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Uncoupling of nonhistone chromosomal protein synthesis and DNA replication in human diploid WI-38 fibroblasts
"Volume34.-number 1
1KEBSI_.ETT~ERS
Aa~gust q973
UNCOUPLING OF NONHIST,ONE CHROMOSOMAL PROTEIN SYNTHESIS AND DNA REPLICATION
IN HUMAN D/I'lL)E) WI-38 FIBROI-,LASTS
G~S. STEIN and C.L. T H R A L L
.DeparZ~enl ~ f J~'ochemistr~. Um'~erM~ o f ~lorida, Gair.'es~ille. .F~. 3260], U 3 A
Received 1 May 1973 Rev~ee. version received 15 May 15~73
1. I n t r o d u c t i o n The g e a o m e o f differen~a~ed .eukaryotic ceils is a nucIe.oprotein complex referred to as chromalhn consisting ,of DNA, RNA, historic, and norflaistone Gazo~ o s o m a l ploteJns. While histories have been shown to be responsible for ths repxessi.on .of DNA-dependen, RNA synthesis I1--2], recent evidence suggests that quantitative [4---7] and .qualitative I8--10] variations iu the transcriptional capaciw o f the genome m a y be mediated b y n0nhistone ch,mmosoma] proteins, The role o f nonhistone .chromosomal proteins in the control ,of gone expression has been the subject o f several recent reviews [11--14]. It has been demonstra*..ed previously ,that in continuously dividing ceils I15--17], as well as in quiescent cells which are stimulated to proliferate 118], histone po]ypepfide ~2nt_hesis is restricted t o the S phase .of ~ e cell cycle and ceases i f DNA replication .is inhibited b y cytosine arabino~ide or h y d r o x y u i e a. Iu continuously d M d i n g .ceils i~ has been ~ho'wn ~aat the synthesis of ~olL1a_istone~nxornosonaal proteins occurs in the cytoplasm ~19_] during all phases o f the ceil ,cycle o n pre-existing ~nd newly t~n" lhesized m R N A ' s [20] and is n o t affected .by ,the inhibition o f D N A replication f ,17]. However, the fleper~dency o f n o n h i s t o n e chromo-;oma] protein synthesis on D N A iep]ication in models of sKmalate d DNA ~ynthesis laas n o t been examined pxeviou.sly. The aim of.the present investigation was to defme - f u r t h e r flue r e l a t i o n s h i p between chromosomal protein synthesis and DNA replication b y studying ~he bios3mthews o f these rnacxomolcbulcs in der~iW=inhibite d human-diploid WI-38 fibroblasts stimulated t o proliferate by a change o f m e d i u m . .2VorthJ~oEanda~ublishing Company -~Amsterdam -
2. Materials and mefla,ods Humar. diploid W1-38 fibzoblasts were ~ o w n to eonBuenee in Eagle's Basal Me,album ,(BME) supplemontreal with 3t3% foetal ,gulf senIm in one it.let Bl~ke ,cu!ture _~qasksin a moisI CO 2 incubator. Seven days after plating, the confi~ent mono]ayers were stimulmed to proliferate by replacmg the old m e d i u m with fresh BME containing 10% foetal eatf serum [6, 2 1 , 2 2 ] . Nuclei and chroma~in were go!areal at 4~C as de~cr,ibed previously by S,l,ein et al, [6], Cnr.omafin was solubitized ~n 1% SDS, 1% ~-mercaptoethmn,ol, O.,O] M so,d~um phosphat,e, p H 5.8; dialyzed against 0 . 1 ~ SDS, 0.1% ~-a-nercap~oelt~.a.-,_ol, 0.01 M sodium phosphate, pH 6.3 and the chlon,~,os~real p r o t o n s were ~ahen s e p ~ a t e d according lo their molecular weight b y SDS polyacrylamide-ge] e2ectroph.oresis [23]. In one ,experiment, ~he histories were separated according to charge, ui".tiring tla~ Mg_h resolution acetic acid urea m e t h o d o f P~nyLrn and Chalkley [24]. -
3. Results a n d disb_ussion Table t indicates Iiaal 16 hr after dens~t-y-ird,fibited h u m a n diploid WI-3$ fibrob]asts are stimulated to prolifexate b y a change o f m e d i u m ~ ~ e cells actively incorporate [3I-1] thymidine. This is !n agi:eement with reports f r o m zevezal laboratories that DNA syn".~esis _.occurs at ~ i s t i m e [6, 2 1 , 2 2 ] . i t is also evidenz f r o m the data ifi-table_l ~h'at ,~jtosine arabin0shde at a ci~n. centratio~, o f 40/~g]ml is effective ha suppresshng ecru. --
_
Volume 34, number I
~EBS L E T T E R S
TaTD]~ 1 Effect of cytosine arabinosifle ,on D N A syntahesiz ha WI-38 hurnmu ,d~plo~df i b r o b h ~ .
5amp]e
Cpm}l O0 VB D N A
,13nstira~ala~d c.on~,o] S-phase ¢oa~,ol ~S.-p]l~_secy~osh~e alabinoside
5 ] 30 42 76:8 3 23]
The rates ,of [9H] flay~a~din=~].neorpozafion ~.~]oDNA were ,de• e;mined L,a density-~lh]bited W/-38 human d~p]oJd fiblcblasts, ~ S-phase cells (1.6-5 hx af'~.ex~he slimula~ion of ,denKP :ty-inlaib]tea 5broB]zsts ~ 10ro]Ke~aieby a ,change ha naedSurnJ, a3 well a5 in S-plla~;: c.~ils/ollo'wir~g 30 naLn e f pxe~IeaLmen±
with 40 ]ag]ard .of ey~.osine arabinoside. One-liier Blake bDtfle~, each .comain~ag 1.0~ cells, wexe l~bele.d fox 30 nain with l~]q]flaymidi~e (0.1 ~CUrnl, 6.0 C~t]rz,M')..Cells veexe harvested, washed twice w]fla cold ,(4'0°C) ]3.3 N pexchloxic ac]fl, and aau-
cleic acid~ were ex~acled wifla hoa (90°.C) ] N perehloxie acid. p]etely this in,creased rate o f D N A synthesis. Cytosine arab~nos]de ~'s a po~enl ~nhibit.or .o£ D N A i,epl~eafion,
August 1'973
noside-treated cells .is ~rtually identical (figs. 1C arrd ID). l i 'should b e n o t e d that there is n o significant Lu. ¢oxpoxafion of lJH]L-tryp.tophma in the regions o f the gel where the h:zt.ones are located. :Elec,Lrophoxes-Ls ,of
purified hist0nes on these gels under similar conditions demonstrates that such po]ypepd.des are ]Smit.ed Io flacfi, ons 62--96 .(data no-t shown). A comparison
,of the distribution o f the [3H]L-tryp.tophma or [3H]L]eu.cine ,(not shown.') radi, oactivRy among ~ae .Ga and S-phase nonhislone chromosomal proteins ~ s o suggests tha~ there are vafia.tions in *_he nonhistone ,chronaosoma] proteins synth.esLzed mad associated with D N A ,during these I w o .d~tinct periods .of the cell
,cycle. An identical .experiment using ~JH]L-]eu.c~ne clearly demonsIraIes tha,~ the irfla~bi~5on,of D N A synth.es~s durhag S phase with .cytosine ~rabinosi,de is ,effective in re.duchag the hacorporafion o f l~H]L-lmacine
int.o proteins 'which migrate in hhe hlstone region of
cytosine arabinoside at a eoneeniration Of 40/a.g]ml does n o t influence the incoz,poration ,of [3]-]] J..-'trypto-
the :gel to ~.e G] level, mad the incorporation .of [3H]L-leueine ~n~.o th,e nonhi~Ione chromosomal pxo.Ieins (fractions 1--61) ~s n,o~ al~ex,ed ,(no~ shown). T o establish con,cl~sive]y that .the synthesis of hist,onesis dependent o n .ooncomltmai D N A ~nth.e~is in hhis ~ystern, S-phase WI-Jg fibroblasts were incubated in the presence o f ,cytpsine arabinoside (40 pg]'m])for 3'0 rnin and t h e n pa,_]se l.abeled for an additional 3 0 rain wilh ~JH]Lqewzine and cytosine arabizaozide, l-lis~ones were extracted 'with 0.4 N H 2 S O 4 froln ~.e ,ch~rolnalin .of ,conIi, ol and ~;yt.o~ine alabinoside-trea~ed ~oBs .mad resolved b y ~ahe bSgh-~e~olution acetic acid--urea method .of Panyirn aM f~~al]dey I24]. Fig. 2 shows that cytosine arabin,oside .completely blocks th~ incorporation o f IJH]L-leuch~e into the major ~la~ses o f hLston~ po!y.pepfides. The presen,t experimems demons~trate that in densityinhibited h u m a n dil:l.oid WI-38 $ibr.oblasts which are stimulated to pxolff~.~rate b y a change in m e d i u m , ~,e synthesis .of nontfist ~ne ,chromosomal proteins w]fich occurs during the S phase o f the cell ay¢le is n.ot affected when DNA ~mthe~i~ ]s blocked by cytosine
phan into the various chromosomal proteins during
arabinoside. This is-m contrast ,to the synthesis of hi~-
the prereplicative phase .of %he cell cycle ,(G1),- eliminat2 ing the pos~Ability that the znfime,tabolite has a direct effect on-the ~Jmthesis ,of these polypeptides, Furthermore, w h e n D N A synthesis ~s :blocked by cytosine arabinoside during S phase, the synthesis of nonhistone chromosomal proteins in ,con.troI and eyt_osine arabi-
tone polypeptides; which ~ restricted ~o the S phase of the .celt .cycle and is inhibited .completely w h e n D N A synthesis is in'~etrupted b y the antirnetabolite. These results ir~ in agreement with previous findings b y this investigator which s h o w e d t h a t ~.he synthesis of nonlfistone chromosomal proteins ~s n o t reduced wtien D N A synthesis is inhibited b~" cytosine arabi-
a n d a~ a ,concentration of 4 0 pg]rrd it has n,o s~gn~fi-
cant effe.e~ .on the bulk rates .of R N A .or p~o~ein synI t l ~ i ~ ~ 0 I.~oIv0 directly the relafi,onship between the syalh.esis o f norda~stone .c.hromosomal p~0 teins and D N A replication in a model of stimulated D N A synthesis, 2 h~ ,(G 1) and 1.6 ,hr .(S) af,te~ .densiW-irflaibited monolayers of h u m a n
,dfip]oid V¢]-38 ~b,xobla~ts
were stim,u]ated ~o proliferale by a change ,of rne~una .the cells wele pretreated wi~a cy~.osLue arzbinos~.de (40 pg]ml) .for 30 rain and then p ~ s e labeled with IBH]L-,tryptophan for 30 rn]n. ,C~,ozine arabinosi.de
was ~ndu.ded in the labe,]ing ~me.dium, ,Chromatia was prepared and 'the t0tgl chromosomal proteins were xeso.~ved a c c o r d i n g t,o m , o l e c u l ~ z weigh% o n S D S p o ] y -
acrylami,de gels I 2 3 ] . The rationale for these rexperi-
ments was that, since his.tones .do not contain tryptophan the distribution o f radi,uactivi,*y t h r o u g h o u t the gels reflects solely *,.he synthesis .of n onhistone chromo~omal proteins. ],t is evJ, den,t f r o m figs. ].A ~a_nd ] ] ] t h a i
~01urn~ 34~ n:~rn,~be~:¢]
F~BS LETTERS
.X~agu~ 1973
.g
~2
o
~o
g
g
~o g _o-,-.
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o
~qdD
37
~]olume 34~ number 1
F]~BS LETTERS
A~gust 1973 -
3000
,~000
~0
~000
~r~ction
Fig~ 2. Ef£ec~ ol cylosin~ ~ab~os~fl~ ~n the p~lya~ry~ana~d~..~e],eleeI~.opho~efiepro.Yi]~of I3H] L/le~xcine-]~be~]edS-phase ~3.4 N HzS'04 ~luble chromosomal p~teins. Eleclzopho~esis was carried ,out a.eco~ding t,o the me~hod ~f Pany~m and Challdey [24]. Cya.osine arzbinoside-trea~t,ed S-phase h~stones ~ o ~ o - - o ) ; ~eoJrtrol~-pha~e histories { . . ~ . - - o ) . noside ,or h y droxynr,ez in cont:,mu,oufly d~v~din,g HeLa S 3 ,cells ~] 7]~ Such findings, taker,~ ~0ge,th er, ]n,dica~e that in .quiescent calls Whireh a~e sfimulat, ed to p~o]it~er.at,e. as well as in ¢ontinaously d~v~,ding cells, the synthesi~ o f non-hist,one chxoynosoma] pxorteins -- ~nlike _ that .of the h~st.ones, which ~ tightly c.oup]ed w~th DNA replication - o c c u r s independently, su!gges.t.~ng the possibility .of a re:gula'.tory £un.e~ion for these acidic chromosomal proteins. ]~eason~.u:g .o£ this nature is ~:onsis~ent w i t h recen,t evidence f r o m :sev.era] ]abora',t,ofies which zugges,ts t h a t ,the n,ordatstone ,eh~omosorn~] proteins .raay play a slgnificant role ~n the regulation o f g e n e exp.xession .in general, and specifically, in ~]ae e0ntro] o f tra.nscription .duzhu:gthe ,cell .cycle l] 1--14]. T h e r e g u h t o r y lanel i o n 'of ,these p~ot.cins is ~up2po:r~e.db y their tissue .and '.species specificity [ 2 5 - 3 0 ] ; their px.esence ~n .greater qu'ant~ties ,in active rather ff~an in ,in'active tissues I3_t]. - a s weli as in aotive..(euchromatiu ) ~rather t h a n i n inac- '_.
38
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~ve .(het.~roch~omat-m) ehroanati~ I32] ; qualit~ativ,e and ,quantitative differences in their iale~ o f synthes~s, ~-nrn,ove.i au,d ffnosphory]ati6n through..out the ¢.etl eyrie I17, 3 3 - 3 7 ] ; and ~,eir, capacity t o regMat.e transcription in a a'nanne.i ~ehar~,c~exist~c oY the tissue o f origin ~8--10.] or the p a r t i c u t ~ sia~e o f the cell cycle 1,6,7]. The specific m~aner "m which n o n h i s t o n e chrom o s o m a l proteLus interact w~'th the g e n o m e ~.emairrs io -be e]u.cidzted.
-Acknowledgements T h e ,~u~a,ors wash t,o ,~xpress ,~h,eir ~ ,apprec~_~tion t o Mrs. ,Gale Iqun:terand Mrs. L e n a Lavie ~or their expert .techmcal .assistance. This ~eseareh was ~uppor~ed b y
grants from the Damon Runyon MemozialFund for : Can, cex :Rese~ch, DRG-] 1,3'8,_fr,o m t h e National- Science F o u n d a t i o n . 'GB-383~9, and the American 'CanceI ~6ciety. F73UF-6..: _k .: ~ _ _
~ o l u m e 34, numbez i
Ang~a~i 3973
17EBS I ~ T I - ~ R S
,'P~.ferences t l ] Huang, R.C. and Bonnet, J. (1965) ]hoe. Nad. Aezd. Sea. UgS. 54, 9 6 E {[2] ~dlfzey, ~,LG., L~aa,, "V. a~_d_~a~zk_~y,A.~. (a963) ~ o = . Nail. Aea& S e i U.S. 49, 414. ~3] Paul, 3..and Gilmore, R,S. {1966) 3. ~I~1. B~ol. 16, 242, ~4] Wang, T.Y. {1968) Exp,/. ,Cell Ri~s. 53, 288. {15] Spelsbexg, T. and ]q~iliea, L. (1969) Bioehim. Biophys. A=la 195, ,63. ~6] S~ein, G.S., Chaudhu~, S. and ~Basez~a,R. (1972) a. Biol. ,Chem. 247, 39!8. I7] S~ein, G.S. and :Farl~r, '3. (a972) Pro~. Nat~. Aead. Sei U.S. 69, 291 $. ~8] Gfirnou:r, R.S. and Pax]a, 3. (1970) ~EBS Le"s~exs9, 435. ~9] Spelsberg, T. :~'axtH_m'lSea,L. (1970) Bioe'heTn. 'J. ]20, 435. I10] ~osllaba, N.C. mad Wang, T.Y. (1972) Bloc]him. ]3iophys. Aela 2'62~ 169. ~111] S~ein, G.S. mad B~serga, R. (1972) Advan. Cmace~ R~, 15, 287. I12] S~_ellwagen,R. and Co]¢, R. (19/599 Ann. Rev. B~oehem. 3~, 951. 113] Spel~tx~zg, T.C., Wilhelm, 2.A. and Bniliea, L.S. 0972) Sub Cell. Biochem. ,I~ 107.
[14] Hnilica, LS. (1972) The stnx~tuze and bi0lo#eal p;op~;"Liesof hiM,ones, C R C Pmess, C!eY~,Ima,d. I15] Robbins~ E. an~1 B o r n , T.I-V. (2967) P~o~:.Raft. A~td. S c i ~.S. 57, 499. '116] Spalding, J., K~ajiwala, X. m'ad Mue]e:r, G. (t966) Pso¢. ~ai]. A1;ad. SeL 13.8. 56, 1535. I17] S~in, GS. and Bonm, T.W. (1972} J. C01Bitfl. 52, 292. | t g ] TaR~, S., Bmma, T.W,, Muchmoze, 3. and Liebermma, L 0 9 6 8 ) Na~uze 2 t 9 , 869.
|19] S~¢hA G.S. and BzsaI~a, R. (197t) Bi0ehem. Biuphy~. Re~. Commu~. 44, 21~. ]20] S~ein, G.S. and M~thews, D.E., S~nce, in p~sss. I21] l~hode, S . L anti ~11em, K.A.O. (1S68) Exptl. C¢]I ~ e s 53, 184. I22] %¥iebel, F. mad ~Basem~a,R. (1969) a. Ce.11~hyaiok ?4, t91. |231 Maize1, 3.¥. ,{1966) Science 151,988. I24] Panyim, S. amd C2az!ldey, R. (19~9) Axch. ~i~chem. Biophys. 130, 337. I~L5] Loeb, 3.1=. and C~reuze%C. (1970) B~a]LSo~:. Ch~,Lm.13it31. 52, !ODT. |26~ MaeG'~llivzay, A J . , C~z.~l~, D. and Pa~l, 3. ~(1971) F E ~ L'e~t*m's~3, 2,04.
[27] K1einsmilh, LJ., He~ama, a. anR CarmK a . (1970) lqa'w,re 226, 1025. I28.~ Temg. C., T~ng, C. an~l A~ICI~y, V. (]972) a. B~oa. Chem. 246, 3597. ~29] ~ g i n , S. ar~.d B~naaea, 3. (1970) B;~,aheafistr_e 9, 4440. t39] Czazian~, S.L. and } t u n g , R.C. (!971) Bio~a~ra'fi~ 10, 4770. |31~ Dingman, C.~'. maa Sporn, M.B. (196¢) a. B~o!. Chem, 239. 3483. |32~ 17rem~ez, J.H. (1965) Naga~e 206, 689. '~33j S't~an, G.S. ~r~fl ~ase_rga, R. (1970) 3 o]~i~,1. Chexn... 2,45, 6095. I34] S~ein, G.S. ~nd Base,g], R. (~970) B~v.hem~ B~phys. Rea. Conarnma. ~ ! , 713. 135] Bo~n, T.W. and S'te'm, G.~ ~1972) 3. Ce~ BioL 52, 308. {1g] Rove~a, G. -~mflBa~rga, 'I~..{~971) 3. Cei! Phy,~oL 77, 20t. ~37] P h i , R.D., S~em, G.S. marl Kleing.m~, L J . {1973) Bio~hem. Biophys. Rcs. Commun. 51,735.
2
=
39
1KEBSI_.ETT~ERS
Aa~gust q973
UNCOUPLING OF NONHIST,ONE CHROMOSOMAL PROTEIN SYNTHESIS AND DNA REPLICATION
IN HUMAN D/I'lL)E) WI-38 FIBROI-,LASTS
G~S. STEIN and C.L. T H R A L L
.DeparZ~enl ~ f J~'ochemistr~. Um'~erM~ o f ~lorida, Gair.'es~ille. .F~. 3260], U 3 A
Received 1 May 1973 Rev~ee. version received 15 May 15~73
1. I n t r o d u c t i o n The g e a o m e o f differen~a~ed .eukaryotic ceils is a nucIe.oprotein complex referred to as chromalhn consisting ,of DNA, RNA, historic, and norflaistone Gazo~ o s o m a l ploteJns. While histories have been shown to be responsible for ths repxessi.on .of DNA-dependen, RNA synthesis I1--2], recent evidence suggests that quantitative [4---7] and .qualitative I8--10] variations iu the transcriptional capaciw o f the genome m a y be mediated b y n0nhistone ch,mmosoma] proteins, The role o f nonhistone .chromosomal proteins in the control ,of gone expression has been the subject o f several recent reviews [11--14]. It has been demonstra*..ed previously ,that in continuously dividing ceils I15--17], as well as in quiescent cells which are stimulated to proliferate 118], histone po]ypepfide ~2nt_hesis is restricted t o the S phase .of ~ e cell cycle and ceases i f DNA replication .is inhibited b y cytosine arabino~ide or h y d r o x y u i e a. Iu continuously d M d i n g .ceils i~ has been ~ho'wn ~aat the synthesis of ~olL1a_istone~nxornosonaal proteins occurs in the cytoplasm ~19_] during all phases o f the ceil ,cycle o n pre-existing ~nd newly t~n" lhesized m R N A ' s [20] and is n o t affected .by ,the inhibition o f D N A replication f ,17]. However, the fleper~dency o f n o n h i s t o n e chromo-;oma] protein synthesis on D N A iep]ication in models of sKmalate d DNA ~ynthesis laas n o t been examined pxeviou.sly. The aim of.the present investigation was to defme - f u r t h e r flue r e l a t i o n s h i p between chromosomal protein synthesis and DNA replication b y studying ~he bios3mthews o f these rnacxomolcbulcs in der~iW=inhibite d human-diploid WI-38 fibroblasts stimulated t o proliferate by a change o f m e d i u m . .2VorthJ~oEanda~ublishing Company -~Amsterdam -
2. Materials and mefla,ods Humar. diploid W1-38 fibzoblasts were ~ o w n to eonBuenee in Eagle's Basal Me,album ,(BME) supplemontreal with 3t3% foetal ,gulf senIm in one it.let Bl~ke ,cu!ture _~qasksin a moisI CO 2 incubator. Seven days after plating, the confi~ent mono]ayers were stimulmed to proliferate by replacmg the old m e d i u m with fresh BME containing 10% foetal eatf serum [6, 2 1 , 2 2 ] . Nuclei and chroma~in were go!areal at 4~C as de~cr,ibed previously by S,l,ein et al, [6], Cnr.omafin was solubitized ~n 1% SDS, 1% ~-mercaptoethmn,ol, O.,O] M so,d~um phosphat,e, p H 5.8; dialyzed against 0 . 1 ~ SDS, 0.1% ~-a-nercap~oelt~.a.-,_ol, 0.01 M sodium phosphate, pH 6.3 and the chlon,~,os~real p r o t o n s were ~ahen s e p ~ a t e d according lo their molecular weight b y SDS polyacrylamide-ge] e2ectroph.oresis [23]. In one ,experiment, ~he histories were separated according to charge, ui".tiring tla~ Mg_h resolution acetic acid urea m e t h o d o f P~nyLrn and Chalkley [24]. -
3. Results a n d disb_ussion Table t indicates Iiaal 16 hr after dens~t-y-ird,fibited h u m a n diploid WI-3$ fibrob]asts are stimulated to prolifexate b y a change o f m e d i u m ~ ~ e cells actively incorporate [3I-1] thymidine. This is !n agi:eement with reports f r o m zevezal laboratories that DNA syn".~esis _.occurs at ~ i s t i m e [6, 2 1 , 2 2 ] . i t is also evidenz f r o m the data ifi-table_l ~h'at ,~jtosine arabin0shde at a ci~n. centratio~, o f 40/~g]ml is effective ha suppresshng ecru. --
_
Volume 34, number I
~EBS L E T T E R S
TaTD]~ 1 Effect of cytosine arabinosifle ,on D N A syntahesiz ha WI-38 hurnmu ,d~plo~df i b r o b h ~ .
5amp]e
Cpm}l O0 VB D N A
,13nstira~ala~d c.on~,o] S-phase ¢oa~,ol ~S.-p]l~_secy~osh~e alabinoside
5 ] 30 42 76:8 3 23]
The rates ,of [9H] flay~a~din=~].neorpozafion ~.~]oDNA were ,de• e;mined L,a density-~lh]bited W/-38 human d~p]oJd fiblcblasts, ~ S-phase cells (1.6-5 hx af'~.ex~he slimula~ion of ,denKP :ty-inlaib]tea 5broB]zsts ~ 10ro]Ke~aieby a ,change ha naedSurnJ, a3 well a5 in S-plla~;: c.~ils/ollo'wir~g 30 naLn e f pxe~IeaLmen±
with 40 ]ag]ard .of ey~.osine arabinoside. One-liier Blake bDtfle~, each .comain~ag 1.0~ cells, wexe l~bele.d fox 30 nain with l~]q]flaymidi~e (0.1 ~CUrnl, 6.0 C~t]rz,M')..Cells veexe harvested, washed twice w]fla cold ,(4'0°C) ]3.3 N pexchloxic ac]fl, and aau-
cleic acid~ were ex~acled wifla hoa (90°.C) ] N perehloxie acid. p]etely this in,creased rate o f D N A synthesis. Cytosine arab~nos]de ~'s a po~enl ~nhibit.or .o£ D N A i,epl~eafion,
August 1'973
noside-treated cells .is ~rtually identical (figs. 1C arrd ID). l i 'should b e n o t e d that there is n o significant Lu. ¢oxpoxafion of lJH]L-tryp.tophma in the regions o f the gel where the h:zt.ones are located. :Elec,Lrophoxes-Ls ,of
purified hist0nes on these gels under similar conditions demonstrates that such po]ypepd.des are ]Smit.ed Io flacfi, ons 62--96 .(data no-t shown). A comparison
,of the distribution o f the [3H]L-tryp.tophma or [3H]L]eu.cine ,(not shown.') radi, oactivRy among ~ae .Ga and S-phase nonhislone chromosomal proteins ~ s o suggests tha~ there are vafia.tions in *_he nonhistone ,chronaosoma] proteins synth.esLzed mad associated with D N A ,during these I w o .d~tinct periods .of the cell
,cycle. An identical .experiment using ~JH]L-]eu.c~ne clearly demonsIraIes tha,~ the irfla~bi~5on,of D N A synth.es~s durhag S phase with .cytosine ~rabinosi,de is ,effective in re.duchag the hacorporafion o f l~H]L-lmacine
int.o proteins 'which migrate in hhe hlstone region of
cytosine arabinoside at a eoneeniration Of 40/a.g]ml does n o t influence the incoz,poration ,of [3]-]] J..-'trypto-
the :gel to ~.e G] level, mad the incorporation .of [3H]L-leueine ~n~.o th,e nonhi~Ione chromosomal pxo.Ieins (fractions 1--61) ~s n,o~ al~ex,ed ,(no~ shown). T o establish con,cl~sive]y that .the synthesis of hist,onesis dependent o n .ooncomltmai D N A ~nth.e~is in hhis ~ystern, S-phase WI-Jg fibroblasts were incubated in the presence o f ,cytpsine arabinoside (40 pg]'m])for 3'0 rnin and t h e n pa,_]se l.abeled for an additional 3 0 rain wilh ~JH]Lqewzine and cytosine arabizaozide, l-lis~ones were extracted 'with 0.4 N H 2 S O 4 froln ~.e ,ch~rolnalin .of ,conIi, ol and ~;yt.o~ine alabinoside-trea~ed ~oBs .mad resolved b y ~ahe bSgh-~e~olution acetic acid--urea method .of Panyirn aM f~~al]dey I24]. Fig. 2 shows that cytosine arabin,oside .completely blocks th~ incorporation o f IJH]L-leuch~e into the major ~la~ses o f hLston~ po!y.pepfides. The presen,t experimems demons~trate that in densityinhibited h u m a n dil:l.oid WI-38 $ibr.oblasts which are stimulated to pxolff~.~rate b y a change in m e d i u m , ~,e synthesis .of nontfist ~ne ,chromosomal proteins w]fich occurs during the S phase o f the cell ay¢le is n.ot affected when DNA ~mthe~i~ ]s blocked by cytosine
phan into the various chromosomal proteins during
arabinoside. This is-m contrast ,to the synthesis of hi~-
the prereplicative phase .of %he cell cycle ,(G1),- eliminat2 ing the pos~Ability that the znfime,tabolite has a direct effect on-the ~Jmthesis ,of these polypeptides, Furthermore, w h e n D N A synthesis ~s :blocked by cytosine arabinoside during S phase, the synthesis of nonhistone chromosomal proteins in ,con.troI and eyt_osine arabi-
tone polypeptides; which ~ restricted ~o the S phase of the .celt .cycle and is inhibited .completely w h e n D N A synthesis is in'~etrupted b y the antirnetabolite. These results ir~ in agreement with previous findings b y this investigator which s h o w e d t h a t ~.he synthesis of nonlfistone chromosomal proteins ~s n o t reduced wtien D N A synthesis is inhibited b~" cytosine arabi-
a n d a~ a ,concentration of 4 0 pg]rrd it has n,o s~gn~fi-
cant effe.e~ .on the bulk rates .of R N A .or p~o~ein synI t l ~ i ~ ~ 0 I.~oIv0 directly the relafi,onship between the syalh.esis o f norda~stone .c.hromosomal p~0 teins and D N A replication in a model of stimulated D N A synthesis, 2 h~ ,(G 1) and 1.6 ,hr .(S) af,te~ .densiW-irflaibited monolayers of h u m a n
,dfip]oid V¢]-38 ~b,xobla~ts
were stim,u]ated ~o proliferale by a change ,of rne~una .the cells wele pretreated wi~a cy~.osLue arzbinos~.de (40 pg]ml) .for 30 rain and then p ~ s e labeled with IBH]L-,tryptophan for 30 rn]n. ,C~,ozine arabinosi.de
was ~ndu.ded in the labe,]ing ~me.dium, ,Chromatia was prepared and 'the t0tgl chromosomal proteins were xeso.~ved a c c o r d i n g t,o m , o l e c u l ~ z weigh% o n S D S p o ] y -
acrylami,de gels I 2 3 ] . The rationale for these rexperi-
ments was that, since his.tones .do not contain tryptophan the distribution o f radi,uactivi,*y t h r o u g h o u t the gels reflects solely *,.he synthesis .of n onhistone chromo~omal proteins. ],t is evJ, den,t f r o m figs. ].A ~a_nd ] ] ] t h a i
~01urn~ 34~ n:~rn,~be~:¢]
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~]olume 34~ number 1
F]~BS LETTERS
A~gust 1973 -
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Fig~ 2. Ef£ec~ ol cylosin~ ~ab~os~fl~ ~n the p~lya~ry~ana~d~..~e],eleeI~.opho~efiepro.Yi]~of I3H] L/le~xcine-]~be~]edS-phase ~3.4 N HzS'04 ~luble chromosomal p~teins. Eleclzopho~esis was carried ,out a.eco~ding t,o the me~hod ~f Pany~m and Challdey [24]. Cya.osine arzbinoside-trea~t,ed S-phase h~stones ~ o ~ o - - o ) ; ~eoJrtrol~-pha~e histories { . . ~ . - - o ) . noside ,or h y droxynr,ez in cont:,mu,oufly d~v~din,g HeLa S 3 ,cells ~] 7]~ Such findings, taker,~ ~0ge,th er, ]n,dica~e that in .quiescent calls Whireh a~e sfimulat, ed to p~o]it~er.at,e. as well as in ¢ontinaously d~v~,ding cells, the synthesi~ o f non-hist,one chxoynosoma] pxorteins -- ~nlike _ that .of the h~st.ones, which ~ tightly c.oup]ed w~th DNA replication - o c c u r s independently, su!gges.t.~ng the possibility .of a re:gula'.tory £un.e~ion for these acidic chromosomal proteins. ]~eason~.u:g .o£ this nature is ~:onsis~ent w i t h recen,t evidence f r o m :sev.era] ]abora',t,ofies which zugges,ts t h a t ,the n,ordatstone ,eh~omosorn~] proteins .raay play a slgnificant role ~n the regulation o f g e n e exp.xession .in general, and specifically, in ~]ae e0ntro] o f tra.nscription .duzhu:gthe ,cell .cycle l] 1--14]. T h e r e g u h t o r y lanel i o n 'of ,these p~ot.cins is ~up2po:r~e.db y their tissue .and '.species specificity [ 2 5 - 3 0 ] ; their px.esence ~n .greater qu'ant~ties ,in active rather ff~an in ,in'active tissues I3_t]. - a s weli as in aotive..(euchromatiu ) ~rather t h a n i n inac- '_.
38
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~ve .(het.~roch~omat-m) ehroanati~ I32] ; qualit~ativ,e and ,quantitative differences in their iale~ o f synthes~s, ~-nrn,ove.i au,d ffnosphory]ati6n through..out the ¢.etl eyrie I17, 3 3 - 3 7 ] ; and ~,eir, capacity t o regMat.e transcription in a a'nanne.i ~ehar~,c~exist~c oY the tissue o f origin ~8--10.] or the p a r t i c u t ~ sia~e o f the cell cycle 1,6,7]. The specific m~aner "m which n o n h i s t o n e chrom o s o m a l proteLus interact w~'th the g e n o m e ~.emairrs io -be e]u.cidzted.
-Acknowledgements T h e ,~u~a,ors wash t,o ,~xpress ,~h,eir ~ ,apprec~_~tion t o Mrs. ,Gale Iqun:terand Mrs. L e n a Lavie ~or their expert .techmcal .assistance. This ~eseareh was ~uppor~ed b y
grants from the Damon Runyon MemozialFund for : Can, cex :Rese~ch, DRG-] 1,3'8,_fr,o m t h e National- Science F o u n d a t i o n . 'GB-383~9, and the American 'CanceI ~6ciety. F73UF-6..: _k .: ~ _ _
~ o l u m e 34, numbez i
Ang~a~i 3973
17EBS I ~ T I - ~ R S
,'P~.ferences t l ] Huang, R.C. and Bonnet, J. (1965) ]hoe. Nad. Aezd. Sea. UgS. 54, 9 6 E {[2] ~dlfzey, ~,LG., L~aa,, "V. a~_d_~a~zk_~y,A.~. (a963) ~ o = . Nail. Aea& S e i U.S. 49, 414. ~3] Paul, 3..and Gilmore, R,S. {1966) 3. ~I~1. B~ol. 16, 242, ~4] Wang, T.Y. {1968) Exp,/. ,Cell Ri~s. 53, 288. {15] Spelsbexg, T. and ]q~iliea, L. (1969) Bioehim. Biophys. A=la 195, ,63. ~6] S~ein, G.S., Chaudhu~, S. and ~Basez~a,R. (1972) a. Biol. ,Chem. 247, 39!8. I7] S~ein, G.S. and :Farl~r, '3. (a972) Pro~. Nat~. Aead. Sei U.S. 69, 291 $. ~8] Gfirnou:r, R.S. and Pax]a, 3. (1970) ~EBS Le"s~exs9, 435. ~9] Spelsberg, T. :~'axtH_m'lSea,L. (1970) Bioe'heTn. 'J. ]20, 435. I10] ~osllaba, N.C. mad Wang, T.Y. (1972) Bloc]him. ]3iophys. Aela 2'62~ 169. ~111] S~ein, G.S. mad B~serga, R. (1972) Advan. Cmace~ R~, 15, 287. I12] S~_ellwagen,R. and Co]¢, R. (19/599 Ann. Rev. B~oehem. 3~, 951. 113] Spel~tx~zg, T.C., Wilhelm, 2.A. and Bniliea, L.S. 0972) Sub Cell. Biochem. ,I~ 107.
[14] Hnilica, LS. (1972) The stnx~tuze and bi0lo#eal p;op~;"Liesof hiM,ones, C R C Pmess, C!eY~,Ima,d. I15] Robbins~ E. an~1 B o r n , T.I-V. (2967) P~o~:.Raft. A~td. S c i ~.S. 57, 499. '116] Spalding, J., K~ajiwala, X. m'ad Mue]e:r, G. (t966) Pso¢. ~ai]. A1;ad. SeL 13.8. 56, 1535. I17] S~in, GS. and Bonm, T.W. (1972} J. C01Bitfl. 52, 292. | t g ] TaR~, S., Bmma, T.W,, Muchmoze, 3. and Liebermma, L 0 9 6 8 ) Na~uze 2 t 9 , 869.
|19] S~¢hA G.S. and BzsaI~a, R. (197t) Bi0ehem. Biuphy~. Re~. Commu~. 44, 21~. ]20] S~ein, G.S. and M~thews, D.E., S~nce, in p~sss. I21] l~hode, S . L anti ~11em, K.A.O. (1S68) Exptl. C¢]I ~ e s 53, 184. I22] %¥iebel, F. mad ~Basem~a,R. (1969) a. Ce.11~hyaiok ?4, t91. |231 Maize1, 3.¥. ,{1966) Science 151,988. I24] Panyim, S. amd C2az!ldey, R. (19~9) Axch. ~i~chem. Biophys. 130, 337. I~L5] Loeb, 3.1=. and C~reuze%C. (1970) B~a]LSo~:. Ch~,Lm.13it31. 52, !ODT. |26~ MaeG'~llivzay, A J . , C~z.~l~, D. and Pa~l, 3. ~(1971) F E ~ L'e~t*m's~3, 2,04.
[27] K1einsmilh, LJ., He~ama, a. anR CarmK a . (1970) lqa'w,re 226, 1025. I28.~ Temg. C., T~ng, C. an~l A~ICI~y, V. (]972) a. B~oa. Chem. 246, 3597. ~29] ~ g i n , S. ar~.d B~naaea, 3. (1970) B;~,aheafistr_e 9, 4440. t39] Czazian~, S.L. and } t u n g , R.C. (!971) Bio~a~ra'fi~ 10, 4770. |31~ Dingman, C.~'. maa Sporn, M.B. (196¢) a. B~o!. Chem, 239. 3483. |32~ 17rem~ez, J.H. (1965) Naga~e 206, 689. '~33j S't~an, G.S. ~r~fl ~ase_rga, R. (1970) 3 o]~i~,1. Chexn... 2,45, 6095. I34] S~ein, G.S. ~nd Base,g], R. (~970) B~v.hem~ B~phys. Rea. Conarnma. ~ ! , 713. 135] Bo~n, T.W. and S'te'm, G.~ ~1972) 3. Ce~ BioL 52, 308. {1g] Rove~a, G. -~mflBa~rga, 'I~..{~971) 3. Cei! Phy,~oL 77, 20t. ~37] P h i , R.D., S~em, G.S. marl Kleing.m~, L J . {1973) Bio~hem. Biophys. Rcs. Commun. 51,735.
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