Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation

Research 350 (1996) 239-246

Protective effect of deoxyribonucleosides on UV-irradiated human peripheral blood T-lymphocytes: possibilities for the selective killing of either cycling or non-cycling cells
Michael H.L. Green *, Alastair P.W. Waugh, Jillian E. Lowe, Susan A. Harcourt, Peter H. Clingen, Jane Cole, Colin F. Arlett
MRC Cell Mutatim

Unit. Su.w.r Univs-sify

Falnw,

Brighton,

BNI

YRR. UK

Accepted 3 June 1995

Abstract Non-cycling human T-lymphocytes from normal subjects show a IO-fold greater sensitivity than fibroblasts to UV-B nm) irradiation from a Westinghouse FS20 lamp, but only a 2.7-fold greater sensitivity to UV-C (254 nm) irradiation. Hypersensitivity is associated with a deficiency in the rejoining of excision breaks. Non-cycling T-lymphocytes have extremely low deoxyribonucleotide pools. Addition to the medium of the four deoxyribonucleosides. each at a concentration of IO- M, substantially increases survival and reduces the persistence of excision-related strand breaks following UV-B or UV-C irradiation (Yew and Johnson (1979) Biochim. Biophys. Acta 562, 240-241: Green et al. (1994) Mutation Res., 31.5. 25-32). UV-resistance of T-lymphocytes is also increased by stimulating the cells into cycle. The addition of deoxyribonucleosides does not further enhance survival of cycling cells and they do not reach the level of resistance achieved by non-cycling cells in the presence of deoxyribonucleosides. We suggest that two opposing effects are in operation. Cells out of cycle can show increased resistance to DNA damage in the absence of division but they also have reduced deoxyribonucleotide pools, which may limit DNA repair. With UV-B irradiation, the exceptionally low dNTP pools in non-cycling T-lymphocytes cause this second effect to predominate. In contrast, with ionising radiation, which forms highly cytotoxic double-strand breaks, non-cyclin g human T-lymphocytes are slightly more resistant than fibroblasts. Non-cycling cells such as T-lymphocytes should be especially sensitive to agents which produce a high proportion of readily excisable damage, but should show normal resistance to agents which produce highly toxic lesions. It may be possible by choice of DNA damaging agent and manipulation of cellular deoxyribonucleotide pools, to choose regimes which will selectively kill either cycling or non-cycling cells and to improve the efficacy of standard therapeutic procedures. Conditions favouring selective killing of non-dividing T-lymphocytes but sparing stem cells may be of value in bone marrow transplantation. Conditions favouring selective killing of dividing cancer cells but sparing non-dividing normal tissue may be of value in cancer therapy.
(280-315

Abbreviations: UDS, Unscheduled DNA synthesis; UV-A, Ultraviolet radiation. nm: UV-C, Ultraviolet radiation, 200-280 nm (commonly 254 nm) Corresponding author. Tel.: +44 (0) 1273 678120: Fax: +44 (0) 1273 678121. 0027.5 107/96/$15.00 SSDl 0027.5107(95)001 0 1996 Elsevier Science B.V. All rights reserved 10-7

315-400

nm; UV-B, Ultraviolet

radiation.

280-315

240

M.H.L. Grew

cf rd. / Mutcrtiorl Rrsrc~rch 3.X (I!861

239-2-M

Keywords: Deoxyribonucleoside; Deoxyribonucleotide Bone marrow transplantation; Radiotherapy

pool: T-lymphocyte:

Human

cell; UV-B radiation;

UV-C radiation:

Cell killing;

1. Introduction The ultraviolet sector of the solar spectrum is commonly divided into three regions, UV-C (200280 nm), UV-B (280-315 nm> and UV-A (315-400 nm). UV-C radiation is totally blocked by the earths atmosphere, and it is the longer wavelengths of UV-B and UV-A which are relevant to skin cancer (Magnus, 1976; Rothman and Setlow, 1979; Rosenstein and Mitchell. 1987; Sterenborg and van der Leun, 1987). It is UV-B radiation which is attenuated by the ozone layer (Russell Jones and Wigley. 1989) and which includes the longest wavelengths that are capable of forming DNA lesions in a direct photochemical reaction (longer wavelengths can induce DNA lesions via interaction with other cellular constituents). The vast majority of ultraviolet research has been into the effects of 254 nm UV-C radiation, although such wavelengths are irrelevant to human carcinogenesis. This is understandable if regrettable, since the pattern of photoproducts formed by UV-C is relatively well-defined, whereas the biological effectiveness and the proportions of different photoproducts formed by UV-B depend on the exact light source used. Such a difficulty can be turned to advantage, however, if UV-B light sources giving photoproducts in different proportions are compared, since it may then become feasible to correlate biological effects with specific photoproducts (Clingen et al., 1995). Non-cycling T-lymphocytes were found to be more sensitive than fibroblasts to UV-C (254 nm) radiation (Arlett et al., 1992). Sensitivity did not appear to be associated with a deficiency in lesion excision but with a defect in rejoining of the resulting break (Green et al., 1992; Yew and Johnson, 1979). Actively dividing T-lymphocytes showed greater resistance to UV-irradiation and less persistence of excision breaks. When similar experiments were carried out with UV-B irradiation, non-cycling human T-lymphocytes demonstrated a quite exceptional hypersensitivity to killing in comparison to fibroblasts (Arlett et al., 1993). (It is essential to define the source of light used in such investigations,

and in that study filtered Westinghouse FS20 lamps were used. The FS20 is a broad spectrum fluorescent tube, with peak emission at 312 nm. We filtered to remove wavelengths below 290 nm.) Arlett et al. (1993) suggested that UV-B and UV-C were not identical in their mode of action, and that UV-B appeared to form a lesion which killed lymphocytes more readily than fibroblasts. They were able to show that this lesion was not the cyclobutane pyrimidine dimer, that it was excisable, and that xeroderma pigmentosum T-lymphocytes were defective in its excision. The predominant lesions formed by UV-C are the cyclobutane pyrimidine dimer and the (6-4) dipyrimidine photoproduct. With UV-B irradiation, a third lesion, the Dewar isomer of the (6-4) photoproduct, is formed with significant yield (Mitchell, 1988; Taylor et al., 1990; Matsunaga et al., 1993). This stable isomer is formed by photoisomerisation of the (6-4) product, most efficiently between 3 15325 nm. Although the lesion can be detected following 254 nm irradiation, the yield is very low (Clingen et al., 1995). It is an open question whether the sole mechanism for formation of the Dewar isomer is by photoisomerisation of existing (6-4) photoproducts or whether it can also be formed directly. Since UV lamps with different spectral outputs produce these photoproducts in different proportions, we have used monoclonal antibodies to specific lesions and image analysis to measure the output of pyrimidine dimers, (6-4) photoproducts and Dewar isomers by a UV-C and two UV-B lamps (Clingen et al., 1995). (For these studies, the Philips TLOl lamp was used in addition to the FS20. It produces nearly monochromatic 312 nm light, although a small peak at 304 nm contributes significantly to damage.) Each lamp produced the three lesions in different proportions so that it was possible to attempt to correlate each of the lesions with cell killing. It would appear that killing of normal human fibroblasts is most closely associated with (6-4) photoproduct formation. In contrast, killing of normal non-cycling Tlymphocytes correlated best with combined (6-4) photoproduct and Dewar isomer formation. The Dewar isomer appears to be the additional lesion formed

M.H.L. Grern et al. /Mutation Research350 (19961239-246

Table I Mean inactivation doses for human T-lymphocytes from normal subjects Cell type Mean inactivation uv-c U/m

241

and fibroblasts

dose (No. subjects) Ionising radiation (Gy)

UV-B (FS20) U/m)

Fibroblast T-lymphocyte Dose reduction factor for T-lymphocytes

4.92 (3) 1.58 (3) 2.72

166.6 (3) 16.39 (3) 10.16

1.38 (33) 1.88 (33) 0.73

cycle and grown in mass culture for several days prior to irradiation. Where appropriate, deoxyribonucleosides were added, singly or in combination, each at lop5 M. For determination of clonogenic survival cells were distributed into multiwell trays. Under the growth conditions used (Green et al., 1994), Tlymphocytes grow to form colonies with high efficiency. The proportion of wells containing colonies was determined after about 14 days and surviving fraction determined as described previously (Green et al., 1994). 2.2. UV irradiation Cells were irradiated with UV-B using Westinghouse FS20 lamps (Arlett et al., 19931, or with UV-C using a Philips 6W (254 nm) germicidal lamp (Green et al., 1994). 2.3. Comet assay DNA strand breaks were measured by the comet assay (single cell gel electrophoresis). using the procedure of Singh et al. (1988). as adapted by Green et al. (1994). Cells were embedded in agar, lysed, placed in alkali. and electrophoresis applied. Under these conditions, DNA containing strand breaks streams out of the nucleus whereas undamaged DNA remains entangled. Slides were stained with ethidium bromide and images captured using an Optomax V system with Insight (Synoptics, Cambridge). At least six fields were measured for each dose point and mean nose to tail comet length determined. Greater comet length is an indication of greater strand breakage. In an individual experiment mononuclear cells were UV-irradiated and incubated for 1 h in growth medium supplemented with combinations of the four deoxyribonucleosides at a concentration of lo- M. Cells were embedded in agar and the comet assay performed. Note that comet length is determined for the mononuclear fraction, which includes mainly T-lymphocytes, but also B-lymphocytes and some monocytes. Our data are not definitive but we have not observed any striking difference in response between these cell types (unpublished observations). In clonogenic survival experiments it is specifically T-lymphocytes which form colonies.

Values are averages for the number of subjects shown. The UV-C and UV-B values are taken from a straight line fit of the data of Clingen et al. (1995). the ionising radiation data from a linear quadratic fit of the data of Green et al. (1991). Survival experiments for lymphocytes were performed as described by Cole et al. (1988) and for fibroblasts as described by Arlett and Harcourt (1980)

by UV-B to which lymphocytes, but not fibroblasts, are sensitive. Although human T-lymphocytes are more sensitive than fibroblasts to UV irradiation, it is a misconception that they are sensitive to all DNA damaging agents. For instance, Green et al. (1991) found that human T-lymphocytes exhibited no greater ionising radiation sensitivity in a clonogenic assay than fibroblasts from the same normal subject. Table 1 shows mean inactivation dose for killing of fibroblasts and T-lymphocytes by UV-C, UV-B (FS20) and ionising radiation. It is clear that the hypersensitivity of T-lymphocytes is to specific types of DNA damaging agent. In this paper. we suggest reasons for these large discrepancies in relative sensitivity.

2. Materials

and methods

2. I. Cell culture Procedures were as described previously (Cole et al.. 1988; Green et al., 1994). Blood samples were taken from volunteer subjects with informed consent. The mononuclear cell fraction was separated on a Ficoll gradient and stored frozen. For an experiment cells were thawed and placed in medium. Non-cycling cells were UV-irradiated prior to being stimulated into cycle. Cycling cells were stimulated into

3. Results 3. I. The effect

of deoxyribonucleosides

As with UV-C, killing of non-dividing Tlymphocytes by UV-B appears to be associated with defective strand rejoining rather than the incision step of repair. It was drawn to our attention by Dr R.T. Johnson that this sensitivity might be related to deoxyribonucleotide pools. These pools are exceptionally low in human T-lymphocytes (Munch-Petersen et al., 1973). Yew and Johnson (1979) have shown that addition of nucleosides to UV-C irradiated T-lymphocytes decreased the level of excisionrelated strand breakage and increased survival as determined by a dye exclusion assay. We therefore tested and found that addition of deoxyribunucleosides (each at 10m5 M) to the medium greatly reduced the level of excision breaks detected in UV-B irradiated T-lymphocytes (Green et al., 1994). suggesting that the efficiency of strand rejoining was enhanced. Ribonucleosides had no effect. The addition of deoxyribonucleosides substantially enhanced survival of non-cycling T-lymphocytes, following either UV-B (FS20) or UV-C irradiation, suggesting that failure to rejoin excision breaks was important in cytotoxicity. Excision-defective xeroderma pigmentosum T-lymphocytes did not show enhanced survival in the presence of deoxyribonucleosides. presumably because they generated fewer excision breaks and thus placed less demand on their deoxyribonucleotide pools. Similarly, cultured primary fibroblasts showed no effect, presumably because their pool sizes were already adequate (Green et al., 1994). These observations suggest a possible explanation of why (6-4) photoproducts and Dewar isomers may be more important than cyclobutane pyrimidine dimers in T-lymphocyte toxicity. (6-4) photoproducts (and we believe Dewar isomers, (Svoboda et al., 1993)) are far more efficiently excised than the majority of dimers (Mitchell et al., 1985). Slow excision of dimers will place less demand on deoxyribonucleotide pools. 3.2. Deo,yvribonucleoside combinations

cytes, we supposed that it might also increase unscheduled DNA synthesis (UDS), which is extremely low in these cells (Freeman and Ryan, 1988; Knudsen et al., 1992). Unfortunately, addition of deoxyribonucleosides affects the measurement of UDS, and in particular the presence of deoxycytidine or thymidine prevents uptake of labelled thymidine from being detected. In an attempt to overcome this problem, we studied the efficiency of strand break rejoining following removal of individual deoxyribonucleosides from the supplemented medium (Fig. 1). It can be seen that removal of any of the four deoxyribonucleosides caused additional strand breaks to persist following UV-B irradiation. Absence of deoxyguanosine had the greatest effect, and deoxyadenosine the least. In order to try to obtain a medium to allow unscheduled DNA synthesis to be observed, we next attempted experiments progressively adding deoxyribonucleosides to the medium (Fig. 2). It was apparent that optimum rejoining was achieved when all four deoxyribonucleosides were present. Again, deoxyguanosine was the most effective single deoxyribonucleoside, and in this instance deoxyadenosine inhibited rather than enhanced rejoining. These results differ slightly from those of Holmberg (1989). who used different combinations

Deoxyribonucleoside

omitted

Since addition of deoxyribonucleosides enhanced strand rejoining and survival in non-cycling lympho-

Fig. 1. Effect on UV-B excision break frequency of omitting individual deoxyribonucleosides from the medium. Greater comet length indicates greater strand breakage. Mononuclear cells from normal subjects received 13 J/m FS20 irradiation (Arlett et al., 1993) and were incubated for 1 h in RPM1 1640 medium, supplemented with all four deoxyribonucleosides at 10-s M or as indicated. Excision breaks were measured by the Comet assay (Singh et al.. 1988: Green et al.. 1994). All values based on the mean of at least two independent experiments.

M. H.L. Green et al. /Mutation

Research 350 (1996) 239-246

243

of deoxyribonucleosides, finding deoxyguanosine alone ineffective, and a combination of deoxyadenosine and thymidine effective in reducing the level of excision breaks in human lymphocytes. We found no deoxyribonucleoside combination which allowed UDS to be readily detected in unstimulated T-lymphocytes. 3.3. Cycling
L~S.

non-cycling

T-lymphocytes
0 100

200

300

The effect of addition of all four deoxyribonucleosides on survival of actively dividing T-lymphocytes is shown in Fig. 3 (data from (Green et al., 1994). It can be seen that survival is not enhanced by addition of deoxyribonucleosides. This is because the pool sizes in cycling cells are large enough not to

UV-B dose (J/m2)

Fig. 3. Effect of deoxyribonucleosides on survival of UV-B -irradiated cycling T-lymphocytes, 0. 0. subject 102; 0. H , subject 159; A, A, subject 314. Open symbols. no deoxyribonucleosides; closed symbols, the four deoxyribonucleosides (each at lo- MJ added to the growth medium following irradiation. Each curve is the mean of 2 experiments. Data are superimposed on results for non-cycling T-lymphocytes from the same subjects (also at least two experiments in each case): -- -, no added deoxyribonucleosides: - -. deoxyribonucleosides (10-s M) added to the culture medium. Reproduced from Green et al. (1994).

60

70 60 50 40 30

limit repair. Although stimulating T-lymphocytes into cycle increases their resistance to UV-B irradiation, it is clear that even greater resistance is achieved by treating non-cycling cells with deoxyribonucleosides. Two opposing effects would appear to be in operation. The norm is for cells out of cycle to be more resistant to DNA damage, but such cells express low levels of ribonucleotide reductase (Elledge et al., 1993). The resulting low deoxyribonucleotide pools may be sub-optimal for repair of DNA damage. In the case of non-cycling T-lymphocytes, pool levels are so low that following UV-B irradiation this effect predominates.
? B W d 2 " = 4:

30

Deoxyribonucleosides

4. Discussion The balance between the cell cycle effect and the pool size effect offers an explanation for the differences in survival of normal human fibroblasts and T-lymphocytes shown in Table 1. Ionising radiation produces highly cytotoxic double-strand breaks or local multiply damaged sites, (Ward, 1988). The total amount of damage is small at doses sufficient to cause lethality. Moreover, much of this damage is

Fig. 2. Effect on UV excision break frequency of addition of successive deoxyribonucleosides to the medium of mononuclear cells from normal subjects. Greater comet length indicates greater strand breakage. Procedures were as for Fig. 1, except that cells were pre-incubated overnight with the combination of deoxyribonucleosides at IO- M. (A) UV-B irradiation from a bank of Westinghouse FS20 lamps. Dark bars, unirradiated: hatched bars, 6 J/m UV-B; shaded bars. 10 J/m UV-B. (BJ UV-C irradiation. Dark bars. unirradiated; hatched bars, 1 J/m UV-C. All values based on the mean of at least two independent experiments.

subject to short-patch repair (Friedberg, 1985; Painter and Young, 1972). Thus at cytotoxic doses relatively little depletion of deoxyribonucleotide pools will occur, and the main determinant for survival will be the inherent resistance of non-cycling cells to DNA damage (Arlett and Priestley, 1984). Indeed, stimulating T-lymphocytes into cycle increases their sensitivity to ionising radiation (Cole et al.. 1988). Since tumour cells are likely to be actively dividing and most normal tissue is not, agents such as ionising radiation are likely to be selectively toxic to tumour cells. In contrast, an agent such as UV-B is only toxic at doses where extensive DNA damage and excision repair occur. In this case, the effect of depletion of deoxyribonucleotide pools on repair predominates, and such treatments are selectively toxic to non-cycling cells such as T-lymphocytes. It is of interest that in addition to ionising radiation. which produces double-strand breaks, a high proportion of DNA-damaging drugs used in cancer chemotherapy are DNA cross-linking agents. These are likely to be agents where toxicity occurs with a relatively small amount of overall DNA damage and demands on deoxyribonucleotide pools are minimal. Agents or treatments such as UV-B irradiation, which cause toxicity only at doses where the total amount of damage is large and extensive excision repair occurs, are likely to offer no therapeutic advantage, or to be selectively toxic to non-dividing cells. In some situations, however, protection of dividing and elimination of non-dividing cells is desirable. In bone marrow transplantation, successful transfer of stem cells is required, but these are contaminated with mature T-lymphocytes which, if viable. may cause graft versus host disease. In this case, UV-B irradiation of donor marrow is an extremely promising strategy (Pamphilon et al., 199 I). whereas ionising radiation might be expected to be ineffective. If psoralen-UV-A treatment were substituted for UV-B irradiation, it would be expected to generate highly cytotoxic crosslinks and to be less effective in selectively killing T-lymphocytes. 3.1. Resistame

of irreparable lesions. It is supposed that in non-cycling cells, although repair is not necessarily more efficient, additional time is available for its completion. An alternative possibility arises from the study of apoptosis and the recognition that it is more advantageous to a multicellular organism that a cell should die than that it should replicate inappropriately (Harrington et al.. 1994). On this model, cells out of cycle do not pose a threat, but cells in cycle can be allowed to survive only if they receive appropriate signals. In the absence of such signals apoptosis results. DNA damage also appears to induce apoptosis, and the success of cancer therapy by DNA damaging agents may be related to the ability of the agent to induce apoptosis in dividing cells of the particular tumour type (Fisher, 1994). It could be argued that it is advantageous to a multicellular organism that a mutated cell should die rather than multiply. One possible explanation for the hypersensitivity of Tlymphocytes to certain types of DNA damage is that this is also advantageous to the organism. Although circulating T-lymphocytes are non-dividing cells, and should be relatively resistant to apoptosis, they have potential for future replication. If this represented a hazard to the organism. it might be preferable for such cells to die. The alternative hypothesis is that T-lymphocyte hypersensitivity arises purely by chance. Circulating T-lymphocytes are continually exposed to nitric oxide released by the vascular endothelium and nitric oxide is an inhibitor of ribonucleotide reductase (Lepoivre et al.. 1991). The sensitivity of T-lymphocytes may simply be a product of their somatic environment. 4.2. Modifiitzg . deo,~~riborlLccleotide pools

qf rlorl-cycling cells to DNA damage

The traditional explanation for resistance of noncycling cells to radiation has been that attempted replication of damaged DNA may lead to formation

We have considered the possibility that further disruption of deoxyribonucleotide pools might increase the selective toxicity of UV-B to T-lymphocytes and other non-cycling cells and one potential procedure is the subject of a patent application. Equally interesting may be the possibility of raising deoxyribonucleotide pool sizes. If the rationale for radiotherapy, and much chemotherapy with DNA damaging agents, is to kill cycling cells while sparing non-cycling cells. then low deoxyribonucleotide pools in non-cycling cells are likely to reduce the

h4.H.L. Grern et al. /Mutation

Research

350 (1996/ 239-246

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selective efficiency of all such treatments. The results of Hennig et al. (this symposium, (Hennig et al., 1996), showing radioprotection by injection of mice with DNA can be explained if this leads to slow release of deoxyribonucleosides and improvement of repair in cells with low pool sizes. It is of interest that bone marrow cells, a dose-limiting tissue in cancer therapy, are effective in scavenging nucleosides and may actually be more dependent on circulating nucleosides than upon de novo synthesis for maintenance of nucleotide pools (Matveenko et al., 1973). 4.3. Conclusiorl Our results with UV light and deoxyribonucleosides suggest a way in which, by choice of DNA damaging agent and manipulation of deoxyribonucleotide pools, it may prove possible to target selectively either cycling or non-cycling cells. This may have relevance both to transplantation and to cancer therapy. The results also suggest one possible explanation for the relative efficacy of certain classes of agent as anti-cancer drugs.

Acknowledgements We thank Dr. R.T. Johnson (Cambridge), Dr. A.D.B. Webster (London), Professor R.C. von Borstel (Edmonton) and our colleagues for helpful advice and discussion. Work supported by CEC grant EVSV-CT9 l-0034.

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