Photochemistry and Photobiology Vol. 35, pp, 275 to 277, 1982 Printed in Great Britain. All rights reserved

003 1-8655/82/020275-03$03.OO/O Copyright 0 1982 Pergamon Press Ltd

RESEARCH NOTE

PHOTOREACTIVATING ENZYME INDUCTION IN HUMAN LYMPHOCYTES

BETSY M. SUTHERLAND* and AMLETO CASTELLANlt

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA and tCNEN Centro Studi Nucleari, Rome, Italy

(Received 17 July 1981; accepted 29 August 1981)

Abstract-Photoreactivating enzyme (PRE) activity was determined in human lymphocytes cultured in the presence or absence of the mitogen phytohemagglutinin (PHA). Photoreactivating enzyme activity increased from 0 to 12pmol/mg/h in about 40h in cells cultured in the presence of PHA; enzyme activity was unchanged in the absence of PHA. The kinetics of induction of PRE and of lactate dehydrogenase, a marker of mitogenic stimulation, were indistinguishable, indicating that PRE induc- tion results from stimulation of transcription and translation by the mitogen, rather than a specific effect of the PHA on PRE production. The level of PRE was also higher in mouse lymphoblasts growing rapidly in RPMI 1640 medium than in the same cells growing more slowly in Dulbecco’s medium.

INTRODUCTION

The photoreactivating enzyme (E.C. 4.1.99.3.) mediates biological repair by the light-dependent monomerization of cyciobutyl pyrimidine dimers, formed by UV irradiation of cellular DNA (for reviews see Cook, 1970; Sutherland, 1981). Although this enzyme occurs almost ubiquitously, the level of photoreactivating enzyme (PRE)$ varies according to the species tissue, age of the organism (Cook and McGrath, 1967) and growth stage of the cells (Boling and Setlow, 1967; Tyrrell et al., 1972; Sutherland et a/., 1974). Photoreactivating enzyme levels for various types of human leukocytes were determined by Sutherland et al. (1974); they found high PRE activity in the monocytes (21.3 pmol/mg/h) and polymorpho- nuclear cells (92.5 pmol/mg/h), but little or no activity in the lymphocyte fractions (0.02-4 pmol/mg/h).

We were thus intrigued by the report of Henderson (1978), who observed photoreactivation of UV-irra- diated Epstein-Barr virus in lymphocytes of xero- derma pigmentosum patients. How could biological photoreactivation be observed in cells with very low P R E levels? Since Epstein-Barr virus infection induces host cell transcription and translation, we thought that other agents which stimulate cellular transcription and translation might also increase PRE synthesis. One such agent is the mitogen phytohe- magglutinin (PHA). We have thus examined the time course of induction by PHA of lactate dehydrogenase (LDH), an enzymatic marker of mitogenic stimu-

*To whom correspondence should be addressed. $Abbreviations: EDTA, ethylenediamine tetraacetate;

LDH, lactate dehydrogenase; PHA, phytohemagglutinin; PR, photoreactivation; PRE, photoreactivating enzyme; XP, xeroderma pigmentosum.

lation, and the production of PRE. We find that PRE activity increases from -0-12 pmol/mg/h, with ap- proximately the same time course as LDH induction. W e have also measured PRE levels in slowly and rapidly growing murine lymphoblasts. Our data sug- gest that in these lymphocytes, unlike other systems, rapidly growing cells contain higher PRE activities.

MATERIALS AND METHODS

Cells. Peripheral human blood was collected from healthy volunteers using heparin-treated springes. Ten mil- litres of blood was mixed with an equal volume of Hanks’ balanced salt solution (minus calcium and magnesium; Gibco), layered gently over 15mt LSM solution (Litton Bionetics), and centrifuged for 40 min at 20°C at 400 g. The lymphocytes at the interface between the plasma and gradient layers were removed by Pasteur pipette and resus- pended in 30 mP of Hanks’ solution. The cells were washed twice by centrifugation for 10 min at 800 rpm, the superna- tant solution removed and resuspensed in medium [RPMI 1640 (Flow Laboratories) plus 20% fetal bovine serum, 2 mM L-glutamine, penicillin (lo3 U/mf) and streptomycin (1 mg/mP)] with or without the addition of PHA (Gibco) 20pl/m8 of medium. Cells were grown at 37°C in a 5% COz atmosphere.

Mouse lymphoblasts were the gift of Dr. R. Daynes, University of Utah. Cells were grown in suspension in either RPMI 1640 medium plus 10% fetal bovine serum of a Dulbecco’s modified Eagle’s medium plus 20% serum.

Enzyme assays. Cells were harvested by centrifugation for 10min at 3000rpm in the SE12 rotor of the Sorvall RC5B. Cell pellets were either frozen or used immediately in the preparation of cell extracts. (Freezing of the cell pellets did not measurably affect the activity of PRE or LDH.) Cells were resuspended in Buffer E (10 mM tris, pH 7.2, 0.1 mM dithiothreitol, 0.1 mM EDTA) at a concen- tration of about 2 x lO’cells/m/. The suspension was sonicated for 45 s at power level 6 7 in a Kontes sonicator.

Photoreactivating enzyme was assayed as previously de- scribed (Sutherland and Chamberlin, 1973). In brief, cell extracts were added to 0.2 me of an assay mixture contain- ing 20mM phosphate buffer (pH 7.2). 0.1 mM EDTA,

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BETSY M. SUTHERLAND and AMLETO CASTELLANI

which varied with the donor and preparation. We next examined the time course of appearance of

C e l l exlract , pI

Figure 1. Photoreactivating enzyme activity in extracts of human lymphocytes cultured in the absence (0) or pres-

ence (A) of PHA.

0.01 m’M dithiothreitol and lOmM MgCl2, plus 1&30pmol of 32P-labeled T7 DNA which had been exposed to 289 nm radiation from a Johns’ monochroma- tor, and contained about 20% of its thymines as dimers. One sample was exposed to photoreactivating light for 30min at 3 7 C . while a duplicate sample was kept in the dark at 37°C. All samples were digested to a mixture of mononucleosides, inorganic phosphate and dimer-contain- ing oligonucleotides (Setlow ef a/., 1964) by the sequential addition of 25 pg of DNase I, then 10 p/ of 1 M tris, pH 8 and SO$ of a mixture containing 1 pg of Escherichia culi alkaline phosphatase and 1oOpg of crude snake venom phosphodiesterase in 50 mM tris (pH 8), 10 mM MgCI,. After I h. the digestion was stopped by the addition of 10pC of 0.062 N :lCl and 1 mk of a suspension of 4% acid-washed norit in 0.1 M sodium phosphate-0.1 M sodium pyrophosphate (pH 6). The mixtures were filtered through Whatman GF/C filters; the radioactivity retained on norit is a different measure of the dimer content of the DNA. Dimer photoreactivation was calculated by sub- tracting the dimer content of the sample exposed to pho- toreactivating light from that of the duplicate sample kept in the dark.

Lactate dehydrogenase activity was determined by the addition of cell extracts to 1 m l of a mixture containing 300ptM sodium pyruvate, 6 0 p M NADH and 30mM NaPO, buffer, pH 7.4. The LDH activity was determined from the rate of decrease in absorbance at 340 nm.

Protein concentrations were determined by the Biuret reaction (Layne, 1957). scaled down to 0.3 md.

RESULTS

We first examined PRE activity in lymphocytes cul- tured for 24 h in the presence or absence of PHA. Figure 1 shows that in extracts of cells cultured in the presence of PHA (+ PHA extract), PRE activity was present at reasonably high levels, while there was little or no PRE activity in extracts from cells cultured without PHA ( - PHA extract). Addition of PHA, or medium containing PHA, directly to the -PHA extract did not increase enzyme activity; mixing ex- periments in which the -PHA extract was added to the f P H A extract did not affect PRE activity in the latter extract. The final specific activity obtained

tude of the increase of PRE activity varied from donor to donor; however, the rate and final level of PRE reflected those of LDH activity.

Since there was considerable variation in the amount and time course of PRE production in the PHA-stimulated human lymphocytes, we examined PRE levels in a continuous line of murine lymphob- lasts, established from mouse lymphocytes by R. Daynes, University of Utah. We first grew these cells in suspension culture in a Dulbecco’s modified Eagle’s medium (Sutherland and Oliver, 1976) which supports the production of high levels of PRE in human fibro- blasts. Although the cells could reach high densities in Dulbecco’s medium, they grew slowly and contained rather low levels of PRE. However, since these cells had originally been established in a different medium (RPMI 1640 plus 10% fetal bovine serum), we trans- ferred cultures to this medium and determined their growth rate and PRE activity levels. The cells grew much more rapidly in RPMI than in Dulbecco’s, (generation times of 19 and 34 h, respectively) and the specific activity of the PRE increased from 4.4 pmol/mg/h to 19.2 pmol/mg/h.

DISCUSSION

Although resting human lymphocytes contain very low PRE activity (Sutherland et al., 1974), higher

+. c .-

- 200: 0 I n -J

- E a

t v1 0

-100 .? c

! { “ A , - 0

0 20 40

Time oftar PHA stimulation, h

Figure 2. Photoreactivating enzyme activity (0) and LDH activity (A) in extracts of human lymphocytes cultured in the presence (filled symbols) or absence (open symbols) of

PHA.

depended on the fraction of the cells stimulated, which varied with the donor and preparation.

We next examined the time course of appearance of PRE activity after PHA addition to the lymphocyte cultures, Figure 2 shows that enzyme activity in- creased for at least 48 h after the addition of PHA to the cells. In the absence of PHA, very little PRE ac- tivity was detected. We also followed the appearance of LDH, a marker for PHA-induced stimulation of protein synthesis (Rabinowitz et al., 1967). The ac- tivity of this enzyme increased with similar kinetics to those for PRE activity. The time course and magni- tude of the increase of PRE activity varied from donor to donor; however, the rate and final level of PRE reflected those of LDH activity.

Research Note 277

levels are present after PHA stimulation (Figs. 1 and 2). The similarity of the kinetics of the increase in PRE activity to that for the induction of LDH implies that the increase in PRE results from mitogenic stimulation of transcription and translation, rather than some induction process specific to PHA and PRE. Our results also show that murine lymphoblasts with generation times of 19 h contain much higher PRE levels than do those growing with a generation time of about 34 h. [Note that in human fibroblasts Dulbecco’s medium supports a high level of PRE pro- duction (Sutherland and Oliver, 1976; Mortelmans et ul., 1977).]

Wagner et a/. (1975) found photoreactivation (PR) of herpes virus in xeroderma pigmentosum (XP) fibro- blasts measured, to have intermediate levels of PRE, but no PR in fibroblasts of an XP patient with very low PRE levels.

Henderson (1978) observed higher survival of UV- irradiated Epstein-Barr virus in lymphocytes of XP patients in the presence of photoreactivating light ad- ministered to the virus-lymphocyte complex. The measurements of Sutherland et a l . (1974), however, indicating little to no PRE activity in resting lympho- cytes, predicted that no biological PR would be ob- served in human lymphocytes, regardless of genotype of the donor. Our results offer an explanation for the data of Henderson: agents like PHA and Epstein- Barr virus which increase cellular transcription and translation may also increase PRE activity. Thus the induced PRE could mediate PR of the UV-irradiated Epstein-Barr virus.

Previous studies have shown that procaryotic and eucaryotic cells contain higher PRE levels in station- ary phase than when growing rapidly. Tyrrell et al . (1972) found more PR in stationary phase E . coli. Boling and Setlow (1967) found 9-35-fold higher PRE activity in stationary phase yeast cells. Sutherland et al . (1974) found higher PRE levels in confluent than in nonconfluent mouse 3T3 fibroblasts. However, our

data show that in human lymphocytes and in murine lymphoblasts, there are much higher PRE levels in PHA-stimulated cells or in cells with shorter gener- ation times. Taken with the biological data of Hen- derson (1978), these data suggest that, unlike pre- viously described systems, PRE levels are higher in actively transcribing and rapidly growing lympho- cytes and lymphoblasts.

Acknowled~ements-This research was supported by grants No. CA23096 and CA26492 from the National Cancer In- stitute of the National Institutes of Health to B.M.S., and by the Department of Energy.

REFERENCES

Boling, M. E. and J. K. Setlow (1967) Biochim. Biophys. Acta 145, 502-505.

Cook, J. S. (1970) In Photophysiology (Edited by A. C. Giese), Vol. V, pp. 191-233. Academic Press, New York.

Cook, J. S. and J. R. McGrath (1967) Proc. Natl. Acad. Sci. U S A 58, 1359-1365.

Henderson, E. E. (1978) Cancer Res. 38, 32563263. Layne, E. (1957) Methods Enzymol. 3, 447-454. Mortelmans, K., J. E. Cleaver, E. C. Friedberg, M. C.

Paterson, B. P. Smith and G. H. Thomas (1977) Mu- tat. Res. 44, 433-446.

Rabinowitz, Y., T. Lubrano, B. A. Willite and A. A. Dietz (1967) Exp. Cell Res. 48, 675-678.

Setlow, R. B., W. L. Carrier and F. J. Bollum (1964) Biochim. Biophys. Acta 91, 446461.

Sutherland, B. M. (1981) In The Enzymes (Edited by P. Boyer), Vol. 15, Part B. Academic Press, New York. fn press.

Sutherland, B. M. and M. J. Chamberlin (1973) Anal. Biochem. 53, 168-176.

Sutherland, B. M. and R. Oliver (1976) Biochim. Bio- phys. Acta 442, 358-367.

Sutherland, B. M., P. Runge and J. C. Suther- land (1974) Biochemistry 13, 471C-4715.

Tyrrell, R. M., S. H. Moss and D. J. G. Davies (1972) Mutat. Res. 16, 345-352.

Wagner, E. K., M. Rice and B. M. Sutherland (1975) Nature 254, 627-628.

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