Vol. 179, No. 4, Supplement, Monday, May 19, 2008 THE JOURNAL OF UROLOGY® 391

including sub-lethal stresses induced by chemotherapeutic agents.

METHODS: To identify novel senescence-inducing compounds, we developed a high-throughput, 96-well, cell-based assay

counting) and effect on other cancer cell lines (LNCaP, PC3) in vitroand in vivo.

RESULTS: In DU145 prostate cancer cells, we have used this assay to screen a library of 4200 known bioactive small molecules and natural products at a concentration of 1 µM. There were 51(1.2%) primary hits. Compounds underwent secondary testing including the ability to induce permanent growth arrest, and the increased expression of the senescence marker genes GLB1, P311, DOC1 and BRAK. The

In vitroa senescent growth arrest via increased expression of the CDK inhibitor proteins p21 and p27kip1

body weight increases SAB-gal activity, p27kip1 protein expression, and

the ability to induce cellular changes consistent with cellular senescence in an in vivo tumor model.

CONCLUSIONS: Our results validate the ability of our assay to identify compounds that induce molecular and cellular responses characteristic of senescence in prostate cancer cells. Moreover, our

in prostate cancer cells in vitro and in vivo (DOD, NIH).Source of Funding: DOD NIH.

1136PLEXIN-B1 MUTATIONS AND PROSTATE CANCERTharani Nitkunan*, Oscar G W Wong, Chun Zhou, Joseph Nariculam, Phillipa Munson, Jason Constantinou, Alex Freeman, John R Masters, Magali Williamson. London, United Kingdom.

INTRODUCTION AND OBJECTIVE: Semaphorins are a large group of proteins, which act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Our objective was to determine whether the semaphorin signaling pathway was harnessed by cancer cells during metastasis.

METHODS: Single strand conformation polymorphism was used to determine whether there were any mutations in Plexin-B1, in prostate cancer cell lines and in clinical samples of prostate cancer. The expression of Plexin-B1 in radical prostatectomy specimens was investigated using tissue microarrays. To determine whether the mutations in Plexin-B1 have an effect on cell function, cDNA constructs were made encoding wild type Plexin-B1 and four mutant versions of the protein using site directed in vitro mutagenesis. Cell collapse assay, time lapse video microsocopy and cell adhesion assays were used to determine whether the mutations altered the function of Plexin-B1. Binding assays were used to determine whether the mutations affected the ability of Plexin-B1 to bind to its downstream effector, Rac.

RESULTS: We report 13 somatic missense mutations in the cytoplasmic domain of the Plexin-B1 gene. Mutations were found in 89% (8/9) of prostate cancer bone metastases, 41% (7/17) of lymph node metastases and 46% (41/89) of primary cancers. 40% of prostate cancers contained the same mutation. Overexpression of the Plexin-B1 protein was found in the majority of primary tumours. The mutations alter the function of Plexin-B1 by increasing cell motility and adhesion, and by decreasing cell collapse. The mutations also inhibit or hinder Rac binding.

CONCLUSIONS: Plexin-B1 is frequently mutated in prostate cancer. Mutations in Plexin-B1 alter cell function. Plexin-B1 is likely to be a key player in cancer metastasis and is a potential target for anti-cancer therapy.

Source of Funding: Royal College of Surgeons of England, South Essex Medical Education and Research Trust, Prostate Research Campaign UK, Prostate Cancer Research Centre.

1137EFFECT OF THE SPECIFIC SRC KINASE INHIBITOR AZD0530 ON OSTEOLYTIC LESIONS IN PROSTATE CANCERJoy C Yang, Lanfang Bai, Hsing-Jien Kung, Christopher P Evans*. Sacramento, CA.

INTRODUCTION AND OBJECTIVE: Advanced prostate cancer becomes androgen-independent and causes bone metastasis with resulting patient morbidity. Although osteoblastogenesis is mostly observed in patients through autopsy, it is believed that bone resorption

to play an important role in tumor growth and metastasis. In this study,

osteolytic prostate cancer cell line PC-3 cells both in vitro and in vivo. METHODS: PC-3 cells were treated with various doses

phosphorylation status of Src kinase and its downstream kinases were

on the expression of IL-8, u-PA and MMP-9, molecules responsible for

vivo study, 2 x 105 PC-3 cells were injected into the tibia of 24 SCID mice. Mice were divided into two groups with the treatment group receiving 25

monitored by X-ray radiography weekly. At the end of eight weeks, mice

staining and the pyridinoline (PYD) crosslink assay.

response manner. Phosphorylation of Src, FAK, paxillin and P130cas

treatment. Secretion of the MMP-9 decreased in PC-3 cells treated with

out of 12 control mice showed osteolytic lesions as compared to only 4 in the treatment group. Both H & E and the Tartrate-resistant acid phosphatase (TRAP) stainings revealed populated osteoclasts in the bone sections of control mice but not in the treated mice. Serum PYDassay showed almost twice the amount of PYD crosslinks detected in the control samples than in the treatment.

inhibits the activities of Src and FAK kinases and their downstream substrates. This inhibition affects cell proliferation and the expression of

invasion into the bone matrix. The animal study further demonstrates

therapeutic advantage in treating patients with prostate cancer.Source of Funding: PCF.

1138A NEW CELL LINE EXPRESSING A NOVEL TYPE OF TMPRSS2-ERG GENE FUSION DERIVED FROM PRIMARY TUMORS OF FAMILIAL PROSTATE CANCER PATIENTHongzhen Li, Bungo Furusato, Jun Miki, Chen Sun, Taduru Sreenath, Albert Dobi, Gyorgy Petrovics, Bharati Hukku, Isabell A Sesterhenn, David G McLeod, Shiv Srivastava, Johng S Rhim*. Bethesda, MD, Washington, DC, and Orion Township, MI.

INTRODUCTION AND OBJECTIVE: Recent studies have established that a high proportion of prostate cancer harbors a gene fusion between the androgen regulated TMPRSS2 gene and the ETSgenes ERG, ETV1 or ETV4. However in vitro model representing primary tumors to study the molecular mechanisms and functional consequences of this important chromosomal rearrangement are currently limited and are greatly needed.

METHODS: The tumor tissue (RC-123T) used for generating the RC-123T/E cell line with HPV-16 E6E7 genes was obtained from a 57 year old familial prostate cancer patient who had adenocarcinoma

phenotypically and cytogenically. Dual color fluorescence in situ

aparts, and for translocation of TMPRSS2 and ERG and ETV1, in both interphase nuclei as well as metaphases. The cells were also

cell markers by immunohistochemistry and RT-PCR.