Background Numerous studies have shown that the intracellular redox potential of

Question

Background

Numerous studies have shown that the intracellular redox potential of the cell is important to cell growth. Cellular redox potential can be determined by the amounts of the reduced coenzyme NADPH, a principal product of the oxidative branch of the pentose phosphate pathway (PPP). The investigators in the study presented here sought to demonstrate links between the activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD) activity, cellular NADPH concentrations, and rates of cell growth. Previous studies have shown that the glucose-6- phosphate dehydrogenase enzyme can be activated on the order of minutes or even seconds, possibly through the action of growth factors that release a bound, inactive G6PD to the cytosol, where, via a mechanism that might involve tyrosine phosphorylation of a membrane-bound receptor, the unbound G6PD translocates to the cytosol and becomes active. NADPH is important to the cell in a variety of ways. The reduced coenzyme can react with potential damaging oxidizing agents, ridding the cell of these agents before they can damage 2 2 important cellular components. For example, hydrogen peroxide, H O , may be reduced to water with concomitant oxidation of NADPH,

In the first experiment reported in this study, the authors stimulated cell growth through a variety of means; then measured corresponding G6PD activity of cultured fibroblasts. (Cells grown in culture are typically grown in a medium containing 10% fetal calf serum, a medium rich in growth factors.) The investigators grew fibroblasts in the absence of serum (serumstarved) to serve as a control. Cells were then treated with 10% fetal calf serum followed by the serum-starvation treatment; other cells were not starved. The results are shown in Figure 32.2. After the enzyme activity was measured, the cells were lysed and analyzed by Western blotting using a G6PD antibody. The results are of the Western blot are shown in Figure 32.3.

QUESTION #3

What is your interpretation of these results? Be quantitative in your assessment of the data for both the activity assays and the Western blot. Why was the enzyme activity of PGD (6-phosphogluconate dehydrogenase) also investigated under the same conditions?

100 E 80 O G6PD P GD 60 40 20 o L yes Serum starved yes n0 Plus Serum yes yes no Figure 32.2: Fibroblasts were (1) serum-starved for 48 hours (2) serum-starved for 48 hours and then stimulated with 10% serum, and (3) actively grown in medium containing 10% fetal calf serum. Enzyme activity was measured in lysates containing the same number of cells (from Tian, et al, 1998). Figure 32.3: Western blot analysis of the same cell lysates as shown in Figure 32.2. Lane 1 shows serum-starved cells; Lane 2 is cells G6PD grown in the presence of serum (from Tian, et al., 1998).

Explanation / Answer

The cellular oxidation reduction environment is influenced by the production and removal of reactive oxygen species (ROS). In recent years, several reports support the hypothesis that cellular ROS levels could function as second messengers regulating numerous cellular processes including proliferation. Periodic oscillations in the cellular reduction environment, redox cycle, regulate cell-cycle progression from quiescence(G0) to proliferation (G1,S,G2, and M) and back to quiescence.A Los in the redox control of the cell cycle could lead to aberrant qualification, a hallmark of various human Pathologies. Desh se review discussion the literature that supports the concept of a redux cycle controlling the mammalian cell cycle, with an emphasis on how this control is relates to proliferation these orders including cancer, wound healing, Fibrosis, cardiovascular diseases diabetes and neurodegenerative diseases. We hypothesize that Re establishing the redox control of the cell cycle by manipulating the cellular redox environnement could improve many aspects of the proliferation disorders.

According to the graph the first one is serum starved but not plus serum. The second one is both serum starved and plus serum. The third one is not serum starved but plus serum.

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