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

NADIN SB; LM VARGAS ROIG; DR CIOCCA

Mendoza, Argentina

Workshop; 3RD International Workshop on Molecular Biology of Stress Responses; 2001

International Cell Stress Society

Doxorubicin is used as a first line chemotherapy in several human tumors. This drug has differente mechanisms of action to damage the tumor cells producing double strand breaks, Topoisomerase II inhibition, DNA crosslinks, and oxidative damage. In previous studies, we have reported that hsp27 and hsp70 may be involved in doxorubicin resistance in breast cancer cells in vitro and in vivo. To advance our knowledge in these hsps and doxorubicin, in this sutdy 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 blood lymphocytes were obtained from peripheral blood of a nonsmoker donor, separated and used in culture. The lymphocytes were incubated in appropiate conditions. The sample was divided in four groups: (1) control, without treatment, (2) treated with 21 nM of doxorubicin for one hour without heat shock with different induction times for hsps after doxorubicin exposure Test 1: 4 h and Test 2: 24 h) or for 24 hours with doxorubicin (Test 3: hsps induction time 24 h), (3) exposed only to a heat shock of 42ºC for 1 hour, and (4) exposed to the heat shock, leave for recuperate for 4 h (Test 1) or 24 h (Test2), and then treated with 21 nM of doxorubicin for 1 hour (Test 1 and 2) or 24 hs (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 expression 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) tahn in the group 2. Finalyy, in group 4 in Test 3 we observed at T2, 40% of cells with total damage and 0% of cells without damage. These preliminary results indicate thata hsps have different forms of action depending on the intensity of the damage induced by doxorubicin 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 heat shock in Test 1 and more significant in Test 2. All cells treated with doxorubicin after heat shock, showed a diminished 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.