1999 WHRG Grant Program

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Apoptotic Signaling Pathways in the Mammary Gland
Kristel Schorr
School of Medicine, University of Maryland, Baltimore

My Ph.D. work centers on the identification of signaling pathways that control induction of apoptotic pathway genes during mammary gland involution. I have reached a point in my research where I need to expand my studies from whole animal models to in vitro systems in which I can manipulate the expression or activation of individual components of specific signaling pathways.

The Bcl-2 family of apoptosis regulators have been implicated in many breast cancers (Zapata et al., 1998), and changes in expression levels of individual Bcl-2 family members can be correlated with prognosis (Sjostrom et al., 1998; Krajewski et al., 1995). To date, the signaling pathways which control expression of the Bcl-2 family during involution are less defined. Our mammary gland involution studies demonstrate a temporal correlation between signal transducer and activator or transcription (STAT) 3 activation and Bcl_x and Bax expression. Additional analyses, however, are required to understand the mechanism of gene regulation and interpret this association.

Hypothesis: Activation of STAT 3 during mammary gland involution regulates expression levels of Bcl_L and Bax, and consequently induces apoptosis in mammary epithelial cells.

Specific Aims:

Establish an in vitro culture system which accurately parallels mammary gland involution in vivo. Compare HC11 cells, primary epithelial cells, and whole mammary gland organ culture.
Determine if changes in phosphorylated STAT 5 and STAT 3 levels regulate expression of Bcl-2 family members during the transition from mammary gland lactation to involution.

Methods: Activity and expression levels of Janus kinase (Jak2), STATs 5 and 3, and epidermal growth factor receptor (EGFR) will be measured by immunoprecipitation and measurement of tyrosine phosphorylation. Western blotting will be used to quantitate the protein expression levels of Bax and Bcl-x_L. Adenovirus vector systems will be used to introduce dominant-negative STAT 5 and STAT 3 genes into our in vitro involution model. The beta-galactosidase adenovirus vector will be used as a control. RNAse protection assays will be used to measure RNA expression levels of Bcl-2 family members. Apoptosis will be evaluated by in situ end-labeling of fragmented DNA.