Overexpression of ribosomal proteins L4 and L5 and the putative alternative elongation factor PTI-1 in the doxorubicin resistant human colon cancer cell line LoVoDxR.

A better understanding of the regulatory network underlying cellular drug resistance and stress response may be helpful to overcome the phenomenon of therapy-induced cross-resistances against a variety of antineoplastic agents. Two new powerful molecular techniques, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) and subtractive suppressive hybridisation were applied for the comparative analysis of the gene expression profile of a doxorubicin resistant and its corresponding sensitive parental colon carcinoma cell line (LoVo H67P). DDRT-PCR generated partial cDNAs from the doxorubicin resistant, sensitive and stress (dexamethasone, doxorubicin, cadmium chloride or heat) exposed sensitive cells, were size-separated on polyacrylamide gels.

Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin-resistant cell lines: comparison of mRNA differential display reverse transcription-polymerase chain reaction and subtractive suppressive hybridization for the analysis of differential gene expression.

Subtractive suppressive hybridization (SSH) and mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) were compared for their ability to detect the expression of drug-resistance associated genes in a doxorubicin-resistant and -sensitive colon carcinoma cell line (LoVo H67P). The expression pattern of more than 9000 bands obtained by DDRT-PCR were identical in both cell lines by more than 95%. Of the remaining differentially expressed DDRT-PCR products, 21 cDNA fragments were further analyzed after cloning.

Increase of P-glycoprotein-mediated drug resistance by hsp 90 beta.

The expression of heat shock proteins hsp27, hsp60, hsp70, hsp90 alpha and hsp90 beta in extracts of three cell lines (LoVo DxR, KBChR8-5 and S180 DxR) expressing the MDR (multidrug resistance) positive phenotype as well as in the sensitive parental lines has been investigated. We present evidence that heat shock protein hsp90 beta is associated with the P-glycoprotein (Pgp or P170) one of the most prominent components of the drug resistance machinery. In the doxorubicin-resistant cell line LoVo DxR, but not in the sensitive parental line, hsp90 beta is expressed constitutively as shown by Northern blotting.

Short technical reports. Quantification of ribozyme-mediated RNA cleavage using silver-stained polyacrylamide gels.

A very sensitive, nonradioactive method for the detection of ribozymes, targets for ribozyme cleavage and cleavage products with subsequent densitometric quantification of RNA, has been developed. Amounts as low as 15 ng--corresponding to 0.16 pmol of a 300-nucleotide RNA molecule--can be visualized in denaturating polyacrylamide (PA) gels.

Improvement of hammerhead ribozymes cleaving mdr-1 mRNA.

Overexpression of the mdr-1 gene is one of the mechanisms involved in therapy induced drug resistance. Gene-specific reduction of mdr-1 overexpression in human cancer using antisense technology may be an efficient tool for the reduction of multiple drug resistance (MDR). The application of catalyticly active RNA species--the so-called ribozymes--represents a possible improvement of this molecular strategy using oligonucleotides due to the catalytic potential of ribozymes.