To produce conditional expression of genes in the mouse epidermis we have generated transgenic mouse lines in which the tetracycline-regulated transcriptional transactivators, tTA and rTA, are linked to the bovine keratin 5 promoter. The transactivator lines were crossed with the tetOlacZ indicator line to test for transactivation in vivo. In the absence of doxycycline, the K5/tTA line induced beta-galactosidase enzyme activity in the epidermis at a level 500-fold higher than controls, and oral and topical doxycycline caused a dose- and time-dependent suppression of beta-galactosidase mRNA levels and enzyme activity. In the K5/rTA lines, doxycycline induced beta-galactosidase activity between 3- and 50-fold higher depending on the founder line, and this occurred within 24-48 h after dosing. Histochemical analysis of all lines localized beta-galactosidase expression to the basal layer of the epidermis and the outer root sheath of the hair follicle, as well as other keratin 5 positive tissues. In several K5/rTA lines, skin-specific transactivation was restricted to the hair follicle. Treatment of these double transgenic mice with 12-O-tetradecanoyl-phorbol-13-acetate caused rapid migration of beta-galactosidase marked cells from the hair follicle through the interfollicular epidermis, demonstrating the usefulness of this specific double transgenic for fate mapping cells in the epidermis. These results show that the tetracycline regulatory system produces effective conditional gene expression in the mouse epidermis, and suggest that it should be amenable to suppression and activation of foreign genes during development and specific pathologic conditions relevant to the epidermis.

NOTCH signaling is an evolutionarily conserved signaling pathway that regulates cell fate during development and postnatal life. It has been increasingly linked to carcinogenesis, although its role in cancer seems to be highly context and tissue specific. Although NOTCH signaling is required for lung development, little is known about its role in lung cancer. In this study, we show that NOTCH signaling, as measured by the ;-secretase cleavage product N IC -1, is active in both normal human and lung tumor samples; however, downstream NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung tumors. Levels of NOTCH signaling components in primary human lung cells reflect observations in tissue samples, yet lung tumor cell lines showed little NOTCH signaling. Because oxygen concentrations are important in normal lung physiology and lung tumors are hypoxic, the effect of low oxygen on these lung tumor cell lines was evaluated. We found that hypoxia dramatically elevates NOTCH signaling (especially NOTCH-1) in lung tumor cell lines and concomitantly sensitizes them to inhibition via small-molecule ;-secretase inhibitors or NOTCH-1 RNA interference. ;-Secretase inhibitorinduced apoptosis of lung tumor cells grown under hypoxic conditions could be rescued by reintroduction of active NOTCH-1. Our data strengthen the role of NOTCH in lung cancer and as a therapeutic target for the treatment of lung and other hypoxic tumor types. [Cancer Res 2007;67(17):7954-9]

Hypoxic microenvironment supports cancer stem cell survival, causes poor response to anticancer therapy and tumor recurrence. Inhibition of Notch-1 signaling in adenocarcinoma of the lung (ACL) cells causes apoptosis specifically under hypoxia. Here we found that Akt-1 activation is a key mediator of Notch-1 pro-survival effects under hypoxia. Notch-1 activates Akt-1 through repression of phosphatase and tensin homolog (PTEN) expression and induction of the Insulin-like Growth Factor 1 Receptor (IGF-1R). The latter seems to be the major determinant of Akt-1 stimulation, since Notch-1 signaling affects Akt-1 activation in PTEN−/− ACL cells. Both downregulation of Insulin Receptor Substrate 1 (IRS-1) and dominant-negative IGF-1R sensitized ACL cells to γ-secretase inhibitor (GSI)-induced apoptosis. Conversely, overexpression of IGF-1R protected ACL cells from GSI toxicity. Inhibition of Notch-1 caused reduced IGF-1R expression, while forced Notch-1 expression yielded opposite effects. ChIP experiments suggested Notch-1 direct regulation of the IGF-1R promoter. Experiments in which human ACL cells were injected in mice confirmed elevated and specific co-expression of Notch-1IC, IGF-1R and pAkt-1 in hypoxic tumor areas.Our data provide a mechanistic explanation for Notch-1 mediated pro-survival function in hypoxic ACL tumor microenvironment. The results identify additional targets that may synergize with Notch-1 inhibition for ACL treatment.

Malignant mesothelioma (MM) is a cancer of the lining of the lungs, heart, and intestine and is known to respond poorly to chemotherapy. Here we show

Inactivation of cellular p53 is a crucial step in carcinogenesis.