| Objective: To investigate TSC genes expression level in newly diagnosed acute leukemia (AL) and its clinical significance, explore the possible mechanisms of the aberrant expression in terms of hypermethylation.Methods: To examin expression levels of TSC mRNA in bone marrow samples from 104 newly diagnosed AL patients by quantitative real-time reverse transcription PCR (RT-PCR). Bone marrow samples from 29 healthy donors for hematopoietic stem cell transplantation were used as control. Western Blot Analysis was performed to test the protein expression of TSC2 in acute leukemia. Methylation Specific PCR (MSP) and sequencing were used to detect the methylation status in AL and a panel of leukemic cell lines, including Nalm-6,Jurkat,U937,and HL-60. Nalm-6 and U937 were treated with various concentrations of 5-Aza followed by detection of changes in TSC2 mRNA expression level by reverse transcription PCR (RT-PCR). Cell apoptosis and differentiation induction by 5-Aza were also observed by both Wright staining and flow cytometry.Results: The expression of TSC2 was down-regulated in AL patients in comparison with the control. The median levels were 19.818 (6.319-63.313) and 84.746 (45.508-278.000) respectively (p<0.001). There were significant differences between the median levels of TSC2 transcript in patients with AML,ALL,BAL and that of normal controls. TSC2 expression levels were not equally distributed among FAB subtypes of AML, the significant differences in TSC2 expression were observed between M3, M5, M6 and normal controls. On the contrary, there were no significant differences between M2, M4 and normal controls. The western blot results revealed that the tuberin protein band around 200kD was observed in healthy controls, while the protein band in patients was undetectable. By MSP, 9 of 10 AL samples present partially methylated TSC2. In contrast, the 2 normal healthy individual samples showed only unmethylated product. Sequencing found that the methylation frequency was higher than the unmethyltion frequency (13/4) in the sequenced region. HL60 and Jurkat showed partial promoter methylation and Nalm-6 showed complete methylation, but U937 demonstrated unmethylation. The expression level of TSC2 transcripts were upregulated in Nalm-6 cells demonstrated by semi-quantitative RT-PCR, while the expression level in U937 cells was unchanged treated with various concentrations of 5-Aza. Apoptosis assay showed that the percentage of apoptotic cells increased significantly after treatment inU937 and Nalm-6 (p<0.05) treated with 1.6μM of 5-Aza.Conclusions: The expression of TSC2 was downregulated in AL patients and the TSC2promoter was hypermethylated which might be an important mechanism for thedownregulation of TSC2 expression. Objective : The human caspase-8 ( FLICE/MACH/Mch5 ) gene encodes an interleukin-1b converting enzyme (ICE)-related cysteine protease, which plays a vital role in the propagation of enzymatic cascade that results in cell apoptosis. Caspase-8 protein possess a long N-terminal prodomain harboring two highly homologous DEDs, DED1 (1-75aa) and DED2 (99-176aa), followed by a C-terminal protease domain that can be divided into two subunits, p18 and p11. This study is to investigate the function of a novel short isoform of caspase-8 (caspase-8s) in apoptosis.Methods: Reverse transcription PCR (RT-PCR) and sequencing were performed to determine the identity of caspase-8s transcript in human bone marrow mononuclear cells (BMMNCs) . Western blot analysis was employed to identify the caspase-8s protein expression in BMMNCs. An expression vector pcDNA3.1 containing the 2DED of caspase-8, caspase-8s and FADD was constructed to transfer the genes into 293T cell lines transiently. In vivo binding and co-immunoprecipitation assay to determine whether caspase-8s can bind to FADD through its incomplete DEDs and whether 'two' intact tandemly repeated DEDs are necessary for interacting between caspase-8 and FADD; HIV-based lentivector expression vector pCDH1-MCS1-EF1-copGFP containing full-length coding sequence (CDS) region of caspase-8s was constructed and stable transfected to overexpress caspase-8s in Jurkat cell lines. Apoptosis assay by flow cytometry, DNA ladder and MTT were performed to determine the function of caspase-8s in Jurkat cell lines induced by the apoptotic stimuli, Fas-agonistic antibody CH11.Results: 1) A novel isoform of caspase-8 in human acute leukemia bone marrow mononuclear cells was identified; compared to the released caspase-8 database [GenBank accession no.NM033355], a 106 bp deletion was identified by RT-PCR and sequencing. Analyses of nucleotide and deduced amino acid sequences revealed that the 106 bp deletion resulted in a frameshift mutation carrying a stop codon and termination of the transcript in advance, with the predicted generation of a 108aa protein, as compared with the 479aa of caspase-8 [GenBank accession no.AAD24962]. The molecular mass of the predicted protein was estimated to be 13KD, which encodes the first DED (Death Effector Domain) and part of the second DED, missing the C-terminal caspase domain. The new caspase-8 transcript was also expressed in BMMNCs from healthy individuals and was named as caspase-8s (caspase-8 short form, GenBank accession no. EU670044). 2) RT-PCR detected transcripts representing caspase-8 as well as caspase-8s in most cell lines such as 293T, Jurkat, MCF-7, U937, K562, HL60, Nalm-6, NB4, KG-1 and the ratio of caspase-8 to caspase-8s varied in the different cell lines. 3) By Western blot assays, we detected the caspase-8s protein product existed in some but not all leukemia samples. 4) Caspase-8s-DED2 can bind to FADD as well as caspase-8-DED2 in vivo. 5) Stably transfected Jurkat cells clones with a lentivector expression vector pCDH1-MCS1-EF1-copGFP encoding caspase-8s were isolated by limited dilution and successful expression of caspase-8s constructs was confirmed by Western blot analysis. 6) The pCDH-caspase-8s Jurkat cell clones JS2, JS3 treated with serial dilutions of CH11 displayed higher apoptosis rates when compared to wild type Jurkat and pCDH-empty vector Jurkat cell clone JP4. 7) DNA fragment formation assay indicated DNA degradation of JS2 cells treated with CH11 comparing to wild type Jurkat cell and JP4 cells. 8) The methyl thiazoleterazolium (MTT) assays showed that the mean growth inhibitory rate of CH11 for JS2 cells was higher than that of wild type Jurkat cells and JP4 cells treated with serial dilutions of CH11.Conclusion: A novel isoform of caspase-8, named caspase-8s, was identified in human bone marrow mononuclear cells. The transcript encodes the first and part of the second stretches of DED but lacks the caspase domain. Caspase-8s transfection may promote FAS-induced apoptosis. In addition, the results also indicate that the first DED was an important structure mediating combination between caspase-8 and FADD. |
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