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We report that ascorbate free radical stimulates onion root growth at 15 °C and 25 °C. The fully reduced form, ascorbate, also stimulates root elongation if culture conditions allow its oxidation. When ascorbate oxidation was inhibited, no stimulation of root growth was found. The effect of the fully oxidized form, dehydroascorbate, was inhibitory. We show also that ascorbate free radical generated by ascorbate oxidation, is reduced back probably by a transplasmalemma reductase. These results are discussed on the basis of an activation of a transplasma membrane redox system likely involved in processes related to cell growth. [ABSTRACT FROM AUTHOR]

Arachidonic acid metabolism is modulated during differentiation induced by 1alpha,25(OH)(2)D(3) in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1alpha,25(OH)(2)D(3)-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1alpha,25(OH)(2)D(3)-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB(2) and PGE(2), suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1alpha,25(OH)(2)D(3). Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1alpha,25(OH)(2)D(3)-induced HL-60 cells.

Ascorbate has been related to the differentiation of several mesenchymal cells including haematopoietic cells. We have previously demonstrated that ascorbate enhances the activity of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) on monocytic differentiation of HL-60 cells. Here, we show that ascorbate-mediated modification of cellular redox state and AP-1 (activating protein-1) DNA binding during early phases are related to the enhancing effect of ascorbate on differentiation. Ascorbate, but not its fully oxidized form, dehydroascorbate, or an ascorbate analogue with a low rate of oxidation, ascorbate-2-phosphate, enhanced the differentiation induced by 1 alpha,25(OH)2D3, modified cytosolic reactive oxygen species levels and mitochondrial redox potential (delta psi m), and modulated AP-1 DNA binding in HL-60 cells. Ascorbate itself increased AP-1 binding to DNA in noninduced cells, whereas it inhibited AP-1 binding in 1 alpha,25(OH)2D3-induced cells. However, ascorbate increased the mRNA levels of c-jun, junB, and c-fos in 1 alpha,25(OH)2D3-induced cells. Taken together, these results suggest that the enhancing effect of ascorbate on HL-60 differentiation induced by 1 alpha, 25(OH)2D3 is related to its effect on the cellular redox state and the modulation of AP-1 activity.

A role for coenzyme Q in the stabilization of extracellular ascorbate by intact cells has been recently recognized. The aim of this work was to study the interactions between reduced ubiquinone in the plasma membrane and the ascorbyl free radical, as an approach to understand ubiquinone-mediated ascorbate stabilization at the cell surface. K-562 cells stabilized ascorbate and decreased the steady-state levels of the semiascorbyl radical. The ability of cells to reduce ascorbyl free radical was inhibited by the quinone analogs capsaicin and chloroquine and stimulated by supplementing cells with coenzyme Q10. Purified plasma membranes also reduced ascorbyl free radical in the presence of NADH. Free-radical reduction was not observed in quinone-depleted plasma membranes, but restored after its reconstitution with coenzyme Q10. Addition of reduced coenzyme Q10 to depleted membranes allowed them to reduce the signal of the ascorbyl free radical without NADH incubation and the addition of an extra amount of purified plasma membrane quinone reductase further stimulated this activity. Reduction was abolished by treatment with the reductase inhibitor p-hydroximercuribenzoate and by blocking surface glycoconjugates with the lectin wheat germ agglutinin, which supports the participation of transmembrane electron flow. The activity showed saturation kinetics by NADH and coenzyme Q, but not by the ascorbyl free radical in the range of concentrations used. Our results support that reduction of ascorbyl free radicals at the cell surface involves coenzyme Q reduction by NADH and the membrane-mediated reduction of ascorbyl free radical.

1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1, 25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1, 25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1, 25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1, 25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1,25-(OH)2D3-induced differentiation of HL-60 cells can be explained by redox regulation of the cAMP pathway.

The synthesis of DNA in CCl 39 cells is inhibited by the presence of the Fe2+ chelator bathophenanthroline disulfonate (BPS) when growth is stimulated by thrombin EGF plus insulin, but not by fetal calf serum. The presence of transferrin and Fe3+ in fetal calf serum can be the basis for lack of BPS effect with serum. The impermeable Fe3+ chelator Tiron does not, by itself, inhibit growth factor induced DNA synthesis, but it induces together with BPS inhibition on fetal calf serum induced DNA synthesis. The combined effect of BPS and Tiron is similar to inhibition of DNA synthesis by impermeable polyvalent DTPA which can chelate both Fe2+ and Fe3+ but does not inhibit ribonucleotide reductase in intact cells. Ferrous iron that bind BPS can relieve the inhibition at stoichiometric concentration. Ferric iron also prevents the inhibition even though it does not bind BPS. BPS does not inhibit DNA synthesis in HeLa cells. BPS reacts with iron from CCl 39 cells but not from HeLa cells. Data show that iron available for impermeable external chelators is in the ferrous state, and that exogenous iron should be reduced before it reverses the inhibition.

We previously showed that thrombin-stimulated DNA synthesis in CCL 39 cells was inhibited by hydroxyurea (HU) and bathophenanthroline disulfonate (BPS) (Proc. Natl. Acad. Sci. USA, in press). A clear difference exists between these two inhibitors. Inhibition mediated by HU was immediate and must be present in the culture medium. BPS was equally effective when it was present in the medium or after preincubation, but it required at least 12 h to achieve maximal effect. The permeable form 1,10 phenanthroline had the same inhibitory effect in short-term incubations that BPS. Moreover, 1,10 phenanthroline was cytotoxic in long-term incubations indicating that the site of BPS inhibition was outside the cell. Further, long-term incubations with HU did not affect the ability of the cell to reinitiate DNA synthesis after removal of the chelator.

Ascorbate, an essential nutrient in humans, primates, and guinea pig, is involved in many cellular functions. Ascorbate also modulates cell growth and differentiation. Ascorbate can reduce or stimulate the growth of tumor cells, depending on the cell type. The inhibitory effect is not specific for the biological active isomer L-ascorbate, and isoascorbate and D-ascorbate are more effective in reducing cell growth than L-ascorbate. These results indicate that ascorbate has a cytotoxic effect by killing cells directly, rather a cytostatic one. However, only L-ascorbate is able to stimulate cell growth, but the mechanism of this stimulation is still unknown. L-Ascorbate stimulates the in vitro differentiation of several mesenchyme-derived cell types by altering the expression of multiple genes as the cell progresses through specific differentiation programs. Stimulation of collagen matrix at gene transcription, mRNA stabilization, hydroxylation, and secretion is a key role for L-ascorbate. L-Ascorbate also prevents cell transformation by stabilization of the differentiated state and cooperates with other agents to induce differentiation in a leukemia cell line.

The effects of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate on morphometric and stereological parameters have been studied using the HL-60 cell line as a differentiation model for the monocytic pathway. Evaluation of the differentiation was carried out by quantification of endoplasmic reticulum, Golgi apparatus, mitochondria and cytoplasmic granules. Changes in both nuclear and cytoplasmic volumes during TPA-induced differentiation led to a decrease of the nucleus-cytoplasmic ratio after 3 days of treatment. Plasma membrane glycoprotein pattern was also determined. The major change in cell surface was the presence of high amounts of glycoproteins containing N-acetyl glucosamine residues that make wheatgerm agglutinin lectin a valuable marker of the monocytic differentiation pathway in HL-60 cells.

Besides its effect in inhibiting proliferation and inducing differentiation of HL-60 cells to macrophage-like cells, TPA also produces a transient increase of transplasma membrane redox activity and pyridine nucleotide levels and a shift in the NAD+/NADH ratio. After 24 h of incubation NADH ferricyanide reductase activity of isolated plasma membranes was significantly higher than that of plasma membrane from non-differentiated cells. This correlated with the enhanced short-term oxidation of NADH in response to ferricyanide by HL-60 cells incubated with TPA for 24 h. Since differentiated cells with similar levels of NADH showed different redox activities, the redox chain itself seems to be modulated during differentiation induced by TPA.

NADH oxidation by pig liver plasma membranes is stimulated by ceruloplasmin (CUP) reaching a maximal value at 50 U/ml of CUP. NADH oxidation activated by CUP is proportional to the amount of protein. Concanavalin A (Con A) which recognizes the glucidic residues of the CUP required for binding to the receptor inhibits the NADH oxidation in a dose-responsive manner. Both adriamycin and bathophenantroline disulfonate (BPS), previously reported as transplasma membrane electron transport inhibitors, also inhibit the CUP-stimulated NADH oxidation of pig liver plasma membranes. Our results show a clear interaction between CUP and the NADH oxidase of plasma membrane, which supports an oxidative role for CUP in its growth effect.

Ascorbate free radical stimulates the growth of human promyelocytic leukemia cells (HL-60) in the presence of a limited amount of serum (1%) when added to the cells under conditions where it is impermeable. Maximum growth stimulation occurs at concentrations from 5 x 10(-9) to 2 x 10(-8) M. Ascorbate mimicks the stimulation effect of its free radical but stimulates at higher concentrations. Autoxidation of ascorbate by oxygen produces its free radical, which apparently causes growth stimulation. Ascorbate could be regenerated by intact cells in vitro, since prevention of autoxidation of ascorbate in the presence of cells is observed. Neither dehydroascorbate nor isoascorbate increases HL-60 cell growth. Short term incubation of cells in the presence of ascorbate free radical induced intracellular NADH oxidation. We propose that the stimulation of growth of HL-60 cells shown here could be caused by activation of the transplasma membrane electron transport system by the ascorbate free radical.