OVEREXPRESSION OF RIBOSOMAL-PROTEINS L4 AND L5 AND THE PUTATIVE ALTERNATIVE ELONGATION-FACTOR PTI-1 IN THE DOXORUBICIN-RESISTANT HUMAN COLON-CANCER CELL-LINE LOVODX(R)

A better understanding of the regulatory network underlying cellular d rug resistance and stress response may be helpful to overcome the phen omenon of therapy-induced cross-resistances against a variety of antin eoplastic agents. Two new powerful molecular techniques, mRNA differen tial display reverse transcriptase polymerase chain reaction (DDRT-PCR ) and subtractive suppressive hybridisation were applied for the compa rative analysis of the gene expression profile of a doxorubicin resist ant and its corresponding sensitive parental colon carcinoma cell-line (LoVo H67P). DDRT-PCR generated partial cDNAs from the doxorubicin re sistant, sensitive and stress (dexamethasone, doxorubicin, cadmium chl oride or heat) exposed sensitive cells, were size-separated on polyacr ylamide gels. The expression patterns of more than 9000 bands of the r esistant, sensitive and stressed sensitive cell populations were ident ical by more than 95%. Of the differentially expressed mRNAs, 20 cDNA fragments were reamplified after isolation from the gel, used as probe s for Northern blot analysis to verify their differential expression a nd sequenced after cloning. Among the differentially expressed cDNAs, homologies of 96% and 87%, respectively, were found to the human proto -oncogene PTI-1 and the human ribosomal protein L4. Subtractive suppre ssive hybridisation revealed overexpression of the ribosomal protein L 5 in the doxorubicin resistant line. These data point to the control o f gene expression at the translational level as an important mechanism involved in cellular stress response. (C) 1998 Elsevier Science Ltd. All rights reserved.