Circulating cell adhesion amidst the high shear environment of the vasculature is central to several physiological and pathophysiological processes, including leukocyte recruitment to sites of inflammation, stem cell homing and cancer metastasis. This process is initiated by selectin-mediated adhesion, the molecular “brakes” that slow cells down relative to bulk fluid flow to facilitate cell-cell signaling and eventual firm cell adhesion. Selectin recognition therefore represents a critical step whereby therapeutic interventions aimed towards the interference of cell homing could be targeted. While the force dependency of these high kon and koff rate interactions has been well described, little understanding exists of the long time- and length-scale interactions of different cell subtypes that would best describe the functional capacity of different cell homing via selectins to systemic peripheral tissues. This limits the adequate description of sustained cell adhesion efficiencies in physiological conditions that predicates the effectiveness of cell homing as well as the design effective therapeutic interventions to selectively attenuate metastasis but not normal cell homing using such criteria. To address this issue, we developed a so-called “cell adhesion chromatography” system, a microfluidic-based device designed for use in conjunction with videomicroscopy for the interrogation of the adhesion behavior of cells over long time- and length-scales. In order to achieve uniform contact of a pulse cell suspension input into a selectin-functionalized parallel plate flow chamber, we designed a feature that enables complete cell settling to the chamber bottom based on Stoke’s flow predictions, increasing contact uniformity of the pulse cell input with the substrate upon entry into the main chromatography channel from ~50 to >95%. Using this configuration, residence time distributions for a pulse input of cells perfused at defined shear stresses were generated based on cell elution times from the cell adhesion chromatography system. Selectin-functionalized substrates delayed cell elution times relative to bovine serum albumin coated-substrates by orders of magnitude in a selectin concentration, shear and cation dependent fashion. Preliminary experiments were also performed to begin to define the differences in efficiencies of healthy (human monocyte) versus malignant (human colon carcinoma) cell adhesion to selectins in shear flow. Our results suggest significant differences in the functional capacity of healthy versus malignant cells to sustain adhesion in shear flow and that cell adhesion chromatography is a new tool that provides unique insight into the process of cell adhesion in fluid flow.

As a posttranslational modification, the sumoylation pathway plays a key role in a wide variety of cellular events such as cell proliferation, differentiation, stress response, DNA repair and apoptosis. Given the important role of protein sumoylation, it is not surprising that alternation of sumoylation will ultimately affect cell growth as well as cancer development. As an essential E2 conjugating enzyme for sumoylation, Ubc9 plays a central role in sumoylation-mediated cellular pathways. In this study, we investigate the role of Ubc9 in drug resistance as well as its regulation in cancer cells. An early gene product, Gam1, encoded by the avian adenovirus CELO, is an inhibitory protein for the sumoylation machinery by degrading E1 and E2 enzymes. Given the suppressive effect of Gam1 on Ubc9, in this study, we use this protein to study the role of Ubc9 in drug resistance as well as its underlying mechanism. Besides showing suppression of Ubc9 expression and sumoylation by Gam1, we found that Gam1 caused significant cell growth inhibition. Of interest, like the Ubc9 dominant negative mutant, Gam1 also sensitized cells to DNA damaging agents such as topotecan and doxorubicin as well as non-DNA-damaging agents such as paclitaxel and vincristine. Furthermore, we elucidated that Gam1-mediated cell growth inhibition was associated with induction of apoptosis. In particular, Gam1 induced caspase-3 activity as detected by immunostaining and Western blot. Taken together, our findings suggest that activation of the caspase pathways is at least in part responsible for the increased apoptosis in Gam1-expressing cells and, thus, contributes to the growth inhibition and enhanced chemosensitivity. Available evidence suggests that Ubc9 is a tumor promoting factor. However, little is known about the regulation of Ubc9. In this study, we first show that Ubc9 is overexpressed in several types of cancers, highlighting its clinical significance. We then investigate the underlying mechanism of Ubc9 upregulation. Of interest, we present evidence that Ubc9 is subjected to the post-transcriptional regulation by microRNAs and the miR-30 family, such as miR-30e, negatively regulate Ubc9 expression. In contrast to Ubc9, miR-30e is underexpressed in tumors. Moreover, ectopic expression of miR-30e suppresses cell growth which can be partially reversed by Ubc9. Finally, using luciferase-Ubc9-3'-UTR reporters, we show that Ubc9 is a direct target for miR-30e by interactions with the putative miR-30e binding sites. Therefore, our study suggests that Gam1 and miR-30e may serve as therapeutic agents for cancer treatment by targeting Ubc9.

published_or_final_version / Medicine / Master / Master of Medical Sciences

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