the effect of halogenated nucleosides on the sensitivity of a mouse lymphoma p388f to x-rays and...

Itlt. J . Cancer: 3, 382-389 (1968)

THE EFFECT OF HALOGENATED NUCLEOSIDES ON THE SENSITIVITY OF A MOUSE LYMPHOMA P388F

TO X-RAYS AND METHYL METHANESU LPHONATE

by

Margaret F o x Patersori Laboratories, Christie Hospital and Holt Radium Institute,

Manchester, England

Populations of the P388F niouse lymphoma were sensitized to the effects of X-rays by prolonged culture in non-toxic doses of 5-iodo-2’-deoxyuridine and 5-bromo-2’- deoxyuridine. A marked decrease in the ability of analogue-exposed cells to recover from fractionated doses of X-rays was found. Thus the mechanism of sensitization may be related to inhibition of repair mechanisms. Replicute cultures of the sanie populations did not show an increase in wrisitivity to the monofunctional alkylating agent methyl methunesulphonate (IMMS) , which suggests that damage by X-rays and M M S may bc by diflererrt mechanisms in the cell line. The results would also suggest that the alkylation o j D N A by M M S is not the only cytotoxic efect of this drug.

The halogenated nucleosides 5-bromo-2‘-deoxyuridine (BUdR) and 5-iodo-2’-deoxyuridine (IUdR) have been shown to enhance the radiosensitivity of many mammalian cell lines in vitro (Delihas et ul., 1962; Mohler and Elkind, 1963; Erickson and Szybalski, 1963) and mouse lymphoma in vivo (Berry and Andrews, 1962). Recently, incorporation of BUdR has also been shown to sensitize the mouse lymphoma P815Y in vitro to the effects of two bifunctional alkylating agents, nitrogen mustard (HN,) and dimethyl niyleran, though to a lesser extent than to radiation (Schindler et al., 1966). These results have been interpreted as indicating that X-rays and the two bifunctional alkylating agents have similar, though not identical, modes of action. Cross-linking of the D N A strands by HN, has been clearly implicated as a major cytotoxic effect in HeLa cells by Crathorn and Roberts (1966) but the mechanism of the cytotoxic action of monofunctional alkylating agents is less clear. Methyl methanesulphonate (MMS) has been shown to inactivate transforming activity in vitro by induction of single strand breaks in DNA

Received: 29 January, 1968. Approved : 20 February, 1968.

(Wahl, 1965), and to react with the nitrogen atom in 7 position of the guanine molecule (Brookes and Lawley, 1961). These events have not however been directly correlated with the cytotoxic effects of the agent in mammalian cells in vitro.

The mechanism of radiosensitization by halogenated base analogues in mammalian cells has still not been finally resolved. Increased fragility of DNA (Delihas et a[., 1962; Mohlerand Elkind, 1963; Erickson and Szybalski, 1963) and effects on repair mechanisms (Lett et al., 1964; Fox and Lajtha, 1967) have both been implicated. In bacteria, however, the majority of evidence suggests that inhibition of repair is the most important mechanism of sensitization (Aoki et al., 1966).

There is some evidence to suggest that the cytotoxic effect of MMS in P388 lymphoma cells in vitro is not directly due to alkylation of DNA ( F o x and Fox, 1967a, b) and absence of sensitization by halogenated nucleosides would support this view. Alternatively, if sensitization to X-rays by lUdR and BUdR is due to inhibition of repair mechanisms, then absence of sensitization could possibly imply that damage by X-rays and MMS

382

SENSITIVITY OF P388F TO X-RAYS A N D MMS

in lymphoma cells is repaired by different mechanisms. The following experiments were designed to test some of these possibilities.

MATERIAL A N D METHODS

The origin and methods for cultivation of the P388F clone have been described (Fox and Fox, 19676). The cells grow in static suspensionculture, in medium described by Fischer and Sartorelli ( 1964) and are maintained in exponential growth by ten-fold dilutions every 2-3 days. Under these conditions, the population doubling time of the cultures is 12-14 hr.

Survival of cells after a single dose of methyl methanesulphonate and single and divided doses of X-rays has been determined by the extrapolated growth curve method described by Alexander and Mikulski (1961). The survival values obtained were corrected for mitotic delay. Mitotic delay for both M MS-treated and X-irradiated cultures has been determined by the method described by Fox and Fox ( 1 9676).

Continuous culture in the presence of growth inhibitory compounds results in an extension of the population doubling time, and under these conditions i t is not possible to use the extrapolated growth curve method for determination of the surviving fraction. It is however possible to grow these cells as colonies in test tubes in a medium solidified by agar, as described by Fox and Gilbert (1966). The colonies are scored by naked eye, and therefore the technique requires that a cell complete 12-14 divisions in 8-9 days before being counted as a survivor. This method has been used to determine the plating efficiencies of control cells compared with those cultured in lUdR and BUdR.

Treatnietit of’cells with IUdR atid BUdR

lUdR and BUdR were dissolved in the growth medium without serum and sterilized by filtration (Millipore, 0.22 p) before addition to the culture medium. At each dilution of the cells, fresh lUdR or BUdR was added. The doses of lUdR and BUdR were chosen so that approximately equi- molar quantities of both drugs were used. Cell numbers were determined after each dilution by haeniocytometer counts, and a t the end of the 15-day culture period, control and treated cells were diluted into drug-free medium and their colony-forming ability on agar was determined.

Treatnient with methylmethane~cilphotiatc ( M M S )

MMS was dissolved in the growth medium without serum and filter sterilized before addition to the cultures. Stock concentrations of MMS were adjusted so that the desired dose could be given by the addition of 0-05-0.2 m1/5 ml culture. After the required exposure at 37 C, cultures were centrifuged at 90 i( g and the drug-con- taining medium was subsequently decanted. Cultures were resuspended in fresh drug-free medium at 37” C and their growth rates determined by replicate counts o n paired tubes at each dose level over the subsequent 12-15 days.

X-ray irradiation

Control, lUdR and BUdR treated cultures, replicate dilutions of those treated with MMS, were irradiated in air on a Resomax machine operating at 300 kvp 2.0 mmCu h.v.1. at a dose rate of 100 R/niin.

Radiation-induced mitotic inhibition was measured for control and treated cultures after doses of 200 rads and 400 rads by the methods described by Fox and Fox (19676).

RESULTS

The mean population doubling time over a series of six experiments was 12.7 hr. Cells cultured for 15 days in the lower doses of lUdR did not show any significant increase in population doubling time or fall in plating efficiency. These lower doses of halogenated nucleosides can therefore be considered to be non-toxic. At the higher doses of IUdR and BUdR, slight toxicity was evidenced by an increase in doubling time and a fall in plating efficiency. These results are summarized in Table I.

TABLE I

POPULATION DOUBLING T I M E A N D PLATING EFFICIENCY OF CONTROL CULTURES

OF P388F CELLS AND OF CELLS GROWN I N 5-10DO-2’ DEOXYURIDINE A N D

5-BROMO-2’ DEOXYURIDINE FOR 15 DAYS

Population Plating doubling efficiency time (hr) in agar (?,J

Control . . . . . 12.7 -!: 0.6 61 l U d R 0 . 1 4 p ~ . , . 13.0 + 0.7 58 IUdR 0.28 ,UM . . . 16.4 t 1.3 46 BUdR 0 . 1 6 ~ ~ . . 13.2 ~+ 0.9 60 BUdR 0.32 [AM . . 15.5 -t 0.6 49

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FOX

I I 100 200 300 400 500

1 1 1 1 1 1 1 1 7 1 1 1 ~ ~

RADS FIGURE I

X-ray dose response curves for control cells 0-0 and for. cells grown in 5-iodo-2’-deoxyuridine (IUdR) for 15 days prior to irradiation. 0-0 0 . 1 4 , ~ ~ IUdR. A-A 0.28 /AM IUdR. Standard error of the mean is shown. The computed regression line has been drawn.

X-ray sensitivity of’ cells grown in IUdR

The survival of control and treated populations after acute doses of X-rays is shown in Figure 1. Points on the exponential part of the survival curve were fitted to a straight line with the help of a computer programme kindly made available by Miss T. Alper (Hammersmith Hospital, London). Experimental points o n the shoulders were not used in computing lines. Cells grown in the lower dose of l U d R (0.14 /LM for 15 days) which had n o effect on population doubling time or plating efficiency showed an increased sensitivity to X-rays due t o a reduction in the D,, value from 88.8 & 4.4 rads to 74.3 4.2 rads and in extrapolation number from 2.52 0.81 to 1.9 5 0.74. Cells grown in the higher level of lUdR (0.28 /(M) which increased the population doubling time from 12.7 & 0.6 hr in the controls to 16.4 4- 1.3 hr also showed sensitiiation. and

in this case the Do value was 64.6 3 3.1 rads and the extrapolation number 1.7

It is possible that s i n e survival was determined by the extrapolated growth curve method, differences in mitotic inhibition after irradiation could account for the change in slope after the higher dose of IUdR. This was measured in cells after 15 days’ culture in 0.28 ,UM IUdR. A mean value for mitotic delay of 1.2 min/rad was obtained after doses of 200 rads and 400 rads to control cultures. The mean value for IUdR grown cells was not significantly different from this. The mitotic index in control non-irradiated cultures was 3.1 & 0.4 and in non-irradiated IUdR grown cultures 2.4 3: 0.7.

X-ray sensitivity of cells grown in BUdR

Cells grown in the lower dose of BUdR (0.16 ~ I M ) which was non-toxic, showed a reduc-

0.72.

1

RADS FIGURE 2

X-ray dose respmse curves for control cells 0-0 and for cells grown in 5-bromo-2’-deoxyuridine (BUdR) for 15 days prior to irradiation. Ci-(O 0.16 ,UM BUdR. A-A 0.32 / iM BUdR. Standard error of the mean is shown. The computed regression line has been drawn.


tion in the D,, value from 101.66 + 6.56 rads to 70.90 4 2.80 rads. The extrapolation number for control cultures was 3.68 5 0.78 and for cells grown in the lower dose of BUdR 4.9 & 0.80. After 15 days’ growth in the higher BUdR concentration which was slightly inhibitory (Table I ) there was no further decrease in the Do value 70.84 i 2.24, but the extrapolation number fell froni 3.68 i 0.78 in control cultures to 1.9 I 0.85. There was no increase in X-ray-induced mitotic delay after this higher dose of BUdR. The mitoic index in non-irradiated BUdR grown cultures was 2.2 + 0.8 % and was thus not significantly difer- ent from that of the control cultures.

There is some discrepancy between the D,, values for the two control populations as presented in Figures 1 and 2. After completion of the IUdR series and some preliminary experiments with BUdR, cells were frozen in liquid nitrogen and stored for approximately 4 weeks at - 196 C i n the presence of 20% horse serum and 10% dimethyl sulphoxide. On thawing, the slightly higher D,, values were obtained (BUdR series Fig. 2). The cells which showed the higher D,, values were diluted 10-fold immediately on thawing and cultured a further I 1 days in normal medium before the first X-ray sensitivity experiment. Dimethyl sulphoxide has been shown to be a radioprotective agent by Vos and Kahlen (1962) but its highest possible concentration in the niediuni during an X-ray experiment would be 0.00001 7,. It is assumed that this concentration would exert no significant radioprotective effect. The reasons for the change in the D,, value are unknown. The dose reduction factors obtained with the lower dose of RUdR in the two series were however very similar, i.e. 1.38 and 1.33 respectively.

Di\lided dose rxpi~riments on cells grown in IUdR and BUdR

The changes in radiosensitivity observed after growth of cells in these low doses of halogenated pyrimidines were to some extent associated with reductions in extrapolation number. The response of such cultures to divided doses of X-rays was therefore determined. Two doses of 150 rads were given, separated by intervals of up to 5 hours and the survival after the divided doses is expressed as a fraction of that after a single dose of 300 rads which is taken as unity. The results are shown in Figure 3. The recovery of cells grown

@ Control

0 BUdR O.Ibl ,M I S d 1 lUdR 0.141,M 1 S d

3

HRS BETWEEN FRACTIONS

FIGURE 3 Response of control cells C-U; cells grown in

lUdR (0.14 / I M 15 days). x - x ; and in BUdR (0.16 ,UM 15 days) 9-G ; to fractionated doses of X-rays. Two doses of 150 R were given and survival after the ’* split ” doses is expressed as a fraction of that after a single dose of 300 R. Results are the mem of three experiments, and standard error of the mean is shown.

in lUdR (0.14 p~ for 15 days) is markedly reduced though not eliminated completely. Corre- spondingly, the extrapolation number on the acute X-ray survival curve was slightly reduced froni 2.5 to 1.9 for cells grown in this concentration of lUdR (Fig. I ) . Growth in the lower concentration of BUdR (0.16 /AM) affected the recovery pattern very little (Fig. 3) and there was correspondingly no reduction in extrapolation number for cells grown in this level of BUdR (0.16 p ~ ) . The higher dose of BUdR (0.32 ~ L M )

reduced the extrapolation number considerably (Fig. 2) and in two or three fractionation experiments no increase in survival between divided doses could be demonstrated.

Sensitivity of cells grown in IUdR and BUcJR to inrrhyl methanesulphonatr

Cells cultured for 15-day periods in two dose levels of lUdR and BUdR have been shown to be more sensitive to X-rays (Figures 1 and 2). Replicate cultures of these cells were simul- taneously tested for sensitivity to the monofunctional alkylating agent methyl methanesulphonate (MMS). The half-time of hydrolysis of this drug is relatively long, 9 hr a t 37” C in saline (Ross and

385

FOX

TABLE 11

SURVIVAL OF CONTROL CELLS A N D CELLS GROWN I N lLdR A N D BUdR FOR 15 DAYS, AFTER TREATMENT WITH METHYL METHANESULPHONATE

Dose of MMS (mM) 1 hour exposure

0.09 0.18 0.36 0.54

Control . . . . . . . . . 71 66.5 1.6 1 .o TUdR 0 . 1 4 , ~ ~ . . . . . . 72 ' 65.2 11.0 1.3 BUdR 0.16 , ~ L M . . . . . . 76.5 64.8 10.5 1.2 Estimated error of the means 75.1 + 1.89 65.4 ?- 2.8 9.7 t 2.08 1 . 1 z 0.44 Mean . . . . . . . . . 75.1 f 1.35 65.4 2.02 9.7 f_ 1.47 1 . 1 t- 0.31

Dose of MMS (mM) 48 hour exposure

0.09 0.13 0.1s

Control I . . . . . . . . . lUdR 0.14 ,UM . . . . . . . BUdR 0.16 /LM . . . . . . Control I1 . . . . . . . . IUdR 0.28 ,UM . . . . . . . BUdR 0.32 / L M . . . . . . Estimated error of the means Mean . . . . . . . . . .

~

24.2 22.4 18.0 27.5 20.3 18.7

21.8 + 1.3 21.8 ~- 3.3

I .43 I .26 2.0 1.43 1.55 I .25

1.48 0.092 1.48 0.037

0.07 0.04 0.06 0.03 0.03 0.06

0.048 2 0.016 0.048 i 0.006

Results are means of three experiments. ' Results of two determinations only.

Davis, 1957) and its toxicity has previously been shown t o depend markedly on the duration of exposure of the cell populations to the drug (Fox and Fox, 1967b). In the present study therefore two dose schedules were used, cells being exposed to the drug for either 1 or 48 hr. In each case the drug was removed by centri- fugation a t the end of the exposure time. The survival levels of cells grown in the two doses of IUdR and BUdR after treatment with M M S are shown in Table 11. At each dose level of MMS the mean survival of control, lUdR and BUdR treated cells showed n o consistent or significant difference from the overall mean, and thus no sensitization t o the effects of MMS was demonstrated. A mitotic delay of 3.5 hr was found after 0.09 mM MMS for 1 hr and of 5 hr after 0.36 mM MMS for 1 hr. Mitotic delay in IUdR and BUdR treated cultures was not significantly different.

DISCUSSION

In several previous studies o n sensitization of mammalian cells to X-rays, cultures have been incubated in relatively high drug concentrations

for short periods of time (Kim rf a/., 1964; Delihas rt al., 1962; Djordjevic and Szybalski, 1960; Lett rt al., 1964). In many cases, toxicity, i.e. prolongation of population doubling time and decrease in platingefficiency, has been evident, and Mohler and Elkind ( 1 963) concluded, after using a wide range of concentrations, that in V791 Chinese hamster cells the cytotoxic effect of BUdR and at least part of its potentiating effect on the X-ray response were closely related.

A considerable range in the degree of replacement of thymine by BU has been reported. One per cent replacement was recorded by Delihas et al. (1962) in HEp cells and this level reduced the plating efficiency from 0.4-0.6 in controls to 0.2-0.5 in BUdR treated cells. On the other hand, Schindler rt al. (1966) reported a n approximately 54 replacement of thymine by BU in P815Y cells after 3 days' growth at 0.03 mM BUdR. Under these conditions, the plating efficiency of the cultures was reduced from 0.96 to 0.33. Many intermediate values have been reported, all of which produce sensitization in different cell lines, but most of them are associated with some degree of toxicity; even one per cent replacement pro-

386

SENSITIVITY OF P388F TO X-RAYS AND MMS

duced a fall in plating efficiency in the HEp cell line used by Delihas ef a/. (1962).

The concept of division probability in non- irradiated and irradiated populations of mammalian cells has been discussed extensively by Whitmore and Till (1964). These workerspoint out that in many of the previously reported studies on radiosensitization by halogenated pyrimidines, the phenomenon may be more apparent than real, since the doses of drugs used caused a decrease in division probability in non-irradiated cells. I t would appear therefore that the most reasonable approach would be to use completely non-toxic doses of IUdR and BUdR. A non-toxic dose is defined as one having no effect on population doubling time or plating efficiency of the treated cells.

Continuous culture of P388F lymphoma cells in relatively low doses of IUdR resulted in an extension of the population doubling time. At doses above 0.28 p ~ , only three doublings were completed at the control rate, after which the population doubling time was increased to 29 hr (Fox and Lajtha, 1967). In the present study 0.14 /LM IUdR and 0.16 p~ BUdR had no effect on population doubling times and the higher dose of both compounds permitted exponential growth at slightly increased doubling times (Table I). Thus the two doses of IUdR and BUdR chosen showed minimal toxic effects.

1 ncreased radiosensitivity has been demonstrated after prolonged growth of the mouse lymphoma P388F in non-toxic doses of lUdR by Fox and Lajtha (1967). In these studies sensitization was shown to develop gradually; it was not possible to demonstrate any increased sensitivity after 3 and 7 days of growth at this low lUdR concentration, but by 16 days sensitization was obvious. This slow development of increased sensitivity would suggest that a thresh- old level of replacement of thymine by 5-iodoura- cil ( IU) was necessary. However, parallel studies showed that P388F cells given a 30 min pulse treatment with a higher but still non-toxic dose of IUdR and subsequently cultured in drug-free medium for up to 50 cell generations still showed increased sensitivity. At the end of 50 population doublings, the possible lUdR content within the cells must be smaller by a factor of a t least 1 O I 2

compared with that after continuous administration. The actual presence of lUdR in the D N A would therefore appear to be unnecessary for

sensitization, which in this case was shown to be largely due to a reduction in the ability of the treated cells to recover between divided doses of radiation.

In view of the above considerations, a 15-day culture period was used in the present study. The use of this relatively long culture period could be criticized on the basis that mutants with different radiosensitivities or with acquired resistance to drugs could be selected. However, the slight changes in growth rates and plating efficiencies observed would suggest that selection is minimal and the occurrence of mutants unlikely.

Radiosensitization was achieved after 15 days’ growth in both l U d R and BUdR (Fig. 1 , 2). The increase in sensitivity was found to be largely due to a reduction in extrapolation number. Also, a pronounced reduction in the ability of cells to recover between divided doses of X-rays was found (Fig. 3). The results of Kim et a/. (1964) indicate recovery of HeLa and HEp cells after growth in BUdR. However, recovery as observed by these authors may in part be artifact as the survival of cells treated with BUdR and X-rays was found to increase with time of irradiation after cell plating (Delihas et a/ . , 1962).

The results of Schindler et al. (1966) would suggest that damage by X-rays and the bifunctional alkylating agents HN2 and dimethyl my- leran is produced by a similar mechanism. The interpretation therefore, based on the absence of sensitization by IUdR and BUdR to the effects of the monofunctional alkylating agent MMS in cells sensitized to the effects of X-rays, is that the primary site of action of X-rays is on the D N A whereas that of MMS is not.

Evidence has been presented (Fox and Fox, 1 9 6 7 ~ ) to suggest that alkylation of DNA by methyl methanesulphonate may not be the only cytotoxic effect. Other factors, including alkylation of ribonucleic acids and interference with cell membrane transport, may play important roles in determining the biological effects observed after treatment of these lymphoma cells with methyl methanesulphonate.

ACKNOWLEDGEMENTS

The author wishes to thank Mr. B. Harrison for excellent technical assistance, Dr. L. G. Lajtha and Dr. B. W. Fox for much helpful discussion and Dr. C. W. Gilbert for help with statistical analysis.

387

FOX

ACTION DES NUCLEOSIDES HALOGENES S U R LA SENSIBILITE DU LYMPHOME P388F D E LA SOURIS AUX RAYONS X ET AU METHANE

SULFONATE DE METHYLE.

Des populations de rellules du lymphome P388F de la souris ont Pt6 sensibilise‘es aux e f f h s des rayons X par ciilture prolongke dans des doses non toxiques de S-iodu-2’- desoxyuridine et de 5-bromo-2’-dksoxyuridine.

On a constatk une diminution marquee de I’aptitude des cellules exposees ci rkcupkrer apr& administration de doses fractionne‘es de rayons X . Le micanisme de sensibilisation pourrait donc gtre lie‘ ir line inhibition des rnkcanismes rkpamteurs.

Des cultures-temoins des mimes populations n’ont present6 auriin accroissement de la sensibilitk au tnithane sulfonate ds mithyle, alcoi‘lanf monofonciionnel, ce qui permet de periser qire les Iksions produites par les rayons X et par ce dernier agent ripondent d des mkcanismes d$&ents.

D’autre part, les rksultats semblent montrer que I‘alcoflation d~ I’A D N par le nikthane siilfotiate cle niithyle rr’est pas le seul effet cytotoxiqiie de cette substance.

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the effect of halogenated nucleosides on the sensitivity of a mouse lymphoma p388f to x-rays and...

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