Primary research

Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells

Krishna Rao¹ , Özge Alper² , Kent E Opheim³ , George Bonnet⁴ , Kristine Wolfe⁵ , Eileen Bryant^6,7 , Siobhan O'Hara Larivee⁷ , Peggy Porter^8,9 and James K McDougall^8,9

¹Southern Illinois University School of Medicine Cancer Institute, P.O. Box 19678, Springfield, IL 62794-9678, USA

²National Institutes of Health, National Institute of Neurological Disorders, Surgical Neurology, Bldg 10, 5D37, Bethesda, MD 20892-1414, USA

³Children's Hospital and Regional Medical Center, Dept. of Laboratories, A-6901 4800 Sand Point Way, NE, Seattle, WA 98105, USA

⁴Cytogenetics Studio, Inc., 41 Myrtle Ave., Cambridge, MA 02138, USA

⁵2511 Boundary St., San Diego, CA 92104-5313, USA

⁶Fred Hutchinson Cancer Research Center, Clinical Research Division, P.O. Box 19024, 1100 Fairview Avenue North, D2-190, Seattle, WA 98109-1024, USA

⁷Seattle Cancer Care Alliance, 825 Eastlake Avenue East, Mailstop G7500, Seattle, WA 98109, USA

⁸Fred Hutchinson Cancer Research Center, Cancer Biology Program, P.O. Box 19024, 1100 Fairview Avenue North, C1-015, Seattle, WA, 98109-1024, USA

⁹Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195, USA

author email corresponding author email

Cancer Cell International 2006, 6:15doi:10.1186/1475-2867-6-15

Published:	26 May 2006

Abstract

Introduction

Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC) transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras.

Methods

HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT). The immortalized cells were passaged in vitro and studied by a combination of G- banding and Spectral Karyotyping (SKY). H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting.

Results

Immortal HMEC alone were not tumorigenic in γ-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor.

Conclusion

Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.