Doxorubicin-induced DNA damage on human normal lymphocytes: effect of heat shock

NADIN, S.B.; VARGAS ROIG, L.M.; CIOCCA, D.R.

Mendoza, Argentina

Workshop; III International Workshop on Molecular Biology of Stress Responses; 2001

Cell Stress Society International

Doxorubicin is used as a first line chemotherapy in several human tumors. This drug has different mechanisms of action to damage the tumor cells producing double strand breaks, Topoisomerase II inhibition, DNA cross-links, and oxidative damage. In previous studies, we have reported that hsp27 and hsp70 may be involoved in doxorubicin resistance in breast cancer cells in vitro and in vivo. To advance our knowledge on these hsps and doxorubicin, in this study we have examined whether normal human lymphocytes are damaged by doxorubicin in vitro, and whether a heat shock can affect this damage and the mechanisms of repair. Normal human lymphocytes were obtained from peripheral blood of a non smoker donor, separated and used in culture. The lymphocytes were incubated in appropriate conditions. The sample was divided in four groups: (1) control, without treatment, (2) treated with 21 nM of doxorubicin for 1 hour without heat shock (with different induction times for hsps after doxorubicin exposure: Test 1: 4 h and Test 2: 24) or for 24 h with doxorubicin (Test 3: hsp induction time 24h), (3) exposed only to heat shock of 42ºC for 1 hour, and (4) exposed to the heat shock, leaved to recuperate for 4 hs (Test 1) or 24 hs (Test 2), and then treated with 21 nM of doxorubicin for 1 hour (Test 1 and 2) or 24 hours (Test 3). In all cases, after treatments, half of the lymphocytes were harvested at time 1 after treatment with doxorubicin (T1) and the other half leaved to recuperate for 24 hs and collected at time 2 (T2). To study the DNA damage we used the Alkaline Comet Assay. The expresion of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1 and hMSH2 was studied by immunocytochemistry. In the group 4 of cells, Test 1 exposed to heat shock and doxorubicin, there were more number of cells with total damage than in the group 3 of cells exposed only to heat shock. In contrast, in the group 4, in Test 2 we observed more cells with score 0 (without damage) than in the group 2. Finally, in group 4 in Test 3 we observed at T2, 40% of cells with total damage and 0% without damage. These preliminary results indicate that hsps have different forms of action on the intensity of the damage induced by doxorubicn and the recuperation time after the heat shock. we observed an increment of hsp27 expression at T2 in Test 1 and 2 induced by doxorubicin, and a nuclear translocation after hrat shock in Test 1 and more significant in Test 2. All cells treated with doxorubicin after heat shock, showed a dimished expression of the hsps in the nucleus, except in the case of Hsp70 and Hsp90 in Test 2. These results correlate with the DNA repair capacity, that in Test 1 was 70.6% and in Test 2 was 83.3%. The nuclear expression of Hsp70, after treatment with doxorubicin in Test 2, correlated with the increased DNA repair capacity.