a modification of the sensitivity of mammalian cells surviving treatment with methyl...

Europ. 07. Cancer Vol. 4, pp. 325-335. Pergamon Press 1968. Printed in Great Britain

A Modification of the Sensitivity of Mammalian Cells Survivin Treatment with Methyl Methanesulphonate

MARGARET FOX and A. H. W. NIAS

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, Great Britain

I N T R O D U C T I O N CONSIDERABLE attention has been focused recently on the study of the growth kinetics of mammalian cells surviving X-irradiation in vitro. Sinclair [1] demonstrated heritable, non-lethal damage in cells surviving X-irradiation, which resulted in the formation of slowly growing "small clones". These "small clones" were also shown to be more radio- sensitive. An analogous situation has recently been described for P388 lymphoma cells growing in vivo in DBA/2JN mice by Berry [2]. A significant increase in the generation time of L1210 leukaemia cells growing in vivo in CDF1 mice and surviving cyclophosphamide treatment has also been described by Johnson et al. [3]. In this case, although the surviving fraction was low, the whole population was affected, and in addition to an increase in doubling time it also showed decreased virulence. Return to control population doubling times and virulence did, however, occur after 4-5 transplant generations. Similar findings have also been reported by Johnson et al. [4] for L1210 cells surviving amethopterin treatment in vivo. In view of the importance of these findings in relation to cancer chemotherapy, it seemed important to determine whether this

phenomenon occurred with other drugs of potential clinical application. Two in vitro tumour systems were chosen for the present study with a view to detailed characterization of surviving populations and clones.

Methyl methanesulphonate (MMS), the drug chosen for this study, has been shown to cause specific lymphopenia in monkeys and rabbits [5], and considerable information exists re- garding its effects on cellular kinetics and macromolecular biosynthesis in the P388F lymphoma in vitro [6, 7].

MATERIALS AND METHODS

The techniques for in vitro culture of mouse lymphoma cells have been described previously by Fischer and Sartorelli [8], and the origin of the P388F clone has been described by Fox and Gilbert [9]. Cells do not attach to glass and grow in static suspension cultures. They were maintained in logarithmic growth by appropriate dilutions every 2-3 days. Under these conditions the population doubling time of the cultures was 12-14 hr.

HeLa cells, cloned in this laboratory, were grown in monolayer culture in 4 oz medical flat bottles using techniques previously des-

325

326 Margaret Fox and A. H. W. Nias

cribed by Nias et al. [10]. The doubling time under these conditions was 25 hr.

Methyl methanesulphonate (MMS) was dissolved in water and sterilized by passage through a membrane filter (pore size 0.22 g Millipore Filter Corp.). The concentrations of stock solutions of the drug were adjusted so that the required dose was given to 5 ml cultures (P388F) or 10 ml cultures (HeLa cells) by the addition of 0. 025-0.2 ml. Cultures were exposed to the drug for varying periods of time during the different experimental proce- dures. In the case of the lymphoma cultures, the drug containing medium was decanted after centrifugation (800 rev/min for 10 min) and replaced by fresh drug-free medium. Drug- containing medium was removed from HeLa cell cultures by aspiration and replaced by fresh drug-free medium.

Cell survival after treatment of P388F cells was determined by the extrapolated growth curve technique described by Alexander and Mikulski [11]. Survival of HeLa cells was determined by the colony-forming techniques described by Puck and Marcus [12].

For determination of cellular multiplicity, 5ml aliquots of a 2000 cell/ml suspension of HeLa cells were plated in Falcon plastic bottles. The monolayer was fixed in formol- saline and subsequently stained in haematoxylin at appropriate times after plating. Counts were made of single and paired cells over a minimum of 100 cells in several separate experiments.

For labelling experiments, replicate samples of 5 ml, 2000 cells/ml were plated in Falcon plastic bottles. Hal f the cultures were treated with M M S 100 gg/ml for 15 minutes at 37°C. At the end of this period, the drug containing medium was removed and replaced by medium containing 8H thymidine 0.1 gc/ml. Similarly, medium was removed from the control cultures and replaced by medium containing 8H thymidine at the same concentration. All bottles were subsequently incubated at 37°C and samples, control and treated, were fixed acetic alcohol at hourly intervals. After staining with acetic-orcein, the bottoms of the plastic bottles were removed and coated with Kodak AR 10 stripping film. Exposure time was 10 days.

All irradiations were carried out at room temperature on a Resomax machine operating at 300 kVp 2" 0 mm Cu h.v.1, with a dose rate of 100 rad/minute. The cells were irraditated in 4 oz medical flat bottles after at tachment to the glass.

R ES U LTS

Effect of single doses of methyl methanesulphonate on P388F lymphoma cells

The half-time of hydrolysis of methyl methanesulphonate in aqueous solution is 9.1 hr at 37°C [13]. The survival of P388F cells after treatment with M M S has previously been shown to fall with increasing time of exposure to the drug [6]. For the present studies, a three-hour exposure period was found to be most convenient. Figure 1 shows the survival curve for lymphoma cells in exponential growth treated with various dose levels of MMS for 3 hours, together with the survival curve for a second treatment. After a first treatment of 40 gg/ml for 3 hours, the surviving fraction was reduced to ~ 30/0 . Eleven to 18 days after the initial treatment, when the population was again growing exponentially with a population doubling time equal to that of the controls, cells were given a second (3 hr) treatment with the same range of MMS doses. The dose response curve for these cells (given an initial treatment of 40 gg/ml 3 hr and subsequently treated a second time) can be compared with that of cells treated once only (Fig. 1). Both survival curves show a similar form with a shoulder and an exponential portion, but the slopes of the exponential part differ by a factor of ~ 2:1.

The results in Fig. 1 represent a mean of 4 experiments, cells receiving the second treatment between 11 and 18 days after the initial treatment. It was of interest therefore to determine how long this increased sensitivity persisted.

A series of replicate cultures of P388F cells were treated with MMS (40 gg/ml 3 hr). Three days later, one of these cultures was given a second similar dose and survival of cells to the second dose was determined. Other replicate cultures were given a second dose at day 6, day 9 and up to 24 days after the initial treatment. The mean survival to the first treatment was 2" 84-0.30/o . The survival of cells given a second treatment between 3 and 24 days after the first is shown in Fig. 2. From 3-18 days survival was less than that for the first dose. The mean survival for all treatments during this period was 0.37-4-0"020/0 . Treatment at 21 and 24 days showed a return of sensitivity to normal levels, i . e .~ 30/o .

The effect of a single dose of 50 gg/ml and 80 gg/ml is included in Fig. 2 for comparison. It would appear therefore that although a large proportion of damage is in fact repaired, there is some residual damage which cannot

Sensitivity of Mammalian Cells 327

I.I

z o I - U

u.. O.

>

i i i i I i i i i i I i i i

Survival of P338 F lymphoma in vitro after treatment with Methyl Methane Sulphonate ( 3hrs )

o Ist treatment Do8.5 Fg/ml

* 2rid treatment Do4.5 ~ /m l

0.01

0.00 0 I0 20 30 40

MM5 /~g / ml

Fig. 1. Sensitivity of populations of P 3 8 8 F lymphoma i n v i t ro to an initial treatment with methyl methanesulphonate compared with the sensitivity of cells surviving a treatment of 40 pg/ml M M S (3 hr)

given 11-18 days earlier. Mean of 4 experiments.

be repaired. The prolonged period of time before normal sensitivity returns suggests that the majority of the surviving populations is affected, and hence a long period of time elapses before the few remaining normal cells overgrow the damaged ones.

Characterization of HeLa cell clones surviving treatment with methyl methanesulphonate

HeLa cultures were exposed to various doses of M M S during the whole of a ten-day culture period. At the end of this time several clones which had survived 2 gg/ml were isolated. These showed changes which were related to the clone size at the time of isolation. Small clones (i.e. those with less than 50 cells) were not viable at all. Medium clones (with less than 200 cells) showed reduced viability and other characteristics described below. Large clones (i.e. control sized with around 1000 cells) also showed these changes to a lesser extent.

Clones M3 and M4 are examples of a large and a medium clone respectively. The cells from these clones were repeatedly sub-cultured over the following 4-5 months. The sensitivity of the two clones to second doses of M M S was

tested at intervals during this period. The survival curves obtained for clones M3 and M4 after a second continuous treatment with M M S are shown in Fig. 3 together with that of a control clone isolated at a similar time. The Do for the control is 3 pg/ml; those for clones M3 and M4 are 1.3 and 1 pg/ml respectively. All three cell survival curves extrapolate to the same point. Both clones M3 and M4 are significantly more sensitive to M M S treatment than control cultures. However, they do not differ significantly from each other with regard to sensitivity. Both clones M3 and M4 showed reduced growth rates and plating efficiencies as well as greater sensitivity to further M M S treatment, Table 1. The clone size distribution (Fig. 4) illustrates the slower growth and increased spread of cell cycle times of these clones; M3 completed a maximum of 6-7 divisions in 10 days and M4, 4-6 divisions during the same period. The control clone completed 9 10 divisions during the same period. There is little overlap between these distributions. The characteristics of reduced growth rate, and plating efficiency, and increased sensitivity have been retained over a period of 4-5 months of continuous subculture.

328

I_

Margaret Fox and A. H. W. Nias

Response of P338 F cells surviving 40/~g/ml (3hrs)of MM$ to a second dose 40/~g/ml at various times afterwards.

Fig. 2.

10 - I

z

V-- 10-:2: O

10-3= re"

10 .4

io -5

t40/,g/ml ° ~ °

o__/°\__o_./ ~50/~g/ml

• Ist dose MMS o 2nd dose MMS

• 80#g/ml

Days after Ist dose of MMS

Time course of return to normal sensitivity of a population of P388F cells surviving an initial dose of 40 pg/ml (3 hr). Survival determined by the extrapolated growth curve technique.

In P388F populations, decrease in exposure time to the drug resulted in decreased toxicity. A similar relationship was demonstrated for HeLa cells. The Do value for 10-day exposure of control populations was 3 .0 pg/ml, that for a 15 minute exposure at 37°C was 30 pg/ml (Fig. 5). Cells were treated with M M S for 15 min either 4 hr or 17 hr after plating. The survival curves obtained had similar Do values but the extrapolation numbers differed consid- erably (Fig. 5). For 17 hr cells the extrapolation number was 9 .0 and for 4 hr cells 5.0. Correction for a cellular multiplicity of 1.51 ± 0 . 0 3 a t l 7 h r f r o m t h a t o f l . 0 9 ± 0 . 1 5 a t 4 h r could account for these differences. One must therefore conclude that there is no major difference in sensitivity of cells treated at these two times after plating. For convenience therefore the experiments described below were commenced on cells 17 hours after plating.

Effects of divided doses of methyl methanesulphonate on HeLa cells

Two 15-minute treatments of 50 gg/ml of M MS at 37°C separated by periods of up to 8

hours were given to a series of replicate HeLa cell cultures starting 17 hours after plating. The surviving fraction was determined and compared with survival after a single dose of 100 gg/ml which was taken as unity. The mean results from four experiments are shown in Fig. 6. Separation of the two 50 gg/ml doses by one hour results in a slight reduction in survival, separation of the two doses by greater time intervals results in a further reduction.

Changes in the survival pattern of cells given two doses of 150 rads X-rays separated by various periods of time, relative to cells given a single dose of 300 rads, are shown for comparison (Fig. 6). A two-fold increase in survival occurred during the first two hours. This is a similar finding to that of Lockhart, Elkind and Moses [14]. Elkind and Sutton [15] have interpreted this increased survival to indicate repair of sub-lethal damage.

The single doses of MMS and X-rays were chosen to give similar surviving fractions. HeLa cells in this laboratory have previously


been shown to have a Do in air of 150 rads. with an extrapolation number of 3 [16].

Treatment of HeLa cell cultures with divided doses of MMS required that the medium be changed twice. Control and X-irradiated cultures were therefore subjected to the same procedure. This handling alone did not affect the total number of colonies which developed.

It is possible that the initial dose of MMS (100 gg/ml 15 min) induces some synchrony in the population and that ceils are in their most sensitive stage when the second dose is given. Continous labelling of the culture with 8H thymidine has shown, however, that the rate of passage of cells from Gz and G~ into S is the same in control and MMS treated cultures (Fig. 7). There cannot therefore be any appreci- able induction of synchrony by MMS.

DISCUSSION The importance of the modification of growth

kinetics of cells surviving drug treatment in vivo has been stressed by Johnson et al. [4]. At that time, however, the additional important factor of increased sensitivity to subsequent drug treatment had not been described. Increased radiosensitivity of small slowly growing clones

surviving radiation is now, however, well established by the work of Sinclair with Chinese hamster cells [1] and has been confirmed for HeLa cells used in the present study. The present results indicate an increase in sensitivity of cells of two mammalian tumour lines in vitro surviving methyl methanesulphonate treatment.

The increased drug sensitivity of HeLa cell cultures is clearly associated with an increased population doubling time and a reduced plating efficiency. This is apparently not the case in P388F cultures. However, the populations of this latter cell line were not cloned, and it is quite possible, therefore, that a slightly reduced growth rate of sensitive cells could be masked by the presence of normal cells with normal growth rates. That normal ceils are present in the surviving populations is indicated by the eventual return of the total population to normal sensitivity (Fig. 2). The stability of the change to increased sensitivity in HeLa cell clones suggests that it is genetic in origin.

Stable changes to drug resistance by somatic cells in culture have been demonstrated for many drugs in many cell lines, e.g., to analogues of folic acid [17], pyrimidines [18, 19] purines [20, 21]. In most cases [17, 22, 23] clones were

EFFECT of METHYL METHANE SULPHONATE upon HeLa CELLS (CONTINUOUSTREATMENT)

g

U-O. I z m > u > ac

0.01

0.001

• CONTROL o CLONE M3

CLONE M4

0 I 2 3 4 5 6

MMS Fg ,'ml

10

Fig. 3. Survival of HeLa cell clones as determined by the "Puck" colony technique after growth in methyl methanesulphonate for a 10 day period. Clones Ms and M4 were slow growing survivors of an initial

treatment with the drug.

33O Margaret Fox and A. H. W. Nias

CLONE SIZE ANALYSIS 40.] of HeLa CELLS at 10 DAYS.

/ • • CONTROL o o Clone M3

x ~ Clene M4

I

DIVISIONS

Fig. 4. Clone size analysis of HeLa cells after 10-days incubation. All clones were cultured in the absence of the drug, clones Ms and M4 show a slower growth rate than the control culture.

Fig. 5.

l I I I ! i I I I I I I I I I

EFFECT of METHYL METHANE SULPHONATE upon HeLa CELLS

( 15 minutes treatment at 37°C)

u. O.i

• 4 Hours after Plating

o 17 Hours after Plating

0.01

o.ool ' ' ' . . . . ~ ' " ' '5 ' ' , 1 25 50 I 0 I 0 200

MMS ~g/m[

The effect of 15-minute treatment of methyl methanesulphonate on the survival of HeLa cells. Treatments were given either 4 hours or 17 hours after plating H 4 hrs, o---o 17 hours.

Sensitivity of Mammalian Cells

Table 1

331

Clone Populat ion Plating doubling t ime efficiency

Control 25 hr 70% M3 38 hr 14% M4 53 hr 7%

isolated which remained stable over long periods of time. Cases of decline of resistance or spon- taneous reversion in cloned populations [ 19] and overgrowth of resistant by sensitive cells in uncloned populations [24] have also been reported. In most of these cases, relatively high degrees of resistance were obtained, e.g., 20,000 fold to amethopterin was reported by Fischer [17]. Mutat ion to resistance has however been shown to be an event of relatively low frequency. Lieberman and Ove [20] gave a figure of 3 . 6 × 1 0 -6 mutation per cell per generation in monkey kidney cells and Szybal- ski and Smith [25] gave a rate of 5 × 10 -4 muta- tions per cell per generation in Detroit D98 cells. Mutat ion to resistance to alkylating agents has less frequently been studied, few reports are available and the degrees of resistance

obtained are very low [26, 27], usually two to threefold. They are thus of the same order of magnitude as the increased sensitivity observed in the present study.

Obviously, therefore, the frequency of occurrence of resistant mutants is low and perhaps a more frequent occurrence is a mixture of surviving normal cells and surviving damaged cells. The damaged ceils may not always show greater sensitivity and inheritance of the change, as this possibly depends on the initial lesion and mode of action of the drug.

The results of the divided dose experiments (Fig. 6) are open to a number of interpretations. Firstly, it is possible that the initial dose of M M S induces some synchrony and cells are therefore in a sensitive phase of the cell cycle when the second dose is given. Preliminary

EFFECT of Split Dosage upon HeLa CELLS

• X--rays_150-1- 150 Rads

o M M S - - 50 q- 50j~/ml.

'~ 2" LIL

N

~- 0." ~ 0.8"

0.7" w 0.6-

o 0.5- t " t I - < 0.4"

"--I 0 I 2 3 4 5 6 7 8

Hours Between Doses

Fig. 6. Effect of divided doses of X-rays and methyl methanesulphonate on survival of HeLa cells. Treatments were started 17 hours after plating the cells. The survival after 300 rad. X-rays and

1 O0 ~g/ml M M S is taken as unity.


J00 ' | | I I I | I I 1 I I I |

9 0 -

8O-

70-

6 0 - LIJ

. . I

• ~ 50-_ o /

40- /

30-

' ~ ' ~ ' ; ' ~ ' ,'o ' ~'2 ' ,'4 ' t'6 '

T Hrs AFTER ADDITIONoF3HTHYMIDINE MMS 100~g/ml 15 mins

I I I I I I t ' ' ! I

o Control • MMS

1'8 ' ~o ' z'2 ' 14

Fig. 7. Rate of increase of labelled cells during the continuous presence of 3H-thymidine in control populations of HeLa cells and in cells exposed to MII/IS (100 gg/ml 15 mins) 17 hr after plating and immedi-

ate~ prior to addition of the aH-thymidine.

experiments in this laboratory using the synchronisation technique described by Tera- sima and Tolmach [28] have shown that cells in G1 show the greatest sensitivity to MMS [29]. The continuous labelling experiments (Fig. 7) indicated however that the rate of progress of cells into S is unaffected by MMS treatment. Induced synchrony would therefore not appear to be responsible for the decline in survival observed in the divided dose experiments.

The two alternative interpretations of this observation are as follows: the fall in survival observed (Fig. 6) is possibly an indication of the early development of increased sensitivity as shown in the surviving HeLa cell clones (Fig. 3) and P388F populations (Fig. 1 and 2). However, in these divided dose experiments, the number of survivors is much higher ~ 50 % (Fig. 4) than in experiments from which sensitive clones were isolated (Fig. 3). The implication in this case would be that all cells surviving an initial treatment would show greater sensitivity. This possibility has not been examined in detail in HeLa cells, although nearly all the (3%) survivors of 40 gg/ml to P388F cells did show increased sensitivity (Fig. 2).

These results also suggest that there is no recovery from alkylation by MMS, but one must consider in this case, and in the previous situation, the possibility that unreacted drug could still be present in the cells after the 15 min pulse treatment. The effect of this in

split dose experiments would essentially be to extend the treatment period, which has been shown to increase the effect of the drug (cfFigs. 3 and 5). However, a thirty-minute treatment of HeLa cells did prove approxi- mately twice as effective as a fifteen-minute treatment at the same dose level implying that the reaction of the drug can be effectively stop- ped by removing it from the medium bathing the cells.

The implications are therefore that in the lymphoma cells some, though not all the damage sustained by cells after treatment with MMS can be repaired, e.g. two doses of MMS 40 IJg/ml (3 hr) separated by 3 days (Fig. 2) resulted in a survival level equivalent to that produced by a single dose of 50 gg/ml (3 hr). Thus, although the two doses are not strictly additive, indicating some repair, some residual damage must remain in the population. In HeLa cells, heritable damage was demon- strable as increased sensitivity and reduced growth rate and plating efficiency of clones Ma and Mr. The divided dose experiments suggest that the increase in sensitivity develops within one hour and increases progressively with time, and also indicate that HeLa cells are unable to repair MMS damage in the same way as X-ray damage is repaired.

A c k n o w l e d g e m e n t s - - T h e authors are grateful for the technical assistance of Mrs. Elisabeth Moore, Mrs. Dorothy Robinson and Mr. J. B. Harrison.


RESUME Des cellules du lymphome P388F et des ceUules HeLa furent traitdes in vitro par des doses uniques d' un agent alkylant monofonctionnel, le mdthyl mdthane sulfonate. La survie ddpendait du temps d'exposition du produit et de la dose. Des ceUules P388F survivantes, et des clones de cellules HeLa, refurent un second traitement, ce qui mit en dvidence une augmentation de la sensibilitL Les clones de cellules HeLa, qui prdsentaient une telle sensibilitd augmentde, avaient aussi un temps de doublement allongd et une possibilitd dYtalement diminude. Ces clones, isolds en sons-culture continue, conservaient leur sensibilitd augmentde, leur croissance retardde, et leur efficacitd dYtalement rLduite, pendant 4 ~ 5 mois. Les cellules P388F retrouvaient leur sensibilitd normale apr~s 21 jours.

La division de la dose en deux fractions Lgales, administrdes ~ un intervalle de 1 ~ 8 heures, produisait, pour les cellules HeLa, une diminution de la survie, par rapport au traitement par dose unique. On compare ces rdsultats ~ ceux obtenus avec des doses fraction- ndes de rayons X qui montrent que la survie est doublde lorsque le traitement est fractionnd en deux doses dgales, administrdes ~ 2 heures d'intervalle.

SUMMARY P388F lymphoma cells and HeLa cells in vitro were treated with single doses of the monofunctional alkylating agent methyl methanesulphonate. Survival was found to depend on duration of exposure as well as dose of the drug. Surviving populations of P388F cells and surviving clones of HeLa cells were given second treatments with the drug and found to show an increased sensitivity. HeLa cell clones showing increased sensitivity also showed increased population doubling time and a reduced plating efficiency. The HeLa cell clones isolated, maintained the characteristics of increased sensitivity and reduced growth rate and plating efficiency over 4-5 months of continuous sub-culture. P388F polulations returned to normal sensitivity after 21 days growth.

Divided dose experiments with HeLa cells showed a decrease in survival, compared with that to a single treatment, when the dose was split into two equal fractions separated by 1-8 hr. These results are compared with those obtained after divided doses of X-rays when survival shows a two-fold increase i f two equal fractions are given two hours apart.

ZUSAMMENFASSUNG P388F Lymphom- und HeLa-ZeUen wurden in vitro mit Einzeldosen des monofunk- tionellen alkylierenden Agens Methansulfonsduremethylester behandelt. Die Uberlebens- zeit der Zellen hing sowohl vonder Dauer der Einwirkung als yon der verwendeten Dosis ab. Die iiberlebenden Populationen yon P388F Zellen und iiberlebende Klone yon HeLa- ZeUen wurden einer zweiten Behandlung mit der Verbindung unterworfen, wobei sie erhO'hte Empfindlichkeit zeigten. Diesen HeLa-Zellen zeigten auch vergr6sste Vermehrungs- geschwindigkeit und verminderte Explantierbarkeit als Einzelzellen. Diese Charakteristiken behielten die Zellen wiihrend einer Weiterziichtung iiber 4-5 Monate. P388F Zellen hingegen wiesen nach 21 tiigigem Wachstum wieder normale Empfindlichkeit auf.

Im Vergleich zur Einzelbehandlung zeigten Versuche mit HeLa-Zellen , bei denen die Dosis in zwei gleichen Fraktionen in zeitlichem Abstand yon 1-8 Stunden gegeben wurde, eine Verminderung an iiberlebenden Zellen. Im Vergleich hierzu ergibt sich bei einer

fraktionierten R6ntgenbestrahlung (zwei gleiche Dosen in zweistiingdigem Abtsand) eine Verdopplung der Uberlebenswahrscheinlichkeit.

1.

2.

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