Date of Award

Spring 2002

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

George L. Wright, Jr.

Committee Member

Timothy J. Bos

Committee Member

Ann E. Campbell

Committee Member

Kenneth D. Somers

Abstract

Prostate cancer (PCA) is the most common cancer and the second leading cause of death among American men. The high mortality is greatly attributed to the lack of early detection tools and effective treatment for metastasis and relapses. Biomarkers that can discriminate benign from malignant tumor and signal the development of androgen independent and metastatic tumor are needed. A biomarker designated prostate specific membrane antigen (PSMA) has the potential to fulfill this need. The objective of this study is to develop a clinically useful immunoassay for quantitation of serum PSMA and to study the molecular mechanism underlying the upregulation of the PSMA gene after androgen deprivation therapy. Surface Enhanced Laser Ionization/Desorption (SELDI) ProteinChip® mass spectrometry was used for the first aim. A baculovirus recombinant PSMA was generated, purified and characterized. After the SELDI immunoassay conditions were optimized, a standard curve was established using rPSMA as the substitute antigen. Serum PSMA levels, as measured by SELDI immunoassay, were able to distinguish benign from malignant prostate disease. To understand the transcriptional regulation of the PSMA gene, the second aim focused on the 14 AP-1 sites in the 5′ region of the PSMA gene. By Northern blot, the PSMA mRNA levels in LNCaP cells were found to be increased by the inducers of AP-1, EGF and TPA, and decreased by androgens. The PSMA promoter activity, as analyzed by luciferase assay, was also induced by EGF and cotransfected AP-1 proteins, but suppressed by androgens. The binding of AP-1 to three putative AP-l sites in the vicinity of the PSMA transcription initiation sites was demonstrated by gel shift assay. The DNA binding was also decreased by androgens and increased by EGF. Gel shift competition further indicated that the AP-1 binding might be inhibited by the interaction between androgen receptors and AP-1. To summarize this study, the serum PSMA level was measured by SELDI immunoassay and was shown to be a more effective biomarker than PSA for differentiating benign from malignant prostate disease. Moreover. PSMA gene transcription is activated by AP-1 and suppressed by androgens possibly through the inhibition of AP-1 binding to the PSMA promoter by androgen receptor.

Comments

A Dissertation Submitted to the Faculties of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/mphs-6p25

ISBN

9780493713045

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