Document Detail


Telomerase immortalization of human myometrial cells.
MedLine Citation:
PMID:  12135889     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Several strategies have been described for the primary culture of human myometrial cells. However, primary cultures of myometrial cells have a limited life span, making continual tissue acquisition and cell isolation necessary. Recent studies have demonstrated that cell culture life span is related to chromosomal telomere length, and cellular senescence results from progressive telomere shortening and the lack of telomerase expression. Transfection of cells with expression vectors containing the human telomerase reverse transcriptase (hTERT) maintains telomere length and effectively gives normal cells an unlimited life span in culture. In addition, hTERT extends the life span of cultured cells far beyond normal senescence without causing neoplastic transformation. In the present study, we developed a cell line from hTERT-infected myometrial cells (hTERT-HM). Cells were isolated from myometrial tissue obtained from women undergoing hysterectomy, and retroviral infection was used to express the catalytic subunit of telomerase in myometrial cells. Cells expressing hTERT have been in continuous culture for >10 mo, whereas the control culture senesced after approximately 2 mo. Telomerase activity was monitored in cells with a polymerase chain reaction-based telomerase activity assay. Telomerase-expressing cells contained mRNA for alpha smooth muscle actin, smoothelin, oxytocin receptor, and estrogen receptor alpha, but the estrogen receptor beta receptor was lost. Immunoblotting analysis identified the expression of calponin, caldesmon, alpha smooth muscle actin, and oxytocin receptor. Although estrogen receptor expression was below the level of detection with immunoblotting, transfection experiments performed with reporter constructs driven by estrogen response elements demonstrated estrogen responsiveness in the hTERT-HM. In addition, treatment of hTERT-HM with oxytocin caused a concentration-dependent increase in intracellular calcium levels, confirming the presence of functional oxytocin receptors. Myometrial cells immortalized with hTERT retained markers of differentiation that are observed in primary cultures of smooth muscle cells. The expression of various smooth muscle/myometrium cell markers suggests that these cells may be an appropriate model system to study certain aspects of human myometrial function.
Authors:
Jennifer Condon; Su Yin; Bobbie Mayhew; R Ann Word; W E Wright; J W Shay; William E Rainey
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biology of reproduction     Volume:  67     ISSN:  0006-3363     ISO Abbreviation:  Biol. Reprod.     Publication Date:  2002 Aug 
Date Detail:
Created Date:  2002-07-23     Completed Date:  2003-01-24     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0207224     Medline TA:  Biol Reprod     Country:  United States    
Other Details:
Languages:  eng     Pagination:  506-14     Citation Subset:  IM    
Affiliation:
Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9032, USA.
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MeSH Terms
Descriptor/Qualifier:
Biological Markers
Calcium / metabolism
Cell Line
Cell Separation
Estrogens / pharmacology
Female
Fluorescent Antibody Technique, Indirect
Gene Expression Regulation, Enzymologic / genetics
Genetic Vectors
Humans
Immunoblotting
Muscle, Smooth / drug effects,  metabolism
Myometrium / cytology,  enzymology*
Oxytocin / pharmacology
RNA-Directed DNA Polymerase / chemistry
Retroviridae / genetics
Reverse Transcriptase Polymerase Chain Reaction
Telomerase / biosynthesis,  genetics,  metabolism*
Telomere / chemistry,  genetics
Transfection
Uterus / cytology,  metabolism
Grant Support
ID/Acronym/Agency:
HD 11149/HD/NICHD NIH HHS
Chemical
Reg. No./Substance:
0/Biological Markers; 0/Estrogens; 50-56-6/Oxytocin; 7440-70-2/Calcium; EC 2.7.7.49/RNA-Directed DNA Polymerase; EC 2.7.7.49/Telomerase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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