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stroma (this presentation), tumor endothelium, and tumor cells.

Methods: Liposomes composed of 66% dipalmitoyl-phosphatidylcholine, 30% cholesterol, 4% distearoyl-phosphatidylethanolamine-polyethyleneglycol contained 0.05-1% ZnPC. Photodynamic efficiency was tested using a bovine serum albumin standard and measuring its oxidation (loss-of-fluorescence; λex:279nm, λem:344nm) following PDT (15J/cm2). ROS-mediated conversion of dichlorodihydrofluorescein to dichlorofluorescein was measured using fluorescence spectroscopy (λex:505nm, λem:525nm). Liposomes were assayed for uptake by human endothelial cells and cholangiocarcinoma cells (Sk-Cha1 cell line) by confocal microscopy and fluorescence spectroscopy. Cell viability following PDT (15 J/cm2, 671nm) was determined using the WST assay.

Results: Fluorescence based assays for ROS-generation and protein oxidation indicated a maximal photodynamic efficiency at a ZnPC:lipid ratio of 0.003. Liposomal uptake by endothelial and cholangiocarcinoma cells was minimal. The liposomes were non-cytotoxic in the absence of irradiation and induced a dose-dependent increase in cytotoxicity when irradiated. Cell viability was reduced to 19±6% with 500μM final lipid concentration measured 8 hours post-PDT compared to light-only treated controls.

Conclusions: This study demonstrates the development and optimization process of phototoxic liposomes. Liposomes were marginally taken up but induced considerable tumor-cell death in vitro following PDT. Future studies will focus on in vivo applications of these liposomes in PDT.

Keywords: Photodynamic therapy, photosensitizer, liposomal drug delivery, in vitro

doi:10.1016/j.freeradbiomed.2012.08.256

[0353]

CD38 expression regulates NAD(H) levels in human leukemia cells

Z.N. Al-Abady*, N.S. Ferguson, A. John Moody, R. A. Billington Plymouth University, UK

Introduction: CD38 is a multifunctional protein that is able to act as an ecto NAD(P)ase on a wide variety of cell types. Its expression is also negatively correlated with prognosis in patients with B-CLL. While much is known about its receptorial functions in B-CLL

pathogenesis, it is not clear whether the enzymatic functions of CD38 play any role in the process.

Methods: We have used the differentiation of the HL-60 cell line as a model for CD38 expression. HL-60 cells were induced to differentiate via the addition of ATRA (1 µM) for 5 days. NAD(H) was measured by an enzymatic cycling assay, CD38 expression by qPCR and CD38 activity was measured using fluorescent NGD assay.

Results: We have measured the consequences of CD38 expression during the differentiation on a number of functions linked to NAD(P). CD38 expression increases significantly after stimulation with all-trans retinoic acid (ATRA) over the first 24 hours and then remains high throughout 5 days of treatment. This expression pattern correlates well with a drop in intracellular NAD levels of c.50% that could be inhibited with the CD38 inhibitor kuromanin. Surprisingly, the NAD+/NADH ratio did not change appreciably, but several NAD-dependent processes (glycolysis, antioxidant status, oxidative damage) were found to be significantly affected by the lowered NAD levels.

Conclusion: These data will allow us to investigate whether such changes in NAD(P) levels induced by increased CD38 expression have any clinical significance for the poor prognosis of B-CLL patients expressing high levels of CD38.

Keywords: human leukemia cells, CD38 expression, NAD(P) levels, glutathione, lactate, NAD/NADH ,TBAR

doi:10.1016/j.freeradbiomed.2012.08.257

[0358]

Growth inhibition of H460 non-small lung carcinoma cells without cytotoxicity by mild hyperoxia under elevated pressure (2ATA)

E. Lee*1, J. Kim1, S. Oh2, D. Kwon3 1Korea University, Republic of Korea, 2Chonbuk National University, Republic of Korea, 3Kwandong University, Republic of Korea

Elevated pressure (EP), an extrinsic mechanical force applied to cells or whole tissues, involves mild hyperoxia under a given condition of air with 2 ATA. H460 non-small lung carcinoma cells were exposed to EP for 48 hours in custom-made pressurized cell incubator. Interestingly, EP inhibited cell proliferation with about 25% decrease of cell growth without DNA damage and apoptosis compared to cells in normal incubator in spite of EP-induced mild hyperoxia. And there was no increase of DNA damage checkpoint genes like p53 and