Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis: A potential therapeutic approach.

We recently demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of de novo cholesterol synthesis, was a potential mediator of the biological effects of retinoic acid on human neuroblastoma cells. The HMG-CoA reductase inhibitor, lovastatin, which is used extensively in the treatment of hypercholesterolemia, induced a potent apoptotic response in human neuroblastoma cells. This apoptotic response was triggered at lower concentrations and occurred more rapidly than had been previously reported in other tumor-derived cell lines, including breast and prostate carcinomas. Because of the increased sensitivity of neuroblastoma cells to lovastatin-induced apoptosis, we examined the effect of this agent on a variety of tumor cells, including leukemic cell lines and primary patient samples. Based on a variety of cytotoxicity and apoptosis assays, the 6 acute lymphocytic leukemia cell lines tested displayed a weak apoptotic response to lovastatin. In contrast, the majority of the acute myeloid leukemic cell lines (6/7) and primary cell cultures (13/22) showed significant sensitivity to lovastatin-induced apoptosis, similar to the neuroblastoma cell response. Of significance, in the acute myeloid leukemia, but not the acute lymphocytic leukemia cell lines, lovastatin-induced cytotoxicity was pronounced even at the physiological relevant concentrations of this agent. Therefore, our study suggests the evaluation of HMG-CoA reductase inhibitors as a therapeutic approach in the treatment of acute myeloid leukemia.

Relationships between the mitochondrial permeability transition and oxidative stress during ara-C toxicity.

The mitochondrial permeability transition and oxidative stress seem to be critical alterations in cellular physiology that take place during programmed cell death. Failure to undergo apoptosis is associated with drug resistance in acute myeloid leukemia and other cancers. Therefore, it is important to establish causal relationships between the physiological changes that take place in apoptosis, because these are potential targets for novel treatment strategies to overcome this form of drug resistance. We describe the use of multilaser flow cytometry methods to make correlated measurements of mitochondrial membrane potential (MMP), the generation of reactive oxygen intermediates, the cellular content of reduced glutathione (GSH), intracellular calcium, and exposure of phosphatidylserine on the cell surface. Using these combined methods, we have mapped a "death sequence" that occurs after treatment of leukemic blasts with clinically relevant concentrations of 1-beta-D-arabinofuranosylcytosine (ara-C). Dual labeling of MMP and cellular glutathione content showed that loss of MMP, indicative of the permeability transition, took place in cells that were depleted of glutathione. The loss of MMP coincided with phosphatidylserine exposure and preceded a state of high reactive oxygen generation. Finally, there was an increase in intracellular calcium. These results demonstrate that the mitochondrial permeability transition takes place during ara-C toxicity but suggest that this occurs downstream of the loss of GSH. Thus, oxidative stress after ara-C-induced toxicity seems to be a biphasic phenomenon, with the permeability transition occurring after a depletion of GSH and preceding a state of high reactive oxygen generation.

Resistance of hepatic nodules to orotic acid-induced accumulation of uridine nucleotides.

It has been hypothesized that orotic acid (OA) promotes rat liver carcinogenesis by a differential mitoinhibitory mode. Consistent with this hypothesis, hepatic nodules are relatively resistant to OA-induced mitoinhibition. OA-induced mitoinhibition is dependent on the metabolism of OA to uridine nucleotides. The present studies investigate the uptake and metabolic pathway of OA, both in vivo and in vitro, as a possible basis for the resistance of hepatic nodules to OA-induced mitoinhibition. Rats bearing hepatic nodules exposed to 1% dietary OA exhibited increased levels of uridine nucleotides in the surrounding non-nodular liver (from 0.44 to 0.70 mg/g liver) but not in the hepatic nodules. Further, following administration of [3H]OA i.p., nodules have significantly lower levels of acid-soluble radioactivity compared to the non-nodular surrounding tissue. Furthermore, most of the acid-soluble radioactivity was present as uridine nucleotides, suggesting that the OA taken up was converted to uridine nucleotides. Similarly, hepatocytes from nodules in primary culture incubated with radiolabeled OA, have significantly lower levels (46-60%) of acid-soluble radioactivity. These results suggest that the decreased uptake of OA by hepatic nodules may be a factor contributing to the observed resistance of hepatic nodules to the mitoinhibitory effects of OA.