The Effects of and Dimethyl Myleran on Murine Leukemia Cell Lines ofDifferent Radiosensitivity in Vitro1

GERALD J. GOLDENBERG2 AND PETER ALEXANDER Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, London, &W.8, England

SUMMARY The effects of nitrogen mustard and dimethyl myleran on 3 mutants of murine leu kemia L5178Y—―radioresistant― and “radiosensitive―diploid cell lines and a tetra ploid line—have been studied. A close parallelism was found in the response of “ra dioresistant― and “radiosensitive― cells to treatment with nitrogen mustard, dimethyl myleran, and X-radiation; in each case the D0 for the “radioresistant― line was twice that of the “radiosensitive―cell line. The tetraploid cell line showed marked differ ences in response to the 3 agents : it showed increased resistance to nitrogen mustard, increased sensitivity to dimethyl myleran, and no change in response to X-radiation, all relative to the response of the “radioresistant―diploid cell line to these same agents. The effect of dimethyl myleran more closely resembled that of X-radiation in that cell division continued for some time after treatment and storage at suboptimal tem perature facilitated post-treatment recovery processes. The monofunctional mustard, dimethyl chloroethylamine, was approximately 35 times less potent in its cytocidal effect than nitrogen mustard. The cyclic ethyleneimmonium ion of nitrogen mustard appeared to retain the full cytocidal action of nitrogen mustard in this system. Attempts to induce mutants of L5178Y more resistant to nitrogen mustard and di methyl myleran by repeated treatment of surviving cells were unsuccessful.

The cytotoxic action of alkylating agents is referred to In vitro studies, if they are to be relevant to the in vivo sit as “radiomimetic― because, like X-rays, these agents give uation, must therefore be carried out with doses of drugs rise to mitotic death (7) ; that is, dividing cells are “steri lower than those that are needed to cause interphase death. lized―in the sense that they are prevented from undergoing Relatively few studies have been made to establish the re indefinite duplication, and an individual cell loses the ca lationship between dose and the fraction of cells rendered pacity to form a clone. After sustaining a lethal injury, incapable of forming a clone in cultures of rapidly dividing defined as an injury causing inability to form a clone, cells mammalian cells. When studies of this type have been may divide several times and can persist and metabolize done, the curves have been of the same type as that found for long periods, during which they are recognized as living for X-rays ; i.e., they depict an exponential relationship by physiologic tests such as dye uptake. To cause out such that the logarithm of cell survival is proportional to right death, called interphase death (1, 7), very much dose after a certain mmimum dose has been passed (5, 9, larger doses are required ; the mechanism involved is en 12, 21, 30). As far as the dose-response curve is concerned, tirely different, and the relative effectiveness of different these agents are truly “radiomimetic―and they fit the substances is not the same for the 2 types of death. When mathematical requirements of a multi-hit curve. But this drugs are used to treat tumors in vivo, the dose levels at terminology implies a target theory type of approach which tamable are such as to kill by the mitotic pathway (1, 7). we feel is not helpful in the elucidation of the basic mech anisms. In an earlier investigation (5) the effect of nitro 1 This work was supported by grants to the Chester Beatty gen mustard, HN2 (CH3N(CH2CH2CL)2), and dimethyl Research Institute (Institute of Cancer Research, Royal Cancer Hospital) from the Medical Research Council, the British Empire myleran, Cancer Campaign, the Anna Fuller Fund, and the National Can (CH,•SO2•O.@H.(CH2)@[email protected]@•CH,) cer Institute of the USPHS. 2 Canadian Hadassah Cancer Research Fellow, National Can @H5 à H, cer Institute of Canada. Present address :Department of Internal Medicine, University of Manitoba, Winnipeg, Canada. on Fischer's murine leukemia L5178Y cells grown in vitro Received for publication February 22, 1965. was determined. These 2 alkylating agents were chosen be 1401

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. 1402 Cancer Research Vol. 25, October 1965 cause both were water soluble and reacted rapidly and be tamed by accident from a culture of the “radioresistant― cause there were considerable differences in the hemato strain that had been maintained in “simplified―medium, logic effects they produced in vivo (14, 15). Differences in in which horse serum is replaced by bovine plasma protein action were seen at the cellular level in vitro (5). HN2 fractions (albumin, a2-globulin, and 7-globulin). This caused immediate cessation of cell division, whereas cells tetraploid line has remained quite stable in culture for a treated with dimethyl myleran showed a tendency to di period of 1 year ; the cells have a doubling time of 16—18hr vide before mitosis stopped. The cytocidal effect of both and morphologically are larger than either the “resistant― agents resembled that of X-rays in causing the formation of or “sensitive―celllines. giant cells (5). The cells used in this iiivestigation can be classified as During the course of culturing the L5178Y cells, a num (a) resistant (R), (b) sensitive (8), or (c) tetraploid-resist ber of mutants that differed in their sensitivity to X-rays ant (T) on the basis of their response to X-rays. The re were isolated, and a tetraploid variant was also obtained. sistant line was obtained by culturing cells from an ascites The principal object of the studies described in this paper tumor that had been established by transplant of a single was to determine whether sensitivity to radiation could be cell into the animal. To guard against the occurrence of correlated with response to HN2 or to dimethyl myleran. sensitive mutants, such cultures were carried for 4 to 6 Similar studies based on the response of grafted tumors weeks only and thereafter a new line was established with (19,23), have been made in vivo,but becausesuch data are cells taken from the mouse. After transfer from in vivo complicated by immunologic and nutritional factors, de the cells grew steadily after 48 hr, and the doubling time ductions concerning sensitivity at the cellular level cannot in culture ranged from 13 to 14 hr. readily be drawn. Hirono (19) has reported, on the basis Two sensitive lines that were isolated at different times of in vivo tests, that a Yoshida ascites tumor resistant to were used ; S63 and 864 both showed the radiation response alkylating agents was not cross-resistant to X-rays. Skip of the sensitive line described by Alexander and Mikuiski per and Schabel (28) found that tumors resistant to one (4) but, as will be seen, are different with regard to their alkylating agent were resistant to other alkylating agents sensitivity to the alkylating agents. The doubling time in and also to mitomycin, urethane, and 6-mercaptopurine culture was 10 hr for both the S63 and the S64 cell lines. ribonucleoside but were sensitive to other anti-metabolites, Treatment of cells with alkylating agents.—The nitrogen X-rays, and cortisone. Calcutt and Connors (10) ob mustard, methyl di-2-chloroethylamine (HN2), and its served a correlat ion between the in vivo response of a van monofunctional equivalent , dimethyl 2-chioroethylamine ety of different grafted tumors to an aromatic nitrogen (HN1), were dissolved in physiologic saline at approxi mustard and their sulfhydryl content. In vitro, Colombo mately 20°C,and 0.5 ml was added to 9.5 ml of a culture et al. (12) found that a variant of a mouse fibroblast that containing approximately 0.5—1X 10@leukemia cells/mi. was cytologically characterized by a greater number of Dirnethyl myleran, (DMI\I) (2 ,5-dimethane sulfonyloxy chromosomes was more resistant to HN2, but not to hexane) was dissolved in 5 % acetone in saline, and 0.5 ml X-rays. of the 5 % acetone solution was added to 9.5 ml of the cells in tissue culture. The acetone introduced a slight growth MATERIALS AND METHODS lag for the 1st 60—80hr. The culture was kept in an in Materials.—HN2 as the hydrochloride was obtained cubator maintained at 37°± 0.5°C, and aliquots were from the Boots Pure Drug Company Ltd. Dimethyl my counted Ill a Neubauer hemocytometer. Following treat leran and the monofunctional nitrogen mustard, (CH3)2N. ment the cell cultures were diluted with fresh medium CH2CH2C1, were synthesized by Mr. J. L. Everett of the every 24 to 48 hr or as often as required to maintain cell Chester Beatty Research Institute. concentration in the range from 4 X 10@to 8 X 10@cells/ Origin of cell lines.—The L5178V leukemia line is car ml. Under these conditions the cells in the untreated con ned in our laboratory from cells given to us by Dr. G. A. trol cultures increased in number at an absolutely constant Fischer (Pharmacology Department, Yale University) in rate. The alkylating agents added react rapidly, and for 1900 an(I continually passaged in their strain of origin, dimethyl myleran the “half-life―at 37°C is 32 mm ;3 that DBA/2 110Cc. They will grown in suspension culture in a is, in this time half of the substance has either reacted by medium containing horse serum, which has been described alkylation (with cell or medium constituents) or been hy by Fischer, and they will grow both in vivo and in vitro drolyzed. The rate of loss of alkylating power of the 2 from an inoculum of a single cell. In the presence of heav nitrogen mustards used depends on the concentration of ily irradiated feeder cells the in vitro cloning efficiency ap substrate, i.e., because they react by a bimolecular reac proaches 100 @;;@.After a period of culture in vitro mutants tion (SN2) as opposed to a unimolecular (SN1) mechanism arise that are more sensit ive to X-rays and divide more for D?tiM (24). In the medium used, the time for half of rapidly. A pure line of these so-called sensitive mutants the alkylating potential to disappear, as measured by the was isolated by cloning from a single cell. Epstein reagent, was 75 mm for HN2 and 210 mm for the “Resistant―and “sensitive―strains both consist largely monofunctional agent. (more than 80 %) of diploid cells (n = 40) , although there Estimation of @rurvivors.—Theeffect of DMM on the are some celLs that have n = 39, 41, or 42. i\Iorphologi growth rate of sensitive cells illustrates a typical experi cally, “sensitive―cellsare larger and more irregular in out ment (Chart 1). The cell number stated is, of course, line than “resistant―cells. In some cultures there is a corrected for a dilution factor, owing to the need to keep very small proportion (usually less than 5 %) of tetnaploid cells; a line consisting of over 93 % tetraploid cells was ob 3 P. Alexander. Unpublished observations.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. GOLDENBERG AND ALEXANDER—EffeCtS of HNfd and Dimethyl Myleran on Leukemia Cells 1403

A BC D E of the outgrowing cultures shown in Chart 1 are all parallel to the control line. By extrapolating these lines back into the ordinate, a value for the number of the survivors is obtained. There are 2 rea.sons why such a figure will be an underestimate of the true situation. 1. The surviving cells, although they have not lost their ability to divide indefinitely, may have sustained a non permanent injury which manifests itself in a delay before they start dividing. Temporary holdup of cells at some stage of interpha.se, which manifests itself as a delay in division (i.e., reduction in growth rate), is a well-known effect of ionizing radiations (11). This delay does not nec essarily lead to an error in the extrapolation method be cause the period of arrest is compensated for by a burst of cell division produced by cells held up in interphase enter ing mitosis (11). The net result is that the total increase

C in cell number over a period of 24 hr or more is not altered significantly by radiation. How completely this second ary wave of division compensates for division arrest by U 0 alkylating agents is not known. 2. Some of the survivors have sustained permanent dam age such that their rate of cell division is slower—or the z doubling time for the cell number is longer—than that of the untreated control cells. Such slowly growing cells would be overwhelmed by the survivors that divide at the usual fast rate and would, in fact , not be seen. The cx trapolation method records only survivors having the same doubling time a.s the untreated controls. Cloning of in dividual cells from treated cultures leaves no doubt that there are slow-growing survivors, and there have been sev eral descriptions of mutants with lower growth rates fol lowing exposure to X-rays (26). Examination of growth curves such a.s those shown in Chart 1 indicates that, in 0 40 80 120 160 200 240 280 the dose range used, the proportion of slow-growing cells TIME (HOURS) is not high, as otherwise it would take much longer before the lines of the treated cultures become parallel to that of CHART 1.—The effect of dimethyl myleran on the growth of the “radiosensitive―(863) mutant of murine leukemia L5178Y cells the control. Dewey et al. (13) have compared the estimate grown in vitro. 1)imethyl myleran was administered in the follow for survivors following X-irradiation of fibroblasts by the ing doses : A , control ; B, 5 @g/ml;C, 10 @zg/ml;D, 15 @g/ml;E, extrapolation technic with that obtained by counting col 20 4ug/ml ; F, 25 ,@g/ml; G, 35 ,ig/ml. onies growing in solid medium. The dose-response curves obtained by the extrapolation technic were lower than, but cell population under 106 cells/mi. This dilution does not, parallel to, those obtained by colony counts, so that al however, exert any bias. From such curves a value for though the ext rapolation number wa.s higher when colony the surviving cells was obtained by the extrapolation counts were used, little difference was observed in the D0 method described by Alexander and Mikuiski (5). The (for definition, see below). assumptions are made that after the reaction of the alkyl With the L5178Y leukemia cells an absolute value for ating agents is complete, a fraction of cells retain their all surviving cells capable of indefinite division cannot be ability to divide and that these “survivors―grow at a nor obtained by colony counts since a method for growing ma! rate. However, the “killed―cellsremain as detectable these cells 011solid medium has not been developed. How cells for 2 or 3 days and under the microscope cannot be dis ever, since one cell is capable of starting a culture, the tinguished with certainty from cells that have retained absolute value of survivors can be determined by a dilution ability to divide indefinitely and that are responsible for technic (4, 20). Experience gained from attempts to in@ the subsequent outgrowth. After doses which “kill―more duce resistant cell lines by repeated treatment of surviving than 99 %, the “survivors―are not readily seen for 2 or 3 cells revealed that the dose of dimethyl myleran needed to days since they represent a small percentage of the cell give 1 survivor out of 10@cells was within 20 % of that de population, and in effect the only cells counted are those termined by extrapolation of the growth curves. Al which have suffered mitotic death, but which remain as though the extrapolation method used in this investigation visible cells. In time the survivors increase in number records only those surviving cells that have retained the until they outgrow the “dead―cellsand give rise to the same rate of cell division as that of the untreated cells, it new population, which is growing at the same rate as the appears that the dose-survival curves obtained are not untreated cells. This is shown by the fact that the lines greatly different from those based on scoring all survivors.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. 1404 Cancer Research Vol. 25, October 1965

In any case comparisons between different cell lines are RESULTS valid. This is shown by the fact that the ratio of D0's for Influence of cell numbers.—Chart 2 shows that the effect 2 mutants determined by the cloning technic (20) is the of HN2 and dimethyl myleran is independent of cell con same as that found by the extrapolation method (4). centration in the range studied (i.e., 43,000—344,000 864 Parameters of the dose-nurvival cui-.—The survival data cells/mi with HN2 and 34,000—340,000with DMM). Pre shown in Charts 3—Sconform apj)roxnnately to a “multi sumably in these dilute suspensions of cells only a minute hit― relationship. The 2 constants n and D0, by which fraction of the alkylating agent reacts with substances in such a curve is defined can be determined from the semi the cell, and the vast majority is used up ineffectively in logarithmic curves. The size of the initial shoulder is reactions with the medium or by hydrolysis. Conse given by the extrapolation number, n, obtained by ex quently, increasing the number of cells does not signifi tending the linear part of the curve to intersect with the cantly alter the amount of the aikylating agent available ordinate. D0 is obtained from the slope of this linear for reactions with the cells. portion and represents the dose needed to reduce survival Comparative sensitivity of different mutants.—Charts 3, 4 by a factor of 1/e (i.e., to reduce survival to 37 % of the show the sensitivity of 3 different L5178Y cell lines to HN2 initial cell population). In other words, once allowance and dimethyl myleran. The reproducibility of the data has been made for the shoulder a dose of alkylating agent is very satisfactory. The dose-survival curves of R cells equal to D0 will kill 63 % of cells present ; a dose of 2D0 (“radioresistant― diploid line) after treatment with HN2 will reduce survival to 37 % of 37 % (or 14 %). The more and DMM are made up of data from 3 separate experi important of these 2 parameters is D0, which can be ments spread over a period of 13 months and involving correctly referred to as a measure of the resistance of the separate cell lines established from an in vivo tumor. The cells to the alkylating agent. The parameter n is experi curves for $63 (“radiosensitive―diploid) and T (the tetra mentally difficult to determine and is likely to be affected ploid strain) after HN2 treatment are also composites from by a number of external factors, such as the presence in the medium of small amounts of substances which have a 2 and 3 separate experiments. Table 1 summarizes the very high competition factor for the alkylating agent. D0 and n values for the separate curves. There is a close Thus the presence of a thiol compound would give rise to a parallelism in the relative sensitivities of R and $63 cells shoulder because it would consume the alkylating agent with X-rays, HN2, and dimethyl myleran; in every case preferentially. the D0 for R is twice that for 863. For T the situation is

0 CONTROL

a 34,000CSII$/mI

108. . 136,IXX@ U 340,000

0 43,000ceIIs/mI A 86,000 I . 172,000 . 34@4,000 E

U 0

Dz 10@

A

a 4;:@ 8@0 1@0 1@0 2&@ 6 4•0 8@0 120 160 200 TIME(HOURS) TIt,@(HOURS) CHART 2.—The effect of variations in cell concentration on the cytocidal action of nitrogen mustard and dimethyl myleran. On the left graph, 4 samples of increasing cell concentration (43,000-344,000cells/ml) were each treated with 0.125 @g/mlof HN2. On the right, 4 samples of increasing cell concentration (34,000—340,000 cells/ml) were each treated with 15 @zg/mlofdimethyl myleran.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. GOLDENBERG AND ALEXANDER—Effects of HNP2 and Dimethyl Myleran on Leukemia Cells 1405

ence in the extrapolation number (n) between $63 and 564 HN2 cells, although a considerable increase in this number oc curs when cells are treated with D1\IM rather than with HN2. SM, like S63, has a doubling time of 10 hr, as com pared with 13—14hr for the R cell line. 564 appears to represent a mutant of the S63 cells that has become more 1o1@ resistant to HN2 and only slightly more resistant to DMM without having changed its response to X-rays. If the x comparison were confined to 564 and R cell lines, it might be concluded that there is a suggestion of cross resistance 0 z between X-rays and dimethyl myleran, but not between > 102 > X-rays and HN2. The behavior of the cells after sustaining “lethal―damage.— The effects of HN2 and DMM on cell growth in the 1st U 40 hr following treatment are shown in Chart 6. The ef 0 z feet of DMM on “radioresistant― cells (R) and the 2 “ra 0 diosensitive― lines (563 and 564) is shown at the top of U Chart 6 and the effect of HN2 on the same 3 cell lines, at the bottom. Obviously more time points are required for a precise statement, but nevertheless a consistent pattern R appears to exist. The straight lines in each of the 6 cases represent untreated controls, and the other curves repre sent cellular growth following treatment with the dose of DMM or HN2 that yields 1 survivor in 10@(that is, the comparisons are made at the same level of cytocidal action for each drug). As reported earlier (5) exposure to HN2 stops cell division of the R cells immediately. Chart 6 shows that this applies equally well to the 563 and 564 0 0.5 1.0 1.5 2.0 2.5

Ht@J2(mpmoles/mI)

CHART 3.—Dose-survival curves of the “radioresistant― (R) and “radiosensitive―(863)diploid cell lines and the tetraploid line (T) of murine leukemia L5178Yafter treatment with nitrogen mustard (HNS) in vitro.

quite different. The increase in ploidy raises the D0 for HN2 by a factor of almost 4, whereas towards dimethyl myleran the tetraploid is more sensitive than the corre sponding diploid R. The radiosensitivities of the R and T C strains are almost identical, and the change from diploid z to tetraploid changes neither n nor D0 significantly. Com > > paring the relative sensitivities of R and T cells to different agents provides an example of every possibility ; the effect of an increase in ploidy can raise or lower D0, or leave it un U 0 affected. z The paradoxical behavior of 563 and S64.—A year elapsed 0 between the 1st and 2nd series of experiments with the U “radiosensitive―diploid cell line. The data for the 1st series, shown in Charts 3 and 4 and referred to as $63, is compared with that of the second series (called $64) in Chart 5. Towards HN2, $64 (D0 = 0.026 @g/mlor 0.135 m@moles/ml) is considerably more resistant than $63 (D0 = 0.015 @zg/mlor 0.078 m@moles/ml), and there is no significant difference between R and $64 as far as this agent is concerned. Towards dimethyl myleran, $64 (D0 = 2.56 @.sg/mlor 9.34 mj.@moles/ml) is only slightly more resistant than $63 (D0 = 2.06 j@g/ml or 7.52 30 60 90 120 DM0. (mpmoles/mI) mzmoles/ml), and the difference in sensitivity between R and S cells is almost fully maintained. Any difference in CHART 4.—Dose-survival curves of the “radioresistant― (B) and “radiosensitive―(865)diploid cell lines and the tetraploid the effect of irradiation with X-rays on 563 and $64 cell line (T) of murine leukemia L5178Y after treatment with dimethyl lines is indistinguishable. There is no significant differ myleran (D.M.M.) in vitro.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. 1406 Cancer Research Vol. 25, October 1965

TABLE 1 A COMPARISONOF THE D0's AND EXTRAPOLATIONNUMBERS FOR “RADIORESISTANT,― “RADIosENsITIvE,― AND TETRAPLOID CELL LINES AVFER TREATMENT WITH HN2, DMM, AND X-RAY

Czu. LINEHN2DMMX-RAY―D0,iD0nD,

rn,sg/mlmMmoles/ml@ag/m1mNmoles/ml“Radioresistant― diploid(R)0.0300.1564.24.2515.514.81082.8“Radiosensitive― diploid 7.52 @ Tetraploid (T)(263)0.015 0.1150.0780.5972.3 1.22.062.12• 7.758.6 3.162 1111.4 2.2

a See Alexander and Mikulski (4). The D0 and n for “radioresistant― and “radiosensitive― diploid cells irradiated with X-ray were obtained from the dose-survival curves, which were based on the extrapolation technic, in this paper. The values obtained by the cloning technic of Lett et at. (20) are somewhat lower. The D0 and n for tetraploid cells were obtained from unpublished experiments (P. Alexander) based on the extrapolation technic.

z Influence of temperature on action of HN2 and DMM.— 0 Attempts were made to induce recovery of the R and 864 cells after treatment by storing them for 24 hr at 30°C. Stapleton et al. (29) showed that survival of various strains of E. coli treated with X-radiation was increased by post treatment storage at temperatures below 37°C, and the optimal temperature for survival of E. coli B/r at any X-ray dose was 18°C. The same procedure was very ef fective with radiosensitive L5178Y cells that had been ir radiated with X-rays (8). In Table 2 a comparison of the S -- - -. ratio of D0 at 30°Cto D0 at 37°Cis made between HN2 and

—0 DMM with the use of R and 564 cells. The results show - ——@0 that the fraction of cells “killed―byHN2 was the same 0 whether they were maintained at 37°Cor at 30°Cfor a z > period after treatment. With dimethyl myeran there was > \ some increase in survival by storage of cells at 30°C a!- \ though the effect was less than with X-rays. These find U \ ings suggest that recovery processes of the type occurring 0 after irradiation with X-rays do not take place with regard z 0 \ to HN2, although they may occur after DMM. U Comparison of a rnornofunctional mustard with HNf2.— \ Haddow et al. (18) found that only nitrogen mustards con taming 2 or more alkylating centers (viz., the .CH2CH2C! group) per molecule were capable of inhibiting the growth of grafted tumors and that monofunctional compounds \ were ineffective in vivo at the maximum tolerated dose. For the production of chromosome abnormalities in plants, bifunctional compounds were in general 50—100 times

4.Ox io@ more effective than the corresponding monofunctional at 127.7 m@ moles lad compounds given in comparable doses (22). For the 2.0 2.5 HN2 induction of gene mutations the effect of polyfunctiona!ity

30 60 90 120 DMM is much less. The D0 of the inonofunctional nitrogen DRUG DOSE (mi, moles I ml) mustard, (CH,)2N . CH2CH2CI, for “radioresistant― cells

CHART 5.—Dose-survival curves of the 2 “radiosensitive― cell was 5.35 m@moles/iiil, which was 35 times greater than lines (863 and 864) of murine leukemia L5178Y after treatment for HN2 (D0 = 0.156 m@tinoles/nil), and the extrapola @ with nitrogen mustard (HN@) and dimethyl myleran (DMM). tion IluIliber, n = 17, 4 times greater. Attempts to induce resistance.—Fischer (16) has been able cells and was also seen with the T cells. On the other to very readily isolate mutants that were more resistant hand, cells treated with dimethyl myleran, like those ir than the original line to amethopterin from the survivors radiated with X-rays, do not stop dividing at once (4, 5). of cultures exposed to this agent. Successive exposure of After treatment the cell population continues to increase the survivors of the R line to X-rays failed to produce a and almost doubles in number before cellular death occurs cell strain that was more radioresistant.3 Attempts were (Chart 6). made to isolate mutants resistant to HN2 and dimethyl

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. GOLDENBERG AND ALEXANDER—Effects of HNP@ and Dimethyl Myleran on Leukemia Cells 1407

myleran by treating 10 ml of a suspension of 10@cells TABLE 2 with a dose of alkylating agent (i.e., 0.3 ,@g/ml of HN2 A COMPARISON OF THE RATIO OF D0 AT 30°C TO D0 AT 37°C and 40 ,@g/ml of dimethyl myleran) sufficient to reduce FOR “RADIORESISTANT―AND “RADIOSENSITIVE―CELL the surviving fraction of cells to approximately 1 in LINES AFFER TREATMENT WITH HN2 AND DMM

10g. The survivors were allowed to grow to a concentra 30'C tion of 10@cells/mi and were treated again with the same D0 37'C concentration of drug ; this procedure was repeated 4 times CELL LINED0 -HN2 in all. The survivors from the last treatment were then DMM“Radioresistant― challenged with a dose that was 50 % greater. There were no survivors from this last treatment, and from this we diploid (B) .08 1.22 “Radiosensitive―diploid (S64)1 0 .96 1.33 conclude that significantly more resistant mutants (i.e., D0 increased by more than 25 %) did not arise under the conditions used. The technic would have detected the TABLE 3 production of 1 mutant in 10's. AGING OF HN2 SoLUTIoN IN H,O (2 @g/ml) AT 20°C Effect of aging a solution of HN2.—In solution, HN2 un AND SUBSEQUENT DETERMINATION OF THE EFFECT dergoes very rapid cyclization to give an ethyleneimmo ON CYTOCIDAL ACTION

R S63 564 Incubation time effect dose of HN2 (mm)Cytocidal(j@g/ml)1 (fraction of cells surviving)Equivalent

X 10' 10 4.4 X 10@ 0.099 375.8 5.1 X 1020.095 0.096

nium ion ; at 37°Cthe half-life of this process is 1—2mm (25). This ethyleneimmonium group is the effective al DMM kylating center of aliphatic nitrogen mustards, and it un dergoes hydrolysis or reacts with substances capable of alkylation. In tissue culture medium, half of the alkyl ating power had disappeared at 37°Cin 75 mm.4 Table 3 shows that HN2 which had undergone cycliza tion to the ethyleneimmonium ion, E tfl CH2 Lu @ U CHR—N\ Cl 0 CH, CH,CH,Cl z by being allowed to stand in water at 20°Cfor 10 and 37 mm was still fully active. After these times 90 % and 99 %, respectively, of the HN2 has cyclized. At first sight this result seems puzzling since the charged ethyleneim monium ion is unlikely to be able to penetrate into cells. However, on addition of the cyclized form to the tissue H N2 culture medium, which contains chloride ions at a concen tration of 0.1 M, the cyclized form will be converted back to the original nitrogen mustard quite rapidly, and there is no need to postulate that the drug enters the cells as the ethyleneimmonium ion. If the HN2 is allowed to stand in aqueous solution for longer periods, then the solution pro gressively loses cytotoxic activity. Clinically, nitrogen mustard is administered as a direct i.v. injection in the treatment of neoplastic disease. The reasons offered for this traditional practice are firstly to minimize the danger of extravenous administration with attendant tissue ne 20 400 crosis and, secondly, to circumvent the loss of HN2 in the TIME (HOURS) i.v. bottle by hydrolysis. The latter explanation appears CHART 6.—The effects of dimethyl myleran (DMM) and nitro gen mustard (HN2) on the growth of “radioresistant―cells(B) to be erroneous in view of our findings of the preservation and the 2 “radiosensitive―celllines (S68 and 864) during the 1st of the cytocidal activity of HN2 stored in water at 20°C 40 hr after treatment. In each case the dose of DMM or 11N2 used (viz., approximately room temperature). Storage of HN2 was such as to yield 1 in 10@cell survivors. The straight lines represent 6 untreated controls. 4 W. Davis and G. J. Goldenberg. Unpublished observations.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. 1408 Cancer Research Vol. 25, October 1965

TABLE 4 marrow and intestinal mucosa. There is no indication AGING OF HN2 SOLUTION IN H2O (2.5 ,@g/ml) AT 37°C that the relative increase in D0 of these cells on going from ANt) SUBSEQUENT ESTIMATION OF RESIDUAL bi- to monofunctional mustard is less than that of the can CY'rocIDAL ACTION cer cells. It seems most likely that pharmacologic effects prevent the monofunctional mustard from being given iri@ @ Incubation time Cytoddal effect do@ of HN2 @ (mm)Equivalentremaining@—(fraction of cells surviving) @g‘ml)HN2 vivo at the dose needed to kill a sufficient fraction of divid ing cells. 0 X 102 The much greater effectiveness of polyfunctional alkyl 3 1.55 X 10-s 0.113 100 ating agents has been attributed to the formation of cross 9 2.25 X 10-2 0.104 92.8 links between important macromolecules (17). Cross 27 3.8 X 102 0.092 82.1 links between DNA molecules in the nucleus were clearly 130 1.1 X 10—' 0.066 58.9 demonstrated in cells treated with HN2, and cell death 3901.6 (1.4 X 10'0.112 0.018100 16.1 was attributed to this reaction. The monofunctional mus tard was shown to react with proteins and DNA, but no cross-links were formed (2, 6). in saline would further prolong the hydrolysis half-life There is very little biochemical evidence to indicate the since chloride ions would inhibit the cyclization to the site of action of dimethyl myleran. In model systems it ethyleneimmonium ion. Table 4 shows that after 130 is difficult to demonstrate any reaction between this al miii at 37°C,41 % of the activity of the mustard has been kylating agent and proteins or nucleic acids because they lost. Since the monofunctional derivative of HN2 has cannot compete with hydrolysis. In aqueous solution di only about 3 % of the cytocidal effect of HN2, activity will methyl myleran is a very poor alkylating agent because the be effectively lost when one of the alkylating groups of relative rate of reaction with water compared with that of, HN2 has been hydrolyzed. say, proteins or nucleic acids is hundreds of times higher than is the case with mustard. On chemical grounds one DISCUSSION might expect dimethyl myleran to alkylate groups in the There is no correlation among the relative sensitivities cell that are in a lipid environment, and this could lead to of the different cell strains towards the alkylating agents a basic difference in the mode of action of dimethyl my and to X-rays. On the whole, dimethyl myleran is more leran and HN2. Alexander and Lett (3) have shown that akin to X-rays than HN2. The only difference found be nitrogen mustard, in concentrations required to inhibit tween DMM and X-rays is that the T strain is more sen mitosis, caused cross-linking of DNA in salmon sperm and sitive than the R straiti to diniethyl niyleran, but the 2 lines other cells but that myleran-type compounds were rela do not differ in radiosensitivity. I he behavior of cells tively ineffective. treated with dimethyl myleran is very similar to that seen The failure in this system to select out mutants that are after X-rays. The existence of postirradiation recovery more resistant than the cell line carried in vivo distinguishes processes by which metabolic processes restore cells that the alkylating agents and X-rays from most other cytotoxic have sustained a lethal degree of damage is well estab agents and may provide an important clue to their mode lished. Postirradiation storage at 30°C assists such repair of action. There may be no alternative biochemical path processes, and the fact that the same procedure promoted ways for by-passing the lesions produced by alkylation or recovery of dimethyl myleran-treated cells suggests that radiation, for if this were the case, resistant mutants should the same repair system is at work (8). It is possible that arise. Damage of DNA or intracellular membranes could the difference in radiosensitivity between the R and the S not be by-passed, and the only way to achieve resistance cells is largely due to the facts that the postirradiation re against such damage is by selection of mutants with greater pair processes are more effective in R cells and that the repair capacity. But this is probably a polygenic charac magnitude of the primary lesion may be the same. The teristic, and improvements are therefore going to be an present data are consistent with the view that the differ extremely rare event. Studies claiming that tumors de ences in D0 for dimethyl myleran may also be related to veloped resistance to alkylating agents have come primar repair. ily from in vivo experiments (19, 23, 28), although Levis Particularly striking is the fact that the tetraploid line (21) has reported a nitrogen mustard-resistant cell line in is so very much more resistant to HN2 than is the diploid. vitro. The failure to produce resistance in vitro also seems If mitotic death were due to the production of lethal muta to be at variance with clinical experience of lymphomas and tions, then on going from a diploid to a tetraploid cell D0 leukemias, which are frequently said to have become re would be expected to remain unchanged, but the size of sistant when they no longer respond to treatment with a! the shoulder (i.e., n) should be doubled. This is not the kylating agents. This resistance may not be the result of case for either X-rays, dimethyl myleran, or HN2, all of the selection of a mutant cell line, but to other, as yet un which respond differently to an increase in ploidy. known factors, which render treatment less effective. In the limited experiments carried out with the mono From a clinical point of view, the exponential type of functional mustard, the mere fact that the D0 is much survival curve formed with the alkylating agents implies higher for the monofunctional mustard does not explain its that the dose needed to achieve a cure—if defined by “kill ineffectiveness in arresting the growth of tumors in vivo. ing―every malignant cell—depends on the number of cells With the bifunctional agents, the dose that can be admin present. This same argument on the relationship between istered is determined by the damage sustained by the bone size of leukemic cell inoculum and chemotherapeutic “cur

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. GOLDENBERG AND ALEXANDER—Effects of HN@ and Dimethyl Myleran on Leukemia Cells 1409 ability― has been advanced in detail by Skipper (27). The peutic Agents and Those of X-rays on the Reproductive maximum tolerated dose may thus cure a lesion consisting Capacity of Mammalian Cells. Ibid., 203: 1150—53,1964. 10. Calcutt, G., and Connors, T. A. Tumor Sulphydryl Levels and of 10@cells but be quite ineffective for the treatment of a Sensitivity to the Nitrogen Mustard Merophan. Biochem. large mass consisting of 1012 cells ; the latter would require Pharmacol., 1@:839—45,1963. approximately twice the dose. In comparing the action 11. Carlson, J. G. Immediate Effects on l)ivision, Morphology of different cytotoxic agents in vitro it is quite useless to and Viability of the Cell. In: A. Hollaender (ed), Radiation Biology, pp. 763-824. New York :McGraw-Hill Book Company, express their activities in terms of the dose needed to kill Inc.,1954. 50 % of the cells (viz., the LD@o). This value lies in the 12. Colombo, G., Levis, A. G., and Mann, G. Final Report Inter shoulder region of the survival curve and provides no guide national Atomic Energy Agency, Contract No. 36 1959—1962, to the dose needed to eradicate a tumor containing millions Institute of Zoology and Comparative Anatomy, University of cells. It is quite possible for one drug to be more effec of Padova, Italy. 13. Dewey, W. C., Humphrey, R. M., and Cork, A. Comparison of tive than another at the 50 % level, while at the more rel Cell Multiplication and Colony Formation as Criteria for evant 0. 1 % level and below the situation is reversed. If Radiation Damage in Cells Grown in Vitro. Intern. J. Radia it is not possible to determine the complete survival curve tion. Biol., 6: 463—71, 1963. and a comparison has to be made at 1 level of kill, then an 14. Elson, L. A. Hematological Effects of the Alkylating Agents. Ann. N. Y. Acad. Sci., 68: 826—33,1958. LD for 99.9 % or greater should be chosen. 15. Elson, L. A., Galton, 1). A. G., and Till, M. The Action of A conclusion that can be drawn from this investigation (CB 1348) and Busulphan (Myleran) on the is that the relative sensitivity of different cells to HN2 and Haemopoietic Organs of the Rat. Brit. J. Haematol., 4: 355— dimethyl myleran is not the same. This provides a basis 74, 1958. for hope that compounds may be found which show selec 16. Fischer, G. A. Increased Levels of Folic Acid Reductase as a Mechanism of Resistance to Amethopterin in Leukemic Cells. tivity for malignant cells. Certainly, our data provide Biochem. Pharmacol., 7: 75—77,1961. support at the cellular level for the clinical observation 17. Goldacre, R. J., Loveless, A., and Ross, W. C. J. The Mode of that some malignant conditions respond better to nitrogen Production of Chromosome Abnormalities by Nitrogen mustards while others respond better to the myleran class Mustards. The Possible Role of Cross-Linking. Nature, 165: of alkylating agents. For example, if the problem were to 667—69,1949. 18. Haddow, A., Kon, G. A. R., and Ross, W. C. J. Effects upon selectively damage T cells in a mixture of R and T, di Tumours of Various Haloalkylamines. Ibid., 16@: 824-25, 1948. methyl myleran would be much more effective than HN2 19. Hirono, I. A Comparative Study of the Mode of Action of (D0 of T/DO of R = 4 for HN2 and 0.5 for dimethyl my X-Rays and Alkylating Agents. Ibid., 176: 1168-69, 1955. !eran). 20. Lett, J. T., Parkins, G., Alexander, P., and Ormerod, M. G. Mechanisms of Sensitization to X-Rays of Mammalian Cells REFERENCES by 5-Bromodeoxyuridine. Ibid.,203:593—96,1964. 1. Alexander, P. Comparison of the Action of Ionizing Radiations 21. Levis, A. G. X-Irradiation Sensitivity of Nitrogen I'sIustard and Biological Alkylating Agents (Nitrogen Mustards) at the Resistant Mammalian Cells in Vitro. Ibid., 198: 498—99, 1963. Cellular and Sub-cellular Level. Sci. Rep. 1st Super Sanita., 22. Loveless, A. Qualitative Aspects of the Chemistry and Biology 1:537—SO,1961. of Radiomimetic (Mutagenic) Substances. Ibid., 167: 338-42, 2. Alexander, P., Cousens, S. F., and Stacey, K. A. Drug Re 1951. sistance in @\1icro-organisms. In: G. E. W. Wolstenholme and 23. Oboshi, S. Effects of X-Irradiation upon the Sublines of C. M. O'Connor (eds.), Ciba Foundation Symposium on l)rug Hirosaki Sarcoma Resistant Respectively to Nitromin, Thio Resistance in Micro-organisms; Mechanisms of Development, TEPA, and Mitomycin C. Gann, 50 (Suppl.): 15-16, 1959. p. 294. London: J. & A. Churchill, Ltd., 1957. 24. Ross, W. C. J. Biological Alkylating Agents. In: Fundamental 3. Alexander, P., and Lett, J. T. The Biological Significance of Chemistry and the Design of Compounds for Selective Toxic the Changes Produced in the Deoxyribonucleic Acid of Cells ity, pp. 4-5. London: Butterworth & Co., Ltd., 1962. Treated with Radiomimetic Alkylating Agents. Biochem. 25. . Ibid., p. 12. Pharmacol., 4: 34-48, 1960. 26. Sinclair, W. K. X-Ray Induced heritable Damage (Small 4. Alexander, P., and Mikulski, Z. B. Mouse Lymphoma Cells Colony Formation) in Cultured Mammalian Cells. Radiation with Different Radiosensitivities. Nature, 192: 572—73,1961. Res., 21: 584—611,1964. 5. - . I)ifferences in the Response of Leukemic Cells in 27. Skipper, Ii. E. Perspectives in cancer : Thera Tissue Culture to Nitrogen Mustard and to J)imethyl Myleran. peutic design. Cancer Res. @4:1295-1302, 1964. Biochem. Pharmacol., 5: 275—82,1961. 28. Skipper, H. E., and Schabel, F. M., Jr. Experimental Evalua 6. Alexander, P., and Stacey, K. A. Effects of Nitrogen Mustards tion of Potential Anticancer Agents. VII. Cross Resistance of and Related Compounds on the Nucleoproteins of the Cell Alkylating Agent-resistant Neoplasms. Cancer Chemotherapy Nucleus. Acta. Unio Intern. Contra Cancrum., 16: 533—39, Rept. No. 22, pp. 1—22,September 1962. 1960. 29. Stapleton, G. E., Billen, D., and Hollaender, A. Recovery of 7. Bacq, Z. M., and Alexander, P. Fundamentals of Radio X-irradiated Bacteria at Sub-optimal Incubation Tempera biology, Ed. 2. Oxford: Pergamon Press, Inc., 1961. tures. J. Cellular Comp. Physiol., 41: 345-58, 1953. 8. Beer, J. Z., Lett, J. T., and Alexander, P. Influence of Tem 30. Walker, I. G., and Helleiner, C. W. The Sensitivity of Cul perature and Medium on the X-ray Sensitivities of Leukaemia tured Mammalian Cells in Different Stages of the Division Cells in Vitro.Nature, 199: 193—94,1963. Cycle to Nitrogen and Sulfur Mustards. Cancer Res., 25: 9. Berry, IL J. A Comparison of Effects of Some Chemothera 734—38,1963.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research. The Effects of Nitrogen Mustard and Dimethyl Myleran on Murine Leukemia Cell Lines of Different Radiosensitivity in Vitro

Gerald J. Goldenberg and Peter Alexander

Cancer Res 1965;25:1401-1409.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/25/9_Part_1/1401

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/25/9_Part_1/1401. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1965 American Association for Cancer Research.