Our previous studies showed that S-adenosyl-methionine (SAM) induced Parkinson's disease-like changes in rat. It caused death to dopamine neurons in the substantia nigra, which appeared shrunken and fragmented, indicative of apoptosis-like changes (Charlton and Crowell [1995] Mol. Chem. Neuropathol. 26:269-284; Charlton [1997] Life Sci. 61:495-502). In this study, we investigated whether SAM causes apoptosis in both undifferentiated PC12 (PC12) cells and nerve growth factor (NGF)-differentiated PC12 (D-PC12) cells. S-adenosyl-homocysteine (SAH), the nonmethyl analog of SAM, was also tested. SAM and SAH (1.0 nM to 10.0 microM) caused lactate dehydrogenase (LDH) release from the PC12 cells and D-PC12 cells; cells with morphological changes and fluorescent DNA fragmentation staining were detected among both PC12 cell and D-PC12 cell. Compared with the PC12 cell, the D-PC12 cell, a postmitotic cell, was more sensitive to the toxic effects of SAM or SAH and presented much greater LDH release, suggesting a lethal effect; surprisingly, the amounts of apoptotic cells did not differ significantly between the two kinds of cells. In medium deprived of exogenous methionine, a decline in LDH release was observed in PC12 and D-PC12 cells. Also, lower levels of intracellular SAM and SAH were observed in the methionine-deleted media, which were reversed by the addition of either SAM or SAH. An antivitamin B(12) monoclonal antibody was added to methionine-depleted medium, resulting in deficiency of both endogenous and exogenous methionine, which caused further decreases in LDH release and reduction in the levels of intracellular SAM and SAH. The preliminary data showed different sensitivities to SAM or SAH between PC12 cell and D-PC12 cells, which suggests that PC12 cell may be more stable as a metabolic model. Apoptosis of PC12 cells was also assessed by PARP cleavage detection, Western blot analysis of Bax and Bcl-2 proteins, and DNA laddering on agarose gel electrophoresis. The proapoptoic protein Bax was dominantly expressed, whereas Bcl-2 was slightly down-regulated by SAM. SAH weakly induced the expression of Bax and slightly decreased Bcl-2 levels. The effects of SAM and its analog, SAH, were demonstrated conclusively to induce apoptosis in PC12 cells.
We demonstrate in this study that both TIMP-1 and TIMP-2 are major serum factors that stimulate the induction of TIMP-1 mRNA in quiescent human gingival fibroblasts (Gin-1 cells) at mid-G1 (6-9 h after serum stimulation) of the cell cycle, but not that of TIMP-2. When we chased the secretion of both TIMP proteins into culture medium containing 10% FCS freed of both TIMPs, TIMP-2 secretion rose to the level in 10% FCS after 24 h, but TIMP-1 secretion remained at a fairly low level even after 3 days, thus reflecting a contrastive difference in the induction of both TIMP mRNAs. The stimulating activity of TIMP-1 on the expression of the TIMP-1 gene switched over to inhibitory activity, when the TIMP-1 concentration in the culture medium exceeded about 30 ng/ml. The depletion of TIMP-1 and TIMP-2 from FCS affected remarkably the induction of c-jun and c-fos mRNAs, but not that of c-ets-1 mRNA. TIMP-1 and TIMP-2-dependent expression of AP-1 protein was further demonstrated by using nuclear extracts of Gin-1 cells in an electrophoretic mobility shift assay.
We first confirmed an earlier immunohistochemical study showing that immunoreactive TIMP-1-like protein accumulated in the nuclei of human gingival fibroblasts (Gin-1 cells), reaching a maximum in the S phase of the cell cycle (Li, H., Nishio, K., Yamashita, K., Hayakawa, T. and Hoshino, T. (1995). Nagoya J. Med. Sci. 58, 133–142). Then we isolated this protein from a nuclear extract of Gin-1 cells and demonstrated it to be identical to human recombinant TIMP-1 by western blotting, by a sandwich enzyme immunoassay for TIMP-1 and by an assay for matrix metalloproteinase inhibition. The amount of TIMP-1 in the cytosolic fraction of quiescent Gin-1 cells after stimulation by fetal calf serum increased continuously for 48 hours, whereas that in the nuclear extract showed a maximum at 24 hours (S phase) and significantly decreased thereafter. Gin-1 cells expressed mRNAs for both TIMP-2 and TIMP-3 together with mRNA for TIMP-1. However, neither TIMP-2 nor TIMP-3 proteins seemed to accumulate in the nuclei of Gin-1 cells. These facts strongly suggest that TIMP-1 accumulates specifically in the nuclei of Gin-1 cells in a cell cycle-dependent manner.
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