Comparison of Cell Deaths Induced by Transmembrane and Secretory TNF-α

Our previous study showed that transmembrane TNF-α （TM-TNF-α） had broader tumoricidal spectrum than secretory TNF-α （s-TNF-α）. This study examined the difference between the two kinds of TNF-α in inducing cells and the relationship between the apoptosis induced by TM-TNF-α and the cell cycle. Bioassay was employed to compare the cytotoxic effect of two kinds of TNF-α on cell lines L-929 and HepG2. TUNEL was used to detect apoptosis and the TdT and PI co-staining were used for determining the phase of apoptotic cells. Our results showed that TM-TNF-α could kill not only s-TNF-sensitive L929 cells but also s-TNF-tolerant HepG2 cells. TM-TNF-α predominantly induced apoptosis while s-TNF could induce both apoptosis and necrosis. The apoptosis of L-929 cells induced by TM-TNF-α mainly occurred in S phase and the apoptosis of HepG2 predominantly took place in G1 phase. It is concluded that the cytotoxic effects of the two TNF differ substantially. Since TM-TNF-α works locally, mainly induces apoptosis and has broader anti-tumor spectrum, it may be more effective for the treatment of tumor than s-TNF.

Correlation of Cytotoxic Effect of Transmembrane and Secretory TNF-α to Cell Cycle

This study was aimed to examine the correlation of the cytotoxic effects induced by two types of TNF-α to cell cycle. Hoechst 33342 and PI were used to detect the morphological changes in the cell death induced by the two types of TNF-α. TdT and PI co-staining was performed to determine the phase of cell cycle of apoptotic cells. L929 cells in different phases of cell cycle were further synchronized and their sensitivity to the two types of TNF-α was observed. Our results showed that the apoptosis of HepG2 cells triggered by tm-TNF-α mainly occurred in G1 phase while in HL-60, Raji and K562 cell lines it mainly took place in S phase. The apoptosis of L929 cells induced by tm-TNF-α mainly occurred in S phase while the apoptosis induced by s-TNF-α mainly appeared in G1 phase. L929 cells were sensitive to s-TNF-α when synchronized in G1 phase (cytotoxicity 49.8%) while their sensi-tivity to tm-TNF-α was highest in S phase (45.7%) and G1/S phase (cytotoxicity 40.6%). It was concluded that tm-TNF-α-induced apoptosis of different target cells took place in different phases of cell cycle. The apoptosis of the specific cell line induced by the two types of TNF-α occurred in different phases of cell cycle. The sensitivity of the specific cell line to the two types of TNF-α was correlated with the phase of cell cycle.

Antitumor Effects of the Fibroblasts Transfected TNF-α Gene and its Mutants

To compare the anti-tumor effects of transmembrane TNF-α(TM-TNF)and secreted TNF-α(S-TNF)in vivo,mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-α(Wt-TNF),TM-TNF mutant(TM-TNFm),S-TNF mutant(S-TNFm).Southern blot,RT-PCR,FACS and bioassay were used to investigate TNF-αgene integration,expression and its biological activity.It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF,the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-αor its mutants and effectively kill H22 in vitro.The transfected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5∶1 or 1∶1(effector/target cells,E/T)after the third day of H22 challenge,respectively.At the E/T=5∶1,the NIH3T3/TM-TNFm induced the highest tumor regression,while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T=1∶1 in vivo.No obvious side effects were noted throughout the course of treatment.The results suggest that both TM-TNF and S-TNF could cause tumor regression.The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.

噬菌体展示TM-TNF-α模拟肽体内杀瘤效应的研究

目的 研究噬菌体展示TM -TNF- α模拟肽的体内杀瘤效应及机制。方法 大量制备所需的噬菌体展示肽,比较不同展示肽对小鼠的肝癌细胞H2 2的体外杀瘤效应,挑取杀瘤效应最好的展示肽并选择其合适的滴度进行体内实验。小鼠接种H2 2细胞3d后,于肿瘤接种部位皮下注射噬菌体展示肽,观察肿瘤的生长情况。结果 噬菌体展示TM- TNF -α模拟肽在体内能明显抑制肿瘤的生长(P <0 .0 1)。且抑瘤率与展示肽之间呈剂量依赖关系。HE染色发现展示肽治疗组肿瘤组织中有大量淋巴细胞浸润,而sTNF- α治疗组除淋巴细胞浸润还可见中性粒细胞、浆细胞浸润。结论 直接注射噬菌体展示TM -TNF -α模拟肽可在体内有效杀瘤,其杀瘤机制可能不同于分泌型TNF -α。