Changes in mRNA levels of poly(ADP-ribose) polymerase during activation of human lymphocytes

Biochimica et Biophysica Acta. 1009(1989) 185-187

185

Elsevier BBAEXP90154

BBA Report

Changes in mRNA levels of poly(ADP-fibose) polymerase during activation of human lymphocytes Ruth McNerney ~, Mannoo TavasoUi 2, Sydney Shall 2, Alison Brazinski and Alan Johnstone I Department of Immunology. St George's Hospital Medical School. London and " Cell and Molecular Biology Laboratory. School of Biological Sciences. Universityof Sussex. Brighton ¢U.K.)

(Received 30 January 1989) (Revised manuscriptreceived24 June 1989)

Key words: Lymphocyteactivation: Eukaryoticdifferentiation:Mitogenstimulation;Poly(ADP-ribose)polymerase; Northern blotting:(Human) The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) po|ymerase (ADP-ribosyltransferase, EC 2A.2.30) was found to be very low in q,~L-,scent human iymghhoeytes and *.o increase at least 10-fold between 1 and 2 dyas after stimu|ation with the mitogen phytohaem~utinin, staying h~gh for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of imly(ADP-ribose) polymemse) but was not affected significantly by aphidieolin. Incubation of activated cells with ¢ydoheximide for 2 h increased the expression slightly. These data demonstrate that, ~luring lymphocyte activation, the level of mP,d~IA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or innmmochemistry.

Addition of mitogens such as phytohaemagglutinin (PHA) to quiescent lymphocytes causes them to increase in size, change their morphology and, after about 48 h, synthesise DNA in preparation for cel| division. This activation is accompanied by an ordered increase in the expression of many genes ranging in t~me f~'om the proto-oncogene c-los at 15 win through to tramferfin receptor after several days [1-3]. The nuclear enzyme poly(ADP-ribose) polymerase (ADPRT) is thought to be involved in eukaryotic differentiation, including the stimulation of quiescent lymphocytes [4-8]. Competitive inhibitors of ADPRT prevent lymphocyte activation but only if present during the first 8-16 h, suggesting an early requirement for the enzyme. On the other hand, resting cells contain only small amounts of ADPRT activity and this does not increase until about 2 days after mitogen stimulation [7,9-13]. In the present study we investigated the expression of the ADPRT gene during witogen stimulation of

Abbleviations: ADFRT, poly(ADP-ribose) polymerase (nuclear ADP-ribosyltransferase,EC 2.4.2.30); PHA, Phaseolusvulgans phytohaemagglutinin. Correspondence: A.P. Johnstone, Department of Immunology,St George's Hospital Medical School, CranmerTerrace, London, SW17 ORE (U.K.).

lymphocytes. Northern blotting revealed only one species oi~ ADPRT nm.,,,~,"" n ~., • that migrated slightly faster than 28 S ribosomal RNA (Fig. ~,). This is consistent with the molecular weight of the ~nzyme (113000) and the size of its full-length cDNA (3.8 kb) [14,15]. The level ¢,f mRNA encoding ADPRT ~,as very low in quiescent ~ymphocytes; after PHA stimulation, the amount increased between 1 and 2 days (by densitometry using a LKB Ultroscan laser densitometer, approx. 10-fold in six experiments) and stayed at an elevated level until at least day ~ tFi~. !) !o ~orne experiments there was a decrease between day 3 and day 5, but this was probably related to variation in growth rates and viability in long-term cultures. No increase in ADPRT mF,NA c.ou~d be detected at 24 h or earlier (Fig. i). Thus the small amount of ADPRT enzyme present in l~sti~, i~mpfica:ytes must be sufficient to accomplish its fu~,:tior:.~_=-1::in the activation process [5-13,16]. 1he kinetics and size of the increase in ADPRT mRNA are fimilar to those of the ADPRT pro~.ein which i~c.r~ases 3- to 10-fold over the period 24 to 72 h after PHA stimulation, measured either by enzyme actiTity [7,9-12] or immunoblotting [13]. Whilst w¢ have not inv.~stigated directly whether the increase in mRNA is due I~ changes in transcription or mRNA turnover, in view bf the stability of the ADPRT mRNA in various cell types (Ref. 13 and see below) it is probable that an

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Fig. 1. Kinetics of increase in ADPRT mRNA following PHA stimulation. In two separate experiments (presented in the two halves of the figure), human peripheral blood lympbocytes [16] ((2-5)- 107/sample) were cultured at 2-I06/ml in RPM! 1640 containing 5~ foetal calf serum and 2 pg/ml PHA (purified, Wellcome) for the times indicated before extracting their RNA as detailed cartier [16] - the yield of RNA was 5.44.0.8/~g/107 resting cells and 13.04-2.8/~g/10 "~stimulated cells (2 days or longer). For longer cultures, fresh medium was added to the cells after 3 days. The content of mRNA encoding ADPRT was analysed in 10 pg portions of total RNA (quantified by A.,60) using Northern blotting [25-27], as detailed earlier [16]. In each ¢~se, a duplicate gel was stained with ethidium bromide to confirm that approximately equal amounts of ribosomal RNA had been loaded onto each track. The probe used was the oligo-32P-labelled 2.1 kb EcoR! fragment of cDNA corresponding to about two-thirds of the human ADPRT coding region [14,15,24]. The migration positions of 28 S and 18 S ribosomal RNA are indicated.

increase in gene expression is responsible for the observed increase in enzyme activity. To our knowledge, this is the only situation in which an increase in measured poly(ADP-r~bose) polyrnera~e activity is attributable to an increase in the level of mRNA and de novo enzyme synthes~s rather than activation of already existing enzyme molecules. The time-course that we observe conflicts whh a recent report appearing after our study was completed [17]. The latter authors observed an increase in ADPRT mRNA within 4 h of PHA stimulation of human lymphocytes that had been enriched for T L'ells by rosetting with sheep erythrocytes (their cell prepa~ation also contained 1~ of the non-rosetting cells added back; Menegazzi, M. and Suzuki, H., personal communication). It is possible that partial activation of the iymphocytes through the CD2 (erythrocyte receptor) pathway [18,19] could account for the accelerated response and also the l~gher mRNA levels in rosetted (T cells) compared with unrosetted cells (monocytes and B cells). The level of ADPRT mRNA that we observe in quiescent peripheral lymphocytes, which are comprised of 70~ T cells (virtually undetectable in most experiments using a high-specific acti~ty probe; (1-2)-109 cpm//tg) is much lower than that observed by Menegazzi et al. [17] in their unstimu|ated T-cell preparation,

Fig. 2. Effect of various chemical inhibitors on the level of ADPRT mKNA in PHA-sdmulated lymphocytes, in two separate experiments (presented in the two halves of the figure), lymphoeytes were prepared and incubated as in Fig. 1 for 48 h with: O, no additions; P, 2 p g / m i PHA; A, 2 ~g/ml PHA and 1 pg/ml aphidicolin; B, 2 p g / m l PHA and 2.5 mM 3-methoxybenzamide: C, 2 p g / m l PHA with 10 p g / m l eycioheximide added for the last 2 h of culture. Then RNA was extracted and analysed for its content of ADPRT mRNA by Northem blotting as in Fig. l. The migration positions of 28 S and 18 S ribosomal RNA are indicated.

especially considering their lower specific activity (nick-translated) probe. The presence of aphidicolin (1 /Lg/ml) throughout the culture period inhibited DNA synthesis by more than 90Fo as assessed by incorporation of [ 3H]thymidine (data not shown), but had no significant effect on the level of ADPRT mRNA analysed 48 h after PHA stimulation (Fig. 2; by densitometry the mean density of the band in aphidicolin compared with untreated cultures in three experiments was 0.97). Hence, the increase in ADPRT mRNA is not dependent upon DNA synthesis, despite the concurrence of the two events. The presence of 3-methoxybenzamide, a competitive inhibitor of ADPRT, throughout the culture period did affect the increase in ADPRT mRNA, causing at 48 h a 40-87~ inhibition (range of three experiments assessed by densitometry; Fig. 2). This inhibitor competes with the enzyme's substrate, NAD +, which is present inside the cell at 0.5-1 raM; therefore, it has been used frequently in mM concentrations. While ~ese concentrations certainly inhibit poly(ADP-nbose) synthesis, the possibility of other enzymes or processes being affected can not easily be excluded. When added to lymphocytes just before PHA, this chemical does not affect raitogen-binding, membrane events such as the hydrolysis of polyphosphatidylinositol, or the rapid increase in expression of the proto-oncogene c-los but does prevent all subsequent events including the increase in c-myc, y-interferon and interleukin-2 receptor expression, as well as total protein and DNA synthesis [5,8,11,16,20]. Hence, it is not surprising that 3-meth-

187 oxybenzamide also inhibited the late increase in ADPRT mRNA levels. The precise mode of action of this chemical remains to be established. There was a slight increase in the level of ADPRT mRNA in activated lymphocytes incubated with cycloheximide for 2 h (Fig. 2; by densitometry, mean 1.51-fold increase, range 0.81 to 2.12, of three experiments), possibly su&~esting that the mRNA is normally degraded by a labile protein. The fact that this increase is considerably lower than the superinduction by cycloheximide of early genes such as c-fos and c-myc [21-23] supports the concept that the products of later genes such as ADPRT are turned over much more slowly (direct evidence for this also exists - Ref. 13; Shall, S. and Miwa, M., unpublished). Menegazzi et al. [17] founc~ that addition of cycloheximide 30 rain before PHA inhibited the increase in ADPRT mRNA 16 h later. This experiment was possible only because of the rapid increase in ADPRT expression in their system (see above); the presence of cycloheximide for 48 h in our cultures caused considerable cell death, so that we could not undertake a comparable investigation of the requirement for earlier protein synthesis. This work was supported by grants from The Wellcome Trust to A.P.J., and from the Medical Research Council and Cancer Research Campaign to M.T. and S.S. We thank Dr C.A. Penning for kindly provit~;ng the ADPRT plasmid and Professor M. Miwa, Dr. M. Menegazzi and Dr. H. Suzuki for helpful discussions. References 1 Reed, J.C., Alpers, J.D., Nowell, P.C. and Hoover, R.G. (1986) Proc. Natl. Acad. Sci. USA. 83, 3982-3986. 2 Granelfi-Pipei:no, A., Andrus, L. and Steinman, R.~vl. (1986) J. Exp. Med. 163, 922-937. 3 Moore, J.P., Todd, J.A., Hesketh, T.R. and Metcalfe, J.C. (1986) J. Biol. Chem. 261, 8158-8162. 4 Farzaneh, F., Zalin, R, Brill, D. and Shall, S. (1982) Nature 300, 362-366.

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