The transcription factor ATF3 acts as an oncogene in mouse mammary tumorigenesis.

BACKGROUND: Overexpression of the bZip transcription factor, ATF3, in basal epithelial cells of transgenic mice under the control of the bovine cytokeratin-5 (CK5) promoter has previously been shown to induce epidermal hyperplasia, hair follicle anomalies and neoplastic lesions of the oral mucosa including squamous cell carcinomas. CK5 is known to be expressed in myoepithelial cells of the mammary gland, suggesting the possibility that transgenic BK5.ATF3 mice may exhibit mammary gland phenotypes. METHODS: Mammary glands from nulliparous mice in our BK5.ATF3 colony, both non-transgenic and transgenic, were examined for anomalies by histopathology and immunohistochemistry. Nulliparous and biparous female mice were observed for possible mammary tumor development, and suspicious masses were analyzed by histopathology and immunohistochemistry. Human breast tumor samples, as well as normal breast tissue, were similarly analyzed for ATF3 expression. RESULTS: Transgenic BK5.ATF3 mice expressed nuclear ATF3 in the basal layer of the mammary ductal epithelium, and often developed squamous metaplastic lesions in one or more mammary glands by 25 weeks of age. No progression to malignancy was seen in nulliparous BK5.ATF3 or non-transgenic mice held for 16 months. However, biparous BK5.ATF3 mice developed mammary carcinomas with squamous metaplasia between 6 months and one year of age, reaching an incidence of 67%. Cytokeratin expression in the tumors was profoundly disturbed, including expression of CK5 and CK8 (characteristic of basal and luminal cells, respectively) throughout the epithelial component of the tumors, CK6 (potentially a stem cell marker), CK10 (a marker of interfollicular epidermal differentiation), and mIRSa2 and mIRSa3.1 (markers of the inner root sheath of hair follicles). Immunohistochemical studies indicated that a subset of human breast tumors exhibit high levels of nuclear ATF3 expression. CONCLUSION: Overexpression of ATF3 in CK5-expressing cells of the murine mammary gland results in the development of squamous metaplastic lesions in nulliparous females, and in mammary tumors in biparous mice, suggesting that ATF3 acts as a mammary oncogene. A subset of human breast tumors expresses high levels of ATF3, suggesting that ATF3 may play an oncogenic role in human breast tumorigenesis, and therefore may be useful as either a biomarker or therapeutic target.

Epidermal hyperplasia and oral carcinoma in mice overexpressing the transcription factor ATF3 in basal epithelial cells.

ATF3 is a highly conserved eukaryotic transcription factor that is ubiquitously upregulated transcriptionally during cellular responses to a variety of stresses, in particular DNA damage. However, the role of ATF3 in the DNA damage response is unclear. Transgenic mice that overexpress human ATF3 in basal epithelial cells under the control of the bovine keratin 5 (K5) promoter were constructed and characterized for epidermal alterations. Strong, nuclear expression of the exogenous ATF3 protein was seen in basal cells of the epidermis, hair follicles, and oral mucosa. Hyperplastic changes in the K5-expressing, outer root sheath (ORS) cells of the hair follicle were observed in young mice, resulting in multiple layers of ORS cells in the mature follicle and large aberrantly shaped follicles. Mild hyperplasia of the interfollicular epidermis was also noted, increasing with age. However, no epidermal tumors were identified in BK5.ATF3 mice observed for 16 mo. At 16 mo of age, most transgenic mice exhibited multi-focal areas of hyperplasia and dysplasia in the oral mucosa, with cellular atypia and underlying acute inflammatory changes. Neoplastic lesions were also seen in the oral cavity of BK5.ATF3 mice, including oral squamous cell carcinoma (60% incidence) and basal cell tumors with follicular differentiation (70% incidence), but not in non-transgenic FVB/N littermates. Heterogeneous nuclear expression (or stabilization) of p53 protein was seen in some oral dysplasias, with a patchy distribution primarily in the least differentiated layers of the lesions. This represents the first indication that ATF3 may have oncogenic properties in epithelial cells.

Quantitative high-throughput measurement of gene expression with sub-zeptomole sensitivity by capillary electrophoresis.

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90-95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells.