J. K. P, A. I G Lu - NASA · PDF fileATP Metabolism after 250 Rads - ... suggests that repair is dependent in part on anabolic processes. ... model 318A high voltage power supply,

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RADIATION -INDUCED NUCLEIC ACID

SYNTHESIS I N L CELLS UNDER

ENERGY DEPRIVATION

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By J. ,L. Sanders(, Glenn V . Dalrymple, Max L. Baker K. P, Wilkinson and Paul A. Dixon

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Distribution of t h i s report i s provided i n the in te res t of information exchange. Responsibility for the contents r e s ides i n the author or organization that prepared it.

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Prepared under Grant No. NGR 04001-014 by the Departments of Radiology and Physiology, University of Arkansas Medical Center , and the Radiology and Radioisotope Services , VA Hospi ta l , Little Rock, Arkansas

Ames Research Center w&ll@ NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

https://ntrs.nasa.gov/search.jsp?R=19680014064 2018-05-09T03:31:22+00:00Z

ABSTRACT

L c e l l s show a br ie f , accelerated uptake of radioactive precursor into both DNA and RNA following relatively low doses of radiation. This response appeared for c e l l s being starved i n a glucose-free s a l t solution and for c e l l s treated with 2,4-dinitrophenol.

Three d is t inc t features character ize the response. F i r s t , a re la t ively fixed postirradiation t i m e delay seems necessary before the acce lera ted labeling of the nucleic acids occurs . Second, the labeling of the DNA fraction c lose ly paral le ls t h e labeling of the RNA fraction. Finally, t h e radioactive label appears to enter and then to leave both nucleic acid fractions.

Although the accelerated labeling was most apparent af ter 100 r a d s , i t a l s o happened at higher d o s e s where severa l cyc les of incorporation and l o s s of label appeared. Since comparable changes were not found under normal growth condi- tions, t h i s response presumably r e su l t s because of the energy-deprived s t a t e . Both continuous labeling and pulse methods were used; t he nucleic a c i d s were labeled with either inorganic phosphate - P32 or radioactive precursors specif ic for a given fraction. Survival s tudies indicated that cells t reated with 2 , 4 - dinitrophenol had an increased resistance to X-rays.

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TABLE OF CONTENTS

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11.

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, Iv.

V.

VI.

SUMMARY

INTRODUCTION

MATERIALS AND METHODS 1. C e l l Culture 2. Radioactive Labeling Methods 3 . Radioisotope Counting 4. Biochemical Analyses 5. Irradiation Procedures \

RESULTS 1. 2.

3 .

4. Labeling with Specific Precursors 5. Prelabeling of Nucleic Acids 6. 7.

The Effect of Radiation on Ce l l s i n Growth Medium The Effect of Radiation on Ce l l s i n Starvation Medium - Pulse Labeling The Effect of Radiation on Ce l l s i n Starvation Medium - Continuous La be lin g

The Effect of Radiation on Ce l l s i n Dinitrophenol Survival Properties under Energy Deprivation

DISCUSS ION

CONCLUSIONS

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VII. REFERENCES 26

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LIST OF ILLUSTRATIONS

FIGURE

1. Schematic Diagram of the Suspension System

2. Cells i n GrG-wth Meditim - Control

3 . C e l l s in Growth Medium - Continuous Labeling

4. C e l l s in Starvation Medium

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C e l l s i n Starvation Medium - Lag Phase

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7.

C e l l s i n Starvation Medium - Continuous Labeling

ATP Metabolism after 250 Rads - Continuous Labeling

8. Comparison of DNA and RNA Specific Activities - Continuous La be ling

I 9. Uptake of Adenosine - C14 into ATP af ter 100 Rads

10. Labeling with Specific Precursors of the Nucleic Acids

11. Prelabeling of Nucleic Acids

1 2 . C e l l s i n Dinitrophenol

13. Survival Properties under Energy Deprivation

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SUMMARY

The resu l t s of these s tudies show that L cells under energy deprivation have a n unscheduled nucleic ac id synthesis a f te r irradiation. A brief acce l - erated uptake of radioactive precursor followed by a subsequent loss of label charac te r izes the response. The labeling of the DNA and RNA fractions closely paral le l each other. This labeling appears to be superimposed on the normal replication. While the effect prevails af ter 100 r a d s , it also occurs a t higher d o s e s . At least 15 minutes must e l apse before the acce lera ted labeling appears . The t i m e de lay , a s well a s the simultaneous DNA and RNA label ing, implies a rigidly controlled cellular mechanism.

Changes i n ATP metabol i sm coincide with the acce lera ted labeling of the nucleic a c i d s af ter 100 r a d s , but not at higher doses . Apparently, ATP is ut i l ized for either energy-requiring reactions or perhaps as a precursor for the increased nucleic acid synthes is .

C e l l s t reated with 2,4-dinitrophenol had a greater sunrival than c e l l s irradiated i n growth medium.

INTRODUCTION

The expanded u s e of radiation, as wel l a s the radiation hazards of s p a c e t rave l , over the last twenty years h a s prompted detai led s tudies on ce l lu la r r e sponses to th i s agent . The cultured mammalian c e l l h a s proved a convenient system for these investigations (1). The cell population is homo- genous a n d c a n be readily controlled. Also, the radiobiologic properties of t h e s e cells a r e similar i n many respec ts to rapidly growing mammalian cells, i n vivo. , --

By studying the postirradiation survival of cultured cells, Elkind et a1 ( 2 , 3 ) demonstrated t h a t mammalian cells c a n modify radiation damage. Using the s ing le cell technique (4) , t hese investigators performed paired-irradiations . The cells were given a n ini t ia l dose which was followed sometime la ter by a second dose. The response to the second dose reflected the damage remaining from t h e f i r s t exposure. I t also indicated how radiation damage var ies as a function of t ime . Although a simple exponential term defined the r is ing survi- val after six hours , t he cells behaved in a rather unusual manner toward the second irradiation during the immediate survival increased to a maximum a t two a t six hours .

postirradiation period. Ini t ia l ly the hours , and then decreased to a minimum

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Elkind (51, Whitmore (6) and Sinclair (7) have postulated the ini t ia l r i s e of the paired-dose curve to be a consequence of ac tua l repair of radiation in- jury. They further speculate that the subsequent decrease in survival is due to the progression of cells through a radiosensit ive period of the life cyc le . Evidence for repair comes from experiments which showed the init ia 1 recovery to be independent of wide temperature variation. The decreased survival , however, was strongly temperature-dependent . Therefore, Elkind (5) contends tha t while ac tua l repair does occur it is probably pass ive . Never the less , in- nibitors of deoxyri'mnucleic ac id (DNA) aiid ribonucleic ac id (?!?:A) syntheses reduce the cell's ab i l i ty to recover from radiation injury (8 , 9) . sugges ts tha t repair is dependent i n part on anabolic processes .

This finding

A continuous and adequate supply of energy must be avai lable for ana- bolic processes . A lack of information, however, prevents a correlation between t h e energic state of the cells and the potential for survival. For th i s r eason , a se r i e s of s tudies were undertaken to es tab l i sh the effect of energy deprivation on t h e radiation response of cultured mammalian c e l l s .

The L-929 cell culture l ine was chosen a s a t e s t system because 1) the cell c a n repair radiation injury (10) and 2) it h a s been studied with respec t t o its metabolism (11-13). The approach to t h i s problem h a s involved measure- ments of DNA a n d RNA syn theses , a s well a s adenosine tr iphosphate (ATP) l eve l s a n d formation, under conditions of normal growth, starvation and chemi- cal inhibit ion. The biochemical ana lyses performed after irradiation have been compared to r e su l t s on s ingle cell survival under corresponding condi t ions.

The invest igat ions have provided evidence tha t , i n energy-deprived cells, relatively low radiation doses induce rapid nucleic ac id synthes is which is paral le led by changes in ATP metabolism. c r e a s e d viabi l i ty for cells under extreme energy deprivation a t t h e t i m e of irradiation.

The survival s tud ies showed a n in-

MATERIALS AND METHODS

C e l l Culture. - Stock cul tures of the L-929 (14) were maintained a s mono- layers a t t ached to the surface of glass bot t les . Eagle's Minimal Essent ia l Medium (15), supplemented with 10% calf serum, supplied the nutrients required for growth. The cell l i ne , a s wel l as the cons t i tuents of the growth medium, was purchased from Microbiological Associates , Bethesda , Md.

Cell survival was quantitated by Puck 's s ingle cell technique ( 4 ) . Usually the percentage of untreated control c e l l s which grew (plating efficiency) was

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between 55 - 75%. cells and their u se in a n experiment allowed time for recovery from the

An 18-hour interval placed between the seedinq of t h e

I transfer process .

Analyses of biochemical changes involved in t h e s e s tud ies required relatively large quant i t ies of c e l l s . A t confluency a monolayer culture con- ta ins approximately lo7 cells. Since a s many as l o 8 cells were needed for some experiments, a means had to be devised for pooling several monolayer cul tures . Figure 1 i l lus t ra tes the essent ia l features of the technique used . Cells from ten to twenty monolayers were detached frorn the glass surface with a rubber policeman, pooled in a dark bot t le , and kept constant ly s u s - pended in the desired medium by the action of a magnetic st irrer. A water bath maintained the temperature at 37OC. This method of operation had the dis t inct advantage of permitting preirradiation and postirradiation samples to be taken from the same homogenous population.

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Radioactive Labelinq Methods. - As shown in Figure 1 , the c e l l s could be pulse labeled with radioactive precursor by transferring a given aliquot of t he suspens ion into a screw c a p tube containing the label. A vacuum, c rea ted by pulling back on the plunger of a syr inge , caused the aliquot to be t rans- ferred. In all in s t ances a 2 . 0 m l volume w a s sampled. The labeling las ted for 4.0 minutes and was terminated by the addition of cold 0 .3 N perchloric ac id . The time points i n all pulse labeling experiments represent the end of t h e incorporation period.

Continuous labeling involved adding the radioactive precursor directly to the cell pool and permitting integral labeling over the course of the experi- ment. A t predetermined t i m e s , s a m p l e s were obtained from the pooled cells a n d label ing was stopped with cold 0 . 3 N perchloric ac id .

The ce l lu la r const i tuents of interest were prelabeled by incubating the cells at 37OC with the appropriate labeled material . The cells were washed with HBSS and then resuspended i n a non-radioactive medium. Here a l s o , samples were taken at predetermined times and labeling was stopped with cold 0 3 N perchloric a c i d .

The radioact ive compounds employed in t h e s e s tud ies a r e l i s ted below with the supplier:

Sodium Phosphate-P32, 25-30 mCi/mg-P Abbott Laboratories, North Chicago, I l l .

Adenosine-8-C14, 32.2 mCi/mM , Schwarz Bioresearch Inc. , Orangeburg, N . Y.

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Thymidine (methyl-T) , 2 0 , 6 0 0 mCi/mM , Nuclear Chicago Corp. , Des P la ines , I l l .

Deoxycytidine-5-T, 14,700 mCi/mM Nuclear Chicago Corp . , Des P la ines , 111.

RadioisotoPe Counting - Phosphorous- 32 from a l l cel lular fractions was detected by a gas-flow ionization chamber (Baird-Atomic model 134 scaler, model 318A high voltage power supply, and model 755 automatic sample changer). Samples containing carbon-14 and t r i t ium were counted with a Pack- ard model 3003 liquid scint i l la t ion counter. After digestion i n hyamine hydrox- ide (Packard Co. , Downer Grove, 111.) , the cel lular material was mixed with 15 ,O ml of a water-based scinti l lat ion fluid. The scinti l lat ion fluid cons i s t ed , by volume, of 8% Liquifluor (T. M. Pilot Chemica ls , W ater town, M a s s .) , 16% toluene, 2% ethanol and 74% dioxane. Finally, 50 grams of napthalene is added to each l i ter of the mixture. The count from a n external radium source indicated the degree of quenching, which was low and qui te cons is ten t for a l l samples of a given experiment.

Biochemical Analyses - The Schmidt-Thannhauser extraction s c h e m e ( 1 6) w a s followed for separat ion of t h e various cell components. This method in- c ludes a co ld perchloric ac id extraction to obtain a low molecular w i g h t fraction containing ATP, a lipid solvent treatment, a lka l i digestion of RNA, and a hot ac id extraction of DNA.

The luciferin-luciferase method described by Addanki, e t a1 (17) represents a n extremely sens i t ive technique for the a s s a y of ATP and was used in t h e s e s tud ie s . Because the measurements a r e based on a chemiluminescent reaction requiring ATP, the photomultiplier system of a Packard model 3003 counter served as t h e detect ion instrument. The samples containing ATP were loaded into v i a l s with the enzyme system, extracted from firefly lanterns (Sigma Chemical Co. , St . Louis , Mo. ) . The luminescence was measured i n the liquid scint i l la t ion counter.

Ion exchange chromatography served to separa te ATP from other adenine nucleot ides (1 8). The res in employed was Dowex-1-formate (Baker Chemical Co. , Phillipsburg , N . J .) with a formic acid-ammonium formate elution system: the recovery of ATP from the columns was 902 5%.

Irradiation Procedures - Irradiations were performed with a Norelco 1 5 0 kvp X-ray machine and a Keleket 3000 curie cobalt-60 unit . When operated a t 150 kvp and 10 m a , the Norelco machine produced a dose ra te of 100 roentgens per m i n u t e at 16 c m f rom the port. The half-value layer of the beam was 6 mm of A l . Tne cobal t -60 beam delivered a dose ra te of 1 3 8 roentgens per minute a t 30 e m from t h e source .

A l l dose measurements were made with a Victoreen R meter. Irradi- a t ions were conducted under conditions of full backsca t te r and electron equilibrium. The roentgen to rad conversion factor is 0.93 for 150 kvp X-rays and 0.975 for cobalt-60 gamma rays (1 9) .

RESULTS

The E f f e c t of ~ Radiation on Cells in Growth Medium - An important point in t h e s e s tud ie s concerned t h e s tab i l i ty of cells in the suspension system used for t h e experiments. Figure 2 indicates t h e biochemical behavior of cells maintained in growth medium for a three hour period. The ATP level remained cons tan t a t about 450 picomoles per 10 c e l l s . Phosphate-P32 incorporation into the ATP, DNA and RNA fractions is expressed a s counts per minute per l o 6 cells, The measurements s tar ted after t he c e l l s had been in t h e suspen- s ion system for 40 minutes. Each t i m e point s ign i f ies t h e end of a 4 . 0 minute label ing period with 12.0 pCi/ml, The mean and standard deviation for t h e poin ts are shown. During t h e course of the experiment, t he variation in t h e measurements did not appear random, but ins tead fluctuated in a cyc l i c manner which is m o s t apparent i n t h e ATP and RNA fract ions.

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Although t h e observations extended up to four hours , a n interval suf- f ic ien t to cover the repair period, radiation d o s e s from 100 rads to 1000 r ads fai led to produce a n y change in the precursor uptake for ATP, DNA and RNA when t h e cells were kept in growth medium.

Figure 3 i l lus t ra tes the incorporation pattern of phosphate-P32 for ce l lu la r f ract ions being labeled continuously. A t 20 minutes prior to i r radiat ion (indicated by t i m e 0) , Phosphate-P32 was added to a f inal con- cent ra t ion of 7.0 pCi/ml. The ATP level is expressed a s percent of t h e f ina l measurement. The uptake into DNA is linear: but t h e labeling of ATP and RNA cannot be described simply. A comparison of the upper and lower pane l s shows no apparent difference in the labeling for irradiated and control cells. Although t h e data come from a n experiment using 500 r a d s , they ref lect t h e r e s p o n s e which is typ ica l for d o s e s of 100 r ads to 1000 r ads .

The Effect of Radiation on C e l l s in Starvation Medium - Pulse Labelinq - Since Hank ' s Balanced Sa l t Solution (HBSS) is a usua l const i tuent of t h e growth medium, a glucose-free preparation of t h i s solut ion served a s the s tarvat ion medium. The effect of suspending the cells i n glucose-free HBSS is shown i n t h e left panel of Figure 4. The measurements a re given a s percent of growth medium control. Each point s tands for t h e end of a 4 .0 minute labeling period with 12.0 pCi/ml of phosphate-P32. Time 0 ind ica tes t he point at which t h e cells were transferred from growth medium to HBSS. Although

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CELLS IN GROWTH MEDIUM- CONTROL

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starvat ion produced a n immediate depression of precursor incorporation into the macromolecular f rac t ions , t h e ATP leve l a s well a s phosphate-P32 uptake into ATP remained high.

The right panel of Figure 4 indicates t he effect of 1 0 0 rads on starved cells. After 50 minutes in HBSS, the cells were irradiated and samples were col lected for another 90 minutes. Again t h e measurements a re standardized to a growth medium control. A period of rapid phosphate-P32 uptake into the ATP, DXA and RXA fractions with a zorrespondir,g dccrezse in the ATP level is t h e most prominent feature . i n logarithmic growth a t t h e t i m e t hey are harvested for the experiment, appeared around 30 minutes postirradiation, but only for low d o s e s . of 250 r ads or more did not produce similar changes.

T h i s r e sponse , charac te r i s t ic for c e l l s

Doses

Cells which a re permitted t o remain in a confluent state for one or two d a y s en ter a lag phase s ince t h e nutrients in the growth medium are rapidly depleted under these circumstances. These "prestarved" cells were s tudied with respec t to the i r nucleic acid synthes is and ATP metabolism after irradiation. Figure 5 compares representative experiments in which t h e cel ls received 100 rads . The e s sen t i a l a s p e c t s d o not differ from those observed for cells in log growth except t he period of accelerated uptake occurs somewhat earlier, The nucleic acid spec i f ic activities i n t he right pane l correspond to the data represented by t r iangles in t h e lef t pane l .

The Effect of Radiation on Ce l l s in Starvation Medium - Continuous Labeling - In a effort to understand better t h e postirradiation act ivi ty of s tarved c e l l s , a continuous label ing technique was used for studying pre- cursor uptake into t h e different cellular f ract ions. The left panel of Figure 6 con ta ins t h e control curves for ATP, DNA and RNA. After t he c e l l s had been s ta rved for 40 minutes in HBSS, phosphate-P32 was added t o a f inal concen- t ra t ion of 10.0 pCi/ml and al iquots of t h e suspension were sampled a t varying t i m e s l a te r . of all t h e va lues . The observed patterns of incorporation c lose ly resemble t h o s e found for cells in growth medium, although the spec i f ic ac t iv i t ies are lower.

The ATP/106 ce l l s is expressed as percent of the average

A s the right panel of Figure 6 shows, 100 rads produces a brief period of nuc le i c ac id synthet ic ac t iv i ty at about 30 minutes postirradiation. concomitant loss of label from the ATP f ract ion at t h i s t i m e probably r e su l t s from t h e reduced ATP content . e n t e r s ATP, continuous labeling effectively prelabels t h e ATP fraction: consequen t ly , t he dec rease is not surprising. The nucleic ac ids are most in te res t ing because t h e phosphate-P32 seems to enter and then immediately to l e a v e both fract ions. After 30 m i n u t e s , however, the labeling returns to a normal pattern.

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A s in the c a s e of pulse labe l ing , continuous lahelinq riiri r8r-,t shqw ATP metabolism to be altered by higher d o s e s . Figure 7 illustrritc,s t!-,e lack of change from control s e e n for ATP metabolism after 250 r ads . The AT? content does not d i f fe r significantly from preirradiation levels ; thc incorpo- ration of phosphate-P32 into ATP follows a pattern similar to that for the control uptake shown in Figure 6.

Unlike t h e r e su l t s with pulse label ing, however, higher dose markedly affected the precursor incorporation into nucleic a c i d s when continuous label ing was used . Figure 8 compares the DNA and RNA spec i f ic ac t iv i t ies for different d o s e s . Labeling s tar ted 20 t o 25 minutes before irradiation with the addition of inorganic phosphate-P32 t o a final concen- tration of 10 +Ci/ml. The r e su l t s a r e presented a s percent of t he final measurement. After 250 and 500 r a d s , the spec i f ic ac t iv i t ies followed a cyclic pattern of incorporation and loss of labe l . The l o s s , however, never fe l l below t h e level of the unirradiated control.

While higher d o s e s produced essent ia l ly the same re su l t s a s those found after 100 r a d s , t h e period of act ivi ty and t h e extent of uptake is grea te r i n t h e la t ter case. The marked similari ty in t h e behavior of DNA and RNA represents another unusual feature of t h e s e d a t a ,

Labelinq with Specif ic Precursors - Although phosphate is a n imined- i a t e precursor for ATP formation, i ts entry into the d e novo syn thes i s of nuc le ic a c i d s begins with glucose-6-phosphate. In order t o e s t ab l i sh more ful ly t h e nature of t h e radiation-induced biochemical changes , labeled pre- cursors spec i f ic for given fractions were used for measuring t h e syn thes i s of ATP, DNA and RNA in starved cells. Because 100 rads produced a notice- able change in ATP metabolism and a period of accelerated DNA and RNA labe l ing centered around 30 minutes postirradiation, t h i s w a s t h e dose employed for t h e s e experiments.

Figure 9 conta ins t h e results for adenosine-C14 uptake into the ATP f ract ion. of 0,010 pCi/ml. A s indicated, t he labeled ATP decreased precipitously around 30 minutes after irradiation, remained low for several minutes, and then recovered completely. The values for the uptake a re ad jus ted to the f ina l measurement.

Continuous labeling s tar ted a t - 20 minutes with a concentration

Figure 1 0 is a compilation of data from experiments designed to spec i f ica l ly label e i ther DNA or RNA. A s shown, continuous labcling with t r i t i a ted deoxycytidine indicated a change in DNA metabolism at 25 minutes post i r radiat ion. The deoxycytidine uptake correlates well with similar observat ions using inorganic phosphate. Again, label appears to en ter and

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FIGURE 7: ATP METABOLISM AFTER 250 RADS - CONTINUOUS LABELING

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FIGURE 10: LABELING WITH SPECIFIC PRECURSORS OF THE NUCLEIC ACIDS l7

and then to leave the DNA fraction. The arrows point out thc pcriotls of reduced ATP content. uptake is expressed a s percent of the terminal value.

The concentration of label was 0 .2 pCi/rnl; the ~

A s seen in Figure 1 0 , the continuous uptake of adenosinc-C14 into ' the RNA fraction agrees well with t h e resul ts obtained using inorganic phos- phate . Label enters and then leaves. The concentration of the labeling medium i n t h i s c a s e w a s 0 . 0 1 0 rJ.Ci/ml.

Prelabelinq of Nucleic Acids - The possibi l i ty of nucleic acid clegra- dation , suggested from the continuous labeling experiments , led to s tudies on the behavior of prelabeled DNA and RNA. Figure 11 shows resu l t s for a 30 minute and a 17 hour prelabeling with inorganic phosphate-P32. Con- centrat ions of 1 . 0 pCl/ml and 0 .10 +Ci/ml were used . After labeling in growth medium, the c e l l s were transferred to HBSS and were allowed to s tarve for 40 minutes before sampling began. Following 100 r ads , n o obvious change from t h e preirradiation level appeared in e i ther nucleic ac id fraction.

The Effect of Radiation on Ce l l s in Dinitrophenol - While starvation in glucose-free HBSS produced a sharp decrease in macromolecular synthes is , it did not lower the ATP content or ATP labeling with inorganic phosphate-P32 much below that observed in growth medium. In order to study the cells under complete energy deprivation, they were irradiated while exposed to DNP prepared to a f ina l concentration of 5 x 10-5 molar i n glucose-free HBSS.

In Figure 12 the resu l t s for the control and irradiated c e l l s , studied by pu l se labeling with 12 .0 pCi/ml of inorganic phosphate-P32 , a re expressed as percent of the growth medium control. A s the left panel shows , DNP produced a precipitous drop in the ATP content and the precursor uptake for all f ract ions. The center panel shows that t he only change from the un- irradiated control after 100 rads is a n increased ATP labeling a t 15 minutes. This finding differs from the resu l t s obtained with HBSS where an increased incorporation w a s also found for the DNA and RNA fract ions, inorganic phosphate enters t he nucleic acid synthet ic pathway a t a point qui te d i s t an t from t h e f inal s t e p , the possibil i ty exis ted that the lack of incorporation resulted from a deficiency of labe l i n the immediate precursor pool. t r i t ia ted thymidine , a precursor specific for DNA syn thes i s . A s s e e n in t h e cen te r pane l , 100 r ads produced a n increased uptake of tri t iated thymidine in to DNA at 15 and 25 minutes postirradiation. This supports the contention tha t a l a c k of labeled precursor, caused by the DNP treatment, is responsible for the failure to see accelerated uptake into the nucleic ac ids when inorganic phosphate is used.

Because

This hypothesis was t e s t ed b y pulse labeling with 4 . 0 +Ci/ml of

18

DNA SPECIFIC ACTlViYY

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19

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Results from continuous labeling with inorganic phosphate-P32 i n DNP a l s o suggest that a lack of labe l in the immediate precursor pool could be the reason for fa i lure to see stimulated DNA and RNA syn thes i s after 100 r ads . The right panel of Figure 1 2 indicates increased uptake around 20 and 40 minutes postirradiation. The concentration of t h e inorganic phosphate-P32 was 5 .0 +Ci/ml. Since the cells had been exposed to the labe l for over 40 minutes, there would have been sufficient t i m e for t h e label to en ter t he precursor pool. As in the case with HBSS, t he label en te r s and then l eaves t h e nucleic acid fract ions. The appearance of two peaks in t h e continuous labeling experiment ag rees with the observation of two peaks of uptake when t r i t ia ted thymidine was used for pulse label ing.

Survival Properties under Enerqy Deprivation - While energy depriva- t ion changes the biochemistry of irradiated c e l l s , its effect on ce l l survival remains to be e s t ab l i shed . The dose-response relat ionships for c e l l s under energy deprivation a re shown in Figure 13. The ordinate is surviving fract ion, and the upper abcissa is t h e dose in rads . After one hour in HBSS, or 30 min- u t e s i n DNP, the cells received d o s e s ranging from 200 rads to 750 r ads . A t two hours postirradiation, t h e HBSS and DNP were replaced with growth medium. Neither treatment reduced the viabi l i ty of the cells. Although cells starved in HBSS did not vary from the control in the i r survival propert ies , cells t reated with DNP did show a significant increase i n survival which became m o s t pronounced at t he higher d o s e s .

The paired-dose r e sponses for cells in growth medium and DNP are also indicated i n t h i s f igure. The lower a b s c i s s a shows the t i m e between two d o s e s of 300 rads e a c h . By 4 hours, t h e survival for c e l l s in growth medium had become three times greater than for the s ingle exposure. Although cells irradiated in DNP had a higher survival , no time-dependent rise in the surviving fraction appeared. Vpon being transferred to growth ,medium, t h e DNP-treated cells acquired a radiosensi t ivi ty charac te r i s t ic of cells in growth medium.

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I ,

DIS CUSS I ON

Although events leading to increased survival of the c e l l s must occur during the immediate postirradiation period, a c l o s e examination of nucleic ac id ' syn thes i s and ATP metabolism under growth conditions did not reveal a n al tered biochemistry in these fractions. The data shown i n Figure 3 represent a n effort t o find a biochemical phenomenon related to recovery from radiation injur-y. lne failure to ficd such a phznsmenon, however, does not ehnina te the possibi l i ty that i t e x i s t s . Perhaps the changes a s soc ia t ed with repair con- s t i tute such a small part of the total metabolism that they cannot be detected with the methods used .

ml

The intent of the present study has been t o determine the effect of energy deprivation on the radiation response of L c e l l s . gat ions indicate that irradiated cells being deprived of energy have unusual patterns of radioactive precursor uptake into the nucleic ac id fractions. A brief, acce le ra ted labeling of DNA and RNA character izes the response. BGth pulse labeling (Figures 4 and 1 2 ) and continuous labeling (Figures 8 and 1 2 ) show the effect . A delayed act ion seems inherent s ince the response W ~ S never detected before 15 minutes postirradiation (Figures 5 and 1 2 ) . There a re two unusual features i n the data. F i rs t , as indicated from the continilous labeling experiments , the radioactive precursor enters and then leaves the nucleic a c i d s . Second, the labeling of the DNA fraction c lose ly paral le ls that of the RIGA frac- t ion. Although th i s behavior prevails a t 100 r ads , i t a l s o happens a t higher d o s e s (Figure 8 ) , where several cyc le s of incorporation and lo s s of label occur .

The resu l t s of tnese invest i -

While these s tud ies give no indication of why a delay is necessary before the acce lera ted labeling of t h e nucleic a c i d s , they do suggest that i t depends on the metabolic state of the cells prior t o their u s e in the experiment. prestarved (Figure 5) or t reated with DNP (Figure 1 2 ) gave a n earlier response than cells i n logarithmic growth (Figures 4 and 6) . Since there were no apparent changes i n the label ing of DNA, RNA and ATP during th i s period, the reason for the de lay remains a n unsolved problem.

Ce l l s which had been

The labeling of RNA i n the present s tudy paral le ls t o a great extent the label ing charac te r i s t ics which have been described for messenger RNA ( 2 0 ) . have a rapid precursor uptake and both show a f a s t turnover. A similar response occurring in the DNA fract ion, however, is most unusual . The loss of labeled DNA following the acce lera ted uptake could indicate 1) a breakdown of the rapidly synthesized DNA or 2) a loss of preincorporated label from other s i t e s in t h e molecule. The evidence strongly favors the first possibil i ty. As indicated

Both

2 3

v

by the experiments i n Figures 8 and 1 2 , the loss of l n l ? c l f:(x;ue,nt!y ~jxceeds the total amount incorporated prior to the accelerated uptake. loss usual ly equals the amount incorporated. F'urthermore, the resu l t s of the prelabeling experiment (Figure 11) do not indicate a breakdown of nucleic ac ids exis t ing before t h e acce lera ted labeling. All t h i s points to a rapid formation and breakdown of the same DNA, which is superimposed on the normal repli- ca t ive process (Figure 8) .

In f G r : t , :ne

While there a r e reports of unscheduled DNA synthes is in X-irradiated cells ( Z l ) , *L:- L L i l a effect has bccn described only for d o e s nf L O ' r a d s nr more, Certain s t ra ins of bacteria a l s o display an unscheduled DNA synthes is after W-irradiation (221, which const i tutes part of the "cut-patch" repair sysrem. This mechanism , however, differs from t h e present biochemical events i n that i t involves degradation of DNA followed by resynthes is . Furthermore, RNA synthes is is not implicated i n the "cut-patch" process .

The c l o s e parallelism in the labeling of DNA and RNA sugges ts :?,e: the two p rocesses a r e re la ted . Although D N A serves a s a template for RNA formation, new DNA synthes is presumably is not required. Poss ib ly , one strand of the double he l ica l DNA is disrupted ei ther directly by radiation or through the act ion of an endonuclease ( 2 2 ) . This break then l eads to the rapid nucleic ac id syn- t h e s i s with the in tac t strand serving a s a template. The newly synthesized nucleic a c i d , be i t DNA or RNA, would code for the synthes is of the other nucleic a c i d . Finally the l a s t nucleic acid formed a c t s t o repair the damaged site of the DNA molecule through a mechanism which, unfortunately, remains unclear. for t he rapidly formed DNA and RNA.

The resu l t s of t hese s tud ies do not es tab l i sh a hierarchy of interaction

The brief interval of accelerated labeling af ter 100 rads could represent nucleic a c i d synthes is a t a local ized point in the genome. Certain regions of t he DNA molecule might be more suscept ible to radiation damage than o thers . S ince 100 rads produces relat ively l i t t le le thal damage, as evidenced by a high surviving fraction for the cells (0.8) , most of the injury a t th i s dose would cen te r i n the sens i t ive region: t he increased nucleic ac id synthes is then indi- cates the c e l l ' s effort t o reverse the damage. Higher doses (250 and 500 rads) affect even more regions of the genome, resul t ing in simultaneous nucleic ac id syn thes i s a t multiple foci. In th i s c a s e , t h e uptake curve a t higher doses re f lec ts a composite of several uptake curves such a s the type seen af ter 1 0 0 r ads . If the synthet ic ac t iv i t ies a t the different foci a re somewhat out of phase , t h e loss of label a t one point would tend t o cance l the uptake of label a t another point. This effect then l eads to the diminished response seen a t the higher d o s e s .

The reduced ATP content for starved c e l l s irradiated with 1 0 0 rads could represent 1) uti l ization of ATP to supply the energy needed for nucleic ac id

24

synthes is and/or 2 ) incorporation of the aden ine moicry into t i , c nuclcir: a c i d s . The increased uptake of inorganic phosphate-P32 3 t th is time p r o h b l y ir-idicatQs new ATP synthes is s ince the level recovers rapldly (Figures 4 cir,ri 5). T h e con- t inuous labeling with inorganic phosphate-P32 (Figure 6) and fidcnosine-Cl4 (Figure 9) suggest that t h i s method effectively prelabels the A T frc;ction. Aqain, i n this c a s e , a n increased utilization of ATP is indicated.

While pronounced changes i n ATP metabolism occurred after 1 :D rads for cells in HBSS, higher d o s e s did not produce differences. This coulrj resul t from the time needed for accelerat ing iabeiing. i f ATP is necessary for i k , e ;;:GI, aiid RNA label ing, then the uptake curve in Figure 8 shows that the requirer;ent must be met rapidly when 100 rads is used. In cont ras t , the labelincj a t hiqher d o s e s seems to extend over a much longer period. Consequent ly , the labeling would not be so extensive i n a given interval. Possibly the cell h a s t i m e to acijilst i t s ATP level when the longer periods of labeling a re involved, but i r cannot meet the demand af ter 1 0 0 rads . Regardless of the si tuation a t higher closes, measurable changes in ATP metabolism do occur after 100 r a d s , even under extreme ATP deficiency (Figure 1 2 ) .

I

The quest ion arises as to why radiation-induced changes appear in energy- deprived cells but not i n c e l l s irradiated in growth medium. The answer coda lie in the relat ive r a t e s of anabolic processes under these conditions. C e l l s s tarved in glucose-free HBSS or t reated with DNP show a reduced macromolecular synthes is compared t o growing ce l l s . Perhaps the radiation-induced changes compete with normal metabolic processes . i f t h i s is the c a s e , energy-depriva- tion might reduce the competition in favor of the former, result ing i n responscs not apparent under normal growth conditions.

A s Figure 13 shows , DNP treatment markedly enhanced the survival of

This increased survival presumably resu l t s from physicochemical or cells. levels. metabolic events occurring after irradiation. Figure 1 2 contains evidence for radiation-induced changes in nucleic acid metabolism under conditions which lead to a n increased survival.

The dose-response curves indicate that the effect is present a t all dose

CONCLUSIONS

The radiation induced changes i n nucleic ac id metabolism reported here are unprecedented, Since comparable responses did not appear for cells in growth medium, t h e s e changes presumably occur because of the energy-deprived stace.

The acce lera ted nucleic ac id labeling appears to be under c l o s e cellul-y a& con- trol a s shown by the relatively fixed delay (25 - 40 minutes for cells i n log growth)

2 5

l and the brief intcrva 1 of labeling (less than 4 . 0 m i n u t e s ) followjnq 190 r ads . Although higher doses gave similar responses I they were not so clear ly seen.

While no hierarchy of interaction can be es tab l i shed from the p rcsen t st'udy, the c l o s e parallelism of labeling for DNA and RNA indicates a cause- effect relationship. The accelerated labeling of RNA h a s the charactc risiics of messenger RNA: the labeling of DNA is novel. The evidence points rc a

The decreased ATP content and increased labeling for ce l l s irradiated with 100 rads furtner indicates that energy-requiring reactions (nucleic ac id synthes ls j a re occurring.

I DNA which is rapidly synthesized and then rapidly degraded.

I Survival s tud ies showed that c e l l s treated with D N P have a hiqher post- irradiation survival than cells i n growth medium. This increased survival may resul t from the metabolic changes observed af ter irradiation.

REFERENCES

1.

2 .

3.

4.

5.

6.

7.

M . M. Elkind and G. F. Whitmore. The radiobiology of cultured mammalian cells. Gordon and Breach, New York, 1967.

M . M. Elkind and H. Sutton. X-ray damage and recovery in mammalian cells i n culture. Nature, 184: 1293-1295 , 1959.

M . M . Elkind and H . Sutton. Radiation response of mammalian cells grown i n cul ture . I . Repair of X-ray damage in surviving Chinese hamster cells. Radiation Res. , 13: 556-593 , 1960.

T . T . Puck and P . I . Marcus. Action of X-rays on mammalian cells. L. Exotl. Med. 103: 653-666, 1956.

M . M. Elkind, H. Sutton-Gilbert, W. B . Moses, T. Alescio and R . W. Swain. Radiation response of mammalian cells grown in culture. V . Temperature dependence of the repair of X-ray damage in surviving cells (aerobic and hypoxic). Radiation Res. 25: 359-376 1965.

G. F. Whitmore , S . Gulyas and J. Botond. t he cell c y c l e and its relationship to recovery. In Cellular Radiation Bioloqy (published for the University of Texas , M . D. Anderson Hospital) pp. 423- 441 , Williams and Wilkins Co. , Baltimore I 1965.

Radiation sensi t ivi ty throughout

W. K . Sinclair and R. A. Morton. Survival and recovery i n X-irradiated synchronized Chinese hamster cells. In Cellular _Radiation Bioloqy (pub- l i shed for the University of Texas , M . D. Anderson Hospital) pp. 418-422 , Will iams and Wilkins Co., Baltimore, 1965.

26

8. R . A. Phil l ips and TA. J . Tolmach. X-irradiated HeLa c e l l s . Radiation - Res. 29: 413-432, 1 9 6 6 .

Repair of potentially lethal damaqc: i n

9. M . M . Elkind, W. B . Moses and F I . Sutton-Gilbert. mammalian cells grown i n cul ture . V I . Protein, DNA and RSA inhibition during the repair of X-ray damage.

Radiation response of

Radiation &. 31: 156-173, 1967 .

10. A . Han, B. Mi le t ic , D. Petrovic and D. Jovic. Survival properties and repair of radiation damage in L cells after X-irradiation. Intern. i. Radiation Biol. 8: 201-211, 1964.

11. M . K . Johnson and E . J . Johnson. Carbohydrate metabolism i n strain L-929 mouse fibroblast cells. P a c . SOC. Exptl. Biol. Med. 111: 149-152, 1962.

12. C . de Luca. Effects of mode of culture and nutrient medium on cyc l ic var ia - t ions in enzyme ac t iv i t ies of mammalian cells cultured vitro. Ex.=~l. Cel l - Res. 43: 39-50 , 1966.

13. A. Zetterberg. Nuclear and cytoplasmic nucleic ac id content and Cytoplasmic protein synthes is during interphase in mouse fibroblasts i n vitro. Exptl. Cell - ESS. 43: 517-525, 1966.

1 4 . K . K . Sanford, W. R . Earle and G. D. Likely. i so l a t ed t i s s u e cells. 1. Natl. Cancer Inst. 9: 229-231, 1948.

The growth in v ~ o of s ingle

1 5 . H. Eagle. Amino ac id metabolism in mammalian ce l l cul tures . Sc ience 130: 432-437, 1959.

16 . G. Schmidt and S . J . Thannhauser. A method for the determination of deoxy- r ibmucle ic ac id , ribonucleic a c i d , and phosphoproteins in animal t i s s u e s . L. Biol. Chem. 161: 83-89, 1945.

17. S . Addanki, J . F . Sotos and P . D. Rearick. Rapid determination of picomole quant i t ies of ATP with a liquid scint i l la t ion counter. Anal. Biochem. 14: 261-264, 1966.

1 8 . R. B . Hurlbert , H. Schmitz, A . F . Brumm a n d V . R , Potter. Nucleotide metabolism. 11, Chromatographic separation of acid-soluble nucleot ides . L. Biol. Chem. 209: 23-39, 1954.

3 19. H. E . Johns. The Physics of Radioloqy, pp. 703 , Charles C . Thomas, Springfield, I l l . , 1961.

27

I 20. E . Volkin and L. Astrachan. Phosphorous incorporation i n w c r r i c h i a c,oli ribonucleic ac id af ter infection with bacteriophage T-2 . Viro1oc;y 2: 1 4 9 - 1 6 1 , 1956.

21. R . B . Painter. Unscheduled DNA synthes is following X-irradiation of HeLa cells (Abstract). Radiation &. 31: 6 0 8 , 1 9 6 7 .

2 2 . R. B. Haynes. The interpretation of microbial inactivation and recovery phenomena. Radiation Res. Suppl. 6: 1-29, 1966.

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. .