[Establishment of a human nasopharyngeal carcinoma drug-resistant cell line CNE2/DDP and screening of drug-resistant genes].
OBJECTIVE
Chemotherapy constitutes one of the chief supplementary methods in the treatment of nasopharyngeal carcinoma (NPC). However, the appearance of drug resistance often causes failure of chemotherapy. For overcoming drug resistance, it is of great importance to screen drug-resistant associated genes so as to identify potential molecular targets. This study was designed to establish a drug-resistant cell line from a human nasopharyngeal carcinoma cell line CNE2, and to screen human nasopharyngeal carcinoma drug-resistant genes by a new strategy based on improved subtractive hybridization.


METHODS
The drug-resistant cell line was established by a program of treating the human nasopharyngeal carcinoma cells CNE2 in the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin. Drug sensitivity was measured by MTT assay. Fluorescence activated cell analysis(FACS) was employed for determining the concentration of fluorescence dye rhodamine 123 within the cells. Cell growth curve, doubling time, and cell morphology were measured and observed. The drug-resistant genes were screened by a new strategy of PCR-based subtractive hybridization. Sequencing and blast analysis were performed after the differentially expressed genes had been verified by reverse dot blotting. The result was further confirmed by RT-PCR.


RESULTS
The resistance indexes of CNE2/DDP to cisplatin (DDP), 5-fluorouracil (5-FU), and vincristine (VCR) were 27.9, 227.9, and 55.5, respectively, indicating its multi-drug resistant property. FACS analysis showed that the concentration of rhodamine 123 was much lower in CNE2/DDP cells than in CNE2 cells (12.98 vs. 243.62). The CNE2/DDP cells appeared smaller, more regularly round, and longer doubling time (26 hours vs. 19 hours) than CNE2 cells. Six differentially expressed sequences were discovered using improved subtractive hybridization; all of them were found to be homologous to known genes after sequencing analysis. Three of them were highly expressed in CNE2/DDP cells. Among them, one sequence, which encodes a 79 amino acid protein,known as DC13 protein (DC13), was a function unknown gene which has certain relationship with malignancy. The other two sequences were ubiquitin C gene and NADH dehydrogenase subunit 2 (ND2) gene, respectively. The other three of the six sequences, whose expression were inhibited in CNE2/DDP cells, were cytochrome C oxidase subunit I(COX1), ribosomal protein L27(RPL27),and ribosomal protein S27 (RPS27) genes, respectively.


CONCLUSIONS
A drug- resistant cell line CNE2/DDP, which showed a typical resistant phenotype to anti-cancer drugs was established. The PCR-based improved subtractive hybridization is an effective approach to identify differentially expressed genes. Many genes, both known and unknown, might contribute to the existence of drug-resistant phenotype, through increasing or decreasing their expression.
Jiang
Run-De
RD
Department of Molecular Tumor Biology,Chinese Academy of Military Medical Sciences, Beijing,100850, PR China.
Zhang
Li-Xin
LX
Yue
Wen
W
Zhu
Yun-Feng
YF
Lu
Hao-Jun
HJ
Liu
Xin
X
Cen
Xin-Tang
XT
Guan
Xin-Yuan
XY
Li
Chun-Hai
CH
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004354:Drug Screening Assays, Antitumor; D005434:Flow Cytometry; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D002945:Cisplatin