Abstract：
:Intracellular calcium (Ca2+) is involved in the regulation of a variety of biological functions in cancer cells, including growth inhibition, tumor invasiveness, and drug resistance. To gain insight into the possible role played by Ca2+ in the development of drug resistance in breast cancer, we performed a comparative high-content analysis of the intracellular Ca2+ dynamics in drug-sensitive human breast cancer MCF-7 cells and five drug-resistant, MCF-7-derived clonal cell lines. Fura-2 single cell ratiometric fluorescence microscopy was used to monitor real-time quantitative changes in cytosolic-free Ca2+ concentration ( [Ca2+]i ) upon addition of phosphoinositol-coupled receptor agonists. While the magnitude and the onset kinetics of the [Ca2+]i rise were similar in drug-sensitive and drug-resistant cell lines, the decay kinetics of the [Ca2+]i increase was found to be consistently slower in drug-resistant than drug-sensitive cells. Such a delay in reestablishing homeostatic [Ca2+]i persisted in the absence of extracellular Ca2+ and was independent of the expression or function of specific drug efflux pumps associated with drug resistance. Moreover, intracellular Ca2+ pools releasable by phosphoinositol-coupled receptor agonists or thapsigargin appeared to be differentially shared in drug-sensitive and drug-resistant cells. In light of the clinical relevance that drug resistance has in the treatment of cancer, the molecular and biochemical relationship between alterations in Ca2+ dynamics and drug resistance demands to be further investigated and tested in a wider array of cell types. Automated microscopy will help greatly in this pursuit by facilitating both sample imaging and data analysis, thus allowing high-content as well as high-throughput screening of large sample sets. A protocol for studying [Ca2+]i kinetics with a commercially available automated imaging platform is described.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
DeBernardi MA,Brooker Gdoi
10.1016/S0076-6879(06)14018-5subject
Has Abstractpub_date
2006-01-01 00:00:00pages
317-35eissn
0076-6879issn
1557-7988pii
S0076-6879(06)14018-5journal_volume
414pub_type
杂志文章abstract：:Hexameric helicases couple the energy of ATP hydrolysis to processive movement along nucleic acids and are critical components of cells and many viruses. Molecular motion derives from ATP hydrolysis at up to six distinct catalytic centers, which is coupled to the coordinated action of translocation loops in the center...
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journal_title：Methods in enzymology
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journal_title：Methods in enzymology
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journal_title：Methods in enzymology
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journal_title：Methods in enzymology
pub_type： 杂志文章
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journal_title：Methods in enzymology
pub_type： 杂志文章
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journal_title：Methods in enzymology
pub_type： 杂志文章,评审
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journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.05.006
更新日期：2016-01-01 00:00:00
abstract：:Ubiquitin-like protein (Ubl) ligation is common to diverse archaea and targets many cellular pathways, including those associated with sulfur mobilization, and also tags proteins as substrates for degradation by the proteasome. Here we highlight protocols to assay proteasome function and Ubl ligation in archaea. A cha...
journal_title：Methods in enzymology
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doi：10.1016/bs.mie.2018.12.036
更新日期：2019-01-01 00:00:00
