Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation.
We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the beta-casein gene promoter (p-344/-1betac-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction (R=0.99, P<0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous beta -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.
Shan
Liang
L
Department of Radiology, Howard University, Washington, DC 20060, USA.
Zhang
Renshu
R
Zhang
Wanghai
W
Lee
Edward
E
Sridhar
Rajagopalan
R
Snyderwine
Elizabeth G
EG
Wang
Paul C
PC
D000818:Animals; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D053614:Janus Kinase 2 / Q000378:metabolism; D008163:Luminescent Measurements; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction
D002364:Caseins; D050799:STAT5 Transcription Factor; D053614:Janus Kinase 2; D019908:Proto-Oncogene Proteins c-raf