Leukemia Research Vol. 13, No. 8, pp. 715-722, 1989. 0145-2126/89 $3.00 + .00 Printed in Great Britain. Pergamon Press plc

M E C H A N I S M OF A C T I O N OF 5-AZA-dC: I N D U C E D D N A H Y P O M E T H Y L A T I O N D O E S NOT L E A D TO A B E R R A N T G E N E

E X P R E S S I O N IN H U M A N L E U K E M I C CEM CELLS

Institut national de la recherche

JACQUES BOUCHARD

scientifique-Sant6, 245 Hymus Boulevard, Pointe-Claire, Qc H9R 1G6, Canada

(Received 27 February 1989. Accepted 1 April 1989)

Abstract--To understand the mechanism of action of 5-AZA-2'-deoxycytidine, a potent antineoplastic agent, we studied its effect in CEM cells on DNA methylation while monitoring coinciding changes in DNA, RNA and protein synthesis. At concentrations near the inhibitory concentrations0, the drug induced a profound reduction of DNA methylation. Effects on DNA and RNA synthesis were also noted at 24 and 48 h of treatment. However, at all concentrations assayed, no change in polypeptide composition as monitored by 2-D gel electrophoresis, were observed. That the cytotoxic effect of 5- AZA-deoxycytidine is due to changes in gene expression induced by DNA methylation reduction is not supported.

Key words: 5-AZA-deoxycytidine, cytotoxicity, methylation, mechanism of action, gene expression.

I N T R O D U C T I O N

5-AZA-DEOXYCYTIDINE (5-AZA-dC) is a triazine base analog of deoxycytidine that is a very potent antileukemic agent in vitro and in vivo [1]. It has recently been introduced in clinical trials against leu- kemia and various other human neoplasia [2-5] and is currently undergoing a phase II clinical trial in refractory stage I I I / IV Hodgkin's lymphoma (EORTC) . Although 5-AZA-dC has been used with some success in human cancer, its mechanism of action remains largely unknown. One requirement for its lethal action is its conversion into a nucleotide triphosphate derivative which is efficiently incor- porated into D N A [6], suggesting that the presence of the fraudulent base 5-AZA-cytosine may hamper some important D N A functions. 5-AZA-dC has not been reported to be mutagenic in mammalian cells [7, 8]; however, the drug is unstable in solution [9]. 5-AZA-cytosine ring scissions are produced in neu-

Abbreviations: 5-AZA-dC, 5-AZA-2'-deoxycytidine; 5- AZA-C, 5-AZA-cytidine; PBS, phosphate buffered saline; TCA, trichloroacetic acid; HPLC, high performance liquid chromatography; 2-D, 2-dimensional; FCS, fetal calf serum; SDS, sodium dodecyl sulphate; SEM, standard error of the mean; ~c50, inhibitory concentrations0; HEPES, (N- [2-hydroxyethyl]piperazine-N'-[2-ethanesul- fonic acid]).

Correspondence to: Dr Jacques Bouchard, Centre de recherche p6diatrique, H6pital Ste-Justine, 3175 C6te Ste- Catherine, Montr6al, Qu6bec H3T 1C5, Canada.

715

tral and basic solutions which suggest that chromo- somal breaks are induced following the incorporation of the abnormal base [10]. Results of D N A alkaline elution experiments realized on 5-AZA-dC treated cells support such a possibility but given the harsh alkaline conditions used in these assays, the con- clusions that can be drawn from these experiments are limited [11]. Another mechanism of action of the analog could involve its powerful inhibitory effect on the level of cytosine methylation into DNA, which has previously been correlated with its cytotoxic action [12]. Since D N A methylation is one of the numerous factors that regulate gene expression [13, 14], it may well be that the hypomethylating action of 5-AZA-dC is responsible for cell toxicity through inappropriate gene expression or induction of cell phenotypic transformation [12, 15].

On the other hand, resistance to 5-AZA-dC has been attributed mainly to a deficiency of cellular deoxycytidine kinase activity [16, 17], although Flatau et al. [18] were able to isolate, under a repeti- tive drug exposure regimen, different clones of 5- AZA-dC resistant cells in which the enzyme is present and active. These cells actively incorporated 5-AZA-dC into DNA, which was hypomethylated to a certain extent, and differences in nuclear binding proteins were identified which could confer cell resist- ance to the drug [19]. As it was impossible to further reduce the level of D N A methylation of these cells, it can be speculated that minimal D N A methylation

716 JACQUES BOUCHARD

is compat ib le with cell survival. These observat ions support the possibility of a direct relat ion be tween 5- A Z A - d C cytotoxici ty and 5 - A Z A - d C induced hypomethy la t ion . By contras t , W o o d c o c k et al. [20] were able, for a different cell line, to isolate r a n d o m clones f rom long- te rm t rea ted cells that exhibi ted only 50% of the methyla t ion of the parental cell lines. However , they did not find increased resistance to 5- A Z A - d C in these cells.

In the present s tudy, to fur ther elucidate the mol- ecular mechan i sm of act ion of 5 - A Z A - d C and to ascertain the role of the D N A hypomethy la t ion activity of the drug in its lethal act ion we have fol- lowed gene expression in h u m a n C C R F - C E M leu- kemic cells exposed to toxic 5 - A Z A - d C molar concentra t ions , by 2-dimensional prote in gel elec- t rophoresis . We were expect ing drastic changes in polypept ide compos i t ion in 2-D gel e lect rophores is which would reflect modif icat ions in gene expression following the hypome thy l a t i on of the D N A or at the very least, we expected specific changes in gene expression (such as H e a t Shock Proteins) that we could isolate and identify. In contrast , we found no changes in po lypep t ide synthesis after 5 - A Z A - d C t rea tment at cytotoxic dosages.

M A T E R I A L S A N D M E T H O D S

Cell lines and maintenance Human leukemic CCRF-CEM cells were obtained from

the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Flow Laboratories, Missis- sauga, Ont.) and 25 mM HEPES. Cells were passaged twice a week, at an initial density of 105 cells/ml. In these conditions, cells were kept in logarithmic phase of growth at a cell population doubling time close to 24 h.

Genomic 5-methylcytosine determinations Cultured cells were recovered by centrifugation

(600 x g, 5 min), washed in phosphate buffered saline (PBS) at 4°C and resedimented. The DNA was precipitated according to already published techniques [18] and was then hydrolyzed to bases with 90% formic acid in sealed ampules at 180°C (30 rain). Formic acid was then evap- orated under a nitrogen stream and hydrolyzates were suspended in the HPLC eluent buffer (75 mM Ammonium phosphate, pH 2.8). Bases were separated by HPLC using a Whatman SCX column (250 x 4 mm). Spectrophoto- metric quantitation of the eluates was performed at 280 nm using a HP3392A integrator.

Radioisotope incorporation Methionine-free RPMI 1640 supplemented with 1% FCS

to avoid methionine depletion, was used in total cell protein labeling with [~SS]methionine or [~H]leucine (ICN, Mon- treal, PQ).

[3H]thymidine and [3H]uridine (ICN, Montreal, PQ) labeling of synthesized nucleic acids was conducted in the complete medium with just 1% added FCS. Radioisotope

incorporations were monitored by lysing and fixing cells onto Whatman GF-A filters, washing them in tri- chloroacetic acid (TCA) (10%) and then counting them by liquid scintillation spectrometry. Labeling duration times were carefully chosen so as to stay in the linear range of radioisotope incorporation.

2-Dimensional gel electrophoresis 2-D gel electrophoresis was performed according to Bio-

Rad Technical Bulletin 1122 included with the Bio-Rad Protean II electrophoresis apparatus. Cells were harvested by low speed centrifugation and rinsed in PBS before they were lysed in an Ampholine (LKB, Montr6al, PQ)-based lysing solution. First, isoelectric focusing in a thin tube gel (2.5 mm) was run at 800 V overnight. Then, at the end, the carefully extruded gel was equilibrated and layered oi1 top of a SDS-slab gel and submitted to electrophoresis at 50mA per gel for 6h. 2-D slab gels were fixed in a methanol : acetic acid : water solution (40 : 10 : 50) and sub- merged in Enlightning (NEN, Montr6al, PQ) before they were dried down on a piece of Whatman 3MM paper and placed against a Kodak XAR-5 film for fluorography.

Analysis of polypeptide composition and synthesis by [3SS]- methionine labeling of cells

[35S]methionine was used to label the newly synthesized polypeptides in a short period of 2 h. Then the ceils were lysed in an isoelectric lysing solution and applied on the top of a thin tube to be submitted to isoelectric focusing overnight. Extruded gel was then applied over the top of the SDS-slab gel and electrophoresed and treated as described previously. Results were expressed as the fluorographs obtained after exposure of radiographic films to the dried gels.

Statistical analysis" The results of labeling studies have been expressed as

linear regression coefficients calculated from 3 h incor- poration determinations +- S.E.M. The significance of the regression coefficients was tested using the t' test described by Sokal and Rohlf [21]. Differences were considered significant at the p < 0.05 level.

R E S U L T S

Determination of the inhibitory concentrationkso

To determine the useful concent ra t ions of 5 - A Z A - dC at which we could look for potent ia l cytotoxic effects in C E M cells, a prel iminary set of exper iments was done to measure the inhibi tory concentrat ions0 0c50) of the analog at 48 h of cell growth. Based on 10 determinat ions , a molar concent ra t ion of 0.46 + 0.06 ~tM of 5 - A Z A - d C (mean +_ S .E .M. ) was established as the Ics0 at 48 h.

We therefore choose to moni tor , at var ious times, the cytotoxic events occurr ing at mola r concen- trations of 5 - A Z A - d C close to the res0 at 48 h. C E M cells were exposed to molar concent ra t ions of 0.1, 1.0 and 10~tM of 5 - A Z A - d C and were carefully assayed at 4, 24 and 48 h for D N A , R N A and protein synthesis using trit iated thymidine, uridine and leu- cine or [35S]methionine. The percentage of methyl-

Mechanism of action of 5-AZA-deoxycytidine 717

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FIG. 1. Effects of different molar concentrations of 5- AZA-dC on [3H]thymidine incorporation in CEM cells (500,000cells/ml). The rate of incorporation of [3H]thymidine was monitored at 4 h (panel A), 24 h (panel B) and 48 h (panel C). The results are expressed as linear regression coefficients calculated from 3 h incorporation determinations -+ S.E.M. Differences are considered

significant at the p < 0.05 level.

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5-AZA-dC (p,M) FiG. 2. Effects of different molar concentrations of 5-AZA- dC on [3H]uridine incorporation in CEM cells (500,000 cells/ml). The rate of incorporation of [3H]uridine was monitored at 4 h (panel A), 24 h (panel B) and 48 h (panel C). The results are expressed as linear regression coefficients calculated from 3h incorporation deter- minations _+ S.E.M. Differences are considered significant

at the p < 0.05 level.

ated cytosine in D N A was also ascertained by H P L C and gene expression was moni tored by 2-D gel elec- trophoresis of labeled cell lysates.

Incorporation of [3H]thymidine Tritiated thymidine incorporat ion into control and

5 -AZA-dC treated C E M cells is shown in Fig. 1. Thymidine incorporat ion was measured each hour for 3 h by T C A precipitation of C E M cell nucleic acids onto G F - A filters. As shown in panel A, no significant changes in the rate of thymidine incor- porat ion were noted at 4 h of t reatment . By 24 h of t reatment with 5 -AZA-dC, a small reduction in the rate of thymidine incorporat ion was noted at 10 ~tM as shown in panel B. A significant increase (p < 0.05) in the rate of thymidine incorporat ion was con- sistently observed at 24 h of t rea tment with a 0.1 #M concentrat ion of 5 -AZA-dC. Panel C shows the results obtained after 48 h of t rea tment with the

drug. A significant reduction was observed at 1.0 and 10.0 ~tM of 5-AZA-dC.

Incorporation of [3H ]uridine Tritiated uridine incorporat ion measured into con-

trol and treated cells is shown in Fig. 2. At 4 h, no changes in the rate of tritiated uridine incorporation were noted (Panel A). However at 24 h, a significant reduction (? < 0.05) was observed for the 1.0 and 10.0 #M concentrations of 5 -AZA-dC (Panel B). The rate at which the tritiated uridine is incorporated into CEM nucleic acids was also reduced for the three molar concentrations of 5 -AZA-dC assayed at 48 h, reduced to less than 50% of the rate measured in control cells for the cells t reated at 10 ~tM (Panel C).

Incorporation of [35S]methionine and [3H]leucine Protein synthesis as moni tored by [35S]methionine

and [3H]leucine incorporation at 24 and 48 h of 5- A Z A - d C t reatment has also been investigated. The

718 JACQUES BOUCHARD

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FIG. 3. Effects of different molar concentrations of 5- AZA-dC on [35S]methionine incorporation in CEM cells (500,000cells/ml). The rate of incorporation of [35S]methionine was monitored at 24 h (panel A) and 48 h (panel B). The results are expressed as linear regression coefficients calculated from 3h incorporation deter- minations -+ S.E.M. Differences are considered significant

at the p < 0.05 level.

results of the [35S]methionine study are given in Fig. 3. The results obta ined at 24 and 48 h for the rate of the [35S]methionine incorporat ion indicate a signifi- cant reduction in isotope incorporation for the cells t reated at 1.0 and 10.0 ~tM of 5 -AZA-dC (shown in panels A and B). No significant changes in the rate of tritiated leucine incorporat ion were noted at any molar concentrat ions of 5 -AZA-dC (results not shown).

Effects of 5-AZA-dC on DNA methylation The effect of C E M cell t rea tment with 5 -AZA-dC

on D N A methylat ion were also measured simul- taneously in separate cell culture plates using the H P L C method of Flatau et al. [18]. Results of these determinations of the percentage of 5-methyl- cytosines relative to the total cytosine content of t reated C E M cell D N A , are presented in Table 1. At 0.1 and 1.0~tM of 5 -AZA-dC, a profound reduction in the percentage of 5-methylcytosine, rela- tive to untreated cells was noted.

The reduction was more important at 48h and reached the threshold levels repor ted for other cell lines after 5 - A Z A - d C t reatment .

Two-dimensional gel analysis Polypeptide composit ion and expression by control

TABLE 1. EFFECT OF DIFFERENT MOLAR CONCENTRATIONS OF 5-AZA-dC ON TIlE PERCENTAGE OF 5-METHYLCYTOSINE

CONTENT OF G E M CELL DNA

Percentage of 5-MC*

Untreated 4.18 -+ 0.157 0.1 p.M 3.78 +- 0.25 1.0 ~tM 1.88 -+ 0.31

* Calculated from integration of cytosine (C) and 5- methylcytosine (5-MC) HPLC peak eluate using the fol- lowing mathematical formula:

5-MC × 100%.

5-MC + C

f Mean +- S.E.M., n = 3.

and 5 -AZA-dC treated CEM cells was also moni- tored using 2-D gel electrophoresis. Fluorographs obtained after exposure of radiographic films to the dried gels, after 4, 24 and 48 h of t rea tment at all the different 5 -AZA-dC molar concentrations did not reveal any change in position nor in the number of identifiable spots in the radiofluorographic patterns. This is illustrated in Fig. 4 for 1 #M 5-AZA-dC. There are, however, some changes in intensity of the labeling. These changes did not appear to be due to the duration of t reatment nor the concentrat ion of 5- AZA-dC . Given that we have counted more than 300 different spots on the fluorographs when we overexposed the films in the presence of the dried gels, we assume that less than 0.3% of the poly- peptide composition is modified in CEM cells after 5 -AZA-dC treatment.

D I S C U S S I O N A N D C O N C L U S I O N

The purpose of this research was to further under- stand the mechanism of action of the potent cytotoxic action of 5 -AZA-dC by monitoring gene expression in t reated cells and at the same time, the inhibition of D N A methylat ion produced by 5 -AZA-dC to see if a correlation between them, could be established. Previous reports had shown that 5 -AZA-dC like 5- AZA-cyt id ine [5-AZA-C] induced expression of

FIG. 4. Effect of 5-AZA-dC treatment on CEM cell gene expression as examined by 2-D gel electrophoresis. CEM cells were treated for 4 (panel a), 24 (panel b) and 48 h (panel c) with 1 ~tM 5-AZA-dC and labeled with [35S]methionine for 2 h. Cell lysates were processed as described in the Materials and Methods section. Control

cells were also labeled (panel d).

D

FIG. 4(a) and (b).

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FIG. 4(c) and (d).

720

Mechanism of action of 5-AZA-deoxycytidine 721

various genes and cell differentiation in treated cells [22]. It was therefore appealing to try to associate inappropriate gene expression and/or cell differ- entiation with the cytotoxicity of 5-AZA-dC [12]. Our results indicate that molar concentrations of 5- AZA-dC just below the I¢50 have either no or very limited effects on DNA, R N A and protein synthesis in treated CEM cells although they significantly reduce the level of 5-methylcytosine in DNA. Higher molar concentrations of 5-AZA-dC have pronounced effects on DNA, R N A and protein synthesis of treated CEM cells, producing a marked reduction of all these parameters after 48 h of treatment. A profound reduction of the level of 5-methylcytosines is also noted. However , none of the molar con- centrations of 5 -AZA-dC tested nor up to 48 h exposure to the drug produced a change in gene expression at a level of detection that is estimated to be less than a 0.3% change. This level of detection was considered to be sufficient to detect the major changes in gene expression expected from a drastic decrease in D N A methylation. Consequently, it is surprising that no expression of genes such as Heat Shock Proteins that could be expected induced when cells are under stress [23], nor the induction of any abnormal or aberrant polypeptides was observed. The results indicate that it is very unlikely that significant changes in gene expression caused by a reduction in DNA methylation are involved in the cytotoxic action of 5-AZA-dC. Using a different human cell line, HL-60, it has been shown that treat- ment with 5-AZA-C induces changes in gene expression which are readily observable by 2-D gel electrophoresis [24]. We were able to repeat these observations in our system (results not shown), further validating the observations made in CEM cells. However , 5 -AZA-C is principally incorporated into RNA and interferes with RNA processing and protein synthesis, although it could also inhibit DN A methylation [22, 25]. Changes seen in gene expression in HL-60 cells induced by the 5-AZA-C treatment may have been due to long-range R N A related effects of the drug. Fur thermore , the changes observed in HL-60 cells are related to the changes obtained after methionine depletion [24]. Never- theless, we have already shown that 5-AZA-dC treat- ment of HL-60 cells is able to induce cell differentiation and to produce significant mor- phological changes at dosages which reduce D N A methylation [26]. However , in a manner similar to the previously reported CEM cell experiments, we have not been able to distinguish any changes in polypeptide composition in HL-60 cells t reated with the same range of molar concentrations of 5-AZA- dC by 2-D gel electrophoresis (data not shown). The

changes picked up by immunofluorescent assay with a monoclonal antibody must have not been detect- able by 2-D gel electrophoresis analysis. Therefore we cannot exclude the possibility that minor and very specific changes in expression were induced by 5- AZA-dC but may not have been detected in CEM cells. On the other hand, the results of our study are in agreement with the observations of Levva et al.

[27] in HL-60 cells resistant to cell differentiation, which did not show a direct relationship between the potent cytotoxic action of 5-AZA-dC and cell differentiation induction. The results are also con- sistent with the observations of Adams et al. [28] with the L929 murine cell line into which 5-AZA-dC induced hypomethylation and cytotoxicity did not correlate.

In conclusion, since 5-AZA-dC is easily incor- porated into D N A [6] and does not perturb the normal DNA, RN A and protein synthesis at con- centrations which profoundly reduce the 5-methyl- cytosine content of the treated cell DNA, the cytotoxic effect of the drug and changes in gene expression induced by it, can clearly be dissociated. It is tempting instead to associate the cytotoxic action of the drug with the instability of the 5-AZA-cytosine ring in solution as suggested by others [11]. Cell death would then result from exhaustion of D N A repair mechanisms and induction of apoptosis by a series of events common to both ionizing radiations and other cytotoxic drugs [29].

Acknowledgements--This work was supported by a grant from l'Institut National de la Recherche Scientifique. The author thanks Mrs Jacinthe Latulippe for her technical assistance, and Drs J. M. Leclerc, Y. Thdor~t and M. C. Walker for critical reading of the manuscript.

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