Destruction of the nuclear morphology of thymic lymphocytes by the corticosteroid cortisol

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Cell Research 52, 349-362 (1968)

DESTRUCTION OF THE NUCLEAR MORPHOLOGY OF THYMIC LYMPHOCYTES BY THE CORTICOSTEROID CORTISOL’ J. F. WHITFIELD, Division

of Radiation

Biology,

A. D. PERRIS and T. YOUDALE

National

Research Council of Canada, Ottawa, Canada

Received December 12, 1967

glucocorticoid, cortisol (hydrocortisone), stimulates the synthesis of ribosenucleic acid in liver cells which is followed by the increased synthesis of a variety of enzymes [5, 9, 20-23, 31, 45, 46, 50, 511. In contrast, when lymphocytes are exposed to cortisol, their nuclear structure is demolished and the cells eventually lyse [ 7, 111. The basis of this nuclear response is the complete disaggregation of all of the highly condensed deoxyribonucleoprotein granules which constitute the characteristic lymphocyte nuclear structure 17, 33, 52, 54-56, 58, 671. Morphologically, the dissolution of nuclear structure following cortisol treatment is identical with that which follows irradiation of lymphocytes [7, 11, 52-641. The radiation-induced loss of nuclear structure (“pycnosis”) is not the result of a direct action of radiation on the lymphocyte’s chromatin structures [37, 39, 52-641. The loss of nuclear structure is indirectly caused by the initiation and operation, during the first hour after irradiation, of a respiration-linked and phosphate-dependent reaction [37, 38, 52-641. A product of this reaction severs the union between deoxyribonucleic acid (DNA) and histones within the nucleoprotein granules and it is the separation of the major components of the nucleoprotein complex which causes the dissolution of nuclear structure [S, 12, 13, 26-28, 30, 37, 38, 52-641. It is not known whether the morphologically identical effect on nuclear structure caused by cortisol is similarly the result of the operation of an abnormal metabolic reaction, or whether the steroid reacts directly with the deoxyribonucleoproteins of the chromatin bodies. Certainly cortisol can form a stable combination with arginine-rich histones [47-491 and might thus disperse the chromatin bodies by combining directly with the histones and drawing them away from their attachment to DNA [5, 9, 461. However, the highly condensed chromatin structures of the lymphocyte nucleus are forTHE

1 Issued as N.R.C. No. 10040. Experimental

Cell Research 52

met1 mainly 1)~ the combination ol 1)9,1 \vith lysine-rich histoncs -3S, A‘~ rather than argininc-rich histoncs for which cortisol has the much greatcl affinity [4T- 49:. In the present study \ve \vill sho\v that cortisol, like ionising radiation, nuclear structure indirectly through the agent) causes the loss of lymphocyte of a metabolic reaction lvhich is both I,hosphate-depentient and linkccl to respiration. MATERIALS

AND

METHODS

The type of lymphocyte used in this study was the thymic lymphocyte. or th)-mocyte. The detailed method of preparation of thymocyte suspensions has been described elsewhere [54,55]. Briefly, thymus glands from young, male Sprague-Dawle>rats (weighing 130 g) were minced in a small volume of a glucose-salts medium such that the resulting concentrated suspension contained about 1 Y 109 cells/ml. A final 5 ml suspension culture was then prepared by diluting this concentrated suspension to contain about 2 x 108 cells/ml. The diluted suspension cultures were usually contained in 25 x 125 mm test tubes which were rotated about their long axes at 30 rpm in an incubator at 37°C. The glucose-salts medium contained 5.5 mA1 glucose, 5.0 rnA1 KCl, 0.63 md1 CaCl,, 1.0 mM MgSO,, and 5.5 miC1 Iris (hydroxymethyl)-aminomethane buffer. Inorganic phosphate was added in the form of Na,HPO, and when its concentration was varied, the total Na+ concentration was maintained at 130 mEq/l by adjusting the concentration of NaCl; for example, in phosphate-free medium the concentration of NaCl was 130 mA1 while in medium containing 50 ml11 Na,HPO, it was reduced to 30 mAf. The pH was always adjusted to 7.2 with HCl. In some of the experitnent reported here, cells were transferred from 1~l~osp~~atcfree to phosphate-containing medium or vicr vma. Phosphate could not be simpl>added to the medium of cell suspensions because of the need to alter the NnCl concentration at the same time. Therefore, cells kvere centrifuged from the 5 ml SLISpension cultures at 500 9 at 22°C and the supernatant medium was decanted. Five ml of the appropriate medium (prewarmed to 37’C) were then added to the tube without disturbing the cellular precipitate and the tube was shaken very gently. These 5 ml were poured off and replaced with an equal volume of the same fresh. warm medium and the cells then resuspended. In other experiments the cells were maintained under anaerobic conditions. The final, dilute cell suspensions (30 ml) were introduced into 250 ml Erlenmeyer flasks. Nitrogen (warmed to 37°C) was passed through the gas phase of the culture flask fol 5 min at a flow rate of 20 litres per min and the flask was then sealed. It must be noted that nitrogen was never passed through the cell suspension itself since the mechanical strain of bubbling and a fragility caused by anoxia would combine to cause lysis of the cells [53]. The oxygen dissolved in the suspension as well as the small amount carried in with the nitrogen was consumed by the cells in about .5 min. The anaerobic cultures were then incubated at 37°C. During incubation, the suspensions were constantly stirred with a tcflon-coated magnetic stirring bar (3.7,? Experimental

(211 Rescwch 52

Action of’cortisol

on lymphocyte

nuclear structure

331

cm long) rotating at about 200 rpm. Aerobic conditions were restored by simply opening the flasks and passing warm (37°C) air (for 5 min at a flow rate of 20 l/min) over the surface of the suspension. A concentrated (137 mM) stock solution of cortisol (hydrocortisone) was prepared by dissolving the steroid in absolute ethanol. This stock solution was then sufficiently diluted with medium to give a final cortisol concentration of 0.27 PM; this concentration was chosen since preliminary experiments showed that it was the lowest one to give a maximum effect on nuclear structure in 6 h. Cellular respiration rates were determined during the first hour of incubation in vitro with a model 53 oxygen monitor by Yellow Springs Instrument Co., Inc. (Yellow Springs, Ohio) which had been modified (in cooperation with Fisher Scientific Co., Montreal, Canada) to simultaneously record the oxygen consumption in four cultures. The rate of appearance of cells with structureless (“pycnotic”) nuclei was determined by removing a few drops from the cell suspensions, fixing the cells in neutral formalin and staining them with Delafields’s haematoxylin according to the procedure of Whitfield rt nl. [55]. RESULTS During the first hour of a continuous exposure to cortisol (at a concentration of 0.27 PM), there was invariably very little, or no, appearance of cells \\-ith structurally homogeneous (“pycnotic”) nuclei in thymocyte populations suspended in medium containing a high concentration of inorganic phosphate (e.g. Fig. 7). This “cytological” lag period was followed by a large increase in the proportion of affected cells (Fig. 7). The rate of accumulation of affected cells very much depended on the level of inorganic phosphate in the medium (Fig. 1). In fact, the proportion of cells which had lost their nuclear structure after being exposed to cortisol for 6 h was linearl? proportional to the phosphate concentration in the medium (Fig. 2). In normal thymocyte populations, on the other hand, the dissolution of nuclear structure was not influenced bp the environmental phosphate level (Figs 1 and 2; see also [B-L]). Althongh cortisol could not cause an extensive loss of nuclear structure in cell populations maintained in phosphate-free medium, it did alter these cells in such a \\-a? that they would rapidly generate structureless nuclei if transferred to a medium containing phosphate. For example, during a 3-h period in phosphate-free medium, there was only a slo\v accumulation of cells \vith structureless nuclei in populations exposed to cortisol (Figs 3 and la=\). \\‘hen these altered cells were transferred to a medium containing both cortisol and 30 or -50 mM Na,HI-‘O,, a rapid dissolution of nuclear chromatin structures began without the characteristic l-h delay ohserved Expe-imenttrl

Cell Reswwch 52

352

,I. F. ll’hitfield,

A. 11. Perris

rrnd 7’. Ihuclnle

\\-hen the cells \vcre exposed to high levels of phosphate from the lqinning oC the experiment (Figs 3 and 12A). Loss of nuclear structure in normal lhymocyle populations was not affected by a similar addition of phosphate after a prolonged sojourn in phosphate-free medium (Fig. -4 and rel’. I(%). It appears that cortisol initiales a reaction \\-hich consists of t\\,o l)hascs.

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Fig. 1. --The accumulation of cells with slructurcless nuclei in normal and cortisol-trcalctl popw lations of thymocytes maintained in media containing different concentrations of inorganic phosphate. 0 - - - 0, The medium contained 2.5 mM Na,HPO,; 0 - 0, the medium contained 15 m&f Ka,HPO,; O--O, the medium contained 30 mM Ka,HPO,. In the medium of the cortisol-treated populations, the cortisol concentration was 0.25 $%I and it was present throughout the incubation period. Abscissa: h of incubation; ordinute: y/, cells with structureless nuclei. Fig. P.-The relations between the generation of structureless nuclei and the inorganic phosphalr concentration in the medium of normal and cortisol-Lreatcd thymocytes. The cortisol concentration was 0.27 ,&I4 and it was present in the medium throughout the incubation period. d bscissu: nuclei at 6 11. Phosphate concentration (m&I) in medium; ordinctfe: o/o cells with structureless 0 __ 0, Cortisol; 0 _~ 0, control.

The first phase can go to completion in the absence of an estraccllular supply of inorganic phosphate. However, the second phase, \\-hich immediately precedes the loss of nuclear structure, cannot begin until an adequate supply of inorganic phosphate becomes available. Once cortisol had initiated the first phase of the reaction, its presence in the medium was no longer needed for the completion of the reaction and the resulting dissolution of nuclear structure in a considerable segment of a thymocyte population. When cells were first incubated for 3 h in a phosphate-free medium containing 0.27 ,&I cortisol and then transferred to cortisol-free medium containing phosphate, there was still a rapid increase of Experimental

Cell Research 52

Action of cortisol on lymphocyte appearance ded on the IVe then sol needed nuclei. To

353

nuclear structure

of cells with structureless nuclei, the magnitude of which depenphosphate supply (Fig. 5). attempted to determine the minimum time of exposure to cortito produce the maximum accumulation of cells with structureless do this, thymocytes were maintained in medium containing 30 7oI

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Fig. 3.--The effect of a confrontation with a high concentration (30 m&f) of phosphate on the generation of structureless nuclei in populations of cortisol-treated thymocytes after a prolonged (3 h) incubation in phosphate-free medium. The cortisol concentration was 0.27 ,L& and it was present throughout the incubation period, Fig. 4.-The responses of the nuclear structure of normal thymocytes to a confrontation with several levels of phosphate after a prolonged (3 h) incubation in phosphate-free medium. 0 - 0 The medium into which the cells were transferred contained no added inorganic phosphate; u - - -17. the medium into which the cells were transferred contained 2.5 mM Na,HPO,; 0 -- - 0, the second medium contained 15 m&f NA,HPO,; 0 - 0, the second medium contained 50 mbf Na,HPO,. Fig. 5.--A demonstration of the ability of a temporary exposure to cortisol to alter thymocytes in the absence of extracellular inorganic phosphate. The cells were incubated for 3 h in phosphatefree medium containing 0.27 p&f cortisol. At 3 h, the cells were transferred to cortisol-free media containing various concentrations of phosphate. O--O, 50 m&i’ PO,; 0 - - - 0, 15 m&f PO,; q ---11,2.5mMP0,; l -•,OmMPO,. Fig. 6.--The abilities of transient exposures of thymocytes to cortisol to initiate the process which causes the dissolution of nuclear chromatin structures. 0 ~ 0, Cells continuously maintained in the presence of 0.27 ,L& cortisol in medium containing 30 m&f Na,HPO,; O---O, cells suspended in medium containing 30 mA4 Na,HPO, and exposed to 0.27 w&f cortisol for only 1 h; q ----n , cells suspended in medium containing 30 m&f Na,HPO, and exposed to 0.27 ,uM cortisol for 2 11; 0 ~ 0, untreated control populations.

mdd Na,HPO, and 0.27 ,uM cortisol for various times after which the cells were transferred to cortisol-free medium. While a l-h exposure of a population to cortisol was sufficient to cause the appearance of a considerable number of cells with structurally homogeneous nuclei, a 2-h exposure was as effective as a continuous exposure (Fig. 6). Experimental

Cell Research 52

A continuous provision of phosphate from the medium throughou I the incubation period was also unnecessarv for the cortisol-inclu~etl tiissol ution of nuclear structure. IVhen phosphate \vas removed front the environ Inent of a thynocyte population after 90 min of csposure to cortisol, a larp. 1)art of this population proceeded to develop structureless nuclei cluring thta Following 4.5 h (Fig. 7). Hmvever, an earlier removal of phosphate from the environment considerably impeded the cortisol-intluwd loss of nuclear structure (Fig. 7). ‘Therefore, both the initial changes incluccti by cortisol anti the subsequent phosphate-dependent events are sufr’icicntly aclv:~ncctl bekveen 1 and 2 h so that neither the removal of cortisol nor phc~~phalr

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Fig. 7. Fig. i.-The generation of structureless nuclei in cortisol-treated thymocylcs aflcr (ranaienl sojourns in medium containing a high concentration (30 mM) of phosphate. (A) 0 0 , The cells were continuously incubated in medium containing 30 mM Na,HPO, and 0.27 /rdl cortisol; 0 ---0, the cells were incubated for the first 30 min in medium containing 30 mJf phosphate and 0.27 ~c.U’cortisol and then transferred to phosphate-free medium containing cortisol: 3 cells maintained for the 1st h in medium containing phosphate and cortisol and then transferred to phosphate-free medium containin, 0 cortisol; O---O, cells maintained for the first 1.5 h in medium containing phosphate and cortisol and then transferred to phosphate-free medium containing cortisol; 0 ~ 0, cells maintained continuously in phosphate-free medium containing 0.27 ,uM cortisol. (B) 0 ~--- 0, Cells maintained in cortisol-free medium containing XI mAI Na,HPO,; C~ 0, untreated cells maintained in phosphate-free medium. Fig. S.-The suppression of the ability of cortisol (0.27 $f) to cause the loss of nuclear structure by incubation of thymocytes under anoxic conditions and the release of this inhibition by the restoration of normal aerobic conditions. (A) O---O, Cells continuously maintained untIeI aerobic conditions in medium containing 30 mM Na,HPO, and corlisol; C---C, cells maintained in the same medium under anoxic conditions for 2 h, after which normal aerobic conditions were restored; O---O, cells were maintained throughout the entire incubation period under anoxic conditions in medium containing 30 mM phosphate and 0.27 1~~11cortisol. (B) l - 0, Normal cells maintained in medium containing 30 mM phosphate, but no cortisol; ~-I:, cells maintained in the same medium for 2 h under anoxic conditions after which normal aerobic conditions were restored. I:igs 3-8. Abscissa: h of incubation; ordinccte: “& cells with structureless nuclei. Experimental

Cell Research 52

dcfion

of corfisol

on lymphocyte

nuclear

3.55

structure

from the cellular environment can prevent the ultimate dissolution of nuclear structure. However, it is apparent from Fig. 7 that the proportion of cortisol-treated cells which eventually developed structureless nuclei even in populations withdrawn from phosphate-containing medium at 90 min was always lower than in populations which had been continuously maintained

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Fig. S.-The persistent effect of a temporary exposure of thymocytes to cortisol under anoxic conditions on the generation of structureless nuclei after the restoration of normal aerobic conditions. 0 -0, Cells continuously maintained under normal aerobic conditions in medium containing 30 mM Na,HPO, and 0.27 @f cortisol; O---O, cells maintained under anoxic conditions for only the first 2 h in medium containing 30 mM phosphate and cortisol; 0 ~ 0, cells maintained under anoxic conditions for 2 h in medium containing phosphate and cortisol after which they were transferred to aerobic conditions in medium containing phosphate but no cortisol. Abscissa: h of incubation; ordinate: y0 cells with structureless nuclei. Fig. lO.-The oxygen consumption of normal and cortisol-treated rat thymocytes maintained in media containing different concentrations of Na,HPO,. O-0, Normal cells; O--O, cells maintained in media containing 0.27 ,rcM cortisol. The oxygen consumptions were measured between 30 and 60 min after the cell populations were suspended in uifro. Abscissa: Medium phosphate concentration (m&f); ordinafe: Cellular oxygen consumption (,~~1/10*cells/h). Fig. Il.-The effects of various concentrations of 2,4 nuclear structure in thymocytes maintained in medium cortisol. O--e, The cell suspension contained no 5 x 1O-5 M DNP; 0 --- 0, the suspension contained contained 1O-5 M DNP.

dinitrophenol (DNP) on the dissolution of containing 30 mM Na,HPO, and 0.27 ,uM DNP; O---O, the suspension contained 2 x 1O-5 M DNP; 0 -~ 0, the suspension

in phosphate-containing medium. This lowering was probably due to variation within the population of the rate at which various groups of cells progressed through the cortisol-induced sequence of changes, or in their rate of uptake of phosphate. Cells which progressed more slowly would not have reached the stage of independence from the external phosphate supply before the phosphate was withdrawn from the environment. Experimentul

Cell Research 52

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J. I+‘. Whitfield,

A. Il. Penis and T. Youdale

So far, the properties of the complex reaction responsible for the loss of nuclear structure in cortisol-treated thymocytes are very similar to those of the reaction initiated by ionising radiation [S’i, 64:. A transient exposure lo either agent starts a series of changes \vhich about 1 h after exposure culmnates in the dissolution of nuclear chromating structures provided thcrc has

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Fig. 12.-The ability of 2,4 dinitrophenol (DSP) to prevent the stimulation of the dissolulion of nuclear structure following the confrontation of cortisol-treated cells with a large level of cnvironmental phosphate after a 3-h sojourn in the absence of extracellular phosphate. (-4) 3 h after incubation in phosphate-free medium containing 0.27 ,uM cortisol, the cells were transferred to a medium containing 50 mM Na,HPO, and cortisol. (B) Cells were transferred, after incubation for 3 h in phosphate-free medium plus 0.27 /AM cortisol, to medium containing corlisol, 50 mA4 phosphate, and 5 x 1O-5 M DNP. Figs 11-12. Abscissa: h of incubation;

ordinate:

“/;, cells with structureless

nuclei.

been an adequate supply of inorganic phosphate from the medium. In the absence of phosphate, the first part of the sequence can be put into operation, but the induction of the subsequent phase must await the appearance of phosphate in the environment. If the processes induced by the two agents are indeed identical, \ve may predict that cortisol will not be able to cause nuclear damage in the absence of oxygen; the metabolic process which causes the loss of nuclear structure in irradiated thymocytes is closely linked to respiration and cannot operate in the presence of respiratory inhibitors or under anaerobic conditions [53, 37, 631. This prediction was confirmed by the observation that an exposure of cortisol-treated thymocytes to anaerobic conditions blocked the dissolution of their nuclear structure (Figs 8 and 9). When normal aerobic conditions were restored after a 2-h incubation under anoxic conditions, the cells raphomogeneous nuclei idly regained their ability to generate structurally (Figs 8 and 9). Despite the fact that the dissolution of nuclear structure could Experimental

Cell Research 52

Action of cods01 on lymphocyte

nuclecrr structure

33 7

not occur under anosic conditions, cortisol was able to inflict its primary damage under these conditions since a large fraction of the population still lost their nuclear structure after the simultaneous restoration of normal aerobic conditions and removal of cortisol (Fig. 9). This excessive loss of nuclear structure after the restoration of aerobic conditions and the removal of cortisol \vas not due to damage inflicted on the cells during their sojourn under anaerobic conditions since incubation of untreated cells under anoxic conditions did not cause an abnormal accumulation of cells with damaged nuclei after the restoration of normal aerobic conditions (Fig. 8; see also [63]). It is no\\- clear that the process by \vhich cortisol causes the loss of nutlear structure requires both respiration and inorganic phosphate. To dctermine \\-hich of the two is more directly involved in the dissolution of nuclear structure, 11-edetermined the cellular respiration rate in the presence of various levels of inorganic phosphate. From Fig. 10 it may be seen that, unlike the loss of nuclear structure (Figs 1 and 2), the respiration rates of normal or cortisol-treated cells during the critical first hour of incubation were not influcncccl by the phosphate concentration. Therefore, while respiration probably provides the necessary energy for the reaction, it is the phosphate \\-hich is directly involved in the loss of nuclear structure. Once again, the action of cortisol closely resembles that of ionising radiation [lil]. The operation of the abnormal radiation-induced reaction responsible for the tlisal)pearance of a thymocyte’s nuclear structure can be impeded by concentrations of 2,4 dinitrophenol (DNP) which do not inhibit its oxygen consumption [62, 64’. If the cortisol-induced reaction is indeed the same, then it should also be opposed by DNP. At a concentration of 5 x 1W5 M, DKl’ reduced both the ability of cortisol to cause the loss of nuclear structure and the cellular oxygen consumption (from a mean normal value of 23.7 lo 19.4 ,uI per 108 cells/h) (Fig. 11). However, at concentrations of 1W5 and 2 x lo-” N, DNP \vas still able to reduce the loss of nuclear structure (Fig. 11) although it did not reduce oxygen consumption (the respective mean OS~~CII consumptions \\-ere 28.4 and 26.6 ,ul per 108 cells/h). The ability of DSP to block the operation of the phosphate-dependent phase of the reaction sequence \vas demonstrated by incubating cells for 3 h in the presence of 0.27 ,uM cortisol in phosphate-free medium and then transferring them to a medium containing cortisol, .50 mAl Na,HPO, and 5 x 1W5 Jf DNP. In th e presence of DXP, the sudden exposure of the cortisol-treated cells (lvhich had completed the first phase of the reaction sequence) to such a high environmental phosphate level could not force them to complete the reaction and generate structureless nuclei (Fig. 12 13). In the 24 ~ 681812

Experimenlul

Cell Resectrch 52

358

.I. F. Whitfield,

A. D. Perris and T. Youdale

absence of DNP, however, this treatment of nuclear structure (Fig. 12A).

caused an acceleration

of the loss

DISCUSSION

We conclude that cortisol causes the dissolution of the highly condensed chromatin granules in the lymphocyte nucleus indirectly by initiating, during the first hour of exposure, a metabolic process which is promoted 1~~ inorganic phosphate from the environment and is closely linked to respiration. Since dinitrophenol (an inhibitor of the intermediate reactions of oxidative phosphorylation) can oppose the action of cortisol without inhibiting respiration, some of the intermediate reactions involved in the transduction of the energy of respiration into lhe structure of adenosine triphosphate (ATP) must also play a role in the disaggregation of the chromatin structures [S, 641. These overall characteristics of the cortisol-induced reaction are identical to those of the chromatin-disaggregating reaction initiated by ionising radiation [52-64!, valinomycin [59!, and parathyroid hormone [G’L The very large chromatin granules in the normal lymphocyte nucleus are formed by the combination of lysine-rich histones with DNA 133, 38:. Conversely, the dissolution of these deoxyribonucleoprotein granules \vith the generation of structureless (“pycnotic”) nuclei occurs \vhen the deoxyribonucleoproteins are dissociated into their constituent histoncs and free DNA [33, 37, 38, 581. Therefore, the reaction initiated by cortisol probably drstrays nuclear structure by generating some product which can separate histones from DNA4. Since the fraction of cells Tvhich develops structureless nuclei in a cortisol-treated population is directly proportional to the level of it is very likely that inorganic inorganic phosphate in the environment, phosphate, or some phosphorylatcd compound, is this nucleoprotein-tiissociating product. It is knolvn that exposure of isolated lymphocyte nuclei to inorganic phosphate causes the complete dissolution of their chromatin structures ::iCrj. Inorganic phosphate probably does not cause the loss of nuclear structure by itself since it is not an effective deoxyribonucleoprotein-dissociating agent [5x]. Hokvever, phosphate is transformed into an extremely polverful chromatin-dispersing and nucleoprotein-dissociating agent when it combines with the hydroxyl groups of serine molecules which are situated in clusters along the length of certain serine-rich nuclear proteins [2, 2-1, 23,
Cell Research 52

Action of cortisol on lymphocyte

nuclear structure

359

phenol [2, 24, 2.51, we suspect that it is a compound such as this which is responsible for the loss of nuclear structure in the present case. This is supported by the facts that other compounds such as phytohaemagglutinin, valinomycin, and parathyroid hormone which cause a disaggregation of chromatin structures share an ability to redirect respiratory energy from ATP synthesis into an increased formation of phosphoprotein [2, 10, 36, 39, 40, 41, 59, 62, 671. Our suspicion is further strengthened by the fact that in the normal lymphocyte nucleus phosphorylated compounds are more concentrated in the structurally diffuse portion of the chromatin and this chromatin fraction contains over three times more phosphoprotein phosphorus than does the highly condensed chromatin in the prominent granular structures [2, 15-18, 421. A major part of the small lymphocyte’s DNA is collected into the several large chromatin granules [2, 15-18, 331. While the DNA is sequestered in these aggregates it is functionally inactive and cannot direct the synthesis of various types of informational RNA [2, 5, 15-18, 331. However, the small lymphocyte is endowed with an ability to respond to an interaction of its surfaces with particular substances (e.g. foreign antigens, phytohaemagglutinin, antibodies in antilymphocytic serum) by rapidly separating histone from DNA in order to initiate the synthesis of RNA and proteins [19, 32, 39, 41, 65, 671. While a compound such as phytohaemagglutinin causes this shift to proceed to only a limited extent and the cell enters into a growth-division cycle [2, 24, 25, 39, 41, 671, exposure to other chromatin-dispersing agents such as cortisol, radiation, valinomycin, and parathyroid hormone cause a more exaggerated version of the same reaction which destroys the cell rather than permitting its growth and proliferation [37, 38, 52-641. The capacity of the small lymphocyte for such rapid and large changes in function probably stems from the adherence of most of the large chromatin granules to the inner surface of a rather special type of nuclear membrane which contains about 50 per cent of the cell’s enzymatic machinery for respiration and the oxidative synthesis of 14TP [l, 3, 4, 26630, 34, 661. Therefore, the chromatin granules are in a particularly intimate contact with enzyme systems which are known to be able to synthesise large amounts of powerful nucleoprotein-dissociating agents if the need should arise [2, 24, 251. C)n the basis of the known metabolic effects of other chromatin-dispersing agents such as phytohaemagglutinin [2, 24, 251, valinomycin [35, 36, 401, and parathyroid hormone [14, 43, 441, we suggest that cortisol might act on the cell by forcing the metabolic reactions in the nuclear membrane to shift from their main task of transducing the energy from respiration into Experimental

Cell Research 52

the structure of *iTI’ to the formation of ahnormal quantities of a phosphaterich, chrornatin-dissocialirlg cornpo~~nd such as l)l~osI)hoI~rotein. The severe nuclear change observed in the cortisol-treated lymphocyte is lnwl~ahly an estrcme expression ol the nuclear change \\.hich I)ermits the initiation of specific HS,k and protein syntheses in liver cells. It has alrcaci\ been suggested that cortisol might stimulate these synlhcses t)y scp:lrating histones from 11S.1, thus dcrepressing the informational activity of the 1)X;\ [:5, 9, 46I. This suggestion has lwen supported by observations that wrlisol can react directly with argininc-rich histones to form stable combinations both in the liver of the nhole animal and in solutions of isolated histoncas [47-49-. HoLyever, Ihc present ohsrrvations suggest that wrtisol can cause fundamentally the same nurleoprotrin changes in another \vay t)y altering the metabolic activity located in certain lipoprotein mcmhranes. The rrlati\-c contribution of these tlifl’erent actions to changes in nuclear structure anal metabolic activity probably depends on lhc type of cell.

SUMMARY Cortisol (hgclrocortisone) at a concentration of 0.27 p.11 causes the cornplete loss of nuclear structure (“pycnosis”) in rat thymocytes maintained ill vitro ty the initiation during the first hour of exposure of a phosphate-clcrespiration-linked reaction. The proportion of cells \vhich lose pendenl, their nuclear structure is directly and linearly proportional to the phosphate concentration in the medium. ITnder anoxic conditions, cells treated \\.illi cortisol cannot dcveIop structureless nuclei. 1)initrophenol can also retlucr or prevent the ~ortisol-inducetl loss of nuclear structure even at co~~cc’iltr:~lions lvhich do not reduce respiration. These properties of the action of cortisol are identical to those of the :I(*tions of ionising radiation, parathyroid hormone and valinomycin. l’ossit)le mechanisms underlying the action of corlisol are discussed in the light 01 available information on the effects of these other agents. REFERENCES

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