Somatic Cell and Molecular Genetics, Vol. 21, No. 3, 1995, pp. 215-218

B R I E F C O M M U N I C A T I O N

Evidence for Gene Silencing by DNA Methylation in Normal Human Diploid Fibroblasts

Robin Holliday and Thu Ho

CSIRO Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, NSW 2113, Australia

Received 17 April 1995--Final 22 May 1995

AbstractNHuman diploid fibroblasts, strain MRC-5, were perrneabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate (5-methyl dCTP) in the S phase of the cell cycle. The frequency of TG n HPRT cells was increased up to 20-fold in comparison to control untreated cultures. Representative TG R clones were unable to grow in HA T, and these were treated with 5-azacytidine (5-aza-CR). In many cases subsequent growth in HA T medium was observed, but in others it is likely that the cells had run out of growth potential. The results provide the first evidence of the silencing and reactivation of a gene in normal diploid mammalian cells.

INTRODUCTION

There are many examples of inactive genes in various mammalian cell lines that can be reactivated by the demethylating agents 5-azacytidine or (5-aza-CR) or 5-aza- deoxycytidine (1, 2). These genes appear to be silenced by de novo methylation of CpG islands on other regions of DNA that are normally unmethylated (3). There are no known examples of the same types of gene being silenced in cultured normal diploid cells.

We previously showed that three genes could be silenced in CHO cells by the uptake of 5-methyl deoxycytidine triphosphate (5- methyl dCTP) into cells permeabilized by electroporation (4). Similar results were also obtained with V79 cells, and, in addition, evidence was obtained that the 5-methyl dCTP was incorporated into DNA (5). Genes inactivated by this procedure could be reactivated by 5-aza-CR. We report here that the same method can be used to inactivate

the X-linked HPRT (hypoxanthine phospho- ribosyl transferase) gene in normal human diploid fibroblasts, strain MRC-5. This fetal lung strain is male, so has only one HPRT gene.

MATERIALS AND METHODS

MRC-5 is a human fetal lung diploid fibroblast strain that is well characterized and has been extensively used in experimen- tal studies (6-8). Cells were obtained from frozen ampoules at the 20-24 population doubling level (PDL) and grown in Eagle's minimal essential medium (MEM from Gibco) supplemented with nonessential amino acids, 10% fetal calf serum (FCS), penicillin and streptomycin (0.06 mg/ml and 0.1 mg/ml, respectively).

Cells were routinely grown in 25-cm 2 or 75-cm 2 flasks at 37~ and 5% CO2. They were harvested with trypsin (0.15% w/v) and versene (0.54 mM) and counted with a Coulter counter. MRC-5 cells are split 1/4 or

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216 Holliday and Ho

1/8 (corresponding to 2 and 3 PDs), and subcultured when confluent. The final life- span of MRC-5 is 50-70 PDs (8).

Cells were initially passaged in HAT medium (MEM with 100 IxM hypoxanthine; 0.4 ~xM aminopterin and 16 txM thymidine by IAc) dilution of a stock from Sigma) to remove any cells resistant to 6-thioguanine (TGa). Populations in S phase were obtained by initial growth in MEM (48 h), removal of FCS (24 h), and readdition of FCS (20-22 h). These cells were subjected to electroporation in the presence of 5-methyl dCTP (Pharma- cia) using the procedure previously described (4), but with a lower voltage pulse (250 or 300 V). 5-methyl dCTP was used either at 0.4 mM or 1.2 mM.

Cells were allowed to recover for four to six days in MEM, and then plated at 5 x 104 cells per 10-cm dish, with a feeder layer, in MEM containing 6 fxg/ml 6TG (Sigma). These cells were also plated at much lower densities in normal MEM with a feeder layer, to determine the plating efficiency, by stain- ing colonies with Giemsa after three weeks of incubation, with weekly change of medium.

Two types of cells were used for the feeder layer, either early passage Lesch Nyhan skin fibrobtasts from a 9-year-old male donor (9), or a clone of T G R cells obtained from early passage MRC-5 cells by the method described here. Growing cells in a 25 cm 2 flask were treated with 20 Ixl mitomycin from a stock of 250 txg/ml. After two days the cells were harvested and plated at 7_5 x 104-105 cells per 10-cm dish, together with the cells treated with 5-methyl dCTP. Plates were incubated in T G medium for three to four weeks, with a weekly change of medium. Representative colonies were isolated using a standard cloning ring and transferred to 1.8-cm wells. Otherwise colo- nies were stained with Giemsa. In each experiment, feeder layer cells, without treated cells, were checked for the absence of growth in 6TG.

TG R isolates were checked for their inability to grow in HAT medium. Growing cells were treated with 1 p.g/ml 5-aza-CR (from a freshly prepared stock of 1 mg/ml) for 24 h and then allowed to recover in normal MEM for five days or confluency. These cells were transferred to 6-cm plates containing 5 ml H A T medium and incubated for 14-21 days with a weekly change of medium, Subsequently they were stained with Giemsa.

RESULTS

MRC-5 is a well-characterized human diploid fibroblast strain that has been exten- sively used in studies of in vitro aging (7). In our hands, the plating efficiency is low, even with a feeder layer. In the experiments reported here we used either a Lesch Nyhan H P R T - strain as a feeder layer or a TG R derivative of MRC-5. This was done to prevent the attached nongrowing feeder cells providing active H P RT through metabolic cooperation. The plating efficiency of MRC-5 ceils with either feeder layer is in the range of 10-20% for treated ceils, but was sometimes lower for untreated controls (Table 1). This was probably due to the fact that the Lesch Nyhan feeder layer cells used in these experiments were at a later passage level.

Before cells were plated on medium containing 6TG, they were previously grown in H A T medium to remove any H P R T - cells. In experiments with untreated MRC-5 cells, we observed a spontaneous frequency of TG R colonies of about 4 • 10 6 (Table 1). In the first series of experiments we set the BioRad Pulser at 250 or 300 V and used 20 Ixl (0.4 mM) 5-methyl dCTP. There was a modest increase in colonies resistant to TG. Subsequently we increased the level of 5-methyl dCTP to 60 ixl (1.2 raM) and applied 300 V. We observed a substantial increase in TG R colonies, which was at least 20-fold greater than the control frequency

Gene Silencing in Human Fibroblasts 217

Table 1. Frequencies of TG R Colonies Obtained After Treatment of MRC Fibroblasts with 5-Methyl dCTP and in Untreated Controls

5-Methyl Cloning efficiency Viable cells T G R

dCTP treatment No. expts. (%) screened colonies Frequency

Control 3 5-11 5.05 x 105 2 ~4 x 10 6 0.4 mM

(250V or 300V) 3 13-21 7.82 x 105 20 2.6 x 10 5 1.2 mM

(300V) 3 9-18 1.04 • 106 95 9.1 x 10 -5

(Table 1). It should be noted that the cells were partially synchronized by serum depriva- tion, so that 20-22 h after adding back serum most cells are in S phase (F. Watt , personal communica t ion) at the time of t r ea tment with 5-methyl dCTP.

The usual test for the existence of an inactivating epimutat ion, due to D N A meth- ylation, is to reactivate such genes with 5-aza-CR. However , h u m a n diploid fibro- blasts have a finite life-span, which makes this test difficult to carry out. The t rea ted cells are already at P D L 24, and the formation of a T G R colony represents a

further 12-15 PDs. These colonies are picked, grown further, and t rea ted with 5-aza-CR. The cells are then incubated for several days before plating on H A T medium. All these steps comprise a fur ther 10-12 PDs. It is also well known that individual clones in a popula t ion of fibroblasts have very variable growth potent ial (10), and many would not survive long enough to produce colonies in H A T med ium after 5-aza-CR treatment . (The final life-span of a populat ion is de te rmined by the longest lived clones). We have found that about half the TG R clones t reated with 5-aza-CR p roduced significant growth in H A T medium. For example, 12 T G R clones f rom one experi- ment in Table 1 (second t reatment , line 3) were tested with 5-aza-CR and six were to some extent reactivated. No growth in H A T was seen when the same T G R clones were not

t rea ted with 5-aza-CR. The most clear cut result we obta ined is shown in Fig. 1.

D I S C U S S I O N

Previous results with the C H O and V79 pe rmanen t lines showed that the enzyme- deficient isolates that were ob ta ined follow- ing 5-methyl d C T P t rea tment of permeabi - lized cells, could be react ivated by 5 -aza -CR (4, 5). The simplest in terpreta t ion of these results is that the genes were silenced by D N A methylat ion of p r o m o t e r sequences. Subsequent ly we have shown in C H O cells that A P R T - isolates, ob ta ined after 5-methyl d C T P t rea tment , have methyla ted p romote r

Fig. 1. Reactivation of a HAT TG R clone of MRC-5. Cells were treated with 5-aza-CR and plated on HAT medium (see Materials and Methods).

218 Holliday and Ho

sequences using the bisulfite genomic se- quencing technique (11) (results to be published elsewhere). By analogy we assume that the TG R isolates obtained in strain MRC-5 are also due to imposed methylation of the H P R T gene. The other evidence that the H P R T gene is inactivated by DNA methylation comes from experiments with 5-aza-CR, which at least in some cases reactivates the gene. In other cases TG R isolates become senescent and so cannot be tested for reactivation.

The control frequency of T G R colonies is comparable to those seen in previous studies. DeMars (12) reported a frequency of 5 x 10 -6 (range 0.5-37), using 8-azaguanine as selective agent. McGregor et al. (13) observed a similar frequency of 5.5 • 10 -6 (range 1.1-14) TG R colonies. Taken together with our controls, the induced frequencies of 2.6 and 9.1 x 10 -5 are highly significant. It is difficult to optimize the conditions of the inducing treatment, because with a low plating efficiency, many dishes must be used for each experiment to obtain reasonable numbers of colonies. For example, in the experiments to screen 1.04 x 106 viable cells (Table 1, line 3), 163 dishes were used. We used serum starvation and replacement to bring most ceils into S phase when they were treated with 5-methyl dCTP, but we have not compared these cells with normal growing populations.

Almost all previous experiments on the inactivation and reactivation of genes by changes in DNA methylation have been carried out in permanent cell lines, which are partially or fully transformed. There is also evidence that inactive genes in transformed cells and hybrids are much more readily reactivated by 5-aza-CR than those in nor- mal diploid cells (14-16). Indeed, it has been stated that inactive X-linked genes in normal cells are refractory to reactivation by 5-aza- CR (14, 17, reviewed in 18). We believe our preliminary results with strain MRC-5 are

important, because for the first time evidence has been obtained that a normal gene in normal cultured cells can be inactivated by DNA methylation and subsequently reacti- vated by a demethylating agent. The results may also indicate that methylation using 5-methyl dCTP is more susceptible to demeth- ylation by 5-aza-CR than the methylated CpG island of the human H P RT gene in the inactive X chromosome (17).

ACKNOWLEDGMENTS

We thank Dr. Fuji Watt for showing us the results of her synchronization experi- ments with MRC-5.

LITERATURE CITED

1. Holliday, R. (1987). Science 238:163-170. 2. Holliday, R. (1991). Mutat. Res. 250:351-363. 3. Antequera, F., Boyes, J., and Bird, A. (1990). Cell

62:503-514. 4. Holliday, R., and Ho, T. (1991). Somat. Cell Mol.

Gen. 17:537-542. 5. Nyce, J. (1991). Somat. Cell Mol. Gen. 17:543-550. 6. Jacobs, J.P., Jones, C.M., and Baillie, J.P. (1970).

Nature 227:168-170. 7. Holliday, R. (1984). Monogr. Dev. Biol. Karger

17:60--77. 8. Holliday, R., Huschtscha, L.I., Tarrant, G.M., and

Kirkwood, T.B.L. (1977). Science 198:366-372. 9. Huschtscha, L.I., Thompson, K S . A , and Holll-

day, R. (1986). Proc. R. Soc. London Ser. B 229:1-12.

10. Smith, J.R., and Whitney, R.G. (1980). Science 207:82-84.

11. Frommer, M., McDonald, E., Millar, D.S., Collis, C.M., Watt, F., Grigg, W., Molloy, P.L., and Paul, C.L. (1992). Proc. Natl. Acad. Sci. U.S.A. 89:1827- 1831.

12. DeMars, R. (1974). Murat. Res. 24:335-364. 13. McGregor, W.G., Maher, V.M., and McCormick,

J.J. (1991). Somat. Cell MoL Gen. 17:463-469. 14. Wolf, S.F., and Migeon, B.R. (1982). Nature

295:667-671. 15. Grant, S.G., and Worton, R.G. (1989). Mol. Cell

Biol. 9:1635-1641. 16. Mohandas, T., Sparkes, R.S., and Shapiro, L.J.

(1981). Science 211:393-396. 17. Migeon, B.R., Axelman, J., and Beggs, A.H.

(1988). Nature 335:93-96. 18. Gartler, S.M., and Goldman, M.A. (1994). Dev.

Genet. 15:504-514.