Suppressive subtractive hybridisation reveals differential expression of serglycin, sorcin, bone marrow proteoglycan and prostate-tumour-inducing gene I (PTI-1) in drug-resistant and sensitive tumour cell lines of haematopoetic origin

Abstract

The development of therapy-induced drug resistance is still one of the most important therapeutic limitations. Nevertheless, an integrating view of the molecular mechanisms underlying resistance development in general is missing. In order to shed some light on the network of this resistance development, we established drug-resistant (doxorubicin (DX), methotrexate (MTX), cisplatin (cisPt), vincristine (Vin)) derivatives of six tumour cell lines (Jurkat, U937, HL60, DoHH-2, K562 and ARH77) of haematopoetic origin. Differential gene expression of drug-sensitive parental cell lines and the drug-resistant derivatives thereof was analysed by suppressive subtractive hybridisation. After dot blot screening for differential expression and sequencing of the cloned PCR fragments, differential expression was confirmed by Northern blot analysis. In an attempt to discriminate for differentially expressed genes only related to one or the other of the investigated drugs, the cDNAs of various resistant sublines (doxorubicin-, methotrexate-, cisplatin-resistant Jurkat cells) were pooled and compared with the sensitive parental cell line. In addition, cDNAs of the resistant derivatives of the different haematopoetic tumour cell lines were pooled and compared with the pooled cDNAs of the corresponding sensitive haematopoetic cell lines to eliminate cell line to cell line variations that were not related to drug resistance. As a result of this screening, the following genes showed a higher (at least 2-fold) or exclusive expression in the drug-resistant variants: serglycin, sorcin, BMPG (bone marrow proteoglycan gene) and PTI-1 (prostate-tumour-inducing gene 1). In addition, elevated expression of hsp90, previously found by our group to be upregulated in the drug-resistant colon carcinoma cell line LoVo H67P was found to be overexpressed in drug-resistant HL60 cells.