- Poster presentation
- Open Access
Characterization of primary mammary epithelial cells with loss of BRCA1at a single cell level
BMC Proceedingsvolume 7, Article number: P58 (2013)
Loss of BRCA1 has been linked to increased cell proliferation in human mammary epithelial cells. To determine if this phenotype is mirrored in the normal-appearing mammary epithelial cells from mouse models of BRCA1 deficiency, time-lapse imaging was performed on primary mammary epithelial cell (PMEC) cultures. Three distinct genetic models were tested to evaluate the role of p53 haploinsufficiency and p53 haploinsufficiency in the background of ERα over-expression to altered cell proliferation.
PMEC cultures were generated from 10-12 month old wild-type, Brca1f11/f11/MMTV−Cre, Brca1f11/f11/MMTV−Cre/p53+/- and Brca1f11/f11/MMTV−Cre/p53+/−/Tet-op-ER/MMTV-rtTA mice. Live cell phase contrast time-lapse imaging performed for 5-7 days immediately after plating. Timm’s Tracking Tool software (http://www.helmholtz-muenchen.de/scd/service/scientific-services/software-downloads/index.html) was used to measure individual cell lifetimes.
Mean cell lifetimes in generations 1-4 were significantly shorter in PMEC cultures from Brca1f11/f11/MMTV−Cre and Brca1f11/f11/MMTV−Cre/p53+/− mice as compared to Brca1f11/f11/MMTV−Cre/p53+/−/Tet-op-ER/MMTV-rtTA and wild-type mice. A higher percentage of dividing cells were found in Brca1f11/f11/MMTV−Cre, Brca1f11/f11/MMTV−Cre/p53+/− and Brca1f11/f11/MMTV−Cre/p53+/−/Tet-op-ER/MMTV-rtTA mice as compared to wild-type mice. Brca1f11/f11/MMTV−Cre/p53+/−/Tet-op-ER/MMTV-rtTA mice showed the highest level of colony formation and lowest numbers of apoptotic cells. Brca1f11/f11/MMTV−Cre/p53+/− mice showed the lowest level of colony formation and highest number of apoptotic cells.
Loss of Brca1 by itself was sufficient to decrease cell lifetime however this was modifiable by exposure to ERα overexpression. An inverse relationship between colony formation and numbers of apoptotic cells was found. In summary, genotype-specific differences in primary mammary epithelial cell behavior were revealed by single cell tracking.
Supported by NCI, NIH RO1CA112176 (P.A.F.), NCI, NIH. R01CA89041-10S1 (P.A.F.), Deutscher Akademischer Austaush Dienst e.V. A/09/72227 Ref. 316 (R.E.N.), Department of Defense W81XWH-11-1-0074 (R.E.N.), NIH IG20 RR025828-01 (Rodent Barrier Facility Equipment), and NIH NCI 5P30CA051008 (Microscopy and Imaging and Animal Shared Resources).