Autologous bone marrow transplantation is used for many patients including those with acute myelogenous leukemias and acute lymphoblastic leukemias who lack appropriate donor marrow. Recurrence of malignancy, however, is a major problem for patients receiving autologous bone marrow. In this study we will explore and optimize the use of hyperthermia to purge leukemic cells from bone marrow specimens in vitro. Heat response of human leukemic cell lines, acute leukemias from patients and normal human bone marrow progenitors (CFU-GM, BFU-E, CFU-GEMM) will be determined using clonogenic assays. Single or fractionated doses of heat with or without chemotherapeutic agents now used in the clinic for in vitro purging of leukemias will be used. Fractionated doses of heat will be used to take advantage of the differential rate of decay of thermotolerance of heat sensitive leukemic cells vs normal bone marrow progenitors. The damage to the bone marrow stem cells and reconstitution of the purged human bone marrow will be measured using long-term bone marrow cultures. To ensure the in vivo reconstitution of the purged bone marrow and to measure the leukemia relapse, a murine leukemia model system will be used. Both AKR leukemia and murine bone marrow will be treated in vitro with single or fractionated hyperthermia with or without chemotherapeutic agents. Furthermore, following purging of leukemia:bone marrow mixtures, both in vivo reconstitution of the bone marrow and leukemia relapse will be measured. Finally, we will test the feasibility of using the expression of the inducible HSP-72 kDa as the predictor of heat response. To determine the expression of the HSP-72 mRNA, in vitro enzymatic amplification of the HSP- 72 mRNA (PCR) will be performed by the synthetic primers. The inducible HSP-72 will then be detected by southern blot analysis using synthetic probes. We will also use 1 & 2 dimensional PAGE to detect the expression of the HSP-72 kDa protein. The expression of the inducible HSP-72 mRNA and proteins will then be correlated with the heat response of human leukemia cells. If there is a positive correlation between the presence of the inducible HSP-72 kDa and thermal resistance, PCR could be used clinically to predict the thermal response of leukemic cells before in vitro purging with heat.
Showing the most recent 10 out of 11 publications
