivity of human lung adenocarcinoma cell line A549.
OBJECTIVE
It is important to overcome gene therapy resistance caused by wt-p53 in non-small cell lung cancer (NSCLC) . The p53 family member p73 is a p53 homolog. This study was to observe the apoptosis and chemosensitivity effect of p53-resistant human lung adenocarcinoma cell line A549 following wild-type p73 gene transfection alone or combined with chemotherapeutic agents.


METHODS
Plasmids pcDNA3-HA-p53 or pcDNA3-HA-p73alpha were transfected into A549 cells with Dosper. Positive cell clones were selected using G418. The exogenous p53 or p73alpha gene expressions were examined by Western blot. MTT assay was used to analyze the response of transfected cells to cisplatin (DDP) or adriamycin (ADM). The drug-induced apoptosis of transfected cells was measured by flow cytometry, TUNEL technique and DNA fragmentation. The biological behavior change of transfected cells was investigated by colony formation assay.


RESULTS
Transfected A549 cells stably overexpressed p53 or p73alpha. Low concentration of chemotherapeutic agents (6.25 micromol/L DDP or 0.25 micromol/L ADM) which had no obvious effects on non-transfected cells, suppressed p73-transfected cell growth significantly; the 50% inhibitory concentration (IC(50)) of DDP for A549 cells decreased from 22.65 micromol/L to 3.75 micromol/L, and the IC(50) of ADM decreased from 4.20 micromol/L to 0.06 micromol/L after p73alpha transfection. p73, but not p53, sensitized A549 cells to DDP and ADM: DDP-induced apoptosis rate was increased from 10.6% to 36.8% (P<0.01), ADM-induced apoptosis rate was increased from 13.0% to 41.1% (P<0.01) after p73 transfection. DDP and ADM significantly suppressed colony formation of p73-transfected cells compared with parental cells (P<0.01). The sensitive enhancement ratios for DDP and ADM were 2.0 and 2.4, respectively.


CONCLUSIONS
Exogenous p73 gene enhances the sensitivity of A549 cells to chemotherapeutic agents by inducing apoptosis through p53-independent pathway. p73 gene could be used to treat p53-resistant tumors.
He
Yong
Y
Thoracic Surgery Center of People's Liberation Army, Daping Hospital/Institute of Surgery Research, The Third Military Medical University, Chongqing, 400042, P. R. China. heyong8998@yahoo.com.cn
Fan
Shi-Zhi
SZ
Jiang
Yao-Guang
YG
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D016158:Genes, p53; D006801:Humans; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004317:Doxorubicin; D002945:Cisplatin