Involvement of Fenton reaction products in differentiation induction of K562 human leukemia cells

Pergamon Leukemia Research Vol. 19, No. 3, 203-212, 1995 pp. Copyright 0 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0145-2126/95 $9.50 + 0.00

01452126(94)00138-3

INVOLVEMENT OF FENTON REACTION PRODUCTS IN DIFFERENTIATION INDUCTION OF KS62 HUMAN LEUKEMIA CELLS

Katalin Nagy,* Gabriella Past&t L&z16 Bene$ and Imre Zs.-Nagy* *Verzar International Laboratory for Experimental Gerontology (VILEG), Hungarian Section; TInstitute of Hygiene; and *Department of Biophysics, University Medical School, H-4012 Debrecen,

Hungary

(Received 13 May 1994. Revision accepted 3 October 1994)

Abstract-ADP-Fez+ (or ATP-Fe*+) complex and H202, components of the Fenton reaction, were added to K562 cells, then cultured for 96h. Ara-C-induced differentiation served as a basis for comparison. Cell numbers, viability, benzidine staining, thymidine incorporation, and cell-cycle distribution by means of flow cytometry were determined. The Fenton reagents reduced the growth rate and thymidine incorporation of leukemic cells in a dose-dependent manner as regards the added HzOz (from 0.01 to 1.0 mM), accompanied by an accumulation of hemoglobin in them. Differentiation of the cells was accompanied by considerable changes in total SOD and catalase activities. Ara-C caused an increase of SOD to 366%, and of catalase to 235%, while the complete Fenton reaction resulted in SOD increase to 705% and catalase decrease to 38% of the untreated control cultures. These shifts in enzyme inductions suggest the existence of a higher H202 flux in the differentiating cells. The results are consistent with the assumption that products of the Fenton reaction, among them OH’ radicals deriving from HzOz by heterolysis, may play a causal role in cell differentiation, whereas an overproduction of these radicals causes aging or death of the cells.

Key words: K562 human erythroleukemia cells, cell differentiation, oxygen free radicals in differentiation, Fenton reaction, Ara-C-induced differentiation, SOD expression in differentiation.

Introduction

K562 human erythroleukemia cells grow unlimited and display only a negligible tendency for spontaneous differentiation under standardized conditions. However, a great number of drugs, including several known antineoplastic agents, induce erythroid differentiation of these cells. During differentiation, K562 cells stop dividing and start to synthesize hemoglobin, that is, become ‘normal’ erythrocytes. The main examples are: l-pD-arabinofuranosylcytosine (Ara-C) [l-4], phorbol esters [5], fagaronine [6], tiazofurin and ribavirin [7], themozolomide [8], pyrizinones [9], and a list of a further 19 drugs is given in Table 1 of Rowley et al. [l]. Some conflicting observations should also be mentioned: for example, the phorbol esters are known as the most potent tumor promoters, yet on hematopoietic cells they may exert either induction

Correspondence too: Prof. Dr. Imre Zs.-Nagy, VILEG Hungarian Section, University Medical School, Debrecen, H-4012, Hungary (Tel:/Fax: (36-52) 418-470).

or inhibition of cell differentiation [lo]. Another fact is that while 1,25-dihydroxyvitamin D3 causes differentiation in HL-60 promyelocytic cells [ 111, the same compound inhibits the Ara-C-induced differentiation of K562 cells [4].

In the case of Ara-C, extensive studies have been carried out to reveal the mechanism of action, and several gene products were shown to be altered by this drug [3]. However, no common explanation is available for the effects of the drugs mentioned above.

One cannot exclude the possibility that cell differentiation might be due to a common process which can be influenced by various chemical entities. A hypothesis has been proposed, assuming that part of the OH’ free radicals derived from H202 (product of superoxide dismutase = SOD) by iron-induced heterolysis may play a physiologically useful, important role in cell growth and maturation [ 12-141. An experimental testing of this hypothesis on HL-60 human promyelocytic leukemia cells gave encouraging results [15]. Therefore, we have decided to extend our studies to K562 cells. Regarding these cells,

203

204 K. Nagy et al.

particular significance can be attributed to the following experimental results.

(i) Desferrioxamine (DFO) is a strong chelator of Fe3 + ; in its presence, the biological system becomes devoid of utilizable iron [16-191. DFO decreases the growth rate of K562 cell cultures without affecting the DNA synthesis (except when high doses are applied); it causes an increase of transferrin receptors in the cell plasma membrane, however, differentiation is not induced by DFO [20]. The iron-saturated form (ferrioxamine = FO) has no effect on the parameters altered by DFO treatment [20].

(ii) A low molecular weight copper complex mim- icking the CuZn-SOD active center (CuPUPY) increases O2 uptake by K562 cells by almost 100% [21]. It is cytotoxic for K562 cells, however, exter- nally added catalase reduces this cytotoxicity very considerably [21]. This may mean that a strongly increased H202 production, and a consequently very intense OH’ radical formation, are probably responsible for the cytotoxicity.

Materials and Methods

Enzymes, reagents and chemicals The following products were used: propidium iodide,

RNAse, SOD, catalase, xanthine oxidase, xanthine, folin- phenol reagent, SDS, Ara-C and Triton-X-100 from Sigma; [6-3H]thymidine from Amersham International. All other reagents were from the Reanal Budapest with the exception of fetal bovine serum which was the product of the Human Institute for Serobacteriological Production and Research, Budapest-Giidiillii, Hungary.

Cell culture The K562 human erythroleukemia cells were cultured

under their established, standard conditions, that is, in RPM1 1640 medium supplemented with 50 IU/ml penicillin, 50 yg/ml streptomycin and 10% fetal calf serum

in a total volume of 20 ml placed in 75 ml tissue culture flasks (Greiner) at 37°C in a humidified, 5% COZ atmos- phere. Cell numbers were counted with a hemocytometer at regular time intervals as described below. Only viable cells were considered, as determined by trypan blue dye exclusion. Non-viable cells occurred in each culture. How- ever, they amounted to a negligible percentage.

Experimental interventions Generation of OH’ free radicals was achieved by the

method of Floyd and Lewis [22], applied also by others [14,15,23,24]. It consists of the addition of ADP-Fe*+ (or ATP-Fe*+) complex and H202. These components were added to various experimental systems in quantities as shown in Table 1. During the experiments, the K562 cells were cultured under otherwise standard conditions for 96 h. The initial concentration of the cell suspensions was 2 X lo5 cells/ml and a volume of 20 ml was applied. After the first 48 h of culturing, the cells were pelleted by cen- trifugation and resuspended in the same volume of fresh RPM1 1640. The experimental cell groups were then treated with identical freshly prepared reaction mixtures as given in Table 1 for another period of 48 h. Control cultures were run in parallel with each experiment. All experiments were repeated at least three times. The determination of cell number and thymidine incorporation assay were performed twice daily. The cell staining, flow cytometric analysis and enzyme assays were accomplished at the end of the 4-gay culture period. Induced differentiation of K562 cells, for purposes of comparison, was produced by addition of 1.8 pmol l-PD-arabinofuranosylcytosine (Ara- C) to the control cultures, according to Feriotto et al. [2].

Benzidine staining The most widely used method for scoring erythroid

differentiation is benzidine staining which reveals the production of hemoglobin; the staining is so specific that it is suitable also for spectrophotometric determinations [ 1,4,6,21,25]. Benzidine dihydrochloride (2 mg/ml) was prepared in 3% acetic acid. H202 (1%) was added immediately before use. The cell suspensions were mixed with the benzidine solution in a 1: 1 ratio and counted in a hemocytometer after 5 min. Blue cells were considered to be positive for hemoglobin. At least 5 X 100 cells were counted per sample.

Table 1. The composition of the cell culture systems tested in the experiments

Group No.

Control Control + Ara-C Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 Exp. 6 Exp. 7

ADP-Fe*+

- - + + + + - - -

Added substances ATP-Fe2+ ADP H202

- - - - - - - - 1.0 - - 0.1 - - 0.01 - - - - - 0.1 + - 0.1 - + -

Final concentrations of ADP or ATP were 2 mM, of Fe*+ was 0.1 mM in each experiment. H20z concentrations (mM) varied as indicated.

Note: The nucleotide-Fe*+ complex was prepared immediately before addition to the culture medium.

Fenton reaction-induced differentiation of K562 cells 205

Time lhoural

Fig. 1. Cell growth of K562 cells during 96 h of culturing. C indicates the control system, whereas numbers l-7 designate the experiments described and numbered identically in Table 1. Data are from one representative experiment. The renewal of the medium after 48 h induced some

changes in cell numbers in each culture.

Thymidine incorporation assay DNA synthesis was assayed [26] by the incorporation of

[6-3H]-thymidine. For this purpose, 100~1 of cell sus- pension was plated in triplicate into 96-well microtiter plates. Cells were then pulse-labeled with 0.2 &i/well [3H]thymidine and cultured for 4 h at 37°C in a humidified incubator with 5% COZ. Cells were then harvested, col- lected on filter paper, and dried. The incorporated thymidine was measured by liquid scintillation counting method.

Fixation and staining of samples for pow cytometry The cells were washed in phosphate-buffered saline

(PBS) and fixed in 70% ethanol at 0°C [9]. Fixed cells were stable for at least 2 weeks at 4°C. The cells were rinsed with PBS and treated with 1 mg/ml of RNAse in PBS at 37°C for 30 min, followed by staining with 50 pg/ml of propidium iodide. The cells were filtered through a 37 km nylon mesh to remove aggregates prior to flow analysis. The cells stained with propidium iodide were analyzed using a flow cytometer (FACS III, Becton Dickinson FACS Systems, U.S.A.) with a 488 nm laser beam as described by Blair et al. [27]. At least 10,000 cells were counted in each sample. The DNA histograms obtained were analyzed to determine the distribution of cells in the Go/G1, S and G2 + M phases of the cell cycle using a suitable computer program.

Protein content and enzyme determinations The cells were washed with phosphate-buffered saline

and disrupted by sonication. Protein content was determined by the method of Markwell et al. [28]. Enzyme determinations were carried out in the control, and Ara-C treated cultures as well as in Experiments 2,.5 and 7 (Table 1) at the end of the 96 h culture period as follows.

Superoxide dismutase (SOD). The method of FlohC and otting [29] was used for total SOD activity. The reduction rate of cytochrome C by 0: radicals is detected at 550 nm using the xanthine-xanthine oxidase system as the source of 0; radicals. Since hemoglobin is known to interfere with the SOD assay, cell suspensions were extracted with a chloroform-ethanol mixture [30]. The mixture was shaken for 1 min at 4°C and centrifuged. SOD activity was measured in the supernatants.

Cutalase (CA). The method of Gaunt and De Duve [31] was applied. Cell suspensions in the presence of 0.1% Triton X-100 were incubated for 30min at 25°C with a known concentration of buffered H202. The reaction was stopped by the addition of titanium oxysulfate. The remain- ing H20, was measured spectrophotometrically at 405 nm.

Results

Cell proliferation

The number of K562 cells, cultured with various experimental additions (Table l), was counted at different intervals up to 96 h. The results are summa- rized in Fig. 1. Cell growth was considerably fast in the control cultures during the observation period;

206 K. Nagy et al.

I

20 I

40 I I

60 60 100

Time C hours I

Fig. 2. [3H]thymidine uptake in K562 cells during 96 h of culturing. C indicates the control system, whereas numbers l-7 designate the experiments described and numbered identically in Table 1. Data are from one representative experiment. Note the logarithmic scale on the vertical axis. The renewal of the medium after 48 h induced some

changes in DNA synthesis in each culture.

the cell numbers displayed a considerable increase as expected. This increase was inhibited by various extents in Experiments l-7. Cell proliferation was completely abolished in Experiment 1 (when the highest hydrogen peroxide concentration was added). In fact, in Experiment 1, the cell number was somewhat higher than the starting value only 8 h subsequent to the seeding, and decreased significantly thereafter (Fig. 1). Decreasing concentrations of added H202 (Experiments 2 and 3) still produced considerable inhibition of cell growth especially during the second half of the observation period. Similar inhibitory effects were observed when ADP-Fe2+ complex, or H202 were added alone, respectively (Experiments 4 and 5). ATP-Fe2+ complex and H202 (Experiment 6) gave the same level of growth inhibition, as observed in Experiment 2, where ADP was substituted for ATP. When ADP was applied alone without iron and hydrogen per-

oxide (Experiment 7), it still caused some inhibitory effect, but this proved to be the most modest level of inhibition among all the experiments.

DNA synthesis The reactants listed in Table 1 resulted in various

degrees of inhibition of the incorporation of [3H]- thymidine into K562 cells (Fig. 2). Due to the large differences in thymidine incorporation data between various experimental models, Fig. 2 depicts the situation by using a logarithmic scale on the vertical axis. The rates of inhibition in various experimental groups followed the patterns found in the cell growth inhibition. The 1 mM H202 concentration (Experiment 1) decreased the uptake below 1000 cpm, that is, the statistical scatter of the results relatively increased. In general, the effects on DNA synthesis are parallel with the effects on cell number.

Table 2 summarizes the numerical values of the

Fenton reaction-induced differentiation of KS62 cells 207

Table 2. Absolute viable cell numbers and their percentage comparison, with the relative [3H]thymidine incorporation in K562 cells at the end of 96 h culturing

(mean % of controls * S.D. of three runs)

Cell number [3H]thymidine

Group No. 104/ml (%) incorp. (%)

Control 194.7 5 16.9 100 2 8.6 100 * 6.1 Ara-C 20.7 k 4.5 10.6 * 2.3 Exp. 1 4.8 ? 2.0 2.4 k 1.0 o.Ei. 1 Exp. 2 15.3 k 8.3 7.8 + 4.3 2.5 2 0.3 Exp. 3 33.5 k 18.6 17.2 k 9.5 8.4 k 0.6 Exp. 4 31.4 + 14.2 16.1 k 7.3 4.8 k 0.1 Exp. 5 45.9 r 11.4 23.5 k 5.8 16.6 2 1.7 Exp. 6 27.8 k 4.3 14.2 k 2.2 4.5 ? 0.2 Exp. 7 107.0 k 13.2 54.9 k 6.7 58.8 t 3.5

Notes: All the experimental results differ significantly from controls at p < 0.001; starting seeded cell number at 0 time was 20 x 104/ml in all cultures; n.m., not measured.

Table 3. Benzidine staining of K562 cells at the end of the 96 h culture period, (mean k S.D. of three runs)

Group No.

Control Ara-C Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 Exp. 6 Exp. 7

Benzidine-positive cells (%)

3.2 -+ 1.0 44.3 k 9.9

Uncountable 27.0 ? 4.3 17.8 5 5.3 7.5 t 0.9 8.3 k 2.0

29.0 AI 6.2 3.0 * 1.4

Notes: The differences between the control and all experimental groups are statistically significant at p < 0.001, except Exp. 7 which is not different from the control. Experiment 1 could not be counted because of the low number of cells.

data concerning cell number and [3H]thymidine uptake at the end of the 96 h incubation time. These parameters are expressed also in relative terms, tak- ing the average control values as lOO%, in order to allow realistic comparisons. It is noteworthy that all the reactants resulted in a significant decrease of both the viable cell number and DNA synthesis, as compared with the controls. Experiments l-3 revealed a dose-dependent decrease in these parameters: the three different concentrations of H202 resulted in significantly increased cell inhibition with higher doses, nevertheless, even the lowest dose was a very efficient inhibitor of both parameters. ADP applied as an iron chelator was about twice as inhibitory as ATP at identical concentrations of H202 (see Experiments 2 and 6 in Table 2).

Benzidine staining The control K.562 cultures display a spontaneous

differentiation (i.e., benzidine staining) in approxi- mately 3% of cells. The benzidine staining increases if more and more cells start to synthesize hemoglobin. Almost all of our reactants induced erythroid differentiation to various extents (Table 3). These inductions proved to be statistically significant at p < 0.001 at the 96th hour of incubation, as compared to the control cultures, with the exception of treatment with ADP alone (Experiment 7), which was practically ineffective. Because of the low number of viable cells in Experiment 1, we could not evaluate the result. There was no difference in the action of ADP- and ATP-chelated iron.

Comparing the effect of Ara-C on differentiation to that of the OH’ free radical generating systems, it appears that Ara-C produces benzidine positivity in 44.3% of cells, and this figure is higher than in any of the experiments.

Flow cytometric analysis of the actual phases of the cell cycle

Flow cytometric analysis was performed after the 96 h incubation. Figure 3 shows some characteristic curves from a representative experiment. Table 4 summarizes the DNA distributions expressed as % of cells in various phases of the cell cycle. In K562 cells Ara-C and the conditions of added compounds in Experiments l-7 induced a very considerable arrest in the G2 + M phase of the cell cycle, as observed with various inducers [S, 321. This arrest in G2 + M phase is statistically significant in all experiments, as compared with control cells. The small difference between Ara-C and Experiment 2 was

208 K. Nagy et al. so0 275 I - Control

--- Are-C

. . . . . . . . . . Exp. 2

125-

0.00 255.75

FluorascenCe intensity

Fig. 3. Flow cytometric DNA distribution histograms in control, Ara-C treated and Experiment 2 in K562 cells.

For further details, see text.

Table 4. Distribution of KS62 cells in various phases of the cell cycle based on flow cytometric determination of the DNA contents (mean % k S.D. of three experiments)

Group No. Go/G S G2 + M

Control 49.0 * 4.3 40.0 2 4.9 10.0 L 0.9 Ara-C 15.9 IL 1.1 15.2 k 2.2 68.8 2 3.2* Exp. 1 Uncountable Exp. 2 19.9 ? 0.2 23.4 2 1.7 56.7 + 1.8* Exp. 3 37.7 ? 1.0 33.9 * 0.9 28.3 k 0.2* Exp. 4 47.7 * 1.5 12.0 +- 2.8 40.2 k 1.3* Exp. 5 45.0 * 3.1 16.7 2 4.3 38.3 A 5.0* Exp. 6 29.2 2 3.7 17.7 k 4.6 53.1 2 3.1* Exp. 7 54.4 * 3.0 28.9 k 1.9 16.6 5 l.l”r

Notes: * and t indicate significant differences in the last column against the control at p < 0.001 or 0.01, respectively.

still significant (p < 0.02), whereas the difference between Experiments 2 and 3 was highly significant (p < 0.001). In the case of Experiment 1 we could not perform flow cytometric analysis because the number of viable cells was very low.

Enzyme levels in treated cultures Total SOD and catalase activities found in control

and treated K562 cells after 96 h of culturing are shown in Tables 5 and 6. These activities can be expressed either per mg protein or per cell number. The significance of these means of expression will be discussed later.

Both means of presentation revealed a very considerable increase in SOD activity both in the Ara- C and the Fenton reactant-treated cultures. The greatest increase (up to 700%) was found in Exper- iment 2 where the complete Fenton reaction was

Table 5. Total superoxide dismutase activities in control and treated K562 cells (mean I S.D. of three experiments)

Superoxide dismutase

Group No. hdw protein) (%) (ng/106 cells) (%)

Control (1) 15.42 t 1.71 100.0 319.7 t 35.4 100.0 Ara-C 31.16 * 2.99 202.1 1172.9 k 112.6 366.9

Control (2) 16.46 + 1.33 100.0 328.5 +- 29.5 100.0 Exp. 2 58.13 -c 2.89 353.2 2316.5 k 201.1 705.1 Exp. 5 35.35 * 1.39 214.8 1277.9 k 67.4 389.0 Exp. 7 30.37 k 1.15 184.5 998.2 * 30.6 303.9

Note: All the values in treated groups are significantly different from control at p < 0.001.

Fenton reaction-induced differentiation of KS62 cells 209

Table 6. Catalase activities in control and treated K562 cells (mean 5 S.D. of three experiments)

Catalase

Group No. (U/mg protein) (%) (U/lo6 cells) (%)

Control (1) 31.8 2 0.9 100.0 13.2 2 0.4 100.0 Ara-C 42.4 k 1.7 133.3 31.0 ? 1.2 234.8

Control (2) 35.5 * 3.1 100.0 18.7 * 4.3 100.0 Exp. 2 17.9 * 1.6 50.4 7.0 * 0.7 37.6 Exp. 5 22.0 2 1.2 62.0 11.8 k 0.7 62.7 Exp. 7 27.7 k 1.6 78.0 16.0 2 0.1 85.4

Note: All the values in treated groups are significantly different from control at p < 0.001.

performed, whereas in the cultures treated with Ara- C or with only one of the Fenton reactants, the increase of SOD was in the range of 3OO-390%, if expressed on a per cell basis (Table 5).

Catalase activity differed from that of SOD. While in the Ara-C treated cultures it increased to 234%, in Experiments 2, 5 and 7 it displayed a decrease to 37-85%) if expressed on a per cell basis (Table 6). In other words, the ratio of relative activities SOD/ catalase, if taken in the controls lOO/lOO = 1, changed to 367/235 = 1.56 in the Ara-C group, and to 705138 = 18.55; 389163 = 6.17; 304185 = 3.58 in Experiments 2, 5 and 7, respectively. These changes indicate a more or less strong shift of the activity ratios in favor of SOD. Although these ratios are numerically different, it is noteworthy that they reach a value of 1.5 even in the Ara-C treated cultures.

Discussion

The increase of total SOD activity during cell differentiation induced either by Ara-C or by the components of the Fenton reaction is similar to the situation described in murine erythroleukemia cells where hexamethylene bisacetamide (HMBA) induced both differentiation and an increased expression of SOD [33-351. It is even more significant that differentiation was induced simply by SOD added in liposomes to the Friend cell cultures [35]. Apparently, a common step of differentiation induction in various cell lines is a strong increase in SOD expression.

Obviously, one has to ask two important questions: (1) HOW the known inducers and the Fenton reactants added to the system may lead to an increased SOD expression’? (2) How increased SOD activity is able to shift cells toward a definitive differentiation?

As regards question 1, the inducers may be able to

cause an alteration in the intracellular redox balance which then stimulates SOD expression. In the case of the added Fenton reactants, one may consider the following known facts.

(a) The ADP-Fe2+ and ATP-Fe2+ complexes reacting with H202 generate various radical species, among them OH’ free radicals [14,22,23,36-381.

(b) ADP or ATP chelate divalent iron. In ADP- Fe2+ or ATP-Fe2+ complexes iron remains divalent at physiological pH and oxygen pressure [14] as shown by their reaction with ferrozine [39], and it also remains available for H,Oz in the Fenton reaction [22,24]. The trivalent iron complexed by nucleotides can also participate in the formation of OH’ free radicals through the Haber-Weiss reaction, however, with a five-fold lower yield [22].

(c) Since OH’ free radicals have an extremely short life-time, those formed in the extracellular medium can hardly reach the intracellular compartments. Nevertheless, intracellular formation of these radicals is also possible in our experimental system, for the following reasons: (i) ADP-Fe2+ complexes are physiological constituents of the cells [40]. (ii) The cells take up such complexes [41,42]. (iii) Since the cells contain ascorbate reducing the oxidized iron in the complex [22], it is quite possible that the ADP- iron complex, once oxidized in the extracellular space, can be reduced within the cell, and may participate repeatedly in the generation of OH’ radicals when reacting with the H20, formed in the mitochondria. Therefore, one can assume that the ADP- iron complex incorporated by the cells becomes a source of OH’ radicals. In fact, ascorbate is a regu- latory factor in the growth of human leukemic [43] and other cells [44,45]. Specially designed further studies may reveal the actual OH’ radical yield in the intracellular space.

The above explanation agrees with the results of incomplete additions of the Fenton reactants. These experiments also produced some inhibitory effects on cell growth and DNA synthesis, and a stimulation of cell maturation, since even if some of the components were omitted, they are physiologically present in the cells. For example, when we added only ADP-Fe2’ (Experiment 4), it could obviously react with the H202 normally produced in the cells. Even ADP added alone (Experiment 7) evoked some minor effects, since its chelating action on Fe2+, formed by naturally present reductive components, such as Vitamin C [44,45], could improve the OH’ radical yield in the cells.

The possible common role of OH’ free radicals in differentiation induction is indicated also by other facts. Namely, Ara-C-induced differentiation of K562 cells is strongly inhibited by desferrioxamine

210 K. Nagy et al.

(DFO) which is a strong chelator of Fe3+ [2]. It is also well known that the iron bound by DFO is not able to participate in any free radical reaction, like lipid peroxidation or OH’ radical formation [ 16-181. On the other hand, it is known that compounds like Ara-C may become di- and triphosphorylated in the cells [46], and like any other di- and triphosphate nucleotides, may chelate iron and catalyze OH’ free radical formation from H202 [35]. Considering the molecular structures, iron chelation is possible in the case of many other compounds of antimitogenic activity, such as fagaronine [6], tiazofurin and ribavirin [7], themozolomide [S], pyrizinones [9], adriamycin [47], etcetera.

Even if the above hypothesis is true, it still remains to be elucidated which product(s) of the increased OH’ radical flux is (are) the direct stimulator(s) of SOD expression.

An attempt to answer question 2 has to consider the consequences of the strong increase of total SOD activity in the cells. An essential characteristic of SOD is that it competes very efficiently with practically all reductive reactions of the available 0; radicals, as revealed by pulse radiolytic techniques [48-501. The consequence of a strongly increased SOD activity, therefore, is a more rapid trans- formation of the 0; radicals into H202, as shown by ESR spin trapping [51].

It is easy to understand that at a higher rate of H202 production the OH’ free radical yield also increases, if sufficient divalent iron is available in the cells. OH’ free radicals are able either to alter the molecular conformation or to cause intermolecular cross-linking. The altered conformation is probably a damaging phenomenon, whereas the cross-linking may also be useful to a certain extent, since it may contribute to the stabilization of subcellular structures. Such an effect may be responsible for the induced cell differentiation in blast-type cells. It should also be stressed that such an interpretation of the effects of Ara-C is not contradictory to any of the results regarding the altered expressions of various genes [3], since all these alterations may represent important intermediary steps of the realization of the differentiatldn mechanism which is induced after all by the OH’ radical flux. It is also relevant to mention here that OH’ radicals deriving from H202 are known to cause single strand breaks in DNA which do not kill the cells [52], and H202 stimulates the expression of cJun genes [53].

The working hypothesis outlined above contains an implicit contradiction against the dogma considering oxygen free radicals as exclusively damaging factors. Some ‘useful’ roles of free radicals have also been suggested by others [54,55]. We believe that the

present experimental findings and numerous other observations support the view that at least a certain portion of OH’ free radicals are directly necessary for cell maturation and differentiation. This concept is more suitable for the interpretation of the great number of contradictory results regarding the role of SOD [12-141. Non-differentiated cells maintain a very low level of OH’ radical production, and undergo differentiation, if the flux of OH’ radicals is increased directly or indirectly. An increased res- piration must be the source of the increased radical flux, through an increased SOD activity. If the cells are already differentiated, a further increase of OH’ free radical formation may be dangerous for their life expectancy, since an exaggerated continuation of the molecular damage and cross-linking results in the aging process. We should like to stress that an increased ability to produce H202 was found also during myeloid cell differentiation [ 15,561. In other experiments a loss of Cu-Zn-SOD gene expression was observed in differentiated cells of myelo-monocytic origin [57], however, the same cells showed an increase of Mn-SOD during their differentiation [581.

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Involvement of Fenton reaction products in differentiation induction of K562 human leukemia cells

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