Mutation Research, 262 (1991) 31-36 Elsevier
31
MUTLET 442
Role of D N A polymerase e and 6 in radiation clastogenesis* M i c h a e l A B e n d e r a, R u t h C. M o o r e b a n d Beatrice E. P y a t t a "Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) and bThe Cancer Institute, Peter MacCallum Hospital. Melbourne (Australia) (Received 8 June 1990) (Revision received 15 August 1990) (Accepted 28 August 1990)
Keywords. DNA polymerases; Radiation clastogenesis; Aphidicolin
We established some time ago, by experiments in which the DNA-synthesis inhibitor aphidicolin (APC) was used as a post-treatment following Xirradiation (Bender and Preston, 1982), the involvement of a DNA polymerase in the repair of DNA damage induced by ionizing radiation in human and other mammalian cells which gives rise, if not repaired, to chromosomal aberrations. We have observed a strong interaction in cells in the Go, G~ and G2 phases of the cell cycle (Bender, 1985, 1989; Moore and Bender, 1987; Moore, RandeU and Bender, 1988), as have others (van Zeeland et al., 1982). Interestingly, despite the large increases in the frequencies of other aberration types, the presence of aphidicolin after irradiation completely prevented the formation of any *Research supported by the U.S. Department of Energy Contract No. DE-ACO2-76CH00016 with Associated Universities, Inc.; accordingly, by acceptance of this article, the publisher and/or recipient acknowledges the U.S. Government's right to retain a r~onexclusive, royalty-free license in and to any copyright covering this paper. Correspondence: Michael A Bender, Medical Department, Brookhaven National Laboratory, Upton, NY 19973-5000
(U.S.A.).
exchange-type aberrations in G2 cells. Because aphidicolin inhibits DNA pol c~, but not pol j3 or pol 7, we originally attributed these effects to pol c~. However, it has since been established both that there is a fourth mammalian DNA polymerase, pol 6, and that this enzyme is also inhibited by aphidicolin, raising the question of the respective roles of the two enzymes in the cytogen~tic phenomena. It has been shown that pol c~ is specifically inhibited by certain butylphenyl and butylamine nucleoside and nucleotide derivatives. In particular, N2-(p-n-butylphenyl)-9-(2-deoxy-~-D-ribo furanosyl)guanine (BuPdG) and 2(p-n-butylanaline)-6-methoxypurine (BuAOMe) have been shown to be effective inhibitors of DNA synthesis in intact mammalian cells (Wright et al., 1980, 1982; Khan et al., 1984; Wright et al., 1987), and the triphosphates have been shown to specifically inhibit pol a, but not to inhibit pol 6 effectively (Lee et al., 1985; Wright et al., 1987). The existence of such specific pol c~ inhibitors offers the possibility of determining the relative roles of pol c~and pol ~5in preventing and/or producing chromosomal aberrations from DNA damage induced by ionizing radiation. We here report the results of ex-
0165-7992/91/$ 03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
32 periments in which human peripheral blood lymphocytes (HPBL) were X-irradiated in shortterm in vitro culture during either the Go or the Gz phases of the cell cycle, and then post-treated with BuPdG or BuAOMe. Materials and methods
Fresh, heparinized, venous blood samples were obtained from healthy adult volunteers. Shortterm lymphocyte cultures were made by adding 1 ml of blood to 10 ml of culture medium RPMI 1640 containing L-glutamine, penicillin and streptomycin and 15% heat-inactivated fetal calf serum (Gibco) in 15 ml, conical, disposable, capped centrifuge tubes. To stimulate cell division, 0.3 ml phytohemagglutinin (PHA; Gibco) was added. Cultures were incubated, slanted, with their caps sealed, at 37°C. Colcemid (Gibco) was added (to a level of 0.1 mg/ml) 2 h prior to fixation. Cultures were fixed in 3:1 methanol:glacial acetic acid following a 15-min hypotonic treatment with 75 mM KC1. Drops of fixed cell suspension were spread on the surfaces of wet glass slides, dried, and stained with 10% Giemsa for 10 min. Scoring and aberration classification followed standard cytogenetic methodology (Bender et al., 1988). BuPdG and BuAOMe were obtained from Dr. George E. Wright of the Department of Pharmacology of the University of Massachusetts Medical School. APC (Sigma) was used as a positive control in some experiments. The drugs were dissolved in dimethyl sulfoxide (DMSO). For cytogenetic experiments, 0.1 ml of 5 X 1 0 -3 M solution of the drug was added per culture, giving a final concentration of 5 × 10- 5 M of drug and 1°70 DMSO, the level also used for solvent controls. Where required (i.e., for Go experiments) the drugs a n d / o r solvent were removed after 2 h by washing twice with prewarmed complete medium and adding P H A prior to reincubation in fresh, prewarmed medium. As a basis for effectiveness comparison, measurements were made of normal DNA synthesis inhibition in H P B L by BuPdG and by
BuAOMe. For these determinations the interphase mononuclear cell layer was harvested from centrifugates of heparinized whole blood from 4 healthy donors in lymphocyte separation medium (Bionetics Laboratory Products), washed twice in phosphate-buffered saline containing 0.3 mM E D T A and suspended in complete culture medium. The suspensions were diluted to give 10 6 cells per ml, and 0.2 ml was inoculated per well into 96-well microtitration plates (Linbro). The plates were incubated at 37°C in a 5% CO2 atmosphere. At 72 h, 2 ~1 of the appropriate 100 x solution of BuPdG or of BuAOMe in DMSO (or of DMSO alone) was added to 3 wells per concentration to be tested. 0.5 h later tritiated thymidine (25 Ci/mM; Amersham) was added to a level of 5 ~Ci per well. 2 h later, the cells were harvested from the wells by collection on glass fiber mats (Flow Laboratories) with a vacuum cell-harvesting device (Skatron, Inc.), washed with water and then absolute methanol, and allowed to air dry. The mats were placed in counting vials with Aquasol II (New England Nuclear) and scintillation counted in a Beckman Model LS7800. Total counts, corrected for background, were expressed as percentage of the corrected counts from PHAstimulated solvent controls. X-Irradiations were done with a G.E. Maxitron 3200 machine operated at 250 KVp and 30 mA, with 0.5 mm Cu and 1.0 mm AI added filtration. Exposure rates were about 85 R per rain, as measured with a Victoreen condenser R meter. Go cultures were exposed to 200 R and G2 cultures to 100 R, in both cases at room temperature. For G2 experiments cultures were treated with drugs or solvent after 69.5 h incubation, allowed to equilibrate at 37°C, and irradiated at 70 h incubation. Colcemid was added immediately and the cultures fixed 2 h later after 72 h in culture. For Go experiments, the drugs or solvent were added prior to P H A addition, the cultures equilibrated at 37°C for 0.5 h and then irradiated. Immediately after, P H A was added and the cultures incubated for 2 h prior to being washed as described above. All cultures except those getting APC plus X-rays were fixed after 52 h, the last two in the presence of colcemid. The APC + X-ray cultures were fixed later,
33
earlier measurements of inhibition of D N A synthesis in H P B L by A P C (Bender and Moore, 1988). The 3 curves do not appear markedly different, although there does appear to be less of a shoulder for BuAOMe. It thus seems that BuPdG, BuAOMe, and A P C all produce inhibition of normal D N A synthesis in H P B L on an equimolar basis, at least in the region of dose of interest here around 5 x 10- 5 M.
\ \ \~
•
\ \
8
PREVIOUS APC PR DATA-SAME MOLAR BASIS
\ \\
\
rr
F-
z
\
8
\
o') 10 o") _ LU -r
\ \,
\ \
I-7
>-
\ \
i
\ \ \
i i
1
\
1 .6 10
~
100.4
10 3
0 2
1
BuPdG or BuAOMe CONCENTRATION ( M )
Fig. 1. Inhibition of, D N A synthesis in H P B L in P H A stimulated culture. Each point is the average of 3 determinations on each of 4 different blood donors. Open circles, BuPdG; closed circles, BuAOMe; the solid and dash-dot lines were drawn by inspection; the dashed line is a similarly-derived curve obtained earlier for H P B L from 9 subjects.
at 54 h, because of our prior experience with division delay in cultures so treated. Results
Inhibition o f normal DNA synthesis. The results for the 4 donors were not significantly different for either drug and so were pooled for each drug. The resulting points are plotted in Fig. 1, together with 'eyeball' lines fitted through the points on the exponential portion of the curve. The dashed line shows the curve similarly fitted for our
G2 experiments with BuPdG. Two separate experiments were done, each with two donors. Two scorers analyzed 150 and up to 100 cells each, respectively, for each donor for each treatment (except for the A P C positive control, which was done in only one of the two experiments). The results for none of the four subjects were quantitatively different. Accordingly, Table 1 presents the pooled results from the two experiments. It will be seen that at 5 x 10 -5 M, a level that produced a reduction of about 80°7o in normal D N A synthesis, there is a small, but statistically significant, elevation in the X-ray-induced frequencies of achromatic lesions, chromatid deletions, and isochromatid deletions in BuPdG post-treated cells over those in X-rayed cells post-treated with D M S O alone. There is no significant increase in aberration frequency with BuPdG alone. The aberration frequencies seen with A P C post-treatment are clearly elevated, just as we have seen in earlier experiments, but the degree of interaction with A P C is clearly much greater than with BuPdG on a molar basis. Also, although the usual total elimination of chromatid exchanges by A P C posttreatment is clearly evident, no such elimination is seen with BuPdG post-treatment. In fact, although there is no numerical increase in their frequency with BuPdG post-treatment, neither is an increase in their frequency over that with DMSO posttreatment of the order of 20°70 excluded. Also, sister union isochromatid-deletion types were seen in the BuPdG post-treated cultures, but none were observed in the A P C post-treated cultures. Go experiments with BuPdG.
Three separate experiments were done, with two donors for one,
34
TABLE 1 INTERACTION
OF 2 h POST-TREATMENT
W I T H 5 × 10 5 M B u P d G W I T H X - I R R A D I A T I O N
(100 R) O F Gz H P B L
Two experiments with two donors each pooled. Frequency of aberrations per cell + Poisson errors. Treatment
Number of cells scored
Chromatid types Achromatic lesions
Deletions
Isochromatid deletions
Exchanges
D M S O solvent control
1000
0 . 0 6 _+ 0.01
0 . 0 2 +_ 0.01
0.01 _+ 0.01
BuPdG
1000
0.13 + 0.01
0 . 0 4 _+ 0.01
0.02 + 0.01
X-ray + DMSO
930
1.47 _+ 0 . 0 4
2.39 _+ 0.05
0.12 _+ 0.01
0.17 +_ 0.01
X-ray + BuPdG
978
1.70 _+ 0 . 0 4
2.75 _+ 0.05
0.17 + 0.01
0.17 _+ 0.01
(1.2x) A P C pos. a control
488
(1.2×)
4 . 8 9 _+ 0 . 1 0
(1.4×)
8.97 _+ 0 . 1 4
(3.3 x )
(1 x )
0.57 +_ 0.03
(3.8×)
(4.8 x )
a R u n o n l y with 2nd E x p t .
and one each in the others. The A P C positive control was done only for the one donor in the second. Both scorers contributed to scoring the first experiment, but only one to the other two. The results for the two scorers agreed closely. Again, the results for the different donors and experiments were not significantly different, and so only the pooled results are shown in Table 2. It will be seen that although we observed the
degree of interaction we have noted before for the chromosome-type aberrations induced by Go Xirradiation, there is no evidence for any increased frequency of aberrations in the cultures posttreated with BuPdG when compared with those post-treated with DMSO. Also, as with the G2 e x p e r i m e n t s , there is no evidence for any clastogenic effect of BuPdG alone.
TABLE 2 LACK OF EFFECT OF 2 h POST-TREATMENT
WITH 5 × 10- 5 M BuPdG FOLLOWING
X-IRRADIATION
(200 R) O F Go H P B L
Three experiments. Four subjects pooled. Frequency per cell _+ Poisson error. Treatment
Number of
Chromatid type
cells scored a
Achromatic
Chromosome type Deletions
Exchanges
Deletions
lesions DMSO control
1000
0.06 _+ 0.01
0.01 _+ 0.01
-
0.01 + 0.01
BuPdG
1000
0.08 +_ 0.01
0 . 0 4 _+ 0.01
-
0.01 ± 0.01
X-ray + DMSO
1000
0 . 0 6 _+ 0.01
0.05 _+ 0.01
X-ray + BuPdG
1000
0.08 _+ 0.01
0.05 _+ 0.01
200
0 . 2 2 _+ 0.03
0 . 0 6 _+ 0.02
APC pos. control b
Dicentrics and rings -
0 . 2 0 _+ 0.01
0 . 2 4 _+ 0.02
0.01 _+ 0.01
0.18 +_+ 0.01
0 . 2 2 _+ 0.02
0.01 _+ 0.01
0.42 _+ 0.05
0 . 5 0 _+ 0.05
(2.3 x )
(2.9x)
a 2 5 0 each donor in E x p t . 1; 300 for the donor in E x p t . 2; 200 for that in E x p t . 3. b R u n concurrently with same donor in E x p t . 2.
35
G2 experiments with BuAOMe. To further test whether the slight increase in G 2 aberration yields with B u P d G post-treatment shown in Table 1 really implicated pol a, we did additional G 2 e x periments with the second pol c¢ inhibitor, BuAOMe. Two experiments were done, one with one donor's lymphocytes and one with lymphocytes from two other donors. 150 metaphases f r o m each donor were scored (by a single observer). The results were not different between donors or experiments, and the data were consequently pooled. The results are shown in Table 3. In contrast to the results with BuPdG, it is clear that there is no increased X-ray-induced yield of either chromatid deletions or isochromatid deletions with posttreatment with 5 x 10-5 M BuAOMe. While the yields of achromatic lesions and of chromatid exchanges were numerically higher with BuAOMe post-treatment, neither difference is statistically significant by Student's t test at the p = 0.01 level. Also, there is no indication of any aberration induction by BuAOMe alone in the treated cells. Discussion The results we obtained with BuPdG seem to indicate clearly that,at least the bulk of the interaction with X-irradiation we have noted earlier for
A P C is attributable to pol 6, not to pol a as we had earlier supposed. The Gz interaction we observe after inhibiting pol a specifically with BuPdG, if real, is very much smaller than that seen with the same level of APC: of the order of 20%, as opposed to approx. 4-fold. The negative results of our G2 experiments with another specific pol o~ inhibitor, BuAOMe, make it seem very likely that the small excess seen with BuPdG is idiosyncratic, possibly representing a tendency of this drug at 5 x 10 -5 M to slightly inhibit pol 6 in G2 (although not Go) cells. It has been suggested that pol o~and pol 6 act during normal 'semiconservative' synthesis as a coordinated complex, with pol o~, which lacks any 3' to 5' exonuclease activity and is not at all processive, synthesizing the lagging strand, and pol 6, which is processive and has a 3' to 5' exonuclease activity, synthesizing the leading strand (Blow, 1987). It has also been shown that pol 6 is complexed with cyclin during the S phase, but not in Go or early G1 (Bravo et al., 1987). Thus if the proposed complex also worked to eliminate whatever class of D N A damage causes the interactions we have observed in G2 cells, it seems not inconceivable that inhibiting one or the other polymerase alone might be much less effective than inhibiting both together. The lack of any sign of prevention of the forma-
TABLE 3 LACK OF EFFECT OF BuAOMe ON CHROMATID 3 DONORS,
ABERRATIONS
IN H P B L G I V E N 100 R O F X - R A Y S IN G2 ( T W O E x p t s ,
150 C E L L S P E R P O I N T )
F r e q u e n c i e s a r e per cell _+ P o i s s o n e r r o r s . Treatment
N cells
DMSO control
450
Achromatic
Chromatid
Isochromatid
Chromatid
Chromatid
lesions
deletions
deletions
exchanges
rings
19 0 . 0 4 + 0.01
BuAOMe
450
11 0 . 0 2 _+ 0.01
DMSO
+ X-rays
450
808 1.80 + 0 . 0 6
BuAOMe
+ X-rays
450
905 2.01 +__ 0.07
4 0.01 _+ 0.01 5 0.01 _+ 0.01 1210 2.69 + 0.08 1201 2.67 +__ 0.08
4 0.01 +_ 0.01 4 0.01 +_ 0.01 41 0 . 0 9 _ 0.01 43 0 . 1 0 +_ 0 . 0 2
0
0
-
-
0
0
-
-
45
0
0 . 1 0 _+ 0 . 0 2 72 0 . 1 6 +_. 0 . 0 2
4 0.01 + 0.01
36
tion of exchange-type aberrations in G2 by inhibition of pol c~ by BuPdG or by BuAOMe seems to implicate pol 6 alone in the production of this effect by APC, although, again, we cannot presently rule out the possibility that the putative complex is required for exchanges. However, the different effect of inhibiting only pol c~ from that observed when both polymerases are inhibited does appear to strengthen the conclusion that they are indeed separate enzymes, despite their similarities. The total lack of evidence for any influence of pol c~ in irradiated Go H P B L in the present experiments was not entirely unanticipated. Recently, Sylvia et al. (1988) showed that the pol c~ present in unstimulated Go H P B L is of very low specific activity. This inactive form of the polymerase is activated by a phosphatidyl inositol 'second messenger' system in which inositol bisphosphate (IP2) is released by phospholipase C when an agonist such as P H A is bound. The IP2 reacts with the low specific activity pol ~, effecting release of diacylglycerol and retention of inositol-l,4-bisphosphate by the enzyme, thus somehow activating it. Sylvia et al. have suggested that the increased specific activity may be a function of increased DNA-binding affinity. If this is so, it is not unexpected that unactivated pol ~ might simply be unable to react with X-ray-induced DNA lesions.
Acknowledgement We thank Dr. George E. Wright for generously supplying us with BuPdG and BuAOMe for these experiments.
References Bender, M.A (1985) Role of DNA polymerase c~ in chromosomal aberration production by ionizing radiation, Am. N.Y. Acad. Sci., 457, 245-254. Bender, M.A (1989) Time course of enhancement of chromosomal aberration production in human lymphocytes by post-treatment with aphidicolin following X-irradiation in G2. Mutation Res., in press. Bender, M.A, and R.C. Moore (1988) Dose effects of aphidicolin in human peripheral blood leukocytes, Mutation Res., 198, 227-281.
Bender, M.A, and R.J. Preston (1982) Role of base damage in aberration formation: interaction of aphidicolin and X-rays, Prog. Mutation Res., 4, 37-45. Bender, M.A, R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch and M.D. Shelby (1988) Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample, Mutation Res., 204, 421-433. Blow, J. (1987) DNA Replication, Many strands converge, Nature (London), 326, 441-442. Bravo, R., R. Frank, P.A. Blundell and H. Macdonald-Bravo (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase-6, Nature (London), 326, 515-517. Khan, N.N., G.E. Wright, L.W. Dudycz and N.C. Brown (1984) Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha, Nucleic Acids Res., 12, 3695-3706. Lee, M.Y.W.T., N.L. Toomey and G.E. Wright (1985) Differential inhibition of human placental DNA polymerase 6 and c~ by BuPdGTP and BuAdATP, Nucleic Acids Res., 13, 8623-8630. Moore, R.C., and M.A Bender (1987) The synergistic effect of aphidicolin on the yield of X-ray-induced chromosome aberrations throughout the cell cycle in JU56 cells, Radiat. Res., 110, 385-395. Moore, R.C., C. Randell and M.A Bender (1988) An investigation using inhibition of G2 repair of the molecular basis of lesions which result in chromosomal aberrations, Mutation Res., 199, 229-233. Sylvia, V., G. Curtin, J. Norman, J. Stec and D. Busbee (1988) Activation of a low specific activity form of DNA polymerase c~ by inositol-l,4-biphosphate, Cell, 54, 651-659. van Zeeland, A.A., C.J.M. Bussman, F. Degrassi, A.R. Filon, A.C. van Kesteren-van Leeuwen, F. Palitti and A.T. Natarajan (1982) Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells, Mutation Res., 92, 379-392. Wright, G.E., E.F. Baril and N.C. Brown (1980) Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha, Nucleic Acids Res., 8, 99-109. Wright, G.E., E.F. Baril, V.M. Brown and N.C. Brown (1982) Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase Ill, Nucleic Acids Res., 10, 4431-4440. Wright, G.E., L.W. Dudycz, Z. Kazimierczuk, N.C. Brown and N.N. Khan (1987) Synthesis, cell growth inhibition, and antitumor screening of 2-(p-n-butylanilino)purines and their nucleoside analogues, J. Med. Chem., 30, 109-116. Communicated by F.H. Sobels
31
MUTLET 442
Role of D N A polymerase e and 6 in radiation clastogenesis* M i c h a e l A B e n d e r a, R u t h C. M o o r e b a n d Beatrice E. P y a t t a "Medical Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) and bThe Cancer Institute, Peter MacCallum Hospital. Melbourne (Australia) (Received 8 June 1990) (Revision received 15 August 1990) (Accepted 28 August 1990)
Keywords. DNA polymerases; Radiation clastogenesis; Aphidicolin
We established some time ago, by experiments in which the DNA-synthesis inhibitor aphidicolin (APC) was used as a post-treatment following Xirradiation (Bender and Preston, 1982), the involvement of a DNA polymerase in the repair of DNA damage induced by ionizing radiation in human and other mammalian cells which gives rise, if not repaired, to chromosomal aberrations. We have observed a strong interaction in cells in the Go, G~ and G2 phases of the cell cycle (Bender, 1985, 1989; Moore and Bender, 1987; Moore, RandeU and Bender, 1988), as have others (van Zeeland et al., 1982). Interestingly, despite the large increases in the frequencies of other aberration types, the presence of aphidicolin after irradiation completely prevented the formation of any *Research supported by the U.S. Department of Energy Contract No. DE-ACO2-76CH00016 with Associated Universities, Inc.; accordingly, by acceptance of this article, the publisher and/or recipient acknowledges the U.S. Government's right to retain a r~onexclusive, royalty-free license in and to any copyright covering this paper. Correspondence: Michael A Bender, Medical Department, Brookhaven National Laboratory, Upton, NY 19973-5000
(U.S.A.).
exchange-type aberrations in G2 cells. Because aphidicolin inhibits DNA pol c~, but not pol j3 or pol 7, we originally attributed these effects to pol c~. However, it has since been established both that there is a fourth mammalian DNA polymerase, pol 6, and that this enzyme is also inhibited by aphidicolin, raising the question of the respective roles of the two enzymes in the cytogen~tic phenomena. It has been shown that pol c~ is specifically inhibited by certain butylphenyl and butylamine nucleoside and nucleotide derivatives. In particular, N2-(p-n-butylphenyl)-9-(2-deoxy-~-D-ribo furanosyl)guanine (BuPdG) and 2(p-n-butylanaline)-6-methoxypurine (BuAOMe) have been shown to be effective inhibitors of DNA synthesis in intact mammalian cells (Wright et al., 1980, 1982; Khan et al., 1984; Wright et al., 1987), and the triphosphates have been shown to specifically inhibit pol a, but not to inhibit pol 6 effectively (Lee et al., 1985; Wright et al., 1987). The existence of such specific pol c~ inhibitors offers the possibility of determining the relative roles of pol c~and pol ~5in preventing and/or producing chromosomal aberrations from DNA damage induced by ionizing radiation. We here report the results of ex-
0165-7992/91/$ 03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
32 periments in which human peripheral blood lymphocytes (HPBL) were X-irradiated in shortterm in vitro culture during either the Go or the Gz phases of the cell cycle, and then post-treated with BuPdG or BuAOMe. Materials and methods
Fresh, heparinized, venous blood samples were obtained from healthy adult volunteers. Shortterm lymphocyte cultures were made by adding 1 ml of blood to 10 ml of culture medium RPMI 1640 containing L-glutamine, penicillin and streptomycin and 15% heat-inactivated fetal calf serum (Gibco) in 15 ml, conical, disposable, capped centrifuge tubes. To stimulate cell division, 0.3 ml phytohemagglutinin (PHA; Gibco) was added. Cultures were incubated, slanted, with their caps sealed, at 37°C. Colcemid (Gibco) was added (to a level of 0.1 mg/ml) 2 h prior to fixation. Cultures were fixed in 3:1 methanol:glacial acetic acid following a 15-min hypotonic treatment with 75 mM KC1. Drops of fixed cell suspension were spread on the surfaces of wet glass slides, dried, and stained with 10% Giemsa for 10 min. Scoring and aberration classification followed standard cytogenetic methodology (Bender et al., 1988). BuPdG and BuAOMe were obtained from Dr. George E. Wright of the Department of Pharmacology of the University of Massachusetts Medical School. APC (Sigma) was used as a positive control in some experiments. The drugs were dissolved in dimethyl sulfoxide (DMSO). For cytogenetic experiments, 0.1 ml of 5 X 1 0 -3 M solution of the drug was added per culture, giving a final concentration of 5 × 10- 5 M of drug and 1°70 DMSO, the level also used for solvent controls. Where required (i.e., for Go experiments) the drugs a n d / o r solvent were removed after 2 h by washing twice with prewarmed complete medium and adding P H A prior to reincubation in fresh, prewarmed medium. As a basis for effectiveness comparison, measurements were made of normal DNA synthesis inhibition in H P B L by BuPdG and by
BuAOMe. For these determinations the interphase mononuclear cell layer was harvested from centrifugates of heparinized whole blood from 4 healthy donors in lymphocyte separation medium (Bionetics Laboratory Products), washed twice in phosphate-buffered saline containing 0.3 mM E D T A and suspended in complete culture medium. The suspensions were diluted to give 10 6 cells per ml, and 0.2 ml was inoculated per well into 96-well microtitration plates (Linbro). The plates were incubated at 37°C in a 5% CO2 atmosphere. At 72 h, 2 ~1 of the appropriate 100 x solution of BuPdG or of BuAOMe in DMSO (or of DMSO alone) was added to 3 wells per concentration to be tested. 0.5 h later tritiated thymidine (25 Ci/mM; Amersham) was added to a level of 5 ~Ci per well. 2 h later, the cells were harvested from the wells by collection on glass fiber mats (Flow Laboratories) with a vacuum cell-harvesting device (Skatron, Inc.), washed with water and then absolute methanol, and allowed to air dry. The mats were placed in counting vials with Aquasol II (New England Nuclear) and scintillation counted in a Beckman Model LS7800. Total counts, corrected for background, were expressed as percentage of the corrected counts from PHAstimulated solvent controls. X-Irradiations were done with a G.E. Maxitron 3200 machine operated at 250 KVp and 30 mA, with 0.5 mm Cu and 1.0 mm AI added filtration. Exposure rates were about 85 R per rain, as measured with a Victoreen condenser R meter. Go cultures were exposed to 200 R and G2 cultures to 100 R, in both cases at room temperature. For G2 experiments cultures were treated with drugs or solvent after 69.5 h incubation, allowed to equilibrate at 37°C, and irradiated at 70 h incubation. Colcemid was added immediately and the cultures fixed 2 h later after 72 h in culture. For Go experiments, the drugs or solvent were added prior to P H A addition, the cultures equilibrated at 37°C for 0.5 h and then irradiated. Immediately after, P H A was added and the cultures incubated for 2 h prior to being washed as described above. All cultures except those getting APC plus X-rays were fixed after 52 h, the last two in the presence of colcemid. The APC + X-ray cultures were fixed later,
33
earlier measurements of inhibition of D N A synthesis in H P B L by A P C (Bender and Moore, 1988). The 3 curves do not appear markedly different, although there does appear to be less of a shoulder for BuAOMe. It thus seems that BuPdG, BuAOMe, and A P C all produce inhibition of normal D N A synthesis in H P B L on an equimolar basis, at least in the region of dose of interest here around 5 x 10- 5 M.
\ \ \~
•
\ \
8
PREVIOUS APC PR DATA-SAME MOLAR BASIS
\ \\
\
rr
F-
z
\
8
\
o') 10 o") _ LU -r
\ \,
\ \
I-7
>-
\ \
i
\ \ \
i i
1
\
1 .6 10
~
100.4
10 3
0 2
1
BuPdG or BuAOMe CONCENTRATION ( M )
Fig. 1. Inhibition of, D N A synthesis in H P B L in P H A stimulated culture. Each point is the average of 3 determinations on each of 4 different blood donors. Open circles, BuPdG; closed circles, BuAOMe; the solid and dash-dot lines were drawn by inspection; the dashed line is a similarly-derived curve obtained earlier for H P B L from 9 subjects.
at 54 h, because of our prior experience with division delay in cultures so treated. Results
Inhibition o f normal DNA synthesis. The results for the 4 donors were not significantly different for either drug and so were pooled for each drug. The resulting points are plotted in Fig. 1, together with 'eyeball' lines fitted through the points on the exponential portion of the curve. The dashed line shows the curve similarly fitted for our
G2 experiments with BuPdG. Two separate experiments were done, each with two donors. Two scorers analyzed 150 and up to 100 cells each, respectively, for each donor for each treatment (except for the A P C positive control, which was done in only one of the two experiments). The results for none of the four subjects were quantitatively different. Accordingly, Table 1 presents the pooled results from the two experiments. It will be seen that at 5 x 10 -5 M, a level that produced a reduction of about 80°7o in normal D N A synthesis, there is a small, but statistically significant, elevation in the X-ray-induced frequencies of achromatic lesions, chromatid deletions, and isochromatid deletions in BuPdG post-treated cells over those in X-rayed cells post-treated with D M S O alone. There is no significant increase in aberration frequency with BuPdG alone. The aberration frequencies seen with A P C post-treatment are clearly elevated, just as we have seen in earlier experiments, but the degree of interaction with A P C is clearly much greater than with BuPdG on a molar basis. Also, although the usual total elimination of chromatid exchanges by A P C posttreatment is clearly evident, no such elimination is seen with BuPdG post-treatment. In fact, although there is no numerical increase in their frequency with BuPdG post-treatment, neither is an increase in their frequency over that with DMSO posttreatment of the order of 20°70 excluded. Also, sister union isochromatid-deletion types were seen in the BuPdG post-treated cultures, but none were observed in the A P C post-treated cultures. Go experiments with BuPdG.
Three separate experiments were done, with two donors for one,
34
TABLE 1 INTERACTION
OF 2 h POST-TREATMENT
W I T H 5 × 10 5 M B u P d G W I T H X - I R R A D I A T I O N
(100 R) O F Gz H P B L
Two experiments with two donors each pooled. Frequency of aberrations per cell + Poisson errors. Treatment
Number of cells scored
Chromatid types Achromatic lesions
Deletions
Isochromatid deletions
Exchanges
D M S O solvent control
1000
0 . 0 6 _+ 0.01
0 . 0 2 +_ 0.01
0.01 _+ 0.01
BuPdG
1000
0.13 + 0.01
0 . 0 4 _+ 0.01
0.02 + 0.01
X-ray + DMSO
930
1.47 _+ 0 . 0 4
2.39 _+ 0.05
0.12 _+ 0.01
0.17 +_ 0.01
X-ray + BuPdG
978
1.70 _+ 0 . 0 4
2.75 _+ 0.05
0.17 + 0.01
0.17 _+ 0.01
(1.2x) A P C pos. a control
488
(1.2×)
4 . 8 9 _+ 0 . 1 0
(1.4×)
8.97 _+ 0 . 1 4
(3.3 x )
(1 x )
0.57 +_ 0.03
(3.8×)
(4.8 x )
a R u n o n l y with 2nd E x p t .
and one each in the others. The A P C positive control was done only for the one donor in the second. Both scorers contributed to scoring the first experiment, but only one to the other two. The results for the two scorers agreed closely. Again, the results for the different donors and experiments were not significantly different, and so only the pooled results are shown in Table 2. It will be seen that although we observed the
degree of interaction we have noted before for the chromosome-type aberrations induced by Go Xirradiation, there is no evidence for any increased frequency of aberrations in the cultures posttreated with BuPdG when compared with those post-treated with DMSO. Also, as with the G2 e x p e r i m e n t s , there is no evidence for any clastogenic effect of BuPdG alone.
TABLE 2 LACK OF EFFECT OF 2 h POST-TREATMENT
WITH 5 × 10- 5 M BuPdG FOLLOWING
X-IRRADIATION
(200 R) O F Go H P B L
Three experiments. Four subjects pooled. Frequency per cell _+ Poisson error. Treatment
Number of
Chromatid type
cells scored a
Achromatic
Chromosome type Deletions
Exchanges
Deletions
lesions DMSO control
1000
0.06 _+ 0.01
0.01 _+ 0.01
-
0.01 + 0.01
BuPdG
1000
0.08 +_ 0.01
0 . 0 4 _+ 0.01
-
0.01 ± 0.01
X-ray + DMSO
1000
0 . 0 6 _+ 0.01
0.05 _+ 0.01
X-ray + BuPdG
1000
0.08 _+ 0.01
0.05 _+ 0.01
200
0 . 2 2 _+ 0.03
0 . 0 6 _+ 0.02
APC pos. control b
Dicentrics and rings -
0 . 2 0 _+ 0.01
0 . 2 4 _+ 0.02
0.01 _+ 0.01
0.18 +_+ 0.01
0 . 2 2 _+ 0.02
0.01 _+ 0.01
0.42 _+ 0.05
0 . 5 0 _+ 0.05
(2.3 x )
(2.9x)
a 2 5 0 each donor in E x p t . 1; 300 for the donor in E x p t . 2; 200 for that in E x p t . 3. b R u n concurrently with same donor in E x p t . 2.
35
G2 experiments with BuAOMe. To further test whether the slight increase in G 2 aberration yields with B u P d G post-treatment shown in Table 1 really implicated pol a, we did additional G 2 e x periments with the second pol c¢ inhibitor, BuAOMe. Two experiments were done, one with one donor's lymphocytes and one with lymphocytes from two other donors. 150 metaphases f r o m each donor were scored (by a single observer). The results were not different between donors or experiments, and the data were consequently pooled. The results are shown in Table 3. In contrast to the results with BuPdG, it is clear that there is no increased X-ray-induced yield of either chromatid deletions or isochromatid deletions with posttreatment with 5 x 10-5 M BuAOMe. While the yields of achromatic lesions and of chromatid exchanges were numerically higher with BuAOMe post-treatment, neither difference is statistically significant by Student's t test at the p = 0.01 level. Also, there is no indication of any aberration induction by BuAOMe alone in the treated cells. Discussion The results we obtained with BuPdG seem to indicate clearly that,at least the bulk of the interaction with X-irradiation we have noted earlier for
A P C is attributable to pol 6, not to pol a as we had earlier supposed. The Gz interaction we observe after inhibiting pol a specifically with BuPdG, if real, is very much smaller than that seen with the same level of APC: of the order of 20%, as opposed to approx. 4-fold. The negative results of our G2 experiments with another specific pol o~ inhibitor, BuAOMe, make it seem very likely that the small excess seen with BuPdG is idiosyncratic, possibly representing a tendency of this drug at 5 x 10 -5 M to slightly inhibit pol 6 in G2 (although not Go) cells. It has been suggested that pol o~and pol 6 act during normal 'semiconservative' synthesis as a coordinated complex, with pol o~, which lacks any 3' to 5' exonuclease activity and is not at all processive, synthesizing the lagging strand, and pol 6, which is processive and has a 3' to 5' exonuclease activity, synthesizing the leading strand (Blow, 1987). It has also been shown that pol 6 is complexed with cyclin during the S phase, but not in Go or early G1 (Bravo et al., 1987). Thus if the proposed complex also worked to eliminate whatever class of D N A damage causes the interactions we have observed in G2 cells, it seems not inconceivable that inhibiting one or the other polymerase alone might be much less effective than inhibiting both together. The lack of any sign of prevention of the forma-
TABLE 3 LACK OF EFFECT OF BuAOMe ON CHROMATID 3 DONORS,
ABERRATIONS
IN H P B L G I V E N 100 R O F X - R A Y S IN G2 ( T W O E x p t s ,
150 C E L L S P E R P O I N T )
F r e q u e n c i e s a r e per cell _+ P o i s s o n e r r o r s . Treatment
N cells
DMSO control
450
Achromatic
Chromatid
Isochromatid
Chromatid
Chromatid
lesions
deletions
deletions
exchanges
rings
19 0 . 0 4 + 0.01
BuAOMe
450
11 0 . 0 2 _+ 0.01
DMSO
+ X-rays
450
808 1.80 + 0 . 0 6
BuAOMe
+ X-rays
450
905 2.01 +__ 0.07
4 0.01 _+ 0.01 5 0.01 _+ 0.01 1210 2.69 + 0.08 1201 2.67 +__ 0.08
4 0.01 +_ 0.01 4 0.01 +_ 0.01 41 0 . 0 9 _ 0.01 43 0 . 1 0 +_ 0 . 0 2
0
0
-
-
0
0
-
-
45
0
0 . 1 0 _+ 0 . 0 2 72 0 . 1 6 +_. 0 . 0 2
4 0.01 + 0.01
36
tion of exchange-type aberrations in G2 by inhibition of pol c~ by BuPdG or by BuAOMe seems to implicate pol 6 alone in the production of this effect by APC, although, again, we cannot presently rule out the possibility that the putative complex is required for exchanges. However, the different effect of inhibiting only pol c~ from that observed when both polymerases are inhibited does appear to strengthen the conclusion that they are indeed separate enzymes, despite their similarities. The total lack of evidence for any influence of pol c~ in irradiated Go H P B L in the present experiments was not entirely unanticipated. Recently, Sylvia et al. (1988) showed that the pol c~ present in unstimulated Go H P B L is of very low specific activity. This inactive form of the polymerase is activated by a phosphatidyl inositol 'second messenger' system in which inositol bisphosphate (IP2) is released by phospholipase C when an agonist such as P H A is bound. The IP2 reacts with the low specific activity pol ~, effecting release of diacylglycerol and retention of inositol-l,4-bisphosphate by the enzyme, thus somehow activating it. Sylvia et al. have suggested that the increased specific activity may be a function of increased DNA-binding affinity. If this is so, it is not unexpected that unactivated pol ~ might simply be unable to react with X-ray-induced DNA lesions.
Acknowledgement We thank Dr. George E. Wright for generously supplying us with BuPdG and BuAOMe for these experiments.
References Bender, M.A (1985) Role of DNA polymerase c~ in chromosomal aberration production by ionizing radiation, Am. N.Y. Acad. Sci., 457, 245-254. Bender, M.A (1989) Time course of enhancement of chromosomal aberration production in human lymphocytes by post-treatment with aphidicolin following X-irradiation in G2. Mutation Res., in press. Bender, M.A, and R.C. Moore (1988) Dose effects of aphidicolin in human peripheral blood leukocytes, Mutation Res., 198, 227-281.
Bender, M.A, and R.J. Preston (1982) Role of base damage in aberration formation: interaction of aphidicolin and X-rays, Prog. Mutation Res., 4, 37-45. Bender, M.A, R.J. Preston, R.C. Leonard, B.E. Pyatt, P.C. Gooch and M.D. Shelby (1988) Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample, Mutation Res., 204, 421-433. Blow, J. (1987) DNA Replication, Many strands converge, Nature (London), 326, 441-442. Bravo, R., R. Frank, P.A. Blundell and H. Macdonald-Bravo (1987) Cyclin/PCNA is the auxiliary protein of DNA polymerase-6, Nature (London), 326, 515-517. Khan, N.N., G.E. Wright, L.W. Dudycz and N.C. Brown (1984) Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha, Nucleic Acids Res., 12, 3695-3706. Lee, M.Y.W.T., N.L. Toomey and G.E. Wright (1985) Differential inhibition of human placental DNA polymerase 6 and c~ by BuPdGTP and BuAdATP, Nucleic Acids Res., 13, 8623-8630. Moore, R.C., and M.A Bender (1987) The synergistic effect of aphidicolin on the yield of X-ray-induced chromosome aberrations throughout the cell cycle in JU56 cells, Radiat. Res., 110, 385-395. Moore, R.C., C. Randell and M.A Bender (1988) An investigation using inhibition of G2 repair of the molecular basis of lesions which result in chromosomal aberrations, Mutation Res., 199, 229-233. Sylvia, V., G. Curtin, J. Norman, J. Stec and D. Busbee (1988) Activation of a low specific activity form of DNA polymerase c~ by inositol-l,4-biphosphate, Cell, 54, 651-659. van Zeeland, A.A., C.J.M. Bussman, F. Degrassi, A.R. Filon, A.C. van Kesteren-van Leeuwen, F. Palitti and A.T. Natarajan (1982) Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells, Mutation Res., 92, 379-392. Wright, G.E., E.F. Baril and N.C. Brown (1980) Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha, Nucleic Acids Res., 8, 99-109. Wright, G.E., E.F. Baril, V.M. Brown and N.C. Brown (1982) Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase Ill, Nucleic Acids Res., 10, 4431-4440. Wright, G.E., L.W. Dudycz, Z. Kazimierczuk, N.C. Brown and N.N. Khan (1987) Synthesis, cell growth inhibition, and antitumor screening of 2-(p-n-butylanilino)purines and their nucleoside analogues, J. Med. Chem., 30, 109-116. Communicated by F.H. Sobels
