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The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma
Chem.-RioI. fnteractionq 11 (1975) 291-299 0 Ebevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands
291
THE BINDING THE YOSHIDA
OF
OF [sH]CHLORAMBUCIL ASCITES SARCOMA
TO NUCLEAR
PROTEINS
PAMELA G. RICHES AND K. R. HARRAP Department of Applied Biochemistry, Institute of Cancer Research, Sutton, Surrey SM2 5PX (Great Rritain)
(Received January dth, 1975) (Revision received March lOth, 1975) (Accepted March 14th, 1975)
SUMMARY
The binding of [sH]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of sH-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.
INTRODUCTION
The alkylating agents are a range of compounds, some of which are used in the clinical management of neoplastic disease. Their chemical and biological properties have been reviewed by Ross1 and more recently by Wheelers who also discussed the problem of cellular resistance to alkylating agentss. The proposals that the alkylating
agents cross-link strands of DNA*-6 and that resistance is attributable to enhanced excision and repair mechanisms in resistant cells, are not entirely compatable with experimental observations in the Yoshida ascites sarcoma, which has been investigated in this laboratory. The alkylating agent-sensitive form of this tumour does not show an inhibition of DNA synthesis as the earliest observed lesion following challenge with chlorambucil [4-p-[bis(2-chloroethyl)amino]-phenyl3-butyric acid]‘. Moreover, the drug-sensitive strain and a strain showing cross-resistance to alkylatingagcnts both excise alkylated bases from DNA and repair the resultant gaps with comparable facilitys. Other workers have suggested that alkylating agents modify the interaction between DNA and protein, thereby changing primer activityPsl0, while the association of nuclear protein with alkylated DNA has also been reportedllslo. We have previously described morphological and biochemical changes in the chromatin of sensitive Yoshida ascites sarcoma cells induced by chlorambucill”, and suggested that these may have occurred either from direct reaction of chlorambucil with nuclear proteins or from the effects of the drug on enzymes concerned with the functional organisation of nuclear proteins. In the present paper we describe investigations related to the first of these possibilities, and report the occurrence of chlorambucil bound covalently and non-covalently to nuclear proteins of tumour cells isolated from animals previously exposed to the drug. MATERIALS AND METHODS
Darvan No. 1 was obtained from R. J. Vanderbilt Co., Inc. (230 Park Avenue, New York 17). Acrylamide, N,N’,-methylenebis-acrylamide and N,N’,N’-tetramethylethylenediamine were purchased from BioRad Laboratories (27 Homesdale Road, Bromley, Kent, U.K.). Other chemicals were purchased from British Drug Houses Ltd. (Poole, Dorset, U.K.), Hopkin and Williams Ltd. (Chadwell Heath, Essex, U.K.) or May and Baker Ltd. (Dagenham, U.K.). Analytical grades were used where available. 3H-labelled chlorambucil, [3H]4-(4-bis(2 chlorethyl)aminophenyl-3,s) butyric acid, was synthesised at the Chester Beatty Research Institute, London, U.K.13. Both alkylating agent-sensitive and -resistant strains of the Yoshida ascites sarcoma were used in this work. Tumours were passaged at weekly intervals by inoculation of 2 - IO6 cells into 6-week-old Chester Beatty Colony bred female Wistar rats. Animals bearing the resistant line routinely received 2 mg/kg melphalan (ClCHtCH~)ZN.GH~CHNH~COOH) on the third day after tumour transplantation. Full details have been given previouslyll. [3H]chlorambucil (2.4 Ci/mmole) was administered to tumour-bearing animals on the fourth day following transplantation, at a dose of 8 mg/kg. The total radioactivity received by each animal was 12 mCi. Animals were killed, by cervical dislocation, 6 and 24 h after drug treatment and tumour cells aspirated from the peritoneal cavity with ice-cold isotonic saline. Nuclei were prepared from the pooled cells from ten animals by the method of Rickwood et all”.
293
Tris-saline soluble proteins were extracted from the nuclei by homogenisation in 50 vol. of 0.14 M NaCl, 0.05 M Tris-HCI (pH 7.4) as previously describedIG. The chromatin pellet was dissociated in 1 mM sodium phosphate (pH 6.8), 2 M NaCl, 5 M urea buffer and chromatographed on freshly prepared hydroxyapatite columns as described by MacGillivray et al. 16. The chromatin proteins were eluted in three fractions Hl, H2, H3 by stepwise increase in phosphate concentration. Hl proteins were eluted in 1 mM phosphate and represented histones; H2 and H3 were non-h&tone proteins eluted with 50 mM and 200 mM phosphate respectivelyl6. The pooled protein fractions were dialysed against 0.1% sodium dodecyl sulphate, lyophilised and dissolved in a solution containing 8 M urea, 1.Oo/, mercaptoethanol, 1.0% sodium dodecyl sulphate, 10 mM Tris-HCI pH 7.4, against which they were dialysed, and then redialysed against a similar solution in which the sodium dodecyl sulphate concentration had been adjusted to 0.1%. The protein fractions were characterised by electrophoresis in 15 % acrylamide-0.1 y0 sodium dodecyl su%hate gels by the method described previously 16.The gels were stained with 1% amido black and destained in ethanol-acetic acid and water (30:7:63 v/v/v) for 36 h using an HSI destainer (Hoefer Scientific Instruments, San Francisco). After scanning the stained gels at 540 nm, using a gel scanning attachment to a Hilger-Gilford Reaction Kinetics spectrophotometer, they were frozen in solid COs and cut into 1 mm slices using a Joyce-Loebel gel slicer. The gel slices were combusted in an Intertechnique Oxymat (Model JAlOl), which resulted in thecompleteoxidation of the gel slice. Tn the following automated sequence, the tritium has condensed to form a small volume of tritiated water to which was added 20 ml of scintillant (dioxan 3500 ml; toluene 1500 ml; naphthalene 100 g; butyl PBD 35 g). The resulting mixture was virtually unquenched and was counted in a /?-scintillation spectrometer (Tntertechnique SL40) with a counting efficiency of at least 48 %. A consistent tritium back-
TABLE
1
BINDING OF [~H~CHLORAMBIJCIL CHLORAMBUCIL
TO WHOLE CELLS,NUCLEI
[5.0jtmoles, 12 mCi] To TUMOUR-BEARING
AND CHROMATIN
AFTER ADMINISTRATION
ok
RATS
Figures are the average of two aliquots from the pooled tumuurs from ten animals counted in duplicate. The values in parentheses are the lower and upper values obtained. Cell line
Hours after treatment
cprn . lo-~ Total ceil binding/ nig protein
Sensitive Yoshida ascites Resistant Yoshida ascites
6 24 6 24
26.47 [23.56-28.75) 20.51 [18.11-20.991 12.02 [l l&5-12.38] 9.83 [ 9.67-10&l]
Total t&ear bindingjnlg proteirr
Total chromatin bindingjmg protein
22.37 [22.13-24.301 15.04 [13.47-16.101
35.49 [32.90-38.081 7.26 [ 6.3% 8.121
[ :::-
8.621
12.64 [12.36-12.911
7.38 [ 6.89-
7.631
4.24 [ 4.w
4.971
294 TABLE II [*~~HUXAMBIJ~ILSEPAR~EDBYD~LYSISAGAIN~T ON FQLYACRYLA~IILX
GEL ELECTROPH~RE~IS
Cell line
Sensitive Yoshida ascites sarcoma cells
Resistant Yoshida ascites sarcoma cells
Hours after treatment
0.1% ~~DIIJMDC)I)ECYLSULPHATE(D~~~ABLE)OR
(NON-COVALENTLY
BOUND)
EXPRESSED AS A PERCENTAGE
Fraction
YOTotal counts dialysable
YONon-dialysable counts noncovalently bound
6
Saline soluble Histone (Hl) NHP (H2) NHP (H3)
18.5 23.9 13.5 3.4
60.1 51.3 16.3 21.5
24
Saline soluble Histone (HI) NHP (H2) NHP (H3)
16.3 25.6 15.0 0.0
4.8 47.0 38.0 31.6
6
Saline soluble Histone (Hl) NHP (H2) NHP (H3)
35.4 16.5 18.8 10.8
31.3 18.4 17.7 8.4
24
Saline soluble Histone (Hl) NHP (H2) NHP (H3)
27.5 13.0 29.4 15.4
0.0 0.0 8.8 2.3
ground of 30 cpm was obtained when non-radioactive gel slices were similarly treated. This method allows an accurate determination of the low counts obtained in the present experiments. Protein was estimated by the method of Lowry et ~1.17;DNA was estimated by the method of Burtonls. RESULTS
Table I gives the values of [aH]chlorambucil associated with whole cells, nuclei and chromatin. All fractions from sensitive cells show higher binding both at 6 h and 24 h after treatment than those obtained from resistant cells. When total chromatin was fractionated on hydroxyapatite columns, the recovery of protein was in all cases 80-90 % of the total applied. However, the recovery of 3H-label in protein and nucleic acids was consistently less than the counts applied. At 24 h after treatment the recoveries of aH-label for sensitive and resistant cell chromatin were 42 % and 47.7 %, respectively; the values at 6 h after treatment were 21% and 20.9 %. The protein fractions collected from the column, HI (histone), H2 and H3 (non-histone proteins) were exhaustively dialysed and unbound chlorambucil would be lost at this stage. The extent of this loss is indicated in Table II, which also shows that a further loss of sH-label occurred following electrophoresis, staining and destaining. Presumably this material was bound non-covalently to the proteins and in Table II
295 this amount has been calculated from the difference between the W-label applied to the gel and its recovery as counts bound to protein. The presence of non-covalent binding was confirmed by running a gel for a short time and then slicing into 1 mm slices and determining the radioactivity associated with each slice. By this method labelfed material could be detected in the region of the dye-marker band (data not shown). Unstained gels were used for this experiment as non-covalently bound material, after electrophoresis, could be removed from the gel by the solvents necessary for destaining. The amount of chlorambucil covalently bound to protein was determined from the amo~t of sH-label associated with the el~trophoretically separated non&stone proteins (see Figs. 1 and 2), after staining and destaining, which would be expected to remove non-covalently bound drug. Approximately three times as much drug was covalently bound to the combined
al
CO
at
30 2.0 1.0 ~
bJ c: 0 x
df
GEL
SLICE
NUMBER
GEL
SLICE
NUMBER
GEL
SLICE
NUMBER
Fig. 1. Binding patterns of [sH]chlorambucil to non-histone chromatin (H2) proteins of alkylating agent-sensitive and -resistant Yoshida ascites sarcoma cells, 6 and 24 h after treatment of tumourbearing animals with ~hlorambu~il (8 mg/kg, 2.4 Ci~mmole). (a) sensitive 6 hr after t~tment; (b) sensitive 24 h after treatment; (c) resistant 6 h after treatment; (d) resistant 24 h after treatment. Slice 1 represents the top (high molecular weight end) of the gel. Fig. 2. Binding patterns of [sH]chlorambucil to non-histone chromatin (H3) proteins of alkylating agent-sensitive and -resistant Yoshida as&es sarcoma cells, 6 and 24 h after treatment of tumourbearing animals with chlorambucil (8 mg/kg, 2.4 Cijmmole). (a) sensitive 6 h after treatment; (b) sensitive 24 h after treatment; (c) resistant 6 h after treatment; (d) resistant 24 h after treatment. Slice I represents the top (high molecular weight end) of the gel. Fig. 3. Binding patterns of [sH]chlorambucil to the Tris-saline soluble nuclear Proteins of alkyiating agent-sensitive and -resistant Yoshida ascites sarcoma cells, 6 and 24 h after treatment of tumourbearing animals with chlorambucil (8 mg/kg, 2.4 Ci/mmole). (a) sensitive 6 h after treatment; (b) sensitive 24 h after treatment; (c) resistant 6 h after treatment; (d) resistant 24 h after treatment. Slice I represents the top (high molecular weight end) of the gel.
296 TABLE III SPECIFIC ACTIVITY OF [~H~CHLORAMBUCIL COVALENTLY IBOUND TO NUCLEAR PROTEINS
Cell line
Hours after treatment
Fraction [cpm - IO-3]/mg protein Saline soluble
Histone (HI)
Non-histone chromatin protein (H2)
Non-histone chromatin protein (H3)
Sensitive Yoshida ascites sarcomacells
6 24
13.96
3.29
10.28
4.64
27.64 10.79
15.71 6.90
Resistant Yoshida ascites sarcoma cells
6 24
2.61 3.40
2.70 3.66
8.23 2.97
9.45 2.69
fractions (saline soluble, HI, H2. H3) of sensitive cells compared with resistant cells and during the period of observation this was reduced to one half in sensitive cells and to one third in resistant cells. In both cell types more than two thirds of the total drug bound to the four protein fractions was associated with the combined non-histone proteins (H2 and H3) (Figs. 1 and 2). Less than 10% was associated with the histone (HI) fraction at 6 h, and by 24 h this had increased slightly. Notably, five times more drug was bound to the Tris-saline soluble fraction of sensitive cells compared with resistant cells at 6 h, and in sensitive cells this was reduced to two-thirds by 24 h (Fig. 3). Since sodium dodecyl sulphate polyacrylamide gels separate; proteins on a mo!ecular weight basis, inspection of Fig. 3 reveals that the binding in sensitive cells was associated with proteins in the high and intermediate molecular weight region of the gel and was virtually absent from this region in resistant cells. While the resolution of a one-dimensional electrophoresis system is limited, one stained band representing more than one protein species, comparison of the electrophoresis profiles of nuclear proteins from sensitive and resistant cells nevertheless reveal no differences. Within the limits of the method we cannot therefore detect differences in the composition of the fractions between the two cell strains before or after treatment. Table III gives the specific activity of the various fractions which have been calculated from the recovery of aH-label covalently bound after polyracylamide gel electrophoresis. The nucleic acids were not extensively studied in these experiments. They were, however, eluted from the hydroxyapatite column in 0.5 M Na-phosphate (pH 6.8), 5 M urea, 2 M NaCl, dialysed and then an estimate made of bound counts/DNA. The values of binding were in the order of 103 cpm/mg DNA. DISCUSSlOh;
The ability of chlorambucil to bind firmly to serum proteins without alkylating them has been previously demonstrated 19. Non-covalent binding of chlorambucil
297
to i~unoglobulins has also been describedze, while Hopwood and Stock have reported binding of alkylating agents to serum ablumin and fibrinogen with retention of alkylating function 21. We have previously reported binding of [aH]chlorambucil to DNA, RNA and protein following irrvivaadministration of high specific activity drug to turnour-daring animals ss. However, these observations need to be reconsidered in relation to the present data which indicate that considerable amounts of non-covalently bound drug are associated with nuclear components. In the present experiments extremes of pH or the use of polar solvents were avoided and an estimate of covalent binding was made by polyacrylamide gel electrophoretic separations, In both soluble nuclear sap and chromatin proteins the covalent and non-covalent binding was greater in sensitive than in resistant cells at corresponding times. Non-covalent binding was practically absent in the resistant cell by 24 h after treatment. The aH-label which was dialysable probably represents free chlorambucil, or a metabolite, since non-covalent binding has been reported to be stable to dialysis at 4” (ref. 23). The nature of the non-covalent binding to nuclear protein fractions has not been determined. It is possible that in the Tris-saline soluble fractions both ionic and hydrophobic binding were present. Any ionic binding to the chromatin proteins, howwever, would be expected to be removed during chromatography on hydroxyapatite. Thus, the non-covalent binding detected in these fractions is probably hydrophobic and was removed by the destaining procedure to which the polyacryiamide gels were subjected. The dialysis of hydrophobic molecules in detergent solutions is inefficient because the former would be expected to bind with high affinity to non-dialysable detergent micelles. The presence of counts in the region of the dye-marker front in unstained gels is consistent with this hypothesis. Covalent binding of drug was greater to non-histone chromatin proteins (H2 and H3 fractions) than to histones (Hl fraction). The nuclear sap proteins (saline soluble} were of particular interest: considerable binding occurred in the high molecular weight regions (Fig. 3, Slices l-10) of sensitive cell proteins, and was almost entirely absent from the resistant fractions at 6 h after treatment. Considerable levels of binding to this fraction from sensitive cells persisted similarly at 24 h after treatment. Nucleic acids were eluted from the hydroxyapatite columns and specific activities determined. Drug binding was of the order of 10s cpmlmg DNA. The calculated amount of bound chloramb~il, assuming ail the aH-label was associated with the drug, was 0.5 pmoles/mg DNA. In previous in vivaexperiments this vahte of bound chlorambucil was 30 pmoles/mg DNA when DNA was acid extracted The molar binding of chlorambucil to the saline-soluble nuclear proteins at 6 h after treatment was of the order of 17.5 pmoleslmg protein; the corresponding covalent binding to this fraction at the same time was 7.0 pmoles~mg protein. This also represents considerably greater levels of drug than that associated with the DNA The ratio of total cell binding 6 h after treatment in sensitive cells to that in resistant cells is approximately 2.0. However, when the nuclear proteins were considered the ratio (i.e. sensitive: resistant) was considerably greater in those groups (i.e.
Tris-saline soluble and non-histone chromatin protein) where binding was detected. It is, therefore, possible that the differences in total cell binding can be attributed to this high nuclear protein binding. This would confirm previous results24 where an autoradiographic study of Yoshida ascites cells treated with chlorambucil showed no significant difference between sensitive and resistant cells in cytoplasmic grain counts but significantly higher grain counts in the sensitive cell nucleus when compared with the resistant cell nucleus. The significance of high nuclear protein binding of chlorambucil in Yoshida ascites sarcoma cells remains to be assessed. The present experiments, however, indicate important differences in binding to protein from sensitive cells compared with resistant cells when high specific activity drug was employed at a pharmacologically effective dose.
REFERENCES 1 W. C. J. Ross, Biological AIkyIating Agents, Butterworth, London, 1962, pp. 3-87. 2 G. P. Wheeler, Alkylating agents, in J. F. Holland and E. Frei (Eds.), Cancer Medicine, Lea and Febiger, Philadelphia, 1973, pp. 791-806. 3 G. P. Wheeler, Studies related to mechanisms of resistance to biological alklyating agents, Cancer Res., 23 (1963) 1334-l 349. 4 P. Brookes and P. D. Lawley, Alkylating agent, Brit. Med. Bull., 20 (1964) 91-95. 5 K. W. Kohn, C. L. Spears and P. Doty, Jnterstrand crosslinking of DNA by nitrogen mustrad, J. Biol. Chem., 19 (1966) 266288. 6 J. 1. Roberts, 7. P. Brent and A. R. Crathorn, The mechanism of the cytotoxic action of alkylating agents on mammalian cells, in P. N. Campbell (Ed.), Symposium of the Interaction of Drugs and Subcellular Components in Animal Cells, Churchill, London, 1968, pp. 5-27. 7 B. T. Hill and K. R. Harrap, The effect of chlorambucil on the incorporation of tritiated precursors into the nucleic acids and proteins of Yoshida ascites sarcoma cells, Neoplasma, 17 (1970) 48-89. 8 C. R. Ball and J. J. Roberts, DNA repair after mustard gas alkylation by sensitive and resistant Yoshida sarcoma cells in vitro, Chem.-Ho/. Interact., 2 (1970) 321-329. 9 R. H. Golder, G. Martin-Guzman, J. Jones, N. 0. Goldstein, S. Rotenberg and R. J. Rutman, Properties of DNA from ascites cells treated in vivo with nitrogen mustard, Cancer Res., 24 (1964) 964-%8. 10 V. J. Strozier and W. L. Nyhan, Effects of cytoxan on the proteins of sensitive and resistant strains of the Ll210 leukaemia, Cancer Res., 22 (1962) 1332-1335. 11 H. Grunicke, K. W. Bock, H. Becher, V. Gang, J. Schnierda and B. Puschendorf, Effect of alkylating antitumour agents on the binding of DNA to protein, Cancer Res., 33 (1973) 1048-1053. 12 P. 6. Riches and K. R. Harrap, Some effects of chlorambucil on the chromatin of Yoshida ascites sarcoma c&s, Cancer Res., 33 (1973) 389-393. 13 B. T. Hill, M. Jarman and K. R. Harrap, Selectivity of action of alkylating agents and drug resistance, 4. Synthesis of tritium-labelled chlorambucil and a study of its cellular uptake by drugsensitive and drug-resistant strains of the Yoshida ascites sarcoma in vitro, J. Med. Chem., 14 (1971) 614-618. 14 K. R. Harrap and B. T. Hill, The selectivity of alkylating agents and drug resistance, Part II: A comparison of the effects of alkylating drugs on growth inhibition and cell size in sensitive and resistant strains of the Yoshida ascites sarcoma, &it. J. Cancer, 23 (1969) 227-234. 15 D. Rickwood, P. G. Riches arid A. J. MacGillivray. Studies of the in vitro phosphorylation of chromatin non-histone proteird in isolated nuclei, Biochim. Biophys. Acta, 299 (1973) 162-171. 16 A. 1. MacGillivray, A. Cameron, R. J Krauze, D, Rickwood and J. Paul, The non-histone proteins of chromatin, their isolation and composition in a number of tissues, Biochem. Biophys. Acta, 277 (1972) 384-402.
299 17 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folic phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
18 K. Burton, A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 19 J. H. Linford, The recovery of free chlorambucil from solution in blood serum, Biochem. Pharmucof., 11 (1962) 693-706. 20 T. Ghose and S. P. Nigam, Antibody as carrier of chlorambucil, Cancer, 29 (1972) 1398-1400. 21 W. J. Hopwood and J. A. Stock, The effect of macromolecules upon the rates of hydrolysis of .( aromatic nitrogen mustard, Chem.-Biol. Interact., 4 (1971/72) 31-39. 22 B. T. Hill and P. 0. Riches, The absorption, distribution and excretion of ~H-chlorambucil in rats bearing the Yoshida ascites sarcoma, Brit. J. Cancer, 25 (1971) 831-837. 23 D. Blakeslee and J. C. Kennedy, Factors effecting the non-covalent binding of chlorambucil to rabbit immunoglobulin G., Cancer Res., 34 (1974) 882-885. 24 M. S. C. Bitbeck, D. Robertson and B. T. Hill, An autoradiograpic study of the binding of 3Hchlorambucil to cells of a drug-sensitive and drug-resistant strain of the Yoshida ascites sarcoma, J. Microsc., (1975) in press.
291
THE BINDING THE YOSHIDA
OF
OF [sH]CHLORAMBUCIL ASCITES SARCOMA
TO NUCLEAR
PROTEINS
PAMELA G. RICHES AND K. R. HARRAP Department of Applied Biochemistry, Institute of Cancer Research, Sutton, Surrey SM2 5PX (Great Rritain)
(Received January dth, 1975) (Revision received March lOth, 1975) (Accepted March 14th, 1975)
SUMMARY
The binding of [sH]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of sH-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.
INTRODUCTION
The alkylating agents are a range of compounds, some of which are used in the clinical management of neoplastic disease. Their chemical and biological properties have been reviewed by Ross1 and more recently by Wheelers who also discussed the problem of cellular resistance to alkylating agentss. The proposals that the alkylating
agents cross-link strands of DNA*-6 and that resistance is attributable to enhanced excision and repair mechanisms in resistant cells, are not entirely compatable with experimental observations in the Yoshida ascites sarcoma, which has been investigated in this laboratory. The alkylating agent-sensitive form of this tumour does not show an inhibition of DNA synthesis as the earliest observed lesion following challenge with chlorambucil [4-p-[bis(2-chloroethyl)amino]-phenyl3-butyric acid]‘. Moreover, the drug-sensitive strain and a strain showing cross-resistance to alkylatingagcnts both excise alkylated bases from DNA and repair the resultant gaps with comparable facilitys. Other workers have suggested that alkylating agents modify the interaction between DNA and protein, thereby changing primer activityPsl0, while the association of nuclear protein with alkylated DNA has also been reportedllslo. We have previously described morphological and biochemical changes in the chromatin of sensitive Yoshida ascites sarcoma cells induced by chlorambucill”, and suggested that these may have occurred either from direct reaction of chlorambucil with nuclear proteins or from the effects of the drug on enzymes concerned with the functional organisation of nuclear proteins. In the present paper we describe investigations related to the first of these possibilities, and report the occurrence of chlorambucil bound covalently and non-covalently to nuclear proteins of tumour cells isolated from animals previously exposed to the drug. MATERIALS AND METHODS
Darvan No. 1 was obtained from R. J. Vanderbilt Co., Inc. (230 Park Avenue, New York 17). Acrylamide, N,N’,-methylenebis-acrylamide and N,N’,N’-tetramethylethylenediamine were purchased from BioRad Laboratories (27 Homesdale Road, Bromley, Kent, U.K.). Other chemicals were purchased from British Drug Houses Ltd. (Poole, Dorset, U.K.), Hopkin and Williams Ltd. (Chadwell Heath, Essex, U.K.) or May and Baker Ltd. (Dagenham, U.K.). Analytical grades were used where available. 3H-labelled chlorambucil, [3H]4-(4-bis(2 chlorethyl)aminophenyl-3,s) butyric acid, was synthesised at the Chester Beatty Research Institute, London, U.K.13. Both alkylating agent-sensitive and -resistant strains of the Yoshida ascites sarcoma were used in this work. Tumours were passaged at weekly intervals by inoculation of 2 - IO6 cells into 6-week-old Chester Beatty Colony bred female Wistar rats. Animals bearing the resistant line routinely received 2 mg/kg melphalan (ClCHtCH~)ZN.GH~CHNH~COOH) on the third day after tumour transplantation. Full details have been given previouslyll. [3H]chlorambucil (2.4 Ci/mmole) was administered to tumour-bearing animals on the fourth day following transplantation, at a dose of 8 mg/kg. The total radioactivity received by each animal was 12 mCi. Animals were killed, by cervical dislocation, 6 and 24 h after drug treatment and tumour cells aspirated from the peritoneal cavity with ice-cold isotonic saline. Nuclei were prepared from the pooled cells from ten animals by the method of Rickwood et all”.
293
Tris-saline soluble proteins were extracted from the nuclei by homogenisation in 50 vol. of 0.14 M NaCl, 0.05 M Tris-HCI (pH 7.4) as previously describedIG. The chromatin pellet was dissociated in 1 mM sodium phosphate (pH 6.8), 2 M NaCl, 5 M urea buffer and chromatographed on freshly prepared hydroxyapatite columns as described by MacGillivray et al. 16. The chromatin proteins were eluted in three fractions Hl, H2, H3 by stepwise increase in phosphate concentration. Hl proteins were eluted in 1 mM phosphate and represented histones; H2 and H3 were non-h&tone proteins eluted with 50 mM and 200 mM phosphate respectivelyl6. The pooled protein fractions were dialysed against 0.1% sodium dodecyl sulphate, lyophilised and dissolved in a solution containing 8 M urea, 1.Oo/, mercaptoethanol, 1.0% sodium dodecyl sulphate, 10 mM Tris-HCI pH 7.4, against which they were dialysed, and then redialysed against a similar solution in which the sodium dodecyl sulphate concentration had been adjusted to 0.1%. The protein fractions were characterised by electrophoresis in 15 % acrylamide-0.1 y0 sodium dodecyl su%hate gels by the method described previously 16.The gels were stained with 1% amido black and destained in ethanol-acetic acid and water (30:7:63 v/v/v) for 36 h using an HSI destainer (Hoefer Scientific Instruments, San Francisco). After scanning the stained gels at 540 nm, using a gel scanning attachment to a Hilger-Gilford Reaction Kinetics spectrophotometer, they were frozen in solid COs and cut into 1 mm slices using a Joyce-Loebel gel slicer. The gel slices were combusted in an Intertechnique Oxymat (Model JAlOl), which resulted in thecompleteoxidation of the gel slice. Tn the following automated sequence, the tritium has condensed to form a small volume of tritiated water to which was added 20 ml of scintillant (dioxan 3500 ml; toluene 1500 ml; naphthalene 100 g; butyl PBD 35 g). The resulting mixture was virtually unquenched and was counted in a /?-scintillation spectrometer (Tntertechnique SL40) with a counting efficiency of at least 48 %. A consistent tritium back-
TABLE
1
BINDING OF [~H~CHLORAMBIJCIL CHLORAMBUCIL
TO WHOLE CELLS,NUCLEI
[5.0jtmoles, 12 mCi] To TUMOUR-BEARING
AND CHROMATIN
AFTER ADMINISTRATION
ok
RATS
Figures are the average of two aliquots from the pooled tumuurs from ten animals counted in duplicate. The values in parentheses are the lower and upper values obtained. Cell line
Hours after treatment
cprn . lo-~ Total ceil binding/ nig protein
Sensitive Yoshida ascites Resistant Yoshida ascites
6 24 6 24
26.47 [23.56-28.75) 20.51 [18.11-20.991 12.02 [l l&5-12.38] 9.83 [ 9.67-10&l]
Total t&ear bindingjnlg proteirr
Total chromatin bindingjmg protein
22.37 [22.13-24.301 15.04 [13.47-16.101
35.49 [32.90-38.081 7.26 [ 6.3% 8.121
[ :::-
8.621
12.64 [12.36-12.911
7.38 [ 6.89-
7.631
4.24 [ 4.w
4.971
294 TABLE II [*~~HUXAMBIJ~ILSEPAR~EDBYD~LYSISAGAIN~T ON FQLYACRYLA~IILX
GEL ELECTROPH~RE~IS
Cell line
Sensitive Yoshida ascites sarcoma cells
Resistant Yoshida ascites sarcoma cells
Hours after treatment
0.1% ~~DIIJMDC)I)ECYLSULPHATE(D~~~ABLE)OR
(NON-COVALENTLY
BOUND)
EXPRESSED AS A PERCENTAGE
Fraction
YOTotal counts dialysable
YONon-dialysable counts noncovalently bound
6
Saline soluble Histone (Hl) NHP (H2) NHP (H3)
18.5 23.9 13.5 3.4
60.1 51.3 16.3 21.5
24
Saline soluble Histone (HI) NHP (H2) NHP (H3)
16.3 25.6 15.0 0.0
4.8 47.0 38.0 31.6
6
Saline soluble Histone (Hl) NHP (H2) NHP (H3)
35.4 16.5 18.8 10.8
31.3 18.4 17.7 8.4
24
Saline soluble Histone (Hl) NHP (H2) NHP (H3)
27.5 13.0 29.4 15.4
0.0 0.0 8.8 2.3
ground of 30 cpm was obtained when non-radioactive gel slices were similarly treated. This method allows an accurate determination of the low counts obtained in the present experiments. Protein was estimated by the method of Lowry et ~1.17;DNA was estimated by the method of Burtonls. RESULTS
Table I gives the values of [aH]chlorambucil associated with whole cells, nuclei and chromatin. All fractions from sensitive cells show higher binding both at 6 h and 24 h after treatment than those obtained from resistant cells. When total chromatin was fractionated on hydroxyapatite columns, the recovery of protein was in all cases 80-90 % of the total applied. However, the recovery of 3H-label in protein and nucleic acids was consistently less than the counts applied. At 24 h after treatment the recoveries of aH-label for sensitive and resistant cell chromatin were 42 % and 47.7 %, respectively; the values at 6 h after treatment were 21% and 20.9 %. The protein fractions collected from the column, HI (histone), H2 and H3 (non-histone proteins) were exhaustively dialysed and unbound chlorambucil would be lost at this stage. The extent of this loss is indicated in Table II, which also shows that a further loss of sH-label occurred following electrophoresis, staining and destaining. Presumably this material was bound non-covalently to the proteins and in Table II
295 this amount has been calculated from the difference between the W-label applied to the gel and its recovery as counts bound to protein. The presence of non-covalent binding was confirmed by running a gel for a short time and then slicing into 1 mm slices and determining the radioactivity associated with each slice. By this method labelfed material could be detected in the region of the dye-marker band (data not shown). Unstained gels were used for this experiment as non-covalently bound material, after electrophoresis, could be removed from the gel by the solvents necessary for destaining. The amount of chlorambucil covalently bound to protein was determined from the amo~t of sH-label associated with the el~trophoretically separated non&stone proteins (see Figs. 1 and 2), after staining and destaining, which would be expected to remove non-covalently bound drug. Approximately three times as much drug was covalently bound to the combined
al
CO
at
30 2.0 1.0 ~
bJ c: 0 x
df
GEL
SLICE
NUMBER
GEL
SLICE
NUMBER
GEL
SLICE
NUMBER
Fig. 1. Binding patterns of [sH]chlorambucil to non-histone chromatin (H2) proteins of alkylating agent-sensitive and -resistant Yoshida ascites sarcoma cells, 6 and 24 h after treatment of tumourbearing animals with ~hlorambu~il (8 mg/kg, 2.4 Ci~mmole). (a) sensitive 6 hr after t~tment; (b) sensitive 24 h after treatment; (c) resistant 6 h after treatment; (d) resistant 24 h after treatment. Slice 1 represents the top (high molecular weight end) of the gel. Fig. 2. Binding patterns of [sH]chlorambucil to non-histone chromatin (H3) proteins of alkylating agent-sensitive and -resistant Yoshida as&es sarcoma cells, 6 and 24 h after treatment of tumourbearing animals with chlorambucil (8 mg/kg, 2.4 Cijmmole). (a) sensitive 6 h after treatment; (b) sensitive 24 h after treatment; (c) resistant 6 h after treatment; (d) resistant 24 h after treatment. Slice I represents the top (high molecular weight end) of the gel. Fig. 3. Binding patterns of [sH]chlorambucil to the Tris-saline soluble nuclear Proteins of alkyiating agent-sensitive and -resistant Yoshida ascites sarcoma cells, 6 and 24 h after treatment of tumourbearing animals with chlorambucil (8 mg/kg, 2.4 Ci/mmole). (a) sensitive 6 h after treatment; (b) sensitive 24 h after treatment; (c) resistant 6 h after treatment; (d) resistant 24 h after treatment. Slice I represents the top (high molecular weight end) of the gel.
296 TABLE III SPECIFIC ACTIVITY OF [~H~CHLORAMBUCIL COVALENTLY IBOUND TO NUCLEAR PROTEINS
Cell line
Hours after treatment
Fraction [cpm - IO-3]/mg protein Saline soluble
Histone (HI)
Non-histone chromatin protein (H2)
Non-histone chromatin protein (H3)
Sensitive Yoshida ascites sarcomacells
6 24
13.96
3.29
10.28
4.64
27.64 10.79
15.71 6.90
Resistant Yoshida ascites sarcoma cells
6 24
2.61 3.40
2.70 3.66
8.23 2.97
9.45 2.69
fractions (saline soluble, HI, H2. H3) of sensitive cells compared with resistant cells and during the period of observation this was reduced to one half in sensitive cells and to one third in resistant cells. In both cell types more than two thirds of the total drug bound to the four protein fractions was associated with the combined non-histone proteins (H2 and H3) (Figs. 1 and 2). Less than 10% was associated with the histone (HI) fraction at 6 h, and by 24 h this had increased slightly. Notably, five times more drug was bound to the Tris-saline soluble fraction of sensitive cells compared with resistant cells at 6 h, and in sensitive cells this was reduced to two-thirds by 24 h (Fig. 3). Since sodium dodecyl sulphate polyacrylamide gels separate; proteins on a mo!ecular weight basis, inspection of Fig. 3 reveals that the binding in sensitive cells was associated with proteins in the high and intermediate molecular weight region of the gel and was virtually absent from this region in resistant cells. While the resolution of a one-dimensional electrophoresis system is limited, one stained band representing more than one protein species, comparison of the electrophoresis profiles of nuclear proteins from sensitive and resistant cells nevertheless reveal no differences. Within the limits of the method we cannot therefore detect differences in the composition of the fractions between the two cell strains before or after treatment. Table III gives the specific activity of the various fractions which have been calculated from the recovery of aH-label covalently bound after polyracylamide gel electrophoresis. The nucleic acids were not extensively studied in these experiments. They were, however, eluted from the hydroxyapatite column in 0.5 M Na-phosphate (pH 6.8), 5 M urea, 2 M NaCl, dialysed and then an estimate made of bound counts/DNA. The values of binding were in the order of 103 cpm/mg DNA. DISCUSSlOh;
The ability of chlorambucil to bind firmly to serum proteins without alkylating them has been previously demonstrated 19. Non-covalent binding of chlorambucil
297
to i~unoglobulins has also been describedze, while Hopwood and Stock have reported binding of alkylating agents to serum ablumin and fibrinogen with retention of alkylating function 21. We have previously reported binding of [aH]chlorambucil to DNA, RNA and protein following irrvivaadministration of high specific activity drug to turnour-daring animals ss. However, these observations need to be reconsidered in relation to the present data which indicate that considerable amounts of non-covalently bound drug are associated with nuclear components. In the present experiments extremes of pH or the use of polar solvents were avoided and an estimate of covalent binding was made by polyacrylamide gel electrophoretic separations, In both soluble nuclear sap and chromatin proteins the covalent and non-covalent binding was greater in sensitive than in resistant cells at corresponding times. Non-covalent binding was practically absent in the resistant cell by 24 h after treatment. The aH-label which was dialysable probably represents free chlorambucil, or a metabolite, since non-covalent binding has been reported to be stable to dialysis at 4” (ref. 23). The nature of the non-covalent binding to nuclear protein fractions has not been determined. It is possible that in the Tris-saline soluble fractions both ionic and hydrophobic binding were present. Any ionic binding to the chromatin proteins, howwever, would be expected to be removed during chromatography on hydroxyapatite. Thus, the non-covalent binding detected in these fractions is probably hydrophobic and was removed by the destaining procedure to which the polyacryiamide gels were subjected. The dialysis of hydrophobic molecules in detergent solutions is inefficient because the former would be expected to bind with high affinity to non-dialysable detergent micelles. The presence of counts in the region of the dye-marker front in unstained gels is consistent with this hypothesis. Covalent binding of drug was greater to non-histone chromatin proteins (H2 and H3 fractions) than to histones (Hl fraction). The nuclear sap proteins (saline soluble} were of particular interest: considerable binding occurred in the high molecular weight regions (Fig. 3, Slices l-10) of sensitive cell proteins, and was almost entirely absent from the resistant fractions at 6 h after treatment. Considerable levels of binding to this fraction from sensitive cells persisted similarly at 24 h after treatment. Nucleic acids were eluted from the hydroxyapatite columns and specific activities determined. Drug binding was of the order of 10s cpmlmg DNA. The calculated amount of bound chloramb~il, assuming ail the aH-label was associated with the drug, was 0.5 pmoles/mg DNA. In previous in vivaexperiments this vahte of bound chlorambucil was 30 pmoles/mg DNA when DNA was acid extracted The molar binding of chlorambucil to the saline-soluble nuclear proteins at 6 h after treatment was of the order of 17.5 pmoleslmg protein; the corresponding covalent binding to this fraction at the same time was 7.0 pmoles~mg protein. This also represents considerably greater levels of drug than that associated with the DNA The ratio of total cell binding 6 h after treatment in sensitive cells to that in resistant cells is approximately 2.0. However, when the nuclear proteins were considered the ratio (i.e. sensitive: resistant) was considerably greater in those groups (i.e.
Tris-saline soluble and non-histone chromatin protein) where binding was detected. It is, therefore, possible that the differences in total cell binding can be attributed to this high nuclear protein binding. This would confirm previous results24 where an autoradiographic study of Yoshida ascites cells treated with chlorambucil showed no significant difference between sensitive and resistant cells in cytoplasmic grain counts but significantly higher grain counts in the sensitive cell nucleus when compared with the resistant cell nucleus. The significance of high nuclear protein binding of chlorambucil in Yoshida ascites sarcoma cells remains to be assessed. The present experiments, however, indicate important differences in binding to protein from sensitive cells compared with resistant cells when high specific activity drug was employed at a pharmacologically effective dose.
REFERENCES 1 W. C. J. Ross, Biological AIkyIating Agents, Butterworth, London, 1962, pp. 3-87. 2 G. P. Wheeler, Alkylating agents, in J. F. Holland and E. Frei (Eds.), Cancer Medicine, Lea and Febiger, Philadelphia, 1973, pp. 791-806. 3 G. P. Wheeler, Studies related to mechanisms of resistance to biological alklyating agents, Cancer Res., 23 (1963) 1334-l 349. 4 P. Brookes and P. D. Lawley, Alkylating agent, Brit. Med. Bull., 20 (1964) 91-95. 5 K. W. Kohn, C. L. Spears and P. Doty, Jnterstrand crosslinking of DNA by nitrogen mustrad, J. Biol. Chem., 19 (1966) 266288. 6 J. 1. Roberts, 7. P. Brent and A. R. Crathorn, The mechanism of the cytotoxic action of alkylating agents on mammalian cells, in P. N. Campbell (Ed.), Symposium of the Interaction of Drugs and Subcellular Components in Animal Cells, Churchill, London, 1968, pp. 5-27. 7 B. T. Hill and K. R. Harrap, The effect of chlorambucil on the incorporation of tritiated precursors into the nucleic acids and proteins of Yoshida ascites sarcoma cells, Neoplasma, 17 (1970) 48-89. 8 C. R. Ball and J. J. Roberts, DNA repair after mustard gas alkylation by sensitive and resistant Yoshida sarcoma cells in vitro, Chem.-Ho/. Interact., 2 (1970) 321-329. 9 R. H. Golder, G. Martin-Guzman, J. Jones, N. 0. Goldstein, S. Rotenberg and R. J. Rutman, Properties of DNA from ascites cells treated in vivo with nitrogen mustard, Cancer Res., 24 (1964) 964-%8. 10 V. J. Strozier and W. L. Nyhan, Effects of cytoxan on the proteins of sensitive and resistant strains of the Ll210 leukaemia, Cancer Res., 22 (1962) 1332-1335. 11 H. Grunicke, K. W. Bock, H. Becher, V. Gang, J. Schnierda and B. Puschendorf, Effect of alkylating antitumour agents on the binding of DNA to protein, Cancer Res., 33 (1973) 1048-1053. 12 P. 6. Riches and K. R. Harrap, Some effects of chlorambucil on the chromatin of Yoshida ascites sarcoma c&s, Cancer Res., 33 (1973) 389-393. 13 B. T. Hill, M. Jarman and K. R. Harrap, Selectivity of action of alkylating agents and drug resistance, 4. Synthesis of tritium-labelled chlorambucil and a study of its cellular uptake by drugsensitive and drug-resistant strains of the Yoshida ascites sarcoma in vitro, J. Med. Chem., 14 (1971) 614-618. 14 K. R. Harrap and B. T. Hill, The selectivity of alkylating agents and drug resistance, Part II: A comparison of the effects of alkylating drugs on growth inhibition and cell size in sensitive and resistant strains of the Yoshida ascites sarcoma, &it. J. Cancer, 23 (1969) 227-234. 15 D. Rickwood, P. G. Riches arid A. J. MacGillivray. Studies of the in vitro phosphorylation of chromatin non-histone proteird in isolated nuclei, Biochim. Biophys. Acta, 299 (1973) 162-171. 16 A. 1. MacGillivray, A. Cameron, R. J Krauze, D, Rickwood and J. Paul, The non-histone proteins of chromatin, their isolation and composition in a number of tissues, Biochem. Biophys. Acta, 277 (1972) 384-402.
299 17 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folic phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
18 K. Burton, A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 19 J. H. Linford, The recovery of free chlorambucil from solution in blood serum, Biochem. Pharmucof., 11 (1962) 693-706. 20 T. Ghose and S. P. Nigam, Antibody as carrier of chlorambucil, Cancer, 29 (1972) 1398-1400. 21 W. J. Hopwood and J. A. Stock, The effect of macromolecules upon the rates of hydrolysis of .( aromatic nitrogen mustard, Chem.-Biol. Interact., 4 (1971/72) 31-39. 22 B. T. Hill and P. 0. Riches, The absorption, distribution and excretion of ~H-chlorambucil in rats bearing the Yoshida ascites sarcoma, Brit. J. Cancer, 25 (1971) 831-837. 23 D. Blakeslee and J. C. Kennedy, Factors effecting the non-covalent binding of chlorambucil to rabbit immunoglobulin G., Cancer Res., 34 (1974) 882-885. 24 M. S. C. Bitbeck, D. Robertson and B. T. Hill, An autoradiograpic study of the binding of 3Hchlorambucil to cells of a drug-sensitive and drug-resistant strain of the Yoshida ascites sarcoma, J. Microsc., (1975) in press.