Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1.
BACKGROUND
The multidrug resistance (MDR) associated with the expression of the mdr1 gene and its product P-glycoprotein is a major factor in the prognosis of hepatocellular carcinoma cell (HCC) patients treated with chemotherapy. Our study was to establish a stable HCC MDR cell line where a de novo acquisition of multidrug resistance specifically related to overexpression of a transgenic mdr1.


METHODS
The 4.5-kb mdr1 cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCI-neo mammalian expression vector, later was transferred by liposome to human hepatocarcinoma cell line HepG2. Then the transfected HepG2 cells resisting G418 were clustered and cultured and the specific fragment of mdr1 cDNA, mRNA and the P-glycoprotein (Pgp) in these HepG2 cells were detected by PCR, RT-PCR and flow cytometry, respectively. The accumulation of the daunorubicin was determinated by flow cytometry simultaneously. The nude mice model of grafting tumour was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla. When the tumour diameter reached 5 mm, adriamycin was injected into peritoneal cavity. The size and growth inhibition of tumour were evaluated.


RESULTS
The mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed. The PCR analysis showed that the specific fragment of mdr1 cDNA in HepG2/mdr1 cells, but not in the control group HepG2 cells. Furthermore, the content of the specific fragment of mdr1 mRNA and Pgp expression in HepG2/mdr1 cells were (59.7 +/- 7.9)% and (12.28 +/- 2.09)%, respectively, compared with (16.9 +/- 3.2)% and (3.07 +/- 1.06)% in HepG2 cells. In the nude mice HCC model, the tumour genes of both groups were identified. After ADM therapy, the mean size of HepG2 cell tumours was significantly smaller than HepG2/mdr1 cell tumours.


CONCLUSIONS
The approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechanism is known. This would provide the experimental basis of MDR research.
Chen
Yong-Bing
YB
Department of General Surgery, Second Hospital, Chongqing University of Medical Sciences, Chongqing, China.
Yan
Mao-Lin
ML
Gong
Jian-Ping
JP
Xia
Ren-Pin
RP
Liu
Li-Xin
LX
Li
Ning
N
Lu
Shi-Chun
SC
Zhang
Jing-Guang
JG
Zeng
Dao-Bing
DB
Xie
Jian-Guo
JG
Yang
Jia-Yin
JY
Yan
Lü-Nan
LN
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D016685:Mitomycin / Q000494:pharmacology / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023041:Xenograft Model Antitumor Assays / Q000379:methods
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016685:Mitomycin; D004317:Doxorubicin