The effects of cis-diamminedichloroplatinum (II) and radiation on the proliferation kinetics of mouse duodenal crypt cells and on a partially synchronized crypt cell population

Int. J. Radmmn Oncology Bid. Phys.. Vol. 12. pi. Printed I” the U.S.A. All n%ts rc~n’ed.

1977-1985

Copy&t

0360.3016186 $3 00 + 00 0 1986 Pergamon Journals Ltd

??Original Contribution

THE EFFECTS OF CI!3-DIAMMINEDICHLOROPLATINUM (II) AND RADIATION ON THE PROLIFERATION KINETICS OF MOUSE DUODENAL CRYPT CELLS AND ON A PARTIALLY SYNCHRONIZED CRYPT CELL POPULATION L. DEWIT, M.D., Y. OUSSOREN AND H. BARTELINK, M.D.. PH.D. Department of Experimental Radiotherapy, Netherlands Cancer Institute, Antoni van Leeuwenhoekhuis, Amsterdam, the Netherlands The effects of c-DDP and radiation, given alone or in combination, on the proliferation kinetics of mouse duodenal crypt cells were investigated by repeated labelling with “H-thymidine. Crypt cells surviving a single dose of X rays or c-DDP alone, appeared to proliferate faster than untreated controls. When both agents were given in combination, the proliferation rate of surviving crypt cells was markedly reduced as a result of a significant increase in the DNA synthesis time. The influence of the proliferation rate of crypt cells on the effects of c-DDP and X rays was analyzed using the crypt microcolony assay. Mice were given single doses or 2 fractions of X rays with 2 different time intervals (4 and 48 hours) either alone or in combination with c-DDP given 30 minutes before the second fraction. The lethal effect of the drug was approximately the same in cells that were in accelerated proliferation after the first irradiation as in unstimulated cells. A tendency for less cell killing by the drug was observed 3.5 hours after the first irradiition. To rule out cell age dependent effects of c-DDP, crypt cell survival was assessed after irradiation was given alone or in combimttion with the drug on a partially synchronized crypt cell population, obtained by repeated injections of hydroxyurea. A predominant lethal effect of c-DDP was found in ceils in the fate Gtiphase. These findings are important to consider both from a mechanistic point of view as for clinical application of the agents. Mouse intestinal crypt cells, Irradiation, c-DDP, Proliferation, Hydroxyurea.

INTRODUCTION In the last 10 years, cis-diamminedichloroplatinum (II) (c-DDP) has been extensively tested as a potential radiosensitizer in various experimental tumor systems.6*‘4*2432g332,4’ The .42.44 interaction of c-DDP and ra-

were addressed: what is the effect of c-DDP and X rays given alone or in combination on the proliferation rate of crypt cells and conversely, and what is the influence of the proliferation rate of these cells on the effects of both agents. Additionally, possible cell cycle phase dependent effects of c-DDP, when combined with X rays, were investigated on a partially synchronized crypt cell population.

diation has also been investigated on acutely responding normal tissues such as skin and intestinal epithelium. Most investigators used single doses of radiation.‘0*‘3.28*32.46 When the drug was given in combination with fractionated irradiation, the overall treatment time was kept short to minimize the influence of stem cell proliferation.1.4,9*28 Stem cell proliferation, however, definitely takes place during daily fractionated irradiation, which is used in clinical practice. This will considerably increase the acute tolerance of the gut to irradiation.5’ We therefore became interested in investigating the relationship between the proliferation rate and the effects of c-DDP and X rays on mouse duodenal crypt stem cells. Two specific problems

used, aged at lo- I4 weeks and weighing 22-27 g. They were irradiated without anesthesia in groups of six through one posterior partial abdominal field, as previously described.12 The radiation characteristics were: 250 kV X rays, 15 mA, filter 0.5 mm Cu, HVL 1.5 mm Cu, focusskin distance 35.5 cm, giving an absorbed dose rate of 1.6 Gy/min, at the level of the duodenum. Dose variations

Reprint requests to: Dr. L. Dewit, Dept. of Radiotherapy of the Netherlands Cancer Institute (Antoni van Leeuwenhoekhuis), Plesmanlaan 12 1. 1066 CX Amsterdam, the Netherlands. Acknowledgements-The authors wish to acknowledge MC Millan Press Ltd. for permission of reproducing the data in Figure I and Table 1 which were previously published.” They also

want to thank Dts. A. C. Berg and F. A. Stewart for their criticism and Mr. A. A. M. Hart for the statistical analysis of the data. Mrs. A. Stuip is acknowledged for typing the manuscript. This work was financially supported by “Het Koningin Wilhelmina Fonds” (the Netherlands Cancer Foundation), grant NKI 84- 1. Accepted for publication 4 June 1986. 1977

METHODS AND MATERIALS Specificpathogen-free female inbred C3H/nu mice were

1978

1. J. Radiation Oncology 0 Biology 0 Physics

at that level were less than 5%. All irradiations were carried out between 2 and 8 p.m. to avoid a circadian variation in radiosensitivity.20 Cis-diamminedichloroplatinum (II) (c-DDP*) was injected intraperitoneally (I.P.) at a concentration of 0.5 mg/ml in normal saline. The same batch was used within one experiment and care was taken to use stainless steel needles with plastic fitting instead of aluminium, to avoid chemical reactions between the two metal compounds.3 In a first experiment, the effects of c-DDP and radiation on the repopulation rate of duodenal crypt cells were assessed. Mice received a single dose of X rays (11 Gy), 18 mg/kg of c-DDP given I.P. or 8 mg/kg of the drug 30 minutes before 9 Gy of X rays. The doses of the agents were chosen based on previous results to obtain similar levels of crypt cell survival.‘0 Starting at 84 hr after treatment, when regenerating crypts can be readily detected, these mice and untreated controls received 0.5 &i/g of ‘H-thymidine (sp ecific activity of 2 Ci/mmolt) at hourly intervals, for a total of nine times. Subgroups of three animals were killed at hourly intervals, 45 minutes after an injection, and the duodenum was excised and fixed in Camoy’s medium, dehydrated, and embedded. Transverse sections of 4 pm thickness were cut at three different levels and prepared for autoradiography by dipping the slides into emulsion$, diluted 1: 1 or 1:7 with distilled water. After 3 weeks of exposure, the slides were developed5 and stained with hematoxylin and eosin. The labelling index was determined by counting at least 1000 cells in untreated or regenerating crypts per animal. Only rarely were fewer cells counted because too few regenerating crypts were present. Care was taken to select those crypts containing a lumen and where the individual cells could readily be distinguished. At the base of the crypt, 5 to 6 cells were not included in the counting as they most likely were Paneth cells with a considerably longer cell cycle than the cells in the proliferative compartment.5’35 This delineation between these two cell types was arbitrary, since the Paneth cells could not be readily detected in our autoradiography slides. In the controls, the cells in the top 6 cell position of a crypt were not counted. After the initial pulse labelling with ‘H-thymidine they were not labelled and therefore were considered to be in a postmitotic non-proliferating state. No such selection criterion was used in the crypts of treated animals, because here after the initial pulse labelling with ‘H-thymidine, the cells in the top of the crypt were labelled, indicating they were proliferating cells. The background was fairly high, on the average, 4 grains per nucleus (presumably due to chemography) and therefore a cell was considered labelled when it contained more than 10 grains per nucleus.

November 1986. Volume 12. Number 1I

In a second experiment, the influence of the repopulation rate on the effects of c-DDP when given in combination with X rays was examined. A group of mice was exposed to single doses or to two equal fractions of radiation with a 4 hr interval. Another group of animals received a priming radiation dose of 8 Gy, followed by graded test doses delivered 48 hr later, at a time when the surviving crypt cells were in accelerated compensatory proliferation, 48 [Withers, oral comm., Sept, 19841. The priming X ray dose was kept constant to trigger the cells to approximately the same degree of accelerated proliferation. Half the animals received, in addition, 8 mg/kg of c-DDP given I.P. 30 minutes before the single dose irradiation, or before the second fraction of the split dose regimens. In this way, any effect by the drug on repair of sublethal radiation damage occurring between the two X ray fractions was avoided. Mice were killed 88 hr after the single dose or the mid-time point of the fractionated irradiation, and the duodenum was excised and conventionally processed for the crypt microcolony assay.50 In a third experiment, the effect of c-DDP given in combination with irradiation was investigated on a partially synchronized crypt cell population. Mice received 0.5 mg/g of hydroxyurea (HU*) given I.P. followed at hourly intervals by four doses of 0.25 mg/g each. This drug is known to kill cells selectively in the S-phase and to cause a G,-S block for at least l-2 hr after each injection.8.‘7*52After the cells are generally released from this block, they will progress, partially synchronized, through the cell cycle. Subgroups of 6 animals received a single dose of X rays at increasing intervals after the last injection of HU, either alone or in combination with 8 mg/kg of c-DDP, given 30 minutes before irradiation. In a separate experiment mice were given graded doses of X rays with or without 8 mg/kg. of c-DDP 2 hr or 7 hr after the last HU injection. After 88 hr the mice were killed and the duodenum was conventionally processed for the crypt microcolony assay.50 To reduce systemic toxicity, all animals that received c-DDP were submitted to a standard hydration protocol, consisting of 1 ml of 2.5% glucose in 0.5 N saline given I.P. 2 hr before and 1 hr after the drug injection, as previously described. lo The mice that received no c-DDP, were injected with equivalent volumes of normal saline.

RESULTS Experiment I The labelling indices (LI) obtained after repeated labelling of duodenal crypt cells with ‘H-thymidine are shown in Figure 1. The single 45 minutes LI’s were higher in the treated animals than in the controls. The LI’s after

* Platinol, Bristol-Myers Company.

9 Kodak D-19, Rochester, N.Y.

7 Amersham International. $ Kodak NTB 2 emulsion, Rochester, N.Y.

* Kindly provided by Squibb Company, Princeton, N.J.

1979

Crypt cell kinetics after c-DDP and X rays 0 L. DEWIT et al

20 -

c -DDP+RX

c -DDP

0

2

4

6

8

time (hours)

1 0

2

4

6

6

Fig. 1. Labelling indices after repeated lahelling with 3H-thymidine of duodenal crypt cells. Each point represents a measurement in one animal. The steep and horizontal parts of the curves as well as the point of inflection are obtained by linear regression analysis. Reproduced with permission from MC Millan Press Ltd. [Dewit et al., Br. J. Cancer, 53 (Suppl. VII), 33-36, 19861.

repeated labelling with ‘H-thymidine increased gradually to reach a plateau at 78 to 9 1%. The point of inflection gives an estimate of the growth fraction (G.F.) in a crypt. Various cell cycle parameters can be deduced from the curves in Figure 1. The time from initial pulse labelling to the point of inflection equals the cell cycle time (T,) minus the DNA synthesis time (T,).47 The time from zero to initial labelling, obtained by back extrapolation of the curve, is equal to T,, whereas the times from zero to 100% Ll and to the point of inflection give an estimate of the cell turn over time (T) and the cell cycle time (T,), respectively. The various curves of repeated labelling are

..-* -16

. ..*

. ..*

..a-

. ..*

. ..-

. ..-

replotted for comparison in Figure 2. and the numerical values of the cell cycle parameters are given in Table 1. It appears from these data that T, was shortened after radiation and even more so after drug treatment compared with the controls, mainly because of reduction in T, - T,, that is the Gr-phase. In contrast, T, was considerably longer after the combination of c-DDP and X rays than in controls, as a result of a significant increase in T, (p = 10-4, variant analysis). 22 When comparing the initial pulse Ll values with the TJT ratio’s, there appears to be good agreement between these values for each subgroup. This would be expected, since

,..*

1

-12

-8

,

-4

0

+4

?? 8

+12

time in hours

Fig. 2. Comparison of curves of repeated labelling with ‘H-thymidine of crypt cells for the various treatment groups. Cell cycle parameters, estimated from these curves, are presented in Table 1.

November 1986. Volume I?. Number I 1

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1980

Table 1. Cell cycle parameters of proliferating

crypt cells Estimates from single pulse labelling

Estimates from repeated labelling curves

Controls RX c-DDP c-DDP + RX

T (hours)

G.F. @)

T, (hours)

T,-T, (hours)

T, (hours)

15.3 + l.Ob 10.5 f 1.2 7.6 + 0.6 23 k3.1

n.ev. 91 78 91

13c* 9.5 6 21

9.5 3 3.1 6

3.5 -+ 0.5 b 6.5 + 1.0 2.9 + 0.4 lS.O-e2.5’

a

T_JT (%) 22.9 62.2 38.4 65.1

k + f +

2.1 b 2.8 2.8 2.3

;, 26.2 + l.Ob 63 kO.7 40 62.4 -+ 2.6

T = time from zero to 100% LI (47); T, = T X G.F. (growth fraction); T, - T, = time from initial pulse labelling to point of inflection; T, = time from zero LI to initial labelling. ’ Average of 3 values. b + 1 standard error. ’ Assuming a G.F. of 85% (36, 39). * Errors on T,-values cannot be given, as the errors on the points of inflection cannot be estimated from the linear regression analysis of the data in Figure 1. ’This value is significantly different from the other Trvalues (p = 10e4, variant analysis - 22).

Reproduced with permission from MC Millan Press Ltd.

LI=X? where X is a correction factor for a non-linear age distribution of the cells in the cell cycle. For the intestinal crypt cell population, it has been shown that X - 1.39 The agreement between the initial LI and TJT values supports the phase length estimates. In these experiments we attempted to use equitoxic doses in the various regimens, since it is well known that the radiation-induced mitotic delay and repopulation rate are dose dependent.7*‘9,26The number of regenerating crypts per circumference after 8788 hr for the irradiated, drug treated, and combined mo-

10

12

I 14

1 16

I 18

I 20 dose (Gy )

dality treated animals were 33.4 & 7.4 (1 SEM), 89.0 f 15.3, and 36.0 + 6.3, respectively. The higher value after c-DDP treatment alone is not surprising in view of the larger variation in crypt survival for a given drug dose compared with after radiation (see figure 4 of reference IO). Experiment 2 The X ray survival curves for crypts after a single dose and 2 fractions of radiation are shown in Figure 3A. The shift of the survival curve for the 2 fractions in 4 hr (2 F4 hr) towards higher doses, compared with the single dose

II ’

I 8

I 10

I 12

I 14

I 16

I 16

I 20 dose (Gy )

Fig. 3. Duodenal crypt cell survival as a function of radiation dose for X rays alone (A) and c-DDP + X rays (B) after single doses (0) or 2 equal fractions of X rays with a 4 hour interval (0) or after a priming radiation dose of 8 Gy followed 48 hours later by graded test doses of X rays (A). C-DDP was given 30 min. before the single dose or the second fraction of the 2 fraction regimen. The curves are obtained by weighted linear regression analysis from the data between 100 and 1 surviving crypt cells per circumference, because points above 100 deviated from linearity. Vertical bars are +I S.E.M.

1981

Crypt cell kinetics after c-DDP and X rays 0 L. DEWIT ef a/

2fr (48h) 100

8

4

-tc

::’ 10

12

14

10

12

14

12

16

14

16

18

20

dose (Gy) Fig. 4. Replot of the data of Figure 3 showing the crypt cell rays alone (closed symbols, full lines) and X rays + c-DDP (0,O); mid-panel: 2 F-4 hr (m, Cl); right panel: 2 F-48 hr (v, (dotted lines) were calculated starting from the level of 50 alone curves.

survival curve, reflects repair of sublethal radiation damage. The additional shift of the survival curve for 2 fractions in 48 hr (2 F-48 hr) compared with the 2 F-4 hr curve is caused by proliferation of surviving crypt cells. C-DDP, when given in combination with irradiation, caused a shift of the X ray survival curve towards lower doses which was larger for the single doses and 2 F-48 hr irradiation than for the 2 F-4 hr schedule (Fig. 3B). The Dose Effect Factor at 10 surviving crypt cells per circumference (DEF,& that is, the ratio of the X ray dose at that level of survival after irradiation alone to that after X rays plus c-DDP, is 1.20 f 0.02 (1 SE) and 1.2 1 +- 0.02 for the single dose and 2 F-48 h radiation, respectively, and 1.10 ? 0.01 for the 2 F-4 h radiation (Table 2). Assuming independent cell killing by c-DDP and X rays, the vertical distance between the survival curves for combination treatment and X rays alone for a given fractionation schedule gives an estimate of the amount of crypt cell killing by 8 mg/kg of c-DDP (Fig. 4). The surviving fractions for the single dose and the 2 F-48 hr irradiation are lower than for the 2 F-4 h regimen: 0.13 f 0.04 and 0.17 f 0.05 versus 0.33 + 0.05. The difference is statistically marginally significant (p = 0.04, variant analysis).22 The slopes of the survival curves after single dose and 2 F-48 hr irradiation are approximately the same, and steeper than those after 2 F-4 hr of X rays (Table 2). These results are. a consequence of our experimental design, since for the 48 hr interval a constant priming X ray dose of 8 Gy was given to trigger the crypt cells to the same degree of accelerated proliferation, whereas for the 4 hr interval graded equal doses of radiation delivered. The more shallow slope in the latter is a result of the dose dependent amount of sublethal radiation damage repair.4y

survival curves as a function of radiation dose after X (open symbols, dashed lines). Left panel: single doses V). The vertical distances between the survival curves surviving crypt cells per circumference on the X rays

Experiment 3 After a single dose of X rays ( 11 Gy), given at increasing time intervals after the last injection of hydroxyurea, the number of surviving crypt cells per circumference remain low for approximately 3 hr and thereafter increased rapidly to a peak value at around 6-8 hr (Fig. 5). This reflects the greater radioresistance of cells in the late S-phase of the cell cycle compared with cells in the late G,-phase.” This is further illustrated in Figure 6, showing radiation dose response curves for crypt cells irradiated 2 hr or 7 hr after the last HU injection, when the cells are in late G, and late S, respectively. The DO and extrapolation values (N) of these dose response curves are given in Table 3. When 8 mg/kg of c-DDP was given 30 minutes before a single dose irradiation of 9 Gy, approximately the same

Table 2. Values of Do and D.E.F. for the various treatment regimens Radiation schedule Single dose

Cis-platinum 8 mtikg

Do GY)

No

1.I4~0.0lt

Yes No

I .02If:0.I 1

Yes No

I .36 + 0.07 I .06 +-0.08

Yes

0.97 + 0. I2

D.E.F.,o*

l.20~0.02f 2 fractions (4 hour interval) 2 fractions (48 hour interval)

I .42 r 0.08 l.lo*o.o1

1.21 LO.02

* D.E.F.,o = Dose of RX alone/Dose of RX, given in c-DDP, at IO surviving crypt cells per circumference. t I standard error.

with

combination

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November 1986. Volume 12, Number 11

dose response curves for synchronized crypt cells which was larger for the cells in late G, than for those in late S: DEFlo of 1.28 + 0.03 and 1.05 f 0.02, respectively (Fig. 6). Assuming independent cell killing by c-DDP and X rays, the vertical distance between the dose response curves for radiation alone and in combination with c-DDP gives an estimate of the amount of cell killing by 8 mg/kg of cDDP. The surviving fraction of cells in late G, after this drug dose is 0.18 + 0.02 and of cells in late S is 0.54 f 0.23. The difference is statistically significant (p = 1.7 X 10e3, variant analysis).”

1’09

I

1

t

I

1

I

DISCUSSION

I

‘I 4 6 8 10 12 14 time after last HU injection (hours)

Fig. 5. Survival of duodenal crypt cells treated with X rays alone (11 Gy) (0) or 8 mg/kg of c-DDP given one half hour before X rays (9 Gy) (0) at increasing intervals after 5 injections of hydroxyurea. The verticle bars are +l S.E.M. When the errors

overlap, only one side of the error bar is shown.

level of crypt cell survival was obtained in the first 3 hr after the last HU injection as for 11 Gy of X rays alone (Fig. 5). The subsequent increase in cell survival as the cells moved through the S-phase, was slightly greater than after irradiation alone. c-DDP caused a shift of the X ray

3

I-I,, late G1

late S

The present results on proliferation kinetics of duodenal crypt cells, assessed by ‘H-thymidine labelling, clearly showed that c-DDP and radiation, when given alone in approximately equitoxic doses, stimulated the surviving cells into accelerated proliferation. When both agents were used in combination the proliferation rate was reduced, apparently because of a significant decrease in DNA synthesis rate (Table 1.). This issue has, so far, rarely been investigated in vim The incorporation rate of ‘H-thymidine in intestine has been measured by liquid scintillation counting after various doses of X rays and c-DDP given either alone or in combination.4.38 It was found that 8 mg,/kg of c-DDP given in combination with 10 Gy of X rays caused some delay in regeneration time of the surviving cells compared with after 10 Gy of X rays alone. The DNA synthesis rate of the surviving cells was not significantly different between the two treatment groups. During the time of regeneration, measurements were, however, performed only once every 24 hours and were done on segments of whole intestine, which may include some factors of inaccuracy in estimating proliferation kinetics of intestinal crypt cells. Porschen et ~1.~~have analyzed the incorporation rate of “51-labelled 5-iodo-2’-deoxyuridine (‘*‘I-UdR) in a mouse adenocarcinoma during the first 70 hours after low doses of c-DDP (4 mg/kg) and irradiation (2.5 Gy). A reduced “‘I-UdR incorporation rate was found from 4 hours onwards when both agents were given in combination at these doses compared with

Table 3. Values of Do, extrapolation values and DEFlo for crypt cells in late G, and in late S IO0

4

I 6

I 8

I

I

1

1

10

12

14

1

I

I

16 18 dose (Gy)

Fig. 6. Duodenal crypt cell survival as a function of radiation dose for partially synchronized cells in late G, (triangles) or late S-phase (circles) after X rays alone (A, 0) or X rays + c-DDP (8 mg/kg) (A, 0). The curves are obtained by weighted linear regression analysis. The vertical distances between the survival curves (dotted lines) were calculated starting from the level of 50 surviving crypt cells per circumference on the X rays alone curve.

Cell age Late G, Late S

c-DDP (8 me/kg)

Do

N’ (X 104)

No Yes No Yes

1.31 +0.09* 1.12kO.18 1.28k 0.22 1.23 f0.17

I .9 1+ 0.09$ 0.95 2 0.09 148 f 19 1182 I2

DEF,ot

I .28 +_0.03$ 1.os f 0.02

N = Extrapolation value (intercept at zero X-ray dose). t D.E.F. = Dose of RX alone/Dose of RX, given in combination with c-DDP at 10 surviving crypt cells per circumference. $1 Standard error. ??

Crypt cell kinetics after c-DDP and X rays 0 L. DEWITef al.

each agent alone. This would indicate a reduced DNA synthesis rate of the surviving tumor cells. Their data could, however, equally well be explained by the larger amount of cell killing obtained with the combination treatment, since no significant difference in the slopes of the curves of ‘251-UdR incorporation were observed.34 Inhibition of the DNA synthesis rate has also been observed after low doses of c-DDP alone in cells in culture’8*45and in vivo in a murine tumor and rat intestinal mucosa.2’*43Again, in these studies, the changes in DNA synthesis rate may reflect only cell killing, as no distinction is made between 3H-thymidine incorporation of sutivor~ and cells which will die. The present findings are therefore clearly different from these reports as they demonstrated a reduced DNA synthesis rate by the drug given in combination with X rays in surviving crypt cells. If this phenomenon also occurs in humans and taking into account the longer intestinal crypt cell turnover times of 1 to 2 days in humans,2~27~30~40 it may cause increased intestinal toxicity in the treatment of abdominal tumors when the drug is administered concomittantly with fractionated irradiation. A single pulse LI of 26% in controls in the present study is relatively low compared with other reports.23*25*36.39 This may, in part, be due to a high background in our autoradiography slides. On the other hand, investigators who reported LI values of more than 40%, used a crypt squash technique, in which the proliferation pool was estimated to be considerably lower than with the method of repeated labelling of unsquashed ~rypt.s.~~,~~,~~ In particular, Kovacs and Potten estimated from “undersquashed” crypts that the proliferation pool was only 54%, and obtained a LI of 46.3% when only cells in this proliferative region were counted versus 28.1% when all the cells in a crypt were included in the counting. They also pointed out that the upper delineation with the non-proliferative compartment in a crypt was rather arbitrary and that up to 10% of all labelled cells might therefore have been excluded from the proliferative pool. Such approaches obviously greatly influence the obtained LI-values. Comparisons of LI-data are therefore valid only if the method of estimating the proliferative pool is well specified. It was previously shown that c-DDP and radiation kill crypt cells mainly be independent cellular mechanisms.’ Taking that into account, the results from the present

1983

second experiment indicate that the amount of cell killing by c-DDP was approximately the same in cells that were in accelerated proliferation as in unstimulated cells. In vitro, a larger cell killing effect of c-DDP has been reported in non-proliferating human colon adenocaFcinoma cells versus exponentially growing cells.16 The lack of proliferation dependence for killing of crypt cells observed here, may be because a large proportion of the unstimulated cells in this tissue are already rapidly proliferating. The lendency for less cell killing by the drug in the 2 F-4 hr interval schedule might be because of cell cycle perturbations, as explained below. The investigations on a partially synchronized crypt cell population demonstrated that c-DDP had a significantly higher lethal effect, by a factor of 3, in cells in the late G ,-phase than in cells in the late S-phase (Fig. 6). This is in good agreement with observations in cells in vitro.‘5,3’.37This difference in lethal effect of the drug in cells in different phases of the cell cycle might account for the less cell killing observed in the 2 F-4 hr irradiation schedule of the second experiment (midpanel of Fig. 4). Indeed, 3.5 hours after the first irradiation, at the time cDDP was administered, the fraction of cells in the G,phase was presumably decreased as a result of the greater lethal effect of X rays in these cells and because of a radiation induced Gz-block. It is likely that c-DDP had a smaller cell killing effect on this cell population compared with unperturbed cells. In conclusion, c-DDP and radiation, given alone, stimulated surviving duodenal crypt cells into compensatory accelerated proliferation. When given in combination, the proliferation rate was markedly reduced due to inhibition of the DNA synthesis rate. The amount of cell killing by c-DDP, when given in combination with irradiation, was not significantly different in crypt cells which were in accelerated proliferation as in unstimulated cells. C-DDP had a predominant lethal effect on cells in the G,-phase of the cell cycle. It would be very interesting to obtain insight in the mechanisms involved on the molecular level to explain the present observations. This requires methods that can visualize in vivo the presence of platinum-DNA adducts in the nuclei of the crypt cells. One of these techniques is a monoclonal antibody linked to these adducts and which can be stained in a tissue section.33 Such a technique is currently being tested in our institute.

REFERENCES I. Baker, D.G., Sager, H., Constable, W., Goodchild, N.: The response of previously irradiated skin to combination of fractionated X radiation, hyperthermia, and cis-diamminedichloroplatinum. Radial. Res. 98: I76- 181, 1984. 2. Bertalan@, F.D., Nagy, K.P.: Mitotic activity and renewal rate of the epithelial cells of human duodenum. Acta Anal. 45: 362-370, 196 1. 3. Bohart, R.D., Ogawa, G., Prestayko, A.W., Cadiz, M.,

Crooke, S.T.: An observation on the stability of cisdichlorodiammineplatinum (II): A caution regarding its administration. Cancer Treaf. Rep. 63: 2117-2119, 1979. 4. Burholt, D.R., Schenken, L.L., Kovacs, CH.J., Hageman, R.F.: Response of the murine gastrointestinal epithelium to cisdichlorodiammineplatinum II: radiation combinations. Int. J. Radiat. Oncol. Biol. Phys. S: 1377-l 38 1, 1979. 5. Caimie, A.B., Lamerton, L.F., Steel, G.G.: Cell proliferation

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studies in the intestinal epithelium of the rat. I Determination of the kinetic parameters. Exp. Cell. Res. 39: 528538, 1965. 6. Carde, P., Laval, F.: Effect of cis-dichlomdiammineplatinum (II) and X rays on mammalian cell survival. Int. J. Radial. Oncol. Biol. Phys. 7: 929-933, 1984. 7. Denekamp, J., Stewart, F.A., Douglas, B.G.: Changes in the proliferation rate in mouse skin after irradiation: continuous labelling studies. Cell Tissue Kinet. 9: 19-29, 1976. 8. Dethlefsen, L.A., Ohlsen, J.D., Riley, R.M.: The combined effects of hydroxyurea and X irradiation on murine duodenal mucosa and host survival. Radiat. Res. 82: 5 18-525, 1980. 9. Dewit, L., Begg, A.C., Kohler, Y., Stewart, F.A.. Bartelink,

H.: Influence of cis-diamminedichloroplatinum (II) on mouse duodenal crypt stem cell survival after multifmction X my treatment. Int. J Radiat. Oncol. Biol. Phys. 11: 18091816, 1985. 10. Dewit, L., Oussoren, Y., Bartelink, H.: Dose and time effects of cis-diamminedichloroplatinum (II) and radiation on mouse duodenal crypts. Radiother. Oncol. 4: 363-372, 1985. 11. D&t, L., Oussoren, Y., Bartelink, H.: The relationship between proliferation rate and the effects of cis-platinum and X rays on mouse duodenal crypt cells. Br. J. Cancer 53(Suppl. VII): 33-36, 1986. 12. Dewit, L., Oussoren, Y., Bartelink, H., Stewart, F.A.: Differences in the acute intestinal syndrome after partial and total abdominal irradiation in mice. Int. J. Radiat. Biol. 48: 745-152,

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