Breast cancer, the most common spontaneous malignancy diagnosed in women, is a classical model of hormone dependency as it is associated with prolonged exposure to female hormones. Different cytoplasmic proteins are important in the transformation of a normal cell to an invasive tumor cell, and these include vimentin and Notch. To investigate the importance of these two genes and proteins in breast carcinogenesis, we used an in vitro breast cancer model system, in which an immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer α particle (150 keV/μm) radiation and subsequent growth in the presence or absence of 17β-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) a parental cell line (MCF-10F), ii) an Estrogen cell line (MCF-l0F continuously grown with estradiol at 10−8), iii) a malignant and non-tumorigenic cell line (Alpha3), iv) a malignant and tumorigenic cell line (Alpha5) and v) a Tumor2 cell line derived from a xenograft of the Alpha5 cell line injected into nude mice. Vimentin and Notch showed greater expression in the Alpha5 and Tumor2 cell lines compared with that in the non-tumorigenic cell lines, MCF-10F, Estrogen and Alpha3. In the present study, positive staining for vimentin was found in 21% of cases. Vimentin and Notch protein expression was negative in noninvasive ductal carcinoma biopsies from breast cancer patients. However, positive cell expression was observed in invasive ductal carcinoma biopsies. These biomarkers can be considered important indicators of breast cancer progression and can be added to the diagnostic panel when overall survival is a primary end-point.