[Inducing effect of cyclosporin A on apoptosis of multidrug resistant cell lines HR20 and HT9].

BACKGROUND & OBJECTIVE: Multidrug resistant (MDR) cells can resist drug-induced apoptosis, which is the functional mechanism for many chemotherapeutic drugs. It is necessary to search for the molecular mechanism underlying anti-apoptosis of MDR cells. However, because of the anti-apoptosis characteristic of MDR cells, it is hard to study the mechanism on their apoptosis pathway. This study was to induce apoptosis in human acute leukemia cell line HL-60, and its MDR cell lines HR20 and HT9 by cyclosporin A (CsA), analyze the differences in apoptosis pathway between MDR cells and sensitive cells by detecting several important apoptosis-related molecules. METHODS: HL-60, HR20, and HT9 cells were treated with 10, and 20 mg/L of CsA, cell apoptosis was identified by cell morphologic changes,electrophoresis,and flow cytometry. Changes of apoptosis-related factors were detected by spectrophotometer and Western blot. RESULTS: HL-60, HR20, and HT9 cells all showed obvious apoptotic characteristics after treated with CsA,such as chromatin condensation, DNA fragmentation factor (DFF) degradation and activation, and DNA fragmentation. However, Caspase-3 activation was only detected in apoptotic HL-60 cells. CONCLUSIONS: CsA may induce apoptosis of HR20, and HT9 cells in a Caspase-3 independent manner, which is different from apoptosis of sensitive cells. In the apoptosis pathway of HR20, and HT9 cells, there may be some factors other than Caspase-3 that can activate DFF. It is postulated that the difficulty of Caspase-3 to be activated may be an important reason for HR20 and HT9 cells to resist apoptosis induced by many chemotherapeutic drugs.

Shedding of TNFR1 in regenerative liver can be induced with TNF alpha and PMA.

AIM: Liver regeneration is associated with apoptosis of hepatocytes, which is mediated via tumor necrosis factor receptor 1(TNFR1). The shedding of TNFR1 in liver regeneration and its mechanism to regulate this shedding were investigated. METHODS: The shedding of TNFR1 in liver regeneration and changes of TNF-alpha, PMA and plasma membrane purified from hepatocytes on this shedding process were measured with Western blot. Then, the relationship between TNFR1 shedding and apoptosis of hepatocytes induced by TNFalpha was studied by detecting apoptotic index. RESULTS: The shedding of TNFR1 began at 4 hours and terminated before 2 months after partial hepatectomy. In culture system, serum from rats at 36 h after partial hepatectomy could also promote this shedding process. With the stimulation of TNFalpha, PMA or purified plasma membrane from hepatocytes at 36 h after partial hepatectomy or from hepatocytes treated with TNFalpha for 2 h, membranous TNFR1 was also shed. With the stimulation of both TNFalpha and plasma membrane from hepatocytes affected with TNFalpha for 2 h or from hepatocytes at 36 h after partial hepatectomy, apoptotic index of hepatocytes decreased from 21 % to 7.52 % and 8.45 %, respectively. PMA could also reduce apoptotic index to 13.67 %. This descent occurred in hepatocytes cultured in serum from rats at 36 h after partial hepatectomy too, but not in serum from rats at 2 months after partial hepatectomy and sham-operated rats. CONCLUSION: Shedding of TNFR1 may help reduce apoptosis of hepatocytes induced by TNFalpha. Membrane-anchored metalloprotases could play a role in shedding membranous TNFR1. At the same time, PKC may take part in regulation of this shedding process.

Effects of Three Protein Phosphatase Inhibitor on A(23187)-induced Apoptosis of HL-60 Cells.

Calcium ionophore A(23187) could increase the intracellular free Ca(2+) concentration and induce apoptosis in some cell lines. In this paper, we reported that A(23187) (1 &mgr;g/ml) could induce apoptosis of HL-60 cells after treating for 4 hours. Pretreatment with the nontoxic concentration of CsA (0.5 3 &mgr;g/ml), an inhibitor of the protein phosphatase 2B (PP2B), could prevent apoptosis induced by A(23187). Neither okadaic acid (OA, inhibitor of PP1, PP2A, PP2C), nor sodium orthovanadate (SoV, inhibitor of tyrosine phosphatase), had such effect. The determination of intracellular Ca(2+) with flow cytometry showed that CsA did not prevent the increase of intracellular Ca(2+) induced by A(23187), which showed that CsA might affect the event in the downstream of calcium increase.