Mutation Research, 233 (1990) 23-30 23 Elsevier

MUT 02852

Regulation of O6-methylguanine-DNA methyltransferase expression in the Burkitt's lymphoma cell line Raji

Peter Ka r r an , Claire S tephenson , Sarah C a i r n s - S m i t h a n d Pe ter M a c p h e r s o n

Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hefts. EN6 3LD 'Great Britain)

(Received 16 November 1989) (Accepted 17 January 1990)

Keywords: O6-Methylguanine-DNA methyltransferase; Burkitt's lymphoma cell line Raji; DNA-repair enzyme; Thymidine kinase; Galactokinase

Summary

We have investigated the expression of the DNA-repair enzyme O6-methylguanine-DNA methyltrans- ferase in the Burkitt's lymphoma cell line Raji. An existing mutant Raji cell line which lacks thymidine kinase activity had previously been shown to be Mex- and to no longer express O6-methylguanine-DNA methyltransferase. We report here that in addition to the methyltransferase and thymidine kinase, a third enzyme with an unrelated function, galactokinase, is also not expressed in Raji cells. The control of thymidine kinase expression is post-transcriptional and it is possible that galactokinase and methyltrans- ferase can share a common post-transcriptional' regulation with thymidine kinase.

Human cells express a DNA-repair enzyme O6-methylguanine (m6-Gua)-DNA methyltrans- ferase, as one of the major defensive strategies against the cytotoxic and mutagenic effects of alkylating agents. In some cell lines (designated Mex-) expression of this enzyme has been lost and Mex- cells are sensitive to killing and muta- tion induction by alkylating (particularly methyl- ating) agents (Day et al., 1980; Sklar and Strauss, 1981; Domoradzki et al., 1984). The sensitivity of Mex- cells is apparently a direct consequence of the absence of an active methyltransferase since it can be corrected by expression of either the ho- mologous E. coli enzyme (Brennand and Margi-

Correspondence: Dr. P. Karran, Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD (Great Britain).

son, 1986; Samson et al., 1986; Ishizaki et al., 1986; Kataoka et al., 1986) or of a truncated version which retains only the ability to repair m6-Gua (and O4-methylthymine) (Ishizald et al., 1987; Hall et al., 1988). A characteristic property of Mex- cell lines is their origin in transformed material: either from tumor biopsies or via trans- formation (Day et al., 1980, 1987) or immortalisa- tion in vitro (Green et al., 1990). The reason for the loss of methyltransferase expression is un- known. The frequency at which Mex- cell lines arise in vitro is too high to be explained by muta- tion and other possible explanations; for example a selective advantage for Mex- cells in tissue culture or an enhanced susceptibility of Mex- cells to transformation, remain largely untested experimentally. The intimate connection of the phenotype with cellular transformation suggests that down-regulation of this important DNA-re-

0027-5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

24

pair enzyme may provide a model for gene regu- lation following transformation and we have been investigating one way in which its expression may be controlled.

The Burkitt's lymphoma cell line Raji, is Mex ÷ whereas a derivative line, Raji TK- is Mex- (Karran, 1985). The simultaneous absence of ex- pression of these two enzymes originally seemed coincidental since there is no obligatory connec- tion between the methyltransferase (mt) and thymidine kinase gene (tk) expression and a num- ber of Mex- human cell lines are TK ÷. Recently, however, we have observed two other examples of this relationship; the first in a human lymphob- lastoid cell line derived from lymphocytes of a normal female (GM1953) and the second in a fibroblast cell line derived from a sun-sensitive individual (46BR) (P. Karran, C. Stephenson, P. Macpherson, S. Cairns-Smith and A. Priestley, Cancer Res., in press). In both of these cell types, the Mex phenotype was unstable and switching occurred between the Mex- and Mex ÷ states. The conversion to Mex- was accompanied in both cases by a simultaneous loss of expression of two apparently unrelated enzymes: thymidine kinase, a pyrimidine salvage enzyme, and galactokinase, the enzyme which catalyses the first step in the metabolism of galactose. In GM1953, the changes in expression of the three enzymes were reversible and coordinate and expression of methyltrans- ferase, galactokinase and thymidine kinase could be altered in a reproducible fashion by altering the conditions under which the cells were propagated. The changes in enzyme levels in GM1953 were abrupt and were not compatible with the selection and colonization of the culture by a limited num- ber of spontaneous mutants with a selective growth advantage.

In contrast to the easy reversibility of the changes in enzyme synthesis in GM1953, the ab- sence of methyltransferase and thymidine kinase expression in Raji Mex- is apparently stable and we have never observed a 'reversion' to a Mex+/TK + state in this cell line. However, in view of the coordinate loss of methyltransferase and thymidine kinase activity we examined the properties of the Mex- and Mex + populations of Raji cells in order to determine whether the regu- lation of methyltransferase and thymidine kinase

in these cell lines was a similar phenomenon to their joint silencing in GM1953 cells.

Materials and methods

Cells and cell culture The Burkitt's lymphoma cell line Raji, obtained

from the American Type Culture Collection, Rockville, MD, and its TK- derivative (Hampar et al., 1972) were maintained in RPMI 1640 tissue culture medium supplemented with 15% fetal calf serum.

Enzyme assays Cells were harvested by centrifugation, washed

in phosphate-buffered saline, and extracted in Ex- traction Buffer (50 mM Tris • HC1, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol, 0.2% Triton X-100) at a concentration of 108 cells/ml. Debris were removed by centrifugation in an Eppendorf centrifuge and the supernatant used to measure methyltransferase, thymidine kinase and galac- tokinase activities. Protein concentrations were de- termined by the method of Bradford (1976).

m6-Gua-DNA methyltransferase activity was measured as previously described (Harris et al., 1983) using a partially depurinated [3H]MNU (Amersham International)-treated DNA substrate with a specific activity of 29 Ci/mmole. One unit of methyltransferase demethylates I pmole m6-Gua in the standard reaction.

Thymidine kinase was assayed as described (Karran et al., 1977) using [3H]thymidine (Amersham International) and unlabelled thymi- dine (Sigma) with a final specific activity of 0.2 Ci/mmole. One unit of thymidine kinase phos- phorylates 1 nmole thymidine per minute under standard conditions.

Galactokinase activity was measured essentially by the method of Soni et al. (1988). Briefly, the assay measured the conversion of [14C]galactose (Amersham International, 60 Ci/mole) to galac- tose 1-phosphate in a reaction mixture (50 /tl), comprising: 0.2 M Tris. HC1 pH 7.8, 6 mM ATP, 6.4 mM MgC12, 3.2 mM NaF, 1 /~Ci/ml [a4C]galactose, 0.29 mM unlabelled galactose (< 0.01% glucose, Sigma). The final specific activ- ity of the [laC]galactose was 5 Ci/mole. Reactions were initiated by addition of cell extract (0-100

/~g in < 6/tl) and transfer to 37 o C. After incuba- tion for 15 rain, reactions were terminated by heating for 1 min at 70°C and 10-#1 aliquots of the reaction mixture were applied to 1.5-cm squares of DE-81 filter paper (Whatman). Un- phosphorylated galactose was removed by wash- ing with water (4 × 500 ml) and the filters were dried and counted in Permablend (Packard). The reaction was linear up to 200 ttg protein. One unit of galactokinase phosphorylates 1 nmole galactose per minute under the standard reaction condi- tions.

Northern and Southern blotting Total genomic DNA and total cellular RNA

were prepared by conventional procedures (Maniatis et al., 1982). EcoRI was obtained from New England Biolabs and DNA digestion was carried out according to the manufacturer's in- structions.

PolyA ÷ RNA and formamide/formaldehyde gel separation were carried out by conventional techniques.

Northern and southern blots were probed with a 32p labelled TK-specific cDNA excised from the recombinant plasmid pTKl l (Bradshaw and Dei- ninger, 1984) kindly provided by Dr. A.R. Leh- mann. Northern blots were additionally probed with a fl-actin probe.

Results

Coordinate silencing of galactokinase expression in Raft cells

We have previously observed that the loss of methyltransferase and thymidine kinase expres- sion by the human lymphoblastoid cell line GM1953 is accompanied by a coordinate loss of the enzyme galactokinase. In Table 1 we present the activities of these 3 enzymes determined by assay in cell-free extracts of the Mex- and Mex ÷ derivatives of Raji. Raji Mex ÷ cell extracts con- tained galactokinase, methyltransferase and thymidine kinase. In contrast all 3 enzyme activi- ties were below the level of detection (all < 0.02 units/mg protein) in extracts of the Mex- Raji cells. In order to establish that the absence of these activities was not a consequence of inactiva- tion of the extracts, we determined the levels of

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TABLE 1

ENZYME ACTIVITY IN CELL-FREE EXTRACTS OF RAJI CELLS

Enzyme Activity (Units/mg protein)

Methyl- TK GalK Uracil-DNA transferase glycosylase

Raji Mex + 0.9 1.5 1.1 35

Raji Mex- < 0.02 < 0.02 < 0.02 35

the non-related DNA-repair enzyme uracil-DNA glycosylase. Cell-free extracts of both Raji Mex ÷ and Raji Mex- cells contained similar levels of this activity. Thus, in addition to methyltrans- ferase and thymidine kinase, Raji Mex- cell ex- tracts are deficient in galactokinase activity. This deficiency is not a consequence of a general in- activation of enzymes during the extraction pro- cess.

Phenotype of Raji M e x - cells As a consequence of the loss of methyltrans-

ferase activity, Mex- Raji cells are more sensitive to MNNG than the wild-type Mex ÷ cells. The hypersensitivity is of a similar magnitude to the reported sensitivities of other Mex- human lymphoid-derived cell lines. In order to establish whether the absence of the two kinase activities from cell-free extracts of Raji Mex- cells was reflected in the intact cell, we examined the sensi- tivity of Raji cells to the two metabolic inhibitors, trifluorothymidine (F3TdR) and 2-deoxygalactose (2-dGal).

Thymidine kinase which catalyses the phos- phorylation of thymidine in the first step in the salvage pathway for thymidine, will also phos- phorylate F3TdR. Trifluorothymidine triphos- phate, the eventual product of this salvage reac- tion is an inhibitor of DNA synthesis (Fujiwara et al., 1970). As a consequence of this ability, F3TdR is cytotoxic to TK + ceils whereas TK- cells are resistant to this compound. In Fig. la, b we pre- sent growth curves of Raji cells grown in the presence of F3TdR. Cultures were initiated in the presence of different concentrations of F3TdR and cells were counted at daily intervals and diluted in F3TdR-containing growth medium as required to maintain exponential growth. The growth of Raji

26

a

o

cJ 1

0 2 D a y s 4 2 D a y s 4

Fig. 1. Sensitivity of Raji cells to trifluorothymidine (F3TdR). Exponentially growing cells were seeded into 24-well plates in full growth medium containing the concentrations of F3TdR indicated. Growth was monitored by daily cell counting. (a) Raji Mex +. ©, control; O, 0.1/~M; II, 0.3/~M. (b) Raji Mex-.

zx, control; v, 3/xM; ×, 10 #M.

b x A ~x / V i A

V.x a J

• ~ , / V x , / x

Mex- cells is unimpaired by the presence of F3TdR at concentrations up to 10 /~M. In contrast, a significant inhibition of growth of Raji Mex ÷ cells is apparent at 0.1/~M F3TdR and no cell prolifera- tion occurs at 0.3/~M.

Galactokinase is the first enzyme in the Leloir pathway for the metabolism of galactose and catalyses the formation of galactose 1-phosphate from galactose. 2-dGal exerts a selective cytotoxic- ity towards galactokinase-expressing cells via the formation of 2-dGal-l-phosphate and inhibition of glycolysis. Cells which do not express galac- tokinase are unable to convert 2-dGal into the toxic metabolite (Schoen et al., 1984). Raji Mex- cells also exhibited a resistance to the cytotoxicity of 2-dGal (Fig. 2a,b). Significant inhibition of proliferation of Raji Mex ÷ cells was observed at 2 mM 2-dGal whereas Raji Mex- cells proliferated at normal rates in the presence of 10 mM 2-dGal and concentrations in excess of 100 mM were

1 0 0

; [

=

2 D a y s 4 6

1 0 o

o- ~ / ~ x

z =

1 2 D a y s 4 6

Fig. 2. Sensitivity of Raji cells to 2-deoxygalactose. Exponen- tially growing cells were seeded into 24-well plates in full growth medium containing the concentrations of 2-dGal indi- cated. Growth was monitored by daily cell counting. (a) Raji Mex ÷. ©, control; O, 2 raM; II, 20 mM. (b) Raji Mex-. ,x,

control; v, 10 raM; X, 100 mM.

required to significantly reduce their survival. The growth curves indicate that the absence of thymi- dine kinase and galactokinase activity in extracts of Raji Mex - cells reflects a failure of the cells to synthesize the active enzymes in vivo and is not a consequence of structural alterations in the en- zymes which render them more labile in cell-free extracts. We therefore conclude that Raji cells do not synthesize detectable amounts of functional methyltransferase, thymidine kinase or galac- tokinase.

Southern blot analysis of the thymidine kinase gene The stable loss of the 3 enzyme activities from

Raji Mex - cells could have resulted from deletion of their genes from this cell line. We tested this possibility for thymidine kinase by examining the tk gene in both Raji Mex - and Raji Mex ÷ cellu- lar DNA. Whole genomic DNA prepared from both derivatives was digested with EcoRI and analysed by southern blotting. Filters were probed with a thymidine kinase cDNA insert excised from the recombinant plasmid pTK11 (Bradshaw and Deininger, 1984). Fig. 3a shows that the tk gene has not been lost by deletion from Raji Mex- cells. T K DNA was present as a single band of high molecular weight (> 12 kb) after digestion with EcoRI. No difference in the intensity of this band was apparent between the Raji Mex ÷ (Lane 1) and Raji Mex - (Lane 2) DNA samples. The size of the T K DNA hybridising band is as ex- pected from the genomic map of the human tk gene (Flemington et al., 1987) in which the only exon-containing EcoRI fragment is > 20 kb (Ex- ons 1 and 2 are contained on a fragment of < 1 kb and would not be detected under the condi- tions of electrophoresis we used). Thus, the failure of Raji Mex - cells to express active thymidine kinase is a consequence of an alteration in gene regulation in this cell line rather than a loss of the tk gene. Although probes are not available to enable us to examine directly the mt and glk genes in Raji cells, in view of the joint regulation of the products of these genes with thymidine kinase in other cell lines, it seems unlikely that their silencing in Raji Mex- is the result of gene loss. More probably, the observed coregulation reflects a common mechanism by which the ex- pression of all 3 genes can be controlled.

27

(~) 1 2

Kb

12 " " ~

4

(b) 1 2

8 - a c t i n

TK

1 .6 ----~

Fig. 3. Analysis of the tk gene in Raji Mex + and Mex- cells. (a) Southern analysis. Whole genomic DNA was prepared from Raji cells, digested with EcoRI and analysed by Southern transfer. Lane 1: Raji Mex +. Lane 2: Raji Mex-. (b) Northern analysis of Poly A + RNA from Raji cells. 2 #g polyA + RNA was separated on a formamide/formaldehyde agarose gel. Lane 1: Raji Mex +. Lane 2:

Raji Mex-. All filters were probed using a tk-specific human eDNA.

T K - m R N A levels in Raft Mex + and M e x - cells The southern b lo t t ing analysis described above

indicates that loss of thymidine kinase expression is no t a consequence of delet ion of the whole tk

gene. We presume that the mt and glk genes are also intact bu t silenced. These data do not, how- ever, rule out the possibi l i ty of genomic rearrange- ments resul t ing in a t ranscr ip t ional si lencing of

28

these normally active genes. In addition, mam- malian cells commonly switch off gene expression through methylation of CpG sequences, particu- larly in the promoter regions of the controlled genes. In Chinese hamster cells, there are numer- ous examples of control of thymidine kinase ex- pression by methylation. Interestingly, in one case a joint regulation by methylation of thymidine kinase with an unrelated gene (metallothionine) has been reported (Gounari et al., 1987). Gene silencing through cytosine methylation is consid- ered to be essentially irreversible unless the cells are exposed to inhibitors of cytosine methylation such as 5-azacytidine or 5-azadeoxycytidine. Fol- lowing treatment with these compounds, reactiva- tion of the silent tk gene is observed at high frequency.

In order to investigate whether cytosine methyl- ation might underlie the changes in expression in Raji cells, we treated Raji Mex- cells with two different concentrations of 5-azadeoxycytidine: 0.1 /~g/ml or 0.3/~g/ml which reduced the survival to around 80% and 10% respectively. After allowing the cells to recover, they were plated at different dilutions in 96-well plates containing HAT medium to select for TK ÷ revertants. No T K + colonies were observed out of a total of > 108 cells plated. We estimate from these data that the reactivation frequency of the T K - phenotype of Raji Mex- cells by 5-azadeoxycytidine is < 10 -5. Reconstruction experiments in which low numbers of Raji Mex ÷ cells were mixed with the Raji Mex- cells indicated that re-expression of thymi- dine kinase at wild-type levels would have allowed us to select HAT-resistant colonies by this proto- col. We conclude that methylation of cytosine probably does not underlie the inactivation of the tk gene in Raji cells.

In order to investigate the possibility that the gene was being controlled at the level of transcrip- tion by a mechanism which was not dependent on 5-azadeoxycytidine-reversible methylation, we measured the steady-state levels of TK-specific mRNA in both Raji Mex- and Raji Mex ÷ cells. Northern blot analysis of polyA ÷ RNA (Fig. 3b) indicated that the steady-state level of T K - m R N A was closely similar in both cell lines and further indicated that there were no gross differences in the size of T K - m R N A between the two cell lines.

Discussion

The events underlying the loss of methyltrans- ferase expression which occurs in transformed hu- man cell lines are unknown. The only indication as to the mechanism of silencing is provided by the high frequency at which enzyme expression is lost. This effectively precludes mutational events and suggests that the methyltransferase might be one of a number of proteins which are regulated by a common mechanism after transformation. The coregulation of methyltransferase with thymidine kinase and galactokinase which we have observed in 3 different cell types, provides some evidence that methyltransferase is subject to this type of regulation.

The 3 enzymes carry out apparently unrelated functions in the cell and their only common fea- ture seems to be that they are non-essential. De- spite the apparent absence of functional similarity, their coregulation is a relatively frequent occur- rence. In fact, thymidine kinase and galactokinase are often coregulated (Wagner et al., 1988) and the observation of a high frequency of co-silencing of these loci in Chinese hamster cells obtained by sib-selection for the T K - phenotype has led to a proposal that this type of coinactivation of two alleles by non-mutational events be termed allelic or Type II gene silencing (Dobrovic et al., 1988). The close proximity of the tk and glk genes on the chromosomes of a number of mammals, in- cluding human and hamster, suggests that their Type II silencing might result from a positional effect. If this is indeed the case, it implies that the mt gene is closely linked to the tk and glk genes. In humans, the tk and glk genes are adjacent on the long arm of chromosome 17 along with p68, a cell cycle-related protein which exhibits extensive homology with a translational initiation protein (Ford et al., 1988) and the proto-oncogene neu or c-erbB-2 (Coussens et al., 1985). Examination of the expression of these proteins in cells in which tk and glk have been cosilenced with mt will be of interest.

We have previously observed that regulation of the tk gene in the human lymphoblastoid cell line GM1953 is not correlated with a regulation in the level of TK-specific mRNA. This observation has now been extended to the Burkitt's lymphoma cell

line Raji. These data indicate that the level of thymidine kinase in these cells is controlled in a post-transcriptional fashion. Numerous observa- tions indicate that the level of thymidine kinase activity in quiescent cells stimulated to divide by serum replacement is correlated with the level of TK-mRNA (Stuart et al., 1985). More recently, it has become clear that the normal regulation of thymidine kinase during the cell cycle is poorly correlated to the level of TK transcripts (Sherley and Kelly, 1988) and instead depends at least partly on an enhanced rate of translation of TK- mRNA, the levels of which remain fairly constant throughout the cell cycle (Gross and Merrill, 1989). In view of the similarity in the levels of TK- mRNA in the Mex- cell lines in which thymidine kinase has been lost by Type II silencing, it seems possible that the regulation of thymidine kinase, galactokinase and methyltransferase activities which we have observed reflects either a reduced rate of translation of their mRNAs or an in- creased rate of protein turnover.

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