The fate of the R7 photoreceptor cell in the developing eye of Drosophila is controlled by the Sevenless (Sev) receptor tyrosine kinase. Sev activates a highly conserved signal transduction cascade involving the proteins Ras1 and Raf and the Rolled/mitogen-activated protein (Rl/MAP) kinase. Here we show that the ETS domain protein encoded by the P2 transcript of the pointed (pnt) gene is a nuclear target of this signalling cascade which acts downstream of Rl/MAP kinase. The PntP2 protein is phosphorylated by Rl/MAP kinase in vitro at a single site and this site is required for its function in vivo. Furthermore, we present genetic and biochemical data suggesting that MAP kinase controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family, Yan and PntP2.

We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

Background: Hepatocellular carcinoma (HCC) typically occurs in patients with chronic liver disease. Such diseases create a demand for liver regeneration due to injury, which in turn promotes upregulation of c-Met, a receptor for hepatocyte growth factor (HGF) involved in wound healing, leading to increased cellular proliferation, survival, and mobilization. c-Met is the product of the proto-oncogene MET, so increased c-Met activity not only effects wound healing but can also contribute to the development and progression of HCC. Aberrant c-Met signaling is associated with rapid tumor growth and aggressively invasive disease in HCC, resulting in poor patient prognosis. We examined c-Met expression and MET gene amplification in 69 procured HCC samples and c-Met expression in adjacent non-tumor tissue. Methods: c-Met expression was assessed using MET IHC MSC2156119J pharmDx anti-c-MET (clone D1C2) rabbit mAb. c-Met expression was scored semiquantitatively on a scale of 0-3+ based on the staining intensity in ≥50% of cells [Koeppen et al. Proc USCAP 2012 (abstract 2001)]; tumors scoring 2+ or 3+ were judged positive. MET gene copy number (GCN) was assessed using the MET IQFISH Kit-111480 with a probe covering a 269 kb segment on chromosome 7q31.2 containing the MET gene. Tumors were considered to be FISH positive if scored cells had a mean MET/CEN7 ratio ≥2.0 or ≥50% of cells contained ≥5 MET signals. Results: All cases showed typical HCC morphology and were classified as low to high grade trabecular, pseudoglandular, or solid with common cytoplasmic features. The tumor cells and cells of the adjacent cirrhotic/non-cirrhotic liver tissue (bile duct epithelia, hepatocytes endothelial cells) showed cytoplasmic and membrane staining of varying extent and intensity. c-Met staining in tumor was heterogeneous and present in 68/68 cases. Semiquantitative analysis showed that 11/67 (16%) tumors were c-Met-positive (3+, 3 cases; 2+, 8 cases). Six out of 69 (8.7%) were amplified by ratio and 3 (4.3%) others had ≥5 MET gene copies in ≥50 % of cells, giving an overall frequency of FISH positivity of 13%. Most interestingly, amplification and/or increased gene copy number was homogeneous throughout the tumor in all cases, in contrast to heterogeneous c-Met staining observed in some of the same tumors with IHC. Five of the 11 c-Met-positive (IHC 2+/3+) tumors were MET amplified, and all 3 of the IHC 3+ tumors were amplified. Conversely, 4 of the 9 FISH-positive tumors were c-Met-negative. Conclusion: These data provide insight into the frequency of c-Met/MET abnormalities in HCC and show there is discordance between c-Met protein expression and MET GCN alterations. This may be of importance for selection of patients with HCC for clinical trials of c-Met inhibitors. Citation Format: Christian Ihling, Sienna Yoast, Matthew DeNicola, Josef Straub, Klaus Dücker, Karsten Nielson, Russell A. Baldocchi, Aaron R. Ellison, Holly Yamada. Analysis of c-Met protein expression by immunohistochemistry and MET gene copy number in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4668. doi:10.1158/1538-7445.AM2017-4668