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

Area of Science:

• * Hematology
• * Molecular Biology
• * Cell Biology

Background:

• * Established two novel erythroleukemia cell lines from R-PK deficient mice infected with Friend leukemia virus.
• * Observed spontaneous apoptotic changes in 10-30% of cells during routine passage.

Purpose of the Study:

• * To investigate the role of mitochondrial inner transmembrane potential (Δψm) in apoptosis in these erythroleukemia cells.
• * To explore potential relationships between pyruvate kinase (PK) activity and apoptotic mechanisms.
• * To utilize these cell lines for studying erythrodifferentiation and signaling networks.

Main Methods:

• * Cell culture and induction of apoptosis.
• * Assays for apoptotic markers: nuclear fragmentation, DNA laddering, propidium iodide (PI) staining, annexin V binding.
• * Measurement of Adenosine 5'-triphosphate (ATP) levels.
• * Assessment of mitochondrial inner transmembrane potential (Δψm) using rhodamine 123 (R123) and diSC3(5) staining.

Main Results:

• * Cells exhibited typical apoptotic features, including DNA fragmentation and annexin V binding.
• * Lower ATP levels were observed in apoptotic cells compared to controls.
• * Crucially, mitochondrial inner transmembrane potential (Δψm) remained largely unchanged until late-stage apoptosis.
• * Difficulty in creating stable R-PK gene transfectants precluded direct correlation with R-PK mutation.

Conclusions:

• * Apoptosis in these erythroleukemia models can occur independently of significant mitochondrial dysfunction.
• * The findings suggest a complex interplay between PK isozymes, mitochondrial function, and apoptosis during erythrodifferentiation.
• * These cell lines provide a valuable tool for dissecting early apoptotic events and PK-isozyme regulation.