THE ROLE OF RECEPTOR TYROSINE KINASE AXL IN PANCREATIC DUCTAL ADENOCARCINOMA AND ITS REGULATION BY HEMATOPOIETIC PROGENITOR KINASE 1

Pancreatic ductal adenocarcinoma (PDA) is one of the most aggressive malignancies with less than 5% of five year survival rate. New molecular markers and new therapeutic targets are urgently needed for patients with PDA. Oncogenic receptor tyrosine kinase Axl has been reported to be overexpressed in many types of human malignancies, including diffuse glioma, melanoma, osteosarcoma, and carcinomas of lung, colon, prostate, breast, ovary, esophagus, stomach, and kidney. However, the expression and functions of Axl in PDA are unclear. We hypothesized that Axl contributes to the development and progression of PDA. We examined Axl expression in 54 human PDA samples and their paired benign pancreatic tissue by immunohistochemistry, we found that Axl was overexpressed in 70% of stage II PDAs, but only 22% of benign ducts (P=0.0001). Axl overexpression was associated with higher frequencies of distant metastasis and was an independent prognostic factor for both poor overall and recurrence-free survivals in patients with stage II PDA (p = 0.03 and 0.04). Axl silencing by shRNA in pancreatic cancer cell lines, panc-28 and Panc-1, decreased tumor cell migration and invasion and sensitized PDA cells to apoptosis stimuli such as γ-irradiation and serum starvation. In addition, we found that Axl-mediated Akt and NF-κB activation and up regulation of MMP2 were involved in the invasion, migration and survival of PDA cells. Thus, we demonstrate that Axl plays an important role in the development and progression of PDA. Targeting Axl signaling pathway may represent a new approach for the treatment of PDA. To understand the molecular mechanisms of Axl overexpression in PDA, we found that Axl expression was down-regulated by hematopoietic progenitor kinase 1 (HPK1), a newly identified tumor suppressor in PDA. HPK1 is lost in over 95% of PDAs. Restoration of HPK1 in PDA cells down-regulated Axl expression. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1, and monensin, suggesting that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway. HPK1 interacted with and phosphorylated dynamin, a critical component of endocytosis pathway. Overexpression of dominant negative form of dynamin blocked the HPK1-mediated Axl degradation. Therefore we concluded that HPK1-mediated Axl degradation was through endocytosis-lysosome pathway and loss of HPK1 expression may contribute to Axl overexpression in PDAs.

Mechanisms for the release of dopamine from turtle retina

The retinal circuitry underlying the release of dopamine was examined in the turtle, Pseudemys scripta elegans, using neurochemical release studies, anatomical techniques, and biochemistry. There was a dose- and calcium-dependent release of dopamine from turtle retinas incubated in $\sp3$H-dopamine after perfusion of the GABA antagonist bicuculline. This indicated that dopamine release was tonically inhibited by GABA. Other putative retinal transmitters were examined. Glutamate antagonists selective for hyperpolarizing bipolar cells, such as 2,3-piperidine dicarboxylic acid (PDA), caused dose- and calcium-dependent release of dopamine from the retina. In contrast, release was not observed after perfusion with 4-aminophosphonobutyric acid, a specific antagonist of depolarizing bipolar cells. This indicated that depolarizing bipolar cells were not involved in retinal circuitry underlying the release of dopamine in the turtle retina. The release produced by PDA was blocked by bicuculline, indicating a polysynaptic mechanism of release. None of the other agents tested, which included carbachol, strychnine, dopamine uptake inhibitors, serotonin, tryptamine, muscimol, melatonin, or dopamine itself produced release.^ The cells capable of the release of dopamine were identified using both uptake autoradiography and immunocytochemical localization with dopamine antisera. The simplest circuitry based on these findings is signal transmission from photoreceptors to hyperpolarizing bipolar cells then to GABAergic cells, and finally to dopaminergic amacrine cells. ^

Synthesis and properties of heterobifunctional photoaffinity reagents for the identification and isolation of receptors

A method employing isotopically- and photoaffinity-labeled probes and polyclonal and monoclonal antibody to the probes for the identification, isolation and recovery of protein receptors is described. Antibody was raised against N-(3-(p-azido-m-($\sp{125}$I) -iodophenyl)) propionate (AIPP) coupled to and photolyzed to BSA. The antibodies specifically bound AIPP-derivatized proteins. An isolation system was developed utilizing this probe and two antigenically identical reversible analogues. N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl)propionyl)amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) reacts with primary amines and N-(((3-p-azido-m-($\sp{125}$I) -iodophenyl)propionyl)amidoethyl)dithiopyridine ($\sp{125}$I-AIPP-PDA) reacts with reduced thiols. The applicability of the system was established by derivatizing known ligands (Transferrin and Interferon-alpha) with one of the probes. The ligand-probe was then allowed to interact with its receptor by incubation with SS5 lymphoma cells and cross-linked by photolysis at 300 nm. The photolyzed ligand/probe/receptor preparation was then recovered with AIPP antibody. Utilization of N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl-propionyl)-amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) allowed the components of the photolyzed complex to be separated by treatment with 2-mercaptoethanol in the SDS-PAGE solubilization buffer. Ligand and receptor labeling were then assessed by Coomassie staining and autoradiography. Results of receptor assays suggest that $\sp{125}$I-AIPP was, indeed, transferred to moieties that represent the receptors for both Transferrin and Interferon-alpha. ^

Maintenance of phosphatidylserine asymmetry in erythroid cells during differentiation, aging, and its significance for macrophage recognition

Phosphatidylserine (PS) is distributed almost entirely in the inner leaflet of the erythrocyte membrane bilayer, and appears to be maintained by a 32 kDa integral membrane protein (PS translocase). The expression of PS on the outer leaflet may serve as a recognition signal for macrophages, since insertion of PS into erythrocytes enhances their adherence to macrophages and clearance from the circulation. Therefore I have hypothesized that erythroid cells display PS on their outer leaflet early in differentiation and upon aging. Analysis of murine erythroleukemia cells (MELC, undifferentiated erythroid progenitor cells) showed high levels of PS on the outer leaflet that decreased during differentiation, correlating with the pattern of macrophage adherence. The activity of the PS translocase during differentiation appears to be unchanged although the equilibrium distribution of PS differs. This difference may be due to qualitative changes in the PS translocase. $\sp{125}$I-Bolton/Hunter-labeled-pyridyldithioethylamine ($\sp{125}$I-B/H-PDA), a radiolabeled probe for the PS translocase, labeled a 32 kDa protein in mature erythrocytes whereas in MELC a 45 kDa protein as well as a 32 kDa protein was identified. The abundance of the 45 kDa protein in relation to the 32 kDa protein declined during differentiation, possibly indicating this protein was a precursor of the 32 kDa protein. Analysis of the 45 kDa protein by N-glycosidase F and endoproteinase cleavage suggested this protein was not a glycosylated form of the 32 kDa protein but appeared to share some structural homology. Aged murine erythrocytes had elevated levels of PS on their outer leaflet, as well as decreased PS translocase activity. $\sp{125}$I-B/H-PDA labeled a 32 kDa protein in both normal and aged erythrocytes. However, the latter cells also contained a 28 kDa protein. Experimental evidence suggests that the appearance of the 28 kDa protein may be due to increased oxidation of aged erythrocytes. Examination of PS distribution showed that the levels of PS on the outer leaflet were elevated early in differentiation, decreased during the mature state, and returned to high levels as the erythrocyte aged. In conclusion,the levels of outer leaflet PS correlated with the differentiation status and macrophage recognition of erythroid cells. ^

The Choosing Healthy Options of Starches (CHOOS) diet and dietary intake self-monitoring for weight loss among adolescent females

Previous research has shown dietary intake self-monitoring, and culturally tailored weight loss interventions to be effective tools for weight loss. Technology can be used to tailor weight loss interventions to better suit adolescents. There is a lack of research to date on the use of personal digital assistants (PDAs) to self-monitor dietary intake among adolescents. The objective of this study was to determine the difference in dietary intake self-monitoring frequency between using a Personal Digital Assistant (PDA) or paper logs as a diet diary in obese adolescent females; and to describe differences in diet adherence, as well as changes in body size and self-efficacy to resist eating. We hypothesized dietary intake self-monitoring frequency would be greater during PDA use than during paper log use. This study was a randomized crossover trial. Participants recorded their diet for 4 weeks: 2 weeks on a PDA and 2 weeks on paper logs. Thirty-four obese females ages 12-20 were recruited for participation. Thirty were included in analyses. Participants recorded more entries/day while using the paper logs (4.10 entries/day ± 0.63) than while using the PDA (3.01 entries/day ±0.75) (p<0.001). Significantly more meals and snacks were skipped during paper log use (0.81/day ± 0.65) than during PDA use (0.23/day ± 0.22) (p=0.011). Changes in body size (BMI, weight, and waist circumference) and self-efficacy to resist eating did not differ significantly between PDA and paper log use. When compared to paper logs, participants felt the PDA was more convenient (p=0.020), looked forward to using the PDA more (p=0.008), and would rather continue using the PDA than the paper logs (p=0.020). The findings of this study indicate use of a PDA as a dietary intake self-monitoring tool among adolescents would not result in increased dietary intake self-monitoring to aid in weight loss. Use of paper logs would result in greater data returned to clinicians, though use of PDAs would likely get adolescents more excited about adhering to recommendations to record their diet. Future research should look at updated communication devices, such as cell phones and other PDAs with additional features, and the role they can play in increasing dietary intake self-monitoring among adolescents.^