Cancer Letters, 63 (1992)

151-

151

157

Elsevier Scientific Publishers Ireland Ltd.

Antitumour imidazotetrazines XXVIII 3-methyladenine DNA glycosylase activity in cell lines sensitive and resistant to temozolomide B. Deans and M.J. Tisdale CRC Experimental Chemotherapy Group, Pharmaceutical Sciences Institute,

Aston University,

Birmingham

B4 7ET

(UK) (Received 12 November 1991) (Accepted 10 February 1992)

Summary The

ability of extracts of a range of cell lines

to release methylated buses from a DNA substrate, which had been modified with [3Hjdimethyl sulphate, lines with differing drug,

has been sensitivity

temozolomide.

High

compared

in cell

to the cytotoxic

performance

liquid

chromatography profiles of the bases released by these extracts showed that the actiuity was specific

for 3-methyladenine. There was little oariation in the leoel of 3-methyladenine-DNA

glycosylase despite a

between the different cell lines difference in sensitiuity to

40-fold

temozolomide and no correlation with the level of 06-alkylguanine-DNA alkyltransferase.

Keyworda: imidazotetrazines; temozolomide; 3-methyladenine-DNA-glycosylase; 06alkylguanine-DNA-alkyltransferase Introduction 8-Carbamoyl-3-methylimidazo[5,1-d]-1,2,3, Correspondence to: B. Deans, CRC Experimental apy Group, Pharmaceutical Birmingham

84 7ET,

0304~3835/92/$05.00

Chemother-

Sciences Institute, Aston University,

UK.

0

1992

Printed and Published in Ireland

5-tretrazin-4-(3H)-one (CCRG 81045; temozolomide) shows pronounced antitumour activity against a range of murine tumours [ 11, and is currently showing some success in the treatment of human gliomas. This compound, which can be considered as a prodrug of the linear triazine 5- (3_methyltriazen- l-yl)-imidazole-4-carboxamide (MTIC) , displays crossresistance to cell lines resistant to both triazenes and nitrosoureas [2,3]. A correlation between an ability to repair 06-alkyl-guanine lesions and cytotoxicity has been demonstrated for the imidazotetrazinones [2], triazenes [4] and nitrosoureas [5]. For the chloroethyl analogues, which are capable of cross-linking DNA [6], it is easy to envisage why such a lesion could be cytotoxic. However, it is more difficult to see why 06-methylguanine should be a cytotoxic lesion, although it can clearly be mutagenic 171. Studies with mutant bacterial strains have identified the purines 3-methyladenine and 3-methylguanine in DNA as being responsible for the lethal effects of simple alkylating agents [8]. Further evidence for a role of other alkylated bases has recently been shown for human glioma cell lines where a mutant resistant to the cytotoxic effect of the haloethylnitrosoureas had levels of 3-methyladenine DNA glycosylase activity (SMAG) 2 - 3-fold higher

El sevier Scientific Publishers Ireland Ltd

152

than the sensitive strain in addition to elevated levels of 06-alkylguanine-DNA alkyltransferase (06AGAT). It was suggested that the increased level of the two enzymatic activities was coordinated in some way and in the present study the level of 3MAG has been determined in a range of cell lines, which have been previously characterized for 06AGAT and sensitivity to temozolomide [2]. Materials

and Methods

[3H]Dimethylsulphate ([3H]DMS) (sp. act. 1.2 Ci/mmole) was purchased from Amersham International, Amersham, Bucks, UK. 7-Methylguanine, 3-methyladenine, calf thymus DNA and bovine albumin were purchased from Sigma Chemical Co., Poole, Dorset, UK. Tissue culture medium and foetal calf serum were purchased from Gibco Europe, Paisley, Scotland. Cell culture The Burkitt’s lymphoma cell line, Raji, GM892A (human lymphoblastoma), K562 (human myeloid leukaemia), MAC13 and MAC16 (murine colon adenocarcinoma) were maintained in RPM1 1640 media containing 10% foetal calf serum. A549 (human lung carcinoma) were cultured in Ham’s F12 media containing 10% foetal calf serum. All cells were maintained under an atmosphere of 5% CO2 in air and were passaged twice a week. Preparation of [3H]methylated DNA t3H]DMS-DNA was prepared as described [9]. The DNA was reprecipitated until a constant activity (5000 counts/min per pg) was obtained. The products of the alkylation were determined by HPLC analysis following depurination of the DNA in 0.1 N HCI at 37OC for 16 h. The principal alkylation products were determined to be 7-methylguanine (81%) and 3-methyladenine (19%) with insignificant 06-methylguanine being formed. Preparation o_f cell extracts Each cell line was grown to confluence

or to

the maximum density obtainable in exponential growth and approximately 400 mg of cells were harvested. All steps in the preparation of cell extracts were carried out on ice. The cells were washed with 0.9% NaCl, centrifuged and resuspended in 50 mM Tris, (pH 8), containing 1 mM EDTA, 100 mM NaCI, 0.1 mM phenylmethylsulphonyl fluoride and 0.03 units/ ml of aprotinin. The NaCl concentration was increased to 700 mM by the addition of 5 M NaCl and the cells were sonicated. The NaCl concentration was then adjusted to 300 mM by the addition of 50 mM Tris, (pH 8), containing 1 mM EDTA, 2 mM 2-mercaptoethanol and 0.1 mM phenylmethylsulphonyl fluoride. The solutions were stirred for 2 h at 4OC, centrifuged at 17 200 x g for 2 h and the supernatants aliquoted and stored at - 70°C. Enzyme assay Cell extracts containing 0 - 150 pg of protein were incubated with 3 pg (15 000 counts/min) of DNA substrate in 20 mM Tris, (pH 8.0), 2 mM 2-mercaptoethanol and 60 mM NaCl in a total volume of 100 ~1 for 30 min at 37OC. The reactions were terminated by cooling on ice and the DNA was precipitated by the addition of NaCl-ethanol for 30 min at - 70°C followed by centrifugation for 20 min at 10 000 x g. The radioactivity in the supernatant was determined directly in Optiphase scintillation fluid (FSA Laboratory Supplies, Loughborough, Leics., UK). The radioactivity released by boiled extracts was subtracted from these figures. identification of excised bases by HPLC In order to identify radioactive bases released by cell extracts, the incubation mixture was scaled up 20-fold and the release by extracts containing 700 pg of protein was determined. After incubation the mixture was passed through a DEAE-Sephadex A.25 column (10 x 1 cm) to remove oligonucleotides, lyophilized, dissolved in HPLC buffer (0.5 ml) and 100 ~1 was analysed on a preparative Lichrosorb C-8 column. The column was

153

eluted at 1 ml/min with 100 mM ammonium acetate, (pH 4.25) and 0.25% acetonitrile and 1 ml fractions were collected and the radioactivity determined. Under these conditions retention times for markers, ring-opened 7methylguanine 0 (r-om’G), 3-methyladenine (m3A), 7-methylguanine (m7G) and 06methylguanine (06mG) (following an increase in acetonitrile to 50% after 20 min) were 7.0, 10.5, 20.8 and 35 min, respectively. Profiles of release for the various cell extracts were compared with those of albumin and boiled cell extracts. Results The cell lines employed in this study show a range of sensitivities to temozolomide (Table 1). Extracts from each of the cell lines were incubated with [3H]DMS-DNA and the ability to excise labelled bases was determined. The protein-dependent release of radioactivity after subtraction of the counts released by boiled extracts is shown in Fig. 1. Extracts from all of the cell lines showed release in a proteindependent manner and there appeared not to be any major differences in activity between the various cell extracts despite there being a

60-fold difference in the 06AGAT activity (Table 1). Comparison of the radioactivity released by 700 g of protein extract with the ID,, values for temozolomide for each cell line showed that there was no correlation between 3MAG and the cytotoxicity of temozolomide (Table 1). Thus the Raji cell line with low sensitivity to temozolomide and high levels of 06AGAT activity showed only slightly higher 3MAG than the GM892 cell line with high sensitivity to temozolomide and low levels of 06AGAT activity. The MAC16 cell line with the lowest sensitivity to temozolomide also appeared to have the lowest glycosylase activity (Table 1). Since the [3H]DMS-DNA substrate contained approximately 4-times as much 7-methylguanine as 3-methyladenine, evidence that the release of radioactivity was the result of 3MAG came from comparison of the distribution of bases released by extracts with that released spontaneously in the presence of albumin or by inactivated extracts. The distribution of radioactivity released by Raji and GM892A cell extracts in comparison with those of control experiments is shown in Fig. 2. In these HPLC profiles the first peak represents ring-opened 7methylguanine, the second 3-methyladenine

Table 1. Release of alkylated bases by cell extracts, sensitivity to temozolomide Cell extract

pmoles

[3H]methyl

r-om’G Raji Raji (boiled extract)

17.7 17.1

GM892A A549 MAC13 MAC16 K562 Albumin

18.1 18.0 17.1 19.1 17.7 17.2

l l

f zt f f

released

+ m’G

m3A

1.0 1.4

26.6 13.2

0.8 0.9 0.5 0.4 l 0.3 +z 0.5

and level of 06AGAT”

h&b temozolomide

+ 0.4 zt 0.4

21.0 f 23.7 zt 22.9 f 18.8 l 21.3 f 13.1 f

0.7 0.5 1.5 0.4 1.2 0.1

’ Results are expressed as means f S.E.M. for at least 3 separate b Concentration required to give 50% inhibition of cell growth. ’ From Ref. 2. d From Ref. 10.

160 zt -

(PM)

4

protein)

634 zt 80’ -

7* 2 299 l 30’ 77 f lod 245 f 8’ 15 f 5’ determinations

06AGAT (fmol/mg

10 f 5’ 391 i 60’ 44zJz 4d 320 f 75’ 87 f 40’ for individual

points.

154

1.5

s r E (3r Y

1.0

1 !

0.5

50

100 pg

150

protein

Protein-dependent release of radioactivity from DNA by extracts of Raji (El ), GM892A (A), KS62 (U), MAC13 ( 0). MAC16 (A) and A549 (0) cell lines.

Fig. 1.

155

2.5

-._

RAJI

GM892A

2 I

1.5

P %

1.0

0.5

0.0 10

0

time

time

(min.)

2.5

BOILED

2.5

EXTRACT

2.0

S z ii E f Y

20

(mln.)

ALBUMIN

2.0

S

s g E Z

1.5 3

2

2

1.5

3

0.5

0.0 10

0

time Fi2. 2.

(mln.)

HPLC profiles of bases released

to the total pmoles

[3H]methyl

groups

time

from [3H]DMS-DNA.

released

Peak 1. r-om’G;

by 700 pg of cell extract

peak 2, m’G;

after incubation

20

(min.)

and peak 3, m”A. The results refer

with 60 Fg DNA for 30 min at 37T.

156

and the third 7-methylguanine. The profiles in Fig. 2 show that a constant amount of 7methylguanine is released by the controls and cell extracts and that the variation of radioactivity released is attributable entirely to the 3-methyladenine peak. These results are quantitated in Table I. Discussion Although considerable experimental data has implicated a role for the 06-lesion in the cytotoxicity of imidazo-tetrazinones and nitrosoureas some authors have suggested that alkylations at the 06-position of guanine are not potential cytotoxic lesions and that defects other than the lack of 06AGAT are responsible for the sensitivity of Mer - cell lines to killing by alkylating agents [ll]. Methylphospotriesters and 04-methylthymine appear to be excluded from consideration as targets for lethality as protein fractions containing 06AGAT activity have little capacity to repair these lesions and so it is unlikely that 06AGAT depletion by 06-methylguanine would alter repair of these sites [12]. Little evidence has been put forward to suggest a lethal role for the major DNA alkylation product 7alkylguanine and other than this and the O6 lesion, the only product produced in significant quantities by temozolomide is S-methyladenine [13]. It can be more clearly understood why 3-methyladenine might be a potentially cytotoxic lesion, as blocks to DNA synthesis, introduced into DNA by N-methylN-nitro-N-nitrosoguanidine (MNNNG), and used as an in vitro template for primed synthesis by the polymerase 1 Klenow fragment, occurred most frequently at the position of adenine residues, showing no particular tendency for 06-methylguanine to cause chain termination [ 141. In view of the suggestion [9] that glioma cell lines deficient in 06AGAT were also deficient in 3MAG we have determined the level of 3MAG in a range of cell lines previously characterized for 06AGAT concentrations, and which were shown to correlate with the

cytotoxicity of temozolomide [2]. The results indicate that the 3MAG activities do not vary between these cell lines and that the levels do not correlate with the level of 06AGAT or with sensitivity towards temozolomide, despite a 60-fold variation towards the latter. This suggests that although 3-methyladenine is a major product of temozolomide alkylation of DNA [13] repair enzyme activity is sufficient in most cells to prevent it from being an important cytotoxic lesion. Acknowledgements This work has been supported by a grant from the Cancer Research Campaign. B. Deans gratefully acknowledges the receipt of a research grant from the SERC TT programme. References Stevens, M.F.G., Hickman, J.A., Langdon, S.P., Chubb, D., Vickers, L., Stone, R., Baig, G., Goddard, C., Gibson, N.W., Slack, J.A., Newton, C., Lunt, E., Fizames, C. and Lavelle, F. (1987) Antitumor activity and pharmacokinetics in mice of 8-carbamoyl-3-methyl-imidazo[5.1-d]-1.2,3,5-tetrazin-4(3H)-one (CCRG 81045; M and B 39831). a novel drug with potential as an alternative to dacarbazine. Cancer Res., 47, 5846 - 5852. Tisdale, M.J. (1987) Antitumour imidazotetrazinones XV. Role of guanine alkylation in the mechanism of cytotoxicity of imidazotetrazinones. Biochem. Pharmacol., 36, 457 - 462. Catapano. C.V., Broggini, M., Erba, E., Ponti, M., Mariani, L., Citti, L. and D’lncalci, M. (1987) In vitro and in vivo methazolastone-induced DNA damage and repair sensitive and in ~-1210 leukemia resistant to chloroethylnitrosoureas. Cancer Res., 47, 4884 - 4889. Gibson, N.W., Hartley, J., France, R.J.L. and Vaughan, K. (1986) Differential cytotoxicity and DNA damaging effects produced in a series of the Mer + and Mer phenotypes by a series of alkytriazenylimidazoles. Carcinogenesis, 7, 259 - 265. Scuderio, D.A., Meyer, S.A., Clutterbuck, B.E., Mattern, M.R., Ziolkowski, C.H. and Day, R.S. (1984) Sensitivity of human cell lines having different abilities to repair 06-methylguanine in DNA to inactivation of alkylating agents including chloroethylnitrosoureas. Cancer Res., 44, 2467 - 2474. Tong, W.P., Kirk, M.C. and Ludlum, D.B. (1982) Formation of the cross-link 1 [N-3 deoxycytidyl]-2-[IV- 1 in DNA treated with deoxyguanosinyll-ethane N,N’-bis(2chloroethyl)N-nitrosourea. Cancer Res., 42, 3102 - 3105.

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10

3-Methyladenine DNA glycosylase activity in a glial cell line sensitive to the haloethylnitrosoureas in comparison with a resistant cell line. Cancer Res.. 51, 1568- 1570. Hepburn, P.A. and Tisdale. M.J. (1991) Antitumour

8

11

imidazotetrazines-XXIV. Growth suppresion by DNA from cells treated with imidazotetratinones. Biochem. Pharmacol., 41, 339 - 343. Karran, P. and Williams, S.A. (1985) The cytotoxic and

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