P388 leukemia sublines were isolated from
leukemia-cell-bearing CD2F1 mice that had been treated in vivo with increasing amounts of
diaziquone (
AZQ). The sublines isolated for in vitro studies were AZQ19 and AZQ30 which corresponded to the 19th and 30th in vivo passages, respectively. The AZQ19 subline displayed a very low degree of resistance to
AZQ (1.5-fold), whereas the AZQ30 subline was sensitive. Both sublines, however, had much higher degrees of resistance to
Adriamycin than to
AZQ (24-fold for AZQ30 cells and 10-fold for AZQ19 cells). Both cell lines were also more resistant to
actinomycin D,
colchicine, and
vincristine than to
AZQ. The AZQ19 line was resistant to the
alkylator thio-TEPA to the same degree that it was to
AZQ, but the AZQ30 line was sensitive to
thio-TEPA. On the other hand, AZQ30 cells were resistant to
hydrogen peroxide with a very low degree of resistance (1.27-fold, P less than 0.05), whereas the AZQ19 line was sensitive.
Drug accumulation experiments indicated that
AZQ-resistant cells differed from the parental line in that they did not accumulate
Adriamycin or
vinblastine. In the case of
AZQ, however, resistant and parental lines accumulated the same amounts of exchangeable
AZQ. Using the immunoblotting technique, no
P-glycoprotein was found in resistant cells. The resistant lines consumed
oxygen at greater rates than the parental line. Oxygen consumption (Mean +/- SD) in sensitive cells was 2.0 +/- 0.4% O2 consumed/min, whereas in resistant cells it was nearly 3.1 +/- 0.6% O2 consumed/min. The increase in oxygen consumption with drug resistance was statistically significant (P less than 0.01). The kinetics of production of
hydroxyl free radicals and of
AZQ free radicals were faster in the resistant lines reflecting, in essence, their increased oxygen consumption. It appears that the two sublines analyzed here show resistance mechanisms that may have been elicited by the two distinct chemical constituents of
AZQ. Therefore, in the AZQ19-resistant line, the alkylating aspect of
AZQ was emphasized, whereas in the AZQ30 line, the
quinone and, thus,
free radical aspect was emphasized. This is consistent with AZQ30 cells being sensitive to the
alkylator thio-TEPA and resistant to
hydrogen peroxide, and the AZQ19 line being resistant to
thio-TEPA and sensitive to
hydrogen peroxide. In addition, the AZQ30 cell line was relatively more resistant than the AZQ19 line to
Adriamycin.