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Polyploidy associated with oxidative injury attenuates proliferative potential of cells

Giridhar R. Gorla1,4, Harmeet Malhi1,5 and Sanjeev Gupta1,2,3,5,*

1 Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
2 Cancer Research Center, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
3 General Clinical Research Center, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
4 Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
5 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, NY 10461, USA
* Author for correspondence (e-mail: sanjvgupta{at}pol.net )



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  		Fig. 1. Hepatocytes showed 8-hydroxyguanine DNA adducts following partial hepatectomy. The liver was immunostained to demonstrate oxidative DNA injury as described in Materials and Methods. (A) No nuclear signal was observed in negative controls when the primary antibody was omitted. (B) Liver removed in a partial hepatectomy showing staining of occasional cell nuclei (arrows). The immunostaining is present in hepatocytes, as well as nonparenchymal cells. (C) Liver from the same animal as in B 5 days after two-thirds partial hepatectomy showing extensive and more intense nuclear staining of hepatocytes (arrows). Nonparenchymal cells also exhibit increased DNA injury following partial hepatectomy. (D) Higher magnification view of the liver in C showing stippled staining of nuclear DNA in hepatocytes. Note presence of polyploid cells with megalonuclei (arrow). The inset shows an apoptotic cell with nuclear fragments containing stained DNA (inset). Oxidative DNA injury was seen in 40-50% of the hepatocytes after partial hepatectomy. p, portal area.

 


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  		Fig. 2. Evidence for oxidative injury in hepatocytes from rats with or without partial hepatectomy. The cells were cultured for 5 days followed by measurement of catalase activity (A), glutathione content (B) and lipid peroxidation (C). Cells from partially hepatectomized livers showed depletion of catalaste and glutathione from the beginning, whereas these activities declined significantly with time in cells isolated from the unperturbed liver. Radiation treatment induced dose-dependent changes in catalase and glutathione activities, which declined progressively in cultured cells. Catalase activity eventually became undetectable in cells from partially hepatectomized livers following radiation. These findings suggest greater lipid peroxidation in cells from partially hepatectomized livers following radiation. All measurements were taken in triplicate and data were reproduced in two independent experiments.

 


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  		Fig. 3. Analysis of proliferative capacity in transplanted hepatocytes. (A,B) Transplanted cell engraftment in animals 2 days after cell transplantation. (A) Occasional transplanted cells from an unperturbed liver in periportal areas (arrows) and (B) irradiated cells from a partially hepatectomized liver. (C-F) Proliferation in transplanted cells 10 days after cell transplantation. (C) Unperturbed hepatocytes from the normal liver with two large transplanted cell foci each containing >50 cells (arrows). (D) Radiation of cells from the normal liver decreased proliferative activity with only 18 cells in the transplanted cell focus. (E) Cells from partially hepatectomized liver forming small foci (arrow). (F) Radiation of partially hepatectomized cells abolished proliferative activity and transplanted cells remained as discrete cells, as seen with three transplanted cells (arrows) in the field.

 


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  		Fig. 4. Flow cytometric evidence for induction of polyploidy in hepatocytes. Cells were isolated from the unperturbed normal rat liver and cultured for 5 days with various treatments as indicated. (A) Flow cytometric profiles from control untreated cells, and cells treated with TGF{alpha}, norepinephrine (NE), vasopressin (VP) and 30 Gy radiation. DNA content is on the x axis with diploid (2C), tetraploid (4C) and octaploid (8C) peaks as shown and the y axis is the number of nuclei analyzed. (B) Analysis of ploidy distribution data from studies shown in A. (C) Laser scanning cytometric analysis of sorted propidium iodide-stained nuclei from diploid, tetraploid and octaploid DNA peaks with columns 1 to 3 showing three representative examples each of sorted nuclei with progressive nuclear enlargement.

 

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