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Increased radioprotection by enhancement of enzymatic mechanisms
Pharmac. Ther. Vol. 39. pp. 287 to 292, 1988
0163-7258/88 $0.00+0.50 Copyright © 1988 Pergamon Press plc
Printed in Great Britain. All rights reserved
Symposium Editors: J. F. WEIss and M. G. SIMI¢
INCREASED RADIOPROTECTION BY E N H A N C E M E N T OF ENZYMATIC MECHANISMS ROBERT B. PAINTER laboratory of Radiobiology and EnvironmentalHealth and Department of Microbiology, Universityof California, San Francisco, California 94143, U.S.A.
1. DNA REPAIR SYSTEMS The principal method by which cells protect themselves from the deleterious effects of ionizing radiation is by means of their DNA repair systems. This is most easily demonstrated in bacteria and yeast, in which very large numbers and many kinds of radiation-sensitive mutants have been tound to be defective in one or more kinds of DNA repair. Higher eukaryotic ceils occasionally show naturally occurring defects in DNA repair; the best-known example is that of patients with the autosomal recessive disease xeroderma pigmentosum (XP). The cells from these patients are sensitive to ultraviolet (u.v.) radiation, not to ionizing radiation, because of their inability to excise u.v. photoproducts from their DNA. Cells from patients with another autosomal recessive disease, ataxia-telangiectasia (A-T), are hypersensitive to ionizing radiation; however, no consistent DNA repair defect has been found for this condition.
1.1. RADIOSENSITIVE MAMMALIAN CELL MUTANTS
In the last few years mammalian cells defective in DNA repair have been isolated in the laboratory. Generally, these mutants are first produced by exposure to a chemical mutagen and then separated from the rest of the population by observing the emergence of colonies that grow more slowly than the surrounding normal ones. Chinese hamster ovary (CHO) cells have been used almost exclusively up to now. The CHO mutants that are hypersensitive to ionizing radiation are, in general, defective in the repair of DNA strand breaks. A series of mutants isolated by Jeggo and collaborators (Jeggo and Kemp, 1983; Kemp et al., 1984) are defective in the repair of double-strand breaks. A mutant isolated by Stamato et al. (1983) is radiosensitive in G1 but not in late S/G2 and is defective in the repair of double-strand breaks in Gi but not in G2 (Giaccia et al., 1985). A mutant isolated by Thompson et al. (1982) is very sluggish in the repair of X-ray-induced single-strand breaks but is only moderately radiosensitive. It is obvious that these mutants will be valuable as tools to isolate and understand repair genes. The fact that mutants are radiosensitive because they have a defect in a repair system has been proved in some cases by complementation tests. For example, when different lines of u.v.sensitive human (XP) cells are fused, the heterokaryon formed often has the ability to carry out the excision of u.v. photoproducts (DeWeerd-Kastelein et al., 1972) and is normal in its resistance to u.v. (Cleaver, 1982). (If the heterokaryon does not regain the capacity for excision repair, it is always assumed that the two cells are in the same complementation group, i.e. that they have exactly the same DNA repair defect.) Similar results have been obtained for some Chinese hamster mutants that are hypersensitive to ionizing radiation (Jones et al., 1987; N.J. Jones, personal communication*). In some cases the normal repair genes were cloned and reintroduced into the mutant, which then attained the normal phenotype (Zdzienicka et al., 1987). Therefore, there is no doubt that these DNA repair enzymes are the principal factors that protect cells from the lethal effects of radiation. *Published with permission of N. J. Jones. 287
288
R . B . PAINTER
4O
,o
o o--'
T
1o,
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1o,
Concentration [3H]dThd(iJCi/ml)
1o,
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FIG. 1. The induction of chromatid breaks (chromatid and isochromatid deletions) in human lymphocytes: (A) breaks induced by exposure to [3H]thymidine alone; ( • ) X-ray-induced breaks after exposure to [3H]thymidine + 1.5 Gy X-rays; (11) X-ray-induced breaks after exposure to [3H]thymidine + 1.5 Gy X-rays + 2 mM 3-aminobenzamide (3AB). Two hundred cells per point were scored. The vertical bars denote standard error of the mean. To calculate the number of induced breaks, the number of background breaks and (for combined treatments) the number of breaks induced by [3H]thymidine or 3AB alone was subtracted from the total number of aberrations scored. (Reproduced from Wiencke et al., 1986, with permission of IRL Press Ltd.)
2. POTENTIAL FOR RADIOPROTECTION BY ENHANCEMENT OF ENZYMATIC MECHANISMS For the purposes of this meeting, I wish to pose the question, is it possible to increase the endogenous activity of repair enzymes or other radioprotective enzymes so that an individual can become more resistant than normal? In theory this could be brought about by any one of several means. The first, and perhaps the most feasible at this time, is by induction of an enzymatic process whose activity can be altered by the manipulation of environmental conditions. An example of this is the so-called adaptive response described first by Olivieri et al. (1984) and elaborated upon by Wolff and collaborators (Wiencke et al., 1986; Shadley and Wolff, 1987). In this case, very small doses of ionizing radiation, administered to lymphocytes either exogenously as X-rays or intracellularly in the form of [3H]thymidine, decrease the clastogenic effect of high doses of X-rays administered later. The adapting effect of radiation can be at doses so low that they themselves cause no measurable increase in aberration frequency (Fig. 1). It is almost certain that this protection is the result of an increase in the amount or activity of some repair system. Because chromosome aberrations are generally considered to be the cause of most radiation-induced cell death, the effect of protection against aberrations would be expected to be manifested as protection from radiation-induced cell killing. However, the first report that has attempted to deal with this suggests that the adaptive response in lymphocytes protects against the mutagenic effects of ionizing radiation rather than the lethal effects (Sanderson and Morley, 1986). Clearly, much more work needs to be done to understand the consequences as well as the molecular basis of this phenomenon.
2 . 1 . GENE AMPLIFICATION
A second possibility for increasing the protection provided by DNA repair systems is to increase the number of repair genes per cell by the process of gene amplification. Here we enter the
Increased radioprotection by enhancement of enzymatic mechanisms
289
domain of pure speculation because at present the only known examples of gene amplification in human cells are in aneuploid ceils and not in the normal diploid cells we would most often want to protect. Moreover, the conditions that are now known to speed up the process of gene amplification (treatment with DNA-damaging agents, hypoxia, etc.) are deleterious to cells. Even so, the idea is attractive because in some cells the amplification has resulted in up to 500 copies (instead of the normal two copies) of the gene per cell with a corresponding resistance to the noxious agent, in this case methotrexate, an inhibitor of dihydrofolate reductase (Heintz and Hamlin, 1982). If it were possible to amplify DNA repair gene(s) in the blood-forming organs, the proportional increase in the gene product(s), i.e. DNA repair enzyme(s), should bring about increased protection. However, that possibility must at this time be considered strictly science fiction.
2.2. DNA TRANSFECTION The last possibility I wish to discuss is that of increasing the number of DNA repair enzymes or other radioprotective proteins by transfer of the appropriate cloned gene(s) into the cells at risk. The technique for doing this, DNA transfection, is well worked out, and the feasibility of repopulating bone marrow with cells containing the increased number of radioprotective genes is very high. A number of genes that control the repair of ionizing radiation-induced DNA damage have been cloned. It is possible that one or more of them could be transfected into the bone marrow cells, some of which are stem cells. These could be reinjected and the marrow repopulated by cells that harbor increased numbers of genes and hence increased amounts of gene products, the radioprotective proteins. The question then becomes, will the increased amounts of radioprotective proteins really result in increased protection for the cells containing them? The definitive experiment that could answer this question has not to my knowledge been done. However, the results of some experiments strongly suggest that the answer is yes. One experiment was done with a bacterial DNA repair gene that was transfected into human (HeLa) cells (Ishizaki et al., 1986). The gene was for the enzyme methyltransferase, which repairs DNA that has been damaged with methylating agents by removing the methyl group on the 06 position of
Fro. 2. Southern hybridization of genomic DNA and whole cellular RNA dot blot hybridization with the 32p-labeled E. coli ada gene DNA. Lane A shows clone 5-1; lane B, clone 7-1; lane C, HeLa MR (mer-). (Reproduced from Ishizaki et al., 1986, with permission of Elsevier Science Publishers.)
290
R . B . PAINTER 100
tT O.o Clone 5-1
\\
10
A
r'l
OHeLo S3 D
\ r'z Clone 7-1
0.1 •
0
HeLo HR
I
I
I
I
1
2
3
4
'
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FIG. 3. Survival of mer + HeLa $3 ( • ), m e r - HeLa MR ( • ) , transformant clone 5-1 (©), and clone 7-1 ([]), after treatment with MNNG for 1 hr at 37°C. (Reproduced from Ishizaki et al., 1986, with permission of Elsevier Science Publishers).
guanine. This bacterial gene was transfected into HeLa cells that are mer-, i.e., they do not contain any methyltransferase of their own. The resulting transfectants were tested for their content of methyltransferase genes and gene transcripts (Fig. 2) as well as their reproductive integrity in the presence of increasing concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a powerful methylating agent (Fig. 3). The results clearly show that the gene is expressed and, more importantly, that its expression endows the recipient cells with a higher resistance to MNNG than is exhibited by HeLa cells that contain the human methyltransferase protein (mer ÷ cells). However, these results fall short of answering the question explicitly because they do not show that the normal resistance of a cell can be increased by the addition of an exogenous repair gene. It is still possible that if the gene were transfected into a cell with its own repair gene functioning normally the resistance would not be increased. It is known that in Escherichia coli, overproduction of the products of the umuC, D gene is toxic, even though this gene normally protects against u.v. light (Marsh and Walker, 1985). Therefore, too many repair genes may sometimes be detrimental, perhaps by initiating repair indiscriminately in DNA.
2.3.
O T H E R POSSIBILITIES FOR ENHANCING PROTECTION
I have been careful to use the term "or other radioprotective protein" in this paper. This is to cover the very strong possibility that the basis of some hypersensitivities to DNA-damaging agents lies in an altered protein that is not itself a DNA repair enzyme. Such a protein might
Increased radioprotection by enhancement of enzymatic mechanisms
291
regulate a cell function, and w h e n the protein is a b n o r m a l the resulting a b n o r m a l function might alter the c e l l ' s sensitivity to the agent. F o r instance, imagine a protein that regulates the time that a cell spends in mitosis. The a b n o r m a l form o f this protein would cause the cell to r e m a i n in mitosis much longer than normal. Exponentially growing cultures of these cells would probably be more sensitive to ionizing radiation than normal cells because they would contain an increased fraction of radiosensitive mitotic cells. I bring this up because it is possible that the radiosensitivity of A - T cells is not due to defective D N A repair. So far no one has been able to identify a D N A repair defect that can explain the A - T s y n d r o m e . There is a good possibility that one o f the defective genes in A - T cells will soon be cloned. If so, it will be interesting to find out if this gene codes for a D N A repair e n z y m e or for some other protein.
3. S U M M A R Y In s u m m a r y , there are theoretically several ways by which a cell's content o f D N A repair genes or other radioprotective proteins can be increased. At present the most feasible way to do so seems to be to search for repair systems, such as the adaptive repair observed in h u m a n lymphocytes, whose activity can be increased by exogenous inducing agents. N o w that this effect has been established in h u m a n lymphocytes, a search for similar systems in other cells should begin. Transfection o f D N A repair genes into bone m a r r o w cells that could be used to make the blood-forming organs more radioresistant is actually very close to being possible; the problem is more a matter o f morality than of feasibility. The practicability of amplification o f D N A repair genes or genes coding for other radioprotective proteins in the cells o f a whole o r g a n i s m is at present close to zero. It is clear, however, that continued research on the basic m e c h a n i s m s o f cellular radiosensitivity could eventually lead to genetically engineered radioresistance in h u m a n s , but o n l y if society approves.
Acknowledgement--This work was supported by the Office of Health and Environmental Research, U.S. Department
of Energy, Contract No. DE-AC03-76-SF01012.
REFERENCES CLEAVER,J. E. (1982) Rapid complementation method for classifying excision repair-defective xeroderma pigmentosum cell strains. Somat. Cell Genet. 8:801-810. I.)EWEERD-KASTELEIN,E, A., KELIZER,W. and BOOTSMA,D. (1972) Genetic heterogeneity of xeroderma pigmentosum demonstrated by somatic cell hybridization. Nature New Biol. 238: 80-83. GIACCIA,A., WEINSTEIN,R., HU, J. and STAMATO,T. D. (1985) Cell cycle-dependent repair of double-strand DNA breaks in a ,,/-ray-sensitive Chinese hamster cell. Somat. Cell Mol. Genet. 11: 485-491. HEINTZ,N. H. and HAMLIN,J. L. (1982) An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc. Natn. Acad. Sci. USA 79:4083-4087. ISH1ZAKI,K., TSUJIMURA,T., YAWATA,H., FUJ10,C., NAKABEPPU,Y., SEKIGUCHI,M. and IKENAGA,M. (1986) Transfer of the E. coli O6-methylguaninemethyltransferase gene into repair-deficient human cells and restoration of cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine. Mutat. Res. 166: 135-141. J EGGO,P. A. and KEMP,L. M. (1983) X-ray-sensitive mutants of Chinese hamster ovary cell line: Isolation and crosssensitivity to other DNA-damaging agents. Mutat. Res. 112: 313-327. JONES, N. J., COX, R. and THACgER,J. (1987) Isolation and cross sensitivity of X-ray-sensitive mutants of V79/4 Chinese hamster cells. Mutat. Res. 183: 279-286. KEMP, L. M., SEIX;WlCK,S. G. and JEGGO, P. A. (1984) X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining. Mutat. Res. 132: 189-196. MARSH, L. and WALgER,G. C. (1985) Cold sensitivity induced by overproduction of umuDC in Escherichia coli. J. Bacteriol. 162: 155-161. OLIVIERI,G., BODYCOTE,J. and WOLFF, S. (1984) Adaptive response of human lymphocytes to low concentrations of radioactive thymidine. Science 223: 594-597. SANDERSON, B. J. S. and MORLEY, A. A. (1986) Exposure of human lymphocytes to ionizing radiation reduces mutagenesis by subsequent ionizing radiation. Mutat. Res. 164: 347-351. SHADLEY, J. D. and WOLFF, S. (1987) Very low doses of X-rays can cause human lymphocytes to become less susceptible to ionizing radiation. Mutagenesis 2: 95-96. STAMATO,T. D., WEINSTEIN,R., GIACCIA,A. and MACgENZm,L. (1983) Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell. Somat. Cell Genet. 9: 165-173. THOMPSON,L. H., BROOgMAN,K. W., DmLEHAY,L. E., CAgRANO,A. V., MAZRIMAS,J. A., MOONEY,C. L. and MtNgLEnJ. L. (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange. Mutat. Res. 95: 427-440.
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WIENCKE,J. K., AFZAL,V., OLIVIERLG. and WOLFF, S. (1986) Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair system. Mutagenesis 1: 375-380. ZDZIENICKA, M. Z., ROZA, L., WESTERVELD, A., BOOTSMA, D. and SIMONS, J. W. I. M. (1987) Biological and biochemical consequences of the human ERCC-1 repair gene after transfection into a repair-deficient CHO cell line. Murat. Res. 183: 69-74.
0163-7258/88 $0.00+0.50 Copyright © 1988 Pergamon Press plc
Printed in Great Britain. All rights reserved
Symposium Editors: J. F. WEIss and M. G. SIMI¢
INCREASED RADIOPROTECTION BY E N H A N C E M E N T OF ENZYMATIC MECHANISMS ROBERT B. PAINTER laboratory of Radiobiology and EnvironmentalHealth and Department of Microbiology, Universityof California, San Francisco, California 94143, U.S.A.
1. DNA REPAIR SYSTEMS The principal method by which cells protect themselves from the deleterious effects of ionizing radiation is by means of their DNA repair systems. This is most easily demonstrated in bacteria and yeast, in which very large numbers and many kinds of radiation-sensitive mutants have been tound to be defective in one or more kinds of DNA repair. Higher eukaryotic ceils occasionally show naturally occurring defects in DNA repair; the best-known example is that of patients with the autosomal recessive disease xeroderma pigmentosum (XP). The cells from these patients are sensitive to ultraviolet (u.v.) radiation, not to ionizing radiation, because of their inability to excise u.v. photoproducts from their DNA. Cells from patients with another autosomal recessive disease, ataxia-telangiectasia (A-T), are hypersensitive to ionizing radiation; however, no consistent DNA repair defect has been found for this condition.
1.1. RADIOSENSITIVE MAMMALIAN CELL MUTANTS
In the last few years mammalian cells defective in DNA repair have been isolated in the laboratory. Generally, these mutants are first produced by exposure to a chemical mutagen and then separated from the rest of the population by observing the emergence of colonies that grow more slowly than the surrounding normal ones. Chinese hamster ovary (CHO) cells have been used almost exclusively up to now. The CHO mutants that are hypersensitive to ionizing radiation are, in general, defective in the repair of DNA strand breaks. A series of mutants isolated by Jeggo and collaborators (Jeggo and Kemp, 1983; Kemp et al., 1984) are defective in the repair of double-strand breaks. A mutant isolated by Stamato et al. (1983) is radiosensitive in G1 but not in late S/G2 and is defective in the repair of double-strand breaks in Gi but not in G2 (Giaccia et al., 1985). A mutant isolated by Thompson et al. (1982) is very sluggish in the repair of X-ray-induced single-strand breaks but is only moderately radiosensitive. It is obvious that these mutants will be valuable as tools to isolate and understand repair genes. The fact that mutants are radiosensitive because they have a defect in a repair system has been proved in some cases by complementation tests. For example, when different lines of u.v.sensitive human (XP) cells are fused, the heterokaryon formed often has the ability to carry out the excision of u.v. photoproducts (DeWeerd-Kastelein et al., 1972) and is normal in its resistance to u.v. (Cleaver, 1982). (If the heterokaryon does not regain the capacity for excision repair, it is always assumed that the two cells are in the same complementation group, i.e. that they have exactly the same DNA repair defect.) Similar results have been obtained for some Chinese hamster mutants that are hypersensitive to ionizing radiation (Jones et al., 1987; N.J. Jones, personal communication*). In some cases the normal repair genes were cloned and reintroduced into the mutant, which then attained the normal phenotype (Zdzienicka et al., 1987). Therefore, there is no doubt that these DNA repair enzymes are the principal factors that protect cells from the lethal effects of radiation. *Published with permission of N. J. Jones. 287
288
R . B . PAINTER
4O
,o
o o--'
T
1o,
"1o
1o,
Concentration [3H]dThd(iJCi/ml)
1o,
,oo
FIG. 1. The induction of chromatid breaks (chromatid and isochromatid deletions) in human lymphocytes: (A) breaks induced by exposure to [3H]thymidine alone; ( • ) X-ray-induced breaks after exposure to [3H]thymidine + 1.5 Gy X-rays; (11) X-ray-induced breaks after exposure to [3H]thymidine + 1.5 Gy X-rays + 2 mM 3-aminobenzamide (3AB). Two hundred cells per point were scored. The vertical bars denote standard error of the mean. To calculate the number of induced breaks, the number of background breaks and (for combined treatments) the number of breaks induced by [3H]thymidine or 3AB alone was subtracted from the total number of aberrations scored. (Reproduced from Wiencke et al., 1986, with permission of IRL Press Ltd.)
2. POTENTIAL FOR RADIOPROTECTION BY ENHANCEMENT OF ENZYMATIC MECHANISMS For the purposes of this meeting, I wish to pose the question, is it possible to increase the endogenous activity of repair enzymes or other radioprotective enzymes so that an individual can become more resistant than normal? In theory this could be brought about by any one of several means. The first, and perhaps the most feasible at this time, is by induction of an enzymatic process whose activity can be altered by the manipulation of environmental conditions. An example of this is the so-called adaptive response described first by Olivieri et al. (1984) and elaborated upon by Wolff and collaborators (Wiencke et al., 1986; Shadley and Wolff, 1987). In this case, very small doses of ionizing radiation, administered to lymphocytes either exogenously as X-rays or intracellularly in the form of [3H]thymidine, decrease the clastogenic effect of high doses of X-rays administered later. The adapting effect of radiation can be at doses so low that they themselves cause no measurable increase in aberration frequency (Fig. 1). It is almost certain that this protection is the result of an increase in the amount or activity of some repair system. Because chromosome aberrations are generally considered to be the cause of most radiation-induced cell death, the effect of protection against aberrations would be expected to be manifested as protection from radiation-induced cell killing. However, the first report that has attempted to deal with this suggests that the adaptive response in lymphocytes protects against the mutagenic effects of ionizing radiation rather than the lethal effects (Sanderson and Morley, 1986). Clearly, much more work needs to be done to understand the consequences as well as the molecular basis of this phenomenon.
2 . 1 . GENE AMPLIFICATION
A second possibility for increasing the protection provided by DNA repair systems is to increase the number of repair genes per cell by the process of gene amplification. Here we enter the
Increased radioprotection by enhancement of enzymatic mechanisms
289
domain of pure speculation because at present the only known examples of gene amplification in human cells are in aneuploid ceils and not in the normal diploid cells we would most often want to protect. Moreover, the conditions that are now known to speed up the process of gene amplification (treatment with DNA-damaging agents, hypoxia, etc.) are deleterious to cells. Even so, the idea is attractive because in some cells the amplification has resulted in up to 500 copies (instead of the normal two copies) of the gene per cell with a corresponding resistance to the noxious agent, in this case methotrexate, an inhibitor of dihydrofolate reductase (Heintz and Hamlin, 1982). If it were possible to amplify DNA repair gene(s) in the blood-forming organs, the proportional increase in the gene product(s), i.e. DNA repair enzyme(s), should bring about increased protection. However, that possibility must at this time be considered strictly science fiction.
2.2. DNA TRANSFECTION The last possibility I wish to discuss is that of increasing the number of DNA repair enzymes or other radioprotective proteins by transfer of the appropriate cloned gene(s) into the cells at risk. The technique for doing this, DNA transfection, is well worked out, and the feasibility of repopulating bone marrow with cells containing the increased number of radioprotective genes is very high. A number of genes that control the repair of ionizing radiation-induced DNA damage have been cloned. It is possible that one or more of them could be transfected into the bone marrow cells, some of which are stem cells. These could be reinjected and the marrow repopulated by cells that harbor increased numbers of genes and hence increased amounts of gene products, the radioprotective proteins. The question then becomes, will the increased amounts of radioprotective proteins really result in increased protection for the cells containing them? The definitive experiment that could answer this question has not to my knowledge been done. However, the results of some experiments strongly suggest that the answer is yes. One experiment was done with a bacterial DNA repair gene that was transfected into human (HeLa) cells (Ishizaki et al., 1986). The gene was for the enzyme methyltransferase, which repairs DNA that has been damaged with methylating agents by removing the methyl group on the 06 position of
Fro. 2. Southern hybridization of genomic DNA and whole cellular RNA dot blot hybridization with the 32p-labeled E. coli ada gene DNA. Lane A shows clone 5-1; lane B, clone 7-1; lane C, HeLa MR (mer-). (Reproduced from Ishizaki et al., 1986, with permission of Elsevier Science Publishers.)
290
R . B . PAINTER 100
tT O.o Clone 5-1
\\
10
A
r'l
OHeLo S3 D
\ r'z Clone 7-1
0.1 •
0
HeLo HR
I
I
I
I
1
2
3
4
'
HNNG(pg/ml)
FIG. 3. Survival of mer + HeLa $3 ( • ), m e r - HeLa MR ( • ) , transformant clone 5-1 (©), and clone 7-1 ([]), after treatment with MNNG for 1 hr at 37°C. (Reproduced from Ishizaki et al., 1986, with permission of Elsevier Science Publishers).
guanine. This bacterial gene was transfected into HeLa cells that are mer-, i.e., they do not contain any methyltransferase of their own. The resulting transfectants were tested for their content of methyltransferase genes and gene transcripts (Fig. 2) as well as their reproductive integrity in the presence of increasing concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a powerful methylating agent (Fig. 3). The results clearly show that the gene is expressed and, more importantly, that its expression endows the recipient cells with a higher resistance to MNNG than is exhibited by HeLa cells that contain the human methyltransferase protein (mer ÷ cells). However, these results fall short of answering the question explicitly because they do not show that the normal resistance of a cell can be increased by the addition of an exogenous repair gene. It is still possible that if the gene were transfected into a cell with its own repair gene functioning normally the resistance would not be increased. It is known that in Escherichia coli, overproduction of the products of the umuC, D gene is toxic, even though this gene normally protects against u.v. light (Marsh and Walker, 1985). Therefore, too many repair genes may sometimes be detrimental, perhaps by initiating repair indiscriminately in DNA.
2.3.
O T H E R POSSIBILITIES FOR ENHANCING PROTECTION
I have been careful to use the term "or other radioprotective protein" in this paper. This is to cover the very strong possibility that the basis of some hypersensitivities to DNA-damaging agents lies in an altered protein that is not itself a DNA repair enzyme. Such a protein might
Increased radioprotection by enhancement of enzymatic mechanisms
291
regulate a cell function, and w h e n the protein is a b n o r m a l the resulting a b n o r m a l function might alter the c e l l ' s sensitivity to the agent. F o r instance, imagine a protein that regulates the time that a cell spends in mitosis. The a b n o r m a l form o f this protein would cause the cell to r e m a i n in mitosis much longer than normal. Exponentially growing cultures of these cells would probably be more sensitive to ionizing radiation than normal cells because they would contain an increased fraction of radiosensitive mitotic cells. I bring this up because it is possible that the radiosensitivity of A - T cells is not due to defective D N A repair. So far no one has been able to identify a D N A repair defect that can explain the A - T s y n d r o m e . There is a good possibility that one o f the defective genes in A - T cells will soon be cloned. If so, it will be interesting to find out if this gene codes for a D N A repair e n z y m e or for some other protein.
3. S U M M A R Y In s u m m a r y , there are theoretically several ways by which a cell's content o f D N A repair genes or other radioprotective proteins can be increased. At present the most feasible way to do so seems to be to search for repair systems, such as the adaptive repair observed in h u m a n lymphocytes, whose activity can be increased by exogenous inducing agents. N o w that this effect has been established in h u m a n lymphocytes, a search for similar systems in other cells should begin. Transfection o f D N A repair genes into bone m a r r o w cells that could be used to make the blood-forming organs more radioresistant is actually very close to being possible; the problem is more a matter o f morality than of feasibility. The practicability of amplification o f D N A repair genes or genes coding for other radioprotective proteins in the cells o f a whole o r g a n i s m is at present close to zero. It is clear, however, that continued research on the basic m e c h a n i s m s o f cellular radiosensitivity could eventually lead to genetically engineered radioresistance in h u m a n s , but o n l y if society approves.
Acknowledgement--This work was supported by the Office of Health and Environmental Research, U.S. Department
of Energy, Contract No. DE-AC03-76-SF01012.
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