- Email: [email protected]
The effects of alkylating agents and DNAase on nucleic acid synthesis in rat-liver nuclei in vitro
THE EFFECTS OF ALKYLATING ACID SYNTHESIS IN RAT-LIVER
AGENTS AND DNAASE ON NUCLEIC NUCLEI IN VITRO
A. k PETERSONAND B. W. FOX m
&&omrorlu, Chris& Hospiral and Ho0 Radium Insrilute, Manchester (Great Brikzin)
(lteccjved Juwary
lath, 1970)
SUMMARY
Adult female rat-liver nuclei have been treated with nitrogen mustard (HN,), methylene dimethane sulphonate (MDMS) and bovine pancreatic DNAase I. DNA synthesis and RNA synthesis in this system have been assayed using the methods of labelled lTP and UTP uptake. The results indicate that low concentrations of MDMS and HN2 stimulate RNA synthesis and that this effect may be simulated by treatment with low levels of DNAase I. However, a similar parallelism between the actions of higher concentrations of MDMS and DNAase does not appear to exist since at such concentrations MDMS does not affect DNA synthesis whereas DNAase stimulates DNA synthesis. At these levels, however, both MDMS and DNAaseinhibit RNA synthesis. It is suggested that low levels of MDMS and HN2 affect RNA synthesis in rat-liver nuclei by inducing breakage of the DNA template, whereas higher levels of these agents could inhibit RNA synthesis in the nuclei by a different mechanism.
INTRODUCTION
Alkylation of DNA in vitro has been shown Is2 to alter its capacity to function as a template for nucleic acid synthesis in cell-free systems. Experiments in which xbbreviation;: ATP, adenosine triphosphate; CTP, cytidine triphosphate; dATP. deoxyadenosine triphosphate; dCTP, deoxycytidine triphosphate; DNAase, bovine pancreatic deoxyribonuclease I; IGTP, daoxy6uanosino triphosphate; dNTP, deoxynucleoside triphosphate; dNTP solution, 0.2 M Tris pH 8.0,0,04 M M6C12, 0.002 M mercaptoethanol containinp 0.18 pmole each of dATP, dGTP, dCTP, ‘ITP and 0.1 @!i [l*CJTTP (Schwartz Bioresearch Inc., New York, U.S.A.); GTP, guano&e triphosphate; HN2, nltroyen mustard, 2,2’-dichloro-IV-methyldiethylamine hydrochloride; MDMS, mathylano dlmethano tulphonato; NTP, nucleoside triphosphate; NTP solution, 0.2 M Tris, pH 8.0. 0.M M Mt~CI~,0.003M morcaptoethanol containing09 pmoleseachofATP, GTP, CTPand0.09 @noles UTP containing 5 j&lS[aH]UTP (0.05 mCi/mM, Radiochemical Centre, Amersham, Bucks. Great Britain); PCA, perehlodc acid; POPOP, l,~~~~~pheoyloxazolyl-2)-benzene; PPO, SS-diphenyioxazole; TCA, trichloroacotlc acid: TMMS suspension, 0.13 M Tris, pH 8.0, 0.007 M Me% 0.0013 M j%nerca#Mhano~, 0.2s M sucrose.; Th& suspension, 0.0s M Tris. pH 7.6, 0.003 M M&12, Ot5 M sucrose; ‘ITP, dooxythymidine triphosphate; UTP. uridine triphosphate. Chem.-Bid. Interactions, 2 (1970) I-8
&R-,B.W.FOX
2
fn vimwith sdkyhhg agentsare eomplkxued by systen& et&e& of the agents or secondary effects on odiufar components other thau DNA3. By the means of nudear transplantation expe&ents, ORD AMY DANIEL&~wen able to obtain toxicity data for the &cts of HNI i&pcndantly on the nucicus and cytopkun of Amorboptw. lb survival curve for the intact organism matched most closely that for the treated rr&e~ strong& supporting the hypothesis6 that c&It& DNA is a primary target for the cytotoxic action. Isolation tzcltniqucsfor rat4iver nuclei bawl ban &crib& which allow ti to continue to undergo RNA ‘*$ and DNA9 synthesis as well as to respond with increased RNA synthesis to hormonal stimulation lo. The aim of this work is to compare the effects of HN,, MDMS and DNAaseI on nucleic acid synthesis in isolated rat-liver nuclei.
whole a~~imalsor ceilsare m&cd
MA,rEIuAu
AND METHODS
Drugs or;d enxynws uwdfw
treatnmt af nudei
HNI was obtained from Roots Pure Drug Co. Ltd., (Mustine hydr~~o~de B.P.) and MDMS was prepared as previously reported”. DNAase I (Koch-fight) was 2000 Kunitz units/mg (batch No. 30874). Nuclei were isohued From the livers of female Wiser albino rats (M-18 weeks old, 190-210 g) by the method of WIDNELLAND TATA’. The isolated nuclei were suspended in TMS,treated and assayed for UTP and TTP uptake on the same day. Aliquots (0.5 ml) of TMS suspension containing l-2*10s nuclei (RNA-synthesis assay) or 2-3*10’ nuclei (DNA-synthesis assay) were treated in IO-mlconical antrifuge tubes with afiquots (0.5 mi) of alkylating agent or enzyme solution in TMS (preparecl immediately hefore use). The tuhes were incubated for 30 mitt at 37q cooled in ice and centrifuged (1200 g) for IO min at So. The supernatants were discarded and the pellets were washed twice for RNA-synthesis assay and once for DNA-synthesis assay, with aliquots Q ml) of coid TMS, For RNA~th~is assays, the washed treated nucki were muspHed in TMS to a volume of 0.5 ml from which aliquots were taken as required. For DNAsynthesis assays the nuclei were resuspended in TMMS to a volume of 150 ~1 and assayed in the same tubes in which they were treated. The method was mod&d from that of WES@: To 150,ul of prewarmed nuclei suspension in TMS containing 2-3*10’ nuclei, was added 150 /tl M1p solution. The reaction mixture was incubated at 37” in a water bath and samples (50 ~1) were withdrawn with mierosyringes at 30 set and various intervals thereafter. ‘Ihe samphs wererapid@transfd to IO-mlcentrifuge tubes containing ahquots (5 ml) of icb cold 10% TCA and 0.01 M sodium pyrophosphatc.Blankswere obtainedby mixing the ingrediits at 0’ and rapidly taking 5 samples into icaoobd TCA-pyrophosphate Ckem.-B&I. fntaocrlorr, 2 (1970) 1-S
NUCtElC ACID syNTHE3IS lb! RAT-LIVER NUCLEl in VitrO
3
solution. After standing for 12 h in iced water, the precipitites were collected by centrifugation, resuspended in 0.2 hi Tris buffer, pH 8.0 (1 ml), containing 20 .~l of 9 N NaOH and 0.01 M sodium pyrophosphate (Tris-NaOH) and reprecipitated by treating with 10% TCA-pyrophosphate solution (2 ml) for 3 h at 0”. This precipitate was collected on glass-fibre discs (Whatman, GF/C) which were washed three times with aliquots (5 ml) of cold 5 “/oTCA-pyrophosphate solution, once with cold 5% TCA (5 ml), once with cold ether (5 ml), dried under infrared lamps and counted in toluene-based scintillant mixture (8 g PPG), 0.2 g POPOP in 2 1 toluene, with a counting efficiency of 10% (ref. 13). The effects of actinomycin D on the uptake of [3H]UTP were checked by including the inhibitor (5.0 p&f) in the assay incubation mixture. In order to check the sensitivity to alkali, the cold TCA-insoluble radioactive product was collected by centrifugation, reprecipitated from Tris-NaOH and washed three times with cold TCA (5%). once with cold ethanol, and once with cold ether. The precipitate was then extracted with NaOH (2 ml, 0.3 N) at 37” for 30 min, reprecipitated with cold TCA (lo%, 4.0 ml), collected by centrifugation and extracted three times with PCA (5 %, 2.0 ml) at 75” for 10 min. The radioactivities in the cold TCA supematant and the hot PCA extract were counted in Triton X phosphors (13:7) and (3:l) with efficiencies for tritium of 20% and 13 % respectively14. DNA-synthesis assay DNA synthesis was measured using a method derived from THOMPSON AND MCCARTHY~. To 150 ,~l nuclei suspension in TMMS containing 2-3-10’ nuclei, was added 150 ~1 dNTP solution. The reaction mixture was incubated at 37” for 60 min and the reaction was terminated by the addition of ice-cold 10% TCA-pyrophosphate solution (5 ml). Blanks were obtained by treating the nuclei with the cold TCA before adding dNTP solution. Following 12 h at 0”, the precipitates were harvested by centrifugation, redissolved in 0.3 N sodium hydroxide (1 ml) containing 0.01 M sodium pyrophosphate and reprecipitated by treating with 10% TCA-pyrophosphate (5 ml). The precipitates were collected by centrifugation, washed twice with 5% TCA, once with 0.5 N PCA and extracted three times with aliquots (2 ml) of 0.5 N PCA at 75” for 10 min. The hot PCA extracts of each sample were pooled, adjusted to 6.0 ml with 0.5 N PCA and 4.0-ml aliquots were added to 6.0 ml of Triton X-100 phosphor (1:l) and assayed for 14C content with an efficiency of 78 % (ref. 14). Chemtcaf assays on nuclei
Nuclei were examined and counted under phase-contrast microscopy. Protein was assayed by the method of LOWRY et al. Is. Nucleic acids were extracted by a modified SCHMIDT-THANHAUSER procedurel’j and assayed by the methods of BURTON” (DNA) and C~tuo’r’W’(RNA).
Ctim.-&of. Inrerucric7ns. 2 (t970) 1%
4
[email protected]
RESULTS
Fllaswontlast examination of tk n&G IBlspm&n shoti that the isolated nuclei are relatively free of cytoplasm. The protciuDNA ratio of the uucki was 3.10fO.23andthe RNA:DNA~owrs0.27f~030.~~oncom~ with those described by other workers7*‘*“.
RNA synthesis [3H]UTp was inhibited by 73 % in the present of actiuomycin D which demonstrates that the uptake is due to the action of a DNAdepeudent polymerase20. Since at least 93% of the radioactive product was aeuaitive to a&&, it is ckat that RNA synthesis mediated by DNAdepeudent RNA polymcrase (EC 2.7.7.7) was being measured in this system.
EJects on RNA synthesis Fig. 1 shows the effects of a ties of treatment levels of HN2 on [3H]UTP uptake into RNA of rat-liver nuclei, and it cau be seen that low levels of HN2 (approximately lo-* mM) appear to stimulate RNA syuthesis but at 10-l mM, almost complete inhibition occurs. The action of MDMS at two different concentrations is shown in Fig. 2. This agent exhibits a consistent bisphasic response, but at low Ieve?*-;L6*10’5 mM), clear ev&oce of the enhancement of RNA synthesis was obtained maximal after approximately 5 min of incubation.
Fig. 1. The e!kcts of HN1 at d&rent concentMoar on RNA ryntbals. RNA ayntheab brine 20 min rltcr trea(auntwith HN2. 8.6IO-‘ n&f, 0-O (I Expt.); 8.640-*mM, A(1 E!Jtpq; 1.3*10-’ In&f, xas ColWol, 0 l-~~-~.(6Expt4,Lu~npared, Fii 2. ?hc ttlcct of MDMS at difkrent COllCCntntjOnaon RNA ayntJWs RNA syathcaia during aalbtafter m with MDMS. 8.64W’ RIM, x -x (2 Expts.), 13404 IBM, o---o (2~~)mdwithTMSrrcontrol,O------.(6~k)&compuod. Ckcm.-B&i. Inmutlonr, 2 (1970) 1%
NUCLEIC ACID SYNTHEUS
IN RAT-LIVER NUCLJ3lin
vitro
5
Fig. 3. The effects of DNAase 1 at different concentrations on RNA synthesis. RNA synthesis during 20 min after treatment with DNAase. 7.6~10~ pg/lO’ nuclei, 0 -0; 7.6W3 )rg/lO’ nuc!ei, X-X ; 7.6*1Wz pg/lO’ nuclei, A -A; and 1.9 &lo’ nuclei, c]13 and with TMS as control, 0 - - - - - -0 is compared. Fig. 4. The effect of DNAase 1 at different concentrations, on RNA and DNA synthesis. RNA synthesis during IO min after treatment (0 -O), and DNA synthesis during 60 min after treatment (M), are compared. (Standard errors, control RNA synthesis, A; control DNA synthesis, B.)
One of the consequences of aikylation of DNA of mammalian cells is considered to be single-strand breakage 21. One reason for the apparent stimulation of [3H]UTP uptake observed at low concentrations of HN, and MDMS may thus be due to an increased availability of template DNA. In order to test this, nuclei were treated with various concentrations of DNAase I. Fig. 3 demonstrates the action of DNAase I at different concentrations, on [“HJUTP uptake. With this enzyme, it can be clearly seen that a marked stimulation of RNA synthesis occurs between 7.6*1O’4 mg/ml (10’ nuclei) and 7.6-10” &ml (10’ nuclei), but at higher concentrations (1.9 rglml), inhibition occurs. Using 0 g0 DNA, it has been shownZ2 that DNAase at approximately 10-j &ml concentrations will stimulate RNA synthesis under in vitroconditions. It is interesting to note that the concentration that produced maximal stimulation of RNA synthesis in the nuclear system was within the same order of magnitude. Fig. 4 shows that the uptake of (14C)TTP is unaEected by concentrations of DNAasc I which produced stimulation of RNA synthesis: but a significant increase of [14C]‘MP uptake occurs, maximal at 1.0 pg/lO’ nuclei. MDMS had no significant effect on [14C)‘lTP uptake at any of the concentratioas tested (Table I).
Contxmuion MDh4s(aM) 3.3-m-3 3.3-10-3 3.3-10-l 6.6*10-’
d
[‘*qmP pmwtage Ironm?l~
agmke a8 qfaolnsd = 9.69YJ
!;j.SiM.S 1I4.0*16.0 n::i.oi 4.0 96.75 l0.25
DISCUSSION
It has been shown that low levels of alkylating agents cause sin@&and breaks in the DNA of treated mammalian cslls’L*~’ and bacteriaas. DNAase I produces double- and single-stmnd breaksa* and stimulates RNA synthesis under in vitro conditionsz2. We have now shown that in isolated nuclei this enzyine will also stimulate RNA synthesis (Figs. 3 and 4). The obe#rvations(Rigs. 1 and 2) that Iow levels of MDMS and HNI stimulate RNA synthesis in this system, are consistent with the observed e&c& of sulphur mustard on E. u& nucleic acid synthesis2s and may be similar to the stimulatoryeffects of dibromodulcitol, a powerfir antitumour agent, on RNA synthesisin sensitive Yoshida sarcoma cells In v&ors. The stimulationof RNA synthesis producedby low levels of DNAase may have resulted from low level breakage of DNA, leading to an increased availability of template for polymerase enzymes22. However, DNA synthesis is not stimulated under such conditions but only after treatment with higher concentrations of DNAase, which may indicate that if there is only a limited amount of template made available, RNA synthesis would be preferentiallystimulated in a manner consistent with the observationsreported by Beno et ul.“. The stimulatoryef&ctsoflow levels of DNAase and these alkyhrtingagents on RNA synthesis in rat-liver nuclei may indicate a common mechanism of action. It is interesting to observe that DNAase also erchibitaantitumour and antimitotic etlixts2s-3e. However, there is no similarity between the e&cts of hi@r levois of DNAase and MDMS on DNA synthesis (Table I), sugSe#ing that inhibition of RNA synthesis observed at high levels of the drug may not relate to its ability to damage DNA, but to o&ts on other sites, e.g. enzymes or membrane transport systemsl,~~4.3’, Tha amount of a!kyRting agent actually respot&ble for its cytotoxic action is generally assumed to be only a small frection of the total administereddose. This would imply that if any of the observed offbcts of these agents in this system arc relat#ltothcircytatoxiF~nismsafIzction,~thbysrsmcMtlilrelytobs observed at low concentrations of drug. It has been 2a that “unbalanced RNAmctaboliem”~ybecsmlatbdwith” ofviabiMydwtounbalance1 gMwthn in bacteria.
NUCLEIC AClD SYNTHESI8 M RAT-LD’BRNUCLD in vitro
7
A similarmetabolic inbalance in a mammaliancell resultingfrom a stimulation of RNA synthesis, could thus be indirectly due to the production of breaks in the nuclear DNA. AcKNowLEDGEMENx3 We thank the British Empire Campaignfor Cancer Researchand The Medical ResearchCouncil for financial support.
REFERENCES R. W. RUDWN AND J. M. JOHNSON, The effects of nitrogen mustard on template activity in puri8ed DNA and RNA polymerase systems, Mol. Phurmucol.,4 (1968) 258-273. W. TROLL, E. RINDLB ANDP. DAY,Eifect on N-7 and C-8 substitution of guanine in DNA on T,,,, buoyant density and RNA polymerasc priming,Biuchim. Biopbys. Acta, 174 (1969) 21I-219. G. P. WHEELER AND J. A. ALEXANDER, Effects of nitrogen mustard and cyclopbosphamide upon the synthesis of DNA in vlvu and in cell free preparations, Cancer Res., 29 (1969) 98-109. M. J. ORDANDJ. F. DANIELW, The site of damage in amoeba exposed to lethal concentrations of methyldi-@-chlorethyB-amine, a “nitrogen mustard”, Qlsorz.J. Microscop. Sci., 97 (1956) 17-28. 5 M. J. ORD, The site of damage in amoeba exposed to low concentrations of caethyldi+ chloroethyl)-amine, a “nitrogen mustard”, Quaff. J. Microscop. Sci., 97 (1956) 39-45. 6 M. OCHOAMD E. Hrasoues~a, Alkylating agents, Exprl. Chemother., 5 (1967) 1-132. 7 C C. W~DNBLL ANDJ. N. TATA, A procedure for the isolation of enzymatically active rat liver nuclei, Bfochem. J.. 92 (1964) 3 13-3 17.
8 A. I. MarsuntAND B. E. TROPP, Studies on r&nucleic acid synthesis in nuclei isolated from rat liver, Bloclum. Biophys. Ada. 174 (1969) 476-490. 9 L. R. THOMPSON AND B. J. MCCARYW, Stimulation of nuclear DNA and RNA synthesis by cytoplasmic extracts in vitro, B&hem. Biophys. Res. Commun., 30 (1968) 166-172. 10 D. GALLI~ITZ AND C. E. Serems, Stimulation of RNA polymerase activity of rat liver nuclei by cortisol In vi&o independent of effects on the acetylation and methylation of histones, FE5S Lerters, 3 (1965) 99-102. II W. D. EVMONS AND A. F. Fmrutrs,Metathetical reactions of silver salts io solution, II. Tk synthesis of alkyl sulpbonates, J. Am. Chem. Sot., 75 (1953) 2257. 12 S. B. Wetsa, Enzymatic incorporation of ribonucleoside triphosphates into the interpolyn~ tide linkages of r&nucleic acid, plot. Narl. Acad. Sci. (U.S.), 46 (1960) 102t&1030. 13 D. M. GILL,Liquid scintillation counting of tritiated compounds supported by solid fUtess, Inter& J. Appl. Ra&arionIsotopes, 18 (1957) 393-398. 14 B. W. Fox, Tbo application of Triton X-100 colloid sciotiilation counting in bioche&try, Inten. 1. Appl. RodiatioonIsotopes, 19 (1968) 717-730. 15 0. H. LOWRY, W. J. R~SENBROUOH, A. L. FARRAND R. J. RANDALL, Protein measurementwith the Folin phenol reagent, J, Bid. Chem., 193 (1951) 265-275. 16 H. N. MUNROAND A. FLITCK.The determination of nucleic acids, MerhodrB&em. Alal., 14 (1966) 113-176. 17 K. BUILIIXV, A study of the conditions and mechanism of the diphenylamine reactioo for the colourimetrlc estimation of deoxyribonucleic acid, 5iochem. J., 62 (1956) 318-323. 18 0. CBU~, Determ&ation of oucleie acids in animal tissues, 1. Bid. Chem., 214 (1955) 59-M. The deoxyribonucleic acid content of the nucleus, in E. CHARAND J. N. 19 R. V~NDRELY, DA(Eds.), The Nuctelc A&&, Vol. 2, Academic Prees, New York, 1965, pp. 155-180. 28 J, Hunwttz AND1. T. Auovsr, The role of DNA in RNA synthesis, Prow. Nmdkk AcLd Res., 1 (1%3) 82-84. 21 8. W. Fox ANDM. Fox, Sensitivity of the newly synthesised and template DNA of l,arpbomr cells to dama8e by methyl metbanesulphonate, and the nature of &ted -pair” m MuratIon Res., 8 (1969) 629-638. Chemd&d. InteracHons, 2 (1970) l-8
8
Ch~m.=Biol.lwractlons, 2 (1970) l-4
aRm?mmmtu,RW.mx
AGENTS AND DNAASE ON NUCLEIC NUCLEI IN VITRO
A. k PETERSONAND B. W. FOX m
&&omrorlu, Chris& Hospiral and Ho0 Radium Insrilute, Manchester (Great Brikzin)
(lteccjved Juwary
lath, 1970)
SUMMARY
Adult female rat-liver nuclei have been treated with nitrogen mustard (HN,), methylene dimethane sulphonate (MDMS) and bovine pancreatic DNAase I. DNA synthesis and RNA synthesis in this system have been assayed using the methods of labelled lTP and UTP uptake. The results indicate that low concentrations of MDMS and HN2 stimulate RNA synthesis and that this effect may be simulated by treatment with low levels of DNAase I. However, a similar parallelism between the actions of higher concentrations of MDMS and DNAase does not appear to exist since at such concentrations MDMS does not affect DNA synthesis whereas DNAase stimulates DNA synthesis. At these levels, however, both MDMS and DNAaseinhibit RNA synthesis. It is suggested that low levels of MDMS and HN2 affect RNA synthesis in rat-liver nuclei by inducing breakage of the DNA template, whereas higher levels of these agents could inhibit RNA synthesis in the nuclei by a different mechanism.
INTRODUCTION
Alkylation of DNA in vitro has been shown Is2 to alter its capacity to function as a template for nucleic acid synthesis in cell-free systems. Experiments in which xbbreviation;: ATP, adenosine triphosphate; CTP, cytidine triphosphate; dATP. deoxyadenosine triphosphate; dCTP, deoxycytidine triphosphate; DNAase, bovine pancreatic deoxyribonuclease I; IGTP, daoxy6uanosino triphosphate; dNTP, deoxynucleoside triphosphate; dNTP solution, 0.2 M Tris pH 8.0,0,04 M M6C12, 0.002 M mercaptoethanol containinp 0.18 pmole each of dATP, dGTP, dCTP, ‘ITP and 0.1 @!i [l*CJTTP (Schwartz Bioresearch Inc., New York, U.S.A.); GTP, guano&e triphosphate; HN2, nltroyen mustard, 2,2’-dichloro-IV-methyldiethylamine hydrochloride; MDMS, mathylano dlmethano tulphonato; NTP, nucleoside triphosphate; NTP solution, 0.2 M Tris, pH 8.0. 0.M M Mt~CI~,0.003M morcaptoethanol containing09 pmoleseachofATP, GTP, CTPand0.09 @noles UTP containing 5 j&lS[aH]UTP (0.05 mCi/mM, Radiochemical Centre, Amersham, Bucks. Great Britain); PCA, perehlodc acid; POPOP, l,~~~~~pheoyloxazolyl-2)-benzene; PPO, SS-diphenyioxazole; TCA, trichloroacotlc acid: TMMS suspension, 0.13 M Tris, pH 8.0, 0.007 M Me% 0.0013 M j%nerca#Mhano~, 0.2s M sucrose.; Th& suspension, 0.0s M Tris. pH 7.6, 0.003 M M&12, Ot5 M sucrose; ‘ITP, dooxythymidine triphosphate; UTP. uridine triphosphate. Chem.-Bid. Interactions, 2 (1970) I-8
&R-,B.W.FOX
2
fn vimwith sdkyhhg agentsare eomplkxued by systen& et&e& of the agents or secondary effects on odiufar components other thau DNA3. By the means of nudear transplantation expe&ents, ORD AMY DANIEL&~wen able to obtain toxicity data for the &cts of HNI i&pcndantly on the nucicus and cytopkun of Amorboptw. lb survival curve for the intact organism matched most closely that for the treated rr&e~ strong& supporting the hypothesis6 that c&It& DNA is a primary target for the cytotoxic action. Isolation tzcltniqucsfor rat4iver nuclei bawl ban &crib& which allow ti to continue to undergo RNA ‘*$ and DNA9 synthesis as well as to respond with increased RNA synthesis to hormonal stimulation lo. The aim of this work is to compare the effects of HN,, MDMS and DNAaseI on nucleic acid synthesis in isolated rat-liver nuclei.
whole a~~imalsor ceilsare m&cd
MA,rEIuAu
AND METHODS
Drugs or;d enxynws uwdfw
treatnmt af nudei
HNI was obtained from Roots Pure Drug Co. Ltd., (Mustine hydr~~o~de B.P.) and MDMS was prepared as previously reported”. DNAase I (Koch-fight) was 2000 Kunitz units/mg (batch No. 30874). Nuclei were isohued From the livers of female Wiser albino rats (M-18 weeks old, 190-210 g) by the method of WIDNELLAND TATA’. The isolated nuclei were suspended in TMS,treated and assayed for UTP and TTP uptake on the same day. Aliquots (0.5 ml) of TMS suspension containing l-2*10s nuclei (RNA-synthesis assay) or 2-3*10’ nuclei (DNA-synthesis assay) were treated in IO-mlconical antrifuge tubes with afiquots (0.5 mi) of alkylating agent or enzyme solution in TMS (preparecl immediately hefore use). The tuhes were incubated for 30 mitt at 37q cooled in ice and centrifuged (1200 g) for IO min at So. The supernatants were discarded and the pellets were washed twice for RNA-synthesis assay and once for DNA-synthesis assay, with aliquots Q ml) of coid TMS, For RNA~th~is assays, the washed treated nucki were muspHed in TMS to a volume of 0.5 ml from which aliquots were taken as required. For DNAsynthesis assays the nuclei were resuspended in TMMS to a volume of 150 ~1 and assayed in the same tubes in which they were treated. The method was mod&d from that of WES@: To 150,ul of prewarmed nuclei suspension in TMS containing 2-3*10’ nuclei, was added 150 /tl M1p solution. The reaction mixture was incubated at 37” in a water bath and samples (50 ~1) were withdrawn with mierosyringes at 30 set and various intervals thereafter. ‘Ihe samphs wererapid@transfd to IO-mlcentrifuge tubes containing ahquots (5 ml) of icb cold 10% TCA and 0.01 M sodium pyrophosphatc.Blankswere obtainedby mixing the ingrediits at 0’ and rapidly taking 5 samples into icaoobd TCA-pyrophosphate Ckem.-B&I. fntaocrlorr, 2 (1970) 1-S
NUCtElC ACID syNTHE3IS lb! RAT-LIVER NUCLEl in VitrO
3
solution. After standing for 12 h in iced water, the precipitites were collected by centrifugation, resuspended in 0.2 hi Tris buffer, pH 8.0 (1 ml), containing 20 .~l of 9 N NaOH and 0.01 M sodium pyrophosphate (Tris-NaOH) and reprecipitated by treating with 10% TCA-pyrophosphate solution (2 ml) for 3 h at 0”. This precipitate was collected on glass-fibre discs (Whatman, GF/C) which were washed three times with aliquots (5 ml) of cold 5 “/oTCA-pyrophosphate solution, once with cold 5% TCA (5 ml), once with cold ether (5 ml), dried under infrared lamps and counted in toluene-based scintillant mixture (8 g PPG), 0.2 g POPOP in 2 1 toluene, with a counting efficiency of 10% (ref. 13). The effects of actinomycin D on the uptake of [3H]UTP were checked by including the inhibitor (5.0 p&f) in the assay incubation mixture. In order to check the sensitivity to alkali, the cold TCA-insoluble radioactive product was collected by centrifugation, reprecipitated from Tris-NaOH and washed three times with cold TCA (5%). once with cold ethanol, and once with cold ether. The precipitate was then extracted with NaOH (2 ml, 0.3 N) at 37” for 30 min, reprecipitated with cold TCA (lo%, 4.0 ml), collected by centrifugation and extracted three times with PCA (5 %, 2.0 ml) at 75” for 10 min. The radioactivities in the cold TCA supematant and the hot PCA extract were counted in Triton X phosphors (13:7) and (3:l) with efficiencies for tritium of 20% and 13 % respectively14. DNA-synthesis assay DNA synthesis was measured using a method derived from THOMPSON AND MCCARTHY~. To 150 ,~l nuclei suspension in TMMS containing 2-3-10’ nuclei, was added 150 ~1 dNTP solution. The reaction mixture was incubated at 37” for 60 min and the reaction was terminated by the addition of ice-cold 10% TCA-pyrophosphate solution (5 ml). Blanks were obtained by treating the nuclei with the cold TCA before adding dNTP solution. Following 12 h at 0”, the precipitates were harvested by centrifugation, redissolved in 0.3 N sodium hydroxide (1 ml) containing 0.01 M sodium pyrophosphate and reprecipitated by treating with 10% TCA-pyrophosphate (5 ml). The precipitates were collected by centrifugation, washed twice with 5% TCA, once with 0.5 N PCA and extracted three times with aliquots (2 ml) of 0.5 N PCA at 75” for 10 min. The hot PCA extracts of each sample were pooled, adjusted to 6.0 ml with 0.5 N PCA and 4.0-ml aliquots were added to 6.0 ml of Triton X-100 phosphor (1:l) and assayed for 14C content with an efficiency of 78 % (ref. 14). Chemtcaf assays on nuclei
Nuclei were examined and counted under phase-contrast microscopy. Protein was assayed by the method of LOWRY et al. Is. Nucleic acids were extracted by a modified SCHMIDT-THANHAUSER procedurel’j and assayed by the methods of BURTON” (DNA) and C~tuo’r’W’(RNA).
Ctim.-&of. Inrerucric7ns. 2 (t970) 1%
4
[email protected]
RESULTS
Fllaswontlast examination of tk n&G IBlspm&n shoti that the isolated nuclei are relatively free of cytoplasm. The protciuDNA ratio of the uucki was 3.10fO.23andthe RNA:DNA~owrs0.27f~030.~~oncom~ with those described by other workers7*‘*“.
RNA synthesis [3H]UTp was inhibited by 73 % in the present of actiuomycin D which demonstrates that the uptake is due to the action of a DNAdepeudent polymerase20. Since at least 93% of the radioactive product was aeuaitive to a&&, it is ckat that RNA synthesis mediated by DNAdepeudent RNA polymcrase (EC 2.7.7.7) was being measured in this system.
EJects on RNA synthesis Fig. 1 shows the effects of a ties of treatment levels of HN2 on [3H]UTP uptake into RNA of rat-liver nuclei, and it cau be seen that low levels of HN2 (approximately lo-* mM) appear to stimulate RNA syuthesis but at 10-l mM, almost complete inhibition occurs. The action of MDMS at two different concentrations is shown in Fig. 2. This agent exhibits a consistent bisphasic response, but at low Ieve?*-;L6*10’5 mM), clear ev&oce of the enhancement of RNA synthesis was obtained maximal after approximately 5 min of incubation.
Fig. 1. The e!kcts of HN1 at d&rent concentMoar on RNA ryntbals. RNA ayntheab brine 20 min rltcr trea(auntwith HN2. 8.6IO-‘ n&f, 0-O (I Expt.); 8.640-*mM, A(1 E!Jtpq; 1.3*10-’ In&f, xas ColWol, 0 l-~~-~.(6Expt4,Lu~npared, Fii 2. ?hc ttlcct of MDMS at difkrent COllCCntntjOnaon RNA ayntJWs RNA syathcaia during aalbtafter m with MDMS. 8.64W’ RIM, x -x (2 Expts.), 13404 IBM, o---o (2~~)mdwithTMSrrcontrol,O------.(6~k)&compuod. Ckcm.-B&i. Inmutlonr, 2 (1970) 1%
NUCLEIC ACID SYNTHEUS
IN RAT-LIVER NUCLJ3lin
vitro
5
Fig. 3. The effects of DNAase 1 at different concentrations on RNA synthesis. RNA synthesis during 20 min after treatment with DNAase. 7.6~10~ pg/lO’ nuclei, 0 -0; 7.6W3 )rg/lO’ nuc!ei, X-X ; 7.6*1Wz pg/lO’ nuclei, A -A; and 1.9 &lo’ nuclei, c]13 and with TMS as control, 0 - - - - - -0 is compared. Fig. 4. The effect of DNAase 1 at different concentrations, on RNA and DNA synthesis. RNA synthesis during IO min after treatment (0 -O), and DNA synthesis during 60 min after treatment (M), are compared. (Standard errors, control RNA synthesis, A; control DNA synthesis, B.)
One of the consequences of aikylation of DNA of mammalian cells is considered to be single-strand breakage 21. One reason for the apparent stimulation of [3H]UTP uptake observed at low concentrations of HN, and MDMS may thus be due to an increased availability of template DNA. In order to test this, nuclei were treated with various concentrations of DNAase I. Fig. 3 demonstrates the action of DNAase I at different concentrations, on [“HJUTP uptake. With this enzyme, it can be clearly seen that a marked stimulation of RNA synthesis occurs between 7.6*1O’4 mg/ml (10’ nuclei) and 7.6-10” &ml (10’ nuclei), but at higher concentrations (1.9 rglml), inhibition occurs. Using 0 g0 DNA, it has been shownZ2 that DNAase at approximately 10-j &ml concentrations will stimulate RNA synthesis under in vitroconditions. It is interesting to note that the concentration that produced maximal stimulation of RNA synthesis in the nuclear system was within the same order of magnitude. Fig. 4 shows that the uptake of (14C)TTP is unaEected by concentrations of DNAasc I which produced stimulation of RNA synthesis: but a significant increase of [14C]‘MP uptake occurs, maximal at 1.0 pg/lO’ nuclei. MDMS had no significant effect on [14C)‘lTP uptake at any of the concentratioas tested (Table I).
Contxmuion MDh4s(aM) 3.3-m-3 3.3-10-3 3.3-10-l 6.6*10-’
d
[‘*qmP pmwtage Ironm?l~
agmke a8 qfaolnsd = 9.69YJ
!;j.SiM.S 1I4.0*16.0 n::i.oi 4.0 96.75 l0.25
DISCUSSION
It has been shown that low levels of alkylating agents cause sin@&and breaks in the DNA of treated mammalian cslls’L*~’ and bacteriaas. DNAase I produces double- and single-stmnd breaksa* and stimulates RNA synthesis under in vitro conditionsz2. We have now shown that in isolated nuclei this enzyine will also stimulate RNA synthesis (Figs. 3 and 4). The obe#rvations(Rigs. 1 and 2) that Iow levels of MDMS and HNI stimulate RNA synthesis in this system, are consistent with the observed e&c& of sulphur mustard on E. u& nucleic acid synthesis2s and may be similar to the stimulatoryeffects of dibromodulcitol, a powerfir antitumour agent, on RNA synthesisin sensitive Yoshida sarcoma cells In v&ors. The stimulationof RNA synthesis producedby low levels of DNAase may have resulted from low level breakage of DNA, leading to an increased availability of template for polymerase enzymes22. However, DNA synthesis is not stimulated under such conditions but only after treatment with higher concentrations of DNAase, which may indicate that if there is only a limited amount of template made available, RNA synthesis would be preferentiallystimulated in a manner consistent with the observationsreported by Beno et ul.“. The stimulatoryef&ctsoflow levels of DNAase and these alkyhrtingagents on RNA synthesis in rat-liver nuclei may indicate a common mechanism of action. It is interesting to observe that DNAase also erchibitaantitumour and antimitotic etlixts2s-3e. However, there is no similarity between the e&cts of hi@r levois of DNAase and MDMS on DNA synthesis (Table I), sugSe#ing that inhibition of RNA synthesis observed at high levels of the drug may not relate to its ability to damage DNA, but to o&ts on other sites, e.g. enzymes or membrane transport systemsl,~~4.3’, Tha amount of a!kyRting agent actually respot&ble for its cytotoxic action is generally assumed to be only a small frection of the total administereddose. This would imply that if any of the observed offbcts of these agents in this system arc relat#ltothcircytatoxiF~nismsafIzction,~thbysrsmcMtlilrelytobs observed at low concentrations of drug. It has been 2a that “unbalanced RNAmctaboliem”~ybecsmlatbdwith” ofviabiMydwtounbalance1 gMwthn in bacteria.
NUCLEIC AClD SYNTHESI8 M RAT-LD’BRNUCLD in vitro
7
A similarmetabolic inbalance in a mammaliancell resultingfrom a stimulation of RNA synthesis, could thus be indirectly due to the production of breaks in the nuclear DNA. AcKNowLEDGEMENx3 We thank the British Empire Campaignfor Cancer Researchand The Medical ResearchCouncil for financial support.
REFERENCES R. W. RUDWN AND J. M. JOHNSON, The effects of nitrogen mustard on template activity in puri8ed DNA and RNA polymerase systems, Mol. Phurmucol.,4 (1968) 258-273. W. TROLL, E. RINDLB ANDP. DAY,Eifect on N-7 and C-8 substitution of guanine in DNA on T,,,, buoyant density and RNA polymerasc priming,Biuchim. Biopbys. Acta, 174 (1969) 21I-219. G. P. WHEELER AND J. A. ALEXANDER, Effects of nitrogen mustard and cyclopbosphamide upon the synthesis of DNA in vlvu and in cell free preparations, Cancer Res., 29 (1969) 98-109. M. J. ORDANDJ. F. DANIELW, The site of damage in amoeba exposed to lethal concentrations of methyldi-@-chlorethyB-amine, a “nitrogen mustard”, Qlsorz.J. Microscop. Sci., 97 (1956) 17-28. 5 M. J. ORD, The site of damage in amoeba exposed to low concentrations of caethyldi+ chloroethyl)-amine, a “nitrogen mustard”, Quaff. J. Microscop. Sci., 97 (1956) 39-45. 6 M. OCHOAMD E. Hrasoues~a, Alkylating agents, Exprl. Chemother., 5 (1967) 1-132. 7 C C. W~DNBLL ANDJ. N. TATA, A procedure for the isolation of enzymatically active rat liver nuclei, Bfochem. J.. 92 (1964) 3 13-3 17.
8 A. I. MarsuntAND B. E. TROPP, Studies on r&nucleic acid synthesis in nuclei isolated from rat liver, Bloclum. Biophys. Ada. 174 (1969) 476-490. 9 L. R. THOMPSON AND B. J. MCCARYW, Stimulation of nuclear DNA and RNA synthesis by cytoplasmic extracts in vitro, B&hem. Biophys. Res. Commun., 30 (1968) 166-172. 10 D. GALLI~ITZ AND C. E. Serems, Stimulation of RNA polymerase activity of rat liver nuclei by cortisol In vi&o independent of effects on the acetylation and methylation of histones, FE5S Lerters, 3 (1965) 99-102. II W. D. EVMONS AND A. F. Fmrutrs,Metathetical reactions of silver salts io solution, II. Tk synthesis of alkyl sulpbonates, J. Am. Chem. Sot., 75 (1953) 2257. 12 S. B. Wetsa, Enzymatic incorporation of ribonucleoside triphosphates into the interpolyn~ tide linkages of r&nucleic acid, plot. Narl. Acad. Sci. (U.S.), 46 (1960) 102t&1030. 13 D. M. GILL,Liquid scintillation counting of tritiated compounds supported by solid fUtess, Inter& J. Appl. Ra&arionIsotopes, 18 (1957) 393-398. 14 B. W. Fox, Tbo application of Triton X-100 colloid sciotiilation counting in bioche&try, Inten. 1. Appl. RodiatioonIsotopes, 19 (1968) 717-730. 15 0. H. LOWRY, W. J. R~SENBROUOH, A. L. FARRAND R. J. RANDALL, Protein measurementwith the Folin phenol reagent, J, Bid. Chem., 193 (1951) 265-275. 16 H. N. MUNROAND A. FLITCK.The determination of nucleic acids, MerhodrB&em. Alal., 14 (1966) 113-176. 17 K. BUILIIXV, A study of the conditions and mechanism of the diphenylamine reactioo for the colourimetrlc estimation of deoxyribonucleic acid, 5iochem. J., 62 (1956) 318-323. 18 0. CBU~, Determ&ation of oucleie acids in animal tissues, 1. Bid. Chem., 214 (1955) 59-M. The deoxyribonucleic acid content of the nucleus, in E. CHARAND J. N. 19 R. V~NDRELY, DA(Eds.), The Nuctelc A&&, Vol. 2, Academic Prees, New York, 1965, pp. 155-180. 28 J, Hunwttz AND1. T. Auovsr, The role of DNA in RNA synthesis, Prow. Nmdkk AcLd Res., 1 (1%3) 82-84. 21 8. W. Fox ANDM. Fox, Sensitivity of the newly synthesised and template DNA of l,arpbomr cells to dama8e by methyl metbanesulphonate, and the nature of &ted -pair” m MuratIon Res., 8 (1969) 629-638. Chemd&d. InteracHons, 2 (1970) l-8
8
Ch~m.=Biol.lwractlons, 2 (1970) l-4
aRm?mmmtu,RW.mx