- Email: [email protected]
Glucocorticoid-induced lymphoma cell death: The good and the evil
1. steroid Eiocltem. Vol. 27, No. l-3, pp. 413-419, Printed in Great Britain. All rights reserved
1987
0022-473 I,‘87 $3.(H) + O.oGt Copyright @ 1987 Pergamon Journals Journals Ltd
Proceedings of the VII International Congress on
Hormonal Steroids (Madrid, Spain, 19x6)
12. The Biology of Neoplasms
GLUCOCORTICOID-INDUCED LYMPHOMA CELL DEATH: THE GOOD AND THE EVIL KLAUS WIELCKENS,* TORSTEN DELFS, ASTRI~ MUTH, VERENA FREESE and HANS-J• ERN KLEEBERG Department of Clinical Chemistry, Hormone and Tumor-Marker
versity of Hamburg, Martinistrasse
Laboratory, Medical Clinic, Uni52, 2000 Hamburg, W. Germany
Sudan-Giucocorticoid hormones and their synthetic derivatives are widely used in therapy due to their strong anti-inflammatory and immunosuppressive potential. While the molecular basis of the anti-inflammatory action is to date at least partially understood, knowledge regarding the mechanism underlying glucocorticoid effects on the immune system is rather fragmentary. The immunosuppression could be attributed to at least two distinct processes: inhibition of the nroduction of growth mediators and glucocorticoid-induced cell death. Tie mechanism of glucocor&oid-induced cell death can be divided into two steps, a reversible growth inhibition and cell lysis. The first step is characterized by many metabolic alterations typical of the catabolic potential of corticosteroids. After a delay of several hours activation of an endonuciease appears to initiate the fytic phase. By the action of this endonuclease the DNA is fragmented. In opposition to the chromatin damage, poly(ADP-ribosyl)ation is activated in order to stabilize the chromatin structure until the antagonistic potential is exhausted and the cells die. Therefore it can be speculated that the lethal event in glucocorticoid-induced cell death is a destruction of the regular chromatin structure.
Clucocorticoids such as cortisol or its synthetic analogs prednisone, prednisolone, dexamethasone, etc. are employed in the clinic because of their strong anti-inflammatory and immunosuppressive properties. The tatter is also the basis of glucocorticoid therapy for various lymphoproliferative diseases. The anti-inflammatory potential of these drugs (or the endogenous hormone) depends at least partially on the inhibition of arachidonic acid release from membrane phospholipids [l, 21 leading to a block of cycle-oxygenase and lipoxygenase pathways. As a consequence the production of two groups of compounds, prostaglandins and leukotrienes, known to be strong mediators of inflammation [3,4] is inhibited. This corticosteroid effect is accomplished by stimulating the synthesis of an inhibitor which blocks for phospholipase A2, the enzyme responsible arachidonic acid release, the rate-limiting step for the production of the inflammation mediators [5,6]. In contrast to the mechanism of the anti-inflammatory action of corticosteroids which is now partially understood, the mechanism of glucocorticoidinduced immunosuppression is still unclear. This may be due to the complexity of glucocorticoid hormone effects on lymphatic tissue. Evidence is accumulating that the mechanism of immunosuppression can be divided into two processes: a block of proliferation and induction of cytolysis of certain cells. Cessation of proliferation is presumably mediated by a block in the production of T cell growth factor [7], but inhibition of leukotriene B, production could also be involved [8], pointing to a con-
*To
whom correspondence
S% 27.113-*a
should be sent.
tribution of the lipoxygenase pa&way to growth regulation in lymphatic cells. Besides the anti-proliferative action of glucocorticoids a direct lethal effect on T lymphocytes has been recognized for many years (cf. Refs[9, IO]). This effect is most dramatic in certain rodents, thymus cells from rats or mice being killed by corticosteroids within hours [lo]. A comparable response of human T lymphocytes to glucocorticoid challenge has been demonstrated but appears to be more delayed than in rodent systems [ lO]. The rapid decrease in the number of blood lymphocytes is therefore not a consequence of glucocorticoid-induced cell death but rather of a rapid redistribution of lymphocytes to extravascular compartments [ 1 I]. The cytolytic action of corticosteroids on lymphoma cells of human origin, both in uivo and in vitro [ 12, 131, substantiates the comparable response of human cells. Glucocorticoid-induced cell death was studied in rat thymocytes and a variety of cultured iymphoma cell lines, e.g. P 1798, W 7, S49 of murine [lo] or CEM C7 of human origin [ 131. Mouse lymphoma lines particularly turned out to be a versatile model to investigate the mechanism of glucocorticoid-medjated cell death. The corticosteroids appear to trigger an active suicide mechanism in these cells apparently depending on the expression of “new” genes [lo]. Glucocorticoid-induced programmed cell death is morphologically distinct from necrosis which proceeds as an unregulated process most probably resulting from a breakdown in energy meta~ljsm~l4]. In contrast to necrosis the ultrastructural changes following corticosteroids, called apoptosis [ 143, primarily affect the cell nucleus and not extranuclear compartments. It is noteworthy that following 413
414
KLAUS WIELCKENSet al.
Table 1. Glucocorticoid-induced alterations cytes and lymphoma cells Metabolic process
in lympho-
References
Inhibition of DNA synthesis Inhibition of RNA synthesis Inhibition of protein synthesis Stimulation of RNA degradation Stimulation of protein degradation Inhibition of glucose transport Inhibition of aminoacid transport Inhibition of nucleoside transport Inhibition of lipid synthesis Inhibition of fatty acid oxidation Inhibition of phospholipid synthesis Inhibition of polyamine synthesis
16,1-l 16-18 16-18 19 20 21,18 18,22 23 ss 26 27
glucocorticoid treatment a labilization of nuclei was reported [ 1.51 pointing to the critical role of nuclear events in corticosteroid-induced cell 1ysis;‘the bio-
chemical basis for these nuclear been characterized.
changes
has not
Many biochemical alterations have been demonstrated in corticosteroid-challenged lymphatic cells
sensitive to the cytolytic action of adrenal steroids (see Table 1) but all changes appear to be the consequence of the antiproliferative and catabolic potential of glucocorticoids and have also been described in non-lymphytic tissue [9] not destroyed by these steroids. Therefore these alterations cannot be involved in the lethal event. Evidence is accumulating, however, that the antiproliferative effect of corticosteroids is permissive for the induction of programmed cell death in sensitive cells. Proliferating lymphoma cells are not killed shortly after the addition of the steroid but following a latency period. During this period cellular metabolism is switched to the state of non-proliferating cells but viability of cells is not affected, as indicated by unchanged ATP [28] levels, which are an excellent parameter for cellular integrity. Accumulation of lymphoma cells in G, of the cell cycle precedes initiation of cell lysis[29] supporting the idea of a permissive role of the antiproliferative steroid effect in glucocorticoid-induced cell death. Additional evidence for this idea can be derived from cell hybridization experiments: the anti-proliferative and the cytolytic potential of glucocorticoids could be separated but no cell line has been identified which responds to corticosteroid challenge by cell death not preceded by growth inhibition. Analyses with the aid of an assay of colony-forming ability suggested the reversibility of the metabolic alterations during the latency period [29], which implies that glucocorticoid-induced cell death is not predetermined until very shortly before the cells die. But what is the lethal event? DNA FRAGMENTATION AND POLY(ADP-RIBOSYL)ATION
In 1980 Wyllie reported the observation that treatment of thymocytes with corticosteroids in-
duced a pronounced DNA fragmentation [30]. The molecular distribution of DNA fragments was dis-
crete as a result of degradation of the internucleosomal linker DNA, possibly by the action of a Ca2+, Mg”-dependent endonuclease known to produce such a digestion pattern [3 11.A comparable response has been found in other cells of lymphatic origin such as S49.1 mouse lymphoma cells (see Fig. 1) which are sensitive to the cytolytic potential of glucocorticoids[32], but was absent from cells derived from other organs[33]. Therefore it was proposed that corticosteroids activate an endonuclease which brings about the dramatic chromatin destruction [30]. This hypothesis, however, poses a problem: a comparable DNA fragmentation could also be detected in cells killed by agents known to initiate necrosis, e.g. fluoride, azide, or by compounds interfering with DNA and protein synthesis, or by overgrowth (Ref. [34] and K. Wielckens, unpublished results). Apparently such an enzyme is permanentIy present in the chromatin since a comparable DNA breakage can be shown in cycloheximide-treated cells where protein synthesis is entirely blocked (Ref.[34] and K. Wielckens, unpublished results). Hence it can be speculated that DNA fragmentation is not involved in the lethal event but occurs post mortem regardless of whether a cell has died by a necrotic or apoptotic process. Recently, however, two observations again support the crucial role of DNA fragmentation in glucocorticoid-induced cell death. Mark Compton and John Cidlowski reported that DNA strand breakage occurred in dexamethasone treated rat thymus cells before the cells were lysed [33]. In our laboratory a slight DNA fragmentation could also be detected in dexamethasone-incubated S49.1 lymphoma cultures when cells were still viable [28] and a concomitant activation of poly(ADP-ribosyl)ation, a nuclear protein modification reaction known to be stimulated by DNA incisions [35, 361. This reaction is catalyzed by poly(ADP-ribose)synthetase, a DNA-dependent enzyme that utilizes NAD and modifies several nuclear proteins covalently, including the synthetase itself 1371, histone H2B [38] and DNA topoisomerase I [39]. Due to the activation of poly(ADP-ribose)synthetase by DNA strand breaks, cell treatments that lead to chromatin fragmentation, e.g. incubation with alkylating agents [40,4 I] or UV irradiation [42], result in a dramatic but transient increase in protein-bound poly(ADP-ribose) and a concomitant decrease in the substrate NAD [40,4 I]. Studies of the turnover of poly(ADP-ribose) revealed an extremely high rate [4 1,431, suggesting an involvement of this protein modification process in fast reactions and probably not comparable to other modification mechanisms, such as phosphorylation, which are known to activate or inactivate certain enzymes. A functional poly(ADPribosyl)ation system is necessary for adaptation of cells to treatment with alkylating agents, suggesting
Glucocorticoid-induced
cell death
Fig. I. DNA fragmentation in dexamethasone-treated 549-l mouse Iymphoma cells. Cells were incubated for 3 days +/- 10m2M dexamethasone. DNA were extracted and analyzed on an agarose gel as described [28]. “DNA-Laengenstandard II” (from Boehringer, Mannheim = EcoRl and Hind III digested lambda DNA) was used as a mol wt marker (fragment sizes: 21.7,5.0,4.2,3.5,2.0,1.9,1.6,1.4, 0.9,0.8, and 0.6 kilobase pairs). Lanes A, B, G and H: marker DNA (5 to 10 pg). Lanes C and D: DNA from control cells. Lanes E and F: DNA from dexamethasone-treated ceil.
415
KLAUS WIELCKENS et al.
416
Table 2. Poly(ADP-ribose) levels in dexamethasone treated and control cells Treatment
Total
activity
None Dexamethasone
1
-
6.7 i-j- 0.2 12.1 -i-l- 1.1
S40.1 mouse lymphoma cells were incubated with or without 10-’ M dexamethasone for 20 h and poly(ADP-ribose) was determined as described 1281.
.c*-i:k., ‘0
Poly(ADP-ribose) (pmol/lON cells)
\ \
Intrinsic
0ctiW
“--o
+ dexomet~asane
r-9
. Control
\ b
+ dexamethasane
Fig. 2. Total and intrinsic poiy(ADP-ribose)synthetase activities in dexamethasone-challenged S 39.1 cells. Cells were treated with 10s7 M dexamethasone for the times indicated and intrinsic and total enzyme activities were determined in digitonin-permeabilized cells as described [28]. Inset: percentage of active enzyme.
an involvement of this process in DNA repair, either directly in the DNA ligase reaction 1441 or indirectly in restoration of chromatin structure [45]. When the poly(ADP-ribose)synthetase was determined in untreated S49.1 mouse Iymphoma cells about 5% of the enzyme was found active and no pronounced change in the intrinsic or total activity could be detected throughout the entire experiment (Fig. 2). Incubation of S49.1 lymphoma cells with dexamethasone led to a 2-fold increase of intrinsic activity after 24 h, while the total .activity decreased slightly. Thereafter, both the intrinsic and the total poty(ADP-ri~se)synthetase activity fell below the values of control cells because of the progressive loss of cells induced by the corticosteroid treatment. Therefore the enzyme stimulation in response to the glucocorticoid challenge must be expressed by the amount of active enzyme (Fig. 2, inset) revealing a S-fold increase of the intrinsic activity on day 3. The stimulation of the poIy(ADFribosyliation system can also be demonstrated by an increase in the protein bound poly(ADP-ribose) (Tabie 2), although only a slight augmentation of the polymer level was detectable when measured 20 h
after the steroid addition. The increase in proteinbound poly(ADP-ribose) was found at a time when all cells were still viable, although the increase was only 2-fold. However, the half-life of the polymer makes it difficult to estimate the real extent of the stimulation. That the activation of the poly(ADPribosyl)ation system occurred at a time where all cells in corticosteroid-treated cultures were still intact was also reflected by a decrease in the substrate NAD [28]. The loss in NAD could be antagonized by an inhibitor of po~y(ADP-ri~s~)synthetase, the nicotinamide analog benzamide [2X], further supporting the observation that glucocorticoid treatment leads to fragmentation of DNA, which in turn activates the poly(ADP-ribose)synthetase, thereby combating the chromatin damage. The antagonistic function of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death can be easily demonstrated by incubation of S49.1 lymphoma cells with dexamethasone in the presence or absence of benzamide: inhibition of poly(ADP-ribose) formation not only increased the number of cells which died by the steroid challenge but also accelerated the velocity of corticosteroid-mediated cell kill (Fig, 3). This observation suggests that the lethal process in glucocorticoid-induced cell death is a competition
1OOr
Day after
dexamethasone
addition
Fig. 3. Effect of the poly(ADP-ribose)synthetase inhibitor benzamide on the number of dead cells in dexamethasonetreated 549.1 cells. Cells were incubated for the times indicated with IV7 M dexamethasone +/- benzamide, and the percentage of dead cells was determined as described [28].
Glucocorticoid-induced cell death between a nuclease destroying the integrity of the chromatin and mechanisms, including poly(ADPribosyl)ation, attempting to restore the intactness of chromatin. Interference with these mechanisms therefore increases the toxicity of the steroid and causes cell death at an earlier time.
417
1OOr
POLY(ADP-RIR~SYL)ATI~NANDCHROMAT~ STRUCTURE
The antagonistic role of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death raises the question of the exact function of this nuclear protein modification process. The involvement of poly(ADP-ribosyl)ation in DNA repair as already mentioned in a preceding section cannot be questioned but the fact that inhibition of poly(ADPribose) formation leads to a higher steady-state concentration of DNA strand breaks in cells damaged with alkylating agents has initiated controversial discussions [45]. The findings that a block in poly(ADP-ribose) formation did not reduce the overall repair rate but led to an increase in the number of incisions and a higher number of unrepairable breaks, however, favoured the idea that poly(ADP-ribosyl)ation could be involved in maintenance or restoration of chromatin structure. This hypothesis was supported by preliminary data showing chromatin alterations in benzamide-treated HeLa cells[45], although it was not clear whether these alterations were restricted to HeLa cells or a general consequence of long-term inhibition of poly(ADPribosyl)ation. When nuclei were prepared from S49.1 cells exposed to benzamide for 24 h by a very gentle technique maintaining full chromatin integrity (on the basis of strand breaks) the sensitivity of the chromatin to endonucleases can be used as a tool to obtain indirect information about structure. Quantitating the number of incisions created by the nuclease treatment in a given time should therefore allow discrimination between states of chromatin compactness. Digestion of chromatin with micrococcal nuclease revealed only a slight increase of the nuclease sensitivity in benzamide vs mock-treated cells (not shown). By contrast, replacement of micrococcal nuclease by DNase I resulted in a pronounced augmentation of enzyme-mediated DNA breakage in benzamide-treated cells (Fig. 4). The sensitivity of the procedure used in this experiment to measure DNA fragmentation allows the detection of several hundred to approximately 20003000 breaks/cell. DNase I hypersensitive sites could therefore be involved in the structural alteration induced by inhibition of poly(ADP-ribosyl)ation. The apparent discrepancy between micrococcal nuclease and DNase I in recognition of changes in the chromatin structure in benzamide-treated cells could be due to the DNase I property of preferential digestion of active chromatin [46]. Micrococcal nuclease also attacks active chromatin with a higher
I
I
0
5
Incubation
I 10 time
(mid
Fig. 4. Effect of benzamide on the DNase I sensitivity of S49.1 cell chromatin. Cells were treated for 24 h +/- 5 mM benzamide and the nuclei prepared and incubated for the times indicated with 0.66 U/ml DNase. DNA fragmentation was determined as described[48]. The log of percentage of double-stranded DNA is in linear relationship to the number of breaks (intact DNA = high percentage of double-stranded DNA).
rate but the difference between active and total chromatin is not as pronounced as with DNase I. Studies with the aid of the nucleoid sedimentation technique [47] led to additional evidence for benzamide-induced changes in nuclear structure, pointing to a more general structural alteration of the chromatin-nuclear-matrix-complex (K. Wielckens and A. Muth, manuscript in preparation). These findings suggest that poly(ADP-ribosyl)ation is necessary for maintaining the regular structure of active chromatin. Introduction of breaks may have drastic consequences for the structure if chromatin is under torsional tension. Although the bulk of chromatin may lack torsional stress [49], recent evidence has supported the idea of torsional stress in active chromatin [SO]. Therefore it is possible that poly(ADP-ribosyl)ation is involved in the restoration of uncoiled regions. This hypothesis would explain why poly(ADP-ribosyl)ation is triggered by DNA incisions as well as the high-energy consumption during poly(ADP-ribose) formation, i.e. restoration of a highly energetic structure. Therefore inhibition of poly(ADP-ribosyl)ation in corticosteroid-treated lymphoma cells should enhance the alterations in chromatin structure caused by the endonuclease mediated introduction of strand breaks. Furthermore, the lethal event in glucocorticoid-induced cell death could be a structural alteration of chromatin interfering with transcription. CONCLUSIONS
Experiments and speculations provided in the preceding sections divided the response of glucocorticoid sensitive lymphoma cells into two steps: a reversible block in proliferation and the lysis of the cells. Although the switch to the nonproliferating state does not represent the lytic event it is per-
418
KLAUS WIELCKENSet al. Stabilization:
Destabilization: Chromatin fragmentation
DNArepair poly(ADP-f-ibosyl)ation
t
I
t
cell
t
survival
death
Fig. 5. Possible mechanism of glucocorticoid-induced
missive for cellular suicide of lymphoma cells. The lytic event is initiated by the activation of a mechanism destabiIizing the regular chromatin structure, i.e. an endonuclease. A DNA repair involving poly(ADP-ribosyl)ation phenomenon occurs in opposition to the chromatin destruction and antagonizes the destabilization until the NAD is consumed and the cellular capacity to counteract is exhausted. Hence glucocorticoid-induced cell death can be described (Fig. 5) as a fight between stabilization (good) and destabilization (evil). The crucial process in glucocorticoid-induced cell death should therefore be the activation of an endonuclease. This enzyme digests the chromatin although the cells are still viable, which is in contrast to necrosis where the same enzyme apparently degrades the DNA post mortem. The nuclease, endonuclease, possibly the Ca’+, Mg2’-dependent appears to be already present in the chromatin and does not need to be induced by the corticosteroid. Since the enzyme is fully active only in the presence of mil~imolar amounts of Ca*’ [.511 and the concentration of free Ca”-ions does not exceed 1 pmol, an elevation of the free calcium-level must also be postulated. Alternatively, it has to be taken into consideration that the early DNA fragmentation in corticosteroid-treated lymphoma cells is not accomplished by this enzyme in contrast to the late DNA breakage which is characterized by the discrete fragment pattern. Not only an increase in the destabilizing capacity but also a decrease in the stabilization appeared to contribute to the cetl lysis, e.g. a decline in the repair capacity (K. Wielckens and V. Freese, manuscript in preparation). Since apoptosis is not only induced by glucocorticoids but represents a general cellular response to a specific physiological cue [14] it is possible that corticosteroids employ a suicide mechanism present in every cell. Thus in glucocorticoid-sensitive lymphocytes and lymphoma cells the glucocorticoid receptor system couid be linked to an yet unknown mediator of the suicide system. REFERENCES 1. Gryglewski R. J., Panczenko B. Grodzinska L. and Ocetkiewica A.: Corticosteroids inhibit Drostaglandin
2.
7 -.
4
5
lymphoma cell death.
release from perfused mesenteric blood vessels in rabbits. Prostuglandins 10 (1975) 343-354. Tashjian A., Yoelkel E., McDonough J. and Levin L,: Hydrocortisone inhibits prostaglandin production by mouse fibrosarcoma cells. Nature, Lond. 258 (197.5) 739-741. Davies P., Bailey P. J., Godenberg M. M. and FordHutchinson A. W.: The role of arachidonic acid oxygenation products in pain and inflammation. Ann. Rev. Immun. 2 (1984) 335-357. Needleman P., T&k J.,‘Jakschik B. A., Morrison A. R. and Lefkowith J. B.: Arachidonic acid metabolism. A. Rev. Biochem. 55 (1986) 69-102. Flower R. J. and Blackwell’G. J.: Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation. Nature, Land. 278 (1978) 456-458.
K., 6. Hirata F., Schiffman E., Yenkatasubramanian Salomen D. and Axelrod J.: A phospholipase A, inhibitory protein in rabbit neutroohils induced bv glucocor%coids. Proc. nam. Acad.’ Sci. U.S.A. i? (1980) 2533-2538. 7. Gillis S., Crahtree G. R. and Smith K. A.: Glucocorticoid-induced inhibition of T cell growth factor production. I. The effect of mitogen-induced lymphocyte proliferation. J. Immun. 123 (1979) 16241631. 8. Goodwin J. S., Atluru D., Sierakowski S. and Lianos E.: Mechanism of action of elucocorticoids. lnhibition of T cell proliferation and iiterleukin 2 production by hydrocortison is reversed by ieukotrien Bq. 1. clin. Inuest. 77 (1986) 1244-1250. 9. Munck A. and L.eung K.: Glucocorticoid receptors and mechanisms of action. In Receptors and Mechanism of Action of Steroid Hormones (Edited by Pasqualini J. R.). Marcel Dekker, New York, Part II (1977) pp. 31 l-397. 10. Munck A. Crabtree G. R.: Glucocorticoid-induced lymphocyte death. In CefZ Death in Biology and Pathology (Edited by Bowen I. D. and Lockshin R. A.). Chapman Hall, London (1981) pp. 329-359. 11 Fauci A. S. and Dale D. C.: The effect of in uiuo hydrocortisone on subpopulations of human lymphocytes. J. clin. Invest. 53 (1974) 240-246. 12. Wolff J. A., Brubaker C. A., Murphy M. L., Pierce M. I. and Sever0 N.: Predisone therapy of acute childh~d leukemia: prognosis and duration of resnonse in 330 patients. J: Pehiat. 70 (1967) 626-631. ’ 13. Norman M. R. and Thompson E. B.: Characterization of a gIucocorticoid-sensi~ve human l~phoid cell line. Cancer Res. 37 (19771 3785-3791.
14. Wyllie A. H.: Ceil deaih: a new classification senarating apoptosis from necrosis. In Cell Death in biology and ~utholo~ (Edited by Bowen I. D. and Lockshin R. A.). Chapman & Hall, London (1981) pp. 9-34. 15. Nicholson M. L. and Young D. A.: Effect of glucocorticoid hormones in vitro on the structural integrity of
Giuc~orticoid-induct
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26
27.
28.
29.
30.
31.
32.
nuclei in corticosteroid-sensitive and -resistant lines of lvmphosarcoma P1798. Cancer Res. 38 (1978) 3673368b. Makman M. H., Dvorkin B. and White A.: Alterations in protein and nucleic acid metabolism of thymocytes produced by adrenal steroids. J. biol. Chem. 241 (1966) 1646-1648. Makman M. H., Dvorkin B. and White A.: Influence of cortisoi on the utilization of precursors of nucleic acids and protein by iymphoid ceils in uifro. J. biol. Chem. 243 (1968) 1485-1497. Rosen J. M., Fina J., Miihoiland R. J. and Rosen F.: Inhibitory effect of cortisol in vitro on 2-deoxyglucose uptake and RNA an protein meta~iism in lymphosarcoma P1798. Cancer Res. 32 (1972) 350-355. Cidlowski J. A.: Glucocorticoids stimulate ribonucieic acid degradation in isolated rat thymic lymphocytes in vitro. Endocrinology 111 (1982) 184-190. MacDonald R. G., Martin T. P. and Cidlowski J. A.: Glucocorticoids stimulate protein degradation in lymphocytes: a possible mechanism of steroid-induced cell death. E~ocr~~o~ogy 107 (1980) 1512-1524. Munck A.: Metabolic site and time course of cortisol action on glucose uptake, lactic acid output, and glucose 6-phosphate levels of rat thymus in vitro. J. biol. Chem. 243 (1968) 1039-1042. Makman M. H., Dvorkin B. and White A.: Evidence for induction by cortisol in vitro of a protein inhibitor of transport and phosphoryiation processes in rat thymocytes. Proc. natn. Acad. Sci. U.S.A. 68 (1971) 1269-1273. Stevens J., Stevens Y. W. and Hoilander V. P.: Substrate requirement and kinetic analysis of the cortisoi effects on uridine uptake and incorporation by mouse iymphoma P1798 cells in t&o. Cancer Res. 34 (1974) 233Cr2337. Foley J. E., Jeffries M. and Munck A.: Glu~ocorticoid effects on incorporation of lipid and protein precursors into rat thymus ceil fractions. J. steroid Biochem. 12 (1980) 231-243. Turneil R. W.. Clarke L. H. and Burton A. F.: Studies on the mechanism of corticosteroid-induced lymphocytolysis. Cancer Res. 33 (1973) 203-211. Story T. M., Standaert M. M. and Melnykovych G.: Gluc~orticoid-induced alterations in phosphatidyicholine metabolism in mouse lymphoma. Cancer Res. 33 (1973) 2872-2877. Russell D. H., Haddox M. K. and Gehring U.: Effects of dexamethasone and cyclic AMP on polyamine synthesizing enzymes in mouse lymphoma c&s. J. cell. Phvsiof. 106 (1981) 375-386. W&kens K.‘ and ‘Delfs T.: Glucocorticoid-induced cell death and ~Iy~ADP-ri~syl)ation: increased toxicity of dexamethasone on mouse S49.1 iymphoma cells with the poly(ADP-ribosyl)ation inhibitor benzamide. Endoirinology 119 (1986) 2383-2392. Harmon J. M.. Norman M. R.. Fowies B. J. and Thompson E. B.: Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line. f. cell. P~ysiol. 98 (1979) 267-278. Wyilie A. H.: Glucocorticoid-induced thyrnocyte apoptosis is associated with endogenous endonuciease activation. Nature, Lond. 284 (1980) 555-556. Nakamura M., Sakaki Y., Watanabe N. and Tagaki Y.: Purification of the Ca*+ plus Mg2+ dependent endodeoxyribonuclease from calf thymus chromatin. 1. Biochem. 89 (1981) 143-152. Sibley C. H. and Tomkins G. M.: Isolation of lymphoma cell variants resistant to killing by giucocorticoids. Cell 2 (1974) 213-220.
cell death
419
33. Compton M. M. and Cidiowski J. A.: Rapid in uiuo effect of glucocorticoids on the integrity of rat iymacid. phocyte genomic deoxyribonucleic Endoctinology 118 (1986) 38-45. 34. Vedeckis W. V. and Bradshaw H. D.: DNA fragmentation in S49 lymphoma cells killed with giucocorticoids and other agents. Mofec cell. Erulocr. 30 (1983) 215-227. Ann. 35. Ueda K. and Hayasin 0.: ADP-riboxyiation. Reu. Biochem. 54 (1985) 7@-5(Ic). 36. Miller E. G.: Stimulation of nuclear polyadenosine diphosphate ribose) polyrnerase activity from Hela cells by endonucleases. B&him. biophys. Acta 395 (1975) 191-200. 37. Ogata N., Ueda K., Kawaichi M. and Hayaishi 0.: Poly(ADP-ribose)synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei. J. biol. Chem. 265 (1981) 4135-4137. 38. Adamietz P. and Rudolph A.: ADP-ribosylation of nuclear proteins in uiuo. f. biol. Chem. 259 (1984) 68416846. 39. Ferro A., Higgins P. and Olivera B.: Poly(ADPribosyi)ation of a topoisomerase J. biol. Chem. 258 (1983) 60006003. 40. Juarez-Saiinas H., Sims H. L. and Jacobson M. K.: Poly(ADP-ribose) in carcinogen-treated cells. Nature, Land. 282 (1979) 740-741. 41. Wielckens K., Schmidt A., George E., Bredehorst R. and Hilz H.: DNA fragmentation and NAD depletion. f. biol. Gem. 257 (fi82) 12,872-12,877. _ 42. Jacobson E. L.. Antol K. M.. Juarez-Saiinas H. and Jacobson M. K.: Poly(ADPlribose) metabolism in ultraviolet irradiated human fibroblasts. J. biol. Chem. 258 (1983) 103-107. 43. Wielckens K., George E., Pless T. and Hiiz H.: Stimulation of poly(ADP-ri~syi)ation during Ehrlich ascites tumor c&Is “starvation” and suppression of CODcorn&ant DNA fragmentation by benzamide. J. biol. Chem. 258 (1983) 4098-4104. 44. Durkacz B. W.. Omidiii. 0.. Grav D. A. and Shall S.: (ADP-ribose), participates in DhA excision repair. Nature, Lond. 283 (1980) 593-596. 45. Wieickens K., Piess T. Schaefer G.: Poly(ADP-ribosyl)ation, DNA repair and chromatin structure. In ADP-Ri~sylado~ of Proteins (Edited by Aithaus F. R., Hiiz H., and Shall S.) Springer-Veriag, Berlin (1985) pp. 252-263. 46. Weintraub H. and Groudine M.: Chromosomal subunits in active enes have an altered conformation. Science 193 (197 d 1848-856. 47. Cook P. R. and Braze11 I. A.: Supercoils in human DNA. J. Cell Sci. 19 (1975) 161-279. 48. Ahrstroem G. and Erixon K.: Measurement of strands breaks bv alkaline denaturation and hydroxyapatite chromatdgraphie. In DNA Repair (Edited by Friedhere E. C. and Hanawait P. C.) Marcel Dekker, New York (1981) pp. 403-418. 49. Sinden R. R., Carlson J. O., Pettijohn D. E.: Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells. Cell 21. (1980) 773-783. 50. Harland R. M., Weintraub H. and McKnight S. L.: Transcription of DNA injected into Xenopus laeois oocytes is influenced by template topology. Narure, Lond. 302 (1982) 38-43.
51. Murphy E., Co11 K., Rich T. L. and Williamson J. R.: Hormonal effects on calcium homeostasis in isoiated hepatocytes. J. biol. Gem. 255 (1980) 6600-6608.
1987
0022-473 I,‘87 $3.(H) + O.oGt Copyright @ 1987 Pergamon Journals Journals Ltd
Proceedings of the VII International Congress on
Hormonal Steroids (Madrid, Spain, 19x6)
12. The Biology of Neoplasms
GLUCOCORTICOID-INDUCED LYMPHOMA CELL DEATH: THE GOOD AND THE EVIL KLAUS WIELCKENS,* TORSTEN DELFS, ASTRI~ MUTH, VERENA FREESE and HANS-J• ERN KLEEBERG Department of Clinical Chemistry, Hormone and Tumor-Marker
versity of Hamburg, Martinistrasse
Laboratory, Medical Clinic, Uni52, 2000 Hamburg, W. Germany
Sudan-Giucocorticoid hormones and their synthetic derivatives are widely used in therapy due to their strong anti-inflammatory and immunosuppressive potential. While the molecular basis of the anti-inflammatory action is to date at least partially understood, knowledge regarding the mechanism underlying glucocorticoid effects on the immune system is rather fragmentary. The immunosuppression could be attributed to at least two distinct processes: inhibition of the nroduction of growth mediators and glucocorticoid-induced cell death. Tie mechanism of glucocor&oid-induced cell death can be divided into two steps, a reversible growth inhibition and cell lysis. The first step is characterized by many metabolic alterations typical of the catabolic potential of corticosteroids. After a delay of several hours activation of an endonuciease appears to initiate the fytic phase. By the action of this endonuclease the DNA is fragmented. In opposition to the chromatin damage, poly(ADP-ribosyl)ation is activated in order to stabilize the chromatin structure until the antagonistic potential is exhausted and the cells die. Therefore it can be speculated that the lethal event in glucocorticoid-induced cell death is a destruction of the regular chromatin structure.
Clucocorticoids such as cortisol or its synthetic analogs prednisone, prednisolone, dexamethasone, etc. are employed in the clinic because of their strong anti-inflammatory and immunosuppressive properties. The tatter is also the basis of glucocorticoid therapy for various lymphoproliferative diseases. The anti-inflammatory potential of these drugs (or the endogenous hormone) depends at least partially on the inhibition of arachidonic acid release from membrane phospholipids [l, 21 leading to a block of cycle-oxygenase and lipoxygenase pathways. As a consequence the production of two groups of compounds, prostaglandins and leukotrienes, known to be strong mediators of inflammation [3,4] is inhibited. This corticosteroid effect is accomplished by stimulating the synthesis of an inhibitor which blocks for phospholipase A2, the enzyme responsible arachidonic acid release, the rate-limiting step for the production of the inflammation mediators [5,6]. In contrast to the mechanism of the anti-inflammatory action of corticosteroids which is now partially understood, the mechanism of glucocorticoidinduced immunosuppression is still unclear. This may be due to the complexity of glucocorticoid hormone effects on lymphatic tissue. Evidence is accumulating that the mechanism of immunosuppression can be divided into two processes: a block of proliferation and induction of cytolysis of certain cells. Cessation of proliferation is presumably mediated by a block in the production of T cell growth factor [7], but inhibition of leukotriene B, production could also be involved [8], pointing to a con-
*To
whom correspondence
S% 27.113-*a
should be sent.
tribution of the lipoxygenase pa&way to growth regulation in lymphatic cells. Besides the anti-proliferative action of glucocorticoids a direct lethal effect on T lymphocytes has been recognized for many years (cf. Refs[9, IO]). This effect is most dramatic in certain rodents, thymus cells from rats or mice being killed by corticosteroids within hours [lo]. A comparable response of human T lymphocytes to glucocorticoid challenge has been demonstrated but appears to be more delayed than in rodent systems [ lO]. The rapid decrease in the number of blood lymphocytes is therefore not a consequence of glucocorticoid-induced cell death but rather of a rapid redistribution of lymphocytes to extravascular compartments [ 1 I]. The cytolytic action of corticosteroids on lymphoma cells of human origin, both in uivo and in vitro [ 12, 131, substantiates the comparable response of human cells. Glucocorticoid-induced cell death was studied in rat thymocytes and a variety of cultured iymphoma cell lines, e.g. P 1798, W 7, S49 of murine [lo] or CEM C7 of human origin [ 131. Mouse lymphoma lines particularly turned out to be a versatile model to investigate the mechanism of glucocorticoid-medjated cell death. The corticosteroids appear to trigger an active suicide mechanism in these cells apparently depending on the expression of “new” genes [lo]. Glucocorticoid-induced programmed cell death is morphologically distinct from necrosis which proceeds as an unregulated process most probably resulting from a breakdown in energy meta~ljsm~l4]. In contrast to necrosis the ultrastructural changes following corticosteroids, called apoptosis [ 143, primarily affect the cell nucleus and not extranuclear compartments. It is noteworthy that following 413
414
KLAUS WIELCKENSet al.
Table 1. Glucocorticoid-induced alterations cytes and lymphoma cells Metabolic process
in lympho-
References
Inhibition of DNA synthesis Inhibition of RNA synthesis Inhibition of protein synthesis Stimulation of RNA degradation Stimulation of protein degradation Inhibition of glucose transport Inhibition of aminoacid transport Inhibition of nucleoside transport Inhibition of lipid synthesis Inhibition of fatty acid oxidation Inhibition of phospholipid synthesis Inhibition of polyamine synthesis
16,1-l 16-18 16-18 19 20 21,18 18,22 23 ss 26 27
glucocorticoid treatment a labilization of nuclei was reported [ 1.51 pointing to the critical role of nuclear events in corticosteroid-induced cell 1ysis;‘the bio-
chemical basis for these nuclear been characterized.
changes
has not
Many biochemical alterations have been demonstrated in corticosteroid-challenged lymphatic cells
sensitive to the cytolytic action of adrenal steroids (see Table 1) but all changes appear to be the consequence of the antiproliferative and catabolic potential of glucocorticoids and have also been described in non-lymphytic tissue [9] not destroyed by these steroids. Therefore these alterations cannot be involved in the lethal event. Evidence is accumulating, however, that the antiproliferative effect of corticosteroids is permissive for the induction of programmed cell death in sensitive cells. Proliferating lymphoma cells are not killed shortly after the addition of the steroid but following a latency period. During this period cellular metabolism is switched to the state of non-proliferating cells but viability of cells is not affected, as indicated by unchanged ATP [28] levels, which are an excellent parameter for cellular integrity. Accumulation of lymphoma cells in G, of the cell cycle precedes initiation of cell lysis[29] supporting the idea of a permissive role of the antiproliferative steroid effect in glucocorticoid-induced cell death. Additional evidence for this idea can be derived from cell hybridization experiments: the anti-proliferative and the cytolytic potential of glucocorticoids could be separated but no cell line has been identified which responds to corticosteroid challenge by cell death not preceded by growth inhibition. Analyses with the aid of an assay of colony-forming ability suggested the reversibility of the metabolic alterations during the latency period [29], which implies that glucocorticoid-induced cell death is not predetermined until very shortly before the cells die. But what is the lethal event? DNA FRAGMENTATION AND POLY(ADP-RIBOSYL)ATION
In 1980 Wyllie reported the observation that treatment of thymocytes with corticosteroids in-
duced a pronounced DNA fragmentation [30]. The molecular distribution of DNA fragments was dis-
crete as a result of degradation of the internucleosomal linker DNA, possibly by the action of a Ca2+, Mg”-dependent endonuclease known to produce such a digestion pattern [3 11.A comparable response has been found in other cells of lymphatic origin such as S49.1 mouse lymphoma cells (see Fig. 1) which are sensitive to the cytolytic potential of glucocorticoids[32], but was absent from cells derived from other organs[33]. Therefore it was proposed that corticosteroids activate an endonuclease which brings about the dramatic chromatin destruction [30]. This hypothesis, however, poses a problem: a comparable DNA fragmentation could also be detected in cells killed by agents known to initiate necrosis, e.g. fluoride, azide, or by compounds interfering with DNA and protein synthesis, or by overgrowth (Ref. [34] and K. Wielckens, unpublished results). Apparently such an enzyme is permanentIy present in the chromatin since a comparable DNA breakage can be shown in cycloheximide-treated cells where protein synthesis is entirely blocked (Ref.[34] and K. Wielckens, unpublished results). Hence it can be speculated that DNA fragmentation is not involved in the lethal event but occurs post mortem regardless of whether a cell has died by a necrotic or apoptotic process. Recently, however, two observations again support the crucial role of DNA fragmentation in glucocorticoid-induced cell death. Mark Compton and John Cidlowski reported that DNA strand breakage occurred in dexamethasone treated rat thymus cells before the cells were lysed [33]. In our laboratory a slight DNA fragmentation could also be detected in dexamethasone-incubated S49.1 lymphoma cultures when cells were still viable [28] and a concomitant activation of poly(ADP-ribosyl)ation, a nuclear protein modification reaction known to be stimulated by DNA incisions [35, 361. This reaction is catalyzed by poly(ADP-ribose)synthetase, a DNA-dependent enzyme that utilizes NAD and modifies several nuclear proteins covalently, including the synthetase itself 1371, histone H2B [38] and DNA topoisomerase I [39]. Due to the activation of poly(ADP-ribose)synthetase by DNA strand breaks, cell treatments that lead to chromatin fragmentation, e.g. incubation with alkylating agents [40,4 I] or UV irradiation [42], result in a dramatic but transient increase in protein-bound poly(ADP-ribose) and a concomitant decrease in the substrate NAD [40,4 I]. Studies of the turnover of poly(ADP-ribose) revealed an extremely high rate [4 1,431, suggesting an involvement of this protein modification process in fast reactions and probably not comparable to other modification mechanisms, such as phosphorylation, which are known to activate or inactivate certain enzymes. A functional poly(ADPribosyl)ation system is necessary for adaptation of cells to treatment with alkylating agents, suggesting
Glucocorticoid-induced
cell death
Fig. I. DNA fragmentation in dexamethasone-treated 549-l mouse Iymphoma cells. Cells were incubated for 3 days +/- 10m2M dexamethasone. DNA were extracted and analyzed on an agarose gel as described [28]. “DNA-Laengenstandard II” (from Boehringer, Mannheim = EcoRl and Hind III digested lambda DNA) was used as a mol wt marker (fragment sizes: 21.7,5.0,4.2,3.5,2.0,1.9,1.6,1.4, 0.9,0.8, and 0.6 kilobase pairs). Lanes A, B, G and H: marker DNA (5 to 10 pg). Lanes C and D: DNA from control cells. Lanes E and F: DNA from dexamethasone-treated ceil.
415
KLAUS WIELCKENS et al.
416
Table 2. Poly(ADP-ribose) levels in dexamethasone treated and control cells Treatment
Total
activity
None Dexamethasone
1
-
6.7 i-j- 0.2 12.1 -i-l- 1.1
S40.1 mouse lymphoma cells were incubated with or without 10-’ M dexamethasone for 20 h and poly(ADP-ribose) was determined as described 1281.
.c*-i:k., ‘0
Poly(ADP-ribose) (pmol/lON cells)
\ \
Intrinsic
0ctiW
“--o
+ dexomet~asane
r-9
. Control
\ b
+ dexamethasane
Fig. 2. Total and intrinsic poiy(ADP-ribose)synthetase activities in dexamethasone-challenged S 39.1 cells. Cells were treated with 10s7 M dexamethasone for the times indicated and intrinsic and total enzyme activities were determined in digitonin-permeabilized cells as described [28]. Inset: percentage of active enzyme.
an involvement of this process in DNA repair, either directly in the DNA ligase reaction 1441 or indirectly in restoration of chromatin structure [45]. When the poly(ADP-ribose)synthetase was determined in untreated S49.1 mouse Iymphoma cells about 5% of the enzyme was found active and no pronounced change in the intrinsic or total activity could be detected throughout the entire experiment (Fig. 2). Incubation of S49.1 lymphoma cells with dexamethasone led to a 2-fold increase of intrinsic activity after 24 h, while the total .activity decreased slightly. Thereafter, both the intrinsic and the total poty(ADP-ri~se)synthetase activity fell below the values of control cells because of the progressive loss of cells induced by the corticosteroid treatment. Therefore the enzyme stimulation in response to the glucocorticoid challenge must be expressed by the amount of active enzyme (Fig. 2, inset) revealing a S-fold increase of the intrinsic activity on day 3. The stimulation of the poIy(ADFribosyliation system can also be demonstrated by an increase in the protein bound poly(ADP-ribose) (Tabie 2), although only a slight augmentation of the polymer level was detectable when measured 20 h
after the steroid addition. The increase in proteinbound poly(ADP-ribose) was found at a time when all cells were still viable, although the increase was only 2-fold. However, the half-life of the polymer makes it difficult to estimate the real extent of the stimulation. That the activation of the poly(ADPribosyl)ation system occurred at a time where all cells in corticosteroid-treated cultures were still intact was also reflected by a decrease in the substrate NAD [28]. The loss in NAD could be antagonized by an inhibitor of po~y(ADP-ri~s~)synthetase, the nicotinamide analog benzamide [2X], further supporting the observation that glucocorticoid treatment leads to fragmentation of DNA, which in turn activates the poly(ADP-ribose)synthetase, thereby combating the chromatin damage. The antagonistic function of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death can be easily demonstrated by incubation of S49.1 lymphoma cells with dexamethasone in the presence or absence of benzamide: inhibition of poly(ADP-ribose) formation not only increased the number of cells which died by the steroid challenge but also accelerated the velocity of corticosteroid-mediated cell kill (Fig, 3). This observation suggests that the lethal process in glucocorticoid-induced cell death is a competition
1OOr
Day after
dexamethasone
addition
Fig. 3. Effect of the poly(ADP-ribose)synthetase inhibitor benzamide on the number of dead cells in dexamethasonetreated 549.1 cells. Cells were incubated for the times indicated with IV7 M dexamethasone +/- benzamide, and the percentage of dead cells was determined as described [28].
Glucocorticoid-induced cell death between a nuclease destroying the integrity of the chromatin and mechanisms, including poly(ADPribosyl)ation, attempting to restore the intactness of chromatin. Interference with these mechanisms therefore increases the toxicity of the steroid and causes cell death at an earlier time.
417
1OOr
POLY(ADP-RIR~SYL)ATI~NANDCHROMAT~ STRUCTURE
The antagonistic role of poly(ADP-ribosyl)ation in glucocorticoid-induced cell death raises the question of the exact function of this nuclear protein modification process. The involvement of poly(ADP-ribosyl)ation in DNA repair as already mentioned in a preceding section cannot be questioned but the fact that inhibition of poly(ADPribose) formation leads to a higher steady-state concentration of DNA strand breaks in cells damaged with alkylating agents has initiated controversial discussions [45]. The findings that a block in poly(ADP-ribose) formation did not reduce the overall repair rate but led to an increase in the number of incisions and a higher number of unrepairable breaks, however, favoured the idea that poly(ADP-ribosyl)ation could be involved in maintenance or restoration of chromatin structure. This hypothesis was supported by preliminary data showing chromatin alterations in benzamide-treated HeLa cells[45], although it was not clear whether these alterations were restricted to HeLa cells or a general consequence of long-term inhibition of poly(ADPribosyl)ation. When nuclei were prepared from S49.1 cells exposed to benzamide for 24 h by a very gentle technique maintaining full chromatin integrity (on the basis of strand breaks) the sensitivity of the chromatin to endonucleases can be used as a tool to obtain indirect information about structure. Quantitating the number of incisions created by the nuclease treatment in a given time should therefore allow discrimination between states of chromatin compactness. Digestion of chromatin with micrococcal nuclease revealed only a slight increase of the nuclease sensitivity in benzamide vs mock-treated cells (not shown). By contrast, replacement of micrococcal nuclease by DNase I resulted in a pronounced augmentation of enzyme-mediated DNA breakage in benzamide-treated cells (Fig. 4). The sensitivity of the procedure used in this experiment to measure DNA fragmentation allows the detection of several hundred to approximately 20003000 breaks/cell. DNase I hypersensitive sites could therefore be involved in the structural alteration induced by inhibition of poly(ADP-ribosyl)ation. The apparent discrepancy between micrococcal nuclease and DNase I in recognition of changes in the chromatin structure in benzamide-treated cells could be due to the DNase I property of preferential digestion of active chromatin [46]. Micrococcal nuclease also attacks active chromatin with a higher
I
I
0
5
Incubation
I 10 time
(mid
Fig. 4. Effect of benzamide on the DNase I sensitivity of S49.1 cell chromatin. Cells were treated for 24 h +/- 5 mM benzamide and the nuclei prepared and incubated for the times indicated with 0.66 U/ml DNase. DNA fragmentation was determined as described[48]. The log of percentage of double-stranded DNA is in linear relationship to the number of breaks (intact DNA = high percentage of double-stranded DNA).
rate but the difference between active and total chromatin is not as pronounced as with DNase I. Studies with the aid of the nucleoid sedimentation technique [47] led to additional evidence for benzamide-induced changes in nuclear structure, pointing to a more general structural alteration of the chromatin-nuclear-matrix-complex (K. Wielckens and A. Muth, manuscript in preparation). These findings suggest that poly(ADP-ribosyl)ation is necessary for maintaining the regular structure of active chromatin. Introduction of breaks may have drastic consequences for the structure if chromatin is under torsional tension. Although the bulk of chromatin may lack torsional stress [49], recent evidence has supported the idea of torsional stress in active chromatin [SO]. Therefore it is possible that poly(ADP-ribosyl)ation is involved in the restoration of uncoiled regions. This hypothesis would explain why poly(ADP-ribosyl)ation is triggered by DNA incisions as well as the high-energy consumption during poly(ADP-ribose) formation, i.e. restoration of a highly energetic structure. Therefore inhibition of poly(ADP-ribosyl)ation in corticosteroid-treated lymphoma cells should enhance the alterations in chromatin structure caused by the endonuclease mediated introduction of strand breaks. Furthermore, the lethal event in glucocorticoid-induced cell death could be a structural alteration of chromatin interfering with transcription. CONCLUSIONS
Experiments and speculations provided in the preceding sections divided the response of glucocorticoid sensitive lymphoma cells into two steps: a reversible block in proliferation and the lysis of the cells. Although the switch to the nonproliferating state does not represent the lytic event it is per-
418
KLAUS WIELCKENSet al. Stabilization:
Destabilization: Chromatin fragmentation
DNArepair poly(ADP-f-ibosyl)ation
t
I
t
cell
t
survival
death
Fig. 5. Possible mechanism of glucocorticoid-induced
missive for cellular suicide of lymphoma cells. The lytic event is initiated by the activation of a mechanism destabiIizing the regular chromatin structure, i.e. an endonuclease. A DNA repair involving poly(ADP-ribosyl)ation phenomenon occurs in opposition to the chromatin destruction and antagonizes the destabilization until the NAD is consumed and the cellular capacity to counteract is exhausted. Hence glucocorticoid-induced cell death can be described (Fig. 5) as a fight between stabilization (good) and destabilization (evil). The crucial process in glucocorticoid-induced cell death should therefore be the activation of an endonuclease. This enzyme digests the chromatin although the cells are still viable, which is in contrast to necrosis where the same enzyme apparently degrades the DNA post mortem. The nuclease, endonuclease, possibly the Ca’+, Mg2’-dependent appears to be already present in the chromatin and does not need to be induced by the corticosteroid. Since the enzyme is fully active only in the presence of mil~imolar amounts of Ca*’ [.511 and the concentration of free Ca”-ions does not exceed 1 pmol, an elevation of the free calcium-level must also be postulated. Alternatively, it has to be taken into consideration that the early DNA fragmentation in corticosteroid-treated lymphoma cells is not accomplished by this enzyme in contrast to the late DNA breakage which is characterized by the discrete fragment pattern. Not only an increase in the destabilizing capacity but also a decrease in the stabilization appeared to contribute to the cetl lysis, e.g. a decline in the repair capacity (K. Wielckens and V. Freese, manuscript in preparation). Since apoptosis is not only induced by glucocorticoids but represents a general cellular response to a specific physiological cue [14] it is possible that corticosteroids employ a suicide mechanism present in every cell. Thus in glucocorticoid-sensitive lymphocytes and lymphoma cells the glucocorticoid receptor system couid be linked to an yet unknown mediator of the suicide system. REFERENCES 1. Gryglewski R. J., Panczenko B. Grodzinska L. and Ocetkiewica A.: Corticosteroids inhibit Drostaglandin
2.
7 -.
4
5
lymphoma cell death.
release from perfused mesenteric blood vessels in rabbits. Prostuglandins 10 (1975) 343-354. Tashjian A., Yoelkel E., McDonough J. and Levin L,: Hydrocortisone inhibits prostaglandin production by mouse fibrosarcoma cells. Nature, Lond. 258 (197.5) 739-741. Davies P., Bailey P. J., Godenberg M. M. and FordHutchinson A. W.: The role of arachidonic acid oxygenation products in pain and inflammation. Ann. Rev. Immun. 2 (1984) 335-357. Needleman P., T&k J.,‘Jakschik B. A., Morrison A. R. and Lefkowith J. B.: Arachidonic acid metabolism. A. Rev. Biochem. 55 (1986) 69-102. Flower R. J. and Blackwell’G. J.: Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation. Nature, Land. 278 (1978) 456-458.
K., 6. Hirata F., Schiffman E., Yenkatasubramanian Salomen D. and Axelrod J.: A phospholipase A, inhibitory protein in rabbit neutroohils induced bv glucocor%coids. Proc. nam. Acad.’ Sci. U.S.A. i? (1980) 2533-2538. 7. Gillis S., Crahtree G. R. and Smith K. A.: Glucocorticoid-induced inhibition of T cell growth factor production. I. The effect of mitogen-induced lymphocyte proliferation. J. Immun. 123 (1979) 16241631. 8. Goodwin J. S., Atluru D., Sierakowski S. and Lianos E.: Mechanism of action of elucocorticoids. lnhibition of T cell proliferation and iiterleukin 2 production by hydrocortison is reversed by ieukotrien Bq. 1. clin. Inuest. 77 (1986) 1244-1250. 9. Munck A. and L.eung K.: Glucocorticoid receptors and mechanisms of action. In Receptors and Mechanism of Action of Steroid Hormones (Edited by Pasqualini J. R.). Marcel Dekker, New York, Part II (1977) pp. 31 l-397. 10. Munck A. Crabtree G. R.: Glucocorticoid-induced lymphocyte death. In CefZ Death in Biology and Pathology (Edited by Bowen I. D. and Lockshin R. A.). Chapman Hall, London (1981) pp. 329-359. 11 Fauci A. S. and Dale D. C.: The effect of in uiuo hydrocortisone on subpopulations of human lymphocytes. J. clin. Invest. 53 (1974) 240-246. 12. Wolff J. A., Brubaker C. A., Murphy M. L., Pierce M. I. and Sever0 N.: Predisone therapy of acute childh~d leukemia: prognosis and duration of resnonse in 330 patients. J: Pehiat. 70 (1967) 626-631. ’ 13. Norman M. R. and Thompson E. B.: Characterization of a gIucocorticoid-sensi~ve human l~phoid cell line. Cancer Res. 37 (19771 3785-3791.
14. Wyllie A. H.: Ceil deaih: a new classification senarating apoptosis from necrosis. In Cell Death in biology and ~utholo~ (Edited by Bowen I. D. and Lockshin R. A.). Chapman & Hall, London (1981) pp. 9-34. 15. Nicholson M. L. and Young D. A.: Effect of glucocorticoid hormones in vitro on the structural integrity of
Giuc~orticoid-induct
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26
27.
28.
29.
30.
31.
32.
nuclei in corticosteroid-sensitive and -resistant lines of lvmphosarcoma P1798. Cancer Res. 38 (1978) 3673368b. Makman M. H., Dvorkin B. and White A.: Alterations in protein and nucleic acid metabolism of thymocytes produced by adrenal steroids. J. biol. Chem. 241 (1966) 1646-1648. Makman M. H., Dvorkin B. and White A.: Influence of cortisoi on the utilization of precursors of nucleic acids and protein by iymphoid ceils in uifro. J. biol. Chem. 243 (1968) 1485-1497. Rosen J. M., Fina J., Miihoiland R. J. and Rosen F.: Inhibitory effect of cortisol in vitro on 2-deoxyglucose uptake and RNA an protein meta~iism in lymphosarcoma P1798. Cancer Res. 32 (1972) 350-355. Cidlowski J. A.: Glucocorticoids stimulate ribonucieic acid degradation in isolated rat thymic lymphocytes in vitro. Endocrinology 111 (1982) 184-190. MacDonald R. G., Martin T. P. and Cidlowski J. A.: Glucocorticoids stimulate protein degradation in lymphocytes: a possible mechanism of steroid-induced cell death. E~ocr~~o~ogy 107 (1980) 1512-1524. Munck A.: Metabolic site and time course of cortisol action on glucose uptake, lactic acid output, and glucose 6-phosphate levels of rat thymus in vitro. J. biol. Chem. 243 (1968) 1039-1042. Makman M. H., Dvorkin B. and White A.: Evidence for induction by cortisol in vitro of a protein inhibitor of transport and phosphoryiation processes in rat thymocytes. Proc. natn. Acad. Sci. U.S.A. 68 (1971) 1269-1273. Stevens J., Stevens Y. W. and Hoilander V. P.: Substrate requirement and kinetic analysis of the cortisoi effects on uridine uptake and incorporation by mouse iymphoma P1798 cells in t&o. Cancer Res. 34 (1974) 233Cr2337. Foley J. E., Jeffries M. and Munck A.: Glu~ocorticoid effects on incorporation of lipid and protein precursors into rat thymus ceil fractions. J. steroid Biochem. 12 (1980) 231-243. Turneil R. W.. Clarke L. H. and Burton A. F.: Studies on the mechanism of corticosteroid-induced lymphocytolysis. Cancer Res. 33 (1973) 203-211. Story T. M., Standaert M. M. and Melnykovych G.: Gluc~orticoid-induced alterations in phosphatidyicholine metabolism in mouse lymphoma. Cancer Res. 33 (1973) 2872-2877. Russell D. H., Haddox M. K. and Gehring U.: Effects of dexamethasone and cyclic AMP on polyamine synthesizing enzymes in mouse lymphoma c&s. J. cell. Phvsiof. 106 (1981) 375-386. W&kens K.‘ and ‘Delfs T.: Glucocorticoid-induced cell death and ~Iy~ADP-ri~syl)ation: increased toxicity of dexamethasone on mouse S49.1 iymphoma cells with the poly(ADP-ribosyl)ation inhibitor benzamide. Endoirinology 119 (1986) 2383-2392. Harmon J. M.. Norman M. R.. Fowies B. J. and Thompson E. B.: Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line. f. cell. P~ysiol. 98 (1979) 267-278. Wyilie A. H.: Glucocorticoid-induced thyrnocyte apoptosis is associated with endogenous endonuciease activation. Nature, Lond. 284 (1980) 555-556. Nakamura M., Sakaki Y., Watanabe N. and Tagaki Y.: Purification of the Ca*+ plus Mg2+ dependent endodeoxyribonuclease from calf thymus chromatin. 1. Biochem. 89 (1981) 143-152. Sibley C. H. and Tomkins G. M.: Isolation of lymphoma cell variants resistant to killing by giucocorticoids. Cell 2 (1974) 213-220.
cell death
419
33. Compton M. M. and Cidiowski J. A.: Rapid in uiuo effect of glucocorticoids on the integrity of rat iymacid. phocyte genomic deoxyribonucleic Endoctinology 118 (1986) 38-45. 34. Vedeckis W. V. and Bradshaw H. D.: DNA fragmentation in S49 lymphoma cells killed with giucocorticoids and other agents. Mofec cell. Erulocr. 30 (1983) 215-227. Ann. 35. Ueda K. and Hayasin 0.: ADP-riboxyiation. Reu. Biochem. 54 (1985) 7@-5(Ic). 36. Miller E. G.: Stimulation of nuclear polyadenosine diphosphate ribose) polyrnerase activity from Hela cells by endonucleases. B&him. biophys. Acta 395 (1975) 191-200. 37. Ogata N., Ueda K., Kawaichi M. and Hayaishi 0.: Poly(ADP-ribose)synthetase, a main acceptor of poly(ADP-ribose) in isolated nuclei. J. biol. Chem. 265 (1981) 4135-4137. 38. Adamietz P. and Rudolph A.: ADP-ribosylation of nuclear proteins in uiuo. f. biol. Chem. 259 (1984) 68416846. 39. Ferro A., Higgins P. and Olivera B.: Poly(ADPribosyi)ation of a topoisomerase J. biol. Chem. 258 (1983) 60006003. 40. Juarez-Saiinas H., Sims H. L. and Jacobson M. K.: Poly(ADP-ribose) in carcinogen-treated cells. Nature, Land. 282 (1979) 740-741. 41. Wielckens K., Schmidt A., George E., Bredehorst R. and Hilz H.: DNA fragmentation and NAD depletion. f. biol. Gem. 257 (fi82) 12,872-12,877. _ 42. Jacobson E. L.. Antol K. M.. Juarez-Saiinas H. and Jacobson M. K.: Poly(ADPlribose) metabolism in ultraviolet irradiated human fibroblasts. J. biol. Chem. 258 (1983) 103-107. 43. Wielckens K., George E., Pless T. and Hiiz H.: Stimulation of poly(ADP-ri~syi)ation during Ehrlich ascites tumor c&Is “starvation” and suppression of CODcorn&ant DNA fragmentation by benzamide. J. biol. Chem. 258 (1983) 4098-4104. 44. Durkacz B. W.. Omidiii. 0.. Grav D. A. and Shall S.: (ADP-ribose), participates in DhA excision repair. Nature, Lond. 283 (1980) 593-596. 45. Wieickens K., Piess T. Schaefer G.: Poly(ADP-ribosyl)ation, DNA repair and chromatin structure. In ADP-Ri~sylado~ of Proteins (Edited by Aithaus F. R., Hiiz H., and Shall S.) Springer-Veriag, Berlin (1985) pp. 252-263. 46. Weintraub H. and Groudine M.: Chromosomal subunits in active enes have an altered conformation. Science 193 (197 d 1848-856. 47. Cook P. R. and Braze11 I. A.: Supercoils in human DNA. J. Cell Sci. 19 (1975) 161-279. 48. Ahrstroem G. and Erixon K.: Measurement of strands breaks bv alkaline denaturation and hydroxyapatite chromatdgraphie. In DNA Repair (Edited by Friedhere E. C. and Hanawait P. C.) Marcel Dekker, New York (1981) pp. 403-418. 49. Sinden R. R., Carlson J. O., Pettijohn D. E.: Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells. Cell 21. (1980) 773-783. 50. Harland R. M., Weintraub H. and McKnight S. L.: Transcription of DNA injected into Xenopus laeois oocytes is influenced by template topology. Narure, Lond. 302 (1982) 38-43.
51. Murphy E., Co11 K., Rich T. L. and Williamson J. R.: Hormonal effects on calcium homeostasis in isoiated hepatocytes. J. biol. Gem. 255 (1980) 6600-6608.