Death-resistant and nonresistant malignant human cell lines under anoxia in vitro

Erythropoietin supports the survival of erythroblasts. We previously demonstrated that 24 malignant human cell lines expressed erythropoietin and its receptor and that erythropoietin secretion was enhanced under anoxia. In this study, we examined the viability of 22 of these cell lines excluding two leukemia cell lines under anoxia. Twenty-two cancer cell lines of various origins were cultured under anoxia or normoxia for 4 days, and their viability was examined at 1-day intervals. The levels of lactate and ATP were measured. The expressions of hypoxia-inducible transcription factor 1alpha (HIF-1alpha) and Bcl-2 family proteins were examined by western blotting analysis. The cellular and mitochondrial features were examined by microscopy. Eleven of the 22 cancer cell lines examined showed 80% to 100% cell viability after 4 days under anoxia; 2 cell lines showed similar viability for 3 days, 3 cell lines showed similar viability for 2 days, and 6 cell lines showed similar viability for 1 day or less. These 11 death-resistant cell lines, which secrete various amounts of erythropoietin under anoxia, produced significantly more lactate during 2 days under anoxia than under normoxia, with ATP levels about 60% of those before anoxia. ATP returned to the normal level when normoxia was restored after 4 days of anoxia. However, the nonresistant cell lines responded to anoxia by yielding significantly more lactate without a reduction of the ATP level. The expression patterns of Bcl-2 family proteins revealed that apoptosis-inhibiting signals predominated over proapoptotic signals in the death-resistant cells under anoxia. The majority of the cancer cell lines examined survived under anoxia in vitro, through the Pasteur effect, in a dormant state without direct support of erythropoietin.