The Denhardt
Laboratory

Nicotine,OPN and Pancreatic Cancer (a collaboration with Dr. Hwyda Arafat, Thomas Jefferson University).

During the past year we have initiated a collaborative project to investigate the hypothesis that nicotine induces (or stimulates the progression of) pancreatic cancer by enhancing OPN expression [Chipitsyna G, Gong Q, Anandanadesan R, Alnajar A, Batra SK, Wittel UA, Cullen DM, Akhter MP, Denhardt DT, Yeo CJ, Arafat HA. (2009) Induction of osteopontin expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells. Int J Cancer. 125:276-85]. The research at Rutgers was begun last year by Tanya Gordonov and Dana Cifelli, and is being continued by Michelle McBride and Jusleen Ahluwalia this year. Dana and Tanya engineered, by permanently transfecting two pancreatic ductal adenocarcinoma cell lines (ASPC-1 and HS766T) with appropriate plasmid constructs, derivatives that exhibited either increased or decreased expression of OPN. Michelle will be entering her senior year this fall; the research will constitute her Honors Thesis. Jusleen, an Aresty Summer Research Scholar (like Tanya, Cassandra Louis and Megha Rajpal before her) is participating with Michelle in the conduct of the research. They will be assessing the extent to which nicotine-induced OPN expression enhances MMP expression and facilitates the ability of the various cell lines to invade Matrigel-coated porous membranes.

Involvement of OPN in Regulating Corticosterone Level and cardiac fibrosis

We have reported that corticosterone levels in the mouse are in part regulated by OPN (Wang et al., 2009). To attempt to understand this surprising finding, Kathryn Jaques-Robinson and Luke Coyle have initiated a study using the murine adrenal cell line AtT-20 [Ciccotosto GD, Schiller MR, Eipper BA, Mains RE. (1999) Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1. J Cell Biol. 144:459-71] to test the hypothesis that OPN stimulates ACTH production, thereby inhibiting by an unknown mechanism the production of corticosterone. ACTH is produced by a controlled proteolysis of POMC; our preliminary results suggest that OPN may be a regulator of this process. Kathryn, a PhD student who began her studies with our colleague Dr. Gary Merrill, is also pursuing studies on how OPN may protect cells from hypoxia/reoxygenation injury. This is a collaboration with Gary that is focused on the left ventricle of the heart and the role of both OPN and acetaminophen in the development of cardiac fibrosis. Luke, a beginning MSc student who joined the lab this summer, is working with Kathryn on these projects.

Continued characterization of anti-OPN monoclonal antibodies

Megha Rajpal has made two unexpected and exciting discoveries in the past year, both concerning the impact of specific anti-OPN mAbs on the ability of cancer cells to form colonies in soft agar. (It is generally thought that OPN stimulates this process.) In one case, several mAbs were found to stimulate the ability of B16F10 mouse melanoma cells to form colonies in soft agar. In a second case, one mAb (so far) has been found to inhibit the formation of colonies in soft agar by the human melanoma cell line Yusiti (courtesy Ruth Halaban,Yale University). Results from these studies are described in a poster she prepared for the 2009 Annual Retreat of the Cancer Institute of New Jersey. Current research is focused on attempting to understand what is going on. We have continued our efforts to define the epitopes recognized by the monoclonal antibodies we have created. Monica Kadia, together with Megha, has been investigating the specificity of binding of certain mAbs to specific peptides present in OPN. Very recent evidence suggests that immunoglobulins may be able to bind OPN via an interaction independent of the antigen binding site.

Kirankumar Patel and Smeet Bhimani, two undergraduates who have only recently begun to participate in our research program, have helped out in many ways including performing ELISAs to assess OPN/mAb interactions and running polyacrylamide gels to characterize the purity and polypeptide composition of preparations of mAbs.