Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase in the mitogen-activated protein kinase signal transduction cascade known to regulate inflammatory pathways. Previously identified as an oncogene, its mutation or overexpression is reported in a variety of human cancers. To address its role in skin carcinogenesis, Tpl2−/− or wild-type (WT) C57BL/6 mice were subjected to a two-stage dimethylbenzanthracene/12-O-tetradecanoylphorbol- 13-acetate (TPA) mouse skin carcinogenesis model. Tpl2−/− mice developed a significantly higher incidence of tumors (80%) than WT mice (17%), as well as a reduced tumor latency and a significantly higher number of total tumors (113 vs 6). Moreover, Tpl2−/− mice treated with TPA experienced significantly higher nuclear factor kappaB (NF-κB) activation, edema, infiltrating neutrophils and production of proinflammatory cytokines than did WT mice. We investigated the role of the p38, JNK, MEK and NF-κB signaling pathways both in vitro and in vivo in WT and Tpl2−/− mice by using inhibitors for each of these pathways. We confirmed that the proinflammatory effect in Tpl2−/− mice was due to heightened activity of the NF-κB pathway. These studies indicate that Tpl2 may serve more as a tumor suppressor than as an oncogene in chemically induced skin carcinogenesis, with its absence contributing to both tumorigenesis and inflammation.

To assess the possible neurobehavioral performance risks to astronauts from living in a space radiation environment during long-duration exploration missions, the effects of head-only proton irradiation (150 MeV/n) at low levels (25-50 cGy, approximating an astronaut's exposure during a 2-year planetary mission) were examined in adult male Long-Evans rats performing an analog of the human psychomotor vigilance test (PVT). The rodent version of PVT or rPVT tracks performance variables analogous to the human PVT, including selective attention/inattention, inhibitory control ("impulsivity") and psychomotor speed. Exposure to head-only proton radiation (25, 50, 100 or 200 cGy) disrupted rPVT performance (i.e., decreased accuracy, increased premature responding, elevated lapses in attention and slowed reaction times) over the 250 day testing period. However, the performance decrements only occurred in a subgroup of animals at each exposure level, that is, the severity of the rPVT performance deficit was unrelated to proton exposure level. Analysis of brain tissue from irradiated and control rats indicated that only rats with rPVT performance deficits displayed changes in the levels of the dopamine transporter and, to a lesser extent, the D₂ receptor. Additional animals trained to perform a line discrimination task measuring basic and reversal learning showed no behavioral effects over the same exposure levels, suggesting a specificity of the proton exposure effects to attentional deficits and supporting the rPVT as a sensitive neurobehavioral assay.

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Tumor progression locus 2 (Tpl2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of serine/threonine kinases. Deletion of the Tpl2 gene is associated with a significantly higher number of papillomas and cutaneous squamous cell carcinomas (cSCCs). Overexpression of hepatocyte growth factor (HGF) and its receptor MET is abundant in cSCC and can lead to increased proliferation, migration, invasion or resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. The aim of this study was to address whether the increased tumor burden in Tpl2 −/− mice is due to aberrant HGF/MET signaling. C57Bl/6 wild type (WT) and Tpl2 −/− mice were subjected to a two-stage chemical carcinogenesis protocol for one year. At the time of promotion half of the mice received 44 mg/kg capmatinib (INC 280), a pharmacological inihibitor of MET, in their diet. Tpl2−/− mice had signficantly higher tumor incidence and overall tumor burden compared to WT mice. Further, carcinogen-intiated Tpl2 −/− mice could bypass the need for promotion, as 89% of Tpl2 −/− mice given only DMBA developed papillomas. v-rasHa -transduced keratinocytes and SCCs from Tpl2 −/− mice revealed an upregulation in HGF and p-MET signaling compared to WT animals. Long-term capmatinib treatment had no adverse effects in mice and capmatinib-fed Tpl2 −/− mice had a 60% reduction in overall tumor burden. Further, no tumors from Tpl2 −/− mice fed capmatinib underwent malignant conversion. In summary targeting MET may be a potential new strategy to combat cutaneous squamous cell carcinomas that result from dysregulation in MAPK signaling.