Unscheduled DNA synthesis in human leucocytes

Mutation Research

393

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

U N S C H E D U L E D DNA S Y N T H E S I S IN HUMAN LEUCOCYTES

W. G. CONNOR AND A. NORMAN

Department of Radiology, Uniw'rsity Hospitals, University of Wisconsin, Madisou, Wis. 53706 ; and Department qf Radiology, UCLA School of 5ledicine, Los Angeles, Calif. 00024 ( U.S./I .) (Received December 24th, 197 o) (Revision received August 5th, I971)

SUMMARY

Polymorphonuclear leucocytes have been induced to synthesize new DNA by exposure to UV light. Preliminary observations (not included) also indicate that 6-MeV electrons and incubation with the radiomimetic agent methyl methanesulfonate (MMS) are effective agents for inducing unscheduled DNA synthesis (UDS). A study of the kinetics of UV-induced DNA synthesis suggests that there are at least two processes operating, one fast and essentially complete within the first 1-2 h and the second lasting at least 8 h. The rapid process can be described by a simple enzyme model using iaH] thymidine and induced lesions as substrates. A kinetic study of the rapid process, following exposure to UV, yielded values for the maximum velocity of 5.I.IO -=' moles/cell-h and 5.5"IO-"° moles/eell-h for the granulocytes and lymphocytes respectively. The rate constants for the rate-limiting steps were 1. 4- Io --~ (min) -* and x.I-IO -~ (min) -~ for granulocytes and lymphocytes respectively. Using these values, the concentration of enzyme molecules are estimated to be 4' I°a molecules/cell for polymorphonuclear leucocytes and 5' IO4 molecules/cell for lymphocytes.

INTRO1) UCTION

Unscheduled DNA synthesis (UDS), first described by RASMUSSEN AND PAINTER~%n, is the incorporation of DNA precursors into the DNA of cells which are not in the normal S-phase of their cell cycles. This incorporation can be induced by UV light4,t°, x~, ionizing radiationsT, *a and radiomimetic agents6, .2, agents that produce damage to the DNA of tile cell and have lethal or mutagenie effects. UDS has been demonstrated in a variety of mammalian cell typeslL It has also been demonstrated in cells in all phases of their life cycles except for the normal S-phase when it is obscured by normal DNA synthesis, or for fully differentiated cells. EVANS ANI) NORMAN4 studied UDS in circulating human blood lymphocytes. SPIEGLH~ AXD NORMANla have demonstrated two types of UDS in lymphocytes, a fast process which Abbreviations: MMS, methyl methanesulphonate; PMN, circulating granulocytes; UDS, unscheduled DNA synthesis.

M.ulation Res., 13 (i97 I) 393-402

394

w. G. CONNOR, A. NORMAN

is essentially complete within the first hour and a slower process which lasts at least 7 h. The rate-limiting step for the fast process can be described by a simple enzyme model in which the substrates are thymidine- and radiation-induced lesions. Hydroxyurea in a concentration of IO-a M, which inhibits normal DNA synthesis, has no effect on UDS while aetinomycin D, which binds to DNA, inhibits both UDS and DNA synthesis. In the present study we have compared UDS in human blood lymphocytes with that of the circulating granulocytes (PMN) after exposure to UV-radiation. These cells differ markedly. Most of the circulating lymphocytes have a long lifespan and are in a Go phase of their life cycles but can be stimulated to transform and divide by a variety of mitogens'L On the other hand, the PMN are unable to divide and have a short lifespan in the circulation before entering the tissues to act as phagocytes. The process of phagoeytosis results in cell death, so these leucocytes are truly cells in a terminal phase. RABINOWITZ"demonstrated that PMN have at least an order of magnitude less polymerase activity than the lymphocytes. If it is assumed that DNA polymerase activity is a prerequisite to UDS, PMN would have no UDS activity or they would show a slower rate of UDS commensurate with their low level of polymerase activity. Our results show the latter to be true, MATERIALSAND METHODS Cell collection and separation Whole venous blood was collected from normal human donors and/or patients with polycythemia into flasks under gravity and defibrinated during collection by constant stirring with glass beads. The PMN and lymphoeytes from patients with polyeythemia behaved as normal cells. Plasmagel (Roger Bellon Laboratories, France), a modified gelatin, was added in the ratio of one part Plasmagel to three parts of defibrinated blood to promote rouleau formation. The blood was allowed to settle in a graduated cylinder at room temperature for about 60 rain and the leucocyterich supernatant was then harvested. The leucocytes were separated by the method of RABINOWlTZs. Columns I cm in diameter were packed loosely to I6 cm in height with glass spheres 2oo/~nl in diameter, which had been thoroughly cleaned, siliconized (Siliclad, Clay-Adams Inc., New York) and sterilized. Columns packed with nylon fibers according to the procedure of WALFORD'" and glass wool columns were also used. Sterile conditions were maintained and the temperature of the column was controlled by circulating preheated (37 °) water continuously through a jacket surrounding the column. The lymphocyte fraction was eluted with serum-supplemented Hanks' solution at a flow rate of about I. 5 ml per min. The column was washed free of lymphocytes and red blood cells and the remaining leucocytes were eluted from the column with EDTA buffer. Ultraviolet light irradiation UV-irradiations were carried out with a Mineralight (Ultraviolet Products, San Gabriel, California). The lamp is a low-pressure mercury discharge lamp equipped with a visible light filter so that most of tile transmitted energy has a wavelength of 2537 ~. Calibration was accomplished with a thermister-operated Wheatstone bridge with an output voltage directly proportional to the radiant energy (USI model RadioMutation Res., 13 (~97I) 393 4°2

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SYNTHESIS IN HUMAN LEUCOCYTES

395

meter, Yellow Springs Instrument Co., Ohio). The samples were suspended in the medium in which they were irradiated, and layered in sterile, siliconized petri dishes and placed 40 cm from the source. The dose rate received by the samples was 2.5 erg/mm ~.sec.

Liquid scintillation counting The cells were pulsed with tritiated thymidine in the incubation medium (a, thymidine-6-T(n), Amersham/Searle, Nuclear Chicago, Calif.; b, methyl-l~H]thymi dine, Schwarz BioResearch Inc., Orangeburg, New York) at various concentrations, immediately after exposure to UV. After the radioactive pulse, the cells were resuspended in incubation medium supplemented with 50/~g/ml of unlabelled thymidine (CalBiochem, Los Angeles) for 15 rain at 37 °. A second wash with Hanks' BSS followed the unlabelled thymidine wash. Following this, the cells were diluted with 2 ml of Hanks' BSS and 4 ml of a 5-g/l cetrimide solution (cetylotrimethyl ammonium bromide, K & K Laboratories, Inc., Hollywood, California). This suspension remained at room temperature for 5 min in order to lyse the cellular membranes and leave a suspension of nuclei. I ml of this suspension was added to 9 ml of Isoton (Scientific Products, Evanston, Illinois) for counting of the nuclei on tile Coulter counter (Coulter Electronics, Chicago). The remaining 5 ml were deposited on millipore filters (HAWP 0.45 ~, Millipore Corp., Bedford, Massachusetts). 5-8 ml each of Hanks' BSS, 5~}/o trichloroacetic acid (Allied Chemical, Morristown, New Jersey) and absolute ethanol were then washed through the filters. The filters were thoroughly dried and placed in IO ml of scintillation flour (5.5 g Permablend I, Packard Instrument Co., per 1 of toluene) and counted on a Packard Tri-Carb Liquid Scintillation Spectrometer (Packard Instrument Co., Inc., Downers Grove, Illinois). The channel ratio method was used to correct the counts.

Autoradiography For autoradiography the cells were fixed in Carnoy's (3 parts ethanol and i part acetic acid) after washing with cold thymidine, and Hanks' BSS. Cell suspensions in a small volume of Carnoy's solution were dried on slides, which were dipped in photographic emulsion (Kodak, Nuclear Tract NTB2) and stored at 4 ° for lO-2O days. The exposed slides were developed for 2 min in Kodak Dektol at 12 °, rinsed and fixed in Kodak rapid fixer for 8 min at 12 °. The developed slides were stained in Harris' hematoxylin for 5-8 nfin, differentiated with one or two dips in acid alcohol (I ml conc. HC1 in IOO ml of 7OYo ethanol) and thoroughly washed. After drying, the slides were cleaned in Xylol for 5 rain and covered with slips mounted in Permount. RESULTS

Preliminary experiments were carried out to evaluate the cell separation technique, to demonstrate the ability of the PMN to carry out UDS, and to measure the effects of hydroxyurea on S-phase cells which m a y contaminate the PMN fraction. The cell separations obtained with our glass bead columns were essentially identical to those obtained by RABINOWlTZ8. The lymphocytes could be completely separated from the rest of the leucocytes. Residual lymphocytes and red blood cells could be decreased to any desired concentration prior to elution of the PMN thus 1?,Iulation ires., 13 (1971) 393-4o2

.~t)6

X.V.G. C O N N O R , A. N O R M A N

eliminating a l y m p h o c y t e c o n t a m i n a t i o n of the PMN fraction. Elution of the PMN could be accomplished with nfinor c o n t a m i n a t i o n b y monocytes. The u p t a k e of aH ]thwnidine b y u n s e p a r a t e d and u n s t i m u l a t e d leucocytes is depressed b y h y d r o x y urea; it is down b y a factor of 7 at io ~ M and a factor ~f 12 at IO 2 M concentrations of h y d r o x y u r e a so t h a t h y d r o x y u r e a was used r o u t i n e l y during these e x p e r i m e n t s to limit the u p t a k e of ! a H ] t h y m i d i n e bv the leucocytes. A u t o r a d i o g r a p h i c studies of UDS in PMN showed t h a t 5o(),~, of the cells exposed to IOO erg/mnl 2 of I,!V and inc u b a t e d with !uH ithymidine for 2 h had b grains or more over their nucleus while Ioo¢~.i, of the u n i r r a d i a t e d controls had three or less grains over their nucleus. These results established t h a t the m a j o r i t y of PMN are capable of [-DS following exposure to UV. Using liquid scintillation counting to measure the UDS in PMN, it was found t h a t MMS a n d ionizing r a d i a t i o n as well as UV s t i m u l a t e d UDS in PMN. The U D S a c t i v i t y of PMN as a function of UV dose is shown in Fig. I. The circles are values o b t a i n e d when the dose was varied from zero to 2oo e r g / m m 2 and

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Fig. [. R a d i a t i o n - s t i m u l a t e d i n c o r p o r a t i o n of [ : ~ l l ! t h y m i d i n c as a f u n c t i o n of I ' V d o s e . I ' M N w e r e i n c u b a t e d w i t h [aH I t h y m i d i m ! (o. 5 • l o ~ 3I, [ 7 C i / m M ) , in p r e s e n c e of [o :~ 31 h y d r ( ) x v u r c a , for 2 h. l;i x. z. l ; n s c h c d u l e d 1 ) N : \ s \ n t h c s i s a s a f u n c t i o n of t i m ~ as m e a s u r e d b y t h e i n c o r p o r a t i o n or' I:~H t h y m i d i n c a f t e r e x p o s u r e to U V . P M N w e r e e x p o s e d t o 5o e r g / n n ] l ~" IT\' a n d i n c u b a t e d c o n t i n u o u s l y f o r v a r i o u s t i m e s w i t h i a H ] t h y m i d i n e (o. 5 • IO " ,ll, 17 C i / m 3 1 ) .

tl/e squares are for a low-dose e x p e r i m e n t when the dose was varied from zero to 3o e r g / m n F . The results show t h a t !a H ! t h y m i d i n e u p t a k e increases r a p i d l y for doses in the range of zero to 25 or 3o e r g / m m L and increases v e r y little with doses in the range of 3 ° 2oo e r g / m m ~. The time dependence of UDS a c t i v i t y for a single UV dose of 5 ° e r g / m m 2 is illustrated in Fig. 2. As can be seen, the u p t a k e increases continuously for at least 8 h and is a linear function of time from a b o u t the I s t or 2nd h. The d a t a show t h a t the rate of u p t a k e within the I s t h is higher t h a n the rate from the 2nd to the 8th h. These d a t a are from two e x p e r i m e n t s (circles and squares), and the control points for the e x t e n d e d time e x p e r i m e n t are p l o t t e d as triangles. The d a t a , as shown however, were corrected for u p t a k e by cells which were not irradiated. The control ( u n i r r a d i a t e d cells) curve is shown to indicate the b a c k g r o u n d level for these experiments. :~lulaliou R e s . , 13 (1971 ) 393 =t° 2

UNSCHEDULED

DNA

397

S Y N T H E S I S IN HUMAN L E U C O C Y T E S

The results illustrated in Fig. 2 suggest that two repair processes are operative, in agreement with the results of SPIEGLER AND NORMAN with lymphocytes exposed to ionizing radiation 13. The first is fast and essentially complete within I or 2 h and the second is slower, continuing for at least 8 h. As suggested by these authors, a simple enzyme model can describe the incorporation of ~aHlthymidine into DNA due to lesions produced by the radiation. The rate-limiting steps of the fast process can then be described by the model using ['~Hlthymidine and induced lesions as substrates for the enzyme system. This is of course an oversimplification because 3Hlthymidine is not a direct substrate for DNA synthesis. Moreover, different kinds of lesions produced in the DNA m a y be repaired in different ways. If one assumes a system analogous to that described for bacterial DNA repair, i.e., an excision repair process, then several steps would be involved in UDS and we would observe only the ratelimiting step. One can describe the kinetics according to the model1: E+N~EN

k2

L + EN ,~ EP k4

h'5

EP-~ E+P where E - ~ free enzyme; N - !3H~thymidine or tile incorporated substrate, concentration of which is assumed to be proportional to that of the exogenous ! 3 H thymidine; L - lesion, concentration of which is assumed to be proportional to dose; P repaired lesion; K1 constants dependent on the rate constants ki's; V =- rate of production of P, assumed proportional to the rate of incorporation of [aHlthymidine. Assuming that the concentrations of N and L are large compared to the number of available enzyme sites, then the bound amount of N and L can be neglected. Under steady state conditions in which the concentrations of enzyme-substrate complexes are not varying appreciably, the differential equations describing their change with time m a y be set equal to zero. This implies that d[ENl/dt = d[EPl/d/ - o and tile resulting expressions with the aid of tile additional equation [Eo~

!E] 4- !EN] + [EP]

where E0 is equal to the total enzyme concentration, m a y be used to derive a rate law for the system. Thus, I

I

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v =-: V,n.x [I-~

K l

K3 ]

K.~

N~ + iL~

+ [N i

where tk" 1 = k S / k l , K 2 = (k4----k5)//~3, I~"3

k2(k4+k5)/klk3,

Vmax =

k 5 [ E o I.

This rate law does not distinguish between mechanisms which obey similar rate laws but does exclude those yielding different rate equations. If the [aH!thymidine concentration is held constant and the lesion concentration allowed to vary, i.e. varying tile dose, then the constants in the simplified Michaelis-Menten equation turn out to be: 3Julation Res., 13 (1971) 3 9 3 - 4 0 2

398

w . G . CONNOR, A. NORMAN

~ 7 Ill }t x

k~Eo[Nj k, + rN]

2

t-,.,[N j~+th'~ IN]

/,'1+

Similar equations are obtained for Vm~x and K,,, when the lesion concentration is held constant and the laH]thymidine concentration is allowed to vary. Our kinetic data show that the model is compatible with the observed results. The Lineweaver-Burk form of the Michaelis-Menten equation is: I

I

I'~ m

The kinetics of UDS were obtained for both PMN and lymphocytes for comparison and analyzed according to the above model. The UV-stimulated UDS activity, in the early phase of the process, is illustrated in Figs. 3 and 4. The curves on the right are plots of uptake velocity vs. concentration

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Fig. 3. Unscheduled DNA synthesis as a function of time and [aHjthynlidine concentration PMN. The PMN were exposed to 50 erg/mm 2 of UV light and incubated for lo, 2o and 30 rain various concentrations of exogenous [aHjthymidine. Fig. 4. Unscheduled I)NA synthesis as a function of tinle and IaHithymidine concentration lynlphocytes. The lymphocytes were exposed to 5 ° erg/nlm 2 of U\; light and incubated for Lo, and 3 ° rain in various concentrations of exogenous aHjthymidine.

in in in 2o

a n d t h e r e c i p r o c a l s of t h e s e v a l u e s as o b t a i n e d f r o m t h e slopes of t h e u p t a k e vs. t i m e c u r v e s . I n Figs. 3 - 6 tile d i f f e r e n t p o i n t s o n t h e u p t a k e vs. t i m e c u r v e s r e p r e s e n t d a t a a t a g i v e n c o n c e n t r a t i o n of ! a H ] t h y m i d i n e or a t a g i v e n d o s e a n d t h e slopes a r e ot)t a i n e d f r o m t h e lines f i t t i n g t h e s e p o i n t s • T h e d a t a in Fig. 3 w e r e o b t a i n e d f r o m P M N ; Fig. 4 s h o w s t h e l y m p h o c y t e r e s u l t s . T h e k i n e t i c s of U V - s t i m u l a t e d U D S as a f u n c t i o n of dose a r e s h o w n in Figs. 5 a n d 6. T h e d a t a for P M N a r e s h o w n in Fig. 5 a n d t h o s e for l y m p h o c y t e s in Fig. 6. As in t h e p r e v i o u s e x p e r i m e n t , l y m p h o c y t e s a n d P M N w e r e o b t a i n e d f r o m t h e s a m e donor and both experiments were performed on the same day. Tim velocity curves w e r e a g a i n o b t a i n e d f r o m t h e slopes of t h e c u r v e s o n t h e left for d o s e s r a n g i n g t o 5 ° e r g / m m 2. Mutation Res., 13 (1971) 393-402

DNA

UNSCHEDULED

399

SYNTHESIS IN HUMAN LEUCOCYTES

The curves shown in Figs. 3-6 are representative of several experiments. Estimates for Vm, obtained by extrapolating the I/v v s . I/dose or I / I3HJthymidine curves back to the ordinate ranged from 3.3 to 7.7" IO-2~ moles/cell-h with a mean of

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Fig. 5- U n s c h e d u l e d D N A s y n t h e s i s as a f u n c t i o n of t i m e a n d UV dose in PMN. The cells were i r r a d i a t e d w i t h v a r i o u s doses of UV a n d i n c u b a t e d in [ a H I t h y m i d i n e (IO "~ M) for IO, zo a n d 3 ° min. Fig. 6. U n s c h e d u l e d D N A s y n t h e s i s as a f u n c t i o n of t i m e a n d UV dose in I y m p h o c y t e s . The cells were i r r a d i a t e d w i t h v a r i o u s doses of UV a n d i n c u b a t e d in EaH]thymidine (IO -6 M) for IO, 2o a n d 3 ° Inin.

5.1 .Io -21 moles/cell-h for PMN and 3.1-8.3. IO 2o moles/cell-h for the lymphocytes with a mean of 5.5' Io-2° moles/cell-h. Another approach is to look at the decrease in the number of lesions as a function of time for low lesion concentrations 14. Assuming the rate constants kl and k3 are large compared to k5 and the concentration of N is essentially constant so that FEN 1 >> FL1, the reaction, according to our model, would go rapidly to EP with essentially all of the L substrate incorporated into the E P complex. Then the decrease in EP would equal that of the number of lesions. It has also been assumed that the reverse reactions with the rate constants ks and k4 can be neglected. Thus, d [EP 1 dt

k5 [EPI

--

and

[EP 1 = IEPloe-~t If we incubate the cells continuously after UV exposure but add the L3HIthymidine at various times after the start of incubation, then the decrease in uptake with time at which the I3Hlthymidine is added reflects the loss of the E P complex as a function of time. Rewriting the expression for EP: In

LEPI [EPI0

--

k~t

we see that a semi-log plot of the data yields a straight line with a slope equal to --ks. The results shown in Figs. 7 and 8 are for a low UV dose. In these experiments, 13H~thymidine was added at various times after the UV exposure and the start of 3Iutation Res., 13 (1971) 393-4o2

400

W . G . CONNOR, A. NORMAN

the 4-h incubation period. Fig. 7 shows the results o b t a i n e d with PMN and Fig. for l y m p h o c y t e s . The plots are on semi-log p a p e r and the decrease in u p t a k e reflects the shorter times t h a t the cells were exposed to the i~Hithymidine. The d a t a have been corrected for zero exposure controls. These, again, are r e p r e s e n t a t i v e d a t a and the e s t i m a t e s f o r / ~ wtried from o.81 . 0 . i o 2 (rain) ~ for both l y m p h o c y t e s and PMN with a mean value of 1 . 3 0 " 1 0 2

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Fig. 7. U n s c h e d u l e d I ) N A s y n t h e s i s a t v a r i o u s t i m e s a f t e r low 1;V doses in PMN. The cells were expo sed to 15 e r g / m m 2 a n d the i n c u b a t i o n s t a r t e d i m m e d i a t e l y t h e r e a f t e r . ~:~H]thymidine (~o (~3]) was t h e n a d d e d a t 2-, 4-, 6- a n d 2o-min i n t e r v a l s a f t e r I;V exposure. The i n c u b a t i o n was t h e n c o n t i n u e d for 4 h when all of the s a m p l e s were prncessed for c ount i ng. The d a t a are p l o t t e d in a r b i t r a r y u n i t s of u p t a k e (m semi Io~ g r a p h paper. I

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Time (min) l;ig. 8. Urlscheduled D N A s y n t h e s i s at v a r i o u s t i m e s afte r low UV doses in l y m p h o c y t e s . The cells were t r e a t e d in the s a m e m a n n e r as t h e p r e v i o u s e x p e r i m e n t .

(min) -1 for PMN and I . I . I O :~ (rain) -1 for lymphocytes. U V doses in tile range of 15 5 ° e r g / m m 2 were used. Subsequent experiments, extending the time of the low dose experiments to (~o min, yielded rate constants within the range of the previous experiments. The ext e n d e d time enabled us to observe the decrease in the E P complex down to 5o}i, of its original value. Finally, considering the expressions for V ..... derived by holding one substrate in each instance constant, they m a v be rewritten: Mulalio*z Rex., 13 (1971) 393 4 °2

UNSCHEDULED

V .....

Vmax

--

DNA

SYNTHESIS IN HUMAN LEUCOCYTES

k~IEol I +K~/[NI ks[E0] I+K2/IN]

401

for c o n s t a n t N, a n d

for c o n s t a n t L.

R e m e m b e r i n g t h a t K~ ks~k1, a n d K 2 - - ( k 4 + k ~ ) / k 3 , a n d our a s s u m p t i o n s t h a t k~ >> ka, a n d k3 >> k5 + k4, we m a y assume t h a t k~/lN] and K2/IL] are negligible c o m p a r e d to one so t h a t : V . . . . = ks[E0~ Using the results o b t a i n e d for k5 a n d Vm~x one can e s t i m a t e the enzyme concentration. The results are: E0 = 5" lO4 enzyme molecules/cell, for l y m p h o c y t e s , a n d E0 - - 4" IO3 e n z y m e molecules/cell, for PMN. DISCUSSION

The results shown in Figs. i and 2 d e m o n s t r a t e t h a t UV s t i m u l a t e s the u p t a k e of EaHithymidine into the m a j o r i t y of PMN. Recently, FREY-WETTSTEIN a n d colleagues 5 published the results of a s t u d y of the U D S a c t i v i t y in n o r m a l a n d leukemic leucocytes. The emphasis of their s t u d y centred on l y m p h o c y t e s from different areas of the l y m p h a t i c system. T h e y did, however, score g r a n u l o c y t e s and were unable to d e m o n s t r a t e U D S in PMN. Their failure to observe U D S in PMN is difficult to unders t a n d since our results show v e r y definitely t h a t at least three quite different agents, UV, ionizing r a d i a t i o n a n d MMS, s t i m u l a t e U D S in the PMN. The kinetic d a t a o b t a i n e d from studies of U V - s t i m u l a t e d U D S in PMN is different from t h a t in l y m p h o c y t e s . A l t h o u g h the rate constants, k~'s, for these cell t y p e s are the same, the Vm~x values were different b y an order of m a g n i t u d e . This implies t h a t the same enzyme was r a t e - l i m i t i n g in b o t h cell types, b u t the n u m b e r of e n z y m e molecules differs m a r k e d l y . W e do not know which enzyme is responsible for the r a t e - l i m i t i n g step in our model b u t the decrease in enzyme c o n c e n t r a t i o n for PMN c o r r e l a t e d well with the observation b y RABINOWITZ 8 t h a t D N A p o l y m e r a s e a c t i v i t y in PMN is down b y an order of m a g n i t u d e when c o m p a r e d to t h a t in the lymphocyte. V . . . . 's o b t a i n e d b y SPlEGLER AND NORMAN 15, for U V - s t i m u l a t e d U D S in l y m p h o c y t e s , in the range 5 - i 0 . I0 -2o moles/cell-h and an e s t i m a t e of Vma× o b t a i n e d from the d a t a published b y EvANs AND NORMAN4 of 2" I0 -2o moles/cell-b, also for U V - s t i m u l a t e d U D S in l y m p h o c y t e s , are in reasonable a g r e e m e n t with the results o b t a i n e d for U V - s t i m u l a t e d l y m p h o c y t e s in the present work. Thus one m i g h t speculate t h a t the different Vm~x'S o b t a i n e d for l y m p h o e y t e s a n d PMN, when using UV to s t i m u l a t e the UDS, is a reflection of a decrease in enzyme concentration in PMN a n d it could well be due to a decrease in D N A polymerase activity. There also a p p e a r s to be a significant difference in the results o b t a i n e d with UV a n d ionizing radiation. In l y m p h o c y t e s SPIEGLER AND NORMAN14 o b t a i n e d a k5 5 ' lO-2 (rain) -1 using 6-MeV electrons to s t i m u l a t e U D S whereas these results v a r i e d between 0.89 a n d 1. 5 . IO-2 (rain) -1 with UV. The r e s u l t a n t half-lives for the loss of the E P complex, according to our model, are 14 rain a n d 63 min, respectively. The d a t a o b t a i n e d for Vmax'S are in closer agreement. As n o t e d earlier, the m e a n Vm~x 3lutation Res., 13 (1971) 393 4 ° 2

402

w. G. CONNOR, A. NORMAN

o b t a i n e d for l y m p h o c y t e s w i t h U V in t h e p r e s e n t w o r k w a s 5.5" IO 21, m o l e s / c e l l - h . O t h e r v a l u e s I5 o b t a i n e d w i t h U V v a r i e d b e t w e e n 5 a n d IO. IO ,,0 m o l e s / c e l l - h . V a l u e s o b t a i n e d f o r Vm~, u s i n g i o n i z i n g r a d i a t i o n as t h e s t i n m l u s i n c l u d e 7" I O 2,, m o l e s / c e l l - h (ref. 13) a n d 7 " I O .~0 m o l e s / c e l l - h (ref. 15). T h e s e r e s u l t s m i g h t i m p l y t h a t t h e r a t e l i m i t i n g s t e p s for t h e r e p a i r of U V a n d i o n i z i n g r a d i a t i o n d a m a g e are d i f f e r e n t , i t is t e m p t i n g t o t h i n k of t h e e n d o n u c l e a s e s t e p as b e i n g r a t e - l i m i t i n g for U V b u t n o t for ionizing radiations. The data from Xeroderma pigmentosum patients", at any rate, s h o w t h a t t h e a b s e n c e of t h e e n d o n u c l e a s e s t e p does n o t s u p p r e s s U D S f o l l o w i n g ionizing radiation. ACKNOWLEDGEMENTS T h i s w o r k w a s s u p p o r t e d in p a r t b y t h e U S A E C a n d a U S P H S T r a i n e e s h i p . W e a c k n o w l e d g e g r a t e f u l l y t h e h e l p of D r . J . SCOTT in o b t a i n i n g t h e b l o o d for t h e s e studies. 11,lLl: I~t),t£NCES [ ALBERTY, I{. A., Enzyme kinetics, A d v a n . Enzymol., 17 11956 ) 1. 2 CLEAVER, J. E., Defective repair replication in xeroderma pignaentosum, Nature, 2I,~ 119681 652. 3 ELVES, M., The Lymphocytes, Year Book Medical Publishers, Chicago, 111., 1967. 4 EVANS, R. G., AND A. NORMAN, Radiation-stimulated incorporation of thymidme into the DNA of h u m a n lymphocytes, Radiation Res., 36 (1968) 287. 5 FREY-WETTSTEIN, M., R. LONGMIRE AND C. G. CRADDOCK, Deoxyribonucleic acid (1)NA) repair replication of ultraviolet (UV) irradiated normal and leukemic leukocytes, .]. Lab. Clin. Ale&, 74 11969) lO9. 6 HAHN, G. M., S. J. YANG AN1) V. PARKER, Repair of sub-lethal danaage and imscheduled I)NA synthesis in mammalian cells treated with monofunctional alkylating agents, Nature, 22o (I968) II42. 7 HILL, H., Non-S-phase incorporation of aH-thynlidine into I)NA of X-irradiated mammalian cells, lntern. J. Radiation Biol., 13 (19671 199. 8 RABINOW1TZ, Y., Separation of lynlphocytes, polynuclear leukoeytes and monocytes on glass columns including tissue culture observations, Blood, 23 (I964) 8i i. 9 RABINOWITZ, Y., DNA polymerase and carbohydrate uactabolizing enzyme content of normal and leukemic lynaphocytes, Blood, *-7 (I966) 47 o. i o RASMUSSEN. R. E., AND R. B. PAINTER, Evidence for repair of ultraviolet damaged (leoxyribonucleic acid in cultured mammalian cells, Nature, 2o 3 11964) 136o. II RASMUSSEN, R. E., AND R. B. PAINTER, Radiation stimulated DNA synthesis in cultured mammalian cells, J. ('ell Hiol., 29 119661 I I. l 2 Si'1EGLER, P., Kinetics of unscheduled DNA synthesis induced by ionizing radiation in h u m a n lymphocytes, Ph. i)..Dissertation, University of California, I,os Angeles, 1969. J 3 SPIEGLER, P., AND A. NORMAN, Kinetics of unscheduled DNA synthesis induced t)v ionizing radiation in h u m a n lymphocytes, Radiation Res., 39 {I969) 4oo. 14 SPIEGLER, P., AND A. NORMAN, Unscheduled DNA synthesis at low X-ray doses, Mutation lees., lo (197o) 379. I 5 SP1EGLER, P., AND A. NORMAN, Temperature dependence of unscheduled I)NA synthesis in h u m a n lymphocytes, Radiation Res., 43 (197 °) J87. 16 WALFORI), R. L., R. GALLAOHER AND P. TAY LAY, Cytotoxicity of pathologic h u m a n sera Ior lymphocytes and neutrophils, Am~. N . Y . Acad. Sci.. t24 (1965) 563 .

Mutation Res., 13 (197I) 393 4 o2