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B and AP-1 Activation1
*
Department of Immunology and Cell Biology, Istituto Ricerche Farmacologiche Mario Negri, Milan, Italy; and
Department of Pharmacology, Pharmacia and Upjohn Research Center, Nerviano, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-
and 
-chains but inhibits IL-2-dependent T cell proliferation. We
have investigated several molecular pathways that are known to be
activated by IL-2 in T cells. We show that PNU156804 does not inhibit
c-myc and bcl-2 mRNA induction. On the other hand,
PNU156804 efficiently inhibits the activation of the NF-
B and AP-1
transcription factors. PNU156804 inhibition of NF-B activation is
due to the inhibition of the degradation of IB- and IB-ß.
PNU156804 action is restricted to some signaling pathways; it does not
affect NF-B activation by PMA in T cells but blocks that induced by
CD40 cross-linking in B lymphocytes. We conclude that the prodigiosin
family of immunosuppressants is a new family of molecules that show a
novel target specificity clearly distinct from that of other
immunosuppressive drugs such as cyclosporin A, FK506, and
rapamycin. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human primary T lymphocytes were obtained from buffy coats of normal volunteers. Briefly, mononuclear cells were separated on a Ficoll-Hypaque gradient (Seromed, Berlin, Germany), and lymphocytes were separated from monocytes by centrifugation onto 50% isoosmotic Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). If necessary, residual monocytes were removed by 1-h adherence onto tissue culture petri dishes (Falcon, Oxnard, England) in complete medium. The resulting lymphocyte population contained 1–2% CD14+ monocytes, 6–10% CD20+ B lymphocytes, and >80% CD3+ T lymphocytes, as determined by indirect immunofluorescence with appropriate mAbs on the FACS. Resting B lymphocytes were purified from fresh human tonsils, obtained from routine tonsillectomies, as described previously (6). Cells were cultured in RPMI 1640 medium (Seromed), supplemented with 10% FCS (HyClone, Steril System, Logan, UT), glutamine (Life Technologies, Paisley, Scotland), and 50 g/ml gentamicin (Life Technologies).
Reagents
PHA (Murex Diagnostics, Dartford, England) was used at 1 µg/ml. Killed Staphylococcus aureus Cowan I (SAC) strain bacteria (Calbiochem) were used at 0.005%. PMA was obtained from Sigma and used at 3 ng/ml. UP and PNU156804 were obtained by a fully synthetic process (D’Alessio et al., manuscript in preparation). Purified anti-CD40 Ab (clone 626.1) was a kind gift of Dr. D. Vercelli (DIBIT, Hospital San Raffaele, Milan, Italy).
Cell stimulations, proliferation, and cytotoxicity assays
Primary lymphocytes were plated at 1–2 x 106/ml in flasks (for RNA and protein extractions) or in quadruplicate in 96-well plates (Falcon). At 48 h, 0.5 µCi [3H]thymidine was added in each well (Amersham, Arlington Heights, IL), and the cells were harvested in a Titertek cell harvester (Skatron, Lyerbyen, Norway) 12–16 h later. For IL-2-dependent T cell proliferation, T lymphocytes were stimulated for 72 h with 1 µg/ml PHA to induce expression of the IL-2R. Cells were then washed and replated in medium containing 1% FCS. After 24 h, recombinant human IL-2 (rhIL-2; Serono, Rome, Italy) was added at 200 U/ml. PNU156804 or UP were added at the indicated concentrations 10 min before IL-2. Nuclear extracts were prepared 30 min or 4 h after addition of IL-2. Thymidine incorporation was measured at 48–60 h.
To measure cell death, T lymphocytes were stimulated as above for 48 h. Cells were then stained with FITC-labeled annexin V and propidium iodide (PI) according to the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany) and analyzed on a FACScan apparatus (Becton Dickinson, Mountain View, CA).
Cell cycle analysis
Monoparametric cell cycle analysis on ethanol-fixed cells using PI was performed as described previously (7). Analysis was done on at least 104 cells using a FACStarPlus (Becton Dickinson). The Baisch method was used to assess the cell cycle phase distribution (7).
RNA extraction and Northern blots
RNA was extracted by standard guanidium isothiocyanate and cesium chloride gradient purification. Twenty micrograms total RNA was run in 1% formaldehyde-agarose gels and blotted onto GeneScreen Plus membranes according to the manufacturer’s instructions. All probes were labeled with 32P by standard nick-translation. The cDNA probes have been described previously (5). The cdk2 and cdk4 plasmids used as probes were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
Western blots
Western blots were performed essentially as described (5). Cytoplasmic extracts were loaded and run in 8–12% SDS/polyacrylamide gels. The gels were electroblotted onto nitrocellulose filters (Schleicher & Schuell, Dassel, Germany) for 3–5 h at 35 V, according to standard procedures. The blots were incubated first in blocking solution (PBS containing 5% nonfat milk powder) overnight, then with rabbit antiserum or mouse monoclonal according to the manufacturer’s instructions. All specific Abs used for Western blots were from Santa Cruz (Santa Cruz, CA). Secondary Abs conjugated to HRP were from Amersham and used at 1/1000. Between each incubation, the blots were washed three times for 10 min in PBS containing 0.5% NP40. Detection was performed using the ECL chemiluminescence system (Amersham).
EMSA
Cells were lysed in buffer A (50 mM KCl, 0.1% NP40, 25 mM HEPES (pH 7.8), 1 mM PMSF, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 100 µM DTT) for 5 min at 4°C and centrifuged 5 min at 2500 rpm at 4°C. The nuclear pellets were lysed in buffer B (500 mM KCl, 25 mM HEPES (pH 7.8), 10% glycerol, and 100 µM DTT) containing protease inhibitors with gentle shaking for 20 min at 4°C. The lysates were then centrifuged at 12,000 rpm for 5 min at 4°C. The nuclear extracts were dialyzed for 2 h at 4°C against buffer C (50 mM KCl, 25 mM HEPES (pH 7.8), 10% glycerol, 1 mM PMSF, and 100 µM DTT).
Double-stranded oligonucleotides carrying a binding site for NF-B
(from the HIV-1 promoter) and for AP-1 (from the IL-8 promoter) were as
follows: NF-B, dGGTCCAGAGGGGACTTTCCGAGAGGC; AP-1,
dGTGTGATGACTCAGGTTTCC.
The oligonucleotides were labeled with [32P]dCTP and klenow enzyme. For binding, 2–5 µg nuclear extract was bound to 0.2 ng probe in binding buffer (40 mM Tris-HCl (pH 7.5), 120 mM KCl, 8% Ficoll 400, 4 mM EDTA, 2 mM DTT, and 2 µg poly(dIdC)) for 30 min. In supershift experiments, 2 µl specific Abs was added.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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UP, the first identified member of the prodigiosin family, showed
too severe side effects upon prolonged treatment in vivo (5) for
development as an immunosuppressant. Screening for prodigiosin
analogues with a more favorable activity led to the identification of
PNU156804, whose structure is shown in Fig. 1
together with that of UP. PNU156804 has
a benzylic substitution in place of methylic substitution on the
oxydrylic group. To determine the activity and mechanism of action of
PNU156804 on human T lymphocytes, we first determined its capacity to
inhibit mitogen-induced T cell or B cell proliferation in comparison
with UP. As shown in Fig. 2A,
PNU156804 inhibited by up to 90% the proliferation of human T cells
stimulated by PHA. The optimal inhibitory dose was 200–300 ng/ml,
compared with 50 ng/ml for UP. Thus a 4- to 6-fold higher concentration
of PNU156804 than UP is required. Of note is that the dose-response
curve of PNU156804 is less sharp than than of UP. PNU156804 also
inhibited the MLR response of T cells, with a similar dose-response
curve (Fig. 2B), showing that inhibition is efficient also
in a more physiological model of T cell response and is not specific
for PHA. Similarly, PNU156804 efficiently inhibited SAC-induced
tonsillar B cell proliferation (Fig. 2C), as well as
EBV-induced B cell proliferation and immortalization (data not shown).
Again the dose response curves were similar to those obtained in PHA
stimulated T cells.
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, PNU156804
at a fully active concentration (300 ng/ml) did not induce a
significant increase in either apoptotic or necrotic cells above that
induced by PHA alone. Less than 4% more dead cells could be detected
in presence of PNU156804 relative to its absence, even when higher
concentrations of PNU156804 were used (600 ng/ml). On the other hand,
incubation of the cells overnight with staurosporine as a control led
to a 3- to 4-fold increase in both apoptotic and necrotic cells.
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To determine the cell cycle phase of inhibition by PNU156804, we
also analyzed the DNA content of human T lymphocytes stimulated with
PHA in the presence or absence of PNU156804. As shown in Fig. 3, entry of the cells in the
S/G2/M phases of the cell cycle, induced by PHA at 48
h, was completely blocked by PNU156804. These data show that PNU156804
inhibits T cell activation before entry into S phase.
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A). Inhibition of cdk2, cdk4,
and cyclin E was confirmed at the protein level in Western blots (Fig. 4B, and data not shown). Finally, we demonstrated that,
similarly to UP, PNU156804 inhibited the hyperphosphorylation of
retinoblastoma protein (Rb), observable as a series of bands of higher
apparent m.w. (p112–114) in Western blots (Fig. 4B).
Finally, PNU156804, like UP, did not inhibit the down-regulation of p27
protein (Fig. 4B).
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PNU156804 blocks IL-2-dependent T cell proliferation
G0-G1 phase progression in T cells is
accompanied by induction of IL-2 and the IL-2R, and signaling by IL-2
drives entry into S phase (8, 9). In these cells, the IL-2R is mostly
regulated by induction of the -chain during activation, allowing
high affinity binding (9). We therefore investigated the effect of
PNU156804 on IL-2 and IL-2R -chain expression. IL-2R mRNA was
strongly induced by PHA and was not inhibited by PNU156804 (Fig. 5). At the protein level, induction of
IL-2R by PHA was slightly lower but was not abolished in the
presence of PNU156804 (Table II). IL-2
expression was not affected at either the protein (Table II) or RNA
level (data not shown), as previously observed for UP (5). Since
expression of the IL-2R -chain also increases during T cell
activation (9), we also investigated its regulation in the presence of
the drug. As shown in Fig. 5, -chain mRNA induction was unaffected
by PNU156804. All together, these data suggest that PNU156804 inhibits
T cell proliferation mostly downstream from IL-2 and IL-2R expression,
in agreement with a block in mid-late G1. To test directly
whether PNU156804 inhibits IL-2-dependent T cell proliferation,
peripheral T lymphocytes were preactivated with PHA for 72 h to
induce IL-2R expression (10). The cells were then washed and starved
for 24 h in the absence of IL-2. rhIL-2 was then added to the
cells in the presence or absence of PNU156804. As shown in Fig. 6A, preactivated starved T
cells required exogenously added IL-2 to proliferate, and this
proliferation was inhibited efficiently by PNU156804. Furthermore the
dose-response curve for inhibition of IL-2-dependent proliferation
(Fig. 6B) was superimposable to that obtained on
primary T cells (Fig. 2
, A and B).
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PNU156804 specifically inhibits activation of NF-B and AP-1
Several signaling cascades are known to be triggered by IL-2 that
lead to the expression of different sets of genes and transcription
factors thought to be important for the proliferative and antiapoptotic
response of T cells (8, 9, 11, 12, 13). To determine in more detail the
mechanism of action of PNU156804 and determine which signaling pathway
is affected by the drug, we have set out to determine the effect of
PNU156804 on the pathways triggered by IL-2, which are thought to be
important for the proliferative response of the cells, in particular
the induction of c-myc, bcl-2, AP-1, and NF-B, using
the IL-2-dependent system. As shown in Fig. 7A, c-myc mRNA
is strongly induced at 3 h after addition of IL-2, and this
induction is not affected by PNU156804. Similarly, bcl-2 expression is
clearly up-regulated with a peak around 8–12 h after stimulation with
IL-2, and this stimulation is not inhibited by PNU156804 (Fig. 7B). Thus, PNU156804 does not affect the signaling pathways
that lead to c-myc and bcl-2 induction in response to
IL-2.
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B. For this purpose, an EMSA was chosen that can
directly measure the activation state of these factors in nuclear
extracts. Oligonucleotides carrying a binding site specific for each
transcription factor were used. As shown in Fig. 8A, both NF-B and AP-1,
which are also strongly induced 4 h after addition of IL-2, were
inhibited by up to 90% by PNU156804. Competition assays with the cold
AP-1 and NF-B oligonucleotides confirmed that the retarded bands
were specific (Fig. 8A).
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B and AP-1 are known to be activated by many extracellular
signals and several second messenger cascades (9, 13, 14, 15). Since
PNU156804 blocks IL-2-driven G1-S proliferation, we
investigated whether PNU156804 could also inhibit PMA-induced
activation of NF-B and AP-1 in resting T cells. As shown in Fig. 8B, PMA strongly activated both transcription factors, but
PNU156804 did not inhibit this activation in any way. Similarly,
PNU156804 showed no inhibitory effect on PHA-induced AP-1 or NF-B
activation (data not shown). We conclude that PNU156804 specifically
inhibits IL-2- and not phorbol ester- or PHA-triggered activation of
NF-B and AP-1 in T cells.
To evaluate the specificity of inhibition by PNU156804 for IL-2
signaling, we also investigated the effect of the drug on a more
physiological signal such as CD40 triggering of a B cell line. As shown
in Fig. 8C, the results show that PNU156804 can inhibit
CD40-triggered activation of NF-B measured by EMSA. We conclude that
PNU156804 blocks NF-B induction by some (IL-2, CD40) but not all
(PMA, PHA) signals.
PNU156804 inhibits NF-B activation through inhibition of IB
degradation
To investigate in more detail by which mechanism PNU156804
inhibited NF-B activation, we first conducted supershift
experiments. As shown in Fig. 9A, anti-p50 Abs
supershifted completely the IL-2-induced NF-B complex, whereas
anti-p65 Abs led only to a partial supershift. This indicates that
the IL-2-induced NF-B complexes contain p50/p65 heterodimers as well
as p50 homodimers or p50 complexed with another c-rel family
member.
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B activation is usually regulated through phosphorylation of
IB, the NF-B-associated inhibitor, with degradation of IB
followed by translocation of free NF-B to the nucleus (15, 16). We
therefore investigated the effect of PNU156804 on cytoplasmic IB-
and IB-ß expression. As shown in Fig. 9B, IL-2 induced
a rapid degradation of IB- and IB-ß, and this degradation
was completely blocked by PNU156804. We conclude that PNU156804
inhibits NF-B activation through inhibition of IB degradation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 transcription
factors induced by IL-2. It has no effect on the activation of the same
factors triggered by phorbol ester or PHA. However, it blocks NF-KB
activation by CD40 on B cells, suggesting that the PNU156804 target is
not restricted to IL-2 signaling. We conclude that PNU156804 targets a
signaling pathway triggered by IL-2 that leads to activation of NF-B
and AP-1. Furthermore we hypothesize that the effect of PNU156804 on
NF-B and AP-1 activation is responsible for the block of T cell
proliferation brought about by the drug. Finally, the data presented
demonstrate that PNU156804 shows a different mechanism of action than
other immunosuppressive drugs such as CsA, FK506, and rapamycin (3, 19, 20, 21). The effectiveness of PNU156804 action, its specificity for
defined signaling pathways, and its diversity from other
immunosuppressants, in particular CsA and FK506, make this drug and
other members of the prodigiosin family good candidate drugs for
combined immunotherapy. Furthermore, the mechanism of action of
PNU156804 defined here makes this family of compounds interesting new
tools to dissect the IL-2-signaling pathways that lead to T cell
proliferation. A simplified scheme of the intracellular signaling
cascades induced by IL-2 and based on this report and available
published data is shown in Fig. 10 (8, 9, 12, 14, 22, 23). The differences in the effects of PNU156804 and
rapamycin on these cascades are also indicated.
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The first part of this report was directed at determining the activity of PNU156804 on human T and B lymphocytes, the point of the cell cycle where the cells became blocked, and at comparing the activity of PNU156804 with that of UP itself, as well as of other known immunosuppressive drugs such as rapamycin. PNU156804 was found to inhibit the proliferation of human T and B lymphocytes by 80–90% at 200–300 ng/ml. Thus, PNU156804 is as effective as UP but at doses 4–6 times higher. This is in agreement with in vivo data in mice. We have also determined that PNU156804, like UP (5), strongly inhibits B cell proliferation stimulated by EBV, as well as EBV-induced immortalization of these cells with the same dose-response curve as inhibition of T cell proliferation (Golay et al., unpublished data). Thus, PNU156804 shows the same biological activity as UP in vitro on human lymphocytes. The capacity to block the immortalization of B lymphocytes by EBV is an important advantage for a candidate immunosuppressive drug due to the frequent occurrence of EBV-induced lymphomas in immunosuppressed patients (3).
Analysis of the cell cycle profile and cell cycle-regulated genes in PNU156804-treated T cells has allowed us to conclude that PNU156804 blocks T cell proliferation in mid-late G1, suggesting that that its target is very similar to that of UP (5). In particular we show that PNU156804 inhibits cells between cyclin D2 and cyclin E expression and that both cdk2 and cdk4 are completely inhibited (5, 24, 25). Furthermore we show that retinoblastoma protein remains hypophosphorylated in the presence of PNU156804 (5, 26). Finally, we demonstrate that PNU156804 does not inhibit the down-regulation of p27 protein, clearly distinguishing it from rapamycin, which on the contrary blocks p27 degradation (19, 27, 28). Thus the prodigiosin family represents a new family of molecules with a common mechanism of action and specific molecular targets.
The second part was aimed at identifying more precisely possible target
proteins of PNU156804 action. We have first demonstrated that PNU156804
blocks IL-2-dependent signaling. It does not inhibit significantly the
induction of IL-2 and of its receptor - and -chains but
efficiently blocks IL-2-dependent T cell proliferation. These data led
to the investigation of the different signaling pathways known to be
triggered by IL-2 in T cells. IL-2 responses are mediated by the IL-2R
complex, which is composed of three subunits, , ß, and (8, 9, 29). The -chain is responsible for high affinity IL-2 binding,
whereas the ß- and -chains mediate signaling (8, 9). Several
non-receptor protein tyrosine kinases such as Jak1, Jak3,
p56lck, and syk interact with the IL-2R and
become activated after IL-2 engagement (8, 9, 19, 29, 30, 31, 32, 33) (Fig. 10).
IL-2 also activates PI3-kinase (9, 19, 22, 34, 35). These different
kinases in turn activate different cascades that lead both to the
induction of c-myc, c-fos, and
c-jun, which form the transcription factors AP-1 and
bcl-2, and to the activation of STAT transcription factors (10, 12, 13, 29, 30, 33, 36, 37, 38, 39). IL-2 has also been shown to activate NF-B (9, 40) (Fig. 10). The serine rich (S) region of the IL-2 ß-chain is
known to associate with Jak1 and syk and to be required for induction
of c-myc and bcl-2 (9, 29, 36). The acidic domain (A) is required for
activation of p56lck, for association of
the adapter protein shc, which may mediate activation of p21ras and of
the MAPK cascades, and for induction of c-fos and
c-jun gene expression (9, 13, 29, 31, 41). The mechanism
of activation of PI3-kinase is still unclear but requires the S region
and leads to activation of protein kinase B (PKB, also called Akt),
which may participate in induction of bcl-2 and down-regulation of p27
(22, 34, 37). The mechanism of activation of NF-B is still unclear
but may involve kinases of the JNK pathway and in particular MEKK-1
(15, 16) (Fig. 10). Evidence has been presented that activation of
c-myc, AP-1, and bcl-2 is important for the proliferative response of T
cells to IL-2 (13, 29, 42, 43).
Here we confirmed bcl-2 and c-myc induction by IL-2 in our
IL-2-dependent system, but we showed that PNU156804 does not affect the
expression of these genes. These data, as well as the lack of
inhibition of p27 down-regulation, suggest that the Syk and PI3-kinase
pathways possibly involved in the signaling of IL-2 to these molecules
are not targets of PNU156804 action (Fig. 10). On the contrary,
PNU156804 very efficiently inhibits both NF-B and AP-1 activation in
response to IL-2. Furthermore, we confirm that a major NF-B complex
induced by IL-2 in our primary T cell system is the p50/p65 (relA)
complex and show that IL-2 signaling leads to rapid down-regulation of
IB- and IkB-ß, as described for many of the other signals that
activate NF-B (15, 16, 44). Finally we demonstrate that PNU156804
inhibits the IL-2-induced down-regulation of IB- and IB-ß.
These data strongly suggest that PNU156804 inhibits NF-B activation
through inhibition of IB- and IB-ß down-regulation.
IB- and IB-ß are thought to undergo phosphorylation by IB
kinase complex(es), and this phosphorylation event renders these
proteins targets for proteasome-mediated protein degradation (15, 16, 44). It is likely that many second messengers can trigger these
phosphorylation events through activation of perhaps several IB
kinases, since many different signals can activate NF-B in different
cells, including TNF, IL-1, LPS, phorbol esters, anti-CD28, etc.
All signals, however, appear to act by degrading IB molecules (15).
Recently, a ubiquination-sensitive IB kinase complex has been
described, and two IB kinases have been cloned that are part of a
different multiprotein complex (45, 46, 47). Little is known about the
second messengers that are triggered by IL-2 and lead to NF-B
activation and about which IB kinase may be involved in IL-2-induced
NF-B activation (9, 40). The data presented here suggest that
PNU156804 inhibits an IL-2-specific signaling pathway that leads to
IB- and IB-ß degradation and which is not shared by PMA or
PHA, since PNU156804 did not inhibit PMA- or PHA-induced NF-B
activation. Furthermore, NF-B is thought to play a role in the
induction of IL-2 and IL-2R transcription (15). The fact that
expression of these two genes was little affected by PNU156804 is in
agreement with a specific inhibition of NF-B activation downstream
from the interaction between IL-2 and its receptor. Numerous signals
can, however, lead to NF-B activation in different cells. The
experiments presented suggest that PNU156804 can also inhibit NF-B
induction by CD40 Abs in B cells. Thus, the inhibition of NF-B
activation by PNU156804 is not specific solely for the IL-2-dependent
signaling pathway. A complete investigation of the different signals
and intracellular cascades that lead to NF-B activation in different
cell types and their sensitivity to PNU156804 will be of particular
interest in identifying a molecular target for PNU156804 but is clearly
beyond the scope of this article.
Although much more information is available on the IL-2-induced
activation of AP-1 activity, the exact signaling pathways have not yet
been clearly identified (8, 13, 14, 29, 31, 35, 48). AP-1 activation is
regulated at different levels, involving transcription of its
components such as c-fos and c-jun and
activation of c-jun through phosphorylation by JNK (13, 14, 48). Furthermore, activation of ras through the
p56lck and the shc, sos, grb pathways and
possibly through the action of other IL-2-associated kinases can lead
to activation of the MAP kinase (ERK) and JNK pathways, which are
required for maximal AP-1 activation (13, 14, 38, 43, 48, 49, 50) (Fig. 10). Thus, the exact target of PNU156804 that leads to AP-1 inhibition
will require a more detailed characterization of its effects on these
kinase cascades, which is beyond the scope of this paper. It is again
of particular interest that PNU156804 specifically blocks the IL-2- and
not PMA-induced AP-1 activation, suggesting a specific effect on a
target downstream from IL-2. Interestingly MEKK-1, a kinase that is
upstream from the JNK cascade and therefore plays a role in AP-1
activation, has been shown to activate NF-B through activation of
one or more IB kinase complexes (15, 23). Thus, the AP-1 and NF-B
pathways appear to be linked by MEKK-1 (Fig. 10). It is tempting to
speculate that a target of PNU156804 could lie in the pathway linking
the IL-2 receptor to such a dual kinase capable of activating both the
NF-B and AP-1 pathways.
We conclude that the prodigiosin family of drugs show a very specific
effect on IL-2-induced signaling, leading to NF-B and AP-1
activation. These two transcription factor complexes appear to be
involved in the control of several cellular functions, including
proliferation and apoptosis (8, 29, 36, 51). Thus, we hypothesize that
inhibition of AP-1 and NF-B may be responsible at least in part for
the proliferation block induced by prodigiosins. The efficacy,
specificity of action, and diversity relative to other known
immunosuppressive drugs make this family of compounds of great interest
for further preclinical studies, as well as interesting tools to
further dissect the signaling pathways triggered by IL-2 in T
lymphocytes. Furthermore, the centrality of IL-2 signaling and AP-1 and
NF-B activation in the biology of T and other cell types makes this
class of compounds of wide interest.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Josée Golay, Istituto Ricerche Farmacologiche "Mario Negri," via Eritrea 62, 20157 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: CsA, cyclosporin A; UP, undecylprodigiosin; PI3-kinase, phosphatidylinositol 3'-kinase; PI, propidium iodide; rhIL-2, recombinant human IL-2; SAC, Staphylococcus aureus Cowan I strain bacteria; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; MEKK-1, MAPK/ERK kinase kinase-1; JNK, c-Jun N-terminal kinase; Jak, Janus kinase; PKB, protein kinase B; cdk, cyclin-dependent kinase.
Received for publication October 13, 1998. Accepted for publication April 5, 1999.
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 S. B. Han, S. H. Park, Y. J. Jeon, Y. K. Kim, H. M. Kim, and K. H. Yang Prodigiosin Blocks T Cell Activation by Inhibiting Interleukin-2Ralpha Expression and Delays Progression of Autoimmune Diabetes and Collagen-Induced Arthritis J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 415 - 425. [Abstract] [Full Text] [PDF]
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S. S. Chuang, H.-T. K. Pham, P. R. Kumaresan, and P. A. Mathew A Prominent Role for Activator Protein-1 in the Transcription of the Human 2B4 (CD244) Gene in NK Cells J. Immunol., May 15, 2001; 166(10): 6188 - 6195. [Abstract] [Full Text] [PDF]
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