Rational Design of Human Prolactin Receptor Antagonists for Breast Cancer Therapy

Abstract

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget,, Washington, DC 20503 AGENCY USE ONLY (Leave blank)2. REPORT DATE October 2000 REPORT TYPE AND DATES COVEREDAnnual (15 Sep 99 -14 Sep 00) TITLE AND SUBTITLE Rational Design of Human Prolactin Receptor Antagonists for Breast Cancer Therapy AUTHOR(S)Wen Y. Chen, Ph.D. There were three tasks proposed for the first year of this project. All three tasks have been accomplished. FUNDING NUMBERSWe have cloned and confirmed by sequencing the cDNAs encoding hPRL, hPRL-G129R and hPRL-BP (task 1). All three cDNAs have been transfected into mouse L cells and stable mouse L cells have been established (task 2).We have also maintaining 12 human cancer cell lines purchased from ATCC(task 3). In addition, we have demonstrated that hPRL-G129R is able to inhibit breast cancer cell proliferation via induction of apoptosis (Clinical Cancer Research, 5:3583-3593, 1999). We are confident that the project will go on as proposed in years two and three. SUBJECT TERMS IntroductionHuman breast cancer is the most predominant malignancy with the highest mortality rate in women from western society. Many risk factors have been identified for this disease. Several lines of evidence strongly linked human prolactin (hPRL) to breast carcinogenesis. In this proposal, two novel approaches have been designed to generate hPRL receptor specific antagonists. First approach is to adopt a sitedirected mutagenesis strategy by which hGH receptor antagonist, hGH-G120R, was discovered, to produce a mutated hPRL, hPRL-G129R, and use it as hPRL receptor blocker. The other approach is to design and produce a soluble form of extra-cellular domain of hPRL receptor namely hPRL binding protein (hPRL-BP), and use it to sequester autocrine/paracrine effects of hPRL. After cloning of hPRL and hPRL-BP cDNAs, mutation will be made in hPRL cDNA to generate hPRL-G129R. Human PRL, hPRL-G129R and hPRL-BP cDNAs will then be used to establish stably transfected mouse L cells. The proteins produced from stably transfected mouse L cells will be purified and used to test its bioactivities in ten different human breast cancer cell lines and two non-breast origin human cancer cell lines (as controls) for receptor binding, inhibition of phosphorylation of the STAT 5 protein induced by hPRL (as an indicator for intracellular signaling), and inhibition of human breast cancer cell proliferation. We hope that these two novel approaches will ultimately result ...