Cloning and characterization of human cDNAs encoding a protein with high homology to rat intestinal development protein OCI-5
Introduction
Chemotherapy plays an important role in the treatment of cancer in clinics. However, the emergence of drug-resistant tumor cells remains a major problem in the use of antineoplastic drugs. Multidrug resistance (MDR), where the treatment of tumor cells with a particular cytostatic compound results in cross-resistance to other structurally unrelated drugs, involves increased drug efflux which has been correlated, e.g., with the enhanced expression of P-glycoprotein (Kartner et al., 1983) or multidrug resistance-associated protein (MRP) (Cole et al., 1992). The available data suggest that mitoxantrone-resistant tumor cell lines, in contrast to many other drug-resistant tumor cells, have an atypical resistance profile displaying only partial cross-resistance to other intercalating agents.
We previously established a mitoxantrone-resistant human gastric carcinoma cell line EPG85-257RNOV (Dietel et al., 1990). This cell line shows an atypical cross-resistance pattern, and no P-glycoprotein or MRP synthesis is detectable. Often this kind of resistance is designated as atypical multidrug resistance (Beck et al., 1987). Several studies demonstrate that there is a wide range of different forms of resistance to mitoxantrone. These mechanisms include changes in the fatty acid composition of the plasma membrane (Burns et al., 1988), overproduction of P-glycoprotein (Jensen et al., 1989), reduction of the cytotoxicity of mitoxantrone by low pH (Jähde et al., 1990), `trapping' of mitoxantrone in intracytoplasmic vesicles (Dietel et al., 1990), reduction of the DNA-topoisomerase II activity (Harker et al., 1991), or expression of cytokeratins 8 and 18 (Bauman et al., 1994). Probably all of these aspects can represent one part of a multicomponent system that leads to mitoxantrone resistance. As part of the search for genes associated with, or responsible for, the mitoxantrone resistance displayed by EPG85-257RNOV cells, in this study a λ-expression cDNA library was constructed from EPG85-257RNOV mRNA and screened by differential hybridization.
Section snippets
Cloning and library screening
The human gastric carcinoma cell line EPG85-257P and its subline EPG85-257RNOV resistant to mitoxantrone were established in our laboratory (Dietel et al., 1990). A λgt10 cDNA library was constructed from the resistant cell line EPG85-257RNOV using oligodeoxythymidine primers for cDNA synthesis. This recombinant phage library was differentially screened. For this procedure the library was dotted onto duplicate nylon filters and hybridized with 32P-labeled cDNA probes reversely transcribed from
Conclusions
- 1.
Five independent cDNAs encoding a protein with high homology to rat OCI-5 were isolated from human gastric carcinoma cells.
- 2.
The clone MXR7 contains the whole ORF which shows 88% homology to the overlapping DNA regions (93% to the derived protein sequence).
- 3.
The polypeptide derived from the amino acid sequence is encoding a 580-amino acid polypeptide with a predicted molecular mass of 66 kDa.
- 4.
Northern blot analysis showed that the expression level of the 2.6-kb mRNA is much more pronounced in the
Acknowledgements
This work was supported in part by grant 10-0922-La 1 of Deutsche Krebshilfe. The authors also would like to thank Gudrun Christiansen and Dr. Dieter Zimmermann (Department of Pathology, University of Zürich) for providing a DNA sequencer.
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