Note: Descriptions are shown in the official language in which they were submitted.
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TITS F OF THE INVENTION
THE USE OF PROTEASOME INHIBITORS FOR
TREATING CANCER, INFLAMMATION, AUTOIMMUNE DISEASE,
GRAFT REJECTION AND SEPTIC SHOCK
~tF~ D OF THE INVENTION
The present invention relates to the use of proteasome
inhibitors for targetting different cellular functions implicated in cancer,
inflammation, autoimmune disease, graft rejection and septic shock.
BACKGROUND OF THE INVENTION
The proteasome is a large protease complex. It is the
main nonlysosomal proteolytic system in the cell, and resides in the
cytoplasm as well as in the nucleus (Jentsch et al.) 1995, Cell $x:881 ).
The proteasome possesses up to five different peptidase activities, in
different catalytic domains (Ciechanover, 1994, Cell X9:13), and the best
characterized ones are chymotrypsin-like, trypsin-like and
peptidylglutamyf-peptide hydrolyzing (PGPH) activities (Orlowski et al.,
1981, Biochem & Biophys. Res. Com. 1Q1_:814; Wilk et al.) 1983, J.
Neurochem ~Q:842). The proteasome is responsible for the degradation
of 70-90% of cellular proteins (Rock et al.) 1994, Cell x$:761 ). Yet its
activity is well controlled and only those destined to be destroyed are
timely digested by the proteasome. It therefore plays a critical role in
irreversibly removing short-lived regulatory proteins, and other types of
proteins. Indeed, the degradation of some important regulators of cell
proliferation such as cyclin 2, cyclin 3, cyclin B, p53 and p2T~P' are
mediated by the proteasome (Deshaies et al.) 1995, EMBO J. X303;
Yaglom et al., 1995, Mol. & Cell. Biol. 1:731; Salama et al.) 1994) Mol.
& Celf. Biol. 14:7953; Seufert et al., 1995, Nature x:78; Scheffner et al.,
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1993, Cell 75:495; Pagano et al., 1995, Science 2:682). The activities
of several important regulators involved in cell activation are also
controlled by the proteasome. For example, the transacting nuclear factor
NF-~cB becomes active after the enzymatic cleavage of its precursor by
the proteasome (Palombella et al., 1994, Cell x:773); IKBa) the inhibitor
of NF-KB, and c-JUN protein are degraded via the proteasome pathway
(Palombella et al., 1994, supra; Treier et al., 1994, Cell x$:787).
According to sedimentation rates, the proteasome could
be purified as 26S and 20S complexes. The 20S proteasome is a
cylindrical proteolytic core composed of multiple a and ~ subunits. Each
subunit is coded by a different gene in high eukaryotic cells and the total
number of subunits varies among different species (Groettrup et al., 1996,
Immunol. Today X7:429). In vitro, the purified 20S proteasomes can
digest small peptides in an ATP-independent fashion) but they are
9 5 inactive on intact folded proteins (Peters, 1994, Trends in Biochem. Sci.
x:377). The 20S proteasome can bind at its ends a 19S regulator and
forms the 26S proteasome, which degrades ubiquitinated protein in an
ATP-dependent fashion (Jentsch et al., 1995, supra). The 20S
proteasome can also complex with an 11 S activator called PA28
(Groettrup et al., 1996, supra). PA28 is a ring-like hexamer or heptamer
composed of a and (i subunits (PA28a and PA28~3), both of which are
about 29KD in size (Realini et al.) 1994, J. Biol. Chem. x:20727; Ahn
et al., 1995, FEES Letters x:37). It is not clear whether the 20S
proteasome can associate both the 19S and 11 S regulators at the same
time.
There are two better characterized mechanisms
regulating the protein degradation via the proteasome pathway. The first
is that of the substrate selection. This process is controlled by a cascade
of enzymes called the ubiquitin-activating enzyme (E1), the ubiquitin-
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conjugating enzyme (E2) and the ubiquitin ligase (E3) (Jentsch et al.,
1995, supra). In addition, the 19S regulator controls the entry of the
ubiquitinated protein into the 20S catalytic core. The second mechanism
is the activity of the 20S proteasome, which is enhanced by the 11 S PA28
(Realini et al., 1994, supra). ft is not clear whether and how the 11 S PA28
exerts its effect on the 26S proteasome, since it and the 19S regulator do
not seem to associate with the 20S at the same time. Moreover) whether
the 20S complex exists in parallel to the 26S complex 'n viv is still an
open question. Nevertheless) it has been shown that overexpression of
PA28a could indeed augment significantly antigen processing by the
proteasome in vivo (Groettrup et al., 1996, supra).
Certain peptide aldehydes such as
N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) and
N-carbobenzyoxyl-L-leucinyl-L-leucinyl-L-norvalinal (MG 115) are
competitive inhibitors of chymotrypsin (Vinitsky et al.) 1992, Biochem.
x:9421; Tsubuki et al., 1993, Biochem & Biophys. Res. Com. ~:11g5).
These agents could effectively block the chymotrypsin-like activity, and
to a lesser extent, the trypsin-like and PGPH activities of the proteasome
(Rock et al., 1994, supra). They have been employed to study the
function of the proteasome in various cellular processes. A caveat of such
studies is that these peptide aldehydes are not specfic to the proteasome
peptidases, and other cellular cysteine proteases such as calpain and
cathepsin B (Rock et al., 1994, supra; Sasaki et al.) 1990) J. Enzyme
Inhib. x:195) are also potently inhibited. This makes some interpretations
less assuring.
Orlowski et al. in US patent 5,580,854 teach the use of
peptidyl aldehydes and their analogues to inhibit proteolysis mediated by
the multicatalytic proteinases complex (MPC) or proteasome. The use of
such compounds is to inhibit intracellular protein degradation) mitosis and
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proliferation of dividing cell population. This reference does not teach any
apoptotic effect of proteasome inhibitors.
Palombella et al. in WO 95/25533 teach a method for
reducing the cellular content and activity of NF-kB, a transcriptional factor
playing a central role in immune and inflammatory response) by using
proteasome inhibitors, peptidyl aldehydes.
Stein et al. in WO 95/24914 teach a method for reducing
the rate of intracellular protein breakdown by inhibiting proteasome
activity. The inhibitor MG 101 given as an example is shown to be an
inhibitor of 26S proteasome. This inhibitory effect may result in inhibiting
destruction of muscle proteins, antigen presentation and degradation of
p53 .
Omura et al. have reported in 1991 the discovery of
lactacystin (LAC) which could induce a neurite outgrowth (Omura et al.)
1991 ) J. Antibiot. X4:113; Ibid., x:117).
Fenteany et al. have subsequently found that LAC is a
proteasome-specific protease inhibitor (Fenteany et al., 1995, Science
x$:72). It inhibits the three major peptidase activities (i.e.,
chymotrypsin-like, trypsin-like, and PGPH activities) of the proteasome)
and the inhibition of the first two is irreversible in in vitro assays. t.AC
does not affect other proteases such as calpain, cathepsin B,
chymotrypsin, trypsin, and papain. Currently, I.AC is the only
proteasome-specific protease inhibitor available.
Schreiber in WO 96/32105 teaches lactacystin and
various analogs to treat conditions that are mediated by the proteolytic
function of the proteasome such as rapid elimination and post
translational processing of proteins involved in cellular regulation,
intercellular communication and immune response, specifically antigen
presentation.
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Griscavage et al. (1996, PNAS x:3308-3312) teach that
proteasome activity is essential for the induction of nitric oxide synthase
and that the proteasome peptidyl aldehyde inhibitors inhibit the induction
of nitric oxide synthase. Nitric oxide production is implicated in initiating
5 and exacerbating symptoms of acute and chronic inflammation (Lundberg
et al.) 1997, Nature Medecine x:30-31 ). Thus the proteasome inhibitors,
peptidyl aldehyde, by inhibiting nitric oxide induction have an anti-
inflammatory activity. There is no teaching of reproducing the same effect
using LAC which is more specific to proteasome than peptidyl aldehydes.
Cui et al. (1997, PNAS X4:7515-7523) had shown that
T-cell hybridoma can be activated using dishes coated with anti-CD3.
Once activated these cells die of apoptosis. It was demonstrated that
lactacystin is an inhibitor of activation induced cell death (AICD) and) in
these activated hybridoma T-cells, lactacystin must be administered
within 2 hours of activation to efficiently block AICD. The same authors
state that at higher doses LAC induces apoptosis in the artificial
hybridoma T cells.
Grimm et al. (1996, EMBO 1:3835-3844) have shown
that proteasome plays a role in thymocyte apoptosis and that peptidyl
aldehyde derivatives that inhibit proteasome and LAC block apoptosis in
some cases. In addition Grimm et al. (supra) reported that the LAC block
of apoptosis was irreversible even when the drug was removed from the
cell media. Imajoh-Ohmi et al. (Bioch. Biophys. Res. Com., 1995,
7:1070-1077), teach that lactacystin induces apoptosis in human
monoblast U937 cells.
The involvement of mitochondria in the apoptotic
process has been described by Kroemer et al. (Immunology Today, 1997)
.x$:44). Teachings relating to the mitochondria) control of apoptosis at the
induction phase that appear to be essential are provided.
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None of these references teach that proteasome
inhibitors eliminate activated normal cells. There is no teachings in these
references of the involvement of proteasome activity in mitochondrial
function. In addition, these references do not describe in mammalian cells
what proportion of the protease activity is derived from the proteasome
and whether there are efficient and simple methods to screen for
additional proteasome inhibitors.
LAC is the most specific inhibitor of proteasome
available. It is mildly toxic and is unstable in aqueous solutions of high
pH. LAC and some of its analogues binds directly to the proteasome and
inhibits three peptidase activities of the proteasome. However) cellular
events downstream of the proteasome are not totally clear. Knowledge
of these down stream events related to proteasome activity will allow
development of strategies and compounds capable of complementing,
synergizing) or substituting the effect of proteasome inhibitors to
maximize their effects and/or to minimize their side-effects.
It therefore appears that there is a need to investigate
the role of proteasome and the effect of LAC or its analogues in the
different cellular processes discussed above) and to develop an efficient
screening method for searching additional proteasome inhibitors.
The present invention seeks to meet these and other
needs.
The present description refers to a number of
documents) the content of which is herein incorporated by reference.
SUMMARY OF TIDE INVENTION
Recent work of the Applicant has revealed that PA28 a
and ~i expression is upregulated during T cell activation, and probably as
a result, the ex vivo proteasome activity is fourtold higher in the activated
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T cells than that in the resting T cells (Wang et al.) 1997, Eur. J. Immunol.
~7: November 1 st, 1997, in press). Such an augmented activity likely
reflects the increased need to destroy short-lived regulatory proteins and
other types of proteins during T cell activation and proliferation.
Consequently, it is logical to hypothesize that blocking the proteasome
activity will interfere with the activation and proliferation of T cells.
The invention demonstrates that proteasome is essential
for progression of T cells from Go to S phase. Taking advantage of LAC's
specificity and potency, this compound was used to investigate the role
of proteasomes in T lymphocyte activation and proliferation. It is
demonstrated that the proteasome is essential for progression of T cells
from the Go to S phase. Probably as a result of blockage of cycling) the
activated but not resting T cells underwent apoptosis when treated with
LAC. It is also shown that the proteasome controls the protein level of
p21 ~'p' and p27"~' as well as the CDK2 activity in the G, phase, and such
control mechanism might be essential in the cell cycle progression. LAC
can effectively inhibit T cell proliferation even if added at the G,IS
boundary. This knowledge is useful in administering LAC to reverse
ongoing graft rejection during the rejection episode.
The present invention further relates to inducing
apoptosis of activated T cells and T cell leukemia but not resting T cells
with LAC or its analogues. Elimination of malignant cells by a proteasome
inhibitor-induced apoptosis is useful in cancer therapy. In addition, normal
T cells that become activated can be induced to undergo apoptosis with
a proteasome inhibitor thus eliminating antigen specific T cells. This is
useful in ameliorating autoimmune diseases and graft rejection by
generating antigen specific tolerance.
The invention further uses the knowledge of the
proteasome involvement in protein degradation and in the steps for the
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induction of nitric oxide synthase and the effect of I.AC or its analogues
on the expression of nitric oxide synthase and the production of nitric
acid. This is useful in the prevention of septic shock and as an anti-
inflammatory.
The present invention also relates to the inhibition of
proteasome activity by LAC or its analogues such that the inhibition
interferes with cell-cell interaction during lymphocyte activation in
mammals and the up-regulation of the adhesion molecule 1CAM-1 is
repressed. This is useful to control undesirable immune responses during
graft rejection, autoimmune diseases and inflammation.
The applicant is the first to show that the electron
transport chain in mitochondria is dependent on the intact activity of the
proteasome. The addition of proteasome - specific inhibitor such as L.AC
reduces the electron transport at the complex IV of the respiratory chain.
The addition of exogenous cytochrome C reverses this effect. The effect
of LAC on mitochondria has potential applications for disorders that relate
directly or indirectly to increased activity of mitochondria) function. As
well, since proliferating cells have a higher energy requirement, inhibition
of mitochondria) respiration could effectively curb the proliferation of
cancer cells and activated T cells by depriving the cells of energy, with
minimal detriment to normal resting cells.
The applicant is further providing a method for screening
proteasome inhibitors by assaying cellular proteinases activity with a
tagged peptide substrate. It is understood that this assay protocol can be
used in a large through-put screening procedure and that any means of
tagging peptide substrates specific to different protease activities of the
proteasome and any means for detection known to a person skilled in the
art, can be used and incorporated into the large through-put procedure.
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All the elements comprising a method for screening proteasome inhibitors
can be incorporated into a kit.
Therefore, in accordance with the present invention it is provided:
The use of a proteasome inhibitor to induce apoptosis
in proliferating cells, wherein said proteasome inhibitor is lactacystin or an
analogue thereof and said proliferating cells are cancerous cells and/or
activated T cells, such that activated T cells are antigen induced. The
above cells are stopped from progressing from Go to G,/M in a cell cycle
as a consequence of proteasome inhibition. As well, CDK2 and the
associated Cyclin E activities are substantially inhibited, whereby said cell
cycle progression is substantially arrested. Additionally, CDK4 cell activity
is not inhibited.
Any one of the use of the above stated provisioned uses
of a proteasome inhibitor, wherein said proliferating cells are eliminated
and cancer progression is arrested and) activated T cells are eliminated.
The use of a proteasome inhibitor to reverse graft
rejection in a patient in need for such a treatment comprising the step of
administering to said patient an apoptotic amount of a proteasome
inhibitor when said patient T cells are activated wherein said patient is in
need of said treatment when an ongoing allograft rejection occurs or at
least 24h after graft transplantation.
The use of a proteasome inhibitor in the making of a
medicament to induce apoptosis in proliferating cells. The use of a
proteasome inhibitor as defined in the above stated provisions, alone or
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in combination with another medication, to eliminate or to reduce antigen-
specific induced T or B cells, and achieve antigen-specific tolerant status
or reduced responsiveness to an antigen in a patient which condition
requires such treatment wherein said condition is selected from the group
5 consisting of: autoimmune disease) graft rejection and inflammation.
A method for screening a compound for proteasome
inhibition activity, which comprises: obtaining a mammalian cell lysate
comprising proteasomes, a partially purified proteasomes preparation or
10 a purified proteasomes preparation; tagging at least one peptide
substrate specific to a known proteasome protease activity; combining
said proteasomes and said at least one tagged peptide substrate;
contacting the so combined proteasomesltagged peptide substrate with
said compound; said at least one tagged peptide substrate fails to release
~ 5 tag if said compound is a proteasome inhibitor, and detecting a decrease
or absence of the released tag in the presence of said compound relating
to the released tag in the absence of said compound as an indication of
proteasome inhibition activity for said compound wherein said at least one
tagged peptide substrate is a fluorogenic peptide and wherein said
proteasome protease activity is trypsin-like chymotrypsin-like or
peptidylglutamyl-peptide hydrolyzing activity.
The use of a proteasome inhibitor to disrupt
mitochondria) function, wherein said inhibitor blocks electron transport in
said mitochondria and, wherein said inhibitor blocks said electron
transport at complex IV in said mitochondria such that mitochondria)
function is disrupted, wherein disruption of mitochondria) function is
corrected by cytochrome C. The use of the afore-mentioned provisions
relating to mitochondria) function to treat a pathological condition wherein
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high mitochondrial activity occurs) said pathological condition is selected
from the group consisting of: cancer, inflammation, undesirable immune
responses and hyperthyroidism.
The use of a proteasome inhibitor to disrupt nitric oxide
synthesis, wherein the proteasome inhibitor inhibits nitric oxide synthase
gene expression.
An apoptotic composition comprising a therapeutically
effective amount of a proteasome inhibitor and a pharmaceutically
acceptable carrier which may additionally comprise a therapeutically
effective amount of an inhibitor to CDK4 activity and/or a therapeutically
effective amount of an inhibitor to CDK2 activity and more particularly to
Cyclin E activity, a therapeutically effective amount of an inhibitor which
prevents p21~~' upregulation blocks the degradation of p27 '''and a
therapeutically effective amount of an inhibitor which prevents CD25
upregulation.
The use of cyclosporin A, rapamycin or FK506 as a
proteasome inhibitor.
A composition for use in inhibiting graft rejection
comprising a therapeutically effective amount of cyclosporin A, rapamycin
or FK506 in combination with a therapeutically effective amount of a
proteasome inhibitor and may be in combination with a therapeutically
effective amount of an inhibitor of /CAM-1 expression.
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A composition for use in inhibiting graft rejection
comprising a therapeutically effective amount of an inhibitor which
suppresses expression ICAM-1 in combination with a therapeutically
effective amount of a proteasome inhibitor.
The use of a proteasome inhibitor to alleviate a disease
or a disorder, wherein adhesion molecule ICAM-1 is upregulated and said
disease or a disorder is graft rejection, autoimmune disease or
inflammation.
The use of a proteasome inhibitor is to alleviate a
desease or a disorder wherein at least one of CDK2, p21~'p', CD25 is
upregufated and/or p2T"P' degraded, wherein said disease or disorder is
graft rejection) autoimmune disease or cancer.
The use of a proteasome inhibitor to alleviate a disease
or disorder, wherein nitric oxide synthase is upregulated and said disease
or disorder is inflammation or septic shock.
The said proteasome inhibitor may be used alone or in
combination with any drugs known in the art for use in treating cancer,
inflammation, autoimmune disease, septic shock or inflammation.
The use of all the afore-mentioned provisions wherein
said proteasome inhibitor is lactacystin or an analogue thereof.
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BRIEF DESCRIPTION OF TIHE DRAWINGS
Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
Figure 1 shows that LAC strongly inhibits T and B cell
proliferation. Lymphocytes were stimulated with various mitogens as
indicated, and LAC at different concentrations was added at the
beginning of the cultures. The cells were pulsed with 3H-thymidine
between 48h and 64h. Samples were in triplicates. All the experiments
were performed at least three times and similar results were obtained.
Representative results are shown.
A: Peripheral blood T cells stimulated with PHA (2 Ng/ml).
B: Peripheral blood T cells stimulated with OKT3 (50ng/ml).
C: Peripheral blood T cells stimulated with anti-CD28 {50nglml) plus
ionomycin (1 Ng/ml).
D: Tonsillar B cells stimulated with SAC (1:15,000 dilution) and IL-2
(100 N/ml).
Figure 2 shows that inhibition of the proteasome activity
results in induction of apoptosis of activated normal cells and leukemic
T cells but not resting normal T cells. Tonsillar T cells (A, B) and D) and
Jurkat cells (C and E) were treated with LAC (10 NM for T cells and 6 NM
for Jurkat cells). LAC was added at the beginning of the culture or 40h
after T cell activation as indicated. The cells were harvested at the time
points as shown. They were evaluated for their viability with trypan blue
exclusion (A, B, and C), and for their mode of cell death according to DNA
fragmentation (D and E).
Figure 3 shows by electron microscopy that the
proteasome inhibitor induced apoptosis in activated T cells and Jurkat
cells.
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A and B: Morphology of resting T cells treated with l..AC. Tonsillar
T cells were culture in the absence (A) or presence (B) of LAC (10mM) for
24h, and the cells were examined by EM.
C and D: Morphology of activated T cells treated with LAC. Tonsillar
T cells were first activated with PHA (2 Nglml) for 40h. The cells were then
cultured in the absence (C) or presence (D) of LAC (10 NM) for additional
24h) and were examined with EM.
E and F: Morphology of Jurkat cell treated with LAC. Jurkat cells were
cultured in the absence (E) or presence (F) of LAC (6 NM) for 24h and
were evaluated with EM. Arrows indicate condensed nuclei.
Figure 4 shows that the effect of I.AC is rapid and
reversible in cell culture.
A. The rapid effect of LAC Peripheral blood T cells were pretreated
with 10 NM LAC in culture medium or in culture medium alone for 3h or
16h. The cells were then washed and recultured in the presence of
2 Ng/ml PHA for 64h. The cells were pulsed with 3H-thymidine for 16h
before they were harvested at 64h. Samples were in triplicates.
B. The inhibitory effect of I.AC on the proteasome activity was
reversible in the cells Jurkat cells were pretreated with I.AC (6 NM) in
culture medium for 3h. The cells were washed and recultured at 0.5 x 10B
cellslml for Oh, 5h or 21 h. The cells were then harvested, washed and
sonicated. The lysate protein (20 Nglsample) was assayed for its
proteinase activity under a condition at which 90% of the activity was
attributed to the proteasome. The samples were in duplicates. The result
is expressed as relative fluorescence intensity at 440nm.
C. The activity of LAC in culture supernatants is short-lived LAC
{6 NM) was added to Jurkat cell culture (0.5 x 106 cellslml). The
supernatants were harvested at 4h) 6h, 16h and 24h. These conditioned
media were used
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to culture fresh Jurkat cells for 3h. The cells were then harvested and
assayed for the proteasome activity as described in Fig. 4B. Samples
were in duplicates.
All the experiments were performed at least three times, and similar
5 results were obtained. Representative data are shown.
Figure 5 shows that LAC inhibits CD25 upregulation
during T cell activation.
Peripheral blood T cells were stimulated with PHA (2 Nglml) for 48h in
the presence or absence of LAC (10 NM, added at the beginning of the
10 culture). CD25 expression on T cells was evaluated by anti
CD25-PE/anti-CD3-FITC two-color flow cytometry. Similar results were
obtained in two independent experiments, and a representative one is
shown. The data are presented as two color histograms in forms of
contours, as well as in an overlay histogram.
15 Figure 6 shows the role of the proteasome in cell cycle
progress.
A. LAC does not inhibit the progress from the GzlM phase to the G,
phase in synchronized Jurkat cells Jurkat cells were synchronized at the
GZ /M phase by 16h nocodazole treatment. For the last 3h of the
treatment, LAC (6 NM) was added to the cultures destined to be treated
by LAC later. The cells were then released by washing out nocodazole,
and recultured in complete medium with or without 6 NM LAC. The cells
were sampled at Oh, 4h and 8h after the GRIM release, stained with
propidium iodide, and analyzed with flow cytometry.
B. LAC slows the cell cycle progress from the G,/S boundary to the
GzM1 phase in synchronized Jurkat cells Jurkat cells were synchronized
at the G,/S by isoleucine starvation followed by a hydroxyurea treatment.
The synchronized cells were released by washing out hydroxyurea and
were cultured in complete medium in the absence or presence of LAC
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(6 NM). The cells were sampled at Oh, 3h) 6h) 9h) 12h, 15h and 24h after
the release, and were stained with propidium iodide and analyzed with
flow cytometry.
C and D. LAC blocks the S phase entry of the mitogen-stimulated
peripheral blood T cells Peripheral blood T cells were stimulated with PHA
(2 N/ml) in the absence or presence of LAC (10 NM, added at Oh, 16h,
24h) or 40h, as indicated in the bottom of the panels). For the flow
cytometry analysis of the cell cycle progress, the cells were harvested at
Oh, 16h, 40h and 64h as indicated on the top of the panels (Fig. 6C). For
3H-thymidine uptake) the triplicated cell samples were pulsed at 48h and
harvested at 64h (Fig. 6D).
The experiments were performed three times, and similar results were
obtained. Representative data are shown.
Figure 7 shows the results of the kinase assays for the
effect of LAC on CDK activity.
Tonsillar T cells were activated with PHA (2 Nglml) for a period as
indicated in each graph. LAC (10 NM) was added once at Oh. The cells
were harvested at 16h) 24h, or 40h as indicated. An equal amount of
lysate protein (40 Nlsample) was precipitated with rabbit anti-CDK4,
anti-CDK2 or anti-Cyclin E antisera (2.5 Ng Ablsample). The immune
complexes were assayed for their kinase activities using histone H1 as a
substrate. (A) CDK4 kinase activity. (B) CDK2 kinase activity. (C)
Cyclin E-associated CDK activity. The membrane in (C) was
subsequently hybridized with anti-Cyclin E (1 Ng/ml) followed by
'251-protein A for the evaluation of the protein level of Cyclin E.
All the experiments were performed three times) and similar results
were obtained. Representative data are shown.
Figure 8 shows the results of immunoblotting analysis
of the effect of l~C on the protein levels of Cyclin E and Cyclin A.
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Tonsillar T cells were stimulated with PHA (2 Ng/m1) for 40h in the
presence of hydroxyurea (1 mM), and these cells were blocked at the G,/S
boundary (G, block). The synchronization was released by washing out
hydroxyurea, and the cells were recultured in medium containing 2 Ng/ml
PHA in the absence or presence of LAC (lOnM, added once at the time
of the release). The cells were harvested at 6h and 22h post the G,/S
block. The cell lysates (40 y~g/sample) were resolved in 10% SDS-PAGE,
and transferred to PVDF membranes. The membranes were hybridized
with rabbit-anti-Cyclin E or anticyclin A antisera followed by '251-protein A.
The Cyclin E level (Fig. 8A) and cyclin A level (Fig. 8B) of representative
experiments are shown. Similar results were obtained in a total of three
independent experiments.
Figure 9 shows the results of immunoblotting analysis
of the effect of LAC on the levels of CDK inhibitors p27wp' and
p21°'p'.
Tonsillar T cells were stimulated with PHA (2 Ng/ml) for 16h, 40 or 64h
in the absence or presence of LAC (10 NM). For the 16h and 40h culture,
LAC was added once at Oh. For the 64h culture, LAC was added once at
40h. The cell lysates were resolved in 10% SDS-PAGE) and blotted onto
PVDF membranes. The membranes were hybridized with rabbit
anti-p2T°p' antisera (Fig. 9A) or with anti-p21 ~'p' antisera (Fig. 9B)
followed by'Z51-protein A. The experiments were performed three times,
and similar results were obtained. Representative data is shown.
Figure 10 shows human peripheral blood mononuclear
cells that were cultured in medium (A), 2 Ng/ml PHA (B), or PHA plus
10 NM lactacystin for 24h. Lactacystin could effectively block the
aggregate formation.
Figure 11 shows mouse lymph node cells that were
cultured in medium (A), 2 Ng/ml Con A (B), or Con A plus 10 NM
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lactacystin for 24h. Lactacystin could effectively block the aggregate
formation.
Figure 12 shows mouse lymph node cells from TCR
transgenic mice named 2C that were cultured in medium (A), 2 pg/ml Con
A (B), or Con A plus 10 NM lactacystin. After 24h and 48h, the cells were
examined for ICAM-1 expression by flow cytometry, using FITC-anti-
ICAM-1 / 1 B2-PE. Monoclonal Ab 1 B2 recognize a clonotypic determinant
on the TCR of the transgenic T cells which are largely CD8 positive
{>75%). Lactacystin could effectively block the upregulation of ICAM-1 on
those CD8 positive T cells.
Figure 13 shows mouse peritoneal exudate
macrophages that were stimulated with 2 Nglml LPS in the presence of
lactacystin at different concentrations. Nitric oxide production by the
macrophages was measured according to the nitrate concentrations in
the supernatants.
Figure 14 shows mouse peritoneal exudate
macrophages that were stimulated with 2 Ng/ml LPS in the presence or
absence of lactacystin (10 NM). Nitric oxide synthase expression was
measured with Northern blot analysis.
Figure 15 shows that Lactacystin blocks electron
transport downstream of Complex I. Respiration of Jurkat cells (JC) or rat
kidney mitochondria (RKM) was measured by OZ consumption using an
oxygen electrode. The function of Complex I of digitonin (Dig)-permeated
Jurkat cells was blocked by rotenone (Rot), and the respiration was
resumed by adding succinate {Suc), which provides electrons to Complex
II directly and thus bypasses Complex I. The maximal respiration was
achieved by adding CCCP (carbonyl cyanide m-chlorophenylhydrazone))
which uncouples the oxidation and phosphorylation. The respiration could
be blocked by antimycin A (Ant)) which inhibits Complex II. Curves 1 and
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6 represent positive controls of rat kidney mitochondria. Curves 2 and 5
represent normals untreated Jurkat cells. Curves 3 and 4 represent Jurkat
cells treated with lactacystin (6 NM) for 2h and 4h, respectively.
Figure 16 shows that Lactacystin blocks electron
transport at Complex IV. Complex 111 in the respiration chain was blocked
at Complex III antimycin (Ant), and the electron flow was resumed by
addiind ascorbate (Asc) and TMPD (tetramethyl-p-phenyl-enediamine).
The maximal respiration was triggered by CCCP, and was totally inhibited
by potassium cyanide (KCN).
Figure 17 shows that Cytochrome completely corrects
the defect at Complex IV caused by LAC. The assay system is identical
to that described in Figure 16. Jurkat cells were treated with LAC for 4h
(curve 3). The decoupling reagent used in this experiment to achieve
maximal respiration is FCCP (carbonylcyanide-p-
trifluoromethoxyphenylhydrazone).
Figure 18 shows that RAPA, FK506, and CsA inhibit
PA28 expression at the mRNA level. Tonsillar T cells (A) and B cells (B)
were cultured in the presence of various reagents as indicated (PHA)
2 Ng/ml) RAPA, 10 nM; FK506, 10 nM, CsA, 1 NM; SAC) 1:10
000 dilution; II-2, 25 U/ml. After 6h, 20h or 40h, the cells were harvested
and total RNA was analyzed by Northern blotting for PA28(3 expression.
The PA28~i message in T cells was also examined by Northern blotting
using a similar condition as for PA28(3 (C). The experiments were
repeated more than three times, and representative ones are shown.
Figure 19 shows that RAPA inhibits PA28(i and PA28a
protein in the activated T cells. (A) An analysis of PA28~ protein by
immunoblotting is shown. Tonsillar T cells were cultured with 2 Ng/ml PHA
or PHA plus 50 nM RAPA for 24h. The cells were harvested and lysed.
Forty micrograms of cleared lysate protein per sample was analyzed by
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immunoblotting using rabbit anti-PA28~ antiserum. (B) An analysis
PA28a and PA28a protein by confocal immunofluorescence microscopy.
Tonsillar T cells were cultured with 2 Ng/m1 PHA or PHA plus 50 nM
RAPA for 24h. The cells were stained with antisera specific for PA28a
5 and PA28~i. Thirteen cells were analyzed for PA28a protein and twelve
cells for PA28~ protein in a blind fashion. The mean + SD of relative
fluorescence intensity per whole cell is presented. Unpaired Student's t
test was employed for statistics. The difference between PHA-activated
sample and PHA plus RAPA-treated samples was highly significant
10 (p = 3.20 x 10-9 for PA28a and p = 5.99 x 10'5 for PA28~).
Figure 20 shows that effect of RAPA on proteasome
activity in human PBMC. Human PBMC were cultured in the absence or
presence of 2 pg/ml PHA or 10 nM RAPA for 16h-70h as indicated. The
cells were then harvested, and the chymotrypsin-like activity of whole
15 cells lysates was assayed in the absence or presence of 20 NM
proteasome inhibitor LAC. The data are presented as arbitrary units of
fluorescence intensity per 20 Ng lysate protein. The experiments were
repeated three times and a representative one is shown. Samples are in
duplicate and the mean t SD is shown. (A) Total chymotrypsin-like
20 activity in the lysate of PBMC. (B) Lactacystin-inhibitable chymotrypsin-
like activity in the lysate of 70h PBMC. Nine micrograms of 20S
proteasome were used as positive controls for the inhibitory effect of LAC
at 10 NM and 20 NM. LAC was always added to the lysates during the
proteinase assay 15 min before the addition of the substrate. The solid
bars represent the activity in the presence of LAC. The net proteasome
activities are calculated as the total activity minus the remaining activity
after the LAC addition.
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Figure 21 shows the elimination of an alloantigen-
specific response by a proteasome inhibitor lactacystin. The C57BU6
spleen cells (H-2b) were stimulated with mitomycin c-treated BALBJc
spleen cells (H-2d). On day 2 when most of the H-2°-specific cells were
activated) the mixed lymphocyte culture (MLR) was treated with
lactacystin (LAC) 8NM) for 3 h. After wash, the cells were put back in
culture for additional 8 days, and then stimulated with either fresh BALB/c
or C3H (H-2k) spleen cells. In MLR treated by LAC, the C57BU6 cells
failed to respond to the BALB/c cells, but respond well to third party C3H
(H-2k) cells. The difference is more pronounced in day three of the
culture.
Figure 22 shows that the LAC-induced DNA
fragmentation is inhibited by a broad spectrum caspase inhibitor
zVAD.fmk. Jurkat cells were treated with LAC (6 NM) in the absence or
presence of different concentrations of zVAD.fms (0.4 NM to 33.3 NM) for
6 h. The cells were harvested and their DNA was analyzed by a DNA
fragmentation assay according to DNA laddering.
Figure 23 shows that preventing the degradation of a
pro-apoptotic Bcl-2 family member Bik is a mechanism for the
proteasome inhibitor-induced apoptosis. Jurkat cells were treated with
lactacystin (6 NM) for 5 h (lanes 2 and 4 of panel A), 4 h (lane 2 of panel
B) or 7h (lane 3 of panel B), lane 1 in panels A and B is untreated control
samples. The cells were separated into mitochondrial (mito in panel A and
mitochondria in panel B) and cytosolic (cytosol in panel A) fractions, and
the lysate of these two fractions analyzed by immunoblotting using goat
anti- Bik, and rabbit anti-Bax) Bak and Bad Ab (alt from Santa Cruz
Biotech, Santa Cruz, CA) followed by enhanced chemiluminescence
(ECL, kit from Amersham).
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Figure 24 shows that overexpression of an anti-
apoptotic Bcl-2 family member Bcl-xL in a B cell line could protect the
cells from apoptosis caused by proteasome inhibition. A human B cell
line Namalwa was stably transfected with an anti-apoptotic Bcl-2 family
member Bcl-xL, and its sensitivity to the proteasome inhibitor-induced
apoptosis tested by the quantitative filter elution assay (Schmitt et al.,
Exp. Cell Res. 240:107, 1998), which detects DNA fragmentation during
apoptosis. The wild type Namalwa and transfected Namalwa cells
overexpressing Bcl-xL were pulsed with'"C- thymidine for 24 h, and then
treated with different concentrations of lactacystin (0.75 NM, 1.5 NM, 3
NM, 6 NM and 10 NM). The cells were harvested at different time intervals
(24-96 h)) and DNA fragmentation measured.
Figure 25 shows that the wild type Namalwa cells have
increased Bik level after treatment with lactacystin and that the Bcl-xL
transfected Namalwa cells have overexpressed Bcl-xL. Jurkat cells, wild
type Namalwa cells and Bcl-xL transfected Namalwa cells were treated
with medium (lanes 1)) staurosporine (0.3 NM) lanes 2) and lactacystin
(6 pM, lanes 3) for 6 H. The proteins from the mitochondrial fraction of
these cells were analyzed by immunoblotting and the amount of Bik, Bcl-
xL) Bax, and Bak evaluated. The same membranes were used
sequentially and probed with different antibodies against these factors.
A nonspecific band recognized by a monoclonal antibody against
cytochrome oxygenase (COX) was used as control for even sample
loading in the lanes.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference to the
accompanying drawings which are exemplary and should not be
interpreted as limiting the scope of the present invention.
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DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention relates to proteasome activities
in cellular processes and any inhibitors of proteasome activities.
Proteasome Activity is Obligatory for Activation and Proliferation of
T and B Cells
The role of proteasome in T cell activation and
proliferation was first examined in PBMC, using the proteasome-specific
inhibitor IJ~C. The PBMC were activated with various stimulants. LAC
was added to the cells in the beginning of the culture (Oh) along with the
stimulants. 3H-thymidine uptake between 48h and 64h of 64h cultures
was used as a parameter for cell proliferation. As shown in Fig. 1) LAC
strongly and dose-dependently inhibited the T cell proliferation induced
by a T cell mitogen PHA (Fig. 1A), by crosslinking TCR with anti-CD3e
(Fig. 1 B), or by Ca" ionophore plus cross-linking of the T cell
co-stimulating molecule CD28 (Fig. 1C). The T cell-independent B cell
proliferation induced with SAC plus IL-2 in tonsillar B cells was also
potently inhibited by LAC (Fig.1 D). In all the four systems employed, LAC
at 5 NM could exert near-to-maximal inhibition. The results suggest that
LAC's effect is not lymphocyte type(T or B cells)-specific nor stimulant-
specific. Rather) it likely affects certain downstream events governing a
more general processes) in lymphocyte activation and proliferation.
LAC Causes Apoptosis in Activated but not Resting T Cells
In one embodiment of the present invention a compound
is provided that induces activated and leukemic T cells to undergo
apoptosis.
Since LAC has been reported to induce apoptosis in
U937 cells (Chen et al., 1996, J. Immunoi. .1:4297), it is crucial to
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examine whether the LAC-induced inhibition of cell proliferation is due cell
death) be it apoptosis or necrosis.
The viability of T cells and Jurkat cells after
LAC-treatment was first evaluated with trypan blue exclusion. Resting
T cells (T cells in medium) or PHA-stimulated T cells were cultured with
NM LAC (LAC added at the beginning of the culture). As shown in
Figure 2A, after 16h culture, the viability of the cells only had minor
decreases (< 12%) in LAC-treated cells compared with those without LAC
{97% vs 92% for cells in medium, and 94% vs 83% for cells with PHA).
10 After a prolonged culture for 64h, the decreases were more prominent
although were still less than 27%{97% vs 79% for cells in medium) and
90% vs 63% for cells with PHA).
There was a tendency that the activated T cells were
more susceptible to LAC than the resting T cells. This became more
evident when LAC was added to T cells 40h after the PHA activation
(Figure 2B). The viability of the activated T cells dropped from 94% to
46% after additional 24h culture, although 9h culture did not change the
viability significantly according to trypan blue exclusion. On the other
hand) the viability of the resting T cells in medium had only a small
decrease (from 98% of the control to 87% of the LAC-treated) after 24h
of LAC presence.
Why did LJ~C added at Oh along with PHA cause less
cell death compared with t.AC added at 40h post PHA stimulation (Figure
ZA vs 2B)? It will be demonstrated that LAC is rapidly degraded in the
cell culture. After 24h in culture medium, LAC lost its activity, and at 40h
when the T cells were fully activated and become more susceptible, there
was no biologically active LAC in the culture. This could explain the
observed difference in terms of viability between the Oh and 40h addition
of LAC to the PHA-activated T cells.
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The effect of LAC on Jurkat cells was quite similar to
that on the activated T cells. Less than 8h exposure to 6 NM LAC did not
induce apparent Jurkat cell death, while about 60% of the Jurkat cells
were trypan blue positive after 24h culture with LAC (Fig. 2C).
5 We next employed DNA laddering to study the mode of
cell death caused by LAC, and the result of this experiment also reflected
the degree of cell death after different treatments. As shown in Fig. 2D,
resting T cells treated with 10 NM LAC for 24h had no apparent DNA
breakdown (lanes 1 and 2). This correlated to the good cell viability as
10 shown in Fig. 2B. On the other hand, clear DNA ladders could be
observed from activated T cells (40h post PHA-stimulation) treated with
LAC for additional 9h (lanes 3 and 4). After 24h of LAC treatment) the
ladders became less discrete, and this probably reflected further DNA
breakdown. For Jurkat cells, DNA fragmentation could be detected as
15 early as 6h after the LAC treatment, and after 16h, the fragmentation
became more prominent (Figure 2E).
Electron microscopy was also employed to examine the
mode of cell death induced by LAC. The resting T cells (cells cultured in
medium, figure 3A)) activated T cells (40h after PHA activation) Figure
20 3C), and Jurkat cells (Figure 3E) were all healthy looking. Occasional
condensed nuclei were observed in medium cultured T cells (Figure 3A)
and this is not unusual. The resting T cells treated with LAC (10 pM)for
24h were still healthy (Figure 3B). However, nuclear condensation, which
is a hallmark of apoptosis, were frequently observed in activated T cells
25 and Jurkat cells after they were exposed to LAC (10 NM and 6 NM,
respectively) for 24h (Figures 3D and F).
Following conclusions are drawn from the results of this
section. 1 ) Resting T cells or T cells in their early activation phase (less
that 24h after PHA-stimulation) are not sensitive to LAC in terms of cell
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viability. Consequently) there are still a signifcant percentage of live cells
after 64h culture should LAC be added once at the beginning. 2) less
than 8-9h of LAC treatment does not affect significantly viability of
activated T cells (40h post PHA activation) or Jurkat cells, according to
trypan blue exclusion. 3) Prolonged treatment (24h) of the activated
T cells or Jurkat cells with LAC causes cell death in the form of apoptosis,
although signs of apoptosis could be detected as early as 9h in T cells
and 6h in Jurkat cells after the LAC treatment.
The data in this section further infer following notions. 1 )
LAC's differential effect on the viability of resting versus cycling cells
suggests that it is not simply nonspecific cytotoxicity, but relates to the
status of the cell cycle. 2) The cell death without doubt contributes to but
cannot solely account for the observed inhibition of proliferation by LAC,
since there are still significant percentage ( about 60%) of live cells at the
end of the culture according to trypan blue exclusion. Moreover, we will
elaborate later that the cell death is a consequence of blockage of cell
cycle progress. 3) Admittedly the trypan blue negative cells includes some
early apoptotic cells, as evidenced by the fact that DNA laddering could
be detected in a largely trypan blue negative population. However, it does
not necessarily mean that the whole population is dead. We will later
demonstrate that most Jurkat cells treated with LAC for 6h to 8h could still
progress normally in cell cycle, in spite that a certain degree of apoptosis
could be detected in these cells. 4) LAC could be used to study the role
of proteasomes in lymphocyte activation and proliferation, as long as the
compound is applied only once in the beginning of activation of the
resting T cells and the experimentation is carried out in 24h-40h, or LAC
is present for less than 8h in the case of cycling cells) since such
treatments do not drastically affect the viability of the cells.
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A specific embodiment of this invention is the ability of
I.AC to induce apoptosis mostly in activated and proliferating cells and
not in normal resting cells. This has value in eliminating cancerous cells
and antigen-specific T cells. The elimination of the latter will create a
specific immune tolerance to alloantigens in transplantation, and to
selfantigens in autoimmune diseases.
The Effect of LAC is Rapid and Reversible
We next investigated how fast and how long LAC could
exert its effects on the lymphocytes, since this information is necessary
to assess the requirement of the proteasome activity for events related
to cell activation and proliferation. PBMC were pretreated with LAC
(10 NM) or medium for a period as indicated in Fig. 4A. The cells were
then washed and recultured in the presence of PHA. The thymidine
uptake was measured 3 days later. It was clearly demonstrated that 3h
preincubation with LAC was sufficient to cause significant inhibition on the
subsequent mitogen-stimulated proliferation in T cells) although 16h
preincubation with I.AC was more effective. This result indicates that LAC
can enter the cells rapidly within 3h.
We used Jurkat cells that have high constitutive
proteasome activity to evaluate the duration of LAC's effect once the drug
entered the cells. Jurkat cells were treated with LAC (6 NM) for 3h, which
was sufficiently long for the compound to enter the cells as shown above.
The cells were then thoroughly washed and recuitured) and they were
harvested at Oh, 5h and 21 h after the wash, and the proteasome activity
in the cell lysates was measured using a chromogenic chymotrypsin
substrate. We have previously established that the proteinase activity
measured by this assay was predominantly (more than 90%) derived from
the proteasome (Wang et al., 1997, Eur. J. Immunol., supra). As shown
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in Fig. 4B, the proteasome activity in Jurkat cells was almost completely
inhibited by 3h preincubation with LAC at 6 NM. Five hours after the LAC
was washed out) the proteasome activity in the cells was still significantly
inhibited but the inhibition was reduced compared with that at Oh. By 21 h,
the proteasome activity returned to a near-normal level. It is to be noted
that the short 3h treatment with LAC did not affect the viability of the
Jurkat cells, and this is also reflected by the normal proteasome activity
of the treated cells at 21 h. The result shows that LAC is not stable and
loses its activity within 21 h in the cells.
We also investigated whether LAC was stable in the
culture supernatant. LAC (6 NM) was added to Jurkat cells culture for 4h,
6h, 16h or 24h. The conditioned medium was harvested and used to treat
fresh Jurkat cells for 3h) and then the proteasome activity in the lysates
of the fresh Jurkat cells was assayed. As shown in Fig. 4C, 4h to 24h
conditioned media without LAC did not affect the proteasome activity of
the fresh Jurkat cells. The media conditioned with LAC up to 6h could still
actively inhibit the enrymatic activity, but after 16h) the LAC-conditioned
media lost their inhibitory effect. The loss of L.AC activity in the 16h and
24h conditioned medium is unlikely due to trapping of lAC by
proteasomes released by dead Jurkat cells, because LAC could rapidly
enter the live cells and the equilibrium of the LAC concentration between
both sides of the cytoplasmic membrane should be established very fast.
Thus, the proteasomes whether released or not should not make a
difference in terms of trapping LAC. Besides, we have also noticed that
LJ~C kept in cell free culture medium at 4°C would lose its activity
within
24h (data not shown). These results indicate that LAC is not only unstable
within the cells, but is also unstable in the supernatant.
LAC's capability to enter the cells to inhibit the
proteasome activity rapidly (less than 3h)) and its short active duration
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within the cell and in the culture media (about 16h) makes the compound
a very useful reagent to evaluate the requirement of the proteasome
activity in various events during cell activation and proliferation, since we
could pinpoint the period when the proteasome activity is critical.
It is an embodiment of this invention, the use of LAC can
be regulated in a time course sequence to be most effective at the period
when proteasome activity is critical to maximise the effect of LAC on cells.
Proteasome Activity is Required for IL-2Ra Upregulation
In the four systems of T and B cell activation and
proliferation studied in the first section, the growth promoting activity of
IL-2 is indirectly (for stimulation by PHA, anti-CD3, and anti-CD28 plus
ionomycin), or directly (for SAC plus IL-2) involved. We then investigated
the role of proteasome in IL-2Ra expression and IL-2 production. As
shown in Fig. 5, CD25 was upregulated in CD3+ T cells 40h after
stimulation with PHA. When LAC (10 NM) was added in the beginning of
the culture, the upregulation was significantly inhibited. On the other
hand) IL-2 production by PBMC 2 to 4 days after PHA stimulation in the
absence or presence of lAC (10 NM, added at the beginning of the
culture) was also examined, but no consistent difference was found (data
not shown). Under the experimental condition used, the viability of the
LAC-treated cell was reasonable (>80% at 40h) as described in the
previous section as LAC was added only once initially. Moreover, no
consistent change of IL-2 production in LAC-treated cells was a functional
indication that the cell viability was reasonable and is not of a concern in
interpreting the data. The results from this section indicate that IL-2Ra
upregulation but not IL-2 production is proteasome-dependent, and the
suppressed IL-2Ra expression likely contributes to LAC's inhibitory effect
on T cells activation and proliferation.
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The Proteasome Activity is Critically Required Between Go and G,IS
Boundary in T Cells
Like normal T cells, the proliferation of Jurkat cells was
also potently inhibited by LAC (data not shown). We used synchronized
5 Jurkat cells to identify the LAC-sensitive phases) of the cell cycle. Jurkat
cells were first synchronized at the G2/M boundary by nocodazole
(Fig. 6A). The cells were released from the blockage by washing out
nocodazole. In the control sample, more than half the cells traversed
through the M phase and arrived at the G: phase within 4h. In the test
10 sample, LAC (6 NM) was added to the culture 3h before the release) so
the compound could have enough time to enter the cells. LAC was also
added to the culture after the release. However, the Jurkat treated with
LAC traversed through the M phase to the G, phase at a similar pace as
the control cells. Since the total duration of the assay was around 7h (3h
15 preincubation plus 4h after the release), LAC was certainly active during
this period. The fact that most of synchronized Jurkat cells could traverse
through G2IM to G1 in the presence of LAC for 7h again suggests that
the viability of the cells thus treated is not a matter of concern. This
result
shows that the G2 to G, progression is not proteasome-dependent.
2p We next studied requirement of the proteasome activity
for the progression from the G,/S boundary to the G~/M phase. The Jurkat
cells were synchronized at the G,/S boundary by HU blockage. The cells
were then released by washing out HU. Within 9-12h, the majority of the
cells progressed to the S and G2IM phase (Fig. 6B). When LAC was
25 added to the culture immediately after the release, it slowed but did not
block the cell cycle progression from the G,IS boundary to the GRIM
phase, as evidenced by the histograms at 6h and 9h post the release. It
is to be noted that although the percentage of cells in the SIG2/M phase
in the LAC-treated sample was similar to that of controls (the inset table
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of Figure 6B), the peak of fluorescence was lagged behind (histogram
array). Beyond 9h, the cells gradually lost their synchronization, the
viability of the cells started to decline and LAC gradually lost its activity)
so the data became difficult to interpret. The result from this part suggests
that the proteasome activity is required for optimal progression from the
G,/S boundary to the GRIM phase, because the progression could still
proceed albeit at a slower pace when the proteasome activity is inhibited.
The result also implies that the absolutely proteasome-dependent window
during the cell cycle, as evidenced by the near-total inhibition of S phase
entry in LAC-treated mitogen-stimulated lymphocytes according to the
proliferation data) must be in the G1 phase before the target point of HU,
which inhibits ribonucleotide reductase in the G./S boundary (Brown et
al., 1996, Cell $x:517).
The cycling Jurkat cells are obviously not the best model
to study the events in the G, phase since the G2IM synchronization
become desynchronized by the time the cells re-enter the S phase, and
there is no appropriate method to synchronize the Jurkat cells at the early
G, phase. We therefore decided to use mitogen-stimulated normal T cells
to study the role of the proteasome in the G, phase.
T cells from PBMC were at Go when isolated. After 16h
stimulation with PHA, they remained before the S phase (Fig. 6C). At 40h,
about 20% of the cells were in the S and G~/M phases. The peak of
3H-thymidine uptake according to a 16h pulse was between 48h and 64h
(data not shown), although at 64h, the cells in the S and G~/M phases
were still about 20% (Fig. 6C). The lack of an increase in percentage of
cells in the S and GZ/M phases at 64h compared with that at 40h was
likely due to the exit of the cells from the S and G2/M phase. It is to be
noted that the cycling T cells in this system never reaches 100%,
because about 15% of the cells were non T cells, and an additional 20%
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were non responsive T cells. Taken the cell proliferation and cell cycle
analysis together, the G,/S boundary of the cycling T cells should be
between about 35h and 48h after the PHA stimulation. The boundary was
broad because the synchronization was not ideal.
In this model, the role of the proteasome in the S phase
entry was examined. As shown in Fig. 6C, LAC added once at 16h could
totally block the S phase entry when examined at 40h. We have noticed
that when the cell viability was evaluated at 40h, there was an increase
of cell death comparing the 16h addition of LAC with the Oh addition
(about 25% vs about 17%, data not shown). The increased cell death was
also reflected in the cells with < 2N DNA in the 40h histogram. However,
such a viability was still reasonable and would not invalidate our
conclusion. According to 3H-thymidine uptake, LAC was strongly inhibitory
even added as late as 40h (Fig. 6D). However, no difference on the
percentage of the population in the S and Gz/M phase was observed at
64h whether or not LAC was added at 40h according to flow cytometry
(Fig. 6C). The discrepancy could~be explained by the fact that the 20%
cells were already in the S and G~/M phases at 40h when LAC was
added. LAC prevented additional cells from entering into the S phase,
therefore the lack 3H-thymidine uptake. At the same time) the drug slowed
the cell cycle progression from the G,/S boundary to the GZ/M phase,
hence the lingering population in the S and GZ/M phases according to
flow cytometry.
It is worth mentioning the inhibition of proliferation by
LAC was a combinatory effect of cell cycle progress and cell death, the
latter possible being the consequence of the former. The later the
compound was added when more T cells are activated, a larger
proportion of the effect should be attributed to cell death caused by LAC.
The extensive cell death for the sample treated with LAC at 40h was not
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fully reflected in the flow cytometry (Fig. 6C) as cells with less than 2N
DNA. This was due to that the histogram was gated on a region of largely
viable cells.
The results from this section indicate that the
proteasome activity is not required from the G2/M to the G, phase. It
optimizes the progression from the G,IS boundary (as defined by the
hydroxyurea target point) to the GRIM phases, and it is absolutely required
for the progression from the Go to the S phase.
In a specific embodiment of this invention LAC is used
to reverse ongoing graft rejection during a rejection episode. Most
immunosuppressive drugs do not have the capability to reverse rejection
once it begun. The use of LAC overcomes the prior art.
The Proteasome Activity is Essential for CDK2 but not for CDK4
Function
Cyclin-dependent kinases (CDK) are critical for cell
proliferation. CDK4 is essential in the early to mid-G, phase to facilitate
the S phase entry (Tam et al., 1994, Oncogene 9_:2663; Lukas et al.,
1995, Oncogene 10:2125) and CDK2 is critical in the fate G, as well as
throughout the S phase for the cell cycle progression (Van der Heuvel et
al., 1993, Science 2:2050). We therefore examined the role of the
proteasome in CDK4 and CDK2 activities in mitogen-stimulated T cells.
In all the models used in this section, LAC was added only once at the
beginning of the culture. Consequently, the viability of the LAC-treated
cells was good for the first 16h and was reasonable at 40h, and was not
a factor that might interfere with the interpretation of the results.
As shown in Fig. 7A, the resting T cells had some CDK4
activity, and the activity reached a plateau within 16h of the activation.
This was in agreement with the critical role of CDK4 in the early G phase.
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Inhibition of the proteasome activity by LAC from 0-16h (LAC added once
at Oh) did not affect the CDK4 activity when examined at 16h and 40h
(Fig. 7A). This indicates that the induction and maintenance of CDK4
activity during the G1 phase is not proteasome-dependent.
In contrast to CDK4, the CDK2 activity was augmented
at 16h but the augmentation was more prominent at a later stage close
to 40h after the mitogen-stimulation (Fig. 7B), and this reflected its
essential role starting from the late G, phase and extending to the early
S phase. The presence of LAC from Oh to 16h (LAC added once at Oh)
significantly inhibited CDK2 activity at 16h and more so at 40h. Therefore,
the proteasome activity during the early activation stage (Oh-16h) is
essential for the activation of the kinase at the G1 phase and early S
phase. The unchanged CDK4 activity in the LAC-treated cells at 40h
served as an internal control for the repressed CDK2 activity and
indicating the latter was not due to the viability problem.
Since at the late G, phase Cyclin E is the predominant
partner of CDK2 (Sherr, 1993, Cell 7:1059), we next examined the
Cyclin E-associated CDK activity. As shown in Fig. 7C, in spite that the
Cyclin E protein was increased after the lr4C treatment (LAC added once
at Oh), the Cyclin E-associated kinase activity was almost completely
inhibited by LAC. These results indicate that the CDK2 activity, and most
likely the Cyclin E-associated CDK2 activity in the late G, phase is
proteasome-dependent. The results also suggest that the inhibition of the
CDK2 activity is probably an important mechanism accountable for the
LAC's effect in blocking the S phase entry.
It is an embodiment of this invention to have elucidated
a downstream target for proteasome activity. That is CDK2, more
specifically Cyclin E-associated CDK2 activity. It is also provided that with
this knowledge, inhibitors of CDK2 can be used alone or in combination
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with proteasome inhibitors. It is further provided that the aforementioned
compositions are of a pharmaceutically effective amount to induce
apoptosis or for any other cellular or physiological effect. Since CDK4
activity is important in Go to G,, progression and it is not affected by
5 proteasome activity, it is conceivable that inhibitors for CDK4 can be used
in combination with proteasome inhibitors of a pharamceutically effective
amount to achieve additive effect in blocking cell proliferation and in any
other relevant cell function.
Inhibitors in this application are defined as any element
10 capable of silencing the activity of a protein at the level of gene
transcription, translation, or post-translational modification of the protein
as well as elements capable of interfering with the protein. These may
include but are not limited to antibody or other ligands) anti-sense or
antagonist molecules.
Degradation of Cyclin E but not Cyclin A is Proteasome-Dependent
It is a specific embodiment of this invention that
contacting LAC with CDK2 is inhibitory to CDK2 activity, more particularity
it is the inhibitory effect of LAC on Cyclin E. The inhibitory effect of LAC
is the disruption of cell cycling.
Oscillation of cyclins during the cell cycle is a mode of
regulation for the CDK activities. Since the CDK2 activity is
proteasome-dependent, and CDK2 associates predominantly with
Cyclin E and cyclin A at the G,/S boundary and during the S phase
respectively (Pagano et al., 1992, EMBO J. ,1:961; Hall et al., 1995,
Oncogene 11:1581), we studied the role of the proteasome in
degradation of these two cyclins. As shown in Fig. 8A, the Cyclin E level
was apparently increased around 40h after PHA stimulation of the T cells,
which were then at the G,/S boundary. If the activated cells were treated
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with HU, the Cyclin E level was significantly enhanced comparing with
those treated with PHA alone (Fig. 8A). This reflects a better
synchronization at the G,/S boundary by HU, and was consistent with our
knowledge that the Cyclin E level peaked at the boundary. After the
boundary, the Cyclin E level started to decline, and the decline was
prevented by LAC (Fig. 8A). This clearly demonstrates that the
degradation of Cyclin E is a proteasome-dependent process, although
whether the increased Cyclin E level contributes to LAC's effect on the
cell cycle is a matter of debate.
For cyclin A, the level was increased around the late G,
phase after the mitogen stimulation as shown in Fig. 8B. The blockage of
the cycle at the G,IS boundary with hydroxyurea did not further increase
the cyclin A level. However, when the cycle passed the boundary and
entered the S phase, the cyclin A level was significantly augmented
(Fig. 8B), consistent with the notion that cyclin A is mainly an S phase
cyclin. Unlike that of Cyclin E, the level of cyclin A did not decline during
the S phase and LAC did not affect the level during this period. This
suggests that the proteasome is not involved in cyclin A degradation, at
least in the G, and S phases) and that LAC's effect on inhibiting cell
proliferation is unlikely mediated via the cyclin A levels. The G,/S phase
synchronized T cells represented activated cells, and prolonged exposure
to LAC would cause significant cell death. However, 6h treatment of LAC
did not apparently affect the cell viability, while the blockage of Cyclin E
degradation but not cyclin A degradation was obvious at that time point.
Moreover, cyclin A could be considered as an internal control for Cyclin E
indicating that the LAC-induced cell death should not affect the
conclusion in this section.
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The Role of Proteasome in Regulating Levels of CDK Inhibitors
p2T"''P' and p21~'P,
In a specific embodiment, LAC is capable of suppressing
the up regulaion of the CDK inhibitor p21~"P' and in blocking the
degradation of the CDK inhibitor p27"'p'
In addition to the cyclin levels, the CDK activities are
also controlled by several low molecular weight inhibitors. We have
examined in this study the effect of the proteasome on the CDK inhibitors
p27"'P' (Hall et al., 1995, supra) and p21 ~'P' (el-Deiry et al., 1993, Cell
75:817). As shown in Fig. 9A, the resting T cells had a high level of p27"~p,
and the level decreased gradually when the cells moved to the G,/S
boundary 40h after the mitogen-stimulation. This is in agreement with
previous reports (Hengst et al., 1996, Science X71:1861; Nourse et al.)
1994, Nature 7~?:570). The presence of t~4C (added once at Oh)
significantly blocked the decrease when assayed at 16h, showing that the
degradation is a proteasome-dependent process. The blockage was less
obvious when assayed at 40h, probably because the gradual loss of LAC
activity during the 40h culture. The result suggests that the blocking of
p27"'P' degradation is a contributing mechanism contributing for the
inhibitory effect of LAC on the CDK2 activity. Unlike p27"'p', p21 ~'P' had
a low level of expression in resting T cells. The level was rapidly
augmented after 16h PHA activation) and the high level was maintained
at the G,/S boundary at 40h (Fig. 9B). Such an induction suggests that
p21~'P' might be required in the G phase for roles other than a CDK
inhibitor. Interestingly, l~C strongly suppressed the upregulation of
p21 ~'P' in the G, phase, indicating that the expression of p21 ~'P' is
proteasome-dependent, and suggesting that the proteasome might
facilitate cell proliferation via its role in p21 ~'p' upregulation during the
G,
phase. In this experiment, LAC was only added once at the beginning of
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the culture, and the viability of the treated cells at 16h was good (83~)
and should not be a concern in drawing the conclusion.
Disruption of Cell-Cell Interaction
Cell-cell interaction is essential in antigen presentation
and in T cell's help to T and B cells. The adhesion molecules are
necessary to establish the cell-cell interaction. Blocking the adhesion
molecules ICAM-1 and LFA-1 is known to inhibit immune responses and
to suppress graft rejection. Our data clearly shows that inhibition of the
proteasome activity will effectively interfere with the cell-cell interaction
during lymphocyte activation in both human (Fig. 10) and mouse (Fig. 11 )
systems, and the upregulation of an adhesion molecule ICAM-1 is
repressed by the proteasome inhibitor lactacystin (Fig. 12). Therefore,
inhibition of the proteasome activity will be a useful way to control
undesirable immune responses during graft rejection, autoimmune
diseases, and inflammation.
Proteasome Activity is Required for Nitric Oxide Production
Nitric oxide (NO) produced by macrophages is involved
in inflammation and septic shock. We have shown that inhibition of the
proteasome activity could effectively repress the endotoxin LPS-induced
NO production (Fig. 13). The usefulness of proteasome inhibitors in
inflammation and in septic shock is implicated. Fig. 14 demonstrates that
proteasome activity is required for NO synthase expression. The addition
of LAC decreases the expression of mRNA for NO synthase.
The Effect of Proteasome on Mitochondrial 1=unction
Mitochondria are pivotal organelles in the cells and their
primary function is to produce ATP via the Krebs cycle coupled to the
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oxidative phosphorylation of the respiratory chain. An intact function of
mitochondria is also required for proper cell viability. Damage of the
mitochondria) membrane potential or release of cytochrome C or other
apoptogenic factors from the mitochondria to the cytosol will induce cell
death via apoptosis.
In our study, we have found that the electron transport
in mitochondria of Jurkat T lymphocytes is dependent on the intact activity
of the proteasome. A proteasome-specific inhibitor lactacystin (LAC)
could rapidly (within 4h) reduce the electron transport at the complex IV
of the respiratory chain, and the effect could be reversed by adding back
exogenous cytochrome C (cytoC).
In Fig. 15, the respiration of Jurkat cells treated with LAC
for 4h (curve 4) but not for 2h (curve 3) could not be resumed by adding
succinate after Complex I blockage, and CCCP failed further to stimulate
the respiration as it could in control Jurkat cells and in rat mitochondria)
preparation (curves 5 to 6, respectively). Adding rat kidney mitochondria
to the blocked reaction results in normal respiration (curve 4), showing the
reagents and the oxygen electrode are functional. The results indicate
that LAC compromises the electron transport after Complex I.
In Fig. 16, Jurkat cells treated with LAC for 2h (curve 3)
had similar OZ consumption after Complex 111, like that of untreated Jurkat
cells (curve 2) and rat kidney mitochondria (curve 1). After 4h LAC
treatment, the 02 consumption of the Jurkat cells could not be resumed
by ascorbate and TMPD to a level similarly high as that of untreated
Jurkat and rat mitochondria, and the decoupling reagent CCCP had no
effect in the treated cells (curve 4). Adding back rat kidney mitochondria
into the assay could resume the 02 consumption, showing a functional
assay system. Curves 5 to 6 are untreated Jurkat cells and rat kidney
mitochondria, respectively, showing normal function of Complex IV. This
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result shows that the LAC treatment caused compromised function in the
electron transport at Complex IV.
in Fig. 17, Jurkat cells treated with LAC (curve 3) have
reduced augmentation of 02 consumption after the addition of ascorbate
5 and TMPD, compared with untreated Jurkat cells (curve 2) and rat kidney
mitochondria (curve 1 ). FCCP could not further stimulate the respiration,
as it could in normal Jurkat cells and rat kidney mitochondria. When
exogenous cytochrome c (CytoC) was added to the LAC-treated cells) the
respiration resumed to a rate similar to that of untreated Jurkat cells and
10 mitochondria. CytoC had no additive effect in stimulating respiration in
normal Jurkat cells and rat mitochondria (curves 2 and 3, respectively).
The implication of aforementioned findings is as follows:
In hyperthyroidism, the mitochondria) activiy is
overactive due to the effect of the thyroid hormone. This results in many
15 symptoms such as excessive body heat, and imbalance of energy uptake
and consumption. The proteasome inhibitors could reduce the rate of
mitochondria) respiration and have therapeutic effect to this disease.
In fast-growing cells such as cancer cells or activated
lymphocytes, the mitochndria are more active than in normal cells in order
20 to meet the energy requirement of a high metabolic activity of these cells.
Consequently, inhibition of the mitochondria) respiration could curb the
proliferation of the cancer cells or activated lymphocytes while have less
detrimental effects to normal resting cells. In addition, apoptosis could be
induced in the cycling cells but not resting cells. Thus, inhibition of the
25 proteasome activity will have therapeutic effect in cancer and in diseases
involving lymphocyte activation and proliferation, such as seen in graft
rejection and autoimmune diseases.
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Rapid Assays for A High Through-Put Screening Procedure to
Identify Additional Proteasome Inhibitors
In our study) we have shown that about 70-80% of the
chymotrypsin-like activity in the lymphocyte lysates is derived from the
proteasome (Fig. 20). In a positive control, LAC at 10 NM could inhibit
90% of the 20S proteasome activity which was in a range similar to that
of the cell lystates. Increasing the concentration of LAC to 20 NM did not
further increase the inhibitory effect, suggesting that the LAC
concentration used was already saturating. The remaining 10% activity
might be derived from non-proteasome proteinases in the 20S
proteasome preparation. When 10 NM LAC was added to the 70-h cell
lysate, it inhibited 73.4%, 76.7% and 86.7% of total chymotrypsin-like
activity in the lysates from medium-, PHA- and PHA plus RAPA-treated
PBMC, respectively, and those percentages represented the portion of
enzymatic activity from the proteasome.
The implication of this finding is that mammalian cell
lysates without other purification could be used as a convenient source
of proteasomes. Tagged substrates specific for the known proteasome
activities, such trypsin-like, chymotrypsin-like, and PGPN activities can be
2o used as displaying parameters. Known compounds could be added into
this enzymelsubstrate system, and the compounds) that inhibits) one or
several aforementioned enzyme activities of the lysate above a certain
threshold (for example 40%) will be identified as proteasome inhibitors.
These assays could be modified to use purified or partially purified 20S
or 26S proteasome as a source of the proteasome enzymes. Since such
assays are simple (only three components) and rapid (only several
minutes of reaction period), they could be adapted for high through-put
screenings) and included in a kit format.
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The Effect of Immunosupressive Drugs on Proteasome Function
Rapamycin (RAPA) is a potent immunosuppressive
drug, and certain of its direct or indirect targets might be of vital
importance to the regulation of an immune response. Seven
RAPA-sensitive genes are known and one of them encoded a protein with
high homology to the a subunit of a proteasome activator (PA28a). This
gene was later found to code for the ~ subunit of the proteasome activator
(PA28~). Activated T and B cells had upregulated PA28~i expression at
the mRNA level. Such upregulation could be suppressed by RAPA,
FK506, and cyclosporin A (CsA). RAPA and FK506 also repressed the
upregulated PA28a messages in PHA-stimulated T cells. At the protein
level, RAPA inhibited PA28a and PA28~ in the activated T cells
according to immunoblotting and confocal microscopy. Probably as a
consequence, there was a fourfold increase of proteasome activities in
the PBMC lysate after the PHA activation. RAPA could inhibit the
enhanced part of the proteasome activity. Considering the critical role
played by the proteasome in degrading regulatory proteins, a proteasome
activator is a relevant and important downstream target of rapamycin, and
that the immune response could be modulated through the activity of the
proteasome.
A lot of efforts have been made to identify direct targets
of RAPA. It is now known that RAPA complexes with a 12KD
FK506-binding protein {FKBP12) (Harding et al., 1989, Nature X1_:371;
Siekierka et al., 1989, Nature X41:755). The RAPA-FKBP12 complex then
binds to cytoplasmic proteins termed TOR1 and TOR2 (target of
rapamycin) in yeast (Kunz et al., 1993) Cell x:585; Helliweil et al., 1994,
Mol. Biol. Cell. 5_:105), and FRAP and RAFT1 in mammalian cells (Brown
et al., 1994, Nature ~:75fi; 11 ). These target proteins have high degree
of homology in their primary sequences, and their C-terminal sequences
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share certain homology with catalytic domains of both PI-3 kinase and
PI-4 kinase.
The mRNA expression of most genes so far studied,
whether they are constitutively expressed or induced after stimulation, are
not sensitive to RAPA (Tocci et al., 1989, J. Immunol. x:718; Shan et
al., 1994, Int. immunol. x.:739). It follows that the genes that are sensitive
to RAPA at the mRNA level have a good probability of being secondary
targets of RAPA and being pivotal in controlling the immune response.
Expression of PA28ø at mRNA and protein levels was found to be
sensitive to RAPA, so was that of the PA28a subunit which shares a high
degree homology with PA28ø. It was found that proteasome activity was
repressed by the drug.
In HeLa cells, PA28ø expression was dramatically
upregulated at the mRNA level by IFNy treatment after 24h. This was
similar to the regulation of PA28a (Realini et al., 1994, J. Biol. Chem.
2.9:20727). When human tonsillar T cells were stimulated by PHA, the
PA28ø expression was augmented after 20h, and the augmentation could
be suppressed by lOnM RAPA as expected (Fig. 18A). In addition, the
expression was also sensitive to CsA (1 NM) and FK506 (10nM). In
tonsillar B cells, SAC and IL-2 upregulated the PA28ø mRNA expression,
and RAPA was inhibitory (Fig. 18B). Similarly, the mRNA expression of
PA28a, which has a high degree of homology with PA28ø, was
upregulated in PHA-activated T cells, and the upregulation was repressed
by FK506 and RAPA (Fig. 18C).
Expression of PA28ø and PA28a at the protein level
was also examined. The result of immunoblotting demonstrated that the
activated T cells had increased PA28ø compared with resting T cells, and
the increase was inhibited in the presence of RAPA (Fig. 19A). Since the
anti-PA28a antiserum did not seem to recognize the denatured proteins,
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we used confocal immunofluorescent microscopy to examine the PA28a
protein as well as the PA28~ protein in the T cells. The experiment was
carried out in an one-way blind fashion, the microscopy operator without
being informed of the treatment of the cells. As shown in Fig. 19B, RAPA
plus PHA-treated T cells had significantly lower levels of both PA28a and
PA28~i proteins compared with T cells treated with PHA atone. We have
noticed that although the difference between the PHA-activated T cells in
the absence and presence of RAPA was highly significant (p<0.0001 ), the
difference of the numeric values of the mean fluorescence intensity
between the two types of cells, especially in the case of PA28~i, was
rather small. However, there was a high standard deviation in the PHA-
treated samples. A closer inspection revealed that about 40% of the cells
treated with PHA alone had elevated PA28(i and PA28a signals while the
rest had basal level expression. This caused the high standard deviation.
Considering that there were 20% non T cells in the T cell preparation) and
that PHA does not activate all the T cells in the culture simultaneously,
those 40% cells with the high signals probably represented the truly
activated T cells. Therefore, the actual difference between the activated
and drug-repressed cells could be much bigger than the data presented
in the histogram.
Taken together, our data indicates that RAPA inhibits
the expression of PA28a and PA28~i at both mRNA and protein levels.
The inhibition of the PA28 mRNAs is a likely cause for the observed
decrease of the corresponding proteins. However, we could not exclude
the possibility that RAPA might also act directly at the translation level for
PA28a and PA28~.
In as much as PHA could upregulate and RAPA could
repress expression of the proteasome activator PA28~ and PA28a in the
T cells) it is logical to examine changes of proteasome activity in these
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cells. PBMC lysates were assayed for their proteinase activity at pH 8.2
which favors the proteasome activity, using a chymotrypsin substrate as
a representative parameter. Forty and seventy hours after stimulation by
a T cell mitogen PHA, the chymotrypsin-like activity in the PBMC
5 increased 2.1 fold and 3.8 fold, respectively (Fig. 20A). RAPA at 10nM
repressed 23.1 % and 41.1 % the activity in the PBMC, respectively, at
these time points.
We then tried to determine the part of enzyme activity
in the lysates conferred by the proteasome. In a positive control, LAC at
10 10 NM could inhibit 90% of the 20S proteasome activity which was in the
range similar to that of the cell lysates (Fig. 20B). Increasing the
concentration of LAC to 20 NM did not further increase the inhibitory
effect, suggesting that the LAC concentration used was already
saturating. The remaining 10% activity might be derived from
15 non-proteasome proteinases in the 20S proteasome preparation. When
10 NM LAC was added to the 70h cell lysate, it inhibited 73.4%) 76.7%
and 86.7% of total chymotrypsin-like activity in the lysates from medium-)
PHA- and PHA plus RAPA-treated PBMC, respectively, and those
percentages represented the portion of enzymatic activity from the
20 proteasome (Fig. 20B). The net proteasome activity increased by 4 fold
from 42.6 x 103 units120 Ng protein in unstimulated cells to 170.3 x 10 3
unitsl20 Ng protein in the PHA-activated cells. In RAPA-treated cells, the
activity decreased to 113.2 x 103 units/20 Ng protein. This equated to
33.6% inhibition of the total activity, or 44.7% of the augmented
25 proteasome activity in the PHA-treated PBMC. It is therefore
demonstrated that RAPA could inhibit the enhanced proteasome activity
during T cell activation.
It is an embodiment of this invention to have identified
known immunosupressive drugs including rapamycin, FK506 and
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cyclosporin A as inhibitors of enhanced proteasome activity. It is therefore
a specific embodiment of this invention for providing these
immunosupressive drugs of a pharmaceutically effective amount and in
combination with specific proteasome inhibitors of a pharmaceutically
effective amount, as an example but not limited to t~C or its analogues
to achieve an additive effect in blocking cell proliferation and any other
relevant cell function. Such combinations as described can be used but
are not limited to the treatment of cancer, graft rejection and autoimmune
diseases.
Elimination of alloantigen-specific response
The results of the functional assay shown in Figure 21
suggests, that there is clonal deletion of BALBIc-specific T cells when
proteasome activity of alloantigen-activated T cells are inhibited for a brief
period. The consequences of this finding suggests that proteasome
inhibitors can be administered when specific T cells are activated, thereby
potentially eliminating the activity of specifically activated T cells while
leaving non-activated T cells intact. It is therefore an embodiment of this
invention to use proteasome inhibitors, particularly lactacystin in
transplantation and autoimmune diseases where certain undesirable
activated T cells can be repressed or eliminated and the rest of the T cell
population is generally unaffected by such inhibitors.
The effect of caspase inhibitor zVAD.fmk. on LAC-induced DNA
fragmentation
The effect of lactacystin as an apoptotic agent in ,lurkat
cells is shown in Figure 22, by the typical apoptotic sign of DNA
laddering. Addition of the broad spectrum caspase inhibitor zVAD.fms
demonstrated an inhibitory effect on DNA fragmentation that is
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concentration responsive. This result indicates that the lactacystin-
induced apoptosis in Jurkat cells is caspase-dependent.
The effect of lactacystin on a pro-apoptotic Bcl-2 family member, Bik
The results shown in Figure 23 panel A, show that Bik,
Bax, Bak, and Bad are predominantly located in the mitochondria)
fraction. Treatment with lactacystin does not appear to have altered the
amounts of Bax, Bak and Bad (Fig. 23 panels A and B). There is
however a demonstable increase in the amount of Bik in the lactacystin
treated Jurkat cells after 4 h, 5 h and 7 h (the first row of panels A and B))
when compared with untreated cells. The results shown in Fig. 23,
suggests that under normal circumstances, Bik is degraded rapidly by the
proteasome. Blocking of this degradation by a proteasome inhibitor,
allows the pro-apoptotic Bcl-2 member to accumulate. The accumulation
of Bik may possibly tip the balance between pro- and anti-apoptotic
factors favoring apoptosis.
The effect of overexpression of Bcl-xL, an anti-apoptotic Bcl-2 family
member
The human B cell line Namalwa stably transfected with
an anti-apoptotic Bcl-2 family member Bcl-xL, was shown to be more
resistant to the proteasome inhibitor lactacystin than the untransfected,
wild type Namalwa cells. The results shown in Figure 24 indicate that the
transfected cells have demonstrably less DNA fragmentation at the
different intervals and lactacystin concentrations tested. This suggests
that the overexpression of Bcl-xL protein has probably counteracted the
effect of the accumulation of the pro-apoptotic Bik. In this manner the
Namalwa cells are somewhat protected from undergoing apoptosis.
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In an additional experiment, Jurkat cells, wild type
Namalwa cells and Bcl-xL transfected Namalwa cells were treated with
staurosporine and lactacystin for 6 H. Proteins from the mitochondria)
fraction of these cells were analyzed by immunoblotting for the amount
of Bik, Bcl-xL, Bax, and Bak. The results summarized in Figure 25, show
that Bik accumulates in the Namalwa cells (panel B, lane 3) and Jurkat
cells (panel A lane 2) after a 6 hour lactacystin treatment. This
accumulation is due to the inhibition of proteasome activity and indicates
that the degradation of Bik via the proteasome is a general phenomenon.
The elevated amount of Bik, is likely a mechanism of lactacystin-induced
apoptosis in the Jurkat and Namalwa cells. The accumulation of Bik was
only observed in the lactacystin-treated but not in staurosporine treated
cells, eventhough staurosporine could equally induce apoptosis in these
cells. The expression of exogenous anti-apoptotic Bc1-2 member Bcl-xL
as expected, was not detected in Jurkat cells and wild type Namalwa cells
(panels A and B). The Bcl-xL overexpression was obvious in the
transfected Namalwa cells (panel C). Moreover, there was an
accumulation of Bcl-xL after lactacystin treatment, showing that under
normal circumstances the degradation of Bcl-xL, like Bic is also rapid and
depends on proteasome activity. These results suggest that the Bcl-xL-
transfected Namalwa cells have two mechanisms to protect them from
proteasome inhibitor-induced apoptosis. First the overexpression of the
anti-apoptotic Bcl-xL changes the balance between pro- and anti-
apoptotic factors and favors the anti-apoptotic factors. Second, after
treatment with lactacystin, there is an accumulation of Bcl-xL which
imparts additional weight to the anti-apoptotic factors.
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Thus, the balance between the pro- and anti-apoptotic
factors in cells is crucial in deciding the fate of these cells. Certain
apoptosis-related factors have a short half life and their degradation is via
the proteasome machinery. Therefore, modulating the proteasome
activity with proteasome inhibitors is a useful way to control the balance
between the pro- and anti-apoptotic factors. This control provides the
means to induce cells into apoptosis or continued survival.
Accordingly, it is an additional embodiment of this
invention to provide the means to balance between pro-apoptotic and
anti-apoptotic factors in a cell using proteasome inhibitors, particularly
lactacystin.
The present invention is illustrated in further detail by the
following non-limiting examples.
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EXAMPLE 1
Reaaen~,g
RPMI 1640) FCS, penicillin-streptomycin, and L-glutamine were
5 purchased from Life Technologies (Burlington, Ontario, Canada).
Lymphoprep was purchased from NYCOMED (Oslo, Norway). PHA,
hydroxyurea, nocodazole, and histone H1 were from Sigma (St. Louis,
MO). Staphylococcus aureus Cowan I (SAC) were obtained from
Calbiochem (La Jolla, CA)) and lactacystin from Dr. E.J. Corey (25).
10 Human rIL-2 was from La Roche (Nutley, NJ), and anti-CD3 mAb OKT3
was from ATCC (Rockville, MD). F1TC-conjugated anti-CD3 mAb(clone
SFCIRW2-8C8) and PE-conjugated anti-CD25 mAb (clone IHT44H3)
were from Coulter (Miami, FL). Anti-CD28 mAb (clone 9.3) was a gift from
Dr. P. Linsley (26). A fiuorogenic chymotrypsin substrate SLLVY-MCA
15 was from Peninsula Laboratories (Belmont, CA). Rabbit antisera against
cyciin A, Cyclin E, p27"'°') p21~'P', CDK2 and CDK4 were purchased from
Santa Cruz Biotech {Santa Cruz, CA). [y-32p]ATP (3000 NCi/mmol) and
['251] protein A {30mCi/mg protein) were ordered from Amersham
(Oakville, Ontario, Canada), and [Methyl-3H] thymidine (2Ci/mmol) was
20 from ICN (Irvine, CA).
Cell culture
Peripheral blood mononuclear cells (PBMC) and tonsillar T cells were
prepared as described before (Luo et al., 1992, Transplantation x:1071;
25 Luo et al., '! 993, Clin. & Exp. Immunol. x:371 ). The cells were cultured
in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.
3H-thymidine uptake was carried out as described previously (Luo et al.,
1992, supra; Luo et al., 1993, supra).
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DNA fragmentation assay
The assay was performed according to a protocol described by Liu et al
(Liu et al., 1997, Cell. $x:175) with some modifications. Briefly, 2-fi
million
cells were re-suspended in 50 ~I PBS followed by 300 NI lysis buffer (100
mM Tris-HCI, pH 8.0, 5 mM EDTA, 0.2 M NaCI. 0.2% w/v SDS, and 0.2
mg/ml proteinase K). After overnight incubation at 37°C, 350 NI of 3M
NaCI was added to the mixture and cell debris was removed by
centrifugation at 13000 g for 20 min at room temperature. DNA in the
supernatant was precipitated with an equal volume of 100% ethanol. The
10 pellet was washed with cold 70% ethanol and then dissolved in 20 NI of
TE containing 0.2 mg/ml RNase A. After incubation at 37°C for 2 h)
the
DNA was resolved on 2% agarose gel and visualized with ethidium
bromide staining.
Electron microscopy
T cells and Jurkat cells were examined by electron microscopy as
described by Tsao and Duguid (Tsao et al., 1987) Exp. Cell Res.
1 f~. 8:365).
Flow cy ometrXfor IL-2Ra
Two-color staining with F1TC-anti-CD3 and PE-anti-CD25 was performed
on tonsillar T cells. The method was described before (Luo et al., 1993,
supra).
Proteinase assay
Jurkat cells were cultured with various treatments and were harvested
and sonicated in 300 NI PBS on ice for 40 sec. Twenty micrograms of
protein per sample from the cleared lysates were supplemented to 100 NI
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with 0.1 M Tris buffer (pH 8.2). The fluorogenic chymotrypsin substrate
sLLVY-MCA was added at a final concentration of 10nM. The samples
were incubated at 37°C for 15 min and the reaction was terminated by
adding 4 NI 2.5M HCI. The samples were then diluted to 2ml with 0.1 M
5 Tris pH 8.2, and measured for their fluorescence intensity by a PTI
fluorometer (Photo Technology International, South Brunswick, NJ). The
excitation wavelength was 380nm, and the emission wavelength 440nm.
Cell cycle synchronization of T cells and Jurkat cells
Tonsillar T cells were cultured in the presence of 2 ~rg/ml PHA and 1 mM
hydroxyurea for 40h. The cells thus treated were synchronized at the
G,IS phase. The synchronization was released by washing out
hydroxyurea, and the cells were cultured in medium for additional 6-22h
according to the need of each experiment. The synchronization of Jurkat
15 cells was described in our previous publication (Shan et al., 1994, int.
Immunol. 6_:739). Briefly, the Jurkat cells were starved in isoleucine
deficient medium for 24h followed by 16h treatment with 2mM
hydroxyurea (HU). Cells thus treated were synchronized at the G,/S
boundary. For synchronization at the G~/M boundary, the G, /S
20 synchronized cells were released from hydroxyurea and cultured in
regular medium for 6h, and then treated with 0.1 Ng/ml nocodazole for
16h. The cells were then synchronized at the GZ/M boundary.
Cell cycle analyrsis
25 Flow cytometry was employed for cell cycle analysis for T cells and Jurkat
cells as described before (Shan et al., 1994, supra) using propidium
iodide staining.
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Immunoblotting
Immunoblotting was employed to evaluate the levels of Cyclin E, cyclin
A, p21~'P' and p27"'p'. The general protocol was described in our previous
publication (Chen et al., 1996, J. immunol. x:4297). Briefly,
5 lymphocytes were lysed in the presence of proteinase inhibitors. The
cleared lysates were quantitated for protein concentrations. An equal
amount of lysate proteins (40 Ng) of each sample was resolved by 10%
SDS-PAGE and was transferred to PVDF membranes (Millipore, Bedford,
MA). The membranes were then blocked with 5% milk, and hybridized
10 with rabbit antisera against Cyclin E, cyclin A, p27K'p' and p21 ~'P' at
dilutions suggested by the manufacturer. The signals on the membrane
were detected by ['251]-protein A followed by autoradiography.
Immunoprecipitation and the kinase assax
15 Lymphocytes were lysed by a lysis buffer as used in the immunoblotting
(Chen et al., 1996, supra), and cleared lysates were quantitated for their
protein content. For immunoprecipitation, 50 pl of rabbit antisera against
CDK2, CDK4 or Cyclin E were added to the lysates equivalent to 20 or
40 Ng protein depending on the experiment. After 2h incubation at 4°C,
20 the immune complexes were recovered by protein A-conjugated
Sepharose (Pharmacia Biotech, Montreal, Quebec, Canada). The
immune complexes bound to protein A-Sepharose were extensively
washed in a lysis buffer without detergents or EDTA) and resuspended
in 50 NI of kinase reaction buffer (100mM NaCI, 20mM HEPES, pH7.S,
25 5mM MnCl2, 5mM MgCl2, 25 NM cold ATP, 2.5 pCi [y-32p] ATP, and 3 pg
histone H1 as a substrate). The reaction was carried out for 10 min at
room temperature, and stopped by adding the SDS-PAGE loading buffer.
After boiling for 3 min, the samples were subjected to 10% SDS-PAGE.
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The proteins were then transferred to PVDF membranes and the signals
were detected by autoradiography.
EXAMPLE 2
Assays Measuring Nitric Oxide Production
Macrophage Preparation and Culture
BALB/c mice were injected i.p. with 3ml of 3% thioglycollate broth. Three
days later, peritoneal exudate macrophages of the mice were harvested
10 and washed at 170 g for 10 min at 4° C. The macrophages were
cultured
in Teflon vials (2cm in diameter) at 4x10s/2ml with various reagents (LPS,
2 Nglml; IFNy, 100u/ml; LAC, 0.62-5 NM for the nitric oxide assay and
5 NM for the Northern blot assay).
Nitric Oxide Measurement
The nitrite concentration in the culture supernatant was measured as a
way to indirectly reflect the nitric oxide level following a method described
by Ding et al ( Ding et al., 1988, J. Immunology X1:2407). Release of
reactive nitrogen intermediates and reactive oxygen intermediates form
mouse peritoneal macrophages: comparison of activation cytokines and
evidence for independent production. Briefly, 100 ~I of supernatants
collected from 48h macrophage cultures was incubated with an equal
volume of the Griess reagent (1 % sulfanimidel 0.1 % naphthylethylene
diamine dihydrochloride/ 2.5% H3P04 )at room temperature for 10 min in
25 96-well microtitration plates, the O.D. was measured at 550nm. Sodium
nitrite of various concentrations were used to construct standard curves.
Northern Blot Analysis of iNOS Expression
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The expression of inducible nitric oxide synthase at the mRNA level was
analyzed by Northern blot as described in our previous publication (Shan
et al., 1994, J., International Immunology f:739). After an overnight
culture, the mouse macrophages were harvested and their total cellular
5 RNA was extracted with the guanidine/CsCI method. The RNA
(10 Ng/lane) was resolved in 1% agarose-formaldehyde gels and blotted
onto nylon membranes. A 562-by fragment corresponding to the mouse
iNOS cDNA (Xie et al., 1992) Science ?x:225) was obtained by reverse
transcription/PCR using the mouse macrophages total RNA as templates.
10 The fragment was labeled with 32P with random primers and used as a
probe for the Northern blot.
15 Res~jr~i~of Jurkat Cells
Pr~a~aration of mitochondria
Rat liver of rat kidney proximal tubules mitochondria were isolated by
differential centrifugation in a medium containing 250 mM sucrose, 1 mM
HEPES-Tris) 250 NM EDTA (pH 7.5). The last washing of the
20 mitochondria was perfom~ed in the same medium without EDTA. Protein
concentration of the mitochondria) suspension was measured after
solubilization of the membranes in 0.1% SDS with the Pierce-BCA
(bicinchroninic acid) protein assay reagent (Pierce, Rockford, IL) USA),
using bovine serum albumin as a standard.
Respiration Measurements
The Jurkat Cells (JC) (30x106/ml) or rat kidney proximal tubules
mitochondria (RKM) (0.5 mg of protein/ml) were incubated in 1 ml
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measuring chamber at 37°C in a respiration buffer containing 200 mM
sucrose, 5 mM MgCl2, 5 mM KH2P04) and 30 mM HEPES-Tris (pH 7.5).
During respiration experiments following substrates and inhibitors were
used: 0.005% Digitonine (Dig); 10 mM Succinate (Suc); 1 mM Ascorbate
(Asc); 0.4 mM tetramethyl-p- phenylenediamine (TMPD); 1 NM CCCP,
1 NM FCCP; 0.1 NM Rotenone (Rot); 50 nM Antimycin A (Anti); 1 mM
KCN; 100 NM Cytochrome C (Cyt C).
The respiration rate of the Jurkat Cells and mitochondria was measured
polarographically with a Clarke oxygen electrode (Yellow Springs
Instruments, Yellow Springs, OH) USA) using 1 ml thermojacketed
chamber. Oxygen concentration was calibrated with air-saturated buffer
using Hypoxanthine - Xanthine Oxidase - Catalase system ("chemical
zero"). Oxygen consumption was continuously recorded using a
"MacLab/8" (Analog Digital Instruments, USA) connected to a Macintosh
SE computer and the MacLab Chart v.3.3.4 software. Rates of oxygen
consumption are expressed as ng-atoms of oxygen/min.
EXAMPLE 4
The effect of immunosuppressive drugs
Cell culture
PBMC were prepared by Lymphoprep gradient as described before (Luo
et al., 1993, Clin. Exp. Immunol. x.4:371; Shan et al., 1994, supra).
Tonsillar T cells were prepared by one cycle of SRBC rosetting and such
preparation contained 80-85% CD3+ cells. The remaining tonsillar cells
were referred to as the tonsillar B cells, which were about 90% CD20+
cells.
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Northern blot ~n~l~
The method is described in our previous publication (Shan et al., 1994,
supra). Tissue or lymphocyte total RNA was extracted with the
guanidine/CsCI method and used in the Northern blot analysis. A 358-by
fragment corresponding to positions -14 to 314 of the PA28~i cDNA (Ahn
et al., 1995, FEBS Lett. x:37) from clone 5F2 was labeled with 32p
using random primers and was used as a probe for PA28~i messages. A
400-by fragment corresponding to positions between 267 and 666 of the
PA28a cDNA (Realini et al., 1994, supra) was obtained with RT-PCR and
10 was used as a probe for PA28a messages. The 5' and 3' primers for the
RT-PCR were GAAGAAGGGGGAGGATGA and
AGCATTGCGGATCTCCAT, respectively.
Immunoblottina
T cell lysates (40 Ng protein/sample) were separated on 12% SDS-PAGE,
and blotted onto PVDF membranes. The membranes were then
hybridized with rabbit anti-PA28(i antiserum (Ahn et al. 1996, J. Biol.
Chem. 271:18237) followed by '251-protein A. Detailed methods were
described previously (Chen et al., 1996, J. Immunol. x:4297).
Confocal immunoflurescent m~croscopv
Cultured tonsillar T cells were stained with rabbit anti-PA28[3 antiserum
(1:1000 dilution) or anti-PA28a antiserum (1:200 dilution) followed by
biotin-conjugated goat anti-rabbit IgG (1:100 dilution, Boehringer
Mannheim, Montreal, QC) and streptavidin-fluorescein. The
immunofluorescence of whole cells was examined and quantified with
confocal microscopy as detailed before (Chen et al., 1997, J. Immunol.
159:905).
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Proteinase assaK
PBMC were cultured with or without PHA {2 Ng/ml) and RAPA (10nM).
After 16h-70h, the cells were harvested and sonicated in 300 NI PBS on
ice for 40 sec. Twenty micrograms of protein per sample ftom the cleared
lysates were supplemented to 100 NI with 0.1 M Tris buffer (pH 8.2). A
proteasome-specific inhibitor lactacystin (Omura et al., 1991, J. Antibiot.
(Tokyo) x:113; Fentenay et al., 1995, Science x,$:726) was added at
a final concentration of 10nM in some samples as indicated. The samples
were incubated on ice for 15 min, and fluorogenic chymotrypsin substrate
sLLVY-MCA was then added at a final concentration of 10nM. The 20S
proteasome, which was prepared as previously described (Friguet et al,
1994, J. Biol. Chem. X9:21639), was used as a positive control in place
of cell lysates. The samples were incubated at 37°C for 15 min and the
reaction was terminated by adding 4 NI 2.5M HCI. The samples were then
15 diluted to 2ml with 0.1 M Tris pH8.2, and measured for their fluorescent
intensity by a PTI fluorometer (Photo Technology International, South
Brunswick, NJ). The excitation wavelength was 380 nm, and the emission
wavelength 440 nm.
Conclusion
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified,
without departing from the spirit and nature of the subject invention. Any
such modification is under the scope of this invention as defined in the
25 appended claims.
