RNA synthesis in Krebs II ascites tumour cells

Life Sciences Vol . 3, pp. 1437-1447, 1984. Pergamon Press, Inc. Printed in the United States .

BNA 8~f1~SIS IN ~S iI ASCITHS TOMfQB GS[.i8 Peter lloNsrraOt Chester Heatt~ Researoh Institnte, Loodon, S .Y .3 .

(Received 15 September 1984) A rapider labelled, high aaleouLr reight RIIA has bha da~onstrated in sW organirs (l, 2,

9.

~~ ~~ ~~ 23~ ~) "

It has bean thought b7 req to

be rss~oger RRA (3), partioularla because is oertaia oases it has beea shorn to teen a high rata of tnrno~er (4) aad the abilitf to hybridise rich DNA (5,ô).

Other ros9ars teen suggested that the rapidly labelled

bast in part, ribososal

RNA

precursor (4).

RNA

is, at

This is based an the fact that

in the presence of actiaosp~cia the rapidly labell ed, heaq RNA disappears aad there is a oonoositant appearaace of radioaatidtf is the ribososal RNAs (7) " The site of gathesia is ~~i{~~ cells Of the rapider Lbelled &U is the nualeos (8). !inch of the aualrr

RRA

corn be entracted rich oald phenol (9) bnt

a certain portion ~arins associated rith the DNA (10) . rich hot phenol and sodiua dodeojlsulphate (ll) . field 16 S aad 23 8 ribosorl

RNA,

This corn be reoond

Yhile both t~pss of eztraotian

aal~ the latter rthod ~rields

RNA

~ioh hu

ra].nas abon 30 . Ia this paper the gath~sis of a rapidly gathesiaed in Yrebs II asaites tumour cells is dranitrated. gnthasis of ribosoaal

BRA

ino:wses with tire .

RNA

Borne aerr L a neoessar~ It eq be that the

is the precursor of the ribosorl RNA.

the caw theta cannot be just a single brealodora of larger srller ones because is both the anolear

rich high 8 *alas

It is aLo shorn that the

svpplrent in the rdiua for this gathasis to occur. rapidl,~ labelled

RNA

Sorter if this is RNA

solecnles into

evctraoted ~ cold phenol and is the address : Dept . o! lbleonlar Biolog, Albort ~++~toi~ Collage of Medioiae, Rw Tork 61 . t Sapported ~ Public Health 8ereioe 6z~ant Ro . 7-~T2-C~A-6x)89-04 froc the Rational Cancer Iastitnte. RNA

1437

1438

RNA SYNTHESIS II~i KREBS II ASCITES TUMOUR CELLS Vol. 3, No.12

o~toplaesio i~ the speoitia aotidt2 of the 16 S BNA is alwgs Greater than that of the 23 S üMA . Data are aLo Giren which indicate that dte of BNA gntheds is the ahrosatia and that the anoleolns oontaias hiGbl~ labelled MIA . Ke 1!u aethods for GrowiaG ICrebs II saris asaites tusour cells were i esseatial~ thoa described b7 Martin ~t a l . (12) .

Ia ediatelt after bareest-

1aG the ae1L were rasped anae is phosphate battered saline and snspmded is the iaanbatiaa asdin" which consisted of sae ball Saa1~'s sedius with 6 g/al Glnooss and ace ball horse terns (Hnrrou~Ghs Yslloose, m-stabilised) .

me cells

were iaoubated at a oa~aoentratioa of aspproziaatel,~ 10? cells per sl . at 3rJ°C. the ooaoentratiaa of ~nridiae ran betvesa 0 .2 ~+o/al and 0 .4 po,/ai. At the end of the iaoubatioa the sells rare sized with as egnal rolnse of partially tros~n Bank's wdina and centrifuged for 3 sinntes at top speed is a retriGerated MSS oentrilYge .

1äe washed cells were treated in ane of the tallowiaG rqs.

BIIA eztraotion trot whale cell : rigorous

S z 109 washed sells were stirred

for three siantes is a siztnre of 30 al .OQit sodius acetate Dntter,

pH 5 .0, oantaiainG 10 y~G washed bentaoite/al (13) and 0.~ n~yythalene-l,5 ei .wi~'te (IIDS) (solntian A), and 50 sl 9~ pheaal eantainiaG 0.1~ 8-lpdras,qu+woh w~ (l4) .

Sh. ~On y,as separated b7 brief oentrübGatioa, the

agneons phase re~ored Gad the interface aGaia eztraoted rith 50 sl of solution A . Säe agneons phases rare oosbiaed and eo~tracted two sure bass rich p~hesol$ iydroaq-gniaoliae.

Sàe &IA w p~reoipitated tsos solution b7 sakiaG it O.3M

with respect to sodius acetate and addiaG two eolnsss of ethanol.

1äe

precipitated BNA was reacted after a halt hour in the cold to pretest oastadaatian of the BIIA ~ the lfDB rbioh precipitates ethanol .

sloth

as siasdiaG is 6rJx

läe iüiA was washed three tines rith 75~ etiasol Gad drained.

Säis

preparstias wou dedgaaLed ilMA I. She iateriaoe and phenol layer trot the eztraotiaa described shore rare added to 50 d

or

0 .~ sodius dodeo~Ln],phate (SDB) is .O1lI acetate batter, ps 5

Vol. 3, No .12

1439

RNA SYNTHESIS IN KREBS II ASCITES TUMOUR CELLS

oontaiaiag 10 yg beata~nite/al. (solution H) .

The BNA is the iaterfaoe ru

eztraoted by heating the rizture to 60oC, stirring vigoroaa~ for three sinutes sad

a~ih

rapidly (11) .

The aqueous phase ras reroved after

aentrülzgation sad the interface again eztraated with solution 8 at 60°C .

The

aosbiaed aqueous phases rere further deproteinimed tro or three tines at 600C rich phenol-8-~drwq-quinoline .

The RNA ws precipitated and rasped as

described above . Isolation of cell fractions :

Nuclei were isolated essenta~ according

to the rethod of Fisher and Harris

250 ss.

(15) " 5

z 109 cells rere suspended in

80

of ice-cold distilled rater aoatain~ O .gd Treen

bentaaite/al .

The suspension ras stirred at

3500

sad

5

y~g

Booth

RP!! ritte a

glass

paddle for 10 ainutes in the sold and then ismediately o~ztrifuged in a refrigerated MBE centrifuge for 2 aia .

The supernatant oontainiag oytoplasrio

raterial x+u poured into a rixture aantainiag 25 rl .

906K

phenol-8~tydrazq-gninoliae .

10~K

Sß8 and

125

rl . of

The aqueous pibase aontainiag aFiopLsaio RNA

ws eztraated in the sold three tires with phenol to reran protein and then precipitated rith sodiu" acetate sad sthaaol . auolei ras iarediately d :ed rüh

50

The pellet containing the

rl . of solution A sad

50

rl . of

90îK

phenol-

8-hFdro~gr-griw~~iwe and treated as described in the seotioa oonceraing RNA extraction iror rhole sells .

The tro HNAs obtained were designated u nuclear

RNA I and aualear RNA II .

Sane aeatrifuaatioa : acetate buffer pH

5.0

Approxirately 0 .8 rg " of RNA dissolved is .0i1M

vas lgered on a linear sucrose gradient of iror 5 - 206

r/v concentration, .O1M is acetate buffer, pS

5"0,

sad sp~m for

15 .5

hours at

21,500 RPM is the B .Y . 25 rotor of the Model L 8pinao nltraoeatrifuge . fraotiaas rere collected iron a needle piercing the honor of the tube . the absorption of the

0.8

The After

rl . fractions had been reasured at 260 sK the 8NA of

each ras quantitatively eztraoted into a rixture of a-butyl alcohol, dihe~glarine sad glacial acetic said (100 :9 :2 ; of the sucrose fraction )

(9)

1 rl . of the orgaaia rizture to

1.8

r1 .

and radioactivity of this top lger coasted is a

1440

RNA SYNTHESLS IN KREBS II ASCITES TUMOUR CELLS Vol. 3, No.12

Pao7card trioarb liquid scintillation speotro~eter .

PreDarwtion of anolesr iraatiaas :

Starting with nuclei prepared ritte

Trees 80, the authod used ws that of Sibatani ~t wl . (16) ezoept fiat the séparatioa of aualeoli iro~ the chro~atia aaterial wia a~ohiered in 19 ~ia . usiag a arooth glana paddle spinning at 3900 EPlI .

Table 1 shors that this

TAB~ 1 The Presaaoe of DNA is Nucleoli after E:iraction rich 1M IiaCl . See Tezt for detail .

Pbaatioa

Thnaiidine is acid iaaoluble tor~ - an~ .

Nnoleoli

j0,000

DNw

9,5~ .~

wtlwd ettectirel~ separates the ~ oontainiag saterial irca tlu mioleoli . 9 z 109 cells rare incubated rith ~0 pa of 3H tlysidins for 30 aia . aliquot oontaiaiag 108 cell wu taiga trot the incubation adstnre . rare cashed fsw of th~idiae, and disrupted b~ sanioatioa .

An The cells

Cold perahlorio

acid (PCA) ws added to the broken cells to a ooaoeatratioa of 10~ .

The

precipitate ras waked free of said soluble radioaotiritf rith 10~ PCA sad eusptaded in 10~ PCJ1 at 90°C for ~ mantes .

~e radioaatirit,~ is this

aalntiaa ws the aeasure o! 3$-th~ae iaoorporatioa iato DNA. rare isolated iron the

The m~oleali

iwiwe cells and rare disrupted sad treated rich PCA

is the rse rq as described aboie for the rhole ae1l .

Table 1 shorn that

less than O .j~ of the oonats in the DNA are associated rith the nndeoli .

Thos

it could appear ttrat the anoleoli are essentially tree of DNA. is Mg . 1 shora the sediseatatioa patterns of NNAs isolated from rhole cells ~ioh had bees iaonbated rich ~-uridiae .

qhe labelled traotica of HNA I

alrys sediaaated olase].~ rith the balk of the HS~A (i`lg . l) .

Ii borne wrw

Vol. 3, No .12

RNA SYNTHESIS IN KREHSII ASCITES TUMOUR CELLS

1441

were left out of the incubation medium the egrathesis of the new RNA wan greatly inhibited (Fig . 1 b) .

A fraction which sedimeated faster than the

bnLc of the RNA was never observed is RNA I . In RNA II a fraction which sedimented faster than the bulk of the RNA was always observed at all the times tested (1, 5, 15, 30, 60 and 120 min.) except 1 minute (Figs . lc and ld) .

Absence of horse serum in the median

caused breakdown of the rapidly sedimenting RNA to a fraction with as S value At all times tested ezaspt one

between 12 and 14 approximately (Fig . le) .

minute the specific activity of BNA II was fiw or six times greater than that of RNA I. Fig . 2 shows the tine course of the labelling of RNAs isolated lY~on the nucleus and the aytoplaem .

~e nuçlear RNA had the highest specific activity

at all times tested, as would be a:petted if RNA II (Fig . 1) is actually nuclear RNA . Fig . 3 shows the sedimentation patterns of RNA iron nuclei and cytoplasm of cells incubated for different times with 3H-uridiae .

~e sedimentation

patterns of oytoplasmic RNA (Figs . 3a sad 3b) and nuclear RNA I (Figs . 3c and 3d) are similar to those of RNA I (Fig . la) . At leant some of the labelling of the 4 b fractions of nuclear RNA I sad cytoplaenic RNA is probably caused by the conversion of uridiae into cytosine and the subsequent terminal addition of cytidine to transfer RNA (17) . FI(i . l

(p" 1442)

Sedimentation pact of RNAa isolated iron whole cells following incubation with 3 ~ uridine/ml . See Methods for more details . (a) RNA I, 120 ad,aute incubation with horse serum ; (b) RNA I, 120 minute incubation without horse aes~ao ; (e) RNA II, 15 minute incubation with horse serum ; (d) RNA II, 120 riaute incubation with horse rerun ; (e) RNA II, 120 minute incubation without horse serin. Full lines, O .D . ; dashed lines, c .p .m . FI(i . 2 (p . 14~42 ) . Tine COYrOe of the eprnthesia of RNA isolated. iron the nndeus sad the cytoplasm. Cells wre incubated with 3 1~ uridine/ml . and horse serum. See Methods for details.

1442

RNA SYNTHESIS IId KREBS II ASCITEB TUMOUR CELLS Yol. 3, No.12

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RNA 3YNTHESIB IN KREHSII ASCITES TUMOUR CELLS

Vol. 3, No.12

The bulk of the radioaatirity is present in nuclear BNA II .

1443

The

aeoimt of radioactirlty present is the Emotion of auolear RNA II (Figs . 30 and 3f) xhioh sedieeats ratipidly deoreassa with ties while the aeoimt of radioaotidty associated with the 23 s peak increases .

The amo~mt

associated with the 16 s peak also increases but at a slower rate than that associated with the 23 s peak .

ibis is in contrast to the labelling pattern

is nuclear BNA I and oytoplaseio RNA in vhioh the specific aotidty of the l6 s peak is a1~rs greater than that of the 23 s peak (Fig " 4) . Table 2 shows the specific aoti~ity of BNAs extracted iroe isolated TABLE 2 Specific Aoti~itiss of RNA Isolated free different parts of the Kreta I~Ascites Tneour Cell . Cells Incubated with 3 Ko Uridin%l.

Fraction

specific 15

Nuclear riboeawl and soluble RNA

aot ieits - om/ux BIü tine - mantes 60 30

64

390

1050

Nuoleolar RNA

304

450

2360

RNA associated with the DNA

555

1165

663

29

193

57

Cytoplaaic RNA

nuclear organelles and the cytoplaee after iacnbaticn of the oella with 3H uridine .

Zt aq be noted that by 60 eiautes the specific aotidty of the

RNA associated with the DNA ~ fallen cad that of the nnoleolar RNA hu ~.g " 3

(p "

1444)

8edieemtatica patterns of BtjA isolated Eros nuclei and aytoplase after incubation with 3 po ~uridia%1. in a eedi~a oontainiog horw serve . (a) cytoplaseio RNA, 15 smuts iaonbatioa ; (c) anolear I BNA, (b) oytoplaseic RNA, 320 smuts iaoubatioa ; 15 eiante iaoubatios ; (d) nuclear I RNA, 120 einnte inoabation ; (f) nuclear II BUIA, (e) nuclear II RNA, 5 einnte incubation ; l20 smuts iacubatioe . 1L11 lines, O .D . ; dashed lines, c .p .e .

1444

RNA SYNTHESIS IN KREHSII ASCITES TUMOUR CELLS

0"

5000

FIG. 7c.

o"

o-s

+ooo

0"

gAOD

o-

as

4

o.D/ ~Y

O.D/ iW .~

0"t

0"

q

TOlF i

dD

SO

Vol . 3, No .12

q

NIf 11,

p

]D

Vol. 3, No .12

RNA SYNTHESIS IN KREHSII ASCITES TUMOUR. CELLS

1445

Minutes

Minutes

FI(i . 4 Tha tiaa dapandaaoa oi tha spaoifio aativit~ oi tha 16 S and 23 S fraatioas of RNA isolatad !~ auolai aad o~topLs, . (a) C7to pLwio RNA, 16 S ; (b) o~rtoplasaio RNA, 23 8 ; (o) nuolear RNA I, 16 8 ; (d) noalaar RNA I, ?,3 8 ; (a) nuolaar RüA II, 16 S ; (f) nuolaar RNA II, 23 8 and haaviar . rises to its hi~ast point .

Thin is consiataat with tiu feat that the

abilitJ of the oall to gathasiaa RNA is vitro daoliaas after etpprozlaatW 1 hour . Diaousaioa wa have psyewastad avidanaa for the gnthoaia o! a rapidly labelled,

high

rolaoular wai.Rht RIiA is 1Croba II asoitu tumor palls .

shove that it is present oa] .~ is the nualai of these palls.

W Lava also Y1~rthar two

Haas of evidaaoa iadioate that this 8NA is gathasia.d an the pall's DNA. PYrst the him rolaoular waiaitt HNA oanaot ba solnbilised mhos it is sa~trwatad with hot phenol oontaiaia6 a dttugea
This has

shown to ba a oharaotaristic of RNA assodatad with ohroratin rtarisl (10) . 8aooadl, when lraoti
This is as indication that the ohroratia is the

RNA SYNTHESIS II~T KRE HS II ASCITES TUMOUR CE LLS

1448

Vol . S, No .12

priaar~ sita of gath .eis ot th. rapidl~ sadiraatias RNL.

or

~. fuaatian vidal~ dieonssad.

rapid~ e.diwntina BNAs fonad in othsr oalls has baan It has bha snsg st.d that it is asw~er RNA (6,8) and

Dara ll aad hie oo-rorlc.rs han sagB.st.d that it is a p~aonrror of ribosomal Onr üPA (4,?) . rasnlts taad to support thia id.a but ~ also an iat .rastiag taat abort th. gnthads of th. o~toplaaio also aaan ia the mork o! othars .

l6

B BNA vhioh is

Althonsh the r~pidl? sedim~atias tatsrLl

is bralora dorra in th . nuolws to 23 8 BNA and thor to l6 8 RNA, ths app.ars tirst in ths o~top7.asa .

rat"

16

8 HNA

It has btia sasswitad tbat this is baoaus .

Ia tha 50 8 ribosmra tnins longar to assaubl. (?) .

ao,~ casa tora ~rork is

aaedad to nudsrstaad the relatioaahip b.t~san t~tu r~pidlT sadiaaating RNA in ths auolnrs and the BNA ot tiaish.d ribosos.s ia tha oJtoplaam. W han also prssaatad stidsao. that tb. rat. ot RNA gnthaais ia Yrabs II asaitss tumour oills daalia.s attar about 60 mianta iaanbatian ~ si~. ~. daalins L partioular~ ma:icad in media oaotaiaias ao horsa s .~ .

Wson

and oo-warlrsrs (1?) tiad omtianiaB gnthesis of rapidl,~ s.diaantin6 Bl4 is th.sa ~ a .lls tor as laos as 440 tiantu ia a tadirr ooataiains ao harea saroa ia both nniaf.atad and rirae int.otsd o .l].s .

~. raason for this

ditt syao . i s not loaam. ~. haaq laballiag ot ths anol.oli aad th. aa~ " +~ ~ of th. saorosa sradiant patt .~s ot nno].solar 8NA to that ot ribosomal RNA l ads support to tha idaa, pnt torwrd b7 a auabar ot vork rs (18), that th. anol.alus is a sonroe of ribosoma .

Zhs faat that at aar].~ times ths ~ aseoeiatsd vith

tha DüA is sosy highlj lsball.d thaa tha anolaalar RIIA arsnas asaiast ths iden ot P.rrt that tha snolsolna ie tha tita of grathwis o! tha rapidl,T s .dir~atins RNA (24) .

~is ianstisatian has ba.n stsaport.d b7 sraats to ths (R
Vol . 3, No.12

RNA SYNTHESIS IN KREBS II ASCITES TUMOUR CELLS

1447

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2.

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3"

F . (iR08, H . HIATr, w . G}II~BE[i'r, C .(i . FûJRLAND, R .w . R7TW~ü(iH and J .D . wAB80N, Natura ~, 581 (1961) .

4.

K . SCi~, H . LATHAlt aad J .E . DARNELL , Proa . Nat . Road . Sri . warb. 4~, 240 (1963)

5"

M . HAYASIiI rad ß . SPI~iEI~AN, Prop . Nat . Aaad . Soi. wash . ~, X564 (1961)

6.

R .P . PERRY, P .R . SRINIYASAN and D .E . KII~LEiY, Soianoa 14~, 504 (1964)

?.

M .aIRARD, ß . PEt~ixAN and J .E . DARNELL, Proo . Nat . Road. Soi . wash . ~1 205 (1964) .

8.

H .H . HIATT, J . Mol . Biol . ~, 217 (1962) .

9.

C . KIDSON, K .B . KIRBY, R .K . RALPH, J . Mol . Biol . ~, 312 (1963) .

â,

10 .

(3 .P . ßHDR(3II:V aad V .L . xANTIE1fA, Bioohia . BiophJe . Aota

ll .

E . wECKNe, Virolo~ ~, 241 (1959) "

12 .

E .x . xARTn~, J . xaLEC, s . svE~Arm aaa J .s . wORK, Bioohao. J . 80 5a5 (1961) .

l3 .

u .z . LITTANxR aaa x . sE~A, Bioohi .. atoahya . Aota 6~, 609 (1962) .

14 .

K .S . KIRBY, Bioohia . BioDh~re . Aota ~, 545 (1962) .

l5 "

H .w . FISI~t aad H . HARRIS, Proo . Ro~. Soo . B , 1~6, 521 (1962)

16 .

A . BIBATANI, 3 .R . da KLOET, V . a . ALLF1iEl! and A .E . MIRSKY, Proo . Nat . Road . Soi . W~h . 48, 471 (1962) .

1? .

R . EASON, M .J . cLn~ aaa R .x .s .

18 .

x .J .H . cHrPCaASE ara x .L . BzaNSTIEi., Proo . Nat . Aoaa. soi . wash . ~0, 1101 (1963) .

19 " 20 .

B .J . xoCAR'i~iY, R .J . BRITrF3~1 rad R .B . ROBERTS, Bio~eioal J . 2 57 (1962) T R . xONIER, Biochir . Bio~e . Aota ~, 1001 (1962) .

21 .

P .-Y . CHL~, Bio~sio J .

22 .

E .F . ZIMlO~lIAN, M . HEETER aad J .E . DARIiELL 1~, 400 (1963) "

23"

x . HOMMA and A .F . aRAHII!!, J . Cß]1 and

24.

R .P . PEttRY, Proo . Nat . Aoad. Soi . Wish . 48, 21?9 (1962) .

â,

AI~IRT "T "TTti ~

153 (1962)

J .Biol .Chae. ~, 3978 (1963)

465 (1962) .

(.OO1D .

Physiol . _, 1?9 (1963) "