High expression of bcl-x(L) in K562 cells and its role in the low sensitivity of K562 to realgar-induced apoptosis.

Arsenic compounds (As(2)O(3 )or()As(4)S(4)) have been used successfully for the treatment of acute promyelocytic leukemia (APL) for quite a long time. It has been noticed that the sensitivity to apoptosis induced by As(2)O(3 )varies among various leukemia cells. It was reported by several groups that As(2)O(3) could induce apoptosis in APL-derived NB4 cells at concentrations of 0.5-1 mumol/l, whereas in other leukemia cells like K562, As(2)O(3) has no effects at the same concentration. K562 cells undergo apoptosis only when the concentration of As(2)O(3 )is greater than 2 mumol/l. Another arsenic compound, realgar (As(4)S(4)), a traditional Chinese mineral medicine, has been used to treat APL effectively and demonstrated to have lower toxicity than As(2)O(3). It would be interesting to know whether NB4 and K562 cells will show different sensitivity to realgar as well and if there is a difference, what is the cellular mechanism of it. In our present study, K562 cells were much less sensitive than NB4 cells to apoptosis induced by realgar. We confirm that the expression of bcl-x(L) is significantly higher in K562 cells than that in NB4 cells and is not downregulated upon realgar treatment. K562 cells become sensitive to realgar at clinically acceptable concentrations when bcl-x(L) expression level is downregulated by transfecting bcl-x(L) antisense RNA vector into the cells. Our results suggest that the increased bcl-x(L) expression in K562 cells contributes to its insensitivity to realgar-induced apoptosis.

[Cloning tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction-based subtractive hybridization].

BACKGROUND & OBJECTIVE: Glioma is a common tumor in central nervous system with no specific clinical therapy. Its pathogenesis is unclear. This study was to clone tumor-related genes and tumor suppressor genes in glioma with polymerase chain reaction (PCR)-based subtractive hybridization, and to explore the molecular biological mechanism of tumorigenesis of glioma. METHODS: mRNA was isolated from a sample of human glioma, and reversely transcribed into cDNA. PCR-based subtractive hybridization was used to clone tumor-related genes and tumor suppressor genes from it. RESULTS: In tumor-related candidate gene group, phospho-protein enriched in astrocytes of 15 (PEA15) and homology of acid fibroblast growth factor (aFGF) were picked up. Whereas, in tumor suppressor gene group, interferon-induced protein 17 and ndr2 were picked up. ndr2 was widely expressed in normal brain tissue, but absent in glioma tissue. CONCLUSION: ndr2 gene is a candidate tumor suppressor gene, and may play a role in tumorigenesis of glioma.

[Cloning and expression of the extracellular domain of calcium-activated chloride channel in mouse airway goblet cells].

AIM: To clone and express the extracellular domain of murine calcium-activated chloride channel (mCLCA3) in airway goblet cell of mouse. METHODS: According to the gene sequence of mCLCA3 the PCR primers for N-terminal, middle and C-terminal extracellular domains were designed. Using recombinant plasmid pcDNA3.1(-)/mCLCA3 as template, the DNAs coding for the three extracellular domains were amplified. And then the DNAs encoding N-terminal and C-terminal extracellular domains were inserted into expression vector pRSET-A, while the middle extracellular domain DNA was inserted into pGEX-T1. E.coli. BL21(DE3) were transformed with the three recombinant plasmids, respectively, and were induced with IPTG for expression. RESULTS: DNA sequencing showed that the cloned DNAs encoding extracellular domains were identical with those in GenBank (GenBank accession No. NM-017474 ). The 3 domains were expressed in E.coli and most of the expressed products existed in the form of inclusion body. CONCLUSION: The expression of three extracellular domains of mCLCA3 lays the foundation for further preparing anti-mCLCA3 antibody and exploring the mechanism of modulation of mCLCA3.

[Inhibitive effect on airway mucus overproduction with DNA vaccine based on xenogeneic homologous calcium-activated chloride channel in asthmatic mice].

OBJECTIVE: To observe the inhibitive effect on airway mucus overproduction with DNA vaccine based on human calcium-activated chloride channel 1 (hCLCA1) in asthmatic mice, who own mCLCA3 being xenogeneic homology of hCLCA1 in airway goblet cell. METHODS: The DNA vaccine was made with hCLCA1 gene inserted into pSecTag2B, and then BALB/c mice were vaccinated by i.m. once every two weeks. When serum antibody showed binding activity to mCLCA3 with ELISA analysis, asthma will be induced with ovalbumin in the vaccinated mice. To detect mucus production, lung sections were PAS stained and their MUC5AC mRNA levels were investigated by reverse transcription polymerase chain reaction (RT-PCR). Mice in control groups were injected with pSecTag2B/mCLCA3, pSecTag2B and saline, respectively. RESULTS: Antiserum of vaccine group after three times vaccination showed good binding activity to three mCLCA3 extracellular domains (ED), and the activity to N-terminal C-terminal ED was stronger than middle-ED. After induced to asthma, the number of goblet cell and MUC5AC mRNA level in vaccine group were lower than these in control group. CONCLUSION: hCLCA1 DNA vaccine can induce mouse to produce serum antibody binding itself mCLCA3, and thus airway mucus overproduction of asthmatic mouse is effectively inhibited.

[Inducible expression of reconstructed human caspase-6 gene in Hela cells].

AIM: To observe the pro-apoptotic effect of RCasp-6 gene in Hela cells. METHODS: RCasp-6 gene was amplified by PCR and cloned into the pIND vector. Hela cells were transfected with pIND-RCasp-6 and then inducced with ecdyson analogue. Expression of target gene was detected by immunocytochemical staining. Changes of the morphology and growth of Hela cells subjected to transfection of target gene were observed by HE staining and viable cell counting. RESULTS: RCasp-6 gene was cloned by PCR and its eukaryotic expression vector was successfully reconstructed. Expression of RCasp-6 gene in the transfected Hela cells leads to the morphological changes of the cells. Many of the transfected Hela cells shrunk and some cells were died. CONCLUSION: The expression of reconstructed human caspase-6 gene can efficiently accelerate death of Hela cells.

Binding of Insulin Receptor Substrate 1 PH Domain to Protein Kinase C.

To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total RNAs were isolated and purified from fresh liver and mRNAs were reversely transcribed into cDNAs. After PCR the fragments of the DNA were cloned into vector pUC19. The sequence of the fusion gene was confirmed by sequencing, and the gene was correctly expressed in E.coli as fusion protein with glutathione S- transferase(GST). The fusion protein was purified by glutathione agarose beads, then was incubated with the lysate of Jurkat cells. After SDS-PAGE, the proteins were transferred to PVDF membrane, and an anti-PKC antibody was used to detect binding between the PH domain with PKC. The sequence of the gene encoding for the PH domains was confirmed to be correct, and the PH domain was successfully expressed in E.coli JM 109 in soluble form. Western blots confirmed the binding of the PH domain with PKC in vitro. In conclusion, purified IRS-1 PH domain GST fusion protein was obtained and its biological activity was confirmed.