[Frontiers in Bioscience E2, 627-640, January 1, 2010]

Characterization of a novel, trastuzumab resistant human breast cancer cell line

Mark Barok1, Margit Balazs2,3,, Viktoria Lazar2, Zsuzsa Rakosy2,3, Eniko Toth1, Andrea Treszl2, Gyorgy Vereb1, Gail T. Colbern4, John W. Park4, Janos Szollosi1,5

1Department of Biophysics and Cell Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, 2Department of Preventive Medicine, Faculty of Public Health, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, 3Public Health Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary, 4Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, USA, 5Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary

TABLE OF CONTENTS

1. Abstract 2. Introduction
3. Materials and Methods
3.1. Patient clinical history
3.2. Maintaining the B585 primary breast cancer xenografts in SCID mice
3.3. Antibodies
3.4. Conjugation of antibodies with fluorescent dyes
3.5. Immunohistochemistry
3.6. Confocal microscopy
3.7. Chromosomal and array comparative genomic hybridization (CGH)
3.8. Gene expression analysis
3.9. Correlation of aCGH and expression data
3.10. Fluorescence in situ hybridization
4. Results
4.1. Immunohistochemical characterization of the B585 xenograft
4.2. Genetic alterations in the original primary breast tumor and the B585 xenograft
4.3. Fluorescence in situ hybridization using centromere and gene specific probes
4.4. Gene expression signature of the B585 xenograft
5. Discussion
6. Acknowledgement
7. References

1. ABSTRACT

HER2-positive breast cancers represent a distinct phenotype and are intrinsically more aggressive than HER2-negative tumors. Although HER2-targeted therapies have been rationally developed, resistance to these treatments represents a process understood poorly. There are few experimental models that allow studying the molecular mechanism of resistance. Our aim was to characterize a trastuzumab resistant breast cancer cell line (B585) that was established from an invasive ductal carcinoma. B585 grows only in immunodeficient mice as a xenograft. CGH and FISH were used to define cytogenetic alterations, gene-expression analysis and immunohistochemistry were applied to detect RNA and protein expression. By array-CGH focused amplifications were identified for C-MYC, EGFR, ErbB2, CCND1 and TOP2-A oncogenes. ErbB2 was co-amplified with TOP2-A. mRNA overexpression was detected for the amplified genes. ErbB2 protein was overexpressed and showed heterogeneous distribution. In summary, molecular cytogenetic analysis and expression profiling of B585 revealed several new alterations. Based on the experiments performed in SCID mice and the genotypic/phenotypic characteristics, this new in vivo breast cancer xenograft is a valuable model to investigate molecular mechanism of trastuzumab resistance.