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DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells.

ABSTRACT: The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Microtubule polymerization inhibitor exhibited a reduced response in shDRG2 cells. Using immunoprecipitation, we show that DRG2 interacts with tau, which regulates microtubule polymerization. Collectively, these data demonstrate that DRG2 may aid in affecting microtubule dynamics in HeLa cells.

SUBMITTER: Dang T

PROVIDER: S-EPMC6315320 | biostudies-other | 2018 Dec

REPOSITORIES: biostudies-other

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DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells.

Dang Thao T Jang Soo Hwa SH Back Sung Hoon SH Park Jeong Woo JW Han In-Seob IS

Molecules and cells 20181114 12

The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Micr ...[more]

PMID: 30453731

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Project description:BackgroundCombination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.MethodsCell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and ?-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, ?-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-?-tubulin antibody and PI, respectively.ResultsWe found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKC?, but not PKC? and PKC?, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing ?- and ?-tubulin protein levels in a PKC-dependent manner.ConclusionsThese results suggest that the synergy between PMA and apicularen A is involved by PKC? activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.

Dataset Information

DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells.

Publications

DRG2 Deficiency Causes Impaired Microtubule Dynamics in HeLa Cells.

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