Genetic Mechanisms of Cytarabine Resistance in Leukemia: Cell Culture and Mouse Transplantation Models.

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Chemoresistance remains a major barrier to successful clinical treatment of acute myeloid leukemia (AML). However, the underlying molecular mechanisms of chemoresistance in AML are largely unknown. Previously, we reported that loss of tumor suppressor genes, Nf1 and Trp53, can confer upon myeloid leukemia cells partial resistance to cytarabine (Ara-C), a mainstay of AML chemotherapy. Microarray gene expression profiling revealed that a group of lysosome function-related genes, and a major regulator of apoptosis, Bcl-xL, were upregulated in in vitro Ara-C resistant myeloid leukemia cell lines. We now report that the lysosome number and size is increased in resistant cells. Also, overexpression of Bcl-xL in both murine and human myeloid leukemia cell lines increased Ara-C resistance to a variant extent in different lines. Furthermore, we found that the key enzyme involved in intracellular activation of Ara-C, Cda, can do so, too, but to a less extent than Bcl-xL. Interestingly, in our Ara-C resistant AML cell lines which were retrovirally induced in BXH-2 stain of mice, we were able to detected new proviral insertion sites which may cause mutations associated with the leukemia resistance feature. We have now examined the possibility of modeling AML chemoresistance in mouse, and through this model, to identify and characterize AML relapse-related genetic abnormalities. During establishing such a model, treatment dosage, intervals, courses, routes of administration, the number of injection, food preparation and supplements have been considered for testing. Ara-C treatment caused a decrease in body weights and peripheral blood cell counts. One major lethal toxicity response, intestine deterioration, appeared to be correlated with high doses of Ara-C. We arrived at a regimen comprised of two 10-day treatment courses at 90 mg/kg/day by one intraperitoneal injection, with a 5-day break between treatment courses. This treatment course was found to give rise to 10~30% lethality due to Ara-C toxicity but it can also suppress the onset of transplanted AML disease in recipient mice for nearly two weeks compared to saline-treated mice as a control. Interestingly, the tumors collected from Ara-C treated transplants displayed higher resistance than saline treated tumors in a methylcellulose colony-forming assay. Testing of apoptosis regulatory genes for Ara-C response in primary tumors is in process.