Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos

Our plain-English summary of Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos, by J.L. Goldberg, C.L. Zanella, Y.M. Janssen, et al (American Journal of Respiratory Cell and Molecular Biology, 1997).

The study

• Authors: J.L. Goldberg, C.L. Zanella, Y.M. Janssen, C.R. Timblin, L.A. Jimenez, P. Vacek, D.J. Taatjes, B.T. Mossman
• Journal: American Journal of Respiratory Cell and Molecular Biology
• Published: 1997;17(3):265-71
• Cited by: 61 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

We developed in situ dual-fluorescence detection techniques for measuring apoptosis and proliferation simultaneously in single dishes of cells. The deoxyribonucleic acid (DNA)-specific labeling method, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), first was used in conjunction with a 4',6-diamidino-2-phenylindole (DAPI) counterstain to detect and measure morphologic characteristics of apoptotic rat pleural mesothelial (RPM) cells isolated from Fischer 344 rats and exposed to 300 microM hydrogen peroxide (H2O2). For this purpose, 100 TUNEL-positive nuclei were measured while being viewed with DAPI counterstaining for area, perimeter, longest diameter, and average diameter, using imaging software and an image-collection apparatus. We then exposed cells to a range of concentrations of crocidolite asbestos and putative apoptotic and mitogenic agents. Exposure to crocidolite asbestos (5 microg/cm2) caused a striking dose-dependent apoptotic response at 24 h, 48 h, and 72 h. The nonfibrous crocidolite analogue riebeckite failed to induce apoptosis. At 24 h, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) caused an increase in apoptotic nuclei. A second method, utilizing an antibody to 5'-bromodeoxyridine (BrdU) and oxazole yellow homodimer (YOYO), showed a dose-dependent increase in proliferation occurring in cells exposed to asbestos (5 microg/cm2) at 48 h and 72 h. In addition, increased numbers of rat pleural mesothelial (RPM) cells exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), TNF-alpha, and epidermal growth factor (EGF) exhibited incorporation of BrdU at these time points, although total numbers of cells per unit area were unchanged. Results indicate a dynamic balance between apoptosis and increased DNA synthesis after exposure of mesothelial cells to asbestos.

In plain English

This laboratory study used cells grown in dishes, not people or live animals. The cells were pleural mesothelial cells (from the lining around the lung) of adult Fischer 344 rats. The authors developed a computer-assisted microscope method to count, in one dish, both dying cells (apoptosis, or programmed cell death) and cells making new DNA. They exposed the cells for 24, 48 and 72 hours to three concentrations of crocidolite asbestos, to riebeckite (a non-fibrous mineral chemically similar to crocidolite), and to TPA, EGF and TNF-alpha. Compared with untreated cells, crocidolite caused a dose-dependent increase in apoptosis at all three times. It also significantly increased DNA-making cells at 48 and 72 hours, as did TPA, EGF and TNF-alpha. Riebeckite caused no apoptotic response and no significant increase in DNA synthesis. TNF-alpha increased apoptosis at 24 hours. Total cell numbers did not change significantly in any group. The authors concluded that asbestos-exposed mesothelial cells showed a dynamic balance between apoptosis and increased DNA synthesis. They added that the riebeckite results support the idea that asbestos's fibrous shape is important.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) cell-culture experiment to develop and apply an imaging method for measuring apoptosis and cell proliferation in the same dish
• Who was studied: Rat pleural mesothelial (RPM) cells isolated from adult Fischer 344 rats, used between passages 5 and 10, at the University of Vermont; experiments were performed in duplicate
• Exposure: Crocidolite asbestos at 0.5, 1.0 and 5.0 per cm2 of culture dish (the scanned text prints 'mg/cm2'; the PubMed abstract gives micrograms per cm2), riebeckite at 5.0 per cm2 as a non-fibrous comparison, TPA (100 ng/ml), TNF-alpha (10 ng/ml) and EGF (5.0 ng/ml), for 24, 48 and 72 hours; hydrogen peroxide was used to establish the criteria for recognizing apoptotic nuclei
• Main results:
  • Compared with untreated controls, crocidolite asbestos caused a dose-dependent increase in apoptosis at 24, 48 and 72 hours (P less than 0.05)
  • TNF-alpha caused significant increases in apoptosis at 24 hours
  • TPA and EGF produced no statistically significant increase in apoptotic cells at any time point, and riebeckite caused no apoptotic response
  • Cells treated with crocidolite asbestos, TPA, EGF and TNF-alpha showed significant (P less than 0.05) increases in BrdU incorporation (DNA synthesis) at 48 and 72 hours compared with untreated controls
  • Riebeckite-exposed cells showed no significant increase in DNA synthesis at any time point
  • Total cell numbers showed no statistical changes in any exposed group compared with untreated controls (data not shown)
  • The formula developed to classify nuclei as apoptotic or normal had a less than 1% chance of misclassification
• The authors' conclusion: The authors concluded that the data indicate a dynamic balance between increased DNA synthesis and cell death by apoptosis after asbestos exposure. They suggested that apoptosis may cause a compensatory increase in cell growth, and that the riebeckite results support the idea that the fibrous geometry of asbestos is important in causing cell death and increased DNA synthesis.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Apoptosis, Mesothelial Cells, Crocidolite, Riebeckite, Tumor Necrosis Factor-alpha (TNF-α).

Related studies

• Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers — C.R. Timblin, G.D. Guthrie, et al. (1998)
• Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos — K.A. BéruBé, T.R. Quinlan, et al. (1996)
• Simultaneous triple fluorescence detection of mRNA localization, nuclear DNA, and apoptosis in cultured cells using confocal scanning laser microscopy — W.P. Davis, Y.M. Janssen, et al. (1997)
• Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis — C.L. Zanella, C.R. Timblin, et al. (1999)
• Effect of simian virus large T antigen expression on cell cycle control and apoptosis in rat pleural mesothelial cells exposed to DNA damaging agents — V. Levresse, S. Moritz, et al. (1998)
• Early mesothelial cell proliferation after asbestos exposure: in vivo and in vitro studies — I.Y. Adamson (1997)

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