Dataset Information

MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

ABSTRACT: The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 decatenation checkpoint response. However, the mitotic defects could be accounted for by a minor defect in the activity of Topo II during G2/M. To test this hypothesis, we have tracked at live single cell resolution the dynamics of mitosis in MCPH1 depleted HeLa cells upon catalytic inhibition of Topo II. Our analyses demonstrate that neither chromosome alignment nor segregation are more susceptible to minor perturbation in decatenation in MCPH1 deficient cells, as compared with control cells. Interestingly, MCPH1 depleted cells were more prone to mitotic cell death when decatenation was perturbed. Furthermore, when the G2 arrest that was induced by catalytic inhibition of Topo II was abrogated by Chk1 inhibition, the incidence of mitotic cell death was also increased. Taken together, our data suggest that the MCPH1 lack of function increases mitotic cell hypersensitivity to the catalytic inhibition of Topo II.

SUBMITTER: Arroyo M

PROVIDER: S-EPMC7231051 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

Arroyo María M Sánchez Antonio A Cañuelo Ana A Heredia-Molina Rosalía F RF Martínez-Molina Eduardo E Clarke Duncan J DJ Marchal Juan Alberto JA

Genes 20200408 4

The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 ...[more]

PMID: 32276518

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Project description:BackgroundPaclitaxel (PTX), a first-line therapy for triple negative breast cancers (TNBC) induces anti-tumor activity by microtubule stabilization and inhibition of cell division. Its dose-limiting toxicity and short half-life, however, pose clinical challenges underscoring the need for strategies that increase its efficiency. RAD6, a E2 ubiquitin conjugating enzyme, is associated with centrosomes at all phases of cell cycle. Constitutive overexpression of the RAD6B homolog in normal breast cells induces centrosome amplification and multipolar spindle formation, indicating its importance in centrosome regulation.MethodsTNBC centrosome numbers were scored by pericentrin immunostaining. PTX sensitivities and interactions with SMI#9, a RAD6-selective small molecule inhibitor, on TNBC cell survival were analyzed by MTT and colony forming assays and an isogenic MDA-MB-468 TNBC model of PTX resistance. The molecular mechanisms underlying PTX and SMI#9 induced cytotoxicity were determined by flow cytometry, immunoblot analysis of cyclin B1 and microtubule associated protein TAU, and dual immunofluorescence staining of TAU and α-tubulin.ResultsOur data show aberrant centrosome numbers and that PTX sensitivities are not correlated with TNBC BRCA1 status. Combining PTX with SMI#9 synergistically enhances PTX sensitivities of BRCA1 wild-type and mutant TNBC cells. Whereas SMI#9/PTX combination treatment increased cyclin B1 levels in MDA-MB-468 cells, it induced cyclin B1 loss in HCC1937 cells with accumulation of reproductively dead giant cells, a characteristic of mitotic catastrophe. Cell cycle analysis revealed drug-induced accumulation of tetraploid cells in S and G2/M phases, and robust increases in cells with 4 N DNA content in HCC1937 cells. TAU overexpression is associated with reduced PTX efficacy. Among the six TAU isoforms, both SMI#9 and PTX downregulated 1N3R TAU in MDA-MB-468 and HCC1937 cells, suggesting a common mechanism of 1N3R regulation. Dual TAU and α-tubulin immunostaining showed that SMI#9 induces monopolar mitotic spindles. Using the isogenic model of PTX resistance, we show that SMI#9 treatment restores PTX sensitivity.ConclusionsThese data support a common mechanism of microtubule regulation by SMI#9 and PTX and suggest that combining PTX with RAD6 inhibitor may be beneficial for increasing TNBC sensitivities to PTX and alleviating toxicity. This study demonstrates a new role for RAD6 in regulating microtubule dynamics.

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MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

Publications

MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

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