ABSTRACT
Objective: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. Methods: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3. 1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. Results: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G 2-M phase increased dramatically after Ad/apoptin(+) infection. Conclusion: Apoptin can induce cell cycle G 2-M arrest and chromatin condensation in cancer cells.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the effect of p21(WAF1) on the proliferation and the sensitivity to Vp16 of leukemia cell line K562.</p><p><b>METHODS</b>A p21(WAF1) retroviral expression vector pLXSN-p21(WAF1) was constructed by FuGENE 6, pLXSN-p21(WAF1) and pLXSN-neo, and transfected into p21(WAF1) defect leukemia cell line K562. After selected with G418, K562-p21(WAF1) cell clones that stably expressed p21(WAF1) were isolated. The ectopic expressions of p21(WAF1) mRNA and protein in K562-p21(WAF1) were identified by RT-PCR and Western b1ot. The cell growth rate was tested by trypan blue dye, the cell cycle by FCM and the sensitivity to Vpl6 by cell count and MTT assay.</p><p><b>RESULTS</b>The expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell as compared with that of the control. The cell number in G(0)/G(1) phase was remarkably increased. The sensitivity to Vpl6 decreased, the IC (50) of K562-neo cells was (56.4 +/- 6.5) microgram/ml, and that of K562-p21(WAF1) cells was (131.0 +/- 8.7) microgram/ml (P < 0.01).</p><p><b>CONCLUSION</b>p21(WAF1) can inhibit the proliferation of leukemia cell and decrease its sensitivity to Vp16.</p>
Subject(s)
Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Blotting, Western , Cell Division , Cell Survival , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins , Genetics , Metabolism , Physiology , Dose-Response Relationship, Drug , Etoposide , Pharmacology , G1 Phase , Gene Expression , K562 Cells , Cell Biology , Metabolism , RNA, Messenger , Genetics , Metabolism , Resting Phase, Cell Cycle , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
For the purpose of thrombocytopenia gene therapy, recombinant RevTet-On and pRevTRE/TPO retrovirus were packaged and transfected to NIH3T3 after selected with G418 and hygromycin, the two inserting recombinant retrovirus cell strain RevTet-On3T3/TPO were established. TPO expression was controlled and regulated by doxycydine (Dox). After using Dox to control the expression of TPO in RevTet-On3T3/TPO cells, the result showed that when Dox is added to the RevTet-On3T3/TPO cells, cell populations expressed TPO highly in the presence of 2 mg/L of Dox, and lowly in the absence of Dox. By using the RevTet-On gene expression system (the retrovirus vector RevTet-On regulation system to control the expression gene by Dox), it could modulate the expression of multiple genes by tetracyline and its derivatives. This system maybe provides a safe and efficacient way for the thrombocy to penia gene therapy.
ABSTRACT
To study the significance of calcium in the apoptosis of HL-60 cells induced by VP-16, the technology of flow cytometry, confocal laser scanning microscopy and Western blot were used. The results showed that VP-16 could induced the apoptosis of HL-60 cells and transient increase of intracellular calcium concentration; EGTA [ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid], that could combine the extracellular calcium, did not prevent the apoptosis of HL-60 cells. BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester], however, a chelating agent of intracellular calcium ions, could prevent apoptosis and the release of cytochrome C from HL-60 cells. It was concluded that the calcium plays an important role in apoptosis and the release of cytochrome C.
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The aim of this work was to test the effect of p16 on the proliferation of leukemic cells and its potential in gene therapy for leukemia. The full-length p16 cDNA was transfered by recombinant retrovirus vector into leukemia cell line K562, which is homozygous p16 deletion and retains functional retinoblastoma (RB) protein. The cell proliferation was tested in liquid and in soft agar culture after transduction of p16 retrovirus. The results showed a strong inhibition of cell proliferation. Phosphorylation of RB protein was also inhibited. The findings demonstrated that p16 (MTS/CDKN2) inactivation is a significant factor in the genesis and progression of leukemia and p16 could be a candidate gene for gene therapy in leukemia.
ABSTRACT
Varied TCM constitutions in the patient of acute cerebral infarction were classifyed by using HLA - DQAl allehc gene to analyze hereditary susceptibility of constitution types and relation among constitution, syndrome and treatment. PCR-SSP technique was used for classification of Yin - deficiency Yang - deficiency, Qi - deficiency, phlegm - dampness and blood stasis constitution in 103 cases of acute cerebral infarction and HLA - DQAl allelic gene was used for gent classification in 99 cases of nomal constituion. Results indicated that HLA -DQA * 0501 gene type in Yin -deficiency constitution was significant higher than that of normal constitution CP