1.Modification of Chinese hamster ovary cells.
Da-Zhi LAI ; Lian-Quan QI ; Chang-Ming YU ; Hai-Tao WANG ; Wei CHEN
Chinese Journal of Biotechnology 2002;18(4):415-419
Chinese hamster ovary cells (CHO) are preferable to prokaryotic, yeast or insect cells as hosts for biopharmaceutical production due to the products are more similar to their natural conformation. However, CHO cells confront tremendous difficulties when cultured in large scale such as mal-adaptation to serum-free medium, apoptosis and over-growth without limitation. So in addition to optimizing CHO system in respect of medium, environment and expression vector, modification of CHO cells themselves has drawn more and more attention. Here the main progress in CHO-modification is reviewed.
Animals
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Apoptosis
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genetics
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CHO Cells
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drug effects
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metabolism
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Cell Cycle
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drug effects
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Cell Cycle Proteins
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drug effects
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Cell Division
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drug effects
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Cricetinae
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Genetic Vectors
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genetics
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Transfection
2.Construction of antisense telomerase hTERT and its effect on K562 cells.
Xiu-xiang MENG ; Ben-li SU ; Li JIA ; Hong-dan SUN ; Zhuo-ran ZHANG
Chinese Journal of Hematology 2003;24(5):245-248
OBJECTIVESTo investigate whether antisense human telomerase reverse transcriptase (hTERT) could inhibit the activity of telomerase and the proliferation of K562 cells.
METHODSThe antisense plasmid was constructed by reverse insertion of hTERT PCR product into plasmid pLNCX-neo. Then the constructed plasmid was introduced into K562 cells by liposomes-mediated DNA transfection. The inhibition effects of telomerase on the proliferation of K562 cells were analyzed by MTT and colony formation assay, the telomerase activity of K562 cells by TRAP-PCR ELISA methods.
RESULTSThe growth rate of antisense hTERT transfected K562 cells was significantly lower than those of the controls, and the colony formation capacity of the transfected cells decreased significantly (P < 0.01), the colony number is (100.33 +/- 7.57)/10(3) cells, (92.67 +/- 5.86)/10(3) cells and (50.33 +/- 6.11)/10(3) cells for control K562 cells, K562 neo cells and antisense hTERT transfected HL60 cells, respectively. The telomerase activity of antisense hTERT transfected K562 cells was significantly inhibited.
CONCLUSIONThe expression of an antisense sequence to the mRNA sequence of telomerase protein subunit can inhibit the activity of telomerase, slow the cell growth and inhibit the capacity of colony formation of K562 cells.
Cell Division ; drug effects ; Humans ; K562 Cells ; Plasmids ; genetics ; RNA, Antisense ; genetics ; pharmacology ; RNA, Messenger ; genetics ; Telomerase ; drug effects ; genetics ; metabolism ; Transfection
3.Synergistic effects of human tumor necrosis factor-alpha gene transfection and interferon-gamma on the growth of tongue carcinoma cells.
Zhennan GAO ; Shengwei LI ; Jiarang GAO
West China Journal of Stomatology 2002;20(1):55-57
OBJECTIVEThe purpose of this study is to investigate the synergistic effects of human tumor necrosis factor-alpha (hTNF-alpha) transfection and interferon-gamma (IFN-gamma) on the growth of tongue carcinoma cells.
METHODSThe cultured Tca8113 tongue carcinoma cells was divided into 2 groups, one group was transferred with hTNF-alpha gene. Each of the 2 groups was then divided into 5 subgroups, and the subgroups were added IFN-gamma until the final IFN-gamma concentrations respectively were 0, 10, 100, and 1000 U/ml. After culturing for 48 hours, the survival rates of the all groups of cells were assayed by MTT enzymatic labeling technique, and the expression of hTNF-alpha in Tca8113 cells was observed with immunocytochemistry.
RESULTSIFN-gamma did not affect the growth of Tca8113 cells without hTNF-alpha, however, the transfection of hTNF-alpha with the above different concentrations of IFN-gamma synergistically inhibited the growth of Tca8113 cells, the concentrations of IFN-gamma were positively correlated with the inhibition effects (r = 0.733, P < 0.01), the transferred Tca8113 cells displayed remarkable overexpression of hTNF-alpha, compared with the non-transferred.
CONCLUSIONIFN-gamma can enhance the inhibition of hTNF-alpha transfection on the tongue carcinoma cells.
Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Drug Synergism ; Genetic Vectors ; Humans ; Interferon-gamma ; pharmacology ; Plasmids ; genetics ; Tongue Neoplasms ; genetics ; pathology ; Transfection ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; genetics
4.Effects of selenium and zinc on rat renal apoptosis and change of cell cycle induced by fluoride.
Rian YU ; Tao XIA ; Aiguo WANG ; Xuemin CHEN
Chinese Journal of Preventive Medicine 2002;36(4):219-221
OBJECTIVEThis study was conducted to study the effects of sodium fluoride (NaF) on rat renal apoptosis and proliferation, the antagonistic effect of selenium-zinc preparation (Se-Zn) to NaF.
METHODSWistar rats were provided with distilled water containing NaF (50 mg/L) and administered by gavage with different dosed of Se-Zn for six months. Kidney cell apoptosis and the cell cycle of proliferation were detected by TUNEL (TdT-mediated dUTP Nick End Labelling) and flow cytometry.
RESULTSNaF caused rat renal apoptosis, reduce the cell number of G(2)/M period in cell cycle and decrease the relative content of DNA significantly. Se-Zn inhibited the effects of NaF on apoptosis and increased the cell number of G(2)/M period in cell cycle, but failed to increase relative content of DNA.
CONCLUSIONIt was suggested that NaF could induce apoptosis and change the cell cycle in rat renal cells and Se-Zn could antagonize apoptosis and the changes of cell cycle induced by NaF.
Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; DNA ; drug effects ; genetics ; metabolism ; Drug Antagonism ; Flow Cytometry ; In Situ Nick-End Labeling ; Kidney ; drug effects ; pathology ; Rats ; Selenium ; pharmacology ; Sodium Fluoride ; pharmacology ; Zinc ; pharmacology
5.The effect of multi-unit ribozymes on the growth inhibition and apoptosis induction of CML K562 cells.
Qi FENG ; Bingzhong SUN ; Kai SUN ; Zhenchuan SHANG ; Sha WANG ; Wei WANG ; Yongtong ZHAO ; Zhen YAN ; Wei HAN ; Yingqi ZHANG
Chinese Journal of Oncology 2002;24(5):435-439
OBJECTIVETo investigate the possibility of multi-unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell's malignant phenotype.
METHODSAs bcr-abl fusion gene plays an important role in CML pathology, three single-unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi-unit ribozymes' in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of (3)H-TdR, RT-PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi-unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis.
RESULTSMulti-unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi-unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi-unit ribozymes could cleave K562 cell's RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells.
CONCLUSIONMulti-unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi-unit ribozymes to purge CML bone marrow. Therefore, multi-unit ribozymes may very well be valuable in the gene therapy of CML.
Apoptosis ; Cell Division ; drug effects ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; K562 Cells ; RNA, Catalytic ; metabolism ; pharmacology
6.Effect and comparison of sodium butyrate and trichostatin A on the proliferation/differentiation of K562.
Chunrui LI ; Wenli LIU ; Fankai MENG ; Wei HUANG ; Jianfeng ZHOU ; Hanying SUN ; Yongdong FENG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2003;23(3):249-253
In order to explore the molecular mechanisms of sodium butyrate and trichostatin A on K562 cell proliferation/differentiation, K562 cells were grown in the absence or presence of sodium butyrate or trichostatin A. The percentage of viable cells was determined by trypan blue exclusion. Differentiation was determined by nitro-blue tetrazolium (NBT) reduction and cell surface adhesion molecules analyzed by FACS. Cell cycle distribution was studied after DNA staining by propidium iodide. Cell cycle regulatory proteins were detected by Western blot and reverse transcription-polymerase chain reaction. The results showed that sodium butyrate blocked cells mainly at the G0/G1 phase of the cell cycle, whereas trichostatin A arrested the cells at G2 phase. Sodium butyrate could down-regulate the mRNA expression of cyclin D1, but not affect its protein expression; down-regulate the protein expression of cyclin D3, but not affect its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3 as sodium butyrate. Both sodium butyrate and trichostatin A could stimulate p21 expression of K562 cells at mRNA and protein levels. It may be concluded that sodium butyrate and trichostatin A could promote the proliferation/differentiation of the K562 cells, which might be contributed to the induced expression of cyclin D3 and p21 proteins.
Butyrates
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pharmacology
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Cell Cycle
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drug effects
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Cell Differentiation
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drug effects
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Cell Division
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drug effects
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Cyclin D
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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biosynthesis
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genetics
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Gene Expression
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drug effects
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Histone Deacetylase Inhibitors
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Humans
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Hydroxamic Acids
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pharmacology
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K562 Cells
7.Arsenic trioxide inhibits proliferation in K562 cells by changing cell cycle and survivin expression.
Xiaofei WU ; Zhichao CHEN ; Zhongping LIU ; Hao ZHOU ; Yong YOU ; Weiming LI ; Ping ZOU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(4):342-353
To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As2O3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As2O3 induced cell apoptosis, K562 cells were cultured with As2O3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As2O3(2-10 micromol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G2/M phase increased in proportion to As2O3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As2O3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As2O3-induced apoptosis.
Antigens, Neoplasm
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biosynthesis
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genetics
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Antineoplastic Agents
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pharmacology
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Apoptosis
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drug effects
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Arsenicals
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pharmacology
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Cell Cycle
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drug effects
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Cell Division
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drug effects
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Humans
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Inhibitor of Apoptosis Proteins
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K562 Cells
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Microtubule-Associated Proteins
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biosynthesis
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genetics
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Neoplasm Proteins
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Oxides
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pharmacology
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RNA, Messenger
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biosynthesis
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genetics
8.Proliferation of hepatocytes after delivery of exogenous hepatocyte growth factor gene.
Yong LIN ; Wei fen XIE ; Wei-zhong CHEN ; Xin ZHANG ; Xin ZENG ; Yue-xiang CHEN ; Xiu-jiang YANG ; Zhong-bing ZHANG
Chinese Journal of Hepatology 2003;11(6):331-333
OBJECTIVETo explore the proliferation of primary cultured rats hepatocytes after delivery of exogenous hepatocyte growth factor (HGF) gene which was inserted into the genome of replication-deficient recombinant adenovirus vector.
METHODSThe recombinant adenovirus-AdHGF which could express HGF was generated by homologous recombination. After the HGF gene was delivered into the hepatocytes, the expression of both HGF and c-met/HGF receptor mRNA in the cells was detected by RT-PCR and the level of HGF in the culture supernatant was also assayed by ELISA. On the other hand, cell proliferation was compared between before and after delivery of the HGF gene by MTS assay and the percentages of cell cycles were analyzed by flow cytometry. In addition, the expression of proliferating cell nuclear antigen (PCNA) was determined by immunocytofluorescent stain.
RESULTS4 x 10(10) efu/ml titer of AdHGF was obtained after recombination, RT-PCR indicated that the expression of HGF mRNA in hepatocytes increased on the third day after infected by the viruses and c-met/HGF receptor mRNA was also up-regulated. The HGF level in the culture supernatant assayed by ELISA was (5,939.0+/-414.39) pg/ml, which was much higher than that in the control (208.1pg/ml+/-37.20pg/ml, F=13.661, P<0.01). In addition, the proliferation of hepatocytes infected with AdHGF increased significantly according to MTS method (F>or=15.158, P<0.01) and more hepatocytes in G0/G1 stages changed into S stage (chi2=41.616, P<0.01), accordingly, PCNA index increased from 6.42+/- 1.88 to 14.56+/-2.85 (F=42.122, P<0.01).
CONCLUSION
THE RESULTSshow that HGF gene delivered into hepatocytes by AdHGF can be expressed with high efficiency in the cells, which can stimulate hepatocytes proliferation. It may be an effective tool for hepatocyte transplantation by gene modified donor hepatocytes.
Adenoviridae ; genetics ; metabolism ; Animals ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Genetic Vectors ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology
9.The effects of wild-type p53 gene transfection on the growth and chemotherapeutic sensitivity of human glioma cells.
Wei XIANG ; Xianli ZHU ; Hongyang ZHAO
Journal of Huazhong University of Science and Technology (Medical Sciences) 2002;22(1):44-46
To evaluate the effects of wild-type p53 gene on the growth and chemotherapeutic sensitivity of human glioma cells, plasmid PC53-SN3 carrying wild-type p53 gene was transfected into U251 cells. p53 gene expression in transfected cells was detected by RT-PCR, the cell growth inhibition and apoptosis in either the absence or the presence of cisplatin was assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT-PCR. MTT showed that p53 gene by itself induced strong inhibition effect on the growth of U251 cells [inhibition rate, IR (79.60 +/- 5.69)%]. The killing effects of cisplatin by itself on U251 cells was not strong [IR (19.40 +/- 6.69)%, (24.41 +/- 2.68)%, (51.84 +/- 13.38)%, (66.22 +/- 5.02)%] and increased with the increase of cisplatin concentration (1, 2, 4, 8 micrograms/ml). When combined treatment of wild-type p53 gene transfection and cisplatin was used, that was significantly increased [IR (91.64 +/- 1.00)%, (94.98 +/- 1.67)%, (95.32 +/- 2.01)%, (95.65 +/- 1.00)%]. The apoptosis rate of U251 cells induced by p53 gene transfection was 17.38%. That induced by cisplatin increased (5.71%, 5.93%, 6.27%, and 6.81%) with the increase of cisplatin concentration (1, 2, 4, 8 micrograms/ml). The apoptosis rate was also significantly increased (23.50%, 23.54%, 23.89%, and 28.88%) after combined treatment of p53 and cisplatin with different concentration (1, 2, 4, 8 micrograms/ml). It is concluded that wild-type p53 gene and cisplatin could result in synergistic inhibition effects on the growth of human glioma cells.
Antineoplastic Agents
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pharmacology
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Apoptosis
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drug effects
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Cell Division
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drug effects
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Cell Line, Tumor
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Cisplatin
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pharmacology
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Drug Resistance, Neoplasm
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genetics
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Gene Expression Regulation, Neoplastic
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genetics
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Genes, p53
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genetics
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Glioma
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genetics
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pathology
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Humans
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Recombinant Fusion Proteins
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physiology
10.p73 gene expression in apoptotic process of acute myeloid leukemia cell line U937 induced by methotrexate.
Guang-Fen XIAO ; Qing LU ; Xiang-Dong YANG
Journal of Experimental Hematology 2002;10(2):104-107
The purpose of this investigation was to study the variation of p73 gene expression in the apoptotic process of acute myeloid leukemia (AML) cell line U937 induced by methotrexate (MTX). Morphological changes of apoptotic cells were observed with microscopy and Wright's + Giemsa staining. DNA ladder and cell cycle were examined by agarose gel electrophoresis and flow cytometry respectively. Using semi-quantitive reverse transcription-polymerase chain reaction (RT-PCR), the expression of p73 mRNA was examined. Results showed that MTX could induce U937 cell apoptosis effectively. Condensed nuclei, fragmentation of chromosome and DNA ladder were seen after 6 hour following treatment of MTX 5 micro mol/L. Sub-G(1) peak and S + G(2)/M arrest were also determined by FCM, but the quantity of p73 expression was generally constant. In conclusion, U937 cell apoptosis induced by MTX did not change p73 mRNA level.
Acute Disease
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Antimetabolites, Antineoplastic
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pharmacology
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Apoptosis
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drug effects
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genetics
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Cell Cycle
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drug effects
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Cell Division
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drug effects
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DNA, Neoplasm
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drug effects
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genetics
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metabolism
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DNA-Binding Proteins
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genetics
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Flow Cytometry
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Gene Expression Regulation, Neoplastic
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drug effects
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Genes, Tumor Suppressor
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Humans
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Leukemia, Myeloid
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drug therapy
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genetics
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pathology
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Methotrexate
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pharmacology
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Nuclear Proteins
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genetics
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RNA, Messenger
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drug effects
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genetics
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metabolism
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Tumor Protein p73
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Tumor Suppressor Proteins
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U937 Cells