Pharmacological Research, Vol. 26, Supplement 2, 1992
5
GLUCOCORTICOID-INDUCED DNA FRAGMENTATION : ROLE OF PROTEINKINASE-C ACTIVITY . G . Migliorati, M.C. Pagliacci*, F. D'Adamio, F. Crocicchio, I . Nicoletti* and C . Riccardi . Istituto di Farmacologia Medica and *Istituto di Clinica Medica I, Perugia University Medical School, 06100 Perugia, Italy . Key words : Glucocorticoids, PKC, Interleukins, Apoptosis, Receptor abuse. ABSTRACT. Glucocorticoid hormones (GCH) and IL-2 induce apoptotic cell death by a PKC-dependent mechanism . IL-4 counteracts apoptosis by inhibiting PKC activity . GCH and IL-2 show antagonistic effects on apoptosis when administered togheter. These data indicate that PKC activation in response to different stimuli can both enhance or reduce thymocyte survival . INTRODUCTION . Glucocorticoid hormones (GCH) are potent antiinflammatory and immunosuppressive drugs . At physiological doses, they modulate the production of a number of cytokines, including IL-1, IL-2 and 1L-4, which play a key role in the control of T-cell development and immune response . GCH are also able to directly kill immature T-cells, such as cortical thymocytes, tumor T-lymphocytes and T-cell hybrids (1) . Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits and condensation of nuclear chromatin, which are the hallmarks of "apoptotic cell death" ( 2) . A similar type of cell death is induced in immature thymocytes by 1-irradiation, anti-CD3 monoclonal antibodies, Ca++-ionophores and PKC-activators, such as phorbol 12-myristate 13-acetate (TPA) . IL1, IL-2 and IL-4 have been reported to inhibit apoptotic cell death in thymocytes and tumor cell lines, but the mechanisms of their protective activity are still a matter of debate (3) . Since thymocyte death produced by either anti-CD3 monoclonal antibodies or GCH is an active phenomenon that requires mRNA and protein synthesis and activation of endonucleases through PKC-dependent mechanisms, ILs and other cytokines could regulate apoptosis by modulating PKC activity . In the present study we demonstrate that DNA fragmentation and programmed cell death induced by GCH in mouse thymocytes are reduced by both inhibition and stimulation of PKC activity . via a mutual esclusion mechanism. MATERIALS and METHODS Cell, suspension: Thymocytes were obtained from 2-4 weeks-old C3H/HeN mice (Charles River, Milan) . The cell suspension was washed, filtered and adjusted to a concentration of 1 .5x106 cell/ml in RPMI-1640 medium supplemented with 5% FCS and 10mM Hepes buffer . Aliquots of 2 ml thymocytes were incubated at 37 ° C with DEX (at concentrations from 10-11 to 10 -7 M), or DEX plus mouse recombinant IL-4 or IL-2 (Genzyme Corporation, Maidstone, UK). In some experiments ZnSO4 (50mM), D-actinomycin (2.5 gg/ml), cycloheximide (50 µg/ml), H7 (50 µg/ml) and staurosporine (30 gg/ml) were also used . At the pre-established times, the cells were centrifuged at 200 g for 10 min ., washed and processed (see below) . DNA labeling tecnique 1d flow cytometric analysis : The 200 g centrifuged cell pellet was gently 1043-6618/92/26110005-05/$03 .00/0 © 1992 The Italian Pharmacological Society
6
Pharmacological Research, Vol. 26, Supplement 2, 1992
resuspended in 1 .5 ml hypotonic fluorochrome solution of Propidium Iodide (PI, 50 µg/ml in 0 .1% sodium citrate plus 0 .1% Triton X-100, Sigma), in 12x75 polypropilene tubes (Becton Dickinson, Lincoln Park, NJ, USA). The PI-fluorescence of individual nuclei was measured using a FACSCAN flow cytometer (Becton Dickinson, Mountain View, CA, USA) as previously described (4) . Membrane associated PKC-activity evaluation : To evaluate PKC-activity, cells untreated, treated with TPA (100 nM) or TPA plus IL-4 (100U/ml) were cultured for 24h . After culturing, cell membranes were separated by centrifugation and the PKC activity evaluated
by measuring the transfer of
(y32P)ATP to a specific PKC substrate, with protein kinase C enzyme assay system (Amersham, UK) .
RESULTS Figure 1 shows the effect of IL-2 and IL-4 on the apoptosis induced by 12 h incubation with 10 -7 M DEX in mouse thymocytes . DEX-treated thymocytes showed an impressive increase in the percentage of apoptotic nuclei (hypodiploid DNA content and enhanced side angle scatter at the FCM)
as
compared to controls . Apoptosis was reduced by IL-2 (100U/ml) and IL-4 (100 U/ml) .
DNA Control
58%
Figure 1 . Computer-drawn flowcytometric profiles of PI-stained thymocyte nuclei after 12h incubation in medium alone or indicated substances . The DNA fluorescence of nuclei is plotted against the respective side-angle scatter (SSC) that express chromatin condensation . Apoptotic nuclei have a reduced DNA fluorescence and an increased chromatin condensation .
The mechanisms of DEX-induced apoptosis can be deduced by the results of experiments shown in Figure 2 . The apoptotic activity of DEX was blocked by endonuclease inhibitors (ZnSO4), inhibitors of RNA (D-actinomycin) and protein (cycloheximide) syntesis, as well as by specific inhibitors of PKC such as H-7 and staurosporine . These data clearly demonstrate that PKC-dependent mechanisms are involved in DEX-induced apoptosis . This hypothesis was further confirmed by analyzing the effect of a direct PKC activation by TPA phorbol-esther on thymocye apoptosis . TPA produced a significant increase in DNA fragmentation of tymocyte nuclei, which was almost completely counteracted by IL-4 (Figure 3, left) . A direct analysis
Pharmacological Research, Vol . 26, Supplement 2, 1992
7
100
k 80 IIJ 0
so .
_ 0
I o L 0 L
Figure 2 : Inhibition of thymocyte apoptosis induced by 10- M DEX . Thymocytes were incubated 12 h at 37°C in medium containing DEX with or without the indicated substances (see materials and methods for doses). Apoptosis was measured by FCM method . Mean±SED of four separate experiments .
40
20
0 I
X
I-
• •
0
< _ _ 1 > + 0 0 + +
0
X
-
a
$
•
n
• H
0
N + +
of PKC activity in parallel experiments, showed that 100 nM TPA increases membrane-associated TPA activity but also demonstrated that IL-4 reduces both basal and TPA-stimulated PKC (Figure 3, right) . These data suggest that the protective effect of IL-4 against thymocyte apoptosis is due, at least
Figure 3 . Left panel : Protective effect of IL-4 on thymocyte apoptosis induced by 100 nM TPA. Mean±SD of three separate experiments. Right panel : Effect of IL-4 on basal and TPA-stimulated membrane associated PKC activity in a parallel representative experiment .
D 0 L D F 2 0 0
i J F 2 0 0
r 0 0
I J f t
0 L
i J
0 0
F Z 0 0
F F
F
a
i J
F . I
0 0
in part, to a PKC inhibition . IL-2, on the contrary, is known to activate PKC via DAG and 1P3 generation . It seems paradoxical, therefore, that IL-2 can protect thymocytes from DEX-induced apoptosis . However, when the effects of increasing IL-2 concentrations were analyzed in thymocytes incubated with and without DEX an intriguing phenomenon was observed (Figure 4) .
8
Pharmacological Research, Vol. 26, Supplement 2, 1992
Figure 4 : Effect of 24h incubation with different IL-2 concentrations, either alone (squares) or with 10-7M DEX (triangles), on thymocyte apoptosis . IL-2 induced a dose dependent increase in DNA fragmentation but afforded dose-dependent protection against DEX-induced apoptosis .
L-2 Wit
High IL-2 doses (above 100 Ulml) produced apoptosis of mouse thymocytes . The apoptotic phenomenon was mediated by PKC-dependent pathways, since it was completely abrogated by PKCinhibitors H-7 and staurosporine (data not shown) . However, the same IL-2 concentrations exerted a clear and dose-dependent inhibition of apotosis induced by 10 -7M DEX . It seems, therefore, that IL2- and DEX-induced apoptosis are mutually antagonistic and that PKC system(s) play(s) a key role in this phenomenon . DISCUSSION. A large proportion of immature thymocytes undergo apoptosis, but the relevance of this phenomenon to T-cell differentiation has remained obscure . T cell precursors are selected both positively and negatively in the thymus, and apoptosis induced by antigen recognition through T-cell receptor (TcR) is an important mechanism of negative selection . Interleukins are physiologically involved in T-cell ontogeny . The demonstration that IL-1 and EL-2 counteract the TcR-mediated apoptosis in immature thymocytes indicates a novel site of action for these cytokines . Recent data suggest that IL-4 also plays a role in T-cell development. IL-4 promotes growth of varios thymocyte subsets, induces growth and differentiation of fetal CD4-CD8- T-cell precursors and protects mouse thymocytes from apoptosis . GCH also have a relevant role in the process of T-cell development, and physiological levels of these hormones regulate apoptotic cell death in thymocytes . Thymic hypoplasia is characteristic of mice exposed to stressful) stimuli and morphine can induce thymic involution in vivo through activation of pituitary-adrenal axis (5). Taken together, these observations indicate that a lot of signals and stimuli, including self and foreign antigens, hormones and cytokines control thymocyte apoptosis . PKC(s) play(s) a pivoltal role in the cellular signalling during thymocyte development, and both IL-i and IL-2 are able to increase DAG and Ca++ levels and to activate PKC . Furthermore, the demonstration that apoptosis can be induced by PKC-activators, such as TPA, and blocked by PKC-inhibitors, indicates
Pharmacological Research, Vol . 26, Supplement 2, 1992 that PKC-dependent mechanisms control programmed thymocyte death. Our results confirm that PKC-dependent pathways are deeply involved in the regulatory mechanisms of thymocyte apoptosis and indicate that PKC may both induce thymocyte death and protect thymocytes from apoptosis produced by other agents . The PKC-inhibitors H-7 and staurosporine were able to completely inhibit apoptosis induced by both DEX and high doses of IL-2, thus confirming that PKC activation is essential to produce apoptotic death of thymocytes . Conversely, IL-4 counteracted the apoptosis induced by both DEX and TPA and reduced both spontaneous and TPAstimulated membrane PKC activity . This suggests that the protective activity of IL-4 against apoptosis is exerted through inhibition of PKC-associated signals . IL-2, which activates PKC through DAG generation (6), was able to both induce apoptosis and counteract the apoptotic activity of DEX . Therefore, whilst IL-2 and DEX produce apoptosis via PKC when used as single agents, they also inhibit one another when administered together . These data are reminiscent of the mutual antagonism between TcR-activation and CGH in inducing thymocyte death (7) and suggest that such an antagonism might occurr at the level of signal transduction via PKC . It can be hypothesized that high IL-2 doses, trough a "receptor abuse"-like phenomenon (8) induce a sustained translocation of PKC on the cell membrane with a reduced PKC availability for the intracellular PKC-dependent pathways (i .e. DEX-induced PKC-mediated apoptosis) . Taken together, our results suggest that PKC plays a critical role in the regulation of apoptotic death in thymocytes. Therefore, while a functioning PKC system is essential for thymocyte apoptosis, different states of PKC activation in response to different stimuli can both enhance or reduce thymocyte survival . Supported by Italian Association for Cancer Research (AIRC) and by PF ACRO, CNR, Italy . REFERENCES . 1) Willie A.H ., Kerr J .F.R., Currie A.R. Cell death: the significance of apoptosis . Int. Rev. Cytol . 1980; 68 :251-265 . 2) Willie A.H . Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation . Nature, 1980 ; 284:555-557 . 3) McConkey D .J ., Orrenius S., Jondal M . Cellular signalling in programmed cell death . Immunol . Today, 1990 ; 11 :120-123 . 4) Nicoletti I ., Migliorati G ., Pagliacci M.C ., Grignani F ., Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry . J. Immunol . Methods, 1991 ; 139 :271-279 . 5) Sey Y ., Yoshimoto K, McIntyre T., Skolnick P ., Arora P.K Morphine-induced thymic hypoplasia is glucocorticoid-dependent . J . Immunol . 1991 ; 146 :194-198 . 6) Eardly D.D ., Koshland M.E . Glycosylphosphatidylinositol : a candidate system for interleukin-2 signal transduction. Science, 1991 ; 251 :78-81 . 7) Zacharchuk C .M ., Mercep M., Chakraborth P .K, Simons S .S .jr., Ashwell J .D . Cell activation and steroid induced pathways are mutually antagonistic . J. Immunol . 1990; 145 :4037-4045 . 8) Favaron M ., Manev H., Siman R ., Bertolino M., Szekely A.M., DeErausquin G ., Guidotti A., Costa E. Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death . Proc. Natl . Acad . Sci . USA, 1990 ; 87 :1983-1987 .
5
GLUCOCORTICOID-INDUCED DNA FRAGMENTATION : ROLE OF PROTEINKINASE-C ACTIVITY . G . Migliorati, M.C. Pagliacci*, F. D'Adamio, F. Crocicchio, I . Nicoletti* and C . Riccardi . Istituto di Farmacologia Medica and *Istituto di Clinica Medica I, Perugia University Medical School, 06100 Perugia, Italy . Key words : Glucocorticoids, PKC, Interleukins, Apoptosis, Receptor abuse. ABSTRACT. Glucocorticoid hormones (GCH) and IL-2 induce apoptotic cell death by a PKC-dependent mechanism . IL-4 counteracts apoptosis by inhibiting PKC activity . GCH and IL-2 show antagonistic effects on apoptosis when administered togheter. These data indicate that PKC activation in response to different stimuli can both enhance or reduce thymocyte survival . INTRODUCTION . Glucocorticoid hormones (GCH) are potent antiinflammatory and immunosuppressive drugs . At physiological doses, they modulate the production of a number of cytokines, including IL-1, IL-2 and 1L-4, which play a key role in the control of T-cell development and immune response . GCH are also able to directly kill immature T-cells, such as cortical thymocytes, tumor T-lymphocytes and T-cell hybrids (1) . Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits and condensation of nuclear chromatin, which are the hallmarks of "apoptotic cell death" ( 2) . A similar type of cell death is induced in immature thymocytes by 1-irradiation, anti-CD3 monoclonal antibodies, Ca++-ionophores and PKC-activators, such as phorbol 12-myristate 13-acetate (TPA) . IL1, IL-2 and IL-4 have been reported to inhibit apoptotic cell death in thymocytes and tumor cell lines, but the mechanisms of their protective activity are still a matter of debate (3) . Since thymocyte death produced by either anti-CD3 monoclonal antibodies or GCH is an active phenomenon that requires mRNA and protein synthesis and activation of endonucleases through PKC-dependent mechanisms, ILs and other cytokines could regulate apoptosis by modulating PKC activity . In the present study we demonstrate that DNA fragmentation and programmed cell death induced by GCH in mouse thymocytes are reduced by both inhibition and stimulation of PKC activity . via a mutual esclusion mechanism. MATERIALS and METHODS Cell, suspension: Thymocytes were obtained from 2-4 weeks-old C3H/HeN mice (Charles River, Milan) . The cell suspension was washed, filtered and adjusted to a concentration of 1 .5x106 cell/ml in RPMI-1640 medium supplemented with 5% FCS and 10mM Hepes buffer . Aliquots of 2 ml thymocytes were incubated at 37 ° C with DEX (at concentrations from 10-11 to 10 -7 M), or DEX plus mouse recombinant IL-4 or IL-2 (Genzyme Corporation, Maidstone, UK). In some experiments ZnSO4 (50mM), D-actinomycin (2.5 gg/ml), cycloheximide (50 µg/ml), H7 (50 µg/ml) and staurosporine (30 gg/ml) were also used . At the pre-established times, the cells were centrifuged at 200 g for 10 min ., washed and processed (see below) . DNA labeling tecnique 1d flow cytometric analysis : The 200 g centrifuged cell pellet was gently 1043-6618/92/26110005-05/$03 .00/0 © 1992 The Italian Pharmacological Society
6
Pharmacological Research, Vol. 26, Supplement 2, 1992
resuspended in 1 .5 ml hypotonic fluorochrome solution of Propidium Iodide (PI, 50 µg/ml in 0 .1% sodium citrate plus 0 .1% Triton X-100, Sigma), in 12x75 polypropilene tubes (Becton Dickinson, Lincoln Park, NJ, USA). The PI-fluorescence of individual nuclei was measured using a FACSCAN flow cytometer (Becton Dickinson, Mountain View, CA, USA) as previously described (4) . Membrane associated PKC-activity evaluation : To evaluate PKC-activity, cells untreated, treated with TPA (100 nM) or TPA plus IL-4 (100U/ml) were cultured for 24h . After culturing, cell membranes were separated by centrifugation and the PKC activity evaluated
by measuring the transfer of
(y32P)ATP to a specific PKC substrate, with protein kinase C enzyme assay system (Amersham, UK) .
RESULTS Figure 1 shows the effect of IL-2 and IL-4 on the apoptosis induced by 12 h incubation with 10 -7 M DEX in mouse thymocytes . DEX-treated thymocytes showed an impressive increase in the percentage of apoptotic nuclei (hypodiploid DNA content and enhanced side angle scatter at the FCM)
as
compared to controls . Apoptosis was reduced by IL-2 (100U/ml) and IL-4 (100 U/ml) .
DNA Control
58%
Figure 1 . Computer-drawn flowcytometric profiles of PI-stained thymocyte nuclei after 12h incubation in medium alone or indicated substances . The DNA fluorescence of nuclei is plotted against the respective side-angle scatter (SSC) that express chromatin condensation . Apoptotic nuclei have a reduced DNA fluorescence and an increased chromatin condensation .
The mechanisms of DEX-induced apoptosis can be deduced by the results of experiments shown in Figure 2 . The apoptotic activity of DEX was blocked by endonuclease inhibitors (ZnSO4), inhibitors of RNA (D-actinomycin) and protein (cycloheximide) syntesis, as well as by specific inhibitors of PKC such as H-7 and staurosporine . These data clearly demonstrate that PKC-dependent mechanisms are involved in DEX-induced apoptosis . This hypothesis was further confirmed by analyzing the effect of a direct PKC activation by TPA phorbol-esther on thymocye apoptosis . TPA produced a significant increase in DNA fragmentation of tymocyte nuclei, which was almost completely counteracted by IL-4 (Figure 3, left) . A direct analysis
Pharmacological Research, Vol . 26, Supplement 2, 1992
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k 80 IIJ 0
so .
_ 0
I o L 0 L
Figure 2 : Inhibition of thymocyte apoptosis induced by 10- M DEX . Thymocytes were incubated 12 h at 37°C in medium containing DEX with or without the indicated substances (see materials and methods for doses). Apoptosis was measured by FCM method . Mean±SED of four separate experiments .
40
20
0 I
X
I-
• •
0
< _ _ 1 > + 0 0 + +
0
X
-
a
$
•
n
• H
0
N + +
of PKC activity in parallel experiments, showed that 100 nM TPA increases membrane-associated TPA activity but also demonstrated that IL-4 reduces both basal and TPA-stimulated PKC (Figure 3, right) . These data suggest that the protective effect of IL-4 against thymocyte apoptosis is due, at least
Figure 3 . Left panel : Protective effect of IL-4 on thymocyte apoptosis induced by 100 nM TPA. Mean±SD of three separate experiments. Right panel : Effect of IL-4 on basal and TPA-stimulated membrane associated PKC activity in a parallel representative experiment .
D 0 L D F 2 0 0
i J F 2 0 0
r 0 0
I J f t
0 L
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0 0
F Z 0 0
F F
F
a
i J
F . I
0 0
in part, to a PKC inhibition . IL-2, on the contrary, is known to activate PKC via DAG and 1P3 generation . It seems paradoxical, therefore, that IL-2 can protect thymocytes from DEX-induced apoptosis . However, when the effects of increasing IL-2 concentrations were analyzed in thymocytes incubated with and without DEX an intriguing phenomenon was observed (Figure 4) .
8
Pharmacological Research, Vol. 26, Supplement 2, 1992
Figure 4 : Effect of 24h incubation with different IL-2 concentrations, either alone (squares) or with 10-7M DEX (triangles), on thymocyte apoptosis . IL-2 induced a dose dependent increase in DNA fragmentation but afforded dose-dependent protection against DEX-induced apoptosis .
L-2 Wit
High IL-2 doses (above 100 Ulml) produced apoptosis of mouse thymocytes . The apoptotic phenomenon was mediated by PKC-dependent pathways, since it was completely abrogated by PKCinhibitors H-7 and staurosporine (data not shown) . However, the same IL-2 concentrations exerted a clear and dose-dependent inhibition of apotosis induced by 10 -7M DEX . It seems, therefore, that IL2- and DEX-induced apoptosis are mutually antagonistic and that PKC system(s) play(s) a key role in this phenomenon . DISCUSSION. A large proportion of immature thymocytes undergo apoptosis, but the relevance of this phenomenon to T-cell differentiation has remained obscure . T cell precursors are selected both positively and negatively in the thymus, and apoptosis induced by antigen recognition through T-cell receptor (TcR) is an important mechanism of negative selection . Interleukins are physiologically involved in T-cell ontogeny . The demonstration that IL-1 and EL-2 counteract the TcR-mediated apoptosis in immature thymocytes indicates a novel site of action for these cytokines . Recent data suggest that IL-4 also plays a role in T-cell development. IL-4 promotes growth of varios thymocyte subsets, induces growth and differentiation of fetal CD4-CD8- T-cell precursors and protects mouse thymocytes from apoptosis . GCH also have a relevant role in the process of T-cell development, and physiological levels of these hormones regulate apoptotic cell death in thymocytes . Thymic hypoplasia is characteristic of mice exposed to stressful) stimuli and morphine can induce thymic involution in vivo through activation of pituitary-adrenal axis (5). Taken together, these observations indicate that a lot of signals and stimuli, including self and foreign antigens, hormones and cytokines control thymocyte apoptosis . PKC(s) play(s) a pivoltal role in the cellular signalling during thymocyte development, and both IL-i and IL-2 are able to increase DAG and Ca++ levels and to activate PKC . Furthermore, the demonstration that apoptosis can be induced by PKC-activators, such as TPA, and blocked by PKC-inhibitors, indicates
Pharmacological Research, Vol . 26, Supplement 2, 1992 that PKC-dependent mechanisms control programmed thymocyte death. Our results confirm that PKC-dependent pathways are deeply involved in the regulatory mechanisms of thymocyte apoptosis and indicate that PKC may both induce thymocyte death and protect thymocytes from apoptosis produced by other agents . The PKC-inhibitors H-7 and staurosporine were able to completely inhibit apoptosis induced by both DEX and high doses of IL-2, thus confirming that PKC activation is essential to produce apoptotic death of thymocytes . Conversely, IL-4 counteracted the apoptosis induced by both DEX and TPA and reduced both spontaneous and TPAstimulated membrane PKC activity . This suggests that the protective activity of IL-4 against apoptosis is exerted through inhibition of PKC-associated signals . IL-2, which activates PKC through DAG generation (6), was able to both induce apoptosis and counteract the apoptotic activity of DEX . Therefore, whilst IL-2 and DEX produce apoptosis via PKC when used as single agents, they also inhibit one another when administered together . These data are reminiscent of the mutual antagonism between TcR-activation and CGH in inducing thymocyte death (7) and suggest that such an antagonism might occurr at the level of signal transduction via PKC . It can be hypothesized that high IL-2 doses, trough a "receptor abuse"-like phenomenon (8) induce a sustained translocation of PKC on the cell membrane with a reduced PKC availability for the intracellular PKC-dependent pathways (i .e. DEX-induced PKC-mediated apoptosis) . Taken together, our results suggest that PKC plays a critical role in the regulation of apoptotic death in thymocytes. Therefore, while a functioning PKC system is essential for thymocyte apoptosis, different states of PKC activation in response to different stimuli can both enhance or reduce thymocyte survival . Supported by Italian Association for Cancer Research (AIRC) and by PF ACRO, CNR, Italy . REFERENCES . 1) Willie A.H ., Kerr J .F.R., Currie A.R. Cell death: the significance of apoptosis . Int. Rev. Cytol . 1980; 68 :251-265 . 2) Willie A.H . Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation . Nature, 1980 ; 284:555-557 . 3) McConkey D .J ., Orrenius S., Jondal M . Cellular signalling in programmed cell death . Immunol . Today, 1990 ; 11 :120-123 . 4) Nicoletti I ., Migliorati G ., Pagliacci M.C ., Grignani F ., Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry . J. Immunol . Methods, 1991 ; 139 :271-279 . 5) Sey Y ., Yoshimoto K, McIntyre T., Skolnick P ., Arora P.K Morphine-induced thymic hypoplasia is glucocorticoid-dependent . J . Immunol . 1991 ; 146 :194-198 . 6) Eardly D.D ., Koshland M.E . Glycosylphosphatidylinositol : a candidate system for interleukin-2 signal transduction. Science, 1991 ; 251 :78-81 . 7) Zacharchuk C .M ., Mercep M., Chakraborth P .K, Simons S .S .jr., Ashwell J .D . Cell activation and steroid induced pathways are mutually antagonistic . J. Immunol . 1990; 145 :4037-4045 . 8) Favaron M ., Manev H., Siman R ., Bertolino M., Szekely A.M., DeErausquin G ., Guidotti A., Costa E. Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death . Proc. Natl . Acad . Sci . USA, 1990 ; 87 :1983-1987 .
