CANCER GENOMICS & PROTEOMICS
Volume 5, Number
6, November-December 2008
CONTENTS
PAGE
The Protein Expression of TRP-1 and Galectin-1 in Cutaneous Malignant Melanomas. Å. BOLANDER, M. AGNARSDÓTTIR, S. STRÖMBERG, F. PONTEN, P. HESSELIUS, M. UHLEN, M. BERGQVIST (Uppsala;
Stockholm, Sweden)
293
Genomic Analysis of Prostate Cancer Stem Cells Isolated from a Highly Metastatic Cell Line. R.A. ROWEHL, H. CRAWFORD, A. DUFOUR, J. JU, G.I. BOTCHKINA (New York, NY, USA)
301
Adenovirus-mediated Thymidine Kinase Gene Therapy and Coxsackie Adenovirus Receptor Expression in Ovarian Cancer Cells. D.G. KIEBACK (Aue, Germany; Maastricht, The Netherlands)
311
Ochratoxin A Lowers mRNA Levels of Genes Encoding for Key Proteins of Liver Cell Metabolism. C. HUNDHAUSEN, C. BOESCH-SAADATMANDI, N. MATZNER, F. LANG, R. BLANK, S. WOLFFRAM, W. BLASCHEK, G. RIMBACH (Kiel; Tuebingen, Germany)
319
Prognostic Utility of Glycosyltransferase Expression in Breast Cancer. N.
PATANI, W. JIANG, K. MOKBEL (Cardiff, Wales; London, UK)
333
CANCER GENOMICS & PROTEOMICS 5: 293-300 (2008)
The Protein Expression of TRP-1 and Galectin-1 in Cutaneous Malignant Melanomas
ÅSA BOLANDER1, MARGRÉT AGNARSDÓTTIR2, SARA STRÖMBERG2, FREDRIK PONTEN2, PATRIK HESSELIUS1, MATHIAS UHLEN3 and MICHAEL BERGQVIST1
1Department of Oncology, Uppsala University Hospital, Uppsala; 2Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala; 3Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden
Abstract: Background: Patients with metastazing malignant melanoma have a poor outcome and determination of thickness of the primary tumor remains as the most important prognostic predictor. The aim of this study was to use an antibody-based proteomics strategy to search for new molecular markers associated with melanoma progression. Two proteins, TRP-1 and galectin-1, were identified as proteins with enhanced expression in cells from the melanocytic lineage. Patients and Methods: Protein profiling of TRP-1 and galectin-1 together with proliferation marker Ki-67 and melanocyte marker Melan-A was performed in normal tissues from 144 individuals and in 216 different tumors using tissue microarrays and immunohistochemistry. The protein expression pattern was further analyzed in a defined cohort of 157 patients diagnosed with invasive cutaneous malignant melanoma. Results: Both TRP-1 and galectin-1 were highly expressed in normal melanocytes and melanoma. The expression of TRP-1 was inversely correlated with tumor stage (p=0.002, (R=-0.28)). Neither TRP-1 or galectin-1 was associated with overall or disease free survival (p>0.14, p>0.46 respectively). Ki-67 was associated with tumor stage and survival (p<0.001). Conclusion: TRP-1 and galectin-1 protein expression patterns were determined in normal and cancer tissues and both proteins were expressed in the majority of the malignant melanomas. There was no correlation between TRP-1 or galectin-1 expression and survival.
Genomic Analysis of Prostate Cancer Stem Cells Isolated from a Highly Metastatic Cell Line
REBECCA A. ROWEHL1, HOWARD CRAWFORD2, ANTOINE DUFOUR3, JINGFANG JU4 and GALINA I. BOTCHKINA5
Departments of 1Microbiology, Cell Culture and Hybridoma Facility, 2Pharmacology, 3Chemistry, 4Pathology, and 5Surgery/Surgical Oncology, Stony Brook University, NY, U.S.A.
Abstract: Background: Tumor-initiating or cancer stem cells (CSCs) were recently isolated from all major human cancers, including prostate cancer. However, the extreme heterogeneity of tumor cells in terms of biological behavior and gene expression patterns and difficulties isolating a pure population of CSCs from tumor tissues significantly impede a comparative analysis of CSCs. Materials and Methods: Different phenotypic populations were isolated from a metastatic derivative of PC-3 cell line, PC3-MM2, and tested for their ability to form tumors in NOD/SCID mice and floating spheroids in 3D culture systems. Results: All tested cell lines possessed minor populations of cells with highest expression of CD133, CD44 and CD166, whereas the vast majority of cells were CD133-negative. Several experimental approaches promoted a higher proportion of CD133-positive cells with increased in vivo tumorigenicity and the ability to produce floating spheres. Genome-wide microarray analysis (Affymetrix; DAVID) of CSC-enriched versus CSC-depleted cell populations revealed 213 genes with 10-100 fold increased activity out of 8994 differentially expressed ones and 87 genes with 5-50 fold decreased activity. Conclusions: The proposed in vitro prostate CSC model allows for reliable isolation and propagation of highly tumorigenic cells. This study may contribute to the identification of novel targets for CSC-targeted prostate cancer treatment.
Adenovirus-mediated Thymidine Kinase Gene Therapy and Coxsackie Adenovirus Receptor Expression in Ovarian Cancer Cells
D.G. KIEBACK1,2
1HELIOS Medical Center, Aue, Germany; 2GROW Research Institute, University of Maastricht, The Netherlands
Abstract: Coxsackie adenovirus receptor (CAR) expression is the main mechanism of adenovirus entry into target cells. It is unclear whether CAR expression itself is influenced by transduction with the adenovirus-Rous sarcoma virus-thymidine kinase (ADV-RSV-TK) gene therapy construct or by the subsequent intracellular accumulation of the TK gene product. Antibody generation and characterization, immunocytochemistry, Western blotting and 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay were performed to investigate the relationship of gene transfer and CAR expression as well as differences in therapeutic susceptibility of MDAH-2774 and OVCAR-3 cell lines to ADV-RSV-TK gene therapy. CAR expression was observed on the membranes but intracellular translocation of CAR also took place dependent on cellular growth patterns. TK gene expression was dependent on multiplicity of infection (MOI) and thus on vector dose in a linear fashion. Neither TK expression nor ADV transduction influenced CAR expression, or ADV-RSV-TK transduction. Differential susceptibility of different cell lines to TK-induced cell killing by acyclovir metabolites was observed. CAR expression appears not to be influenced by adenoviral transduction or by the accumulation of the TK gene product. Differences in therapeutic sensitivity are most likely mediated by intracellular mechanisms and not by modulation of CAR expression.
Ochratoxin A Lowers mRNA Levels of Genes Encoding for Key Proteins of Liver Cell Metabolism
CHRISTOPH HUNDHAUSEN1, CHRISTINE BOESCH-SAADATMANDI1, NICOLE MATZNER2, FLORIAN LANG2, RALF BLANK3, SIEGFRIED WOLFFRAM3, WOLFGANG BLASCHEK4 and GERALD RIMBACH1
Institutes of 1Human Nutrition and Food Science and 3Animal Nutrition and Physiology, and 4Department of Pharmaceutical Biology, Institute of Pharmacy, Christian Albrechts University, Kiel; 2Department of Physiology, Eberhard Karls University, Tuebingen, Germany
Abstract: Ochratoxin A (OTA) is a nephro- and hepatotoxic mycotoxin that frequently contaminates food and feedstuffs. Although recent studies have indicated that OTA modulates renal gene expression, little is known regarding its impact on differential gene expression in the liver. Therefore a microarray study of the HepG2 liver cell transcriptome in response to OTA exposure (0, 0.25, 2.5 µmol/l for 24 h) was performed using Affymetrix GeneChip technology. Selected microarray results were verified by real-time PCR and Western blotting as independent methods. Out of 14,500 genes present on the microarray, 13 and 250 genes were down-regulated by 0.25 and 2.5 µmol/l OTA, respectively. Reduced mRNA levels of calcineurin A beta (PPP3CB), which regulates inflammatory signalling pathways in immune cells, and of the uncoupling protein 2 (UCP2), which has been suggested to control the production of reactive oxygen species (ROS), were observed in response to 0.25 µmol/l OTA. A particularly strong down-regulation due to 2.5 µmol/l OTA was evident for the mRNA levels of insulin-like growth factor binding protein 1 (IGFBP1) and tubulin beta 1 (TUBB1) which have been demonstrated to function as a pro-survival factor in hepatocytes and as an important cytoskeletal component, respectively. In addition, many genes involved in energy and xenobiotic metabolism, including phosphoglycerate kinase 1 (PGK1), stearoyl-Coenzyme A desaturase 1 (SCD), and glutathione S-transferase omega 1 (GSTO1), were down-regulated by OTA. Furthermore, OTA significantly inhibited the capacitative calcium entry into the HepG2 cells, indicating an alteration of calcium homeostasis. Overall, OTA dose-dependently affects multiple genes encoding for key proteins of liver cell metabolism.
Prognostic Utility of Glycosyltransferase Expression in Breast Cancer
NEILL PATANI1, WEN JIANG2 and KEFAH MOKBEL1,2
1The London Breast Institute, The Princess Grace Hospital, London; 2Cardiff University, Cardiff, Wales;3St. George's University of London, London, U.K.
Abstract: Background: The post-translational modification of proteins, including glycosylation, is known to differ between normal and tumour cells. In this study, the expression profile of two glycosyltranferases, UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) and á6-sialyl-transferase-I (ST6GalNAc-I) was assessed, in a cohort of women with breast cancer. Patients and Methods: Breast cancer tissues (n=127) and normal background tissues (n=33) were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analyzed against conventional pathological parameters and clinical outcome over a 10 year follow-up period. Results: Significantly higher levels of ppGalNAc-T6 were found in the breast cancer specimens compared to the background tissue (p=0.015). There was a non-significant trend for levels to increase with the Nottingham Prognostic Index (NPI) and TNM stage and those who died from breast cancer. ST6GalNAc-I expression was associated with better prognosis, reaching significance when comparing patients who remained disease free to those with distant recurrence (p=0.0096). The relationship approached significance when comparing NPI 2 to NPI 3 (p=0.058) and disease free patients to non-disease free patients (p=0.052) or those who died of breast cancer (p=0.060). For both enzymes a significant association with ductal type was found. Conclusion: Expression of ppGalNAc-T6 is significantly higher in breast cancer compared to 'normal'/benign breast tissue samples. ST6GalNAc-I expression in breast cancer is associated with better prognosis.
