Gastric cancer cells in collagen gel matrix: three-dimensional growth and differential expression of adhesion molecules (CD44s, CD54, E-cadherin).

To characterize the growth of human gastric cancer cells in collagen gel matrix and adhesive status of the cells in comparison with conventional monolayer cells. Three kinds of human gastric cancer cell lines (BGC823, SGC7901, and MKN28) were cultured alone or co-cultured with normal human fibroblasts in collagen gel matrix, and their cell cycle, metabolic function, and the expression of adhesive molecules (CD44s, CD54, and E-cadherin) were analyzed by flow cytometry or other methods. Two of three cell lines (BGC823 and SGC7901) and their co-cultures showed multilayer growth in collagen gel matrix, and their growth and metabolism rate became slow and the cell adhesion molecules (CAMs) expression was down regulated. Gastric cancer cell alone or with fibroblasts in collagen gel matrix showed distinct growth feature when compared with monolayer cells, which represent two kinds of different experimental models. BGC823 and SGC7901 cells growing in three-dimension may recur some characteristics of their original solid tumor in vivo with the invasive or metastatic ability. According to different aims, it should pay great care in choice of experimental model to get more reasonable results.

[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.

Shedding of Fas ectodomain that affects apoptosis of hepatocytes occurring in regenerative liver.

BACKGROUND: On the basis of comprehending several membrane proteins undergoing ectodomain shedding, including tumor necrosis factor (TNF) alpha receptors, we want to know if Fas (CD95/APO-1), which belongs to the TNF receptor (TNFR) superfamily and transduces signals resulting in apoptosis upon binding of Fas ligand (L) or agonistic anti-Fas antibody, is shed on its ectodomain during liver regeneration METHODS: After purification of the total membrane protein of hepatocytes, we analyzed Fas ectodomain shedding, using Western blotting, and determined the effect of Fas shedding on the apoptosis of hepatocytes by a statistical method relevant to apoptosis. RESULTS: Fas protein ectodomain shedding occurred at 2, 12, and 36 h after partial hepatectomy, and its level decreased at 2 months after partial hepatectomy. The same results were gained from in-vitro cultured hepatocytes induced by serum from rats after partial hepatectomy. The sensitivity of hepatocytes to agonistic anti-Fas antibody increased significantly after they were treated with serum from rats after partial hepatectomy. However, the expression of Fas mRNA and Fas protein did not affect the liver regeneration. CONCLUSIONS: Fas ectodomain shedding might be an important mechanism in controlling hepatocyte apoptosis during liver regeneration.

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.