Nucleoprotein changes and initiation of RNA synthesis in PHA stimulated lymphocytes

Experimental Cell Research56 (1969) 418-424

NUCLEOPROTEIN

CHANGES AND INITIATION OF RNA SYNTHESIS IN PHA STIMULATED LYMPHOCYTES Z. DARZYNKIEWICZ,

L. BOLUND

and N. R. RINGERTZ

Institute for Medical Cell Research and Genetics Medical Nobel Institute, Karolinska Institutet, 10401 Stockholm 60, Sweden

SUMMARY Stimulation of human lymphocytes with phytohaemagglutinin in vitro induces marked changes in the properties of the nuclear deoxyribonucleoprotein complexes. These changes manifest themselves in an increased capacity to bind acridine orange, acetylation of histones andan altered affinity for actinomycin D and can be observed before RNA synthesis is initiated. An increased capacity to bind acridine orange is observed in all cells but only part of the lymphocyte population appears to acetylate histones and to show an enhanced actinomycin binding. Double emulsion autoradiography after labelling with both “C-acetate and ‘H-uridine suggests that it is those cells which acetylate histone which will also initiate RNA synthesis. It is suggested that the phenomena studied mainly reflect different stages of dissociation of the nucleoprotein complex and are part of a multistep preparatory mechanism for initiation of transcription.

The human peripheral lymphocyte offers a good model for studying the mechanisms of genome activation. When cultured in vitro the cells are resting in interphase but after stimulation by phytohaemagglutinin (PHA) or by certain antigens some of them undergo marked metabolic changes and are transformed into large, proliferating, pyroninophilic cells. (For a review see reference [9].) Before a cell starts to synthesize RNA [3,4,7] profound changescan be observed in its deoxyribonucleoprotein (DNP) complex, such as an increased ability of DNA to bind acridine orange (AO) [8] and actinomycin D (AMD) [6, 151,a changed stainability of histones [l, 2, 181and an acceleration of histone acetylation [I I]. These changes may reflect changes necessary for the transformation of inactive chromatin into an active template for RNA synthesis. The present study was undertaken as an attempt to distinguish the sequence of events during genome activation in PHA stimulated lymphocytes. Exptl Cell Res 56

MATERIAL AND METHODS Suspensions of human peripheral blood leucocytes were prepared as described in detail elsewhere [6,15]. All incubations were carried out in an atmosphere of 10 % CO, in air. PHA-M (Difco) was used at a final concentration of 50 ~1 of standard solution per 1 ml of culture medium.

A. Acridine orange binding The cells were cultured in TC 199 medium suunlemented with 20% autochthonous plasma on Btirker-hemocytometer coverslips in plastic Petri dishes (2.5-5.0 x l@ cells per 5 ml of medium) for 1 h before PHA was added. The cultures were then incubated for 1 h in the presence of PHA. The cells were kept at either 37”C-or 4°C throughout the experiment. Control cultures were incubated under the same conditions but without PHA. After incubation the coverslips were washed in saline and fixed in ethanol: acetone (1: 1) for at least 30 min at room temperature and then stored in the fixative at 4°C until stained. Staining with A0 was performed according to Rigler [13]. Measurements of fluorescence at 530 nm were made in a Zeiss fluorimeter.

B. Histone acetylation and RNA synthesis One ml cultures of leucocytes (0.5-1.0 x 10s cells) in Ringer solution supplemented with 20 % autochtonous plasma were incubated in glass centrifuge tubes at 37°C. All cultures were treated with puromycin (Sigma) at a final concentration of 15 pg/ml and 20 min later 14C-

Initiation Table

of RNA synthesis in lymphocytes

1. Effect of PHA stimulation at +37” and +4” in AO-binding

to leucocyte DNP

Lymphocytes FS3,,+_S.E.

Polymorphonuclear cells Fhao4 S.E.

Expt 1 Control 37°C PHA-treated 37°C

33.5 TO.9 (n = 75) 84.0 + 1.O (n = 75)

39.3 2 1.7 (n = 25) 90.5 5 2.1 (n = 24)

Expt 2 Control 37°C PHA-treated 37°C

34.8 f 1.4 (n = 20) 90.8 AZ2.4 (n = 20)

29.141.6(0=8) 82.2 3~3.0 (n = 10)

Expt 3 Control 4°C PHA-treated 4°C

38.4 + 1.4 (n = 20) 70.4 f 3.8 (n = 20)

34.21- 1.3 (n= 10) 60.2 +4.7 (n = 10)

Expt 4 Control 4°C PHA-treated 4°C

11.4f1.6 (n=lO) 63.Ok4.1 (n = 10)

acetate (Amersham, sp. act. 50 mC/m mole), was included at a final concentration of 2 &/ml (at the same time PHA was added). Incubation with r4C-acetate was carried out for 20 min and some cultures were then harvested. When harvested, the cultures were always diluted to 10 ml with TC 199 medium +20 % bovine serum and washed twice with this medium. The cells were then smeared onto gelatinized slides. Other cultures were incubated in TC 199 + 20 y0 bovine serum (after r*Cacetate had been removed by changing the medium) in the presence of puromycin and PHA for an additional time- period of 2-or 4 h-before being smeared. In control cultures PHA was excluded. It has been shown by Pogo et al. [ll] that acetate, when used at the conditions described, is incorporated into histones by the process of acetylation rather than being utilized for labelling of other cell components. Protein synthesis is completely inhibited by puromycin at the dose used (15 yg/ml) and synthesis of nucleic acids does not start within this short time (20 min after PHA administration). Furthermore most of the lipid material which might have incorporated isotope is extracted by ethanol fixation. The incorporation of 14C-acetate observed in our experiments is-therefore assumed to reflect mainly the nrocess of histone acetvlation. Cuhures parallel to the ones described above (already labelled with 14C-acetate, and with PHA and puromycin present) were incubated with 3H-uridine (Amersham, sp. act. 5 C/mmole at a concentration of 10 &/ml for 20 min before harvesting, i.e. 2 and 4 h after the removal of r4C-acetate. This was done in order to correlate histone acetylation to the initiation of RNA synthesis in the individual cells with the aid of double emulsion autoradioaranhv (see below). As a &p&ate measure of RNA synthesis other cultures were labelled for 20 min with onlv ‘H-uridine. 2 and 4 h after PHA administration (no prelabelling with r4Cacetate). In control cultures PHA was excluded. All smears from a particular labelling procedure were prepared on the same slide to make the conditions of fixation, washing and autoradiography identical and thus the results comparable. The smears were fixed either in absolute ethanol, or in 10 % neutralized formalin followed by absolute ethanol, or in case of labelling with SH-uridine alone in ethanol: acetic acid (9: 1). After 27 -

691803

419

fixation the smears were washed in 70 % ethanol for 2 h and then in water before being subjected to autoradiography. Some of the “‘H-uridine” smears were passed through 5 % TCA for 5 min at 0°C and then washed in water. In case of single labelling the conventional AR-10 (Kodak) stripping film has been applied. The double labelled smears were first coated with AR-10 stripping film and after drying in air (2 h) a thick layer of NTB-3 (Kodak) emulsion was applied on top of the AR-10 film. This was done by pipetting 0.5 ml of NTB-3 emulsion (melted at 43°C and-diluted 3 : 1 with 2 % glycerine in distilled water) on to the surface of the precooled (to O’C) slides. When the emulsion had gelified the autoradiograms were predried in the airstream of a fan (1 h) and then dried in dessicators under continuous flow of dry CO, for 4 h at room temperature. The autoradiograms were exposed for 5 (“double labelled” and “IHuridine”) or 20 days (“14C-acetate”) in CO* atmosphere at 4°C.

C. Actinomycin binding One ml cultures of leucocytes in TC 199 medium with 20 % autochthonous plasma were incubated with *HAMD in glass centrifuge tubes at 37°C. 8H-AMD (Schwarz Bio Research, Orangeburg, N.Y., original snecific activitv 3.38 Clmmole) was used at final concentration 2.5-pg/ml and specific activity 530 mC/mmole (adiusted with unlabelled AMD). Unlabelled AMD was a &t from Merck, Sharp & Dohme Res. Lab., Rathway, N.J. In one set of cultures *H-AMD and PHA were added at the same time and the cultures were harvested 20 and 60 min later. Other cultures were treated for 20 and 60 mitt with *H-AMD 4 h after PHA administration. Control cultures incubated without PHA were labelled with aH-AMD in a similar way. The cells were harvested, fixed in absolute ethanol and subjected to autoradiography as described above. Single emulsion autoradiograms were processed according to standard procedures and counterstained with hematoxylin and eosin. Grains were counted over 200 lymphocytes and 100 polymorphonuclear leucocytes in each smear. Exptl Cell Res 56

420

Z. Dariynkiewicz

10

20

30

40

50

et al.

60

70

80

90

loo

110

10

20

30

40

50

60

Abscissa: FGS,,;ordinate: number of cells ( %). Fig. 1. Frequency distribution histograms showing variation in AO-binding of normal human lymphocytes (A) and polymorphonuclear leucocytes (C) in control cultures maintained for 1 h. Addition of PHA to parallel cultures increases markedly the acridine orange binding capacity of both lymphocytes (B) and polymorphonuclear leucocytes (0). All cells appear to react. Acridine orange binding was measured by microfluorimetry at 530 nm on fixed and stained cells. The double emulsion autoradiograms were soaked with Kodak D19 developer at + 4°C for 15 min and then transferred to the same developer at 18°C for 10 min. After developing, the autoradiograms were briefly washed in a 1% solution of acetic acid and then fixed for 30 min in Kodak acid fixer. Double emulsion autoradiograms were mounted in glycerine and grains and tracks were counted above the same cells in the first (AR 10) and second (NTB) layer of emulsion respectively. Only those tracks were counted that consisted of 4 or more grains and were coming from individual cells. For a detailed description of track autoradiography, counting conditions, background etc, see [5,17]. Grains and tracks were counted over 100 lymphocytes from each smear. All main experiments were performed at least twice.

RESULTS A. Acridine orange binding

DNP-changes. In experiments with hen erythrocytes we have previously [14] obtained data which suggestthat this type of DNP-changes can occur also at low temperatures and in the presence of enzyme inhibitors. At +4”C the change in the stainability of lymphocytes is somewhat less dramatic and the intercellular variation bigger. However, in all experiments the difference between control and PHA treated cells is statistically highly significant. Analysis of the frequency distribution histograms reveals that within both lymphocyte and PMN cell populations all cells respond to the PHA treatment (fig. 1).

Results from two experiments are presented in B. Histone acetylation and RNA synthesis table 1 and fig. 1. Within 1 h after PHA ad- Table 2 shows the results of 14C-acetateincorministration both lymphocytes and polymor- poration by lymphocytes and PMN leucocytes. phonuclear (PMN) cells markedly increase their A 20 min pulse of 14C-acetatelabels 17 % of the ability to bind AO, when cultured at 37°C as lymphocytes and about 70 % of the PMN cells. well as at 4°C. The incubation at +4”C was car- When the cells are treated with PHA the proporried out in order to test whether or not the in- tion of lymphocytes taking up label becomes creased AO-binding may reflect non-enzymatic 39%, and the number of heavily labelled cells Exptl Cell Res 56

Initiation

Table 2. Effect of PHA stimulation on W-acetate

of RNA synthesis in lymphocytes

421

incorporation into human leucocytes

Percentage of labelled cells Time

Control

after the addition of PHA

Unlabelled

3- 10

1l-20

220

Unlabelled

3-10

11-20

>20

83 79 74

10 13 17

4 6 4

3 2 5

61 55 41

26

3

::

10 11 15

s6

60

5

6

21

65

7

7

PHA-treated

Grains/cell

Grains/cell

Lymphocytes

20 min 2h 4h Polymorphonuclear

20 min

leucocytes

29

a 20 min pulse labelling, puromycin block.

increases.l Only a slight increase in the number of labelled PMN cells is observed at the same time. Lymphocytes which were treated with 14Cacetate and then after rewashing cultured for 2 and 4 h, as a control for the double labelling experiment did not show any decreaseof labelling. On the contrary, quite a number of cells became labelled during this time in spite of the lack of external 14C-acetateand a relatively high concentration of unlabelled acetate in the medium. This is probably due to the fact that the 14C-acetateincorporated comesfrom the internal pool, still markedly enriched with labelled preTable 3. Effect of PHA stimulation on the incorporation of SH-uridine into leucocyte RNA Percentage of labelled cells Grains/cell Unlabelled

3-10

1l-20

220

81 66 41

10 18 30

i 11

6 10 18

41 28

10 7

1:

Lymphocytes

Control 2 h after PHA 4 h after PHA Polymorphonuclear

Control 4 h after PHA

leucocytes

43 52

1 While this work was being completed Mukherjee & Cohen [lo] reported that only a fraction of the lymphocyte population begins to incorporate 14C-acetate after PHA stimulation, which agrees with our results.

cursor from the 20 min pulse. After 2 and 4 h there were 45 % and 59 % labelled lymphocytes in the PHA-treated culture. No significant difference was found between preparations fixed in absolute ethanol or 10 % formalin which was then exchanged for ethanol to avoid negative chemiography. The results of 8H-uridine labelling are presented in table 3. 34 % and 59 % of the lymphocytes became labelled 2 and 4 h respectively after PHA administration, as compared with 19 % in the control. Similar to the result obtained with 14C-acetate, no marked difference was noted between the 3H-uridine labelling of PMN cells in control and PHA treated cultures. Preparations washed in 5 % TCA after fixation were as heavily labelled as those which were only washed in water. This fact justifies the procedure used in the double labelling experiments where acid was avoided in order to diminish the risk of histone extraction. The results of the double labelling of lymphocytes with 14C-acetateand 3H-uridine are shown in table 4. Two hours after PHA treatment 34 % of the lymphocytes are not labelled; neither grains in the first layer of emulsion nor tracks in the second one could be detected.24 % unlabelled cells were found 4 h after PHA administration. There seemsto be a strong correlation between the intensity of the two types of labelling of individual cells. The results are analyzed further in the discussion. Exptl Cell Res 56

422

2. Dariynkiewicz

et al.

Table 4. Double emulsion autoradiography on PHA-stimulated and 3H-uridine 2 h after addition of PHA

lymphocytes labeled with 14C-acetate

4 h after addition of PHA

Grains/cell (AR-lo)’ Tracks/cell (NTB)” Unlabelled 2-5 6-10 >lO

Unlabelled 34 A 0

Grains/cell (AR-lo)’

3-10

1l-20

>20

13 19 3 4

4 4 3 2

2 2 1 8

Tracks/cell (NTB)” Unlabelled 2-5 6-10 >lO

Unlabelled

3-10

1l-20

>20

24 2 i

12 14 4 2

2

1 4 4 12

7 5

a SH-uridine labels only the AR-10 emulsion. * W-acetate labels both the AR-10 and the NTB emulsions. Results are expressed as percentage of cells showing different degrees of labelling.

DISCUSSION

C. Actinomycin binding

Killander & Rigler [8] reported that, within the very first min of the action of PHA, it produces a striking increase in the ability of DNA of stimulated lymphocytes and PMN leucocytes to bind the basic dye acridine orange. They suggested that this is due to a liberation of phosphate groups in DNA (probably previously bound to histones) which then become available to the dye. Our results confirm their finding that all cells in these populations undergo this change in stainability (fig. 1). This is remarkable since PMN cells do not significantly change their synthetic activity in the presence of PHA, and

Only 8 % and 20 % of the control lymphocytes were labelled after incubation with 3H-AMD for 20 and 60 min, respectively (table 5). When PHA is added at the same time as 3H-AMD and the cells are harvested 20 min later, 17 % of the lymphocytes (8 % in the control) became labelled. After 60 min, 36 % of the mononuclear cells bind AMD in the PHA treated cultures (20% in the control population). Similarly, if lymphocytes are cultured with PHA for 4 h and then treated with 3H-AMD for 20 or 60 min, 15-18 cells more per 100 bind the drug than in the control culture (table 5).

Table 5. Effect of PHA on SH-AMD binding of human IeucocyteY 20 min; 8H-AMD

60 min; 3H-AMD

Grains/cell

Grains/cell

Unlabelled

3-10

11-20

>20

Unlabelled

3-10

11-20

>20

92 83 77

4 9 13

1 3 4

3 5 6

80 64 62

14 27 28

2 3 2

4 6 8

23 30

41 38

26 15

10 17

Lymphocytes Control PHAb PHA’ 4 h

PMN leucocytes Control Not tested PHAC 4 h

a Results are expressed as percentage of cells showing various degrees of labelling. * PHA and SH-AMD were included into cultures at the same time and the cells were incubated for 20 and 60 min. c Cells were pretreated with PHA for 4 h and then “H-AMD was given for 20 and 60 min. Exptl Cell Res 56

Initiation of RNA synthesis in lymphocytes 423 since only part of the lymphocyte population is stimulated to undergo transformation. (30-40 % of the cells, which after transformation and multiplication might become60-90% of the total mononuclear population in 3-day-old cultures.) A similar change in stainability, although somewhat less pronounced, was found when the cells were kept at 4°C. This observation together with results obtained on other cell systems [14] suggests but does by no means prove that the DNP-changes reflected in an increased AO-binding are triggered by non-enzymatic mechanisms. Using biochemical methods Pogo et al. have observed that PHA stimulated lymphocytes show an increased rate of histone acetylation [l 11. These authors could also demonstrate that human PMN leucocytes did not respond to PHAstimulation by an increased acetylation of histones. The present experiments-labelling with 14Cacetate or 3H-uridine alone (tables 2 and 3) as well as double labelling with these isotopes (table 4)-indicate that the proportion of lymphocytes increasing the rate of acetylation is similar to that starting RNA synthesis,and moreover, that the cells which start to synthesize RNA have incorporated 14C-acetate.The assumption that 14C-acetateis mainly incorporated into proteins by the method of acetylation is based on the results of Pogo, but the incorporation into lipids or RNA under these conditions has not been estimated biochemically. Further proof is clearly needed before it can be stated that the 14C-acetateincorporation reflects nothing but histone acetylation. The double labelling, double emulsion autoradiography permits a distinction of cells which are unlabelled with both isotopes, and with somewhat less certainty those labelled only with tritium. The thickness of the first AR-IO emulsion and its gelatine layer completely prevents the weak energy beta particles of tritium to reach the second layer [17]. The thick (ca 30 ,LJ) second layer of NTB3 emulsion on the other hand creates much better conditions to detect the tracks of carbon 14 beta particles (max. energy 155 keV) which can penetrate the emul-

sion to a distance of about 80-100 ,u [17]. Thus, the number of cells labelled with tracks in the second layer of emulsion is representing those which have incorporated 14C-acetate.In the first layer, however, both 3H and 14Cbeta particles are responsible for the image. Detection of 34 % and 24 % of the cells unlabelled with both isotopes, when at the same time (2 and 4 hours after PHA treatment respectively) 45 % and 59 % have incorporated 14C-acetate(table 2) and 34 % and 59 % are labelled with 3H uridine (table 3), permits the conclusion that mainly those cells which became acetylated have started RNA synthesis. This is also supported by the good correlation between the intensities of labelling in both emulsions above individual cells (table 4). AMD has a very high affinity for the guanine residues in double stranded DNA and it has been suggestedthat this drug binds to some of the sites normally occupied by RNA polymerase during the transcription process [12]. The AMD binding properties of isolated nucleoprotein have been reported elsewhere [16]. In previous communications [6, 151 we have reported an increased ability of PHA stimulated lymphocytes to bind AMD. The stimulated cells bind the drug at a much higher rate than control cells at 2 and 4 h after PHA treatment. In the present experiments we have observed that, within as short a period of time as 20 min after PHA administration, some lymphocytes acquire an increased ability to bind AMD. The increase in AMD-binding capacity appears to be fully established already after 60 min. Thus it seems that also the increase in AMD binding to the chromatin in time precedes the start of RNA synthesis which cannot be detected with autoradiographic methods earlier than 2 h after PHA treatment [4]. Similarly to the histone acetylation and the rise in RNA synthesis and contrary to the A0 binding, no PMN cells and only some of the lymphocytes respond to PHA treatment with an increased binding of AMD to their chromatin. On the basis of the results presented and the literature one could try to distinguish some successivesteps or processesduring genome activaExptl Cell Res 56

424

Z. Dariynkiewicz

et al.

tion. The increased ability of DNP to bind A0 [S] and the altered histone stainability [l, 2, 181 seemto precede in time the increased 14C-acetate and 3H-uridine incorporations as well as the increase in AMD-binding. (The plateau of the A0 reaction is reached within the first 15 min after PHA administration.) The increased AObinding and the altered histone staining also appears to be less specific since all cells are affected. It is probably a non-enzymatic phenomenon perhaps of ionic nature [14]. It might reflect the first stage of DNP dissociation (i.e. a weakened binding between protein and DNA), which is extensive enough to create conditions for the small dye molecules to penetrate into the complex and to bind to the phosphate groups of DNA. The next stage of DNP dissociation is probably initiated by histone acetylation. This process,preceding in time the rise of RNA synthesis [Ill, is much more specific since it is observed mainly in those cells which later start to synthesize RNA. Acetylation of histones as suggested by Pogo et al. would lower the affinity of histones for binding to DNA, thereby making possible the transcription process. It would appear that it is this step rather than the first step which is reflected in the increased capacity of the DNP molecules to bind 3H-AMD.. Clearly more evidence must be collected before this conclusion can be drawn, however. The genome activation could thus be regarded as a multi-step process. All cells pass the first steps in the chain but the number of cells proceeding becomes restricted by each step until only a minority starts RNA synthesis and undergoes transformation. There is, however also the possibility that only a small number of lympho-

Exptl Cell Res 56

cytes directly react to PHA but that a stimulatory effect then spreads to other cells (via the releaseof substances)and forces them to undergo those DNP changes which express themselvesin an increased AO-binding capacity. The cells which initially reacted to PHA proceed to subsequent steps in the genome activation process whereas the “indirectly” stimulated cells fail to do so. This work was supported by grants from the Swedish Natural Science Research Council, The Swedish Cancer Society, and Reservationsanslaget, Karolinska Institutet. The development of the biophysical instruments used in this investigation was supported from the Swedish Medical Science Research Council to professor T. Caspersson.

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Black, M M & Ansley, H R, J cell biol 35 (1967) 619. Burton, D W, Exptl cell res 49 (1968) 300. Cooper, H & Rubin, A, Blood 25 (1965) 1014. Dariynkiewicz, Z, Krassowski, T & Skoptiska, E, Nature 207 (1965) 1402. Dariynkiew&z, i?, Rogers, A W & Barnard, E A, J histochem cvtochem 14 (1967) 915. Dariynkiewici, Z, Bolunb, i & Ringertz, N R, Exptl cell res. 55 (1969) 120. Kay, J & Korner, A, Biochem J 100 (1966) 815. Killander, D & Rigler, R, Exptl cell res 39 (1965) 701. Ling, N R, Lymphocyte stimulation. North Holland, Amsterdam (1968). Mukherjee, A B & Cohen, M, Exptl cell res 54 (1969) 257. Pogo, B G T, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 55 (1966) 805. Reich, E & Goldberg, I H, Progr. nucl acid res & mol biol 3 (1964) 183. Rigler, R, Acta physiol stand 67 (1966), Suppl. 267. Ringertz, N R & Bohmd, L, Exptl cell res 55 (1969) 205. Ringertz, N R, Dariynkiewicz, Z & Bolund, L, Exptl cell res. 56 (1969) 411. Ringertz, N R & Bohmd, L, Biophys biochim acta 174 (1969) 147. Rogers, A W, Techniques of autoradiography. Elsevier, Amsterdam, London, NY (1967). Zetterberg, A & Auer, G, Exptl cell res. 56 (1969) 122.

Received January 15, 1969