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Title: p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts

Abstract

This study focused on the activation of cell cycle checkpoint responses in diploid human fibroblasts that were treated with cadmium chloride and the potential roles of ATM and p53 signaling pathways in cadmium-induced responses. The alkaline comet assay indicated that cadmium caused a dose-dependent increase in DNA damage. Cells that were rendered p53-defective by expression of a dominant-negative p53 allele or knockdown of p53 mRNA were more resistant to cadmium-induced inactivation of colony formation than normal and ataxia telangiectasia (AT) cells. Synchronized fibroblasts in S were more sensitive to cadmium toxicity than cells in G1, suggesting that cadmium may target some element of DNA replication. Cadmium produced a dose- and time-dependent inhibition of DNA synthesis. An immediate inhibition was associated with severe delay in progression through S phase and a delayed inhibition seen 24 h after treatment was associated with accumulation of cells in G2. AT and normal cells displayed similar patterns of inhibition of DNA synthesis and G2 delay after treatment with cadmium, while p53-defective cells displayed significantly less of the delayed inhibition of DNA synthesis and accumulation in G2 post-treatment. Total p53 protein and ser15-phosphorylated p53 were induced by cadmium in normal and AT cells. The p53 transactivationmore » target Gadd45{alpha} was induced in both p53-effective and p53-defective cells after 4 h cadmium treatment, and this was associated with an acute inhibition of mitosis. Cadmium produced a very unusual pattern of toxicity in human fibroblasts, inhibiting DNA replication and inducing p53-dependent growth arrest but without induction of p21{sup Cip1/Waf1} or activation of Chk1.« less

Authors:
 [1];  [2]; ;  [3];  [3];  [4];  [5];  [2];  [6]
  1. Dept. of Toxicology, School of Public Health, Medical Center of Fudan Univ., Shanghai (China)|[Dept. of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Center for Environmental Health and Susceptibility, Univ. of North Carolina, Chapel Hill, North Carolina 27599 (United States)
  2. Dept. of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Center for Environmental Health and Susceptibility, Univ. of North Carolina, Chapel Hill, North Carolina 27599 (United States)
  3. Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Center for Environmental Health and Susceptibility, University of North Carolina, Chapel Hill, North Carolina 27599 (United States)
  4. Department of Toxicology, School of Public Health, Medical Center of Fudan University, Shanghai (China)|[Dept. of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Center for Environmental Health and Susceptibility, Univ. of North Carolina, Chapel Hill, North Carolina 27599 (United States)
  5. Dept. of Toxicology, School of Public Health, Medical Center of Fudan Univ., Shanghai (China)
  6. Dept. of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, and Center for Environmental Health and Susceptibility, Univ. of North Carolina, Chapel Hill, North Carolina 27599 (United States). E-mail: wkarlk@med.unc.edu
Publication Date:
OSTI Identifier:
20976851
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 218; Journal Issue: 2; Other Information: DOI: 10.1016/j.taap.2006.10.031; PII: S0041-008X(06)00411-X; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CADMIUM; CADMIUM CHLORIDES; CARBOXYLIC ACIDS; CELL CYCLE; CHLORINATED ALIPHATIC HYDROCARBONS; COLONY FORMATION; DEOXYURIDINE; DNA; DNA REPLICATION; FIBROBLASTS; INHIBITION; MITOSIS; PROTEINS; SODIUM; STRAND BREAKS; SULFATES; SYNTHESIS; TIME DEPENDENCE; TOXICITY

Citation Formats

Cao Feng, Zhou Tong, Simpson, Dennis, Zhou Yingchun, Boyer, Jayne, Chen Bo, Jin Taiyi, Cordeiro-Stone, Marila, and Kaufmann, William. p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts. United States: N. p., 2007. Web.
Cao Feng, Zhou Tong, Simpson, Dennis, Zhou Yingchun, Boyer, Jayne, Chen Bo, Jin Taiyi, Cordeiro-Stone, Marila, & Kaufmann, William. p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts. United States.
Cao Feng, Zhou Tong, Simpson, Dennis, Zhou Yingchun, Boyer, Jayne, Chen Bo, Jin Taiyi, Cordeiro-Stone, Marila, and Kaufmann, William. Mon . "p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts". United States. doi:.
@article{osti_20976851,
title = {p53-dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts},
author = {Cao Feng and Zhou Tong and Simpson, Dennis and Zhou Yingchun and Boyer, Jayne and Chen Bo and Jin Taiyi and Cordeiro-Stone, Marila and Kaufmann, William},
abstractNote = {This study focused on the activation of cell cycle checkpoint responses in diploid human fibroblasts that were treated with cadmium chloride and the potential roles of ATM and p53 signaling pathways in cadmium-induced responses. The alkaline comet assay indicated that cadmium caused a dose-dependent increase in DNA damage. Cells that were rendered p53-defective by expression of a dominant-negative p53 allele or knockdown of p53 mRNA were more resistant to cadmium-induced inactivation of colony formation than normal and ataxia telangiectasia (AT) cells. Synchronized fibroblasts in S were more sensitive to cadmium toxicity than cells in G1, suggesting that cadmium may target some element of DNA replication. Cadmium produced a dose- and time-dependent inhibition of DNA synthesis. An immediate inhibition was associated with severe delay in progression through S phase and a delayed inhibition seen 24 h after treatment was associated with accumulation of cells in G2. AT and normal cells displayed similar patterns of inhibition of DNA synthesis and G2 delay after treatment with cadmium, while p53-defective cells displayed significantly less of the delayed inhibition of DNA synthesis and accumulation in G2 post-treatment. Total p53 protein and ser15-phosphorylated p53 were induced by cadmium in normal and AT cells. The p53 transactivation target Gadd45{alpha} was induced in both p53-effective and p53-defective cells after 4 h cadmium treatment, and this was associated with an acute inhibition of mitosis. Cadmium produced a very unusual pattern of toxicity in human fibroblasts, inhibiting DNA replication and inducing p53-dependent growth arrest but without induction of p21{sup Cip1/Waf1} or activation of Chk1.},
doi = {},
journal = {Toxicology and Applied Pharmacology},
number = 2,
volume = 218,
place = {United States},
year = {Mon Jan 15 00:00:00 EST 2007},
month = {Mon Jan 15 00:00:00 EST 2007}
}
  • Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore,more » Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.« less
  • Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes andmore » induces nuclear aberrations and a G{sub 1}/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the mature centriole present at microtubule foci, indicates that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities.« less
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  • Synchronous human diploid fibroblasts were exposed to 1000 rad of X rays at various times in S phase, and thymidine incorporation, an indicator of DNA synthesis, was measured throughout the cell cycle. When cells were irradiated just before the time when thymidine incorporation peaked in control cells, the peak was strongly suppressed. Irradiation after appearance of an incorporation peak in control cells resulted in some depression of thymidine incorporation but had much less effect than irradiation before the peak. These results are in agreement with the interpretation that, in human diploid fibroblasts, most replicons initiate during relatively restricted times inmore » S phase, leading to the multiple thymidine incorporation peaks observed in these cells.« less