Down-regulation of bcr-abl and bcl-x(L) expression in a leukemia cell line and its doxorubicin-resistant variant by topoisomerase II inhibitors.

K562 cells are usually resistant to apoptosis induction, probably because of the expression of bcr-abl, the hybrid gene characteristic of the Philadelphia chromosome (t 9;22). However, we have previously shown that amsacrine and, to a lesser extent, doxorubicin, could induce apoptosis in the doxorubicin-resistant variant of this cell line. In order to elucidate the role of bcr-abl in triggering apoptosis, we investigated the effect of the topoisomerase II inhibitors doxorubicin, amsacrine, and etoposide on the expression of several genes that may be related to apoptosis induction in both cell lines. This was done using a technique of reverse transcription-polymerase chain reaction coupled with HPLC of the amplified fragments to obtain semiquantitative evaluations. We showed that amsacrine, at pharmacologically relevant concentrations, was able to decrease the expression of bcr-abl down to 20% of the basal value in the doxorubicin-resistant variant only, whereas doxorubicin and etoposide were unable to do so. No effect of these drugs was seen on the expression of the normal abl gene. In addition, there was an effect of amsacrine on the expression of bcl-x(L) in the resistant cell line only, but at concentrations higher than the IC(50) of this drug. Our results emphasize the role of bcr-abl in protecting cells from apoptosis and the possible involvement of specific topoisomerase II inhibitors in overcoming resistance to apoptosis.

Doxorubicin-induced alterations of c-myc and c-jun gene expression in rat glioblastoma cells: role of c-jun in drug resistance and cell death.

We studied the effect of doxorubicin on the expression of c-myc and c-jun in the rat glioblastoma cell line C6 and its doxorubicin-resistant variant C6 0.5, at equitoxic exposures. For quantitation, the mRNA levels of these oncogenes were related to those of two domestic genes, beta-actin and glyceraldehyde phosphate dehydrogenase. After a transient overexpression of the genes during the first hour of incubation, there was a selective, dose-dependent down-regulation of both genes by doxorubicin in the sensitive cells. In the resistant cell line, c-myc expression was also decreased in response to doxorubicin incubation, but the expression of c-jun remained unchanged over the whole range of concentrations. In contrast, vincristine had no effect on the amounts of c-myc and c-jun mRNAs in either line. The effect of doxorubicin on the mRNA levels of c-jun was also observed on the JUN proteins by immunoblotting, but the MYC protein levels remained unchanged upon doxorubicin treatment. There was a significant correlation between the levels of c-myc and c-jun gene expression and the degree of growth inhibition induced by doxorubicin. In addition, doxorubicin induced a fragmentation of DNA in sensitive cells, but not in resistant cells, thus revealing a resistance to apoptosis in this line. Doxorubicin-induced cell death did not appear to be mediated by p53 in either cell line.

Transcriptional down-regulation of c-myc expression in an erythroleukemic cell line, K562, and its doxorubicin-resistant variant by two topoisomerase II inhibitors, doxorubicin and amsacrine.

We have evaluated the effect of two topoisomerase II (Topo II) poisons, amsacrine and doxorubicin, on the expression of the c-myc oncogene, both at the mRNA and protein levels, in the leukemia cell line, K562, and its doxorubicin-resistant counterpart, K562 DoxR. We report in this study a concentration-dependent decrease in c-myc mRNA levels upon exposure of both cell lines to amsacrine and doxorubicin, with a more pronounced effect for amsacrine in the resistant line. In either case, c-myc down-regulation closely paralleled the drug-induced growth inhibition. We have also used the technique of PCR stop-assay to detect the occurrence of DNA breaks within the P2 promoter of the c-myc gene. We have shown that Topo II-mediated breaks induced by amsacrine are probably responsible for the down-regulation of c-myc in the resistant line. In addition, amsacrine induced apoptosis only in the resistant line while doxorubicin did not induce apoptosis in any cell line. These results suggest that c-myc is not involved in the resistance of K562 DoxR cells, but can induce the apoptosis pathway in these cells, while no drug-induced apoptosis could be detected in the sensitive line.