Induction of apoptosis in murine B lymphocytes by a plasmacytoma cytotoxic factor:comparison between normal and tumor cells

• Main Author: 尤寶慶
• Other Authors: 曾哲明
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/11112941081779701705

碩士 === 國立臺灣師範大學 === 生物研究所 === 88 === The tumoricidal mechanisms of macrophages include cell-cell contact and secretion of cytotoxic agents.Our previous report indicated that the culture supernatant of P388D1 murine macrophage-like cell line was cytotoxic to MOPC-315 plasmacytoma.One of the cytotoxic factors in culture supernatant , which was designated PCF was characterized.PCF killed the tumor cells by induction of apoptosis. This study further identified other factors in P388D1 culture supernatant which might be cytotoxic to other tumor cells or normal lymphocytes. Protein components in P388D1 culture supernatnat were fractionated by 0-20% (Ⅰ) ,20-40% (Ⅱ) and 40-60% (Ⅲ) saturation of ammonium sulfate. Each fraction was assayed for its cytotoxic activity against MPC-11 plasmacytoma , MOPC-315 plasmacytoma and normal spleen lymphocytes by MTT method. Results suggested that Fraction Ⅰ was cytotoxic to the normal lymphocytes. The same fraction had less cytotoxic to MPC-11 cells. Fraction Ⅱ was also cytotoxic to normal cells but less extent to MPC-11 cells.Fraction Ⅲ showed predominant cytotoxicity against MOPC-315 , and less extent to normal lymphocytes and MPC-11 cells. To study further the possible involvement of apoptosis during the process of cytotoxicity , the TUNEL assay was used to monitor the generation of DNA fragmentation.Results suggested that Fraction Ⅰ induced DNA fragmentation in normal lymphocytes , Fraction Ⅱ predominantly induced DNA fragmentation in MPC-11 cells , and Fraction Ⅲ induced DNA fragmentation in MOPC-315 cells. Induction of apoptosis in three types of target cells was further confirmed by detection of phosphatidylserine translocation by Annexin V assay. TNF-α alone induced apoptosis in L929 cells but had no effect on MPC-11, MOPC-315, and normal lymphocytes.In addition , an anti-TNF-α antibody failed to neutralize the cytotoxicity of P388D1 culture supernatant against three types of targets cells.An anti-PCF antibody CB7.C2 failed to neutralized fraction Ⅱ- induced apoptosis in MPC-11 cells , suggesting that cytotoxic factor against MPC-11 in fraction Ⅱ is distinct from TNF-α or the PCF characterized by previous papers.