1.Establishment of HSP90 overexpressing cell line and effects on cell proliferation.
Chinese Journal of Applied Physiology 2004;20(4):376-379
AIMTo establish a HSP90 highly expressing cell line and study the effect of high level of HSP90 on cell stress response.
METHODSThe recombined plasmid pSmycHSP, which contained the full length DNA coding for human HSP90 B, was introduced into mouse fibroblast cell line NIH-3T3 by electroporation after being subcloned, purified and identified by limited enzyme digestion. After screened by G418, the positive clones were selected and identified by immunocytochemistry and Western-blotting. Contrasted with NIH-3T3 cells transfected with empty plasmid, the effect of high-level HSP90 on cell proliferation and cell cycle was analyzed by MTT method and flow cytometry.
RESULTSThe rising level of HSP90 was shown by immunocytochemistry and Western-blotting. Compared with control, the growth of HSP90 highly expressing cell line slowed down and the DNA content of S phase was lower.
CONCLUSIONThe NIH-3T3 derived cell line, which stably expressed high level of HSP90 was established. The effect of high-level HSP90 on cell proliferation was to retard cell growth by affecting cell cycle.
Animals ; Cell Cycle ; Cell Proliferation ; HSP90 Heat-Shock Proteins ; metabolism ; Mice ; NIH 3T3 Cells
2.Expression of heat shock protein 90β and its regulation in the reproductive system of male mice.
Chengting RONG ; Junling HAN ; Zhenning DU
Journal of Southern Medical University 2013;33(4):491-495
OBJECTIVETo study the expression and regulation of heat shock protein 90β (HSP90β) in the testis, epididymis and sperms of mice.
METHODSThe localization and expression of HSP90β mRNA and protein were investigated in the testis, epididymis and sperms of mice, and the regulation of HSP90β in the male reproductive system was explored.
RESULTSHSP90β was expressed at a higher level in the epididymis than in the testis. In the sperms of the mice, HSP90β was localized in the acrosome area. The expression of HSP90β in mouse epididymis decreased after castration and recovered the normal level after testosterone treatment. HSP90β expression in the testis and epididymis also underwent changes during the postnatal development of the mice.
CONCLUSIONHSP90β may play an important role in spermiogenesis and fertilization, and its expression pattern in the epididymis after castration and during the postnatal development suggests its regulation by hormones and development.
Animals ; Epididymis ; metabolism ; HSP90 Heat-Shock Proteins ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Spermatozoa ; metabolism ; Testis ; metabolism
4.Palmitoylation of heat shock protein 90 in mouse sperm.
Rui LI ; Kun LI ; Yue YANG ; Pei-Bei SUN ; Ai-Jun CHEN ; Ya NI
Acta Physiologica Sinica 2017;69(3):298-304
Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.
Animals
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Epididymis
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HSP90 Heat-Shock Proteins
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metabolism
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Lipoylation
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Male
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Mice
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Palmitic Acid
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chemistry
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Sperm Capacitation
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Spermatozoa
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metabolism
5.Cryopreservation-induced decrease in heat-shock protein 90 in human spermatozoa and its mechanism.
Wen-Lei CAO ; Yi-Xin WANG ; Zu-Qiong XIANG ; Zheng LI
Asian Journal of Andrology 2003;5(1):43-46
AIMTo study the protein changes of spermatozoa associated with sperm motility during sperm cryopreservation and its mechanism.
METHODSIn 18 healthy men, the seminal sperm motility and HSP90 levels were studied before and after cryopreservation using SDS-PAGE, Western blotting and computerized image analysis.
RESULTSThe sperm motility declined significantly after cryopreservation (P<0.01). The average grey level and the integrated grey level of sperm HSP90 before cooling were 34.1+/-3.2 and 243.0+/-21.6, respectively, while those after thawing were 23.2+/-2.5 and 105.7+/-28.5, respectively. Both parameters were decreased significantly (P<0.01). No HSP90 was found in the seminal plasma before and after cryopreservation.
CONCLUSIONHSP90 in human spermatozoa was decreased substantially after cryopreservation. This may result from protein degradation, rather than leakage into the seminal plasma.
Blotting, Western ; Cryopreservation ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Male ; Semen Preservation ; Sperm Motility ; Spermatozoa ; cytology ; metabolism
6.Protective effect of heat preconditioning on NIH-3T3 fibroblast.
Xue-mei CHEN ; Si-ze CHEN ; Fei ZOU
Chinese Journal of Industrial Hygiene and Occupational Diseases 2006;24(4):232-234
OBJECTIVETo establish stress adaptation model of mouse fibroblast cell line NIH-3T3, to provide a group of parallel object for stress adaptation research, and to explore the function and mechanism of HSP90 in stress adaptation.
METHODSA stress-adapted cell model was established by thermal preconditioning (42 degrees C, 20 minutes), and the adaptation result was evaluated by observing the change of the membrane injury and the damage of DNA induced by the heat stress for the second time (44 degrees C, 20 minutes). The HSP90 content was detected by Western blot.
RESULTSAccording to the membrane injury and HSP90 synthesis induced by the heat stress for the second time, it was primarily confirmed that 6 hours after thermal preconditioning were the optimum stress protection time. When cells underwent heat stress for the second time 6 hours after thermal preconditioning, the membrane injury (15.4% +/- 2.6% vs 41.2% +/- 5.1%), damage of DNA (15.1% vs 26.3%) were decreased compared with the control group in which there was no preconditioning. The OD(HSP90)/OD(control) value indicated that the cellular HSP90 contents was decreased immediately after heat stress (44 degrees C, 40 min). The content of HSP90 was 0.82 +/- 0.18 in the heat stress group, 1.70 +/- 0.52 in the preconditioning group and 1.41 +/- 0.16 in the heat stress after preconditioning group.
CONCLUSIONWith the preconditioning for the NIH-3T3, the time point for the stress protection is confirmed, the model for the cellular stress adaptation is established and the protective effect of HSP90 is primarily confirmed in this model.
Adaptation, Physiological ; physiology ; Animals ; DNA Damage ; HSP90 Heat-Shock Proteins ; biosynthesis ; Heat Stress Disorders ; metabolism ; physiopathology ; L-Lactate Dehydrogenase ; metabolism ; Mice ; NIH 3T3 Cells
7.Mechanism of C-reactive protein on proliferation of multiple myeloma U266 cells.
Yi-Rong YANG ; Ling-Juan HUANG ; Yan-Ping MA ; Yu-Jin LU ; Lin-Hua YANG ; Yong-An ZHOU
Journal of Experimental Hematology 2009;17(5):1242-1245
This study was purposed to investigate the mechanism of C-reactive protein (CRP) on proliferation of U266 cells. The human multiple myeloma cell line U266 was incubated with human CRP (0, 5, 10, 20 mg/L) for 24 hours, then the proliferation level of U266 cells was detected by using blood analyser. The mRNA expressions of survivin and HSP90alpha were examined by RT-PCR. The results showed that the proliferation ratio was increased, as compared with the control group (p<0.05); furthermore, the mRNA levels of survivin and HSP90alpha were up-regulated in proportion to the increased CRP concentrations. There was significant correlation between expression of survivin and HSP90alpha (r=0.737, p<0.0001) in incubated cells. It is concluded that CRP can stimulate the proliferation of MM cells directly by up-regulating the expression of survivin and HSP90alpha in MM cells. CRP can be regarded as a potential target for MM treatment.
Apoptosis
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C-Reactive Protein
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metabolism
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Cell Line, Tumor
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Cell Proliferation
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HSP90 Heat-Shock Proteins
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metabolism
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Humans
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Inhibitor of Apoptosis Proteins
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metabolism
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Multiple Myeloma
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metabolism
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pathology
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RNA, Messenger
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genetics
8.Expression and significance of heat shock proteins in esophageal squamous cell carcinoma.
Jun-Hui CHEN ; Li-Ming CHEN ; Li-Yan XU ; Ming-Yao WU ; Zhong-Ying SHEN
Chinese Journal of Oncology 2006;28(10):758-761
OBJECTIVETo investigate the expression and significance of HSP27, HSP60, HSP70 and HSP90 alpha in esophageal squamous cell carcinoma (ESCC) and tissues along the incision margin (TIM).
METHODSThe presence and the level of expression of HSP27, HSP60, HSP70 and HSP90 alpha were determined in 168 specimens from ESCC and 42 from tissues along TIM by EnVision immunohistochemistry and Western blotting, to compare their positive staining rates and explore the correlation between their expressions and clinicopathologic features in ESCC.
RESULTSThe positive staining rates of HSP27, HSP60, HSP70 and HSP90 alpha in ESCC and TIM were 62.0% and 42.1%, 92.7% and 63.2%, 57.9% and 22.2%, and 33.7% and 18.5%, respectively. There was very significant difference between the expression of HSP60 and HSP70 in ESCC and TIM (P < 0.01), but not significant about HSP27 and HSP90 alpha (P > 0.05). The positive staining rate of HSP27 declined with the lower grade of differentiation of ESCC (P < 0.05).
CONCLUSIONThe present findings suggest that the expression of HSPs of different molecular weight in ESCC and TIM is a common event. The level of expressions of HSP60 and HSP70 are higher than those in TIM. HSP60 and HSP70 expression correlated with the biological behavior of ESCC. The expression of HSP27 was positively correlated to the grade of differentiation of ESCC. Overexpression of HSP27 may be associated to the differentiation of squamous cell carcinoma.
Blotting, Western ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Differentiation ; Chaperonin 60 ; metabolism ; Chi-Square Distribution ; Esophageal Neoplasms ; metabolism ; pathology ; Esophagus ; chemistry ; pathology ; HSP27 Heat-Shock Proteins ; HSP70 Heat-Shock Proteins ; metabolism ; HSP90 Heat-Shock Proteins ; metabolism ; Heat-Shock Proteins ; metabolism ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism
9.Expressions of heat shock protein in cervical cancer and precancerosis.
En-Feng ZHAO ; Lei BAO ; Long LIANG ; Dongping LI
Chinese Journal of Applied Physiology 2006;22(2):250-253
AIMTo investigate the expressions of main subtypes of heat shock protein (HSP) in cervical cancer and precancerosis tissues.
METHODSAccording to the pathological diagnosis, 478 cases of cervical biopsy specimen were divided into invasive carcinoma of cervix group (63 cases), cervical intraepithelial neoplasia group (CIN, 106 cases), chronic cervicitis group (293 cases) and normal uterine cervix (16 cases).The expression levels of HSP70, HSP90a and HSP90beta3 mRNA were detected by quantitative RT-PCR with specific complex cRNA as internal control.
RESULTS(1) The expressions of HSP70, HSP90a and HSP90beta mRNA were significantly down-trended stepwise in invasive carcinoma of cervix, CIN, chronic cervicitis and normal cervix tissue (P < 0.01, respectively). (2) In the invasive carcinoma of cervix group, the expression level of HSP90beta mRNA was higher in advanced stage (FIGO II b) compared with incipient(FIGO-II a) carcinoma of the cervix (P < 0.05). (3) The expressions of HSP70 and HSP90 mRNA were each higher in poorly differentiated tumor than in well-differentiated tumor (P < 0.05, respectively). (4) The expression levels of all three HSP mRNA had no significant differences, it was observed with different histological types of cervical cancer (P > 0.05).
CONCLUSIONHeat shock protein may play some important roles in malignant transformation of cervix cell and aggravation of cervix cancer. HSP70 and HSP90alpha may promote cancer cell transition and proliferation, and HSP90beta may participate in cell differentiation.
Carcinoma ; metabolism ; pathology ; Female ; HSP70 Heat-Shock Proteins ; metabolism ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Precancerous Conditions ; metabolism ; pathology ; Tumor Cells, Cultured ; Uterine Cervical Neoplasms ; metabolism ; pathology ; Uterine Cervicitis ; metabolism ; pathology
10.Influence of heat shock protein 90 on protein serine threonine kinases expression in hypoxic cardiomyocytes.
Xiao-hui ZHAO ; Yi-zhi PENG ; Yuan-yuan WANG ; Yue-sheng HUANG
Chinese Journal of Burns 2007;23(4):265-268
OBJECTIVETo explore the effect of endogenous heat shock protein 90 (HSP90) on the AKT signaling pathway of hypoxic cardiomyocytes.
METHODSThe hypoxia model of neonatal rat cardiomyocyte was established. The cells were randomly divided into normal control, hypoxia, Geldanamycin (GA, with hypoxia after Geldanamycin treatment) groups. The myocardial cell activity and the expression of endogenous HSP90 and AKT were determined with MTT and Western blotting, respectively at 1, 3, 6, 12, 24 and 48 post-hypoxia hours (PHH). The apoptotic index (AI) of cardiomyocytes were determined with TUNEL method at 24 PHH.
RESULTS(1) At 24 and 48 PHH, the activity of cardiomyocytes in hypoxia group and GA group were evidently lower than that in control group (P < 0.05). The activity of cardiomyocyte in GA group began to decrease at 12 PHH, and it was obviously lower than that in hypoxia group at 48 PHH (P < 0.05). (2) At 24 PHH, the AI in hypoxia group (10.7 +/- 1.2)% was obviously higher than that in normal control group [(1.9 +/- 0.3)%, P < 0.05], while it was obviously lower than that in GA group [(26.3 +/- 5.3)%, P < 0.01]. (3) The expression of endogenous HSP90 and AKT in hypoxia and GA groups were markedly increased compared with that of normal controls at 12 PHH, and it decreased at 24 PHH in hypoxia and GA groups, especially in the latter.
CONCLUSIONEndogenous HSP90 plays important roles in maintaining the cardiomyocyte activity, and its level might affect the expression of AKT.
Animals ; Cell Hypoxia ; Cells, Cultured ; Disease Models, Animal ; HSP90 Heat-Shock Proteins ; metabolism ; Myocytes, Cardiac ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Signal Transduction