**3. Proteomics Studies**

Proteomics provides important qualitative information on post-translational modifications to proteins and quantitative data on protein expression in response to a particular stimulus. This information is particularly important when it provides data on early cellular events, such as the stimulus and signaling cascades triggered independently of protein neosynthesis. In accordance with its proposed pro-oxidant activity, vitamin C-mediated reduction in the GSH/GSSG ratio correlated with an intracellular H2O2 increase in the NB4 cell line [17,19]. This type of change in regional oxidation state could cause changes in the cellular milieu that could result in changes in protein structure. This is especially true of the oxidation state of cysteine sulfur, which is important for determining the tertiary structure of proteins. The immediate effects of cell stimuli are associated with protein post-translational modifications, such as phosphorylation, glutathionylation and cysteine oxidation. To study these early modifications, NB4 human leukemia cells were treated with 0.5 mM vitamin C and then analyzed by two-dimensional analysis. Approximately 240 different spots that were focused in a pH range of 4–7 were detected per sample.

After exposing cells to vitamin C, we observed one new spot, three intensified spots and five attenuated spots [19]. Each of these spots were excised, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Peptide mass fingerprint analysis and non-redundant sequence database matching allowed the unambiguous identification of all of the analyzed species [19]. An important protein identified from the proteomic analysis was a thiol/disulfide exchange catalyst, protein disulfide isomerase (PDI), which was a marker of the effect of vitamin C on NB4 cells [19]. PDI belongs to the superfamily of thiol/disulfide exchange catalysts, which act as protein-thiol-oxidoreductase enzymes, sharing sequence homology with thioredoxin [28]. PDI is composed of four domains, which have similarities with thioredoxin folds (*i.e.*, a-b-b′-a′) [29]. In our study [19], the intensity of the spot corresponding to the PDI b subunit was decreased in vitamin C-treated cells as compared with control cells. These results demonstrated that thiol/disulfide exchange proteins are regulated in NB4 cells after vitamin C exposure. This is consistent with our study showing that intracellular GSH/GSSG exchange occurs shortly after vitamin C exposure [17].

When we measured cysteine uptake in leukemia cell lines exposed to vitamin C by using 35S-labeled-L-Cys in the media, the time-dependent rate of cysteine uptake in the cell culture increased significantly. The rate of uptake, determined under conditions without vitamin C, was very low. The glutathione synthesis inhibitor, buthionine sulfoximine, potently inhibited this increase, suggesting that incorporation of cysteine that corresponded to the amount of increased glutathione was mediated by glutathione synthesis. Overall, our results indicated that vitamin C-induced glutathione synthesis was accompanied by intracellular cysteine uptake.

Glutathione-S transferases (GSTs) are enzymes that catalyze the conjugation of electrophilic substitution to GSH, which protects cells by removing reactive oxygen species and regenerating *S*-thiolated proteins [30]. Intracellular total GSH levels in AML cells incubated with vitamin C peaked around 3 h, then declined, while the increase in incorporated [35S]-L-Cys peaked at 3 h and remained high. These results showed that [35S]-L-Cys transported into cells through cysteine uptake was incorporated and transferred intracellularly, strongly suggesting that the sulfhydryl transfer system is affected by vitamin C [30].

We therefore hypothesize that the biochemical pathway leading to thiol/disulfide redox regulation could be activated by vitamin C. Interestingly, of the proteins whose expressions changed by vitamin C treatment, immunoglobulin heavy chain binding protein (BiP, identical to Hsp70 chaperone) [19], like PDI, is also a multi-domain chaperone. BiP associated with the α-subunit of prolyl 4-hydroxylase (P4-H) by a disulfide bond [31]. P4-H consists of two distinct polypeptides, the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme, PDI [31]. Thus, BiP associated with the α-subunit of P4-H, which is another partner of PDI. The interaction of PDI with its substrates was due to a change in disulfide bonds, indicating that intrachain disulfide bonds between domains and substrates had been reduced [19]. Taken together, these results suggested that vitamin C oxidizes intracellular-reduced glutathione and affects disulfide bond formation in proteins [30].

Tropomyosin was also identified as a marker of the oxidative effect of vitamin C in NB4 cells. The spot corresponding to tropomyosin was positioned at an isoelectric point (pI) of approximately 5.0 and was attenuated in vitamin C-treated cells. In addition, a new spot having almost the same molecular weight was detected, which was positioned at a pI of 4.9 [19]. This new spot was also identified as tropomyosin, suggesting that post-translational chemical modification had affected its pI value. This result is consistent with previous data showing that the extracellular signal-regulated kinase (ERK)-mediated phosphorylation of tropomyosin-1 promoted cytoskeleton remodeling in response to oxidative stress [32]. The acidic shift of the spot with pI 5.0 to the phosphorylated tropomyosin spot by treatment with vitamin C was found to be abrogated by co-treatment with PD98059 [19], demonstrating that phosphorylation of tropomyosin was responsible for the observed acidic shift.

The significance of this observation may be related to differences in the regulation of the actomyosin contractile system in non-muscle cells as compared with that present in muscle cells. In addition, proteins that specifically reacted with sera from chronic myeloid leukemia patients included structural proteins, such as β-tubulin and tropomyosin isoforms [33]. Although the function of these proteins in myeloid leukemia needs further investigation, tropomyosin may have value as a leukemia-associated antigen and as a molecular target in antigen-based immunotherapy. In this regard, it is important to note that vitamin C causes a tropomyosin isoform to be modified during the immediate early response.
