Thesis
The effect of mitogen-activated protein kinase phosphatase-2 (MKP-2) overexpression in prostate cancer cell function and its clinical prostate cancer progresion
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2013
- Thesis identifier
- T13621
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- MAP kinase signals have been reported as a crucial cancer chemopreventive and chemotherapeutic target due to their involvement in tumour cell growth, proliferation, apoptosis and survival. They consist principally of extracellular signal-regulated kinase (ERK), c-jun NH2- terminal kinase (JNK) and p38 MAP kinase, which has been correlated with a more malignant phenotype in several tumour models and in vivo. A key regulatory off switch for the MAP kinases is the dual specificity phosphatase, DUSP-4, also known as MAP kinase phosphatase-2 (MKP-2). This phosphatase is a type one DUSP, located in the nucleus, induced in cells in response to a number of extracellular stimuli and selective for ERK and JNK in vitro. This study was designed to examine the role of MKP-2 in the prostate cancer cell lines LNCaP Androgen sensitive (AS) and Androgen Insensitive (AI) in proliferation and apoptosis in vitro and cancer development in vivo. MKP-2 was found to be expressed endogenously in both (AS) and (AI) cells. Adv.MKP-2 was then used as a tool to study the effect of MKP-2 overexpression in LNCaP (AS) and (AI). Immunofluorescent staining revealed strictly nuclear expression of the MKP-2 adenovirus, with more than 90% of the cells infected. In LNCaP (AS), ERK phosphorylation in response to EGF was transient whilst in LNCaP (AI) cells it was sustained. In contrast, JNK phosphorylation was sustained in both LNCaP (AS) and (AI) in response to Ultraviolet (UV) light C (60 j/m2). Infection of both LNCaP (AS) and (AI) cells with Adv.MKP-2 significantly inhibited the phosphorylation of ERK and JNK. The kinetics of cell cycle progression was established. Stimulation with FCS over 48 h in LNCaP (AS) and 24 h in LNCaP (AI) caused a marked increase in cells both in S phase and G2/M phase. Following infection with Adv.MKP-2 progression inhibited cell numbers in both S phase and G2/M phase where they were reduced by over 50%. This correlated with a reduction in the expression of both cyclin B and D1. In contrast, Adv.MKP-2 did not modify apoptosis in both LNCaP cell lines stimulated by several agents including; UV-C, Doxorubicin and X-ray, These results indicate the potential for MKP-2 to influence cancer cell function proliferation but not apoptosis. To specify the affects are mediated via dephosphorylation of ERK and/or JNK, we used a form of MAP kinase which has a triple substitution of arginine within positions R74, R75 and R76 to alanine and is unable to bind to ERK. Using this mutation further specifies the role of ERK or, in particular, JNK in regulating cellular proliferation, Adv.MKP-2-NLS1 caused inhibition of either EGF or UV-C ERK and JNK phosphorylation respectively in both LNCaP cells, whilst Adv.MKP-2-CI was not effective, suggesting that Adv.MKP-2-NLS1 can still bind and deactivate ERK. However, both NLS1 and CI MKP-2 adenoviruses inhibited cell cycle progression. The effect of either WT-MKP-2 or CI-MKP-2 on histone H3 phosphorylation was also assessed and both were found to cause substantial inhibition, suggesting the potential of a phosphataseindependent function for MKP-2 in the regulation of cell cycle progression. Finally, work using human prostate tissue confirmed that MKP-2 is expressed in invasive prostate tumours and localised in the nucleus, and staining intensity was positively correlated with PSA below 10 ng/ml levels and in early grade of Gleason score. These findings clearly suggest the potential for MKP-2 to be used as a possible gene tool in prostate cancer, which requires not only the inhibition of LNCaP cells proliferation in vitro, but PSA below 10 ng/ml levels and in early stage of Gleason score survival in vivo.
- Resource Type
- DOI
- Date Created
- 2013
- Former identifier
- 1004027
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