Differential Effect of Ionizing Radiation on the ... · after radiation, we studied the effects of X-ray exposure on cyclin B expression, both the mRNA and the protein, in Hela cells

[CANCER RESEARCH 5.1. 1128-1135. March I. 1993]

Differential Effect of Ionizing Radiation on the Expression of Cyclin A and Cyclin Bin Hela Cells1

Ruth J. Muschel,2 Hong Bing Zhang, and W. Gillies McKenna

Departments nf Pathology Â¡R.J. M., H. B. Z./ and Radiation Oncology Â¡W.G. M.I. University of Pennsylvania. Philadelphia, PA 19104

ABSTRACT

Ionizing radiation induces a G2 delay in eukaryotic cells. Since mitoticcyclins are required to trigger the transition from G2 into and throughmitosis, we chose to investigate their expression after irradiation in Helacells. In normally cycling Hela cells, both cyclin A and B mRNA andprotein levels rise dramatically in !.j/\l and rapidly fall coincident withthe completion of mitosis. The rise of cyclin A mKNA at the S/(., boundaryslightly precedes that of cyclin B mKNA. Although the peaks of expressionof each of these molecules overlap, cyclin A mRNA and protein diminishbefore cyclin B. After irradiation in S, cyclin A mRNA and protein levelsrose with the same kinetics as in the controls, but ultimately exceeded thelevels seen in the control population. Cyclin A mRNA and protein levelsremained high throughout the G2 delay induced by irradiation. In contrast, cyclin B mRNA and protein levels did not rise as the irradiated cellsentered (. ,/\l. Only later, before the irradiated cells exited from G2/M,did levels of cyclin B reach the levels seen in the unirradiated controls. Thedecreased amount of cyclin B mRNA and protein was inversely proportional to the dose of radiation. These data indicate that irradiation thatresults in a G2 delay appears to block cells at a point after production ofcyclin A but before cyclin B can be fully expressed and that cells do not exitfrom the delay until cyclin B is again expressed. Thus, cyclin A and cyclinB expression respond differentially to radiation, with cyclin A rising at thesame time as the control and to even higher levels than that seen in thecontrols, whereas cyclin B shows a temporal delay in expression.

INTRODUCTION

A universal effect of ionizing radiation on growing eukaryotic cellsis induction of a division delay. The most characteristic of theseeffects is the prolongation of the G2 phase of the cell cycle (1-8). Thiseffect on G2 phase is also seen after exposure of cells to some che-motherapeutic drugs and other DNA-damaging agents (9-11). There

is some evidence that links these perturbations in G2 phase to thesensitivity of the cells to the killing effects of ionizing radiation. Themutant, rad9, of Sacchammyces cerevisiae, which is highly sensitiveto radiation, is also characterized by an absence of a radiation-inducedG2 delay (12). Conversely, in the system of oncogene-transfected

primary rat embryo cells, which we have developed for studyingradiation resistance in H-ra.c-transfected rat embryo cell lines, the

radioresistant phenotype is associated with a prolongation in theradiation-induced G2 delay in these same cell lines (13, 14).

In recent years, a number of the biochemical events that appear totrigger mitosis have been identified. Much of the work has been donein yeast, both Sacchammyces pombe and 5. cerevisiae, and in oocytesof various species, but a number of the identified mechanisms alsoapply to mammalian cells (15, 16). In all of these systems, the levelsof proteins called cyclins rise in late S or G2 before mitosis is initiated,and then fall after mitosis has been completed (17-19). The linkage of

this rise to the initiation of mitosis is based upon the following

Received 7/22/92; accepted 12/21/92.The costs of publication of this article were defrayed in part hy the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by an American Cancer Society Junior Faculty Research

Award (R. J. M.). a Clinical Oncology Cancer Development Award (W. G. M.), and GrantsCA 46830, 51149. and GM47439.

2 To whom requests for reprints should be addressed, at Room 520, Clinical Research

Building, University of Pennsylvania. Philadelphia. PA 19104.

experimental evidence, (a) Injection of surf clam cyclin A mRNA intoresting Xenopus oocytes triggered their meiotic M phase (20). (b)Blockage of cyclin synthesis can block mitosis (21). (c) Fission yeastwith a conditional lethal mutation in a gene homologous to the cyclinsof higher organisms is blocked in cell division (22, 23). This evidencetogether indicates that the mitotic cyclins are necessary for the triggering of mitosis.

In human cells, 2 mitotic cyclins, cyclin A and B, have beenidentified based on their homology to sea urchin cyclins A and B ( 16).Cyclin mRNA levels rise in Hela cells at late S phase and peak in G2.Unlike oocytes, in which cyclin levels are regulated solely throughtranslational controls, levels of mitotic cyclins appear to be regulatedin Hela cells through mRNA levels as well as at the protein level.There are differences in the behavior of the 2 cyclins in Hela cells. Theprotein for cyclin A begins to accumulate earlier and also declines atan earlier point in the cycle in Hela cells and in Xenopus oocytes(24, 25). Thus, cells blocked in metaphase in mitosis by the drugnocodazole have high levels of cyclin B but baseline levels of cyclinA (18, 25).

The cyclins mediate their action through a complex with p34cdc2

kinase, which is highly active as a histone HI kinase and whoseactivity peaks in M phase (15, 16). Mitosis in oocytes as well as awide variety of other cells can be triggered by maturation promotingfactor, which was first identified because injection of the contents ofan egg about to undergo mitosis into a resting oocyte would triggermitosis. This material has been highly purified, and its activity isassociated with 2 proteins, one a cyclin and the other p34cdc2 kinase

(15, 16). Thus, the suggestion is that part of the signal triggeringmitosis involves a rise in cyclin levels, and that this cyclin then bindsto p34cdc2 kinase, altering the activity of the kinase. In addition, theactivity of p34cdc2 kinase is regulated by a series of phosphorylation

and dephosphorylation events events (26-33). Degradation of cyclin

is required to complete mitosis (34, 35). In Hela cells, cyclin Bcomplexes with p34cdc2, whereas cyclin A is also found in a complex

with a different molecule that shares some antigenic determinants withp34cdc2 (18). The actual role of both of the cyclins in human cells is

not yet defined, but in Drosophila it has been demonstrated thatcyclins A and B are both required for completion of the cell cycle (36).In oocytes depleted of cyclin A, mitosis is uncoupled from DNAsynthesis, suggesting a role for cyclin A as a regulatory element (37).It is not known at this time whether one cyclin is required to activatethe complexes formed by the other in a sequential fashion or if bothare activated and are both independently required.

Lock and Ross (38) have shown that radiation with X-rays ortreatment with etoposide leads to substantially decreased p34cdc2 H l

kinase activity. In an attempt to understand the phenomena of G2 delayafter radiation, we studied the effects of X-ray exposure on cyclin B

expression, both the mRNA and the protein, in Hela cells (39). Twoeffects were seen after exposure of synchronized cells to doses ofX-rays ranging from 6 to 10 Gy. If the cells were exposed to radiation

in the S phase of the cell cycle, a delay was seen in the expression ofthe mRNA for the cyclin B gene. If however, the cells were exposedto X-rays in the G2 phase of the cell cycle, when mRNA levels were

already rising, the effect on mRNA expression was much smaller, buta significant delay was seen before cyclin B protein levels started to

1128

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MITOTIC CYCI.IN EXPRESSION AFTER Hela IRRADIATION

rise. The results imply that ionizing radiation can effect a delay incyclin B expression at both the mRNA and protein levels, dependingon the phase of the cell cycle at which cells are exposed.

In this study, we extend these experiments to show that the effect ofradiation on cyclin B expression is seen not only after exposure tohigh doses of X-rays, but also at the lower levels of radiation, which

are used in the treatment of cancer in the clinical setting. Furthermore,we show that the effect of delaying expression seems to be specific tocyclin B, since initiation of cyclin A expression is not delayed byradiation. Indeed, cyclin A accumulation reaches even higher thannormal levels during the radiation-induced G2 delay. These data allow

us to better focus on the points within G2 at which radiation may exertits effects.

MATERIALS AND METHODS

Synchronization of Hela Cells. Hela cells (1 x IO6) were plated inDulbecco's modified Eagle's medium supplemented with \(V7cfetal calf serum

on 100-mm tissue culture dishes and cultured at 37Â°Cin a CCK incubator. The

cells were synchronized using a modification of the procedure developed byHeintz et al. (40). Two days after plating. 2 m.w thymidine was added to themedium for 12 h. It was washed out and the cells were cultured for another 18h in the presence of 20 UM Ihymidine and deoxycytidine. Then 5 mg/mlaphidicolin was added for 12 h and washed out. Thirty min after release fromthe aphidicolin. the experimental cells were exposed to the indicated doses ofX-rays using a Siemens therapeutic X-ray machine operated at 250 kV. 15 inAwith a 2-mm Al filter (effective energy. 70 kV). The control cells were mock-

irradiated for the same time. Immediately after irradiation and at various timesthereafter, cells were harvested for DNA content analysis, for extraction ofRNA, and for lysates to be used for immunoblotting. Distribution of cellsthrough the cell cycle was measured in each experiment by flow cytometryafter fixation of the cells in 70% ethanol. The methods used were as describedby Gohde et al. (41 ), and calculation of the percentage of cells in each phaseof the cell cycle was performed using software supplied by Partek.

Preparation of Polyclonal Antibody to Human Cyclin B. Based on ananalysis of the sequence of the human cyclin B as published by Pines andHunter (25) in comparison to the sequences of the rat (Rattus norvegicus) andChinese hamster cyclin B cDNAs from our own laboratory.' we chose to raise

an antibody against a region that diverged significantly between the species.The sequence used was KKEAKPSATGKVIDKKL. An analysis of its hydro-

philicity suggested it was likely to lie on the surface of the molecule, and thehuman sequence appeared to contain a 6-amino acid insert in this region that

was not found in either of the rodent species, suggesting that this sequence waslikely to be immunogenic in a rabbit. This peptide was synthesized with aC-terminal cysteine by the Protein Chemistry Laboratory at the University ofPennsylvania and coupled to maleimide-activated keyhole limpet hemocyanin

(Pierce, Rockford, IL). Antisera to the coupled peptide were raised in rabbitsby injection with complete Freund's adjuvant (Cocalico Biologicals. Ream-

stown, PA). These antisera reacted in enzyme-linked immunosorbent assay to

the peptide and in Western blot to Hela cells with a major band of about M,60,000 in size, which was competed by excess peptide (data not shown).

RNA Blotting. Total cellular RNA was extracted using the commercialadaptation (RNAzol; Cinna/biotecx, Houston. TX) of the method of Chomc-

zynski and Sacchi (42). Ten ug of RNA were denatured with formaldehyde andlonnamide and electrophoresed through a \rk agarose gel containing formal

dehyde (43). The RNA was transferred to a Zeta Probe filter (Schleicher andSchuell) by capillary blotting in 20X SSC.4 After baking for 2 h at 80Â°C,the

filter was prehybridized in 5x SSC, 100 mg denatured herring sperm DNA,4X Denhardt's solution (2<7cbovine serum albumin. 27c Ficoll. and 2% poly-

vinylpyrrolidone). and then hybridized in the same solution at 65Â°Covernight

using a cyclin B probe made by random primer labeling of the 1.6-kilobasecyclin B cDNA or a cyclin A probe from the 2.2-kilobase cyclin A cDNA (verygenerously supplied by Dr. Pines and Dr. Hunter) to about 5 x l()*cpm/mgDNA and then washed with 2x SSC followed by 0.5 X SSC at 65Â°C.The

cyclin A probe reacted with 2 bands, most intensely with the band at about 2.2kilobases. The other band, which had a lesser intensity, did not appear afterhybridization at higher temperatures, thus, it appears to be a species with

considerable homology. but different from cyclin A. Filters were exposed at-70Â°C with an intensifying screen using XAR-5 film and developed at 3 to 5

days. Filters were reprohed with probes made from a human ÃŸ-actincDNA

(kindly supplied by R. Taub) or by a rat ribosomal 18S clone (supplied by thelaboratory of Dr. Schmickel). Extent of hybridization was quantified using aLKB densitometer equiped with Gelscan software. Three passes of the beam

were used through each band.Immunoblotting. Immunoblots were performed on samples derived from

5 x IO5 cells as described by Pines and Hunter (18, 25). Twenty ug of total

cellular protein were loaded into each lane. Antisera to cyclin B were the

antibody to the synthetic peptide as described above and used at dilutions of1:500. A LKB laser scanning densitometer equipped with Gelscan softwarewas used to quantify the intensity of the cyclin bands on the autoradiograph as

above. The linearity of the response of this antibody was determined byelectrophoresing different amounts of a Hela cell extract from Hela cellsblocked in G2/M for 24 h with 0.4 jjg/ml (Sigma) nocodazole. The resultant gelis shown in Fig. \A, and the plot of the densitometry scan of that gel in Fig.\B. Thus, this antibody used in the conditions of these experiments will

provide a linear response. Antisera to cyclin A were a gift from Dr. Pines andDr. Hunter. Filters were exposed at -70Â°C to Kodak X-AR film.

50 25 12.5 6.3 3.2

B

3 D. A. Markiewicz et Â«/., unpublished observations.4 The abbreviation used is: SSC, standard saline-citrate.

O 10 20 30 40

Amount of LysateFig. 1. Effect of the amount of lysate on the cyclin B signal in immunoblots. Hela cells

were arrested in melaphase with 0.4 ug/ml nocoda/.ole as described in "Materials andMethods." The extract was diluted so that 50 ul contained 30 ug of total protein. The

indicated amounts of lysate were electrophoresed. and imniunohlotting was performed. A,resultant immunoblot; B. densitometry scan of thai blol.

1129



MITOTIC CYCLIN EXPRESSION AFTER Hela IRRADIATION

RESULTS

Dose Response of Cyclin A and B to X-Rays. To determine thedose response of mitotic cyclin levels to X-rays, Hela cells were

irradiated in S and then harvested in G2 for flow cytometry, mRNA,and protein analysis. Hela cells were synchronized using an S phaseblock with thymidine followed by a block with aphidicolon as described in "Materials and Methods." Three h after release of the

aphidicolin block, when the majority or the cells had entered S phase,the cells were irradiated with various doses of X-rays. To monitor theeffects of X-rays on cell cycle progression, the position of the cells in

the cycle was monitored using flow cytometry for DNA content 8, 10,and 12 h after irradiation. Fig. 1A shows the cell cycle progression at8 and 10 h after the initial release from aphidicolin after irradiation inS phase with doses from 2 to 12 Gy. By 8 h, 80% of the control, mockirradiated, cells were in G2/M phase. At 10 h, the G2 delay induced byirradiation is apparent in that from 70 to 90% of the cells that hadreceived from 2 to 12 Gy remained in G2, whereas 80% of the controlcells had re-entered G, at that time (Fig. IB). The length of the G2delay induced by radiation is dose dependent (1-8). Thus, by 12 h, the

cells that had received the lowest dose, 2 Gy, had exited from G2/Mwith 70% of the cells in G, at that time, whereas the cells that had

*G2/M8hr100

B

5 10

Dose (Gy)

%G2/M 12hr

100

5 10Dose (Gy)

is

Fig. 2. Cell cycle progression after varying doses of X-rays. Hela cells were synchronized as described in "Materials and Methods" and subjected to from 2-12 Gy of X-rays

3 h after release from the aphidicolin block. At 8, 10. and 12 h after irradiation, sampleswere taken for DNA content analysis, RNA extraction, and Western blotting. A. DNAcontent analysis at 8 (----) and 10 h (â€”), indicating the percentage in Gi/M ' â€¢¿�.O) orS (A, A) plotted against dose, fi, percentage in GÃ¬â€¢¿�D) and G2/M (â€¢.O) plottedagainst dose at 10 (â€”) and 12 h ( ).

5 10Dose (Gy)

B30 T

10Dose (Gy)

Fig. 3. Cyclin A mRNA and protein levels plotted against dose of X-rays. Afterirradiation in S, Hela cells were synchronized as described in "Materials and Methods"

and the legend to Fig. 2. Total RNA was extracted at each time point for each dose andlysates were also prepared for immunoblotting. The RNA was used in RNA blotting forcyclin A, and the gels scanned with the densitometry signal plotted against dose in A forboth 8 (â€¢)and 10 (O) h after irradiation. B, result of densitometry scanning of theimmunoblot for cyclin A using 50 ug of total protein for each point. O. results at 8 h afterirradiation: â€¢¿�.at 10 h.

received higher doses from 6-12 Gy still remained in G2/M. At the

lower doses, 2 and 4 Gy, although the G2 delay is shorter, it is stillevident at 10 h with 70 and 85% of the irradiated cells in G2/M phase,whereas the controls had exited into G, at that time. Regardless of thelength of the delay induced, cells given from 2-12 Gy in S were in anX-ray-induced G2 delay at 10 h after release from the block in this

experiment.Although the division delay induced by radiation is predominantly

in G2 phase, some cells, including Hela cells, do undergo smallerdelays in S phase (44). This feature can be seen in this experiment. At8 h, the cells given the higher doses from 6 to 12 Gy remained in S,although the controls and the cells given lower doses are in G2/M (Fig.2). However, by 10 h the majority of the irradiated populations hadentered G2. Thus, we chose to compare cyclin levels at 10 h in theirradiated samples when most of the irradiated cells were in G2/M.

Cyclin A mRNA levels and protein levels were measured on RNAblots and immunoblots from the samples obtained after irradiation inS, and then collected at 8, 10, and 12 h after irradiation. These data areshown for the time points at 8 and 10 h after release in Fig. 3. At 8 h,the unirradiated sample had a readily detectable level of cyclin AmRNA. The cells that had been irradiated with from 2 to 10 Gy hadvirtually identical amounts of cyclin A mRNA. Cyclin A mRNA levelsfrom the 10-h time point, when all of the samples except the unirradiated controls were in G2/M, is plotted against dose of X-rays in Fig.

3A. These data indicate that there is no decrease in cyclin A mRNA1130




levels with doses from 2 to 12 Gy of X-rays at 8 or 10 h. The RNA

blot for cyclin A mRNA 10 h after irradiation is shown in Fig. 4. Thecontrol for RNA loading is shown in Fig. 5, when the blot wasreprobed with a 28S ribosomal RNA probe. Thus, at both 8 and 10 h,the mRNA levels for cyclin A are high and do not decrease withincreasing doses of X-rays up to 12 Gy. The cyclin A protein levels as

measured on immunoblot are also equivalent at all doses at 8 h, asshown in Fig 3B. At 10 h, the control cells have entered G, and so at10 h at 0 dose, the cyclin A protein levels are low. At 2 and 4 Gy, thecyclin A levels somewhat exceed the control values, but at the higherdoses the levels of cyclin A are equivalent to those at control values.The immunoblot for the 10-h time point is shown in Fig. 4. Thus, both

the cyclin A mRNA and protein levels are essentially unaffected afterradiation in S phase at doses from 2 to 12 Gy, and in fact the cyclinA levels exceed those seen in the control cells at 2 and 4 Gy.

In contrast, cyclin B levels showed a dose-dependent decrease after

radiation in S phase as seen in Figs. 5 and 6. Both the mRNA and theprotein levels decrease in a fashion inversely proportional to dose. Thesame samples collected after release from the S block and then irradiated with doses from 2 to 12 Gy of X-rays were subjected to analysis

for cyclin B mRNA and cyclin B protein. At 8 h, the amount of cyclinB mRNA and protein is considerably less than at 10 h (data notshown), and again illustrates that cyclin A expression precedes that ofcyclin B. Thus, much of the data for cyclin B were evaluated at 10 h.Cyclin B mRNA, but not cyclin B protein, was detected at 10 h afterrelease from S in the unirradiated cells, even though the control cellshave passed from G2/M into G, at that time. This shows that the cyclinB mRNA persists after the protein has been destroyed. We coulddetect diminished amounts of cyclin B mRNA even at 2 and 4 Gy. At10 Gy, the cyclin B mRNA level is depressed 4.5-fold (Figs. 5 and 6).

However, the levels of 28S ribosomal RNA detected on the RNA blotare essentially the same at all doses, and the cyclin A mRNA levels arealso not depressed. The cyclin B protein levels have dropped in thecontrol cells at 10 h as they are in G(, but the protein levels otherwisereflect the decreased amounts of mRNA with decreased amounts ofcyclin B being detected on Western blots with the decrease inverselyproportional to dose. Thus, our data indicate that cyclin B levels butnot cyclin A levels are depressed in a dose-dependent fashion by

irradiation.Effect of X-Rays on Cyclin A and B Protein and mRNA Levels

with Time after Irradiation in S Phase. Hela cells were synchronized using a double block of thymidine followed by aphidicolin asdescribed in "Materials and Methods." We irradiated cells at 3 h after

release into S with 6 Gy and monitored samples at the indicated timesfor DNA content and cyclin A and B mRNA and protein levels. In Fig.IB, the percentage of cells in G2/M after the release from the aphidicolin block is plotted against time. The peak of G2/M was reached at8 h after irradiation in S in the control population, and the majority of

0 2 4 6 8 10 12 Dose(Gy)

0 2 4 6 8 10 12 Dose (Gy)

Protein

mRNA

Fig. 4. Cyclin A mRNA and protein evaluated after irradiation in S. The samplesdescribed in Fig. 3 at 10 h after irradiation were subjected to immunoblotting for cyclinA and the mRNA for RNA blotting using cyclin A as a probe. Those blots are shown. Theresults of reprobing of the RNA blot for 28S ribosomal RNA are shown in Figs. 5 and 6.

Cyclin B mRNA

Cyclin B Protein

28S RNA

Fig. 5. Cyclin B mRNA and protein levels after irradiation in S with varying doses ofX-rays. The samples described in Figs. 3 and 4 were also used in RNA blotting for cyclin

B mRNA and immunoblotting for cyclin B protein at 10 h after release from the aphidicolin block. The same blot used for cyclin A and B mRNA analysis was reprobed usinga 28S ribosomal probe. These blots are shown.

â€¢¿�30

o s 10Dose (Gy)

Fig. 6. Densitometric analysis of cyclin B mRNA and prolein levels after irradiation inS with varying doses of X-rays. The relative levels of cyclin B mRNA and cyclin B proleinwere determined using densitometric scanning of the blots shown in Fig. 5. â€¢¿�.cyclin BmRNA plot against doses of from 2-12 Gy O. cyclin B protein.

these cells had entered G, by 10 h. The irradiated population enteredG2/M at 8 h, but due to the G2 delay, up to 50% of the populationremained in G2/M for up to 16 h after the release from S. The G2 delaydoes not lead to a delay of exactly the same time in each member ofan irradiated population so that cells will exit from the block asyn-

chronously. When cyclin A mRNA levels are compared to the percentage of cells in G2/M from this experiment, it is apparent that thecontrol cyclin A mRNA levels peaked at 8 h, coincident with the G2/Mpeak (see Figs. 7, 9, and 10). The cyclin A mRNA levels in theirradiated population began to rise at the same time as in the controlpopulation, but these levels continued to rise as the cells remain in theblock. Cyclin A levels in the irradiated population overshot theamounts seen in the control population and only fell again as the cellsentered G,. Reprobing of this blot with a 28S ribosomal RNA probeindicated that the loading of RNA was equal in all lanes (Figs. 7 and8). Cyclin A protein levels followed this same pattern, with cyclin Alevels rising at the same time as in the unirradiated control population

1131




oâ€¢â€”â€¢¿�1.^Oâ€”oâ€”o^0"â„¢"*"'"""^â€¢-â€¢-â€¢sI

|Â«-0-0~Â«--0.^â€¢"'1 1 1-15-10-5

5 10 15 20 25 30Time (hours)

B

--80

--60 ?

--40 Â£

S--20 O

J UO 5 10 15 20 25 30

Time (hours)

Fig. 7. Analysis of the effect of irradiation on cyclin B mRNA and protein levels withtime. Hela cells were synchronized as described in "Materials and Methods." Cells were

irradiated with 6 Gy at 3 h after release from the aphidicolin block, while control cellswere taken out of the incubator for the same time. Samples were harvested every 2 h, andprocessed for DNA content analysis, RNA extraction, and Western blotting. A. densito-metric tracing for the RNA blot using a cyclin B probe (top plot), and result of reprobingof the blot used for cyclin B analysis with a 28S ribosomal probe (bottom plot). Therelative levels of cyclin B mRNA are plotted against the time after irradiation. O, controls;0. irradiated cells. B. densitometric analysis of the cyclin B protein immunoblots again(top pht) and results of DNA content analysis with the percentage in G2/M plotted againstthe time after release from the aphidicolin block (bottom plot). O, controls; â€¢¿�),irradiatedsamples.

and the cyclin A levels remaining high until 14-16 h after irradiation

as the cells exit from G2/M (Figs. 9 and 10). In the control population,cyclin A protein levels peaked at 8 h, coincident with the percentageof cells in G2/M. In the irradiated population, cyclin A levels rose atthe same time as the control cells and reached the maximum level seenin the controls at the same time, but continued to increase in amountabove the highest levels seen in the controls as the cells remained inG2. The cyclin A levels peaked at 12 h along with the percentage inG2/M and fell as the percentage in G2/M declined. Thus, cyclin Aappeared in the G2/M cells at the same time in the controls and theirradiated cells, but continued to rise in the irradiated cells to still

higher levels and remained easily detectable after the cyclin A wasabsent in the control population.

Cyclin B mRNA and protein levels showed a different response inirradiated cells. Cyclin B mRNA levels peaked in the control cells,coincident with the peak of cells in G2/M (Figs. 7 and 8). In contrast,the irradiated population showed a delay in the rise of cyclin BmRNA, with the peak levels seen at 10-12 h instead of at 8 h as in the

control. The cyclin B protein levels also were lower than that seen inthe irradiated cells when a similar percentage of cells was in G2/M(Figs. 7 and 8). The peak of cyclin B occurs at 12 h after radiation asthe irradiated population begins to exit G2/M and to enter G,. Thus,cyclin B expression in contrast to cyclin A lags behind the expectedamounts seen in the unirradiated cells.

Cyclin B mRNA0246 8 1012 1416 18 2022

0 Gy Â«â€¢Â»-

6 Gy

28S RNA

0246 8 1012 1416 18 2022

0 Gy

6 Gy

Cyclin B Protein0 2 4 6 8 10 12 14 16 18 20 22

0 Gy

6 GyFig. 8. Effect of irradiation on cyclin B mRNA and protein levels with time. The

samples shown here were collected as described in Fig. 7. Shown is the RNA blot on thesesamples for cyclin B mRNA, the same blot reprobed with a 28S ribosomal probe and animmunoblot for cyclin B with the time in hours after release from the aphidicolin blockindicated above the lane for the sample taken at that time. The controls (0 Gy) are shownabove the irradiateti samples (6 Gy).

1132




3J

I(0

O 5 10 15 20

Time (hours)

Fig. 9. Analysis of the effect of irradiation on cyclin A mRNA and protein levels withtime. The samples from the experiment described in Fig. 7 were also analyzed for cyclinA mRNA and protein by reprobing of the RNA blot and immunoblotling with an anti-cyclin A antisera. Top plot, relative levels of cyclin A mRNA plotted against the time ofrelease of the aphidicolin block; bottom plot, relative cyclin A protein levels as determinedby densitometric analysis of the blots shown in Fig. IO. O. controls; â€¢¿�irradiated samples.

subsequent radiation-induced G2 delay. In our previous experiments,

we examined the effect of irradiation with IO Gy during S phase oncyclin B expression in Hela cells (39). This dose produced a muchlonger G2 delay than that seen in the experiments we report here. Inthese previously reported experiments, it was shown that the S phasedelay was clearly over and all the cells had progressed into theradiation-induced G2 delay before cyclin B expression rose. Further

more, the data presented here indicate that when the majority of thecells (65% G2/M, 10% S; Figs. 3 and 7) had progressed past the S/G2boundary into G2 cyclin B level still remained low. Thus, we concludethat the delay in cyclin B expression has a temporal relationship to theradiation-induced G2 delay and is not a simple consequence of a

prolongation of the S phase. The argument is strengthened by notingthat cyclin A expression started to rise at the same time in the irradiated and control cells, indicating that the cells had indeed entered G2since this gene reaches maximal expression in G2/M.

Datta et al. (45) have found similar results in that after irradiationwith 5 or 10 Gy of X-rays, cyclin B levels were decreased in U 937and HL-60 cells. However, at 20 Gy cyclin A, and cdc2 mRNA levelsas well as cyclin B RNA levels were depressed through a posttran-

scriptional mechanism.The pattern of expression of cyclin A and B that we see in the

irradiated cells is consistent with Ruderman's suggestion that it is the

appearance of cyclin B that is the signal for the destruction of cyclinA (46). Since cyclin A started to accumulate in both irradiated andunirradiated cells at the same time and since the appearance of cyclinB is delayed in the irradiated population, cyclin A continues to accumulate in the irradiated cells and rises to supernormal levels untilcyclin B appears.

These data further indicate that irradiation has some specificity inits effects on both cyclin B mRNA and protein levels, as cyclin A wasnot so affected. Although some genes have been found to be induced

Cyclin A mRNA

0 246 8 10 12 14 16 18 20 22

DISCUSSION

The patterns of expression of cyclin A and cyclin B were examinedafter irradiation in Hela cells. We found that cyclin B undergoes adose-dependent decrease in levels of expression in G2 after irradiation

in S, whereas cyclin A does not. In following the expression of these2 cyclins at increasing time after irradiation in S phase with 6 Gy, wenoted that cyclin A rose at the same time and with the same kineticsas in the control unirradiated population. With increasing time as theirradiated cells were blocked in G2, the level of cyclin A continued torise and exceeded that seen in the controls. The level of cyclin A didnot decline until the cells exited from G2/M. In contrast when comparing the level of cyclin B to the unirradiated controls, the level inirradiated cells rose only after a delay, not at the same time as thecontrols, and the levels did not exceed that seen in the controls. Thus,there were coincident peaks in the expression of both cyclins in theirradiated and unirradiated cells, but the pattern of expression of the 2genes was considerably different.

Radiation produces perturbations in all phases of the cell cycle(1-8). In exponentially growing unirradiated cells, cyclin B message

is first seen to rise at or near the S/G2 boundary. In the experiments wereport here and in previously reported data, it is evident that irradiation in S phase produces a short S phase delay as well as a longer G2delay (2). One question that arises from these data is the extent towhich the apparent delay in cyclin B expression might be attributableto an S phase delay after irradiation and thus might be unrelated to the

0 Gy

6 Gy

Cyclin A Protein

02 4 6 8 10 12 14 16 18 20 22

0 Gy

6Gy

Fig. 10. Effect of irradiation on cyclin A mRNA and protein levels with time. The blotsdescribed in Fig. 9 are shown, with the time in hours after irradiation indicated ahove thelane with that sample. The blots of the controls (0 Gy) are shown ahove ihe irradiatedsamples (6 Gy). The 0 time point is shown only for the control for the cyclin A mRNA blot,but 0 time points were run for both the control and the irradiated samples on the cyclin Aprotein immunoblot.

1133




in cells after irradiation, including members of the heat shock genefamily, metallothioneins, c-fos, c-jun, the transcription factor KB, typeI collagenase, ÃŸ-polymerase, and a series of genes called DDI (DNAdamage-inducible), which were identified because of their increase in

level after DNA damage, others have been shown to be unaffected(47-51 ). Only cyclin B has been specifically reported to be depressed

at the doses considered here. Holland et al. (52) examined the patternsof protein synthesis after irradiation in G2 using one-dimensional gel

electrophoresis and found only one band at M, 170,000 to be reduced,the others were not changed. Boothman el al. (53) examined proteinsynthesis using 2-dimensional gel electrophoresis after irradiation in

plateau phase. While the great majority of proteins appeared unaltered, 8 spots increased in intensity and 2 decreased. Thus, the effectson cyclin B mRNA and protein are not a general consequence ofirradiation on protein or RNA synthesis and the pattern of their alteredexpression, while not unique, is unusual.

These data suggest that there is a cellular mechanism that recognizes DNA damage and transduces this signal to result in alteredcyclin expression. This mechanism cannot yet be identified becausethe controls that allow cells to recognize DNA damage and the signalsthat transduce this recognition are not well understood. The rad9 genein yeast is one such gene, but its mammalian counterparts have notbeen identified. Furthermore, it appears to act posttranslationally (54).The RCCl gene is another potential candidate. tsBN2 cells, whichNishimoto (55, 56) and his group isolated as temperature-sensitive for

DNA replication, undergo mitotic events without completion of DNAreplication. The wild type gene was cloned by complementation, andtsBN2 proved to have a point mutation in that gene (55, 56). Thismammalian gene, which is homologous to the yeast pirn-1, functionsto regulate p34cdc2 histone HI kinase activity after the complex of

p34cdc2-cyclin B has formed. Thus, it is unlikely to be involved in the

events described in this study, but would appear to function throughregulation of the sequence of phosphorylation and dephosphorylationevents influencing the activity of p34cdc2. The data presented here

suggest that one target of the mechanisms regulating G2 transitionafter DNA damage may involve controls on the levels of cyclin BmRNA.

These data are consistent with but do not prove the idea that the G2delay in cells that have been irradiated in S may be associated with atime in G2/M after cyclin A has been expressed, but before the initiation of cyclin B expression. There are other G2/M checkpoints forX-ray-induced DNA damage. Tobey (57) identified several points inG2 at which DNA-damaging agents might produce G2 delays. In our

previous work, we irradiated cells in G2/M at a time when the mRNAand protein levels for cyclin B were just beginning to rise. Cyclin BmRNA levels then continued to rise but did not achieve quite the samelevels as normal. However, cyclin B protein levels did not rise afterradiation despite apparently high levels of cyclin B mRNA until theG2 delay was over. Thus, there appears to be an additional regulatorymechanism affecting mainly cyclin B protein after irradiation in G2.Aberrations in p34cdc2 phosphorylation status at a time when p34cdc2

is complexed with cyclin B could also lead to G2 arrest and may be thepoint at which the gene mcJ9 exerts its control (54). Any of thesealterations could potentially led to the loss of p34cdc2 HI kinase

activity, which Lock and Ross (38) have demonstrated occurs aftereither ionizing radiation or treatment of cells with etoposide. O'Con

nor et al. (44) treated lymphoma cell lines with nitrogen mustard andfound that the G2 delay induced in these cells was correlated withaccumulation of phosphorylated p34cclc2in the presence of cyclin B.

Lock (58) has also reported similar results after treatment of CHOcells with etoposide, and Tsao et al. (59) found that camptothecintreatment of Hela cells also led to reduced dephosphorylation ofp34cdc2. They also found cyclin B levels to be unaltered, although they

noted synthesis of cyclin B to be reduced after camptothecin treatment. Thus, the supposition in these cases is that the dephosphorylation processes have been blocked as a consequence of the DNAdamage leading to a G2 delay. The ultimate loss of p34cdc2 HI kinase

activity would be anticipated to lead to a division delay as is seen afterDNA damage. Thus, the induction of G2 arrest after DNA damageappears likely to be a complex series of events at the molecular levelinvolving alterations in gene expression at both the RNA and proteinlevels and alterations in enzyme activation via phosphorylation anddephosphorylation. The experiments reported here on the regulationof cyclin mRNA expression may contribute to our understanding ofthis complex phenomenon.

ACKNOWLEDGMENTS

We thank George Iliakis for his advice and encouragement. We are especially grateful to Dr. Pines and Dr. Hunter for supplying us with their anti-

human cyclin A antisera and the cDNAs for cyclin A and B.

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1993;53:1128-1135. Cancer Res Ruth J. Muschel, Hong Bing Zhang and W. Gillies McKenna Cyclin A and Cyclin B in Hela CellsDifferential Effect of Ionizing Radiation on the Expression of

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Differential Effect of Ionizing Radiation on the ... · after radiation, we studied the effects of X-ray exposure on cyclin B expression, both the mRNA and the protein, in Hela cells