scholarly journals Estrogen Up-Regulates Mismatch Repair Activity in Normal and Malignant Endometrial Glandular Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (10) ◽  
pp. 4863-4870 ◽  
Author(s):  
Tsutomu Miyamoto ◽  
Tanri Shiozawa ◽  
Hiroyasu Kashima ◽  
Yu-Zhen Feng ◽  
Akihisa Suzuki ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Mismatch Repair ◽  
Proliferating Cells ◽  
Endometrial Cells ◽  
Estrogen Exposure ◽  
Estrogen Receptor Positive ◽  
Ishikawa Cells ◽  
Glandular Cells ◽  
Repair Activity

Impaired mismatch repair (MMR) is reportedly crucial in the early stages of endometrial carcinogenesis. Although estrogen exposure is considered an important risk factor for endometrial carcinoma, the relationship between estrogen and MMR activity remains undetermined. The present study was undertaken to elucidate the effect of estrogen on MMR activity in normal and malignant endometrial cells. The expression of MMR proteins, hMLH1 and hMSH2, and its correlation with estrogen was examined using immunohistochemical and immunofluorescent techniques. The effect of estradiol (E2) on the expression of hMLH1/hMSH2 protein/mRNA and in vitro MMR activity using two types of heteroduplex (G/T mismatches, 2-base insertion-deletion loops) was examined in cultured normal endometrial glandular cells and estrogen receptor-positive endometrial carcinoma Ishikawa cells. Immunohistochemical expression of hMLH1 and hMSH2 in normal endometrial glands was positively correlated with the serum E2 levels. The expression of hMLH1/hMSH2 protein and mRNA was increased in normal endometrial glandular and Ishikawa cells by E2 treatment. In vitro MMR activity was up-regulated by E2 in both types of cell and heteroduplex. Immunofluorescent analysis demonstrated that E2 enhanced proliferation and hMLH1/hMSH2 expression in both cells; however, proliferating cells without hMLH1/hMSH2 expressions implying high-risk cells were more frequently observed under low E2 concentrations. Collectively, the E2-induced up-regulation of MMR activity in endometrial cells suggests that high estrogen levels act as an intrinsic defense against endometrial carcinogenesis, whereas the imbalance between cell growth and MMR under low E2 environment as seen at postmenopause is vulnerable to carcinogenesis.

scholarly journals Expression of NR1H3 in endometrial carcinoma and its effect on the proliferation of Ishikawa cells in vitro

2019 ◽  
Vol Volume 12 ◽  
pp. 685-697
Author(s):  
Fang Fang ◽  
Dawei Li ◽  
Lu Zhao ◽  
Yue Li ◽  
Teng Zhang ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Ishikawa Cells

scholarly journals Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology

Reproduction ◽  
2007 ◽  
Vol 134 (3) ◽  
pp. 525-534 ◽  
Author(s):  
S Guay ◽  
A Akoum
Keyword(s):  
Interleukin 1 ◽  
Receptor Type ◽  
Type Ii ◽  
Endometrial Cells ◽  
Functional Changes ◽  
Empty Vector ◽  
Ishikawa Cells ◽  
Empty Expression Vector

Our previous studies showed a marked deficiency in interleukin 1 receptor type II (IL1R2) in the endometrial tissue of women with endometriosis, particularly in epithelial cells. We believe that such a deficiency in IL1R2, a potent and specific IL1 inhibitor, makes endometrial cells more sensitive to IL1 and less capable of buffering the cytokine’s effects, which may lead to functional changes that favor endometriosis development. The main objective of our study was to stably inhibit IL1R2 expression in endometrial cells in order to evaluate the role of IL1R2 deficiency in endometriosis pathophysiology. Stable clones of Ishikawa adenocarcinoma endometrial cells transfected with IL1R2 antisense and showing downregulation of IL1R2 protein expression, or with the empty expression vector alone and showing no noticeable difference in IL1R2 expression, were selected. The downregulation of IL1R2 expression in IL1R2 antisense transfectants when compared with control cells was confirmed by ELISA, Western blot and immunofluorescence. In these cells, IL1R2 expression was markedly reduced, compared with non-transfected cells or cells transfected with the empty vector, and there was a significant increase in the basal and the IL1-β (IL1B)-induced levels of matrix metalloproteinase (MMP)-2 and MMP-9 secretion. Furthermore, a significant decrease in IL1B-induced secretion of tissue inhibitor of MMPs-1, a known MMP-9 inhibitor, was observed. These in vitro data make plausible a role for IL1R2 deficiency in the capability of endometrial cells to invade the host tissue and develop in ectopic locations.

DNA mismatch repair detected in human cell extracts

1987 ◽  
Vol 7 (1) ◽  
pp. 218-224
Author(s):  
P M Glazer ◽  
S N Sarkar ◽  
G E Chisholm ◽  
W C Summers
Keyword(s):  
Hela Cell ◽  
Mismatch Repair ◽  
Plasmid Dna ◽  
Coli Strain ◽  
Dna Polymerization ◽  
E Coli ◽  
Cell Extracts ◽  
Repair Activity ◽  
Supf Gene

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

scholarly journals DNA mismatch repair detected in human cell extracts.

1987 ◽  
Vol 7 (1) ◽  
pp. 218-224 ◽  
Author(s):  
P M Glazer ◽  
S N Sarkar ◽  
G E Chisholm ◽  
W C Summers
Keyword(s):  
Hela Cell ◽  
Mismatch Repair ◽  
Plasmid Dna ◽  
Coli Strain ◽  
Dna Polymerization ◽  
E Coli ◽  
Cell Extracts ◽  
Repair Activity ◽  
Supf Gene

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.

scholarly journals Heteroduplex DNA formation is associated with replication and recombination in poxvirus-infected cells.

Genetics ◽  
1991 ◽  
Vol 129 (1) ◽  
pp. 7-18 ◽  
Author(s):  
C Fisher ◽  
R J Parks ◽  
M L Lauzon ◽  
D H Evans
Keyword(s):  
Mismatch Repair ◽  
Direct Detection ◽  
Dna Viruses ◽  
Dna Recovery ◽  
High Frequencies ◽  
Repair Activity ◽  
Infected Cells ◽  
Dna Substrates ◽  
Heteroduplex Formation

Abstract Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells and recombine at high frequencies. Calcium phosphate precipitates were used to cotransfect Shope fibroma virus-infected cells with different DNA substrates and the recombinant products assayed by genetic and biochemical methods. We have shown previously that bacteriophage lambda DNAs can be used as substrates in these experiments and recombinants assayed on Escherichia coli following DNA recovery and in vitro packaging. Using this assay it was observed that 2-3% of the phage recovered from crosses between point mutants retained heteroduplex at at least one of the mutant sites. The reliability of this genetic analysis was confirmed using DNA substrates that permitted the direct detection of heteroduplex molecules by denaturant gel electrophoresis and Southern blotting. It was further noted that heteroduplex formation coincided with the onset of both replication and recombination suggesting that poxviruses, like certain bacteriophage, make no clear biochemical distinction between these three processes. The fraction of heteroduplex molecules peaked about 12-hr postinfection then declined later in the infection. This decline was probably due to DNA replication rather than mismatch repair because, while high levels of induced DNA polymerase persisted beyond the time of maximal heteroduplex recovery, we were unable to detect any type of mismatch repair activity in cytoplasmic extracts. These results suggest that, although heteroduplex molecules are formed during the progress of poxviral infection, gene conversion through mismatch repair probably does not produce most of the recombinants. The significance of these observations are discussed considering some of the unique properties of poxviral biology.

A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

2010 ◽  
Vol 23 (6) ◽  
pp. 496-501 ◽  
Author(s):  
Shi-Ying LI ◽  
Xiang-Yu ZHANG ◽  
Xin ZHANG ◽  
Yan LAN ◽  
Zi-Chun HUA
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Dna Mismatch ◽  
Repair Activity

Hormonal modulation of ishikawa cells during three-dimensional growth in vitro☆

1998 ◽  
Vol 5 (4) ◽  
pp. 217-223 ◽  
Author(s):  
D PINELLI ◽  
J DRAKE ◽  
M WILLIAMS ◽  
D CAVANAGH ◽  
J BECKER
Keyword(s):  
Three Dimensional ◽  
Ishikawa Cells ◽  
Hormonal Modulation ◽  
Dimensional Growth

scholarly journals Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues

2021 ◽  
Vol 22 (15) ◽  
pp. 7920
Author(s):  
Myroslava Mytsyk ◽  
Giulia Cerino ◽  
Gregory Reid ◽  
Laia Gili Sole ◽  
Friedrich S. Eckstein ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Therapeutic Potential ◽  
Stromal Vascular Fraction ◽  
Human Adipose Tissue ◽  
Bioreactor Culture ◽  
Proliferating Cells ◽  
Angiogenic Potential ◽  
Engineered Tissues

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

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