Apoptotic changes precede mitochondrial dysfunction in red cell-type pyruvate kinase mutant mouse erythroleukemia cell lines

Two erythroleukemia cell lines have been established from the splenic lesions of red blood cell-type pyruvate kinase (R-PK) activity-deficient mice of CBA/N origin infected with a polycythemic strain of Friend leukemia virus complex (FVp). Ten to 30 % of the cells of these cell lines undergo apoptotic changes in routine passage, as shown by nuclear fragmentation, DNA laddering, DNA content (propidium iodide (PI) staining), and annexin V binding assay. In these cells, however, although adenosine 5'-triphosphate (ATP) levels were lower than in the control cells, the mitochondrial inner transmembrane potential (Delta psi(m)), detected by rhodamine 123 (R123) and diSC(3)(5) staining, remained unchanged until the final stage of apoptosis, No evidence was obtained to relate this finding to R-PK mutation due to difficulty in cloning stable, conditionally inducible R-PK gene transfectants. However, low Delta psi(m), in the apoptotic cell population of the control T3-K-1 (K-l) and T3-Cl-2-0 (2-0) Friend erythroleukemia cells supports a possible relationship, as do results obtained in two Friend erythroleukemia cells recently isolated from normal CBA/N mice. These cell lines are expected to be useful for clarifying both the primary apoptotic changes independent of mitochondrial dysfunction and the PK-isozyme changes during erythrodifferentiation, for example, the decreased muscle type 2 (M2) PK level. Modification of growth signals in these cell lines may modulate differentiation and/or apoptosis and allow further elucidation of the signaling networks.