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p150 ADAR1 isoform involved in maintenance of HeLa cell proliferation.

Wang H, Hou Z, Wu Y, Ma X, Luo X - BMC Cancer (2006)

Bottom Line: Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150.Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth.The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, School of Pharmacy, the Fourth Military Medical University, Xi'an 710032, PR China. wang.haifang@gmail.com

ABSTRACT

Background: RNA-specific adenosine deaminase ADAR1 is ubiquitously expressed in a variety of mammalian cells and tissues. Although its physiological importance in non-nervous tissues has been confirmed by analysis of mutation phenotypes, few endogenous editing substrates have been identified in numerous peripheral tissues and biological function of ADAR1 has not been fully understood.

Methods: A conditional site-specific, ribozyme-based gene knock-down strategy was utilized to study the function of full-length isoform of ADAR1 (p150 protein) in HeLa cell. Double-stable HeLa cell lines were developed by transfecting HeLa Tet-On cells with a pTRE-derived plasmid that can express a hammerhead ribozyme against mRNA of p150 ADAR1 isoform under induction condition. Semi-quantitative RT-PCR and Western blotting were performed to measure the expression of p150 in selected cell clones. Cell proliferation was evaluated by means of MTT assay and growth curve analysis. Cellular morphological changes were observed under light microscope. Flow Cytometry was used for cell cycle analysis. Growth rate of cell transplants in BALB/c nude mice was also investigated.

Results: Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150. Additionally, cell cycle analysis showed that cell progression from G1 phase to S phase was retarded in the ADAR1 p150 suppressed cells.

Conclusion: Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth. The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes.

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Cell cycle analysis of HeLa-Rz cells. Propidium iodide staining was performed and samples were analyzed by flow cytometry. A. Cell cycle of untreated cells. B. Cell cycle is altered after 48 h of Dox (2 μg/mL) treatment.
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Figure 4: Cell cycle analysis of HeLa-Rz cells. Propidium iodide staining was performed and samples were analyzed by flow cytometry. A. Cell cycle of untreated cells. B. Cell cycle is altered after 48 h of Dox (2 μg/mL) treatment.

Mentions: HeLa-Rz cells were grown in medium containing 2 μg/mL Dox for 48 h, causing the expression level of ADAR1 mRNA to decrease. Flow cytometry analysis detected a constant increase in G1 DNA content (from 38.87 ± 0.11 % to 46.53 ± 0.10 %, p < 0.05) in comparison with the control group. The percentage of cell DNA in replication phase S slightly decreased, but statically the change was not significant (39.94 ± 0.08 % vs 37.49 ± 0.07 %, p > 0.05). These data suggest that Dox treatment, acting through knockdown of the p150 ADAR1 mRNA, could lead to an arrest of cells in G1 phase, a delayed transition into S phase, and eventually restrained proliferation of cells (Fig 4). Under our experimental condition, Dox did not induce apoptosis.


p150 ADAR1 isoform involved in maintenance of HeLa cell proliferation.

Wang H, Hou Z, Wu Y, Ma X, Luo X - BMC Cancer (2006)

Cell cycle analysis of HeLa-Rz cells. Propidium iodide staining was performed and samples were analyzed by flow cytometry. A. Cell cycle of untreated cells. B. Cell cycle is altered after 48 h of Dox (2 μg/mL) treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764903&req=5

Figure 4: Cell cycle analysis of HeLa-Rz cells. Propidium iodide staining was performed and samples were analyzed by flow cytometry. A. Cell cycle of untreated cells. B. Cell cycle is altered after 48 h of Dox (2 μg/mL) treatment.
Mentions: HeLa-Rz cells were grown in medium containing 2 μg/mL Dox for 48 h, causing the expression level of ADAR1 mRNA to decrease. Flow cytometry analysis detected a constant increase in G1 DNA content (from 38.87 ± 0.11 % to 46.53 ± 0.10 %, p < 0.05) in comparison with the control group. The percentage of cell DNA in replication phase S slightly decreased, but statically the change was not significant (39.94 ± 0.08 % vs 37.49 ± 0.07 %, p > 0.05). These data suggest that Dox treatment, acting through knockdown of the p150 ADAR1 mRNA, could lead to an arrest of cells in G1 phase, a delayed transition into S phase, and eventually restrained proliferation of cells (Fig 4). Under our experimental condition, Dox did not induce apoptosis.

Bottom Line: Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150.Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth.The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, School of Pharmacy, the Fourth Military Medical University, Xi'an 710032, PR China. wang.haifang@gmail.com

ABSTRACT

Background: RNA-specific adenosine deaminase ADAR1 is ubiquitously expressed in a variety of mammalian cells and tissues. Although its physiological importance in non-nervous tissues has been confirmed by analysis of mutation phenotypes, few endogenous editing substrates have been identified in numerous peripheral tissues and biological function of ADAR1 has not been fully understood.

Methods: A conditional site-specific, ribozyme-based gene knock-down strategy was utilized to study the function of full-length isoform of ADAR1 (p150 protein) in HeLa cell. Double-stable HeLa cell lines were developed by transfecting HeLa Tet-On cells with a pTRE-derived plasmid that can express a hammerhead ribozyme against mRNA of p150 ADAR1 isoform under induction condition. Semi-quantitative RT-PCR and Western blotting were performed to measure the expression of p150 in selected cell clones. Cell proliferation was evaluated by means of MTT assay and growth curve analysis. Cellular morphological changes were observed under light microscope. Flow Cytometry was used for cell cycle analysis. Growth rate of cell transplants in BALB/c nude mice was also investigated.

Results: Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150. Additionally, cell cycle analysis showed that cell progression from G1 phase to S phase was retarded in the ADAR1 p150 suppressed cells.

Conclusion: Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth. The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes.

Show MeSH
Related in: MedlinePlus