Funded Research

The McCord Research Foundation is the philanthropic arm of McCord Research, the parent company for Pinnaclife. The Foundation endows fellowships for promising scientists in free radical, anatomy and cell biology through the University of Iowa Foundation. Funding for the research shared on this site was provided to the Free Radical and Radiation Biology Program; Department of Radiation Oncology; University of Iowa; Iowa City, Iowa; Prabhat Goswami Laboratory.

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Abstract

MnSOD activity regulates the hydroxytyrosol-induced protection of chronological life span
(PDF - 15.11 MB; ZIP - 14 MB)

Maneesh G. Kumar¹, Amanda L. Kalen¹, Monali Goswami¹, Garry R. Buettner¹,
Prabhat C. Goswami1, and Ehab H. Sarsour¹

¹The University of Iowa

Cellular aging is a combination of both replicative senescence and chronological life span. Chronological life span is characterized as the ability of quiescent cells to reenter the proliferative cycle. Although replicative senescence is widely studied, the mechanisms governing chronological life span are poorly understood. Previously, we showed that MnSOD activity protects the chronological life span of normal human fibroblasts (NHFs) from age-dependent loss, suggesting that the cellular redox environment regulates chronological life span. Hydroxytyrosol (HT), a naturally occurring polyphenolic compound found in olives and dates with antioxidant properties, was used to investigate the hypothesis that MnSOD activity regulates HT-induced protection of chronological life span. We observed a significant increase in MnSOD activity in HT-treated quiescent NHFs, while there were no changes in the activities of CuZnSOD, catalase, and GPx. Flow cytometry measurements of Mitosox fluorescence demonstrated that treatment with HT suppresses the age-related increase in cellular ROS levels; this suppression was associated with the ability of HT-treated quiescent NHFs to reenter the proliferative cycle (approximately 29% S+G2+M compared to 9% in control). Cell population doubling was 29 h in HT-treated vs. 37 h in control cells. Interestingly, overexpression of a dominant negative form of MnSOD eliminated the protective effect of HT on chronological life span. Results from electron paramagnetic resonance spectroscopy demonstrated the formation of HT and superoxide radicals, supporting the hypothesis that HT-induced increase in superoxide levels may activate MnSOD expression. An increase in MnSOD activity protects the chronological life span of NHFs. These results are of significance in many issues in human health, including aging and wound healing. (McCord Research Foundation and NIH CA111365)

Abstract

SOD2 activity regulates a metabolic switch facilitating transition from proliferative to quiescent growth state
(PDF - 1.95 MB)

Ehab H Sarsour¹, Amanda L Kalen¹, Monali Goswami¹,
Maneesh G Kumar¹, and Prabhat C Goswami¹

¹The University of Iowa

Proliferating cells are known to rely on aerobic glycolysis, "the Warburg effect", whereas quiescent cells utilize oxidative phosphorylation to generate energy. We hypothesize that SOD2 activity regulates a metabolic switch; higher activity supports quiescence with a decrease in aerobic glycolysis and lower activity supports proliferation with an increase in aerobic glycolysis. We observed that mouse embryonic fibroblasts (MEFs) carrying wild type SOD2 exit the proliferative cycle; SOD2 heterozygous (+/-) and homozygous (-/-) knockout MEFs continue to proliferate. SOD2 activity was low in proliferating vs. quiescent cells. Overexpression of a CMV-promoter driven SOD2 cDNA in SOD2 (-/-) MEFs demonstrated that a posttranslational modification of SOD2 regulates its activity during proliferative and quiescent growth. In synchronized fibroblasts, a decrease in SOD2 activity during S-phase was associated with an increase in glucose and oxygen consumptions, and superoxide levels. Glucose consumption was higher in exponential cultures of SOD2 wild type MEFs, which decreased dramatically as cells enter quiescence; such a transition was absent in SOD2 (+/-) and (-/-) MEFs, consistent with their inability to exit the proliferative cycle. Results from microarrays showed significant increase in c-Fos (15-fold), MAPK (4-fold), p15 (26-fold), and p57 (15-fold) in exponential cultures of SOD2 (+/-) vs. (+/+) MEFs; these results were consistent with higher levels of cyclin D1 and B1 proteins in proliferating vs. quiescent fibroblasts. Our results support the novel hypothesis that periodic changes in SOD2 activity influence glucose metabolism, "metabolic switch", regulating transitions between proliferative and quiescent growth. A loss in such a metabolic switch could facilitate aberrant proliferation. SOD2 represents a novel molecular player of the Warburg effect. (NIH CA111365)

Abstract

MnSOD activity protects mitochondrial morphology of quiescent human skin fibroblasts from age-associated abnormalities
(PDF - 1.2 MB)

Ehab H Sarsour¹, Amanda L Kalen¹, Monali Goswami¹,
Maneesh G Kumar¹, and Prabhat C Goswami¹

¹The University of Iowa

This study investigates the hypothesis that MnSOD activity protects the mitochondrial morphology of quiescent normal human skin fibroblasts (NHFs) from age-associated damage and maintains the proliferative capacity of aged quiescent cultures. Quiescent NHFs cultured for 30-60 days exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles compared to 10-day cultures. Adenovirus mediated overexpression of MnSOD in quiescent NHFs suppressed the age-related increase in ROS levels and maintained the normal morphology of mitochondria. Consistent with these results, flow cytometry measurements of Mitotracker fluorescence showed approximately 15 and 50% decrease in MnSOD heterozygous and homozygous knockout mouse embryonic fibroblasts (MEFs) compared to wild-type MEFs. Likewise, measurements of MitoSOX fluorescence exhibited 75 and 130% increase in MnSOD heterozygous and homozygous knockout MEFs compared to wild type MEFs. Sixty to eighty day quiescent NHFs cultured in 21% oxygen environment showed significant damage in mitochondrial morphology compared to cells cultured in 4% oxygen environment. Cell population doubling was 44 h in NHFs cultured in 4% vs. 54 h in 21% oxygen environment. These results clearly demonstrate that MnSOD activity protects mitochondrial morphology and proliferative capacities of quiescent NHFs from age-related abnormalities. These results support the hypothesis that ROS dependent mitochondrial retrograde signaling regulates the proliferative capacity of quiescent normal fibroblasts. (McCord research foundation and NIH CA111365)

Abstract

N-acetyl-L-cysteine enhances the recruitment and proliferation of quiescent human fibroblasts during wound healing
(PDF - 2.2 MB)

Monali Goswami¹, Abby E Wilbanks², Amanda L Kalen¹,
Prabhat C Goswami¹, and Ehab H Sarsour¹

¹The University of Iowa, ²Iowa State University

Wound healing is a complex process that has three major overlapping stages: inflammation, proliferation, and maturation or closure. The rebuilding of the connective tissue as a base for wound maturation requires the recruitment of fibroblasts to the wound area as well as reentry of quiescent fibroblasts to the proliferative cycle. N-acetyl-L-cysteine (NAC) is a small molecular weight thiol antioxidant that is widely used in medicine and research. We investigate the hypothesis that NAC facilitates wound healing by modulating the cellular antioxidant system. A novel uni-directional wound healing assay was used to measure migration in 15 day quiescent cultures of control and NAC treated normal human skin fibroblasts (NHFs). Control cells exhibited a migration rate of 28 μm/hr, while 1 and 5 mM NAC treated cells showed a migration rate of 24 and 19 μm/hr, respectively. The number of cells recruited into the wound area increased 40-70% in NAC treated cells compared to controls. Fifteen day quiescent control and NAC treated NHFs were replated at a lower density and cell numbers counted at different days post-plating. While control cells showed cell population doubling of 48 h, 1 and 5 mM NAC treated cells exhibited 24 and 28 h doubling time, respectively. Interestingly, the NAC treatment increased protein and activity levels of MnSOD without a corresponding change in MnSOD mRNA levels, suggesting that a post-translational mechanism regulates MnSOD activity in NAC treated cells. These results demonstrate that NAC enhanced the recruitment of quiescent NHFs into the wound area and facilitated the proliferation of NHFs following reentry into the proliferative cycle. Furthermore, our results also suggest that the wound healing properties of NAC is due to its ability to enhance MnSOD activity. (McCord Research Foundation and NIH CA111365)