טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAdi Ayala
SubjectSignaling Mechanisms of Growth Hormone in Human Prostate
Cancer Cells
DepartmentDepartment of Medicine
Supervisor Assistant Professor Ronnie Barkey


Abstract

This thesis demonstrates initial functional responses to GH in LNCaP cells, based on the study of GH-induced signaling. Western analysis of lysates showed that a 120 kDa protein, identified as Janus tyrosine kinase (JAK2), was phosphorylated in a time- and dose-dependent manner. GHR appeared as a broad band of 111-120 kDa. JAK2 appears to be constitutively associated to GHR. GH also induced phosphorylation of a 97 kDa protein, identified as signal transducer and activator of transcription (STAT5a). No phosphorylation of 93 kDa STAT5b was detected. GH is also known to activate the mitogen-activated protein kinase (MAPK; Erk1/2) in some cell types. Erk1/2 were phosphorylated in a dose- and time-dependent manner. The PI3K plays an important role in proliferation and survival of cells by activating protein kinase B (PKB/AKT). AKT was shown in LNCaP cells, as a 60 kDa protein, which was phosphorylated in a dose- and time-dependent manner following exposure to hGH.

In order to demonstrate the key role of JAK2 as the initial activation step in the signal transduction of hGH, we made use of a JAK2-specific inhibitor, AG-490. Exposure of LNCaP cells to AG-490 reduced hGH-induced JAK2 phosphorylation by up to 50%. AG-490 reduced by up to 65% the phosphorylation of Erk1/2 and reduced by up to 80% the hGH-induced phosphorylation of AKT. In order to demonstrate the possibility of cross-talk between the PI3K pathway, the activation of its substrate, AKT, and the phosphorylation of Erk1/2, we made use of a PI3K-inhibitor, wortmannin. Exposure of LNCaP cells to wortmannin prevented the hGH-induced phosphorylation of AKT and reduced basal AKT phosphorylation by up to 95%. Wortmannin also caused a reduction of up to 75% in the Erk1/2 phosphorylation. In contrast, the protein kinase C (PKC) inhibitor GF109203x had no effect on the hGH-induced Erk1/2 activation, ruling out the involvement of the PKC pathway in the activation of Erk1/2 in response to hGH.