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Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion

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Abstract

Serum starvation has recently been shown to cause cell death of cardiac fibroblasts and increased synthesis of extracellular matrix proteins in the surviving cells. In the present study, events occurring in the dying cells were investigated. Cultured adult rat cardiac fibroblasts were exposed to serum-free medium. Cell number was measured using a Coulter Counter Channelyzer. The activity of the extracellular signal-regulated or mitogen-activated protein kinases (ERK1/2, p42/p44MAPK), the p38 kinase (p38MAPK), the c-Jun N-terminal kinases (p46/p54JNK), and Akt kinase was assessed by Western blotting and phospho-specific antibodies. Caspase 7-cleavage was investigated by Western blotting and specific antibodies. Caspase 3 activity was measured by detection of its cleaved substrate. The appearance of necrosis was studied by inclusion of trypan blue. Apoptosis was assessed by DNA ladder formation. The mRNA expression of Bax and Bcl-2 was investigated by quantitative real-time PCR. Serum withdrawal led to the death of 26% of cultured isolated cardiac fibroblasts during the first 5 h. The activity of the p42/p44MAPK as well as of Akt kinase was partially reduced. For p46/p54JNK and p38MAPK, elevated phosphorylation was measured. Inhibition of p46/p54JNK and p38MAPK activity by SB202190 did not affect the decrease in cell number. Cleavage of caspase7 was detected after 90 min. However, no activation of caspase 3 was measured. DNA fragmentation was not found after serum depletion. Trypan blue staining, however, was observed in 16% of the cells after 5 h. The mRNA levels of both Bax and Bcl-2 were increased after 30 min. These results indicate the appearance of necrosis during serum starvation in cardiac fibroblasts. However, some processes typical of apoptosis were also detected.

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Authors and Affiliations

  1. Carl-Ludwig-Institute of Physiology, University of Leipzig, Leipzig, Germany

    Monika Leicht, Grit Marx, Doris Karbach & Heinz-Gerd Zimmer

  2. Institute of Physiology, University of Wuerzburg, Wuerzburg, Germany

    Michael Gekle

  3. Roboscreen® Gesellschaft für molekulare Biotechnologie mbH, Leipzig, Germany

    Thomas Köhler

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  1. Monika Leicht
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Leicht, M., Marx, G., Karbach, D. et al. Mechanism of cell death of rat cardiac fibroblasts induced by serum depletion. Mol Cell Biochem 251, 119–126 (2003). https://doi.org/10.1023/A:1025446302759

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