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B Activity and Apoptosis in Chronic Lymphocytic Leukemia B Cells1
,
Divisions of
*
Hematology-Oncology and
Allergy and Immunology, Department of Medicine, Weill Medical College and
Program in Immunology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B proteins. Based on observations that in some CLL cases, the
tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we
proposed a model for CLL pathogenesis due to CD40 ligation within the
tumor. To evaluate this issue, we used freshly isolated CLL B cells to
examine constitutive and inducible NF-B activity by electrophoretic
mobility shift assay. We consistently observed high levels of nuclear
NF-B-binding activity in unstimulated CLL B cells relative to that
detected in nonmalignant human B cells. In each case examined, CD40
ligation further augmented NF-B activity and prolonged CLL cell
survival in vitro. The principle NF-B proteins in stimulated CLL
cells appear to be quite similar to those in nonmalignant human B cells
and include p50, p65, and c-Rel. In a CD154-positive case, blocking
CD154 engagement by mAb to CD154 resulted in inhibition of NF-B
activity in the CLL cells. The addition of anti-CD154 mAb resulted
in accelerated CLL cell death to a similar degree as was observed in
cells exposed to dexamethasone. These data indicate that CD40
engagement has a profound influence on NF-B activity and survival in
CLL B cells, and are consistent with a role for CD154-expressing T and
B cells in CLL pathogenesis. The data support the development of novel
therapies based on blocking the CD154-CD40 interaction in
CLL. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation (7, 8), proliferation
(9), differentiation (10, 11), and survival
(12, 13). The physiologic ligand for CD40 is CD154 (CD40
ligand, T cell-B-activating molecule), a TNF-related molecule that is
transiently expressed at the surface of activated
CD4+ T cells (14, 15). We and others
have reported that in some cases of CLL, the tumor B cells express
CD154 (16, 17, 18). Based on the fact that CLL B cells express
CD40 and under some circumstances express CD154, we proposed a model
for CLL tumor growth and pathogenesis due to an autocrine or paracrine
stimulatory effect within the tumor, which might depend on NF-B
(17, 19).
The NF-B proteins are key regulators of differentiation and survival
in B cells (10, 20). In mammals this protein family
includes p50, p52, p65 (RelA), c-Rel, and RelB (21, 22, 23).
In the inactive state, NF-B proteins occur as homodimeric or
heterodimeric complexes in the cytoplasm bound to IB proteins. After
appropriate stimulation, IB is phosphorylated, ubiquinated, and
degraded, which allows translocation of NF-B to the nucleus and
transcription of NF-B target genes (24, 25, 26). Induction
of NF-B activity protects cells from apoptosis induced by a variety
of stimuli including exposure to TNF-
, chemotherapy, and ionizing
radiation (27, 28, 29). Recent studies have elucidated some
mechanisms by which NF-B inhibits apoptosis, such as induction of
c-IAP1 and c-IAP2 (30), bcl-xL, and
bfl-1 (31).
Our model predicts that CD40 ligation, delivered either by
CD154+ T cells in the host, or by
tumor-expressing B cells in a subset of cases, would result in
augmentation of NF-B activity and CLL cell survival
(17). To test this hypothesis, we examined constitutive
and inducible NF-B activity in freshly purified CLL B cells. We
evaluated the impact of CD40 ligation on nuclear NF-B and survival
in CLL cells using electrophoretic mobility shift assays (EMSAs) and
fluorescence flow cytometry. Through these investigations, we
determined that unstimulated CLL cells have high constitutive levels of
NF-B activity, and that CD40 engagement enhances this activity and
promotes survival in vitro. These results indicate that CD40-CD154
interactions may be central in CLL pathogenesis and offer new targets
for therapy in this disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood CLL samples were obtained from untreated CLL
patients whose diagnosis was confirmed at The New York Hospital-Cornell
Medical Center based on standard criteria (3). For studies
in nonmalignant human B cells, we used buffy coat preparations of human
peripheral blood leukocytes from The New York Blood Center (four
samples) or tonsillar B cells obtained from waste surgical specimens.
For all samples, the mononuclear cells were isolated by Ficoll-Hypaque
density centrifugation and the T cells depleted by rosetting with sheep
erythrocytes (Colorado Serum, Denver, CO) according to standard
techniques. For evaluation of NF-B activity in unstimulated cells,
nuclear and cytosolic lysates were prepared from B cells immediately
following purification, without culture.
Cell culture reagents
Purified B cells were cultured at 37°C with 5%
CO2 in RPMI 1640 media supplemented with 10% FCS
(Gemini Biological Products, Calabasas, CA), penicillin, streptomycin,
and L-glutamine, and the following reagents, as indicated:
IL-4 (10 ng/ml; R&D Systems, Minneapolis, MN), anti-CD40 mAb (2
µg/ml, clone M3, murine IgG1, R&D Systems), anti-CD154 mAb (3
µg/ml, TRAP1 clone, murine IgG1; PharMingen, San Diego, CA),
anti-CD23 (3 µg/ml, EBVCS; American Type Culture Collection,
Manassas, VA), and dexamethasone (10-7 M; Sigma,
St. Louis, MO). In the time course experiment, anti-CD30 mAb (2
µg/ml, murine IgG1; PharMingen) was used as an isotype-matched
control Ab. For some experiments, we used two Jurkat mutant cell lines
in which the cells constitutively express (clone D1.1) or cannot
express (clone B2.7) CD154, in coculture with target B cells after
irradiation (2000 rad) of the T cells, using a T:B cell ratio of 1:4.
The Jurkat lines were the kind gift of Dr. Seth Lederman (Columbia
University College of Physicians and Surgeons, New York, NY)
(32).
Preparation of cytosolic and nuclear lysates
Nuclear and cytosolic fractions were prepared as described previously (10). For each circumstance, a minimum of 2 x 107 cells were washed in cold PBS and resuspended in buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT) with 0.1% Nonidet P-40 and protease inhibitors (leupeptin, aprotinin, trypsin-chymotrypsin inhibitor, pepstatin A, and PMSF) at 0°C. For each sample, lysis of the plasma membrane was confirmed by trypan blue uptake before centrifugation, removal of the supernatant (cytosolic extract), and resuspension of the nuclear pellet in buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM DTT) with protease inhibitors at 0°C. The nuclear material was sonicated briefly before rotation at 4°C for 30 min. To remove insoluble debris, cytosolic and nuclear lysates were centrifuged before storage at -70°C. The protein content of each lysate was determined according to the Bradford assay (Bio-Rad, Hercules, CA).
EMSA and Ab inhibition (supershift) assay
EMSAs were performed as described previously (10)
using a T4 kinase end-labeled probe with the NF-B-binding sequence
from the Ig light chain promoter region (5'-GGGACTTTCC-3'). For
each EMSA, the nuclear lysates were incubated in DNA-binding buffer (10
mM Tris, 50 mM KCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol), poly(dI-dC)
(100 ng/µl), Nonidet P-40 (0.25%) and 20,000 cpm of
32P-labeled probe for 15 min at room temperature
before electrophoresis in a 6% native polyacrylamide gel. The gels
were dried and exposed to film at -70°C, and the autoradiographs
were analyzed by densitometry (SigmaGel; Jandel, San Rafael, CA). For
supershift EMSAs, 10 µg of nuclear protein were incubated with 6 µg
of polyclonal rabbit Ab to p50, p52, p65, c-Rel, or RelB (Santa Cruz
Biotechnology, Santa Cruz, CA) for 30 min at 0°C before addition of
labeled probe.
Flow cytometry and immunofluorescence analyses
Cells were washed in PBS and incubated with Abs according to standard techniques. Fluorescence was measured using a Becton Dickinson FACScan (Becton Dickinson, San Jose, CA) and analyzed using the CellQuest program (Becton Dickinson). Abs used for two-color analyses included anti-CD3-PE (Immunotech, Westbrook, ME), anti-CD5-PE (Coulter Pharmaceutical, Hialeah, FL), anti-CD19-PE (BioSource International, Camarillo, CA), anti-CD154-PE (clone 89-76; Becton Dickinson), anti-CD40-FITC (R&D Systems), and anti-CD19-FITC (Immunotech). For measurement of apoptosis, cells were washed in annexin-binding buffer (Chemicon International, Temecula, CA) and exposed to annexin V-FITC (Chemicon) before analysis by flow cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity that is
augmented by CD40 ligation
We purified B cells from peripheral blood samples of healthy
donors and CLL patients, and exposed the cells in vitro to IL-4, CD40
ligation, or dexamethasone before evaluation of nuclear NF-B-binding
activity by EMSA (Fig. 1
). In this set of
experiments, CD40 ligation was achieved using an agonistic, soluble
anti-CD40 mAb. We chose also to examine the effects of IL-4,
because this cytokine is an established growth factor for CLL, and in
some circumstances acts in concert with CD40 ligation to favor B cell
survival (13, 33). Dexamethasone exposure was used for
purposes of comparison, as it is known to inhibit NF-B activity and
promote CLL cell apoptosis in vitro (34).
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A, CD40
ligation increased NF-B activity in both normal and CLL B cells, and
high levels of NF-B activity were readily detected in nuclear
extracts prepared from CLL cells as compared with those of nonmalignant
human B cells isolated and analyzed in the same manner. To demonstrate
that the high levels of NF-B activity we observed in unstimulated
CLL cells were not a function of culture in media, we examined NF-B
activity in four normal peripheral blood and four CLL B cell samples
immediately after purification (Fig. 1B). As shown, the
difference in signal intensity for NF-B activity in unstimulated CLL
cells was significantly greater (p = 0.004) and
2.5-fold increased, relative to that for normal B cells purified by
the same method and analyzed in the same autoradiograph. These data
indicate that CLL B cells have high levels of constitutive
NF-B-binding activity compared with normal human peripheral blood B
lymphocytes.
Fig. 2 summarizes the composite results
for all of the EMSAs in which NF-B activity was measured in CLL
cells after 6 h of stimulation. To circumvent some of the inherent
difficulty in assessing the effects of stimulation on NF-B activity
among a heterogeneous group of clinical samples, we used densitometry
to measure the EMSA results for each experimental circumstance. As
shown, the results represent percent values relative to the signals
observed for cells of the same case exposed to media. The 6-h time
point was chosen for these studies based on experiments in which we
observed maximal changes in NF-B activity between 6 and 9 h
after initiation of treatment (Fig. 3,
below). In 11of 12 cases examined at the 6-h time point, NF-B
activity was augmented in CLL cells subjected to CD40 ligation by
anti-CD40 mAb.
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B activity, but this was
not the case for the majority of the samples. In four of eight cases
analyzed after exposure to IL-4, there was an increase in NF-B
activity in the CLL cells. Although the effect was not statistically
significant, based in part on the small number of samples analyzed for
this effect, there were four cases in which CD40 ligation and IL-4
together appeared to induce NF-B activity to a greater extent than
either agent alone. As anticipated, and consistent with the known
capacity of glucocorticoids to induce apoptosis of CLL cells via a
proteosome-dependent mechanism (34), exposure to
dexamethasone reduced NF-B activity in all but 1 of 11 cases.
CD40 ligation is a potent inducer of NF-B activity in CLL cells
To investigate the kinetics of CD40-mediated NF-B induction in
CLL B cells, we analyzed nuclear NF-B immediately after purification
or after exposure to media only, anti-CD40 mAb, or an
isotype-matched control Ab for various time intervals (Fig. 3). As
shown, NF-B was activated within 3 h of CD40 ligation, peaked
within 9 h, and was sustained at 21 h. Based on this and
similar experiments, we conclude that NF-B activity peaked between 6
and 9 h after CD40 ligation and was maintained for as long as
72 h after stimulation (data not shown). This observation is
consistent with reports of sustained NF-B activity induced by CD40
ligation in nonmalignant human B cells (8). It is
noteworthy that we observed clear and reproducible NF-B induction in
these experiments in which the CLL cells were stimulated only by
soluble Ab to CD40, a relatively weak effector of CD40 ligation. These
observations indicate that CLL B cells are extremely sensitive to
CD40-mediated transcriptional stimulation.
Characterization of specific NF-B components in normal human and
CLL B cells
Cross-linking of CD40 by anti-CD40 mAb in CLL B cells resulted
in an approximate doubling of NF-B activity in the cells (Fig. 2).
In similar assays performed using four preparations of nonmalignant
human peripheral blood B cells, we observed 3-fold induction of
NF-B activity (data not shown). To characterize which are the
predominant NF-B components in CLL B cells, we performed supershift
EMSAs using Abs to specific NF-B proteins with nuclear extracts
prepared from untreated or CD40-stimulated CLL B cells. The data in
Fig. 4 demonstrate that the dominant
NF-B components in stimulated CLL cells include p50, p65, and c-Rel.
These results are quite similar to those observed using nuclear
extracts prepared from human tonsillar B cells subjected to CD40
ligation by anti-CD40 mAb in vitro, and suggest that the regulation
of NF-B activity in CLL cells is similar to that of nonmalignant
human B cells.
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B activity and reduced survival in vitro
CLL B cells express CD154 in only a subset of cases (17, 19). To investigate the consequences of blocking CD154-CD40
interaction in a CD154+ case, we purified the B
cells from the peripheral blood of a patient whose cells we had
previously characterized in our laboratory and determined to be
CD154+. After isolation and purification of the
cells, CD154 expression was evaluated by FACS analysis. In this case,
>99% of the purified cells coexpressed CD5 and CD19, and 16% of the
B cells expressed CD154 (data not shown).
As was evident by EMSA (Fig. 5), NF-B
activity in the CLL cells of this case was inhibited by anti-CD154
mAb to a similar extent as it was upon exposure of the cells to
dexamethasone. In this particular case, mAb to CD154 also favored CLL
cell death in vitro (Table I).
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To determine whether CD40-mediated NF-B induction was
associated with CLL cell survival, we measured apoptosis in cells
exposed to the anti-CD40 mAb, or to anti-CD154 mAb, by annexin
V binding and fluorescence flow cytometry (Fig. 6). In this case, there was some baseline
expression of CD154 in the CD19+ B cells (9%)
which was augmented after exposure to anti-CD40 mAb (17%). This
result is similar to what we have reported previously regarding
CD40-mediated induction of CD154 in CLL B cells (17). As
shown, there was only a modest survival benefit in CLL cells exposed to
anti-CD40 mAb, which was typical of the results for similar
experiments using cells of other cases. In our laboratory, we have
observed much more dramatic prosurvival effects of CD40 ligation in CLL
B cells when the stimulus to CD40 is cell-bound CD154 (Fig. 7, below) as compared with anti-CD40
mAb. However, there was a dramatic reduction in survival among cells
placed in culture with an Ab to CD154 (TRAP clone), which blocks
intercellular CD40-CD154 interaction, such that 77% of the CLL cells
bound annexin V at this time point. Strikingly, the degree of cell
death induced by anti-CD154 mAb was similar to that observed in cells
exposed to dexamethasone, which is concordant with the inhibition of
NF-B activity upon exposure to anti-CD154 mAb documented for
another case in Fig. 5. Taken together, these data suggest that
CD40-CD154 interactions are crucial in CLL B cell survival, and that
soluble Ab to CD154 can accelerate CLL cell death in cases that express
CD154.
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A shows a representative experiment in which we
evaluated apoptosis in CLL cells after coculture with irradiated
CD154-expressing T cells in the presence or absence of blocking Ab to
CD154. In this case, there was considerable baseline apoptosis in the
cells cultured with media alone, such that only 45% of the cells were
viable at 48 h. Survival was improved (60%) after exposure to
CD154-deficient Jurkats, possibly due to soluble factors such as IL-4
or other cytokines provided by these T cells. However, the proportion
of surviving CLL cells was greatest after coculture with
CD154-expressing T cells (72%). This prosurvival effect of the T cells
was completely abrogated in the presence of Ab to CD154 (14%), but not
in the presence of an isotype-matched control Ab to CD23 (62%). Fig. 7B indicates the compiled results for this and three similar
experiments, which demonstrate that CD154-expressing T cells promote
the survival of CLL B cells in coculture via a CD154-dependent
mechanism. The mean survival value among CLL cells exposed to
irradiated CD154+ T cells in this system was
71%, as compared to 31% among cells exposed only to media
(p = 0.0028, Student’s t test). In
cells exposed to CD154+ T cells in the presence
of anti-CD154 mAb, there was a significant reduction in the mean
value for the surviving cells (38%, p = 0.039), as
compared with the results for the same cells without blocking
mAb.
We have reported that CD154 expression in CLL B cells diminishes over
the initial days in culture, such that it is usually undetectable
within 72 h after isolation of the cells in a positive case
(17). In the example shown in Fig. 7A, the
degree of apoptosis that occurred in the stimulated cells exposed to
anti-CD154 mAb exceeded that which occurred in cells exposed to
media only, and was similar in degree to that observed in the
experiment shown in Fig. 6. In our laboratory, we have observed that
CD154 expression is readily apparent at the cell surface in 15% of
CLL cases and can be detected at some level in up to one-third of cases
(17, 19). The data included in Figs. 6 and 7 suggest that
anti-CD154 mAb can block endogenous CD154 expressed by tumor B
cells. In cases that express CD154, impedance to CD154 results in
inhibition of NF-B activity and CLL cell death. In cases that do not
express CD154, anti-CD154 mAb might impede survival signals
conferred to CLL cells via CD154-expressing T cells or possibly by
other cells such as endothelial cells in the host
(35).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity relative to nonmalignant human B cells and
that this activity is significantly induced by CD40 engagement. The
dominant NF-B components in CLL cells appear to be p50, p65, and
c-Rel, which is similar to the results reported by Romano et al.
(36). Augmented NF-B activity upon CD40 ligation was
associated with improved CLL cell survival. In a
CD154+ case, the addition of anti-CD154 mAb
resulted in dramatic inhibition of NF-B activity in the CLL cells to
a similar degree as did dexamethasone exposure. In the majority of
cases examined, enhanced NF-B activity upon CD40 engagement was
associated with CLL cell survival, and blocking CD40-CD154 interactions
by anti-CD154 mAb resulted in accelerated CLL cell death in vitro.
Taken together, our results indicate that the CD40-CD154 interaction
can have profound effects on transcriptional regulation and tumor cell
survival in CLL.
Previous investigators have identified NF-B activity in lymphocytic
tumors associated with transforming viruses. For example, the Tax
protein of the human T cell leukemia virus-1, which occurs in certain
human T cell leukemias and lymphomas, interacts with the IB kinase
complex, resulting in enhanced IB phosphorylation, degradation, and
induction of NF-B (37). Recently, high levels of
NF-B activity were detected in EBV-associated posttransplant B cell
lymphoproliferative disorders (38). Other investigators
have observed NF-B activity in cell lines derived from B cell tumors
(39) and Hodgkin’s disease (40, 41). Our
observation that NF-B activity is constitutively turned on in
primary CLL B cells is significant because these cells are not infected
by a virus. These findings implicate an alternative mechanism for
NF-B induction in this tumor.
We have reported that in some CLL cases the B cells coexpress CD40 and
CD154 (17, 19). However, the data do not support that
levels of NF-B activity in the tumor cells correlate with B
cell-derived CD154. Rather, we have observed relatively high levels of
constitutive NF-B activity in most CLL cases examined, whether CD154
positive or negative. There are three broad possible explanations for
the lack of correlation: 1) that the high levels of NF-B activity
are due to CD154-independent mechanisms in some cases; 2) that most CLL
B cell express CD154 in vivo, which exerts powerful effects, but that
the molecule is shed rapidly upon isolation of the cells such that it
is usually not detected; or 3) that the observed activity occurs as a
function of CD40 engagement in vivo by CD154 derived from nonmalignant
cells in the host such as CD4+ T cells.
Despite that B cell-derived CD154 appears to be a factor in only a
minority of cases, our data are consistent with the hypothesis that
constitutive NF-B activity in CLL cells is due, at least in some
cases, to the continuous engagement of CD40 by its ligand in vivo.
These results are consistent with and build upon those recently
reported by two other groups, who have identified that CD40 ligation
inhibits chemotherapy and dexamethasone-induced apoptosis in CLL B
cells, and that this effect may be dependent on NF-B (36, 42). We would emphasize that the CD154-CD40 interaction is
unlikely to be the primary pathogenic mechanism in CLL. Rather, our
data support that this molecular interaction promotes growth of a tumor
that has already arisen due to a separate, transforming event. In CLL
cases that express both CD40 and CD154, autoligation of CD40 within a
tumor cell, or ligation of CD40 by a nearby CLL cell, would result in
NF-B activation in vivo. In cases that do not express CD154, CD40
ligation might occur due to tumor-infiltrating
CD4+ T cells. Such a mechanism for NF-B
induction and CD40 engagement via CD154 on T cells would be consistent
with clinical observations regarding the efficacy of adenosine
analogues in this disease. These chemotherapeutic agents effectively
deplete the host’s CD4+ T cells for prolonged
periods (43) and could function, at least in part, by the
elimination of T cell "help" that drives the B cell tumor
(19).
Finally, these results offer two possible targets for new therapies in
this disease. First, we have demonstrated that in some cases blocking
the CD154-CD40 interaction results in inhibition of NF-B activity
and CLL cell death. Therefore, new approaches might include the use of
Ab to CD154, such as is being tried in patients with autoimmune
diseases such as systemic lupus erythematosus and idiopathic
thrombocytopenic purpura. This treatment might be targeted to cases in
which the CLL cells express CD154, or cases in which the patients
suffer from associated autoimmunity. Second, our results also support
the investigation and trial of specific inhibitors to NF-B, which
might in themselves cause apoptosis of the malignant cells, or would
facilitate death in CLL cells exposed to conventional chemotherapy
agents.
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Acknowledgments |
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Footnotes |
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2 H.-C.L. and E.J.S. contributed equally to the work.
3 Address correspondence and reprint requests to Dr. Elaine Schattner, Division of Hematology-Oncology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. E-mail address:
4 Abbreviations used in this paper: CLL, chronic lymphocytic leukemia; IB, inhibitor of NF-B; EMSA, electrophoretic mobility shift assay; dex, dexamethasone.
Received for publication July 20, 1999. Accepted for publication December 3, 1999.
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A. Petlickovski, L. Laurenti, X. Li, S. Marietti, P. Chiusolo, S. Sica, G. Leone, and D. G. Efremov Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells Blood, June 15, 2005; 105(12): 4820 - 4827. [Abstract] [Full Text] [PDF]
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A. Rodriguez, N. Martinez, F. I. Camacho, E. Ruiz-Ballesteros, P. Algara, J.-F. Garcia, J. Menarguez, T. Alvaro, M. F. Fresno, F. Solano, et al. Variability in the Degree of Expression of Phosphorylated I{kappa}B{alpha} in Chronic Lymphocytic Leukemia Cases With Nodal Involvement Clin. Cancer Res., October 15, 2004; 10(20): 6796 - 6806. [Abstract] [Full Text] [PDF]
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J. Houldsworth, A. B. Olshen, G. Cattoretti, G. B. Donnelly, J. Teruya-Feldstein, J. Qin, N. Palanisamy, Y. Shen, K. Dyomina, M. Petlakh, et al. Relationship between REL amplification, REL function, and clinical and biologic features in diffuse large B-cell lymphomas Blood, March 1, 2004; 103(5): 1862 - 1868. [Abstract] [Full Text] [PDF]
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M. A. Sepulveda, A. V. Emelyanov, and B. K. Birshtein NF-{kappa}B and Oct-2 Synergize to Activate the Human 3' Igh hs4 Enhancer in B Cells J. Immunol., January 15, 2004; 172(2): 1054 - 1064. [Abstract] [Full Text] [PDF]
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N. W. C. J. van de Donk, D. Schotte, M. M. J. Kamphuis, A. M. W. van Marion, B. van Kessel, A. C. Bloem, and H. M. Lokhorst Protein Geranylgeranylation Is Critical for the Regulation of Survival and Proliferation of Lymphoma Tumor Cells Clin. Cancer Res., November 15, 2003; 9(15): 5735 - 5748. [Abstract] [Full Text] [PDF]
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