|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
Journal of Cell Science, Vol 107, Issue 5 1169-1179, Copyright © 1994 by Company of Biologists
JOURNAL ARTICLES |
GV Kulkarni and CA McCulloch
Faculty of Dentistry, University of Toronto, Ontario, Canada.
Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)
This article has been cited by other articles:
|
|
 |
|
  J. G. Crowston, X. Y. Wang, P. T. Khaw, H. Zoellner, and P. R. Healey Human Serum Reduces Mitomycin-C Cytotoxicity in Human Tenon's Fibroblasts. Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 946 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Park, A. Helip-Wooley, and J. Thoene Lysosomal Cystine Storage Augments Apoptosis in Cultured Human Fibroblasts and Renal Tubular Epithelial Cells J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2878 - 2887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Bordone and C. Campbell DNA Ligase III Is Degraded by Calpain during Cell Death Induced by DNA-damaging Agents J. Biol. Chem., July 12, 2002; 277(29): 26673 - 26680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Shefer, T. A. Partridge, L. Heslop, J. G. Gross, U. Oron, and O. Halevy Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells J. Cell Sci., January 4, 2002; 115(7): 1461 - 1469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Okuya, K. Tanabe, Y. Tanizawa, and Y. Oka Leptin Increases the Viability of Isolated Rat Pancreatic Islets by Suppressing Apoptosis Endocrinology, November 1, 2001; 142(11): 4827 - 4830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Henaff, S. N. Hatem, and J.-J. Mercadier Low Catecholamine Concentrations Protect Adult Rat Ventricular Myocytes against Apoptosis through cAMP-Dependent Extracellular Signal-Regulated Kinase Activation Mol. Pharmacol., April 13, 2001; 58(6): 1546 - 1553. [Abstract] [Full Text]
|
|
|
|
 |
|
 B. Beltran, A. Mathur, M. R. Duchen, J. D. Erusalimsky, and S. Moncada The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death PNAS, December 19, 2000; 97(26): 14602 - 14607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. CANTIN, B. PAQUETTE, M. RICHTER, and P. LARIVEE Albumin-mediated Regulation of Cellular Glutathione and Nuclear Factor Kappa B Activation Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): 1539 - 1546. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Massari, Y. Ho, and L. M. Wetzler Neisseria meningitidis porin PorB interacts with mitochondria and protects cells from apoptosis PNAS, August 1, 2000; 97(16): 9070 - 9075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ruhl, E. Sahin, M. Johannsen, R. Somasundaram, D. Manski, E. O. Riecken, and D. Schuppan Soluble Collagen VI Drives Serum-starved Fibroblasts through S Phase and Prevents Apoptosis via Down-regulation of Bax J. Biol. Chem., November 26, 1999; 274(48): 34361 - 34368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Riou, H. Tonoli, F. Bernier-Valentin, R. Rabilloud, P. Fonlupt, and B. Rousset Susceptibility of Differentiated Thyrocytes in Primary Culture to Undergo Apoptosis after Exposure to Hydrogen Peroxide: Relation with the Level of Expression of Apoptosis Regulatory Proteins, Bcl-2 and Bax Endocrinology, May 1, 1999; 140(5): 1990 - 1997. [Abstract] [Full Text]
|
|
|
|
|
|
I Korichneva and U Hammerling F-actin as a functional target for retro-retinoids: a potential role in anhydroretinol-triggered cell death J. Cell Sci., January 8, 1999; 112(15): 2521 - 2528. [Abstract] [PDF]
|
|
|
|
|
|
N. Mizuno, H. Yoshitomi, H. Ishida, H. Kuromi, J. Kawaki, Y. Seino, and S. Seino Altered bcl-2 and bax Expression and Intracellular Ca2+ Signaling in Apoptosis of Pancreatic Cells and the Impairment of Glucose-Induced Insulin Secretion Endocrinology, March 1, 1998; 139(3): 1429 - 1439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhang, S. Fan, and E. V. Prochownik Distinct Roles for MAX Protein Isoforms in Proliferation and Apoptosis J. Biol. Chem., July 11, 1997; 272(28): 17416 - 17424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bjorkerud and S. Bjorkerud Contrary Effects of Lightly and Strongly Oxidized LDL With Potent Promotion of Growth Versus Apoptosis on Arterial Smooth Muscle Cells, Macrophages, and Fibroblasts Arterioscler. Thromb. Vasc. Biol., March 1, 1996; 16(3): 416 - 424. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. Day, B. Galgoci, and V. Irish Genetic ablation of petal and stamen primordia to elucidate cell interactions during floral development Development, January 9, 1995; 121(9): 2887 - 2895. [Abstract] [PDF]
|
|
|
|
|
|
R. Jakobi, E. Moertl, and M. A. Koeppel p21-activated Protein Kinase gamma -PAK Suppresses Programmed Cell Death of BALB3T3 Fibroblasts J. Biol. Chem., May 11, 2001; 276(20): 16624 - 16634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Wang, K. S. Ko, A. Kapus, C. A. G. McCulloch, and R. P. Ellen A Spirochete Surface Protein Uncouples Store-operated Calcium Channels in Fibroblasts. A NOVEL CYTOTOXIC MECHANISM J. Biol. Chem., June 15, 2001; 276(25): 23056 - 23064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Ko, P. D. Arora, and C. A. G. McCulloch Cadherins Mediate Intercellular Mechanical Signaling in Fibroblasts by Activation of Stretch-sensitive Calcium-permeable Channels J. Biol. Chem., September 14, 2001; 276(38): 35967 - 35977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. M. Hickson-Bick, G. C. Sparagna, L. M. Buja, and J. B. McMillin Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of ROS Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H656 - H664. [Abstract] [Full Text] [PDF]
|
|
