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Cell Growth & Differentiation Vol. 13, 19-26, January 2002
© 2002 American Association for Cancer Research

Control of Differentiation and Apoptosis of Human Myeloid Leukemia Cells by Cytokinins and Cytokinin Nucleosides, Plant Redifferentiation-inducing Hormones1

Yuki Ishii, Yuko Hori, Shingo Sakai and Yoshio Honma2

Saitama Cancer Center Research Institute, Saitama 362-0806 [Y. I., Y. Hor., Y. Hon.], and Institute of Biological Science, Tsukuba University, Tsukuba, Ibaraki 305-8572 [Y. I., Y. Hor., S. S.], Japan


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We examined the effects of various adenine analogues on the growth and differentiation of human myeloid leukemia HL-60 cells. Some of these analogues inhibit growth and induce nitroblue tetrazolium reducing activity in HL-60 cells. Cytokinins such as kinetin, isopentenyladenine, and benzyladenine were very effective in inducing nitroblue tetrazolium reduction and morphological changes in the cells into mature granulocytes. On the other hand, cytokinin ribosides such as kinetin riboside, isopentenyladenosine, and benzylaminopurine riboside were the most potent for growth inhibition and apoptosis. Cytokinin ribosides greatly reduced the intracellular ATP content and disturbed the mitochondrial membrane potential and the accumulation of reactive oxygen species, whereas cytokinins did not. When the cells were incubated with cytokinin ribosides in the presence of O2- scavenger, antioxidant or caspase inhibitor, apoptosis was significantly reduced and differentiation was greatly enhanced. These results suggest that both cytokinins and cytokinin ribosides can induce granulocytic differentiation of HL-60 cells, but cytokinin ribosides also induce apoptosis prior to the differentiation process.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Nucleic acid bases are essential for a wide spectrum of metabolic processes, not the least of which is nucleic acid synthesis. Derivatives of nucleic acid bases and nucleoside/nucleotides play important roles in energization, cell division, senescence, and defense reactions. Cytokinins are important purine derivatives that serve as hormones that control many processes in plants (1) . Cytokinins were discovered as factors that promote cell division in tobacco tissue cultures (2) and have been shown to regulate several other developmental events, such as de novo bud formation, release of buds from apical dominance, leaf expansion, delay of senescence, promotion of seed germination, and chloroplast formation (3) . Naturally occurring cytokinins are mainly adenine derivatives, such as IPA3 and trans-zeatin, and synthetic cytokinins include several adenine analogues, such as kinetin and 6-benzyladenine (4) . Although cytokinins are known to have pronounced effects on plant development, little is known about their precise mechanisms of action. Cytokinins induce callus to redifferentiate into adventitious buds (5) . Callus are clusters of dedifferentiated plant cells that are immortal and proliferate indefinitely in a disorganized manner, just like human cancer cells (6) . Because there are some similarities in the biological phenotypes of cancer cells and callus cells, cytokinins may also affect the differentiation of human cancer cells through a common signal transduction system.

AML cells do not undergo the cell differentiation that normally leads to mature functional blood cells. Instead, they are arrested at immature stages of development, and genes important for myeloid differentiation are often affected by chromosomal changes in AML. The arrest of maturation in myeloid leukemia cells can sometimes be reversed, based on the results of studies of established cell lines and leukemia cells in primary culture. Differentiation has been induced by retinoids, vitamin D3, cytokines, and various chemicals (7) . There are many ways to induce differentiation in leukemia cells, because differentiation inducers act by different mechanisms to induce the production of more or less identical end-stage cells. This may reflect flexibility in the differentiation program. However, there may be common final pathways that mediate maturation in malignant cells by bypassing genetic defects, thus reversing the malignant phenotype. The human myeloid leukemia cell line HL-60, derived from the peripheral blood leukocytes of a patient with AML, can be induced to mature toward either the granulocytic (8, 9, 10) or monocytic (11, 12, 13) phenotype when they are exposed to various compounds. These cells have provided a useful model system for the study of human myelomonocytic cell differentiation.

Regulators that play an important role in the differentiation and development of plants or invertebrates may also affect the differentiation of human normal and malignant cells through a common signal transduction system and might be clinically useful for treating some cancers. Some nucleoside analogues have been found to induce differentiation of several human myeloid leukemia cells (14, 15, 16) and might be effective as differentiation inducers to control AML cells. Modified adenine or Ado derivatives are mainly found in tRNA and originate from cellular RNA breakdown. These derivatives are found in urine taken from both patients with lung carcinoma and healthy subjects (17 , 18) . In this study, we examined the effects of various cytokinins and their derivatives on the proliferation and differentiation of HL-60 cells to determine what structures are involved in the growth-inhibiting and differentiation-inducing effects on the cells and to understand the mode of action of cytokinins in HL-60 cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Effects of Various Adenine Analogues on the Growth and Differentiation of HL-60 Cells.
We examined the abilities of various adenine analogues containing cytokinins to inhibit the growth of HL-60 cells. The cells were cultured with various concentrations of the analogues for 5 days, and concentrations of the analogues that inhibited the number of viable cells by 50% (IC50) were calculated (Table 1)Citation . The growth-inhibitory ability of purine was greater than that of adenine or methyladenine. However, IPA, kinetin, and 6- dimethyladenine were more potent than purine or other adenine analogues in inhibiting cell growth. IPA, kinetin, and benzylaminopurine are well-known cytokinins in plants (19) . Zeatin is also a naturally occurring cytokinin (19) , but its inhibitory effect is much weaker than those of other cytokinins. With respect to growth inhibition of HL-60 cells, the riboside derivatives such as kinetin riboside, IPAR, and benzylaminopurine riboside were more potent than the respective riboside-lacking compounds (Table 1)Citation . On the other hand, adenine was more effective than its nucleosides, Ado and dAdo. Similar results were obtained in other myeloid leukemia ML-1, NB4, and U937 cells (data not shown), indicating that adenine derivatives with cytokinin activity, except zeatin, are potent inhibitors of the proliferation of myeloid leukemia cells.


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Table 1 Effects of adenine analogues on growth of HL-60 cells

Cells were cultured with various concentrations of the analogues for 5 days. Means of three separate experiments are shown. The IC50 is the concentration of compound required for 50% inhibition of cell growth.

 
Next, we examined the abilities of these analogues to induce the differentiation of HL-60 cells (Fig. 1)Citation . NBT-reducing activity, a typical marker of myelomonocytic differentiation, was significantly induced by several adenine analogues. Among the methyladenine analogues, 6-methyladenine was the most potent in inducing NBT reduction of HL-60 cells (Fig. 1A)Citation . Because N6 modification potentiated the adenine analogues in inducing NBT reduction, we examined the effects of various N6-modified derivatives on NBT reduction (Fig. 1B)Citation . Although all of the derivatives except zeatin were very potent in inducing NBT reduction, IPA was the most potent (Fig. 1B)Citation . On the other hand, the differentiation-inducing effects of cytokinin ribosides were much weaker (Fig. 1C)Citation , although the growth-inhibitory activities of cytokinin ribosides were much greater than those of their cytokinins (Table 1)Citation . These results indicate that cytokinin ribosides are highly effective in inhibiting cell growth, whereas cytokinins, except for zeatin, are effective at inducing NBT reduction of HL-60 cells.



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Fig. 1. Effects of various analogues of adenine on induction of NBT reduction in HL-60 cells. Cells were incubated with various concentrations of the compounds for 6 days. Values are means of four separate experiments. A, effects of purine and its derivatives: {diamondsuit}, purine; {triangleup}, 2- aminopurine; {circ}, adenine; {bullet}, 2,6-diaminopurine; {square}, 1-methyladenine; {blacktriangledown}, 3-methyladenine; {blacksquare}, 6-methyladenine, and {diamond}, olomoucine. B, effects of various cytokinins: {blacksquare}, isopentenyl adenine; {bullet}, kinetin; {blacktriangledown}, trans-zeatin; {triangleup}, 6-benzyladenine; {square}, 6-methyladenine; {blacktriangleup}, 6-dimethyladenine; {triangledown}, 6- n-hexylaminopurine; {diamondsuit}, 6-anilinopurine; , 6- methoxyaminopurine; {circ}, adenine. C, effects of cytokinins and cytokinin nucleosides: {blacksquare}, isopentenyladenine; {square}, isopentenyladenosine; {bullet}, kinetin; {circ}, kinetin riboside; {blacktriangleup}, 6-benzyladenine; {triangleup}, 6- benzyladenosine; {diamondsuit}, 2,6-diaminopurine; {diamond}, 2,6-diaminopurine deoxyriboside; {blacktriangledown}, adenine; {triangledown}, adenosine; and , deoxyadenosine.

 
To investigate the differential effects of cytokinins and cytokinin ribosides on the growth and differentiation in HL-60 cells, we examined the morphological changes of cells treated with IPA and IPAR (Fig. 2)Citation . HL-60 cells that had been treated with IPAR for 24 h showed some characteristics of apoptosis (chromatin condensation, nuclear fragmentation, and the formation of apoptotic bodies; Fig. 2BCitation ). On the other hand, IPA induced the morphological differentiation of HL-60 cells into granulocytes without apparent apoptosis after 6 days (Fig. 2C)Citation . The IPA-induced differentiation of HL-60 cells was confirmed by examining other differentiation-associated properties, such as lysozyme activity and CD11b expression (Fig. 3)Citation . IPAR-induced apoptosis was confirmed by Annexin V-FITC binding and DNA ladder formation on gel electrophoresis of the DNA from cells exposed to IPAR (Fig. 4)Citation . Untreated cells showed no Annexin V staining, whereas the cells progressively became Annexin V-positive when treated with IPAR (Fig. 4A)Citation or kinetin riboside (data not shown). Significant DNA fragmentation attributable to internucleosomal cleavage occurred <12 h after exposure to IPAR, but not to IPA, in HL-60 cells (Fig. 4B)Citation . Activation of caspase-3 was observed within 6 h after treatment with IPAR or kinetin riboside but not with IPA or kinetin (Fig. 4, C and D)Citation . Kinetin and IPA significantly induced NBT reduction in other human myeloid leukemia cells, such as ML-1, NB4, and U937 cells, and kinetin riboside and IPAR effectively induced apoptosis of these cells (data not shown). These results show that the effects of cytokinins and cytokinin ribosides are distinguishable from their ability to induce differentiation or apoptosis in human myeloid leukemia cells.



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Fig. 2. Morphological changes in HL-60 cells by IPA and IPAR. Cells were cultured with (B) or without (A) 5 µM IPAR for 1 day. C, cells treated with 0.1 mM IPA for 6 days.

 


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Fig. 3. Effects of IPA on the induction of lysozyme activity (A) and CD11b expression (B) in HL-60 cells. A, cells were cultured with 0 ({blacksquare}), 0.05 ({bullet}), 0.1 ({blacktriangleup}), or 0.15 ({diamondsuit}) mM IPA for various times. B, cells were treated with various concentrations of IPA for 6 days. Means of three separate experiments are shown; bars, SD.

 


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Fig. 4. Induction of Annexin V binding (A) and DNA fragmentation (B) by IPAR. HL-60 cells were cultured with 2.5 µM IPAR for various times or with various concentrations of IPAR for 12 h. C, induction of caspase-3 activity by cytokinin ribosides. Cells were treated with various concentrations of IPA ({blacksquare}), kinetin ({bullet}), IPAR ({diamondsuit}), or kinetin riboside ({blacktriangleup}) for 12 h. D, time course of caspase-3 activation by IPAR. Cells were cultured with 0 ({bullet}), 2 ({blacksquare}), or 4 ({blacktriangleup}) µM IPAR. Means of three separate experiments are shown; bars, SD.

 
Effects of Inhibitors of Ado Receptors on Growth and Differentiation of HL-60 Cells.
Different types of Ado- specific receptors are located on the surface of mammalian cells, and some effects of Ado are mediated by these receptors (20) . We measured the effects of Ado receptor antagonists on growth inhibition and differentiation of HL-60 cells. Extracellular Ado can either bind to specific cell surface receptors A1 and A2, which activate the G-protein/cyclic AMP pathway (21) , or can enter cells through nucleoside transporters (22) and be phosphorylated by Ado kinase. We examined the effects of 3,7-dimethyl-1-propargyl-xanthine, an inhibitor of Ado A2 receptor, and 8-cyclopentyl-1,3-dipropylxanthine, an inhibitor of Ado A1 receptor, on cytokinin-induced growth inhibition and cell differentiation and cytokinin riboside-induced apoptosis and found that they did not have any significant effects on the differentiation and apoptosis induced by cytokinins and cytokinin ribosides (data not shown). Thus, the effects of cytokinin and cytokinin ribosides on HL-60 cells are unlikely to be mediated by extracellular Ado receptors.

Effect of 5'-Amino-dAdo, an Inhibitor of Ado Kinase, on the Growth Inhibition and Differentiation Induced by Kinetin or Kinetin Riboside in HL-60 Cells.
The effects of cytokinins or cytokinin ribosides may be at least partly associated with Ado metabolism. The effect of 5'-amino-dAdo, an inhibitor of Ado kinase (23) , on the growth and differentiation of HL-60 cells treated with kinetin or kinetin riboside was examined. This inhibitor completely counteracted the growth-inhibitory and apoptosis-inducing effects of kinetin riboside (Fig. 5)Citation . 5'-Amino-dAdo also reduced the growth inhibition and differentiation induced by kinetin (Fig. 5, A and B)Citation , although it was less effective than in the case of kinetin riboside-treated cells (Fig. 5C)Citation . These results indicate that the apoptosis- and differentiation-inducing effects of cytokinin ribosides and cytokinins are closely related to Ado metabolism, which requires phosphorylation by Ado kinase.



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Fig. 5. Reduction by 5'-amino-dAdo of the growth-inhibitory (A and C) and differentiation-inducing (B) effects of kinetin (A and B) and kinetin riboside (C). Cells were treated with various concentrations of kinetin or kinetin riboside in the presence of 0 ({diamondsuit}), 1 ({bullet}), or 2 ({blacktriangleup}) µM 5-amino-dAdo for 5 days. The values are means of triplicate determinations; bars, SD.

 
Changes in the Intracellular ATP Content by Kinetin Riboside.
To examine the possibility that the actions of cytokinins and/or cytokinin ribosides may be involved in ATP metabolism, we examined the intracellular ATP content of HL-60 cells treated with IPA and IPAR (Fig. 6)Citation . Intracellular ATP was maintained in cells treated with IPA, whereas the ATP content dramatically decreased within 2 h in cells treated with IPAR, and this decrease was concentration dependent (Fig. 6B)Citation . Similar results were obtained when the cells were treated with kinetin or kinetin riboside (data not shown). These results suggest that the apoptosis-inducing effect of cytokinin ribosides is closely related to ATP depletion.



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Fig. 6. Effects of IPA and IPAR on intracellular ATP levels and mitochondrial membrane potential in HL-60 cells. A, cells were treated with 0.1 mM IPA ({diamondsuit}) or 2 µM IPAR ({bullet}), and ATP contents were measured at the indicated times. B, cells were treated with various concentrations of IPA ({diamondsuit}) or IPAR ({bullet}) for 6 h. Each point represents the ratio of ATP in treated cells to that in untreated cells at the time indicated. C, cells were treated with various concentrations of IPAR ({blacksquare}), IPA ({blacktriangleup}), or kinetin ({bullet}) for 9 h. D, cells were cultured with ({blacksquare}) or without ({bullet}) 4 µM IPAR for various times. Percentages of cells with reduced membrane potential are given. Means of three separate experiments are shown; bars, SD.

 
Mitochondrial Transmembrane Potential and ROS Generation in HL-60 Cells Treated with IPAR.
Because mitochondrial dysfunction has been proposed to represent a point of no return during cell death (24) , cytokinin riboside-induced changes may be closely related to disruption of the mitochondrial membrane potential. IPAR produced a dose- and time-dependent decrease in the mitochondrial transmembrane potential (Fig. 6C)Citation . On the other hand, neither kinetin nor IPA essentially affected the membrane potential (Fig. 6C)Citation . Disruption of the mitochondrial transmembrane potential is usually associated with increased mitochondrial ROS production and mitochondrial membrane damage (25) . To confirm the accumulation of ROS in IPAR-treated cells, we used hydroethidine, a compound specifically converted by ROS to highly fluorescent ethidium, to measure the cellular ROS content by flow cytometry analysis. Fig. 7ACitation shows that treatment of HL-60 cells with IPAR caused a significant increase in ROS, whereas treatment with IPA did not (Fig. 7B)Citation . This ROS accumulation was completely blocked by treatment with 5'-amino-dAdo (data not shown).



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Fig. 7. Effects of IPAR on ROS accumulation and differentiation of HL-60 cells in the presence of O2- scavenger, antioxidant, or caspase inhibitor. ROS accumulation in HL-60 cells treated with IPAR (A) or IPA (B). Cells were treated with 5 µM IPAR or 60 µM IPA for 10 h. Dotted line, untreated cells. C, effect of N-acetyl cysteine (NAC) on morphological changes in IPAR-treated cells. Cells were treated with various concentrations of N-acetyl cysteine in the presence or absence of 5 µM IPAR for 4 days. , apoptotic cells, {blacksquare}, differentiated cells. D, effect of ambroxol on NBT reduction of IPAR-treated cells. Cells were cultured with various concentrations of IPAR in the presence of 0 ({blacksquare}), 10 ({bullet}), or 100 ({blacktriangleup}) µM ambroxol for 4 days. E, effect of caspase inhibitor on the expression of CD11b of HL-60 cells treated with IPAR. Cells were cultured with IPAR in the presence of 0 ({blacksquare}), 1.5 ({bullet}), or 4.5 ({blacktriangleup}) µM caspase inhibitor (FK-011) for 2 days. Means of three separate experiments are shown; bars, SD.

 
Induction of Differentiation of HL-60 Cells by Cytokinin Ribosides Along with Antioxidants and Caspase Inhibitors.
Cytokinins and cytokinin ribosides are similarly effective as plant redifferentiating hormones in most plant cells (19) . Therefore, it is possible that cytokinin ribosides also induce the differentiation of human myeloid leukemia cells when apoptosis is prevented. When HL-60 cells were incubated with IPAR in the presence of the O2- scavenger ambroxol or the antioxidant N-acetyl cysteine, IPAR-induced apoptosis was significantly reduced and differentiation was greatly enhanced (Fig. 7, C and D)Citation . On the other hand, neither ambroxol nor N-acetyl cysteine significantly affected IPA-induced NBT reduction. Morphological changes, CD11b expression, and lysozyme induction in addition to NBT reduction confirmed differentiation. The differentiation-inducing effect of IPAR was also supported by experiments with caspase inhibitors. When HL-60 cells were incubated with kinetin riboside or IPAR in the presence of inhibitors of caspase-3, apoptosis was significantly reduced, and pronounced differentiation was induced (Fig. 7E)Citation .


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Adenine derivatives have various biological and biochemical functions. 1-Methyladenine is a potent inducer of oocyte maturation in star fish (26) , but it is ineffective in inducing the differentiation of human myeloid leukemia cells. CDKs are conserved regulators of the eukaryotic cell cycle, with different isoforms controlling specific phases of the cell cycle. Some adenine derivatives are known to inhibit CDKs. Olomoucine specifically inhibits CDC2, CDK2, and mitogen-activated protein kinases, and IPA is a relatively nonspecific protein kinase inhibitor (27 , 28) . IPA, but not olomoucine, is potent in inducing the differentiation of myeloid leukemia cells. Several inhibitors of protein kinase can induce the differentiation of leukemia cells (29) . These results suggest that the inhibition of protein kinase(s) other than CDC2, CDK2, and mitogen-activated protein kinases may be involved in inducing the differentiation of myeloid leukemia cells. However, the significance of kinase inhibition on plant redifferentiation is not yet understood. Among the adenine analogues tested, cytokinins are the most potent inducers of the differentiation of HL-60 cells, suggesting that there are some common signal transduction pathways between the differentiation of human myeloid leukemia cells and the action of plant redifferentiation-inducing hormone. Interestingly, the effects of zeatin on growth inhibition and differentiation of HL-60 cells were much weaker than those of other cytokinins (Table 1Citation and Fig. 1BCitation ). Zeatin has less of a positive inotropic effect than kinetin or benzyladenine in rat atria (30) . These results suggest that animal cells do not recognize or metabolize zeatin as a regulator of growth and differentiation, because zeatin is a naturally occurring cytokinin that exists only in plants. This implies disparity, rather than commonality, between the mechanisms of action of cytokinins in plants and mammalian cells. Further experimentation is necessary to explain fully these observations.

The growth inhibition of human leukemia HL-60 cells by these adenine analogues was in the order of benzyladenine riboside > kinetin riboside > IPAR >> IPA > kinetin > benzyladenine. Moreover, 6-methyladenine was more potent than 3-methyladenine or 1-methyladenine in inhibiting cell growth and inducing NBT reduction (Table 1Citation and Fig. 1Citation ). These results indicate that the growth-inhibitory activity is enhanced by the transfer of relatively long side-chains such as isopentenyl, benzyl, and furfuryl residues at the N6 position of adenine and greatly enhanced by the addition of ribose or deoxyribose. These modified Ado and dAdo analogues are potent inducers of apoptosis, although Ado and dAdo themselves are not. Cytokinins, such as IPA, kinetin, and benzyladenine, are potent inducers of differentiation; however, their nucleosides did not effectively induce differentiation. Morphological and biochemical examinations revealed that cytokinins induced granulocytic differentiation, and cytokinin ribosides induced apoptosis in HL-60 cells. In plants, cytokinin ribosides have almost the same biological effects as cytokinins. Although the effects of cytokinins on HL-60 cells are clearly different from those of their ribosides, cytokinin ribosides induce granulocytic differentiation in HL-60 cells in the presence of antioxidant, O2- scavenger, or caspase inhibitor. Similar results were obtained when other human leukemia cells were treated with cytokinins or cytokinin ribosides, indicating that these different effects are not restricted to HL-60 cells. Cytokinins may act directly as antioxidants or indirectly as regulators of antioxidants (31, 32, 33) . These properties help explain why cytokinins have differentiation-inducing activity without apoptosis. These results suggest that cytokinin nucleosides have the same differentiation-inducing activity as cytokinins in human leukemia cells, as in the case of plants, and that cytokinin ribosides induce mitochondrial disruption whereas cytokinins protect against mitochondrial disruption and apoptosis in leukemia cells.

5'-Amino-dAdo, a potent inhibitor of Ado kinase (23) , counteracted the effects of cytokinins and cytokinin ribosides on cell growth and differentiation. This inhibitor partially reduced the growth inhibition by cytokinins such as kinetin and IPA, whereas it completely counteracted the growth-inhibitory effects of kinetin riboside or IPAR. Moreover, it also blocked the induction of NBT reduction by cytokinins. On the other hand, Ado receptor inhibitors hardly affected the actions of cytokinins and cytokinin ribosides on HL-60 cells. These findings suggest that the mechanism of action of cytokinins and cytokinin nucleosides is closely related to adenosine metabolism, which requires phosphorylation by adenosine kinase. The causal connection between Ado metabolism and the mechanism of action of cytokinin-induced differentiation remains to be elucidated.

Cytokinin ribosides greatly reduced the intracellular ATP content and mitochondrial membrane potential and induced caspase-3 activity and apoptosis of cells. It has been reported that the conversion of adenosine derivatives by phosphorylation seems to be the most common intracellular mechanism for the transformation of nontoxic compounds into toxic ones (34 , 35) . Mlejnek and Kuglik (36) suggested that the intracellular phosphorylation of benzylaminopurine riboside within cells is necessary for the manifestation of its cytotoxicity. The pathways of the apoptosis induced by cytokinin nucleosides have not yet been described. The generation of ROS has been suggested to be a primary regulatory component, followed by the activation of caspases and caspase-dependent loss of the mitochondrial membrane potential (37 , 38) . However, in the present study, ROS levels significantly increased after the appearance of caspase-3 activation or reduction of the membrane potential and ATP content, suggesting that ROS accumulation might not be involved in the upstream events of cytokinin riboside- induced apoptosis. The previous report by Johnson et al. (37) has unequivocally demonstrated that in p53-induced apoptosis, ROS serves as an early mediator acting upstream of the changes in mitochondrial membrane potential, as well as the activation of caspase-9, which is thought to act earlier than caspase-3. ROS levels significantly increased after the appearance of caspase-3 activation or reduction of the mitochondrial membrane potential and ATP. Therefore, there is a possibility that apoptosis might well be triggered by a slight accumulation of ROS, long before one could recognize its significant increase, in the cytokinin riboside-induced apoptosis.

Cotylenin A has been isolated as a plant growth regulator from the metabolites of a simple eukaryote and exhibits cytokinin-like activity (39) . Although it is structurally different from cytokinins, it also induces the differentiation of human myeloid leukemia cells (13) . These results suggest that there is an association between the action of plant redifferentiation-inducing hormones and the mechanisms of differentiation of human leukemia cells, although there may not be the same signal transduction pathways between them.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
All of the cytokinins and other adenine analogues, RPMI 1640, N-acetyl cysteine, ambroxol, and NBT were purchased from Sigma Chemical Co. (St. Louis, MO), and luciferin and luciferase were purchased from Wako Chemicals (Osaka, Japan). Inhibitors of caspase-3, Z- Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK (FK-010) and BOC-Asp(OMe)-CH2F (FK-011), were obtained from Enzyme System Products (Livermore, CA).

Cell Line and Cell Culture.
The HL-60 cell line, derived from a patient with acute promyelocytic leukemia (8) , was maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 80 µg/ml gentamicin at 37°C in a humidified atmosphere of 5% CO2 in air. The other human leukemia cells were also cultured under the same conditions (40) .

Assay of Cell Growth and Properties of Differentiated or Apoptotic Cells.
Suspensions of cells (0.5 x 105 cells/ml) in 2 ml of culture medium were incubated with or without the test compounds in multidishes (Costar, Cambridge, MA). Cell numbers were counted in a Model ZM Coulter Counter (Coulter Electronics, Luton, United Kingdom). Superoxide-generating oxidase was determined by the ability of the cells to reduce NBT upon exposure to 12-O-tetradecanoylphorbol-13-acetate (15) . Cells were incubated in 1 ml of RPMI 1640 containing NBT (1 mg/ml) and 12-O-tetradecanoylphorbol-13-acetate (100 µg/ml) at 37°C for 50 min. The reaction was stopped by adding 5 M HCl (1 M, final concentration). The suspension was kept at room temperature for 20 min and then centrifuged. Formazan deposits were solubilized in DMSO, and the absorption of the formazan solution at 560 nm/107 cells was measured in a spectrophotometer. Lysozyme activity was determined by the lysoplate method described previously (15) . The expression of CD11b was analyzed by indirect immunofluorescent staining and flow cytometry using an Epics XL flow cytometer (Beckman Coulter) as described elsewhere (40) . Morphological changes were examined in cell smears stained with May-Grünwald-Giemsa solution.

Annexin V Binding.
Cells were labeled with FITC-labeled Annexin V (Genzyme Co., Cambridge, MA) for 30 min on ice, as described previously (41) . FITC-conjugated murine IgG monoclonal antibodies of unrelated specificity were always used as controls. After staining, cells were washed and analyzed by flow cytometry.

Assay for Caspase-3 Activity.
Caspase-3 activity was assayed with the fluorogenic substrate acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-MCA; Peptide Institute, Inc., Osaka, Japan), as described previously (42) . Enzyme activity was expressed as pmol aminomethylcoumarin/min/mg protein.

DNA Fragmentation Assay.
After exposure to drugs, cells were collected and lysed in DNA isolation buffer [10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.65 M NaCl, 1% SDS, and 0.2 mg/ml proteinase K]. DNA was extracted with phenol/chloroform and then precipitated with ethanol. After treatment with 0.1 mg/ml RNase A for 1 h at 37°C, equal amounts of DNA were analyzed by electrophoresis on 1.5% agarose gels stained with ethidium bromide.

Determination of Intracellular ATP Content.
Suspensions containing 1 x 107 cells were washed three times with cold PBS. The cells were centrifuged at 1,000 x g for 5 min. The supernatant was discarded, and 1 ml of 10% (w/v) perchloric acid was added. The mixture was allowed to stand for 20 min at 4°C. The cell lysates were centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was neutralized with 1 M KOH and then centrifuged at 10,000 x g for 10 min at 4°C. The clarified sample (100 µl) was mixed with 100 µl of luciferase-luciferin reagent. Luminescence from the reaction at room temperature was measured with a luminometer. The blank was subtracted from the raw data, and the ATP concentration was determined from a log plot of the standard curve data.

Measurements of Mitochondrial Transmembrane Potential and Intracellular ROS.
Mitochondrial membrane potential was quantitated by the flow cytometric analysis of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide)-stained cells. We assayed using a DePsipher kit (Trevigen, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. For measurement of ROS, cells were incubated with 20 ng/ml hydroethidine (Molecular Probes, Inc., Eugene, OR) in PBS at 37°C, washed twice with PBS, and then analyzed as soon as possible by flow cytometry using the red laser channel. This assay is based on the chemical properties of hydroethidine, a weak blue fluorescent dye that is selectively converted by O2- to ethidium with a bright red fluorescence (43) .


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, Technology, Sports and Culture and by a grant for Cancer Research from the Ministry of Labor, Health and Welfare, Japan. Back

2 To whom requests for reprints should be addressed, at Saitama Cancer Center Research Institute, 818 Komuro, Ina, Saitama 362-0806, Japan. Phone: 81-48-722-1111; Fax: 81-48-722-1739; E-mail: honma{at}cancer-c.pref.saitama.jp Back

3 The abbreviations used are: IPA, isopentenyladenine; AML, acute myeloid leukemia; Ado, adenosine; dAdo, 2'-deoxyadenosine; IPAR, isopentenyladenosine; ROS, reactive oxygen species; CDK, cyclin-dependent kinase; NBT, nitroblue tetrazolium. Back

Received for publication 7/ 3/01. Revision received 11/ 5/01. Accepted for publication 11/12/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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