Mitomycin C may inhibit mitosis by reducing “G2” RNA synthesis

CURRENTS

IN

MODERN

B,OLooY

2 (1968)

254-263. NORTH.HOLLAND

PUlmSH,NG

COMPANY.

AMSTERlMM

When tile vertebrate lens b explanted to a suitable culture me:dium the epithelial cells aie stimulated to divide (Harding, Rothstein and Newman, 1962; Rothstein, lauder and Weinsieder. 1965). Though of the stbmdusfor reaction remaim unknov~ it has been revealed in our laboratory and elsewhere that active mitosis is pieceded by reasonably well de-

Following experinmntal wniptdation, the lenses were incubated in %J “ridine 00.0 c/mM: 5 u&d). 3H thymidine (6.0-l 1.2 c/n% S&nl). 38 deoxycytidrne (32 c/mM; Z&nl), or 38 wcanstituted protein hydrolysate ** (1.135 c/mM, 2 w/ml). The “u:t~ rial was fixed in 3: &hol:acctic a~. and

fmed periods of RNA, profein and DNA sy”thc8is (Reddan, Crotty and Harding, 1966; Rothstein, Fortin and Youngerman, 1966; Rothstein, Bagchi nnd Girthy, 1967; Bagchi, Rothstein and Stein, 1968). Shwld the manufacture of any of the above molcculhrrpeciss be reducedbelow a critical level, mitotic activity fails to occue (Rothstein, Fortin and “%nne&“r”, 1966; Rothstein and Bagchi. in preparation). In some rew,nt studies we have found that the antibiotic mitomycin C blocks mitosis in cultured fr”g lenses eve” when it ia added to the system after peak DNA synthesis. This effect was rather surprising. inw much as mitomycin has been generally considered to be a rather specilic ilhibitor of DNA synthesis (Shiba,

preparedfor autoradiogaphy M sch~tillationcounting as describedin earlierwnununicatio~ (Rothtleh~ Fortin and Youngemtan.1966) ***.

ture

this

thema-

Terawaki,‘faguchi urd Kawunata, 1959). Ap the mrults presented below will illustrate, mitotic inhibition produced by the antibiotic may be due to a” effect on the production of RNA. Lenses of adult bullfrogs (&w~ catesbeionn) were isolated from enucleated eyes and cultured accord@ to a previously described techrlique (Rothstein. Lwder and Weinrieder, 1965). After varying periods of tbne the tissue was expwed to either mitomycin C (IO-‘-

.

,-

I absolute

At 24% peak mitotic activity in cultured fm tcnrr usually takes place 87 hr after is”lati”n. When co”th”l”urly exposed to co”ce”lratiau of “lit”myein above lWz ,&I& however, tit”& fwrn do IWI appear (table I). This “@ht have been anticipated in view of the drug’s well Imown effect on DNA synthcsia (Shiba, Tcrawaki,Tagwhi and Kawa-

nuta, 1959& And. indeed, as fw 1 md table 1 rhwr, “dtunyci” Ldgnifiiuy diiinLha incorporlti0” of 58 thymidineinto lens epithclium.This rppesr to be true for wmparatively short as well as long term expwures. For eXampIe, wh.Xl “Iitmnyci” 00-t &nl)is presentir. tne.culturemediumfmm 46-52 l

A giftfro”~Dr.W.E.Scotl. Hoffmmm4a Rocke.Inc.. Nutly, New Irruy.

ICFFBCT Or: UITOEYCIN

Irpibhor wth cancenlratian and time of cnposutc

CON

RNA SYNTtlESIS

% lnhlbltion olexpcnmrntal

255

n\ compared to unttralcd controls -Mitosis Yrotcin ryntbcrir

RNA Synthesis

DNA Synthesis .-.-._.

loll 63 + 3.0 a) 65 ? 2.8 “)

WDR (10-6 44.52 hr 64.72 hr

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64.87 hr

? f 3.8

24.X7 hr

100

The dala pertaining to natopc incorporatinn were ubtaincd and was expord -0 isotope during the Iart hour of cuIture. C) baxd 8) based on uptskc of 3tl ihymidmc d) bawl b) based on uotakc of 3H dcoxvcrtidine

from vintillntion counts.Tirsuc was iwcd at the btcst time indirated Standard c’rrors ML‘ mcludcd. on uptaku UT %I uridmc on uprrkc of311 rccunrt,tutrd protein hydrolyutc.

hr DNA synthesrs is reduced hy approximately 63%. A> lig. 2 indicst:,s the peak time fur thymidine incuporation oxurs about 52 hr following cxplantatim. It was therefore something of a surprise to find that mitosis at 87 hr could be inhibited even when lenses were exposed to mitomycin from 64-72 hr (in o,her words, during the G1 phase of those cells that are in division at 87 hr). Fig. 2 makes it pbio however that there is a significant aroount of DNA synthesis between 64 and 72 hr and it coil!d be maintained that the mitomycin blockade is really due to a supprenion of DNA syn-

unreasonable to speculate that mitomycin inhibits mitosis in cultured let s cpithelium because it luwers the level of RNA (and therefore protein) synthesis below some minimally essential level. We have pointed out in several earlier accounts actinomycin D (Roth-

that

stein, Fortin and Sonneborn. IY66: Rothstein, Fortin and Youngerman, 1966) can also block cell division when added to the medium duringG1. Moreover, measurements of 3H uridine incorporation show that RNA synthesis peaks at two intervals;one peak occors prior to the burst of DNA synthesis (in C, J and the

thesis owr thif period. That this argument is invalid was shown by cxpcriments with fluorodcoxyuridine (FLIDR). It is well known that FUDR Inhibits DNA synthc$18Nithoot itself becoming incorporated into the

other one wxrs before maximum mitosis (G,) (Rothstein, Fortin and Youngerm:r., 1966). The fact that FUDR, which is quite competent in rapidly stoppmg, almost completely, the uptake of dcoxycylidinc, produces almost no effect on the mitotic index when present between 64 and 87 hr proves

nuclex acid. The Inhibition Las been traced to an

that DNA synthesis is unnecessary over that interval.

interference with the mcthylation of dUMP (Hsrtman and Hcidclhcrger, 1961). The autoradiograms shown in fig. 3 revwi that IO 6 M FUDR effectively stifles incorporation of ‘H deoxycytidine into lenr cpithclial no&i. Thus it may be taken that FUDR prevents DNA synthesis in our system. It is therefore of interest that even when IO -6 hi FUDR is supplied

Since mitotic activity can be completely eliminated at 87 hr when inhibitors of DNA synthesis are added bcfore or during S it appears Ecrtain that amphibian lens epithelial cells comprise a “C,” population. The finding that those experimental manipulations which interfere with G1 RNA synthesis (e.g. addition

to that system from 6457 hr, mitosis is not inhibited to any significant extent (fig. 4). On the other hand complete inhibition can be produced by incubating lenses in FUl?R f:om 20-87 hr (table 1). That these long exposure:; to the wimctabolite are not detrimental to the cells has been show by reversal experiments. In most casesthe cells will enter mitosis if the tissue is simply exposed lo a hundredfold excess of thymtdine. Wha then ih the diffcrcncc in the actions of FUDR and mitomycin? As shown above both drop are folly capable of stopping DNA synthesis in bullfrog lens

in press) arrest the mitotic activation provides UEwith B reason to suggest that G, RNA synthesis is essential for the mitotic activity occurring in bullfrog lenses at 87 hr. Whet is more, the data presented in table show that mitomycin C can also block such synthesis.

epithclium. Yet the former dots not prevent the ap pearawe ul’-;ll!osis when added in G2 (even at lobs M) while t’ie Iutter does. Table I mey provide a partial cxplai etiou af~hew results. It will be noted that IXJDR ho:, virtwlly no effect upon the incorporation of ‘H uri6inc. This quite in contrast to the action

i

of mitamycin C which. as table

I indicates. reduces

the incorporation of the isotope by l7-34%:moreover, protein synthesis is lowered to approximately 50% of the control Icvcl. In view of the results rcportcd it does not seem

of actinomyein, U.V. irradiation) (Stein and Rothstein.

I

Iyer and Szybalsk: (1964) indicate that mitomycin may act by cross linking the two strands of DNA. On general grounds such an action might be expwted to influence the RNA as well as the DNA polymerase reaction. However, et present there is some conflict in the litrrature on this point. Thus in their workon phage X and E.co/iK 12. Ricer and Weissbach(1964) failed to detect any effect of mitomycin on the RNA polymerase reaction. By contrast Kuroda and Furuyama (1963) do report ‘5 moderate action” on Hel_a cells which were exposed to 0. I fig/ml mitomycin. Certainly the latter system is more comparable to ours than is that of Pricer and Weissbach. It could be that the actual effect of mitomycill on RNA synthesis in a particular system is determined by the base frequcnties of the relevant DNA template. Our data from the mitomycin experiments reported

A

.

WFECT

OF MlTOhlYCIN

C’ON RNA SYNTllESJS

EFFECT OF WITOMYCIN

here and

alro

stein, Fortin uld

from studies with actmomycin and Sonneborn,

Youngemtan,

RNA

1966)

1966;

Rothstein,

show that G2 DNA

synthesis must precede mitosis.

D (RothFortin primed

In work with

Enger et al. (1968) have revsatedthat 18.5(ribosomal)RNA is synthesizedmore in:enrivelyin late than in early interphase.‘T&sobservationis in keepingwith our recent findings.Since alI RNA synthesisis primedby DNA one question that arisesia whether G2 RNA synthesis is primedexcb~sivelyby “old” DNA. by that formed duringthe immediatelyprecedingS phase or by some combinationof the two. This matterwill be discussedin a subsequentreport. cultured

ovarian cells of the Chinese hamster,

This work was supported by United States Public He&h ServiceGrant NB 0542504 from the National Institutefor NeurologicalDiseasesand Blindness.

REFERENCES

Bag&i. ht.. Rotbrtcin, H aad G.S. SIC,& 19611, Synthesis of macromokcukz in epithclW c:uI of the cultured nmpbi. bim kns, II. Effecta of pummyein md actinomycin Don ~10tcb1 wntkesis. Exe. CeU Rer. 50.454. Eqer. M.D., R.A. Tobey and A.G. Saponam, 1968, RNA syn. ,herirblChinez hamucr tear. 1. Diimmlial synmctic rate for IibauxnJ RNA in ev,y and late i”lerphuo. J. cdl 810,. 36,583. Hudi”g,C.V.. H. Rolhrtsin and MS. Ncwmsn, 1962. The wttwtian of DNA synthcrir and cell division in rabbi; knrin ~,a. Exp,,. Eye Rer. t. 457.

.

I

~..

263

C Oh RN?, SYNTHESIS

tb’tman, K.-U. and C. Heidelberger. ,961, Studies 00 xlrarinatw pyrimidincr. XIII. Inhibition of rhymidylato synthe fw. i. Biot. Chcm. 236.3006. ,yer.V.N.

md W. Szyb&ki,

19&1, A molecular mechanism

of mitomycin action: Linking of complementary DNA stmndr,Proc. Nstl. Acad. Sci. SO, 355. Kurodn, Y. and J. Fumyama, 1963. Physiological and hi& cllsmiral rtudlcrofcffectl ofmitamyein C on strain HaLa cells in a” cuit”re,Car.ecr Rep. 23,682. Pdcer, WI. and A. Weissbach. 1964, The effect of lyrogenic induction with mitomycin C on the DNA and DNA po,y_ merasc of&&ri&ia oafi, Biwhem. Biophys. Rcr. Corn. rnllll. t4,9,. Red&,,,, J ht. Qotty and C.V. Hud.,g. 1’366. Chamctetiatb n of RNA synthesis in cultured rabbit tenses. 1. Cc,, Bi1,t.3,,,56& Bottutein. H.. M. Blgchi and N. Fmemsn.. 1968, Inhibition of mitotir activation in bullfrog lens epitrlellum by puromycin arId cyclohcxbnide, 1. Cell Biol., in pma. Rothrtein H., M. Bagchi and Gierthy, 1967. b~olvoment of ptratcin synthesis in the in vim “actiwtion” of lens epitheBill cell0.l. CeU Bio,. 35,185A. Rothpein, H., J. Fortin and D. Sonncbo,“, L966. inhibition of DNA $yntbc~is and cell division by wetinamycin D, Expertent,a 22,294. Roth:tcm, H., 1. Fartim and M.L. Youngerman, 1966. Synthesi of macmmoleculer in epifbplial ce,b of the cultured amphibian lonr. 1. DNA uld RNA, Exp. Ce,, Rer. 44.303. Rothstein, H.. J.M. Lauder and A. Wrinsicder. ,965. In vilro culture of amphibian knw. N&turn 206.1267. Shrr, S., A. Tcrawki. T. Taguchi and 1. Kawsmata, ,959, Selective inhltlfl”n or fwmll,iOll aP de”xydbon”FlEic ai* :n L-rcllPrich, co6 by m,tomycin C. Natwe 183. ,056. b.
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