Induction of chromosomal aberrations in the CHO mutant EM9 and its parental line AA8 by EcoRI restriction endonuclease:
electroporation experiments
Felipe Cortrs and Trinidad Ortiz Department of Cell Biology, Faculty of Biology, E-41012 Seville (Spain)
(Received 18 May 1990) (Revision received 16 July 1990)
(Accepted 27 July 1990)
Keywords: Chromosomal aberration; EcoRI; Electroporation; CHO cells
Summary
EcoRI restriction endonuclease (RE), which produces cohesive-ended double-strand breaks (dsb) in DNA, was tested in the ethyl methanesulfonate- and X-ray-sensitive CHO mutant EM9 and its parental cell strain AA8 for its chromosomal aberration-inducing effect. The RE was efficiently introduced by electroporation into AA8 cells, while the mutant cells showed a very high sensitivity to electroporation, which consistently resulted in cell death. Nevertheless, the incubation of EM9 cells in the presence of EcoRI , without electroporation, was sufficient to induce about three times the chromosome damage observed in the electroporated parental cell line AA8 for any given dose of the RE.
The CHO ethyl methanesulfonate (EMS)-sensi- tive mutant EM9 is also hypersensitive to killing by X-rays, and has a very high baseline sister- chromatid exchange (SCE) frequency compared to its parental line, AA8 (Thompson et al., 1982; Dillehay et al., 1983). At the molecular level, it has been shown that EM9 has a defect in the rate of rejoining DNA-strand breaks after treatment with X-rays, EMS or MMS (Thompson et al., 1982). Nevertheless, both DNA-ligase (Chan et al., 1984) and apurinic/apyrimidinic endonuclease activities (La Belle et al., 1984) appear normal ha EM9.
Restriction endonucleases (RE) that produce
Correspondence: Dr. F. Cortrs, Department of Cell Biology, Faculty of Biology, E-41012 Seville (Spain).
either 'blunt-' or 'cohesive'-ended double-strand breaks (dbs) in DNA have been a useful tool to analyze the nature of lesions responsible for in- duction of chromosomal aberrations (Natarajan and Obe, 1984; Bryant, 1984). It has been shown that RE behave like ionizing radiation in their clastogenic action, i.e., they belong to the direct- acting (S-phase-independent) chromosome-break- hag agents (Natarajan and Obe, 1984; Obe and Winkel, 1985).
In spite of some reports proposing that blunt- and cohesive-ended dsb are potentially equally effective (Gustavino et al., 1986; Winegar and Preston, 1988) the current view is that RE that produce blunt-ended DNA dbs are much more efficient inducers of chromosome aberrations than those which generate cohesive-ended dbs (Natara-
jan and Obe, 1984; Bryant, 1984; Bryant and Christie, 1989; Winegar et al., 1989).
EeoRI is a well-characterized RE which recog- nizes G $ AATTC sequences in DNA and makes cohesive dsb (for review, see Kessler, 1987). Morgan et al. (1988) cloned the EcoRI coding sequences into an expression vector under the control of the mouse metallothionein gene promo- ter and reported that CHO cells transfected with this plasmid showed a high yield of chromosome aberrations. While the most commonly used meth- ods of introducing RE into cultured mammalian cells included permeabilization by inactivated Sendai virus, trypsinization, and osmotic shock (Natarajan and Obe, 1984; Bryant, 1984; Obe et al., 1985; Winegar and Preston, 1988), more re- cently, the cell electroporation method has been shown to yield a much higher efficiency and re- producibility (Winegar et al., 1989; Morgan et al., 1989). Using electroporation, a moderated cyto- genetic effect of EcoRI in CHO cells has also been demonstrated (Morgan et al., 1990).
Since the CHO-cell mutant strain EM9 has been reported to be sensitive to DNA-strand breaks induced by X-rays, we have made use of electroporation to compare the cytogenetic effect of the restriction endonuclease EcoRI in EM9 with that induced in the parental line, AA8.
Materials and methods
Cell culture The parental line AA8 and mutant EM9 were
kindly provided by Dr. L.H. Thompson (Lawrence Livermore National Laboratory, U.S.A.). Both cell lines were grown as monolayers in McCoy's 5A medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and the antibiotics penicillin (50 uni ts /ml) and streptomycin (50 /~g/ml). Cells were cultured at 37 °C in 5% CO2 in air.
Treatment of cells Exponentially growing cells were trypsinized
and treated with different doses of EcoRI (Boeh- ringer Mannheim) either directly in the same buffer used for electroporation (see below) or, alternatively, they were electroporated using a
Bio-Rad Gene Pulser to introduce efficiently the enzyme into the cells. The electroporation proce- dure was carried out according to Winegar et al. (1989). In short, the trypsinized cells were washed once with HEPES-buffered saline (21 mM HEPES, pH 7.05, 137 mM NaCI, 5 mM KCI, 0.7 mM N azH P O 4, 6 mM glucose) and resuspended later on in the same buffer at a final concentration of 2 x 106 cells/ml. 800 /~1 of this cell suspension was placed in a disposable electroporation cham- ber (Bio-Rad Gene Pulser cuvette, 0.4 cm) with EcoRI and storage buffer (SB) (50 mM KCI, 10 mM Tris, 0.1 mM EDTA, 1 mM dithiothreitol. 0.5 m g /m l bovine serum albumin, 50% glycerol pH 7.5) up to a final volume of 20 /~l, except the control cells that only received 20 /L1 of storage buffer. The total volume in the chamber (cell suspension + RE + SB) was 820 /xl. Our electro- poration conditions were: room temperature, field strength 750 V / c m (indicated voltage of 300, 0.4 cm electrode gap), and capacitance set at 960/~F.
After electroporation, cells were held on ice for 5-10 min and, subsequently, the cell suspensions were removed from the electroporation chamber and placed into 6-well Falcon plates with 5 ml of fresh medium. The cells were cultured for 20 h after the treatment with the restriction enzyme and, subsequently, exposed to Colcemid (2 x 10 v M final concentration) for 3 h. Cells were treated with hypotonic 0.075 M KC1 (2 min) and fixed in 2 washes with methanol-acetic acid (3:1). Pre- parations were stained finally in 3% Giemsa for 5-7 rain.
For aberration yields 100 metaphase spreads were analyzed, and chromosome a n d / o r chro- matid aberrations were scored. Achromatic lesions (gaps) were not included.
Results and discussion
It has been shown that EcoRI is able to induce a moderate frequency of chromosomal aberrations when introduced into mammalian cells with or without a permeabilizing agent (Bryant et al., 1987; Zhang and Dong, 1987; Darroudi and Natarajan, 1989; Bryant and Christie, 1989). Recently, it has been reported that cell electroporation is a rapid and highly efficient method for introducing RE into CHO cells (Winegar et al., 1989). Using elec-
TABLE 1
INDUCTION OF CHROMOSOMAL ABERRATIONS BY EcoRI IN AA8 CELLS
B', chromatid breaks; B", isochromatid breaks. a Electroporation experiments.
t ropora t ion , it has been also demons t r a t ed that E c o R I has cytogenet ic effect in C H O cells (Morgan et al., 1990).
As can be seen in Table 1, E c o R I did not induce ch romosoma l aber ra t ions in the pa ren t a l cell l ine A A 8 when the cells were exposed to di f ferent concent ra t ions of the enzyme (50, 100 and 150 units) in s torage buffer wi thout electro- pora t ion . However , when the enzyme was in t ro- duced into cells b y e lec t ropora t ion , the result was a concen t r a t ion -dependen t increase in the per- centage of abe r ran t metaphases . The aber ra t ions observed were bo th of the ch romosome type (di- centrics, r ings and ch romosome delet ions) and of the ch romat id type (chromat id breaks and ex-
changes). This m o d e r a t e increase in the yield of c h r o m o s o m a l abe r ra t ions induced by E c o R I in e l ec t ropora t ed A A 8 cells is consis tent with that r epo r t ed b y M o r g a n et al. (1990) in another Chinese ha ms t e r ovary cell l ine ( C H O - K 1 ) under s imilar exper imen ta l condi t ions , except that in our exper iments capac i t ance was set at 960 /~F ins tead of 1600/~F.
The results for the repa i r -def ic ien t EM9 cell l ine are p resen ted in Tab le 2. As can be seen, the s imple incuba t ion of t ryps in ized EM9 cells in the presence of E c o R I in the buffer was sufficient to induce abou t 3 t imes the c h romosome damage for any given dose of the enzyme observed in the pa ren ta l cell l ine A A 8 af ter e lec t ropora t ion . Elec-
TABLE 2
INDUCTION OF CHROMOSOMAL ABERRATIONS BY EcoRI IN EM9 CELLS
Treatment Percent Aberrations per 100 metaphases
aberrant B' B" Exchanges Dicentrics Rings HD metaphases Total
troporation of the mutant cells with or without the presence of EcoRI, on the other hand, con- sistently resulted in cell death.
Some reports have shown that RE can induce chromosomal aberrations in CHO cells or cultured human lymphocytes even in the absence of any permeabilizing agent (Obe et al., 1985, 1086; Obe and Natarajan, 1985). Vasudev and Obe (1988) obtained evidence for a surface receptor-mediated endocytosis of AluI in CHO cells. Nevertheless, the high degree of variability usually found in these experiments as well as in those using the traditional permeabilizing agents has made elec- troporation an interesting alternative (Winegar et al., 1989).
In spite of the high efficiency achieved by elec- troporation to introduce EcoRI in the parental cell line AA8 observed by us, which supports the idea of the usefulness of this method, the absence of mitosis consistently found after electroporation of EM9 mutant cells seems to indicate that there are important differences between different cell lines concerning their response to electroporation. In our opinion, on the other hand, the high dose- dependent frequency of chromosomal aberrations observed in EM9 cells in response to EcoRI without electroporation (Fig. 1) compared with the lack of any effect in the parental line AA8 could be due to either a much easier penetration of the RE into the mutant cells or important differences existing in the capacity to repair the
Fig. 1. EM9 cell showing multiple exchange-type aberrations induced by 100 U of EcoRI.
initial damage induced in DNA, or both. Never- theless, if we consider that electroporation is a highly efficient method for introducing RE into cells (Winegar et al., 1989) and that, for any given dose of the enzyme, the non-electroporated EM9 cells showed about 3 times the chromosome damage observed in the electroporated AA8 cells, our results seem to indicate that the mutant EM9 is highly sensitive to EcoRI.
Bryant et al. (1987) reported on the sensitivity of the X-ray-sensitive mutant xrs 5 to chro- mosome damage induced by PvuII and EcoRV (which induce blunt-ended dsb), on the one hand, and to BamHI and EcoRI (which induce cohe- sive-ended dsb), on the other. The result was that xrs 5 showed higher frequencies of CA after PvuII, EcoRV and BamHI treatments than did its nor- mal parental line C H O K1, while no differences were observed after EcoRI treatment. More re- cently, however, Darroudi and Natarajan (1989) have shown that both X-ray-sensitive mutant cell strains xrs 5 and xrs 6 are more sensitive to cytogenetic damage (1.5-2-fold) produced by RE which induce either blunt-ended D N A dsb (HaeIlI and AluI) or cohesive-ended dsb (CfoI, EcoRI and HpaII) and concluded that such an increase in the frequency of chromosomal aberra- tions is likely to be due to a defect in the rejoining of induced dsb in these mutants.
EM9 has also been reported to be highly sensi- tive to killing by X-rays and has been shown to be defective in the rejoining of the DNA-strand breaks induced by X-rays (Thompson et al., 1982). Though there are no data available on the propor- tion of the X-ray-induced dsb having blunt- or cohesive-ended structures, cohesive-ended dsb are considered to be likely to dominate since the prob- ability of frank breakages of the D N A comple- mentary strands at sites exactly opposite one another would be very low (Bryant and Christie, 1989). In our opinion, this proposal makes our results on the effectiveness of EcoRI in the X- ray-sensitive CHO mutant EM9 specially signifi- cant, because they show a correlation between the induction of chromosome damage by either RE or X-rays and an apparent defect in the repair of cohesive-ended dsb.
The molecular mechanism of repair of either radiation-induced or RE-induced dsb in mam-
m a l i a n cel ls is l a rge ly u n k n o w n , b u t i t is poss ib le
t ha t c o h e s i v e - e n d e d dsb c o u l d be c o n v e r t e d in to
b l u n t - e n d e d ones v ia e x o n u c l e a s e d iges t ion ( C o x
et al., 1984, 1986; D e b e n h a m et al., 1987). I t
b e c o m e s e v i d e n t tha t m o r e ex tens ive s tudies at
b o t h the c y t o g e n e t i c a n d the m o l e c u l a r level m a k -
ing use of m a m m a l i a n m u t a n t cel ls sens i t ive to R E
a re needed .
Acknowledgements
W e thank Drs . W i l l i a m F. M o r g a n ( U n i v e r s i t y
o f Ca l i fo rn ia , San F r a n c i s c o ) and L a r r y H.
T h o m p s o n ( L a w r e n c e L i v e r m o r e N a t i o n a l L a b o -
ra to ry) for the i r s u p p o r t a n d e n c o u r a g e m e n t . T h e
t echn ica l ass i s tance of J .A. R o j a n o is ve ry m u c h
apprec i a t ed .
Th is r e sea rch was s u p p o r t e d by funds f r o m the
J u n t a de A n d a l u c i a (Spain) .
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