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Prepublished online as a Blood First Edition Paper on May 24, 2002; DOI 10.1182/blood-2001-11-0073.
NEOPLASIA
From the Department of Pathophysiology, Department of
Embryology, Department of Hematology, and Department of
Immunopathology, Medical University of Gdansk, Poland.
Decreased susceptibility to apoptosis and impaired proliferative
control are thought to be responsible for prolonged life span and
accumulation of chronic lymphocytic leukemia (B-CLL) cells. The
activity of calpains (calcium-dependent, neutral proteases, active in
the cells responding to signals inducing a rise of cytoplasmic Ca++) is involved in the regulation of apoptosis of some
cell types by interaction with caspase-3. This work verifies the
hypothesis of the abnormal activity of calpains and its role in reduced
apoptosis of the B-CLL cells. Casein zymography, reverse
transcriptase-polymerase chain reaction, and Western blotting were
used for identification and quantification of the activity and
expression of calpains in B-CLL cells and purified normal B
lymphocytes. The activity and expression of µ-calpain (requiring
micromolar Ca++ for activation) are significantly higher in
the leukemic than in nonmalignant cells. Contrarily, the activity and
expression of m-calpain (requiring millimolar Ca++) as
well as the expression of calpastatin (an endogenous inhibitor of
calpains) are unchanged or reduced in the B-CLL lymphocytes. Correspondingly, the activity of caspase-3 is many times lower in the
B-CLL cells than in normal B lymphocytes. Inhibition of overexpressed
µ-calpain in living B-CLL cells in vitro results in doubling of the
proportion of the cells undergoing spontaneous apoptosis. This
observation suggests a possible role for calpains in longer survival of
the B-CLL cells and may open new therapeutic possibilities.
(Blood. 2002;100:1802-1809) Chronic lymphocytic leukemia (CLL) Causes of B-CLL are still not very well understood; the search
concentrates around possible genetic aberrations on one
side4-7 and biochemical alterations on the other. On
average, the life span of a B-CLL cell is significantly longer than
that of a normal B lymphocyte, whereas the proliferation rate in the
leukemic population is similar or even decreased compared with the
nonneoplasmic ancestors.1 These features directed
researchers toward resistance to apoptogenic signals as a possible
cause of prolonged survival of the B-CLL cells and, in fact, the cause
of the disease.8
We propose here that the prolonged survival, reduced apoptosis, and a
decreased proliferation rate of B-CLL cells might all depend on an
impaired function of common, neutral, cytoplasmic thiol (cysteine)
proteases called calpains. Calpains are involved in the control of the
level and duration of the transduction signals leading to either
proliferation or apoptosis in multiple cell systems (reviewed by Suzuki
et al,9 Sorimachi et al,10 and Ono et
al11). The role of calpains in the development of
leukemias has not been postulated before. Ubiquitous members of the
calpain family that can be detected in blood cells are defined
according to the concentration of Ca++ required for full
activity (millimolar Ca++-dependent [m-] and micromolar
Ca++-dependent [µ-] calpains,
respectively).9-11 Substrates of calpains include protein
kinase C (PKC) and transcription factors such as c-fos, c-jun, nuclear
factor of activated T cells (NF-AT), nuclear factor- Calpains are supposedly involved in the regulation of apoptosis of
various types of cells. However, their role in the development and
course of apoptosis is controversial. Although most of the data seem to
indicate a level of collaboration between calpains and caspases in the
induction of apoptosis,10,22-24 some groups observed
apoptosis induction when cellular calpains were selectively inhibited.16,25,26 We have shown recently that the
activity of µ-calpain increases in T cells of old mice that express
high activities of a membrane transporter P-glycoprotein (J.M.W. and Miller, manuscript in preparation, 2002). These
P-glycoprotein-positive T cells exhibit simultaneously decreased
proliferative capability and lower cytokine production that also may
suggest lower susceptibility to apoptosis.27
Thus, it is possible that intrinsic changes of the activity of
calpain(s) in the B-CLL lymphocytes modulate their signal transduction, leading to impaired susceptibility to apoptosis and thus prolonging their effective life span in an organism. Therefore, we ask if the
activity of calpain(s) in the B-CLL differs quantitatively and/or
qualitatively from that characteristic for the non-CLL B lymphocytes.
Also, we attempt to answer (at least preliminarily) a question of the
possible role of calpain activity in the reduced apoptosis of B-CLL cells.
Patients
Cells
Peripheral blood lymphocytes of healthy controls were enriched
by elimination of monocytes by plastic adhesion. Further enrichment of
"healthy" B cells was done by magnetic separation. Briefly, cell
suspension was incubated for 45 minutes on ice with mouse antihuman CD3
(DAKO, Glostrup, Denmark) alone or together with mouse
antihuman CD16 (Becton Dickinson, San Jose, CA) monoclonal antibodies.
Then, appropriate numbers of antimouse Ig-conjugated magnetic beads
(Advanced Magnetics, Cambridge, MA) were added and the mixture was
incubated for another 45 minutes on ice. Strong magnetic field was then
applied, and the suspension of cells that did not bind the beads
(CD3 Epstein-Barr virus (EBV)-transformed human B cells were derived from PBLs of healthy people at the International Institute of Cellular and Molecular Biology in Warsaw, Poland. Estimation of calpain activity Casein zymography was applied to compare the activities of endogenous calpains in B-CLL cells, PBMCs, and enriched normal B lymphocytes.28-30 The cells were suspended at 8 × 107/mL, and 25 µL aliquots were prepared in either a lysis buffer (50 mM Tris-HCl, 1 mM dithiothreitol [DTT], 5 mM EGTA [ethyleneglycoltetraacetic acid]) or in calcium- and magnesium-free phosphate-buffered saline (PBS) with protease inhibitor cocktail (10 µg/mL aprotinin, 10 µg/mL leupeptin, 1.8 mg/mL iodoacetamide, and 1 µM phenylmethylsulfonyl fluoride [PMSF]; Sigma). Cell lysis was performed by 5 cycles of liquid nitrogen (LN2) flash-freezing, followed by rapid thawing of the sample in a 37°C water bath. Cell nuclei and debris were then removed by short spin at 10 000g, and the resulting lysates were flash-frozen in LN2 and stored at 70°C. Electrophoretic separation of the enzyme from calpastatins influencing its activity was
done in a nonreducing, detergent-free buffer in a 12% polyacrylamide gel containing 0.2% milk casein (Sigma) at 126 V. The amount of lysate
corresponding to 2 × 106 cells was loaded onto each
lane. The activity of calpains in the gel was induced by overnight
incubation in a buffer containing 2 mM CaCl2 and 10 mM DTT
in 20 mM Tris-HCl, pH 7.4, at room temperature. Casein proteolysis by
calpains produced clear, transparent bands in the Coomassie
blue-stained gels. The identity of the bands with the µ- and
m-calpains was confirmed by simultaneous running of a sample containing
a mixture of purified calpains (0.1 µg porcine µ-calpain and 0.1 µg rabbit muscle m-calpain; Calbiochem, Laufelfingen, Switzerland) in
the lysis buffer. Casein-digesting activity could only be seen at the
levels corresponding to the band positions of these "standard" µ-
and m-calpains. The intensity of the bands was quantified by
densitometry28-30 after digitization using the UVP gel
documentation system (UVP, Cambridge, United Kingdom) and GelBase
software (UVP). Densitometric analysis was performed with the
ImageQuant 3.3 (Molecular Dynamics, Sunnyvale, CA) software and the raw
results were collected as number of "white" pixels in a band. These
data were then converted to arbitrary units of the calpain activity,
based on the signal obtained from the known amount of standard enzymes.
Preliminary experiments had shown a linear dependence between the
density of digested bands and the amount of standard calpain in the
range of 10 to 250 ng per lane (Figure
1). In control samples, 20 µg/mL
calpain inhibitor II (N-Acetyl-Leu-Leu-Met-al; specific for both
isoforms of the enzyme; Calbiochem); or 20 µg/mL m-calpain
inhibitor rabbit skeletal muscle calpastatin, inhibitory mostly toward
m-calpain (calcium-activated neutral protease [m-calpain] inhibitor;
Sigma)31; or 1 µM calpeptin (benzyloxycarbonyldipeptidyl
aldehyde Z-Leu-Nle-CHO, specific for µ- and m-calpain; Calbiochem)
were added and the mixture was incubated on ice for 15 minutes before
loading onto the gel.
Specificity of the test was confirmed by the ability of all 3 calpain inhibitors used to decrease the activity of standard µ-calpain (Figure 1) and by significant reduction of the bands digested at the level of µ-calpain and lack of visible m-calpain activity in the lanes containing calpain inhibitor II-treated samples (compare Figure 3). The effects of calpeptin and rabbit calpastatin were similar (not shown). No proteolytic activity could be seen in the gels incubated in activation buffer without Ca++. Estimation of µ-calpain contents by Western blotting Lysates of cell samples containing 2 × 106 cells each were prepared in a denaturing buffer containing Nonidet P-40 and Triton X-100 detergents. Lysate proteins were electrophoretically resolved using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose. Rabbit anticalpain serum, shown to be cross-reactive with human and porcine µ-calpain (a kind gift from Dr M. Spencer, The Scripps Institute, CA), was used to detect µ-calpain in the blots. After second incubation with peroxidase-conjugated donkey antirabbit Ig (Amersham, Little Chalfont, United Kingdom), the µ-calpain-containing bands were visualized using the enhanced chemiluminescence kit (ECL, Amersham). Luminescent bands were recorded on the x-ray film and analyzed by densitometry. Samples containing 0.1 µg porcine µ-calpain standard instead of cell lysates were used as positive specificity control. To confirm the uniform loading of cell lysates in each lane, the levels of actin expression in the same volumes of lysates were determined by an analogous Western blot protocol using antiactin antibody (Sigma).Comparison of the expression of calpains and calpastatin genes in B-CLL and normal B cells Total RNA was isolated from samples of 1 × 106 B-CLL cells using TRI-Reagent (Sigma) according to the manufacturer's protocol. Isolated mRNA was immediately converted to cDNA by reverse transcription with Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) (Promega, Madison, WI) and oligo-dT as starter of the RT reaction. Amplification of gene products of the µ-calpain, m-calpain, and calpastatin genes was performed by polymerase chain reaction (PCR) using 1 µg cDNA as an amplification template in each case. The following primer pairs were used: for µ-calpain: 5'-CCTGCTTGAGAAGGCCTATG-3' (forward), 5'-GGTCCACGTTGTTCCACTCT-3' (reverse, product size 389 bp); for m-calpain: 5'-AGGCATACGCCAAGATCAAC-3' (forward), 5'-GGATGCGGATCAGTTTCTGT-3' (reverse, product size 306 bp); and for calpastatin: 5'-AAGACCTCGATGATGCCTTG-3' (forward), 5'-TCACTGGTTTGTCCTGTCCA-3' (reverse, product size 201 bp). In addition, -actin (a
"housekeeping" gene), was amplified from each cDNA sample, and the
amount of product of its amplification (determined densitometrically)
served as basis for normalization of the PCR results.
Comparison of the activity of caspase-3 in B-CLL, B-ALL, and EBV-transformed normal B cells The Caspase-3 Assay Kit (Sigma) was used. Briefly, cell lysates were incubated with the peptide substrate acetyl-Asp-Glu-Val-Asp-(p)nitroanilide (Ac-DEVD-pNA) with or without specific inhibitor of caspase-3. The activity of caspase-3 was calculated from the amount of p-nitroaniline (pNA) released from the substrate, against a standard curve obtained for known amounts of the pNA. Final results were indexed against these obtained for the samples of B-CLL cells.Analysis of the influence of calpain inhibitors on the spontaneous apoptosis in B-CLL cells B-CLL lymphocytes were suspended at 1 × 106 cells per milliliter in RPMI 1640 medium, supplemented with 10% fetal calf serum and 2 mM L-glutamine, and incubated with 20 µM membrane-permeable calpain inhibitor (calpeptin; Calbiochem), or 20 µg/mL rabbit muscle calpastatin (calcium-activated neutral protease [m-calpain] inhibitor; Sigma), or protease inhibitor cocktail (10 µg/mL aprotinin, 10 µg/mL leupeptin, 1.8 mg/mL iodoacetamide; Sigma), or without any additions for 24 hours at 37°C. Harvested and washed cells were then stained with the apoptosis detection kit containing fluorescein isothiocyanate (FITC)-annexin V and propidium iodide (PI) (a kind gift from the Nexins Research, The Netherlands) according to the manufacturer's instructions and analyzed by flow cytometry. Cells showing strong binding of the annexin V and no PI staining were considered apoptotic, whereas those with strong PI signal regardless of their annexin staining were considered necrotic.
B-CLL lymphocytes express the activities of both µ- and m-calpain Samples of 2 × 106 B-CLL lymphocytes were lysed in either a lysis buffer containing EGTA and DTT or in plain PBS and processed for casein zymography. In both types of lysates only the proteolytic activity of calpains could be detected, at the levels identical to the bands digested by standard µ- or m-calpain (Figure 2).
The activity of µ-calpain was strong; partially inhibited by calpain
inhibitor II (Figure 3), calpeptin, or
rabbit calpastatin (not shown); and somewhat higher in the samples
lysed in PBS than in the lysis buffer (Figure 2). On the other hand,
the activity of m-calpain was detectable (as digested bands) only in
the lysates prepared in PBS and absent in the lysates made in the EGTA-
and DTT-containing medium. This activity was completely absent from the
samples treated with calpain inhibitor II (Figure 3), calpeptin, or
rabbit calpastatin (not shown).
The activity of µ-calpain is higher and that of m-calpain is lower in the B-CLL cells than in nonmalignant B lymphocytes When the activity of calpains was simultaneously assessed in the lysates of B-CLL lymphocytes and of age-matched PBL and CD3 (B-enriched) cells of healthy people, the activity of
µ-calpain was significantly higher in the leukemic cells than in
either control (Figures 3 and 4A). The
activity of m-calpain was significantly lower in the chronic leukemia
cells than in matched, nonmalignant B lymphocytes (CD3-depleted cells),
whereas it was similar in B-CLL lymphocytes and in the age-matched PBL
cells (Figures 3 and 4B). There was no difference between the results
obtained for the CD3 and
CD3CD16 cells, indicating that
participation of the natural killer (NK) cells in the overall activity
of calpains in human lymphocytes is negligible (not shown). Therefore,
the 2 proteolytic activities of µ- and m-calpain behave reciprocally
in the B-CLL cells compared with normal B-cell control. No differences
in the activity of either calpain were observed in relation to the
advancement of the disease (Rai classification).
Lysates of normal, nonmalignant peripheral blood lymphocytes
had shown the presence of both µ- and m-calpain activities
(Figure 5). However, when the PBLs were
depleted of T (CD3+) cells by magnetic separation, the
activity of µ-calpain disappeared and that of m-calpain remained at
only a slightly lower level. The removal of CD16+ cells
resulted in no further change in m-calpain activity (Figure 5A).
Interestingly, when the activity of µ-calpain was assessed for the
PBLs of healthy people, it was strong for young people aged 25 to 34 years, clearly decreased for middle-aged individuals aged 40 to 50 years, and almost none for the elderly above 65 years (Figure 5B),
whereas the proportion of T:B:NK cells among PBLs of people of various
age did not differ. These results suggest that in nonmalignant
lymphocytes the activity of µ-calpain is associated almost
exclusively with T cells (for which it is clearly age dependent),
whereas it is very low in the B lymphocytes regardless of the age of
the individual. On the other hand, the activity of m-calpain is present
at a similar level in all populations of PBLs regardless of age
(not shown).
The amount of µ-calpain protein is higher in B-CLL cells than in normal CD3 lymphocytes The calpain protein amount was estimated by Western blot in detergent lysates from the same numbers of cells as used for casein zymography. The amount of µ-calpain protein was evidently increased in the B-CLL cells compared with the B (CD3) lymphocytes
from age-matched healthy individuals (representative blot is shown in
the Figure 6A). When the amount of the
enzyme was estimated in the same amount of total cellular protein and compared in B-CLL samples versus normal PBLs and
CD3 cells, not only was the difference between B-CLL and
CD3 cells highly significant, but total PBLs also
contained less than half the amount of the enzyme present in the B-CLL
(Figure 6B).
The expression of µ-calpain gene is significantly higher in B-CLL than in normal B lymphocytes Analysis of the expression of genes for µ- and m-calpain as well as for calpastatin was done using RT-PCR. The amount of the amplification product of µ-calpain gene from B-CLL cells was twice as big as that obtained from cDNA derived from normal B cells, whereas the expression of m-calpain and calpastatin genes did not differ (Figure 7).
Inhibition of µ-calpain increases the level of spontaneous apoptosis in B-CLL cells The level of apoptosis was determined by flow cytometry using FITC-annexin V to detect the apoptotic and PI necrotic cells. Upon treatment with calpeptin resulting in the significant inhibition of µ-calpain (Figure 1), the proportion of apoptotic (annexin+) cells increased twice compared with untreated B-CLL cells and this increase was highly statistically significant (Figure 8A). Inhibition of m-calpain by rabbit calpastatin had a reciprocal, weaker (but still significant) effect: a decreased percentage of cells binding annexin. Inhibition of other cellular proteases by a protease inhibitor cocktail had no effect on the same parameter (Figure 8A). Neither calpeptin (specific for both µ- and m-calpain) nor rabbit calpastatin (calcium-activated neutral protease inhibitor, relatively specific for m-calpain under experimental conditions used) had a significant effect on the viability of B-CLL cells (proportion of PI+ cells), whereas protease inhibitor "cocktail" very significantly reduced their viability (Figure 8B). Thus, action of calpeptin and calpastatin influencing the susceptibility of B-CLL cells to apoptosis seems to be related to their modulation of activities of calpains in these cells.
The activity of µ-calpain in normal and leukemic B cells reciprocally correlates with the activity of caspase-3 Comparison of caspase-3 activities in B-CLL cells, acute leukemia B lymphocytes, and EBV-immortalized B cells shows that the activity of this caspase is almost absent in B-CLL lymphocytes, clearly detectable in the ALL cells, and relatively very high in the EBV-transformed, vigorously proliferating B lymphocytes (Figure 9). This result suggests a reciprocal relation between the activity of caspase-3 and µ-calpain in normal B and B-CLL lymphocytes.
Activities of 2 isotypes of calcium-activated neutral
proteases The level of transcription of calpastatin gene in the B-CLL cells was
in our study similar to that exhibited by normal B cells. This
observation suggests that the increased activity of µ-calpain does
not depend on lower endogenous inhibition. Interestingly, the activity
of m-calpain in B-CLL cells is much lower than that of µ-calpain and
can only be demonstrated in the lysates prepared in the PBS without the
addition of calcium chelator EGTA or DTT. This effect is most probably
due to the requirement of a relatively high (supraphysiological and
available only in vitro) concentration of Ca++ Our understanding of changes of the proliferation control in the B-CLL is still elusive and remains multifaceted. Suggested causes of the derailment of the cell cycle include various mutations, especially in the molecules controlling the course of the cell cycle, such as p53, c-myc, Rb, and others.32,33 Proliferation control of B-CLL cells comprises both the mechanisms of decreased division rate and prolonged survival. The latter may be due to the cells escaping the signals for apoptosis. In fact, decreased susceptibility of the B-CLL cells to apoptosis has been recently discussed as one of the factors deciding of their accumulation during the disease.34,35 Proteolytic enzymes are clearly involved in the induction and progression of apoptosis; of these, apopains (caspases) are the best-known group.23 Participation of calpains in programmed cell death of blood cells (although not of the B-CLL cells so far) is also postulated.23,24 Our observation of increased apoptosis in B-CLL cells treated with a calpain inhibitor (calpeptin) suggests an antiapoptotic role of the enzyme activity. It also must be stressed that currently there are no inhibitors specific for either µ- or m-calpain. Thus, calpeptin inhibits both µ-calpain at ID50 (median effective dose) 52 nM and m-calpain at 34 nM; calpain inhibitor II acts in a similar way. Rabbit muscle calpastatin is described as inhibitory against mostly the m-calpain, although specificity of calpastatins against specific isoforms of calpains depends to some extent on their phosphorylation level and cleavage.36 On the other hand, we show in this work that an overexpression and overactivity of the µ isoform predominates in the B-CLL cells over much weaker activity of m-calpain. It is thus conceivable that the proapoptotic effect of calpain inhibitors that we observe depends mostly on the inhibition of µ-calpain and not of the minute activities of m-calpain in these cells. Most of the current reports suggest a positive relation between the activity of calpains and the induction of apoptosis. Thus, for example, calpains were found to participate in the apoptosis of human leukemic cell lines, where their activity was necessary for increased GADD153 gene expression at the onset of apoptosis.37 GADD153 encodes CHOP 10, a nuclear protein acting as a negative modulator of CCAAT (enhancer of expression of some transcription factors), thus inhibiting cell cycle progression and inducing apoptosis.38 On the other hand, Juin et al38 find no participation of calpains in nuclear apoptosis induced by 5 mM Ca++ (a concentration of calcium sufficient to activate both µ- and m-calpain). Considering the spectrum of calpain substrates, including inter alia
PKC and transcription factors c-fos, c-jun, NF-AT, NF- One of the causes for lower susceptibility of the B-CLL cells to
apoptosis might be an increased Bcl-2/Bax ratio, due to significantly and reciprocally altered levels of both factors, observed in the malignant cells of both treated and untreated B-CLL
patients.41 In a recent work, Wood et al show that calpain
in the leukemic HL-60 cell line can selectively cleave the Bax
protein.42 Thus, acting that way in the B-CLL cells,
calpain could increase the Bcl-2/Bax ratio and actually
prevent rather than induce apoptosis. Recent reports indicate that
apoptosis in the B-CLL is prevented mostly by increased levels of the
Bcl-2 with a simultaneous decrease of bax.34,35,43 The
level of Bcl-2 can be increased by Finally, our results indicate very low immediately available activity of the caspase-3 in the B-CLL cells. The activity of caspase-3 (and apoptosis) in/of the B-CLL cells can be induced by multiple factors, suggesting the possibility of impaired regulation of the gene for caspase-3 but not of its defect.47,48 The possibility of caspases being directly inhibited by calpain action had been recently demonstrated.49,50 Thus, it is feasible that calpain in B-CLL cells is either directly or, more likely, indirectly responsible for their lack of caspase-3 activity and, consequently, reduced apoptosis. Another question related to the possible participation of
calpains in the prevention of apoptosis in B-CLL cells is posed by our
observation of reciprocal behavior of the µ- and m-calpains. Interestingly, we have demonstrated here a decrease in the activity of
the m-calpain that requires millimolar Ca++ for activation
and is probably only active in the cells in which calcium is (almost)
freely entering the cytoplasm (as in the case of late apoptotic and
necrotic cells). It cannot be said at the moment if these 2 isoforms of
the enzyme could reciprocally control apoptosis in the B-CLL cells Although modulation of p53 antioncogene had been shown not to participate directly in the B-CLL resistance to drug-induced apoptosis,31 it was recently shown that mutations and impaired function of p53 occur in a proportion of chronic B-cell leukemias.52 Calpain has been shown to cleave p53, and thus it could prevent this pathway of apoptosis induction in the B-CLL.16 Garban et al reported recently that the PKC is down-regulated in the B-CLL and that this reduced activity is accompanied by lack of Ca++ signal, at least in response to stimulation through HLA-DR.53 This modulation of the PKC may be attributed to increased calpain activity; it has been shown that both µ- and m-calpain cleave and ultimately down-regulate PKC in other cell types.9-11,17 Increased activity of µ-calpain that we observe in B-CLL cells may be related to at least some of the mutations found in the genome of these leukemic cells. Genetic disorders associated with B-CLL concern mostly the 11 and 13 chromosomes, whereas the genes for µ- and m-calpains are located, respectively, in the 11th (11q13) 5-8,54 and first55 chromosome. In fact, Brizard et al find in the B-CLL rare translocations and deletions precisely in the 11q13 band of the 11th chromosome containing the calpain gene.56 Translocations in this chromosome region of B-CLL cells were also reported by Cuneo et al.57 Thus, the relation between mutations typically seen in the B-CLL and modulation of calpain gene expression in the B-CLL cells cannot be excluded. In conclusion, our observation of significant increase of the available activity of µ-calpain in the B-CLL lymphocytes is in agreement with both the cells' lower proliferation and increased survival phenotype and with genetic markers of the disease. The actual role of high activity of the protease and the exact mechanism(s) through which it may control the survival of B-CLL cells remain to be investigated. Finally, our finding of the increased spontaneous apoptosis level upon µ-calpain inhibitor treatment may suggest a possibility for potential therapeutic action of specific calpain inhibitors in chronic B-cell leukemia.
Submitted November 27, 2001; accepted April 13, 2002.
Prepublished online as Blood First Edition Paper, May 24, 2002; DOI 10.1182/blood-2001-11-0073.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jacek M. Witkowski, Department of Pathophysiology, Medical University of Gdansk; Debinki 7, 80-211 Gdansk, Poland; e-mail: jawit{at}amedec.amg.gda.pl.
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© 2002 by The American Society of Hematology.
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L. Xu and X. Deng Suppression of Cancer Cell Migration and Invasion by Protein Phosphatase 2A through Dephosphorylation of {micro}- and m-Calpains J. Biol. Chem., November 17, 2006; 281(46): 35567 - 35575. [Abstract] [Full Text] [PDF]
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T. Wada, S. H. Schurman, G. J. Jagadeesh, E. K. Garabedian, D. L. Nelson, and F. Candotti Multiple patients with revertant mosaicism in a single Wiskott-Aldrich syndrome family Blood, September 1, 2004; 104(5): 1270 - 1272. [Abstract] [Full Text] [PDF]
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R. W. Neumar, Y. A. Xu, H. Gada, R. P. Guttmann, and R. Siman Cross-talk between Calpain and Caspase Proteolytic Systems During Neuronal Apoptosis J. Biol. Chem., April 11, 2003; 278(16): 14162 - 14167. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
B (NF-
). According to Rai classification, stage I
was found in 4 patients, stage II in 7, stage III in 3, and stage IV in
1 patient. Thirteen patients participated in this study at
diagnosis, that is, before the onset of chemotherapy; for the remaining
2, the therapy-free period was 6 and 18 months, respectively. Neither
participant suffered from any infectious disease at the time of
blood drawing.
-interferon.
-interferon was found
to increase the level of calpain mRNA and protein in monocytic cell
lines U-937 and THP-1.