Chem.-Biol. Interactions, 14 (1976) 217-232 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

ACTION OF A PLATINUM COMPLEX [ CIS-DICHLOROBIS (CYCLOPENTYLAMINE)-PLATINUM (H)] ON CHINESE HAMSTER OVARY CELLS IN VITRO

IRENA SZUMIEL and A.H.W. NIAS

l

Department of Radiobiology and Health Rotection. Institute of Nuclear Research, 03-195 Warsaw (Poland), under Fellowship of th? International Atomic Energy Agency in the Glasgow Institute of Radiothempeutics. and l Glasgow Institute of Radiotherapeuks, Belvidere Hospital, Glasgow G 31 4PG (Great Britain) (Received October 14th, 1975) (Accepted February 12th, 1976)

SUMMARY

cis-Dichlorobis(cyclopentylamine)platintim (II) (DBCP) treatment of Chinese hamster ovary (CHO) monolayer cell cultures was found to cause: (1) growth inhibition due to w impaired G 1+ S transition and formation of a noncycling compartment; (2) delayed cell death (with a maximum at 72 h after treatment); (3) decrease of [“C] TdR incorporation ints DNA. Part of the surviving cell population was subject to non-lethal damage. The D, valu the dosesurvival curves were 9.2 and 14 yg/ml in two kinds of media differing in Na’ and Cl- concentrations. There was no sparing effect o tionation and no cell cycle phase specificity. In the dose range corres the shoulder region of the dose-sun&al curve, the lethal effec treatment was directly related to the decreased rate of DNA syn DBCP-treated cells. __

_.._ _ __ __._____.

INTRODUCXON

Platinum coordination complexes are a were first described in 1969 by Rosenbe six years much work has been done on Abbreviations: CHO, Chinese hamster (ovary); DBCP, c&Ii platinum (II); cis-DDP, cis-dichlorodiammineplatinum (II); HEPES, N-!&hydroxyethylpiperazine-N’-ethanesulphonie acid ; saline.

complexes and the relationship between their chemical structure and chemotherapeutic activity. Most of that work was recently reviewed in detail [ 21. In spite of progress in understanding the pharmacological properties of Pt complexes, however, relatively little is known of their effect at the cellular level. Most of the studies of the mechanism of action of Pt complexes have been carried out using the best known compound - c&DDP - the only one which has been put into clinical trial so far. However, a number of organic analogues of c&DDP have been synthesized by Connors et al. [3,4] which show higher activity (against ADJ/PCGA plasma cell tumour in mice) than the original inorganic complex. One of these is DBCP. The structure of this coordination complex consists of #-chlorides, planar platinum (II) and two dative coordinate bonds whereby the lone pairs on the primary amines are donated into the metal’s orbitals (Fig. 1).

Fig. 1. Structure of DBCP.

A therapeutic index of 235.7 was obtained for DBCP, a value nearly 30 times higher than that for c&DDP [ 31. This high value encouraged studies of the effect of DBCP at the cellular level. In this paper we describe the action of DBCP on CHO cells in monolayer culture. Additional studies of the interaction in CHO cells of DBCP and ionising radiation have been reported elsewhere [ 171. MATERIALS

AND METHODS

ft complex. cis-Dichlorobis(cyclopentylamine)platinum (II) was kindly provided by Dr. T.A. Connors and by Johnson Matthey and Co. Ltd. Cell line. CHO cells were cultured as a monolayer in Falcon tissue culture plastic flasks (T75 for stock cultures and chromosome experiments, T30 for all other experiments). HEPES-buffered (20 mM) Ham’s F12 medium supplemented with 16% calf serum, non-essential amino acids and 2 mM glutamine was prepared with two different concentrations of Na’ and Clions, using Ham’s F12 concentrated (10 X) solution. All medium components were supplied by Gibco-Biocult Ltd. During the period of these studies the concentration of Na’ and Cl- ions in Ham’s F12 solution was altered by Gibco-Biocult Ltd. The original medium (A) contained 106 mM Na+ and 108 mM Cl-, the new medium (B) contained 119 mM Na’ and 120 mM Cl- with no other change in constituents. In both kinds of media CHO cells were 218

maintained in log~ithmic growth phase with a doub~g time of ~1.5--12, h. Methods of cell culture were described in detail ~~~ou~y [5]. vestment of CHC cells with DBCP. cells w~tfe expo period of 1 h at 37°C. 2 h after plating lo3 cells per T30 volume of DBCP solution was added to 5 ml of DBCP solution was prepared by d~ut~g 1 : 4 urn B) with cold (+QOC) medium, immediately trated solution of DBCP in DMSG. This was pre -2O”C, at the concentration 1 mg/ml for exporimen 2 mg/ml for experiments with medium 8. ConQcol cells DMSO only and this did not change the plating e~~~jeu~y 70% in both DMSG-treated and untreated cell cultures. The bottle conning diluted DBCP ~luti~n w last aliquot was added. Samples (in triplicate) recew set intervals, and after exactly 1 h, medium was ch same sequence. After 5 days’ ~cubation, cultures and stained with carbol fuchsin (Gurr, diluted 1 : 10 number of clones containing more than 50 cells were ing microscope. Split-dose treatment with DBCP. The split-dose experim formed three times in medium A (26 &g/ml of DBCP) and twice in m B (62 pg/ml of DBCP). All the samples were treated with the or 62 gg/ml, I h, 37°C); the second dose (26 or 62 @g/ml, 1 given ~mediately after the change of medium (0 h interval) or 3 h, 4 h and 24 h incubation of the cell cultures at 37°C. In experiments performed in medium B, 5 - lo3 cells per T30 flask were plated. In all earnments seven T30 flasks with lo4 cells were plated and stied at 0,l 3 h, 4 h and 24 h intervals for cell multiplicity correction of the s data. Half-l;ie of DBCP. For this determination a solution of 200 Erg/ml DBCP was prepared using warm (37°C) medium for d more concentrated DBCP solution in DMSO (cf. ~‘~a~~t of CI-I with PBCP”). Concentrations used for cell culture treatment w &g/ml in medium A, and 62 and 90 &g/ml in medium B. The ples were treated immediately after preparation of DBCP ~luti~n, subsequent ones at 15, 30, 45 and 60 min, 2, 3, 4, 5 and 24 h. The solution was kept during all that tie at 37’C. After 5 days’ ~cubation of the treated cultures th mined and from these data and the previously obtained a plot was constructs of DBCP concentration versus t 37”C, from which the half-life was read. The experiments medium were carried out twice. Clone size a~ulysis. For clone size ~alysis the num 100-300 clones was counted using an inverted micro clone size analysis was described porously [5].

Viability. Control and DBCP-treated cells were checked for the presence of non-viable cells by the dye exclusion test. Trypan Blue (0.4% in BSS from Gibco-Biocult Ltd.) was used in a final concentration 0.07%. After 5 min at room temperature the number of dyed and undyed cells were scored during the following 10 min; at least 300 cells were scored. Synchronization. Experiments on synchronized CHO populations were performed using the mitotic selection method [6] . The degree of synchronization was estimated from the mitotic index at zero time (this was usually 95% or higher) and from average cellular multiplicity and labelling index, determined at 1 h intervals. Autoradiography. For autoradiography lo4 cells per T30 flask were plated. ( [3H] TdR; specific Pulse-labelling was performed using [Me-3H] thymidine activity 5 Ci/mmole, TRA UO, The Radiochemical Centre, Amersham, Bucks.) for 15 min at the concentration of 5 pCi/ml; continuous labelling was performed with 0.3 pa/ml [ 3H] TdR for l-30 h. After labelling was completed, cells were washed with PBS and fixed twice with Carnoy’s fixative for 15 min. The bottoms of T30 flasks were subsequently cut off, washed thoroughly in tap water and coated with Kodak NTB 3 emulsion. Plates were exposed for 5 days at room temperature, developed and stained by dipping for 3 set in carbol fuchsin solution. For labelling index determination 500-1000 cells/plate were counted. DNA synthesis. DNA synthesis experiments were performed by labelling 2 - 10’ cells/T30 flask (2 flasks per sample) with [2-‘“Cl thymidine (0.15 &i/ml, specific activity 750 mCi/mmole, CFA 219, The Radiochemical Centre, Amersham) for 3 h and measuring the radioactivity of 5% trichloroacetic acid-insoluble fraction by the liquid scintillation method [73 . Results were expressed in counts per min per lo4 cells and as percent of incorporation into control cells. Chromosome preparations. For the analysis of chromosomal aberrations synchronized cell cultures were used. Mitotic cells were harvested, plaied in twelve T75 flasks (1.6 + 10’ cells per flask) and, after 1.5 h, treated with DBCP (46 Ccg/ml, 1 h, 37°C). One flask was left untreated for the cont;ol preparation. Mitotic cells were subsequently shaken off at the first and second mitosis after DBCP treatment; this was carried out after treating the first six cell cultures with colchicine (1 I.cg/ml) between the 11th and 14th hour of the experiment (the control culture - between the 10th and 13th hour), and the second six cell cultures - between the 22nd and 25th hour of the experiment. Mitotic cells were harvested, washed once with PBS, given a hypotonic treatment in 0.7% sodium citrate [8] for 13 min at 37”C, fixed twice with cold Carnoy’s fixative for 15 min, spread on chilled, wet microscopic slides, dried over a micro-burner flame for about 1 min, stained with 10% Giemsa for 1 h, and made permanent in Canada balsam. RESULTS

Dosesurvival

relationship

Fig. 2 shows that the response of CHO cells to DBCP treatment 220

is signifi-

cantly influenced by the composition of cell culture medium: in medium A (106 mM Na’, 108 mM Cl-) DBCP reduces cell survival more effectively than in medium B (119 mM Na’, 120 mM Cl-); the respective survival cmve parameters being -D, 9.2 and 14.0 pg/ml, Do 17.5 and 27.5 pg/ml. Both curves are clearly bi-phasic with the extrapolation number about 7. In the search for an explanation of the different dose-response of CHO cells, determination of the half-life of DBCP in both kinds of media was carried out. In medium A the half-life was approx. 165 min, while in medium B it was only approx. 45 min. Split-dose survival data As shown in Fig. 2 both dosesurvival curves have a “shoulder” and an exponential part. In radiation-dose response it is usual to explain the presence of a shoulder in terms of the ability of cells to accumulate and repair sub-lethal damage. However, dose-survival curves for alkylating agents are known where the sparing effect of dose splitting is absent [9). This has also been found for CHO cells submitted to split-dose DBCP treatment. The data presented in Table I indicate that there is no split-dose recovery in cells treated with both low and high doses of DBCP; the relative survival values calculated as described in [9 ] were close to 1 for all intervals studied. TABLE I EFFECT OF FRACTIONATED OF CHO CELLS Dose E.cg/ml, 1 h, 37’C, medium B

Interval between doses (h)

DOSES OF DBCP ON THE PERCENTAGE

Fractionated A

26

26 + 26

dose B

55.0 0 1 2 3 4 24

62

62 + 62

Relative survival B/A

Percentage survival f S.E. of mean a Single dose

SURVIVAL

22.9 + 3.02

1 22.9 22.3 24.3 13.2 19.8

f 3.07 %0.92 It 0.71 + 3.45 + 0.84

t.97 1.06 0.79 0.36

10.5 0 1 2 3 4 24

0.103 + 0.025 0.114 0.114 0.094 0.102 0.112

+ 0.030 + 0.030 + 0.028 ?r0.013 b

1 1.10 1.10 0.91 0.99 1.09

a Mean values from 2 separate experiments. b Single determination.

221

Surviving Fraction IQ-

l-

o+

00)

a001

I

I

I

1

10

30

50

70

DBCP Dose(ug/ml.

I 90

lh.37”~)

Fig. 2. Dose-urvival curves for CHO cells treated for 1 h at 37’C with DBCP; a, in medium A, b, in medium B. Mean values from 3-20 experiments are shown; standard error indicated if larger than the point drawn.

Cell cycle phase-sensitivity

The effect of DBCP on synchronized CHO populations treated at different points of the cell cycle is shown in Fig. 3. The data obtained indicate that for doses reducing survival to 26% and 5% there is no cell-age-dependent sensitivity to DBCP. This is in accord with the only similar data available for l-h treatment at 37°C in vitro for a Pt complex, namely, that published by Drewinko et al. [lo] ; these authors found no pronounced phase sensitivity in human lymphoma cells in vitro treated with cis-DDP. Chromatid aberrations. The formation of chromosomal aberrations by DBCP was studied in synchronous CHO cell cultures. As shown in Table II only chromatid aberrations were found. An interesting feature of DBCP action at the chromosomal level is that gaps and breaks are found in the first mitosis after treatment, while chromatid exchanges are present only in cells in the second mitosis after treatment. 222

04-

Surviving Fraction

o.,_

72 uglml

0.02 1

I

I

,

IO

14

/Gz

/

Average Cellular Multiplicity

%

(0)

60

Lobelled Cells (0)

0

2

Time After

6

Cell

10

14

Horvest(hours)

Fig. 3. Survival data for synchronized for 1 h at 37’C. Mean values from larger than the point drawn.

CHO cell populations treated with 2 doses of DBCP 2 experiments are given; standard error indicated if

DNA synthesis Platinum complexes are known to inhibit DNA synthesis (for review see ref. 2). DBCP is no exception in this respect. As may be seen in Table III inhibition of [14C] TdR incorporation into DNA during the first 3 h after DBCP treatment is related to the dose. In independent experiments it was found that [‘“Cl TdR uptake measured at l-h intervals was limar until 4 h in control as well as in DBCP-treated cells. Comparison of the incorporation data and those obtained from continuous and pulse labelling indicates that DBCP causes a decrease not only in the rate of DNA replication but also in the number of cells entering S phase. 24 h after DBCP treatment incorporation of [14C]TdR is restored to the normal level, with the exception of the highest dose-treated cells. The discrepancy between these data and the labelling indices which are lower than those for control cells, is probably caused by the experimental procedure: before trypsinization of cell cultures preceding their counting and preparation of trichloroacetic acid-insoluble fraction every flask was thor. oughly washed with PBS to wash out the excess [ “C]TdR. This treatment could detach some of the cells damaged by DBCP which would be unable

223

E

24-27

[f 4C]TdR incorporation ?I:SE. Labelling index after continuous 24 h labelling (W)

Fraction of control

$ontroI)

98.7

1

75.4

1

3.0

4.3