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MAPK mediates RAS-induced chromosome instability
H I Saavedra1, K Fukasawa, C W Conn
1Department of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA.
Abstract:
The generation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic material. The involvement of the MAPK pathway in mediating v-ras-induced micronuclei in NIH 3T3 cells was examined by inhibiting MAPK activation. Conversely, the MAPK pathway was constitutively activated by infecting cells with a v-mos retrovirus. Micronucleus formation was inhibited by the MAPK kinase inhibitors PD98059 and U0126, but not by wortmannin, an inhibitor of the Ras/phosphatidylinositol 3-kinase pathway. Transduction of cells with v-mos resulted in an increase in micronucleus formation, also consistent with the involvement of the MAPK pathway. Staining with the anti-centromeric CREST antibody revealed that instability induced by constitutive activation of MAPK is due predominantly to aberrant mitotic segregation, since most of the micronuclei were CREST-positive, reflective of lost chromosomes. A significant fraction of the micronuclei were CREST-negative, reflective of lost acentric chromosome fragments. Some of the instability observed was due to mitotic events, consistent with the increased formation of bi-nucleated cells, which result from perturbations of the mitotic spindle and failure to undergo cytokinesis. This chromosome instability, therefore, is a consequence of mitotic aberrations, mediated by the MAPK pathway, including centrosome amplification and formation of mitotic chromosome bridges.
Insights
The MAPK pathway mediates micronucleus formation, a sign of DNA damage. Inhibiting this pathway reduces micronuclei, while its activation increases them, indicating its role in chromosome instability.
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Micronuclei formation indicates DNA damage, mitotic defects, and genetic material loss.
- The Mitogen-Activated Protein Kinase (MAPK) pathway is implicated in cellular stress responses.
Purpose of the Study:
- To investigate the role of the MAPK pathway in mediating v-ras-induced micronucleus formation in NIH 3T3 cells.
- To determine if constitutive MAPK activation influences chromosome instability.
Main Methods:
- Inhibition of MAPK activation using specific kinase inhibitors (PD98059, U0126).
- Constitutive activation of the MAPK pathway via v-mos retrovirus infection.
- Analysis of micronuclei using CREST antibody staining to identify chromosome loss and segregation errors.
Main Results:
- MAPK kinase inhibitors PD98059 and U0126 significantly reduced micronucleus formation.
- Wortmannin, a Ras/phosphatidylinositol 3-kinase inhibitor, did not affect micronucleus formation.
- Constitutive MAPK activation by v-mos increased micronucleus formation, predominantly CREST-positive (lost chromosomes) and some CREST-negative (acentric fragments).
- Observed instability included bi-nucleated cells, centrosome amplification, and mitotic chromosome bridges, indicating mitotic aberrations.
Conclusions:
- The MAPK pathway plays a crucial role in mediating micronucleus formation and chromosome instability.
- Constitutive MAPK activation leads to chromosome loss and segregation errors, primarily through aberrant mitosis.
- Mitotic aberrations, including spindle perturbations and cytokinesis failure, are key mechanisms underlying MAPK-mediated chromosome instability.