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Mitochondrial antisense RNA for cytochrome C oxidase (MARCO) can induce morphologic changes and cell death in human
N Shirafuji1, S Takahashi, S Matsuda
1Department of Hematology/Oncology, and the Department of Oncology, The Institute of Medical Science, The University of Tokyo, Japan.
Abstract:
To identify essential molecules capable of inducing terminal morphologic maturation and cell death of myeloid progenitor cells, we isolated cDNA clones by functional expression cloning using a library constructed from all-trans retinoic acid (ATRA)-treated human promyelocytic HL-60 cells. Clones which induced morphologic changes in HL-60 cells from blastic cells to mature neutrophilic granulocytes were selected. The isolated positive cDNA clone was demonstrated to encode an antisense RNA for cytochrome c oxidase/serine tRNA derived from a mitochondrial gene (MARCO). When MARCO was expressed in HL-60 cells with the lac switch system, blastic cell morphology became neutrophilic after 48-hour incubation with IPTG, and cell death was observed after 3 days. Also, high molecular weight DNA fragmentation was observed after 36 hours in culture. Similar results were observed using transformants from human K562 cells and CMK cells. RT-PCR analysis revealed that MARCO was transcribed in both ATRA and TNF-alpha systems, and also in human blood neutrophilic granulocytes. Following transfection with cytochrome c oxidase expression plasmids, TNF-alpha-induced high molecular weight DNA fragmentation in U937 cells and HL-60 cells was inhibited in these transformants. These results indicate that maturational changes in hematopoietic cells and the process of cell death may be induced by mitochondrial respiratory insufficiency, and also that the mitochondrial gene MARCO may be used as one of the candidates for gene supplementation therapy for the acute leukemias.
Insights
Researchers identified MARCO, a mitochondrial gene, that induces maturation and cell death in myeloid progenitor cells. This discovery offers potential gene therapy for acute leukemias by targeting mitochondrial function.
Area of Science:
- Molecular Biology
- Cell Biology
- Hematology
Background:
- Myeloid progenitor cells require specific molecular signals for terminal differentiation and apoptosis.
- All-trans retinoic acid (ATRA) is known to induce differentiation in certain leukemia cell lines.
Purpose of the Study:
- To identify novel molecules that induce terminal differentiation and cell death in myeloid progenitor cells.
- To investigate the role of mitochondrial genes in hematopoietic cell maturation and apoptosis.
Main Methods:
- Functional expression cloning using a cDNA library from ATRA-treated HL-60 cells.
- Induction of MARCO expression using the lac switch system in HL-60, K562, and CMK cells.
- Analysis of morphologic changes, cell death, and DNA fragmentation.
- RT-PCR to detect MARCO transcription.
- Inhibition studies using cytochrome c oxidase expression plasmids.
Main Results:
- A cDNA clone encoding antisense RNA for mitochondrial MARCO was isolated.
- MARCO expression induced neutrophilic differentiation and cell death in myeloid leukemia cell lines.
- High molecular weight DNA fragmentation, indicative of apoptosis, was observed.
- MARCO is transcribed in ATRA and TNF-alpha treated cells and in mature neutrophils.
- Cytochrome c oxidase expression inhibited TNF-alpha-induced DNA fragmentation.
Conclusions:
- Mitochondrial respiratory insufficiency can induce maturational changes and cell death in hematopoietic cells.
- The mitochondrial gene MARCO is a potential candidate for gene supplementation therapy in acute leukemias.