Elizabeth A. Kingsley

Methylation of a CpG island within the promoter region of the KAI1 metastasis suppressor gene is not responsible for down-regulation of KAI1 expression in invasive cancers or cancer cell lines

The molecular basis for downregulation of the KAI1 metastasis suppressor gene in invasive and metastatic human cancers is unknown. We have used bisulphite methylation analysis of DNA from paraffin-embedded invasive bladder tumour samples and from bladder cancer cell lines to determine if hypermethylation of a CpG island within the KAI1 promoter is responsible for this effect. Representative invasive tumour cell lines were also exposed to 5-aza-2-deoxycytidine. We found no evidence for hypermethylation of the CpG island and suggest that mechanisms other than promoter hypermethylation are responsible for reduced KAI1 expression in invasive bladder tumours and tumour cell lines.

Inverse correlation between KAI1 mRNA levels and invasive behaviour in bladder cancer cell lines

We have previously shown that levels of KAI1 mRNA are dramatically reduced in invasive human bladder cancers. To further investigate the role of KAI1 in bladder cancer, we have examined the relationship between KAI1 mRNA levels and cell behaviour in 18 bladder cancer cell lines and a virus-transformed uro-epithelial cell line. We found that low KAI1 mRNA levels correlated with increased in vitro invasive ability, reduced Ca(2+)-dependent and -independent cell-cell adhesion and reduced adhesion to fibronectin. These data support the idea that loss of KAI1 expression is an important factor in tumour cell invasive behaviour.

Molecular profiling of bladder cancer : Involvement of the TGF-β pathway in bladder cancer progression

A human bladder cancer model of nine cell sublines derived from the BL17/2 cell line was used to evaluate genes related to disease progression. Molecular profiling of sublines that were non-tumorigenic and invasive in nude mice was performed and identified 1367 differentially-expressed genes. Quantitative real-time PCR analysis of six transforming growth factor-beta (TGF-beta) pathway genes using the entire panel of nine cell lines was performed. Bone morphogenetic protein-2 expression was significantly associated with in vivo tumorigenicity of the cell lines (p=0.0228, Mann-Whitney); inhibin-betaB was related to their invasiveness (p=0.0468, Mann-Whitney). Analysis of conditioned medium showed TGF-beta1 production to be significantly associated with the phenotype of the cell line. The study shows the possible involvement of the TGF-beta pathway in bladder cancer progression.

Relationship between expression of KAI1 metastasis suppressor gene, mRNA levels and p53 in human bladder and prostate cancer cell lines

The molecular basis for the loss of KAI1 expression in invasive and metastatic tumors and tumor cell lines is not understood. Recently, identification of a sequence with homology to the consensus p53-binding motif in the promoter of the KAI1 metastasis suppressor gene, has led to a proposal that transcriptional regulation by p53 controls expression of KAI1, and that a dramatic down-regulation of KAI1 mRNA levels in invasive tumors and many tumor cell lines, is directly due to loss of p53 function. We have tested this hypothesis by assessing KAI1 mRNA levels in a series of 22 cell lines derived from bladder and prostate cancers, in which we confirmed the p53 gene sequence and characterized the functional status of the endogenous p53 protein. We anticipated that cell lines expressing p53 capable of transactivation should express high levels of KAI1 mRNA compared with cell lines expressing defective p53, or which were p53-null. KAI1 mRNA levels were determined by northern analysis using a full-length KAI1 cDNA probe, and varied widely between cell lines examined. However, there was no association between these levels and p53 status. Furthermore, transfection of representative cell lines with wild-type p53, or exposure to DNA damaging agents, had no effect on KAI1 mRNA levels. Our data suggest that p53 is not a major factor regulating levels of KAI1 mRNA in bladder and prostate cancer cell lines.

Prostate cancer methods and protocols

Epidemiological Investigation of Prostate Cancer Graham G. Giles Human Prostate Cancer Cell Lines Pamela J. Russell and Elizabeth A. Kingsley Growth of Prostatic Epithelial and Stromal Cells In Vitro Donna M. Peehl Prostate Epithelial Stem Cell Isolation and Culture David L. Hudson and John R. W. Masters Generation of Immortal Human Prostate Cell Lines for the Study of Prostate Cancer Johng S. Rhim Spheroids of Prostate Tumor Cell Lines George Sgouros, Wei-Hong Yang, and Richard Enmon Animal Models of Prostate Cancer Pamela J. Russell and Dale J. Voeks Transgenic Mouse Models for Prostate Cancer: Identification of an Androgen-Dependent Promoter and Creation and Characterization of the Long Probasin Promoter-Large T Antigen (LPB-Tag) Model Susan Kasper, William Tu, Richard L. Roberts, and Scott B. Shappell In Vivo Models of Human Prostate Cancer Bone Metastasis Julie M. Brown Effects of Fixation on Tissues Elin Mortensen and Julie M. Brown Background, Methods, and Protocols for the Histopathological Diagnosis of Prostate Carcinoma Warick Delprado Realizing the Potential of Ejaculate/Seminal Fluid in Detecting and Predicting Natural History R. A. Gardiner, Michelle Burger, Judith A. Clements, and Martin F. Lavin Bisulfite Methylation Analysis of Tumor Suppressor Genes in Prostate Cancer from Fresh and Archival Tissue Samples Susan J. Clark, Douglas S. Millar, and Peter Molloy Production and Characterization of Antipeptide Kallikrein 4 Antibodies: Use of Computer Modeling to Design Peptides Specific to Kallikrein 4 Tracey J. Harvey, Ying Dong, Loan Bui, Russell Jarrott, Terry Walsh, and Judith A. Clements The Androgen Receptor CAG Repeat and Prostate Cancer Risk Peter E. Clark, Ryan A. Irvine, and Gerhard A. Coetzee Studies on Androgen Receptor Mutations and Amplification in Human Prostate Cancer Zoran Culig, Alfred Hobisch, Martin Erdel, Georg Bartsch, and Helmut Klocker Proteomics in the Analysis of Prostate Cancer Soren Naaby-Hansen, Kohji Nagano, Piers Gaffney, John R. W. Masters, and Rainer Cramer Application of Gene Microarrays in the Study of Prostate Cancer Colleen C. Nelson, Douglas Hoffart, Martin E. Gleave, and Paul S. Rennie Enhancer Trap Method Using a Green Fluorescent Protein Reporter Plasmid for Cloning Tissue-Specific Enhancers Active in Prostate Cells Fujiko Watt and Peter Molloy Targeted Alpha Therapy of Prostate Cancer Barry J. Allen, Yong Li, Syed M. A. Rizvi, and Pamela J. Russell Phenotypic and Functional Differences of Dendritic Cells Generated Under Different In Vitro Conditions Stephanie E. B. McArdle, Selman A. Ali, Geng Li, Shahid Mian, and Robert C. Rees Flavonoid Compounds in the Prevention and Treatment of Prostate Cancer Graham E. Kelly and Alan J. Husband Index

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components hadRF values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.

Preclinical studies of monoclonal antibodies for intravesical radioimmunotherapy of human bladder cancer

Eighty percent of bladder cancers present as superficial disease. Many are multifocal, and apparently successful treatment is frequently followed by recurrence. The use of monoclonal antibodies (MAbs) to target radiotherapy to these tumors offers great potential, especially since they can be administered directly into the bladder (intravesically) bypassing many of the side effects encountered to date with systemic MAb-based therapy. Implantation of human bladder cancer cell lines in the bladder wall of nude rats results in tumor formation, providing an excellent model to test this. Tumor size can be monitored by X-ray analysis after administration of urograffin. Comparative studies of two murine MAbs, BLCA-8, IgG3, and C1-137, IgG1, against malignant human bladder cancer cells have been performed. Radio-immunoconjugates produced with125Iodine (125I) have been used for biodistribution studies following administration directly into rat bladder. Radioiodinated intact MAbs or Fabs administered intravesically into nontumor bearing rats did not leak into the systemic circulation and were stable in urine for up to 100 h. Biodistribution studies carried out following intravesical administration of radio-immunoconjugates to tumor-bearing nude rats indicate better tumor uptake of C1-137 than BLCA-8. Further studies to test two-step intravesical administration of biotinylated MAb followed by radioiodinated streptavidin are in progress. Our studies indicate that the C1-137 MAb may have considerable potential for intravesical radioimmunotherapy of patients with superficial bladder tumors.