ABSTRACT
BACKGROUND: Disparities of Minor H antigens can induce graft rejection after MHC-matched transplantation. H60 has been characterized as a dominant antigen expressed on hematopoietic cells and considered to be an ideal model antigen for study on graft-versus-leukemia effect. METHODS: Splenocytes from C57BL/6 mice immunized with H60 congenic splenocytes were used for establishment of H60-specific CTL clones. Then the clones were characterized for proliferation capacity and cytotoxicity after stimulation with H60. Clone #14, #15, and #23 were tested for the TCR binding avidity to H60-peptide/H-2Kb and analyzed for TCR sequences. RESULTS: H60-specific CTL clones showed different levels of proliferation capacity and cytotoxic activity to H60-stimulation. Clones #14, #15, and #23 showed high proliferation activity, high cytotoxicity, and low activities on both aspects, respectively, and have TCRs with different binding avidities to H60-peptide/H-2Kb with t 1/2 values of 4.87, 6.92, and 13.03 minutes, respectively. The TCR usages were Valpha12D-3-01+Jalpha11-01 and Vbeta12-1-01+Dbeta1-01+J2-7-01 for clone #14, Valpha13D-1-02+Jalpha34-02 and Vbeta13-1-02+Dbeta2-01+Jbeta2-7-01 for clone #15, and Valpha16D+Jalpha45-01 and Vbeta12-1-01+Dbeta1-01+Jbeta2-5-01 for clone #23. CONCLUSION: The results will be useful for modeling GVL and generation TCR transgenic mouse.
Subject(s)
Animals , Mice , Clone Cells , Graft Rejection , Histocompatibility , Histocompatibility Antigens , Mice, Transgenic , TransplantsABSTRACT
Paclitaxel (taxol) is known as effective drug inhibition of cell cycle encouraging activity in human ovarian and metastatic breast cancers and malignant melanoma. It is an antimicrotubule agent that is believed to mediate its antineoplastic effects by inducing mitotic arrest followed by apoptosis. The relation between phorbol 12 myristate 13 acetate (PMA), protein kinase C (PKC) activator, and taxol-induced apoptosis is not well understood until now. This study was performed to investigate the effects of PMA on the signal transduction pathways of taxol-induced apoptosis in MCF-7 human breast carcinoma cells. Taxol-induced apoptosis is attenuated by curcumine, JNK inhibitor, and pyrrolidine dithiocarbamate (PDTC), inhibitor of NFkB. Pretreatment with PKC activator (PMA) or protein kinase A (PKA) activators (forskolin and dibutyryl cAMP) inhibited taxol-induced apoptosis in MCF-7 cells. In addition, thapsigargin, a specific inhibitor of endoplasmic reticulum(ER) Ca(2+)-ATPase and CaCl2, also blocked the activation of caspases by taxol. From these results suggest that taxol-induced apoptosis may be mediated via JNK or NFkB pathway and PKC activation.
Subject(s)
Humans , Apoptosis , Breast , Breast Neoplasms , Caspases , Cell Cycle , Curcumin , Cyclic AMP-Dependent Protein Kinases , MCF-7 Cells , Melanoma , Myristic Acid , Paclitaxel , Protein Kinase C , Signal Transduction , ThapsigarginABSTRACT
Nitric oxide (NO) elevates intracellular calcium. But the actions of calcium in NO-induced cell death are not well understood. This study was carried out to investigate the signal transduction pathways of calcium and NO-induced cytotoxicity in H9c2 cardiac myoblasts by using NO donor compounds such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP). Pretreatment of intracellular calcium chelating agent (BAPTA/AM) or L-type calcium channel blockers (nicardipine, nifedipine, diltiazem and veraparmil) or T-type calcium channel blocker (flunarizine) blocked SNP-induced cytotoxicity respectively only in a three hours. However, thapsigargin (TG), which inhibits endoplasmic reticulum dependent Ca(2+)-ATPase and thereby increases cytosolic Ca(2+), augmented SNP-induced cytotoxicity. The protective effect of BAPTA/AM was inhibited by treatment of protein synthesis inhibitor, cyclohexamide. In addition, pyrrolidine dithiocarbamate (PDTC), NF-kB inhibitor, attenuates the protective effect of BAPTA/AM against SNP-induced cytotoxicity. It is indicated that the protective effect of BAPTA/AM against NO-induced cytotoxicity might be due to the expression of protein related to activation of NFkB. From these results, it is concluded that SNP-induced cytotoxicity is mediated by calcium in a 3 hours via down regulation of protein expression rleated to activation of NFkB.