Only the adenoma or carcinoma things compartment was scored, except in the reduction mammoplasty group, where the entire section was analyzed. Statistics Unless otherwise noted, all results are presented as means SEM. Paired t tests were conducted on IHC quantification and in vitro assays. Tumor incidence was compared between WAP Brk and wt mice using Fishers exact test. Tumor latency was estimated using Kaplan Meier methodology and curves compared between WAP Brk and wt mice using the Wilcoxon test. A chi squared test was used to compare the association of tumors staining positive for phospho p38 MAPK with tumors staining positive for Brk. All statistical tests were conducted at a significance level of 0. 05.
Results The WAP Brk transgene is expressed in the mammary gland To determine the effects of inducible Brk expression in the normal mammary gland in vivo, a Brk cDNA encod Inhibitors,Modulators,Libraries ing the full length wild type protein kinase was put under the control of the WAP promoter, which directs transgene expression predominantly to the luminal epithelium in response to the hormones of pregnancy and upon lactation. Founders were gen erated and successfully bred to establish two indepen dent lines, transgene presence was verified by PCR. Expression of Brk protein was confirmed by both Western blotting of purified mammary epithelial cells and IHC analysis of formalin fixed paraffin embedded mammary tis sues. Total p38 MAPK, a ubi quitous member of the MAP kinase family, served as a loading control in Western blotting experiments.
Total p38 levels were somewhat lower in MEC purified from mammary glands of both virgin and pregnant animals, but remained rela tively constant during involution Days 1 to 6. In lactat ing transgenic but not wild type animals, Brk protein expression Inhibitors,Modulators,Libraries was readily detectable at involution Inhibitors,Modulators,Libraries Day 1 and this persisted to Day 6 of mam mary involution. Brk expression was signifi cantly reduced by Day 14 of mammary involution, variable weak expression of Brk also occurred in pregnant transgenic animals, but remained consistently high during lactation. IHC of FFPE tissues also demonstrated Inhibitors,Modulators,Libraries significant Brk protein expression in mammary epithelial cells in both transgenic lines, we designated these independently derived transgenic lines Brk97 and Brk83. Brk was undetect able in wild type controls by both protein detection methods.
WAP Brk expression alters Inhibitors,Modulators,Libraries the kinetics of mammary gland involution Based on in vitro studies of Brk overexpression in HB4a mammary epithelial cells, we hypothesized that Brk may confer a proliferative and or pro survival phenotype to normal MEC in vivo. Additionally, numerous studies have demonstrated similar phenotypes upon selleck chemical Rapamycin expression of human breast oncogenes in the mouse mammary gland. Therefore, we investigated whether Brk expression in the mouse mammary gland resulted in proliferative or inhibitory effects.