PMC

H1299-GFP and H1299-N3 were mock-treated, or treated with 3 μM MRK-003 for 8 days. (A) Relative Notch pathway activity, as determined by Hes1 expression by qRT-PCR (all reactions are normalized to β-actin) (B) 3×10³ viable cells were plated in a 12-well plates in triplicate, allowed to form colonies for 7 days, and were then stained with crystal violet (p=0.003 for the difference between cell lines).

Downregulation of Notch signaling using a selective γ-secretase inhibitor (MRK-003; Merck) was observed in two NSCLC cell lines: (A) NCI-H1299 and (B) NCI-H1435. Cells were grown to 50% confluence and treated with MRK-003 at the indicated doses for 96 hours. Relative expression of Hes1 was evaluated using qRT-PCR normalized to β-actin. NT: not treated.

(A) Lung sections stained with hematoxylin/eosin reveal similar tumor histologies from mutant KRAS and KRAS/DNMAML mice. (B) Total tumor burden, calculated as a fraction of total lung area, is similar in KRAS only and double transgenic KRAS/DNMAML mice. No tumors were observed on control wildtype or DNMAML only littermates. Analysis was performed at 8 weeks after infection.

(A) Strong DNMAML gene expression was seen by qRT-PCR in double transgenic (DNMAML/KRAS) tumors, but not in tumors from KRAS only transgenics. DNMAML only transgenic lungs did not form tumors, and lower level expression is seen, reflecting that only a fraction of epithelial cells are infected. (B) Immunohistochemical staining with an anti-GFP antibody of tumors from KRAS only and KRAS/DNMAML tumors. The anti-GFP antibody recognizes the GFP component of the DNMAML fusion protein. (C) Relative expression of intratumoral Hes1 from KRAS and KRAS/DNMAML mice.

Quantitative PCR demonstrates relative expression levels of genes encoding Notch receptors, Hes1, and Notch ligands in four NSCLC cell lines compared to those of normal human bronchial epithelial cells. Relative expression was evaluated using Taqman probes (ABI). All reactions were normalized to β-actin (Notch4 levels were undetectable in all lung and cell line samples).

LSL-KRAS^G12D transgenic mice were given inhaled adenoviral Cre, leading to oncogene activation in the transgenic animals. Lungs were harvested from cohorts of mice pretreatment and at 1, 3, 6 and 10 weeks post-infection. Top: hematoxylin/eosin stain. Bottom: immunohistochemistry for Hes1. A transient induction of Hes1 in seen in airway epithelial cells at 1 week, with scattered positive cells in subsequent growing tumors. Representative images are shown. No Hes1 induction over baseline staining was observed in concomitantly treated non-transgenic littermate controls (not shown).