Cancer Letters

Cancer Letters

Volume 185, Issue 2, 28 November 2002, Pages 211-218
Cancer Letters

Differentially expressed gene profiles between multidrug resistant gastric adenocarcinoma cells and their parental cells

https://doi.org/10.1016/S0304-3835(02)00264-1Get rights and content

Abstract

To better understand the molecular mechanisms of multidrug resistance (MDR) of human cancers, we isolated differentially expressed genes from drug-resistant human gastric adenocarcinoma cell lines using a polymerase chain reaction-based subtractive hybridization technique. Sixty three genes were identified to be differentially expressed in the drug-resistant human gastric adenocarcinoma cell lines. Among the 63 up-regulated genes, 27 were known genes, of which two have been reported to be associated with MDR. The remaining ones were undefined genes or expressed sequenced tags. The results suggest that this strategy is efficient for large-scale cloning of differentially expressed genes in drug-resistant cells. Further characterization of these genes will shed more light on the understanding of molecular mechanisms of MDR of human cancer cells.

Introduction

Multidrug resistance (MDR) describes a variety of strategies that tumor cells develop to evade the cytotoxic effects of anti-cancer drugs. Tumor cells that have become MDR show decreased cellular sensitivity to the drugs employed in chemotherapy as well as a broad spectrum of drugs without obvious common targets or structural homology. MDR is a major obstacle to the successful treatment of tumors.

The mechanisms that have been found to confer drug resistance, include high expression of drug efflux pump, altered cellular drug pharmacokinetics, increased drug detoxification, increased DNA damage repair, suppression of drug-induced apoptosis, upregulation of lipids and other biochemical changes [1], [2], [3], [4], [5], [6], [19]. However, to date, no mechanism can be generalized, and preclinical and clinical evidence from interventions aimed at any single resistance mechanism has been disappointing. Therefore, it is not surprising that two or more mechanisms often co-exist to confer MDR phenotypes [20], [21]. In addition, the increasing evidence of genetic diversity in drug-resistant cell lines and clinical resistance [22], [23] allows us to assume that there is an intricate molecular network controlling the sensitivity of tumor cells to chemotherapeutic agents.

These concepts suggest that searching for more molecules associated with MDR will be necessary in order to understand the mechanisms of MDR and further design new therapeutic regimens to circumvent drug resistance. So, we used a modified subtractive hybridization method to identify up-regulated genes from vincristine (VCR) or adriamycin (ADR) resistant human gastric adenocarcinoma cell lines that derived from the cell line SGC7901. Although these two drug-resistant cell lines were selected with a single anti-cancer drug, they also displayed cross-resistance to other anti-cancer drugs such as cisplatin, etoposide, mitomycin C and 5-fluorouracil (5-FU) [7], [8].

Section snippets

Cell culture

VCR resistant human gastric adenocarcinoma cell line SGC7901/VCR and ADR resistant SGC7901/ADR were maintained in RPMI1640 (GIBCO) containing 10% heat inactivated fetal calf serum (FCS), the final concentrations of VCR and ADR were 1 and 0.3 μg/mL, respectively.

Isolation of polyA mRNA from cells

Cells were grown to 80% confluence, and mRNA was isolated from 107cells using Promega polyATract 1000.

Preparation of driver cDNA and tester cDNA

The driver mRNA from drug sensitive cell line (human gastric adenocarcinoma cell line SGC7901) was reverse transcribed with oligodT18,

Differentially expressed genes

To isolate genes that are differentially expressed in the MDR gastric adenocarcinoma cells, we used streptavidin magnetic beads mediated subtraction cloning method. We obtained 200 clones containing cDNA insertion fragments from the two drug-resistant gastric adenocarcinoma cell lines. After restrictive enzyme digestion, 86 clones containing cDNA fragments of 0.5–1.5 kb were identified. Among the 86 clones, 63 (23 clones from SGC7901/VCR cells; 40 clones from SGC7901/ADR cells.) displayed

Discussion

Development of MDR prevents successful chemotherapy of many tumors, including gastric adenocarcinoma. Although those known mechanisms such as drug efflux pumps have been shown to play an important role in the MDR phenotype, evidence for other contributing molecules prompts us to use subtractive hybridization method to search for differentially expressed genes in the MDR human gastric adenocarcinoma cell lines.

The subtractive hybridization method in our study used the cap-finder method,

Acknowledgements

This project is supported by National Foundation of Outstanding Young Scientists, People's Republic of China (No. 39525020).

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