Instability of Mex− phenotype in human lymphoblastoid cell lines

Mutation Re~earch, 208 (1988) 167-172 Elsevier

167

MTRL 0125

Instability o f M e x -

p h e n o t y p e in h u m a n l y m p h o b l a s t o i d cell lines

Izumi Arita 1, Kouichi Tatsumi ~'2, Akira Tachibana t'2, Mariko Toyoda ~ and Hiraku Takebe 1'2 'Department of Molecular Oncolog.v and 2Department of b2rperimental Radiology, Faculty of Medicine, A]voto University, Sakvoku Kvoto 606 /Japan) (Accepted 7 April 1988)

Kew'ords." O6-Alkylguanine-l)NA alkyltransferase; Lymphoblastoid cell line; Mex ; l-(4-Amino-2-methyl-5-pyrimidinyl)methyl3-(2-chloroethyl)-3-nilrosourea hydrochloride (ACNU)

Summary Three lymphoblastoid cell lines (LCLs) had extremely low activities of O6-alkylguanine-DNA alkyltransferas,." (O6-AGT), and were classified as M e x - . They were highly sensitive to cell killing by l-(4-amino-2methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), whereas NMO2, a M e x LCL with a high O6-AGT activity, was resistant to the agent. Small fractions of these M e x - LCI.s survived the treatment with 10 #g/ml of A C N U for 24 h, and the surviving cells were found to be resistant to subsequent treatments with the agent. In addition, they contained O6-AGT activities comparable to that of NMO2 and were therefore regarded as Mex +. These results suggest that the M e x - phenotype in LCLs is unstable.

O6-Methylguanine (O6-MeG: guanine methylated at the 06 position) has been implicated as a major mutagenic lesion in cellular DNA among various alkylated bases produced by alkylating agent~ (Pegg, 1984). The residue mispairs with thymine (Abbott and Saffhill, 1979), and

Correspondence: 1. Arila, Department of Molecular Oncology, I.aculty of Medicine, Kyoto University Sakyo-ku, Kyoto, 606 (Japan).

Abbrev:ations: I.CL, lymphoblastoid cell line; PE, plating efficiency; O6-MeG, d'-methylguanine; O6-AGT, O6-alkylgua nine-DNA alkyltransferase; CNU, chloroethylnitrosourea; ACNL;, 1-(4-amino-2-met hyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride; MNN(.I, N-methylN'-nitro-N-nilrosoguanidine; MNU, N-methyl-N-nitrosourea; EBV, Epstein-Barr virus; SV40, simian virus 40.

presumably causes a G C - A T transition mutation (Bhanot and Ray, 1986). Day et al. (1980a,b) found that about one-fourth (9/39) of human tumor cell strains and some human fibroblast lines transformed with simian virus 40 (SV40) were unable to reactivate adenovirus treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and they designated such cells Mer . Sklar and Strauss (1981) showed that approximately one-third (7/23) of Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines (LCLs) were incompetent in their abilities to remove O6-MeG residues produced by MNNG. They termed those strains M e x - (methyl excision minus) and LCLs which were capablc of removing the rcsidue Mex + M e r - cells are phenotypically M e x - (Day et al., 1980b). O6-Alkylguanine residues including O6-MeG and O6-cthylguanine

0165-7992/88/S03.50 • 1988 Elsevier Science Publishers B.V. (Biomedical Division)

168 are removed by O6-alkyiguanine-DNA alkyltransferase (O6-AGT) (Harris et al., 1983; Yarosh et al., 1983; Pegg et al., 1984). M e x - LCLs (Harris et al., 1983) and M e r - cells (Yarosh et al., 1983) contained little or no O6-AGT activity. Mer- cell strains were much more sensitive than Mer + cell strains to cell killing by alkylating agents including chloroethylnitrosoureas (CNUs) (Scudiero et al., 1984). The cytotoxicity of CNUs is largely due to the formation of interstrand crosslinks following the initial chloroethylation of the 0 6 position of guanine in DNA (Brent, 1985). O6-AGT suppressed the formation of DNA interstrand cross-links in vitro by one CNU, 1,3bis(2-chloroethyl)-l-nitrosourea (BCNU) (Brent et al., 1987). It was also reported that the sensitivity of tumor cell strains to another CNU, l-(4-amino2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) (Yoshida, 1982), inversely correlated with the O6-AGT activities in crude extracts of cells (Tsujimura et al., 1987). The mechanisms of the differentiation into M e x - or M e r - including the role of EBV or SV40 remain to be elucidated. Mex" may be incompletely dominant since hybrid cells between Mex* and Mex - had an intermediate level of O6-AGT activity (Ayres et al., 1982). Sklar and Strauss (1983) reported that both Mex* and M e x - LCLs were established by EBV-mediated transformation of a single blood sample from a normal male individual. It has also been reported that Mex + fibroblasts and Mex - LCLs (Shiloh et al., 1983) or Mer* fibroblasts and M e r - tumor cell strains (Day et al., 1980b) can be obtained from the same individual. Except for tumor cell strains, the above-mentioned results were obtained with polyclonal human cell populations. These reports, therefore, suggest that the distinctions between Mex" and M e x - or Mer + and M e r - characteristics may be epigenetic, a n d / o r that the Mex- and M e r - phenotypes of cell population may be unstable. We examined the activities of O6-AGT in the polyclonally established LCLs and their sensitivities to the cytotoxic effects of A C N U , and found

that the transition of population phenotype from M e x - to Mex + took place in some LCLs after treatment with ACNU. Materials and methods

Cell lines LCLs were established by infecting lymphocytes with EBV. XP7NI, derived from a xeroderma pigmentosum (complementation group A) patient, was provided by Drs. T. Hashimoto and J. Furuyama, Hyogo College of Medicine, Nishinomiya (Tatsumi et al., 1987). A T I - I (Okano et al., 1985) and GM2783 (Tatsumi and Takebe, 1984), derived from 2 unrelated patients with ataxia telangiectasia, were obtained from Dr. T. Osato, Hokkaido University, Sapporo and H u m a n Genetic Mutant Cell Repository, Camden, N J, respectively. NMO2 was established in this laboratory from the peripheral blood of a healthy donor. Culture condition Cells were grown in R P M I I 6 4 0 medium supplemented with 4 mM L-glutamine, 1 mM uketoglutaric acid, and 17°70 fetal calf serum. Cells in a 25-cm z culture flask were incubated at 37°C in a humidified CO2 incubator. Cytotoxicity assay based on the back-extrapolation o f growth curves 10 ml of cell suspension (4-5 × 105 cells/ml) was irradiated by UV-light or gamma-rays as previously described (Tatsumi and Takebe, 1984; Tatsumi et al., 1987) or treated with 10 #g/ml of ACNU (Sankyo Co., Tokyo) for 24 h. Cell density was measured daily or every 2 days with a Coulter counter (Coulter Electronics, Ltd., FL) and diluted to 3-5 × 105 cells/ml. Relative cell numbers after the beginning of the treatment were plotted on a semi-log scale and the log-linear portion of the growth curve was extrapolated to day 0. The intercept was used as the estimate of the fraction of surviving cells. Cytotoxicity assay based on the clonogenicity Cell suspensions containing various concentra-

169

tions of ACNU were dispensed into wells of microtiter plates (Corning Glass Works, New York). The microtiter plates were incubated in a CO2 incubator for 2-4 weeks. Positive wells with growing clones were scored by visual inspection. The plating efficiency (PE) was estimated as described elsewhere (Tatsumi and Takebe, 1984). The survival fraction was obtained by dividing PE of treated cultures by PE of untreated control.

0 6 - A G T assay The assay followed the method of Myrnes et al. (1984) with slight modifications. The substrate DNA containing methyl-3H-labeled O6-MeG was prepared by incubating calf thymus DNA with [3HJN-methyl-N-nitrosourea ([3HIMNU: sp. act. 1.0 C i / m m o l e , New England Nuclear, MA) at 37°(7 for 3 h, followed by heating at 80°C for 16-18 h in order to selectively remove major a l k y a t e d products other than O6-MeG. The final substrate contained approximately 150 d p m / # g DNA. About 3-6 × 107 cells were resuspended in 200 # l of T r i s - E D T A buffer (50 mM Tris-HCl pH 7.8, I mM E D T A and 1 mM dithiothreitol) and sonicated followed by centrifuging at 12 000 rpm for 10 min at 4°C. The supernatant was used as crude extract. The reaction mixture containing 50 #g of the substrate DNA and 45 ul of the crude extract was incubated at 37°C for 1 h. The reaction was then stopped by adding 500 #1 of 5% (w/v) trich~oroacetic acid and the mixture was heated at 80°C for 30 min to hydrolyze DNA containing radioactive O6-MeG. The mixture was then filtered and washed with 5°7o trichloroacetic acid and with ethanol on a G F / C filter (Whatman Ltd., England). The radioactivity remaining on the filter was counted with a liquid scintillation counter. The non-specific binding of 3H to protein was estimated by replacing crude extracts with bovine serum albumin. This background count was subtracted from the actual radioactivity in each sample. Results and discussion

As shown in Table I, GM2783, ATI-1, and

TABLE I O6-AGT ACTIVITY OF L Y M P H O B L A S T O I D CEI.L LINES Cell line

O%AGT activity'~ ( p m o l e / m g protein)

NMO2

0.3(12 + 0.026 b

GM2783 GM2783-ACN1.J'-I (;M2783-ACNU'-2

0.029 _+ 0.009 0.385 _- 0.054 0.449 _+ 0.075

A TI-I ATI-I-ACNUr-I ATI-I-ACNU'-2

0.015 _+ 0.003 0.142 ± 0.019 0.273 _+ 0.053

XP7NI XP7NI-ACNU'-I XP7NI-ACNUr-2

0.028 ~_ 0.018 0.216 _+ 0.030 0.198 ± 0.029

aO6-AGT activity is expressed as pmole of methyl group removed from [~H]MNt'-treatcd DNA per h per mg of protein in the cell extracts. h Mean t SI) calculated from triplicate samples.

XP7NI were found to have marginal activities of O6-AGT, while NMO2 had a high activity. The former 3 LCLs were, therefore, classified as Mexcell lines. NMO2 was used as a representative Mex" LCL throughout this study. We compared the sensitivities of 3 M e x - LCLs to ACNU with that of a Mex + LCL. The estimated surviving fraction of NMO2 after the treatment with ACNU (10 #g/ml) for 24 h was 700/0 (Fig. la). Because cell density of GM2783 did not increase for 10-14 days following A C N U treatment, we replaced half of the medium every 2 days during this period without measuring cell density. The estimated surviving fraction of ACNU-treated GM2783 was 0.065°7o (Fig. lb), while those of other M e x LCLs, A T I - I and XP7NI, were 0.12% and 0.045%, respectively. These indicate that M e x - ICLs are highly sensitive to cell killing by ACNU. The small fractions of GM2783, A T I - I , and XP7NI that survived the first treatment with ACNU were designated GM2783ACNU~-I, ATI-I-ACNUr-I, and XP7NIACNU~-I, respectively. To recognize the difference in sensitivity to ACNU between ACNUr-1 sublines and their

170

(a)

(b)

(c)

(d)

j

Y

tOo

°t g,q 10.2

o

l0

15

o

5

~o 15 r, Days

Fig. 1. The effect of ACNU on the growth of human lymphoblastoid cells. (?ells were exposed to 0 ( 0 - - 0 ) or I0 (~, ~-~ ) ~g/ml of ACNU during day 0 to day I for 24 h. (a) NMO2, (b) GM2783, (c) GM2783-ACNU'-I that survived the first treatment with ACNU as depicted in (b), (d) GM2783-ACNUL2 that survived the second treatment with ACNU as depicted in (c). The dotted lines denote the back-extrapolation of the log-linear portion in growth curves.

parental Mex- LCI..s, regrowing A C N U L I cells were treated again with 10 #g/ml of ACNU for 24 h. The estimated surviving fraction of GM2783-ACNUL1 was 41% (Fig. lc), and essentially similar results were obtained for A T I - I ACNUr-I and XP7NI-ACNU~-I sublines (data not shown). Cells which survived the second ACNU

(a)

(b)

(c)

l

10-1-

0

10

,

1

20

0

,

,

10

,

20

,

0

,

,

i

10

,

20

ACNU cor'w~r~ation ~9-~t )

Fig. 2. The cytotoxic effects of ACNU in terms of the loss of clonogenieity in parental cell lines (0---0) and their corresponding ACNU'-2suhlines (L.---~)of(a) XP7NI, (b) A T I - I , and (c) GM2783. PE values of untreated XP7NI, XP7NIACNU'-2, ATI-I, ATI-I-ACNU'-2, GM2783, and GM2783-ACNU'-2 cells were 48%, 38%, 62%, 38%, 13%, and 7%, respectively. The bars denote 95% confidence intervals.

treatment were designated ACNUr-2. TO reconfirm the resistance to ACNU, GM2783-ACNUr-2 cells were treated with ACNU (10 p.g/ml) for 24 h, and the estimated surviving fraction was 46°7"0 (Fig. ld). To verify the change in ACNU sensitivities of M e x - LCLs, we analyzed the cytotoxicity of ACNU in these LCLs using the clonogenic assay (Fig. 2). The doses of ACNU yielding 10% survival were 1.9, 2.9, and 2.7 t~g/ml for GM2783, A T I - I , and XP7NI, while those for their corresponding ACNUL2 sublines were 19, 15, and 20 #g/ml, respectively. We assayed the O6-AGT activities of ACNUr-I and ACNU~-2 sublines to determine whether the changes in the sensitivities to ACNU killing were accompanied by changes in their O6-AGT activities. O6-AGT activities of GM2783-ACNU~-2, A T I - I - A C N U L 2 , and XP7NI-ACNUr-2 were 15.5, 18.2, and 7.1 times higher than those of their corresponding parental cell lines, respectively (Table 1). The activities of O6-AGT in these ACNUr-2 sublines were comparable to that in M e x - NMO2 cells. These results indicate that ACNUr-1 and ACNUr-2 sublines are Mex ~ . In order to rule out the possibility of crosscontamination of cell lines as a cause for the change from M e x - to Mex + as population phenotype, we examined H L A types and the sensitivities of cells to UV-light or gamma-rays. The H L A types of ACNU~-2 sublines were identical with those of their parental LCLs (data not shown). XP7NI was sensitive to the cytotoxic effect of UV-light as described earlier (Tatsumi et al., 1987), while NMO2 was rather resistant. XP7NI-ACNUr-2 and XP7Ni showed essentially similar sensitivities to UV-light (Fig. 3a). A T I - I ACNU~-2 was as sensitive to g a m m a irradiation as ATi-1, whereas NMO2 was rather resistant (Fig. 3b). Thus, the data collectively indicate that the small fractions of 3 M e x - LCLs which survived the treatment with 10 ug/ml of ACNU were resistant to ACNU and had high O6-AGT activity. One possible explanation for this conversion is that the cell populations were originally heterogeneous and contained a small fraction of pre-existing M e x '

171

(a)

(b)

rhabdomyosarcoma grown as xenograft in nude mice could be eradicated by a single treatment with I -(2-chloroethyl)-3-(trans-4-methylcyciohexyl)-

> Q:

O.t-

W

0.0'. uJ CE

0

I

2

3

UV close ( ~m2 )

r-ray

dose (Gy)

Fig. 3 Cytotoxic effects of UV-light and gamma-rays in h u m a n lympkoblastoid cells. (.'ell killing was assayed by backextraFolating the growth curve. (a) UV-light, XPTNI (O----O), X P 7 N I - A C N U ' - 2 (, ,..-,"i), NMO2 ( , a - - , ' , ) ; (b) gamma-rays, A'I'I-I ( I1--11 ), A T I - I - A C N U L 2 ( 13--13 ), NMO2 ( A - - A ).

cells when they were treated with ACNU. Our method of drawing survival curves by fluctuation test (:lid not permit us to estimate accurately the survival below 10 ~, and only a slight indication of the heterogeneity is suggested by the breaks in the survival curves at around 10 3 (Fig. 2). The alternative interpretation may be that ACNU induced phenotypic change of a Mex- cell to Mex ~ . The frequency ot" appearance of Mex ÷ cells in the ACNU-treated Mex- cells was approximately 5 x 10 -4 or higher estimated by the levels of survival after the treatment (Fig. lb). The frequency could be too high to be totally attributed to induced mutation. Another possible cause is that the phenotypes were interchangeable by some unknown epigenetic factor(s) as mentioned in the introduction. A method has not been developed yet to determine Mex phenotypes of each individual cell. This prevents us from deciding the precise mechanisms underlying the present findings with polyclonally established L C L s . Further studies on the responses of monoclonal Mex - cells to ACNU are in progress in this laboratory. Since primary untransformed cells from normal tissue are Mex ' or Mer + (Mex -~/Mer ÷) without exception (Day et al., 1980a), we can reasonably expect the higher therapeutic index where a tumor is M e x - / M e r - . For instance, a Mer- human

1-

nitrosourea (MeCCNU), whereas Mer + tumors were resistant to this treatment (Brent et al., 1985). However, we still do not know whether M e x - / M e r - ceils stably retain their original phenotype after chemotherapy. Hayward and Parsons (1984) reported that the treatment of a human M e r melanoma cell strain with the activated form of 5-(3' ,3' -dimethyl- l-triazeno)imidazole-4-carboxamide (DTIC) in vitro resulted in the conversion to Mer + phenotype, suggesting the possible instability of M e r - phenotype of human tumor ceils. During the preparation of this article, Satoh et ai. (1987) reported that V79 Chinese hamster cells acquired resistance to ACNU and increased their capacity to remove O6-alkylguanine after treatment with the agent. Although the underlying mechanism is not yet clear, these reports together with our data, indicating transition of M e x - / Mer- to Mex ~/Mer ~, may imply the difficulties in annihilating M e x - / M e r - tumor by repeated treatments with CNUs.

Acknowledgements The authors are grateful to Dr. T. Tsujimura and Dr. P. Karran for helpful discussions. This work is supported by a Grant-in-Aid for Special Project Research on Cancer Bio-Science from the Ministry of Education, Science and Culture of Japan.

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Yoshida, .I., N. Shibuya, T. Kobayashi and N. Kagcyama (1982) Sensitivity to l-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (A('NU) of glioma cells in vivo and in vitro, Cancer, 50, 410-418. Communicated by F.H. Sobels