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Clinical Trials |
Department of Medicine, Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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INTRODUCTION |
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Here we report the laboratory findings in a single patient with CLL in a Phase I clinical trial. This trial was designed to treat patients with Bryo-1, followed by 2-CdA. We examined the change in cell differentiation and the molecular pathways of cell death by apoptosis associated with sequential treatment of Bryo-1 and 2-CdA in a patient.
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MATERIALS AND METHODS |
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Chemotherapy Protocol.
Bryo-1 (252 µg; 120 µg/m2) in 0.9%
benzo-alcohol-preserved saline was infused through infus-a-port for
72 h followed by 2-CdA (26.4 mg; 0.06 mg/kg) by continuous
infusion over 5 days. The patient underwent three cycles of
chemotherapy during November 17–25, 1998; December 15–23, 1998; and
January 19–27, 1999. During each cycle, blood samples were collected
for the isolation of lymphocytes at the following time points: before
Bryo-1 administration (pre-Bryo); at the completion of Bryo-1
administration; before 2-CdA administration (post-Bryo); and at the
completion of 2-CdA administration (post-2CdA). Bryo-1 (NSC 339555) was
supplied by the National Cancer Institute Division of Cancer Treatment
and Diagnosis.
Peripheral Blood Mononuclear Cells.
Blood samples were collected at the time points indicated above via
venipuncture in heparinized tubes. Lymphocytes were isolated by a
density gradient using Histopaque (Sigma Diagnostics Inc., St. Louis,
MO). The lymphocyte layer was recovered and washed once, and cells were
prepared for cytospin, flow cytometry, Western blotting, and enzymatic
analysis.
Assessment of Lymphocyte Morphology.
Cytocentrifuge smears (Cytospin II centrifuge; Shandon, Southern
Instruments, Inc., Sewickley, PA) were prepared at different time
points. Smears were air-dried and stained with tetrachrome. Cell
morphology was then analyzed by light microscopy (Nikon, Garden City,
NY). A total of 200 cells were counted by two different individuals. We
examined the proportion of differentiated and nondifferentiated cells
among these viable cells. Nondifferentiated cells are smaller in size
and have a low nuclear:cytoplasmic ratio, whereas the differentiated
cells are relatively larger in size with cytoplasmic processes and a
high nuclear:cytoplasmic ratio.
Expression of Differentiation Cell Markers.
Peripheral blood mononuclear cells from the patient were obtained by
Ficoll-Hypaque separation at pre-Bryo, post-Bryo, and post-2CdA time
points of each chemotherapy cycle. Cells were stained simultaneously
with three antibodies directly conjugated to peridinin chlorophyll
protein (CD20), phycoerythrin, and FITC. CD20+ cells were
gated as total B cells of interest, within which the proportion of
cells that expressed CD5 alone (CD20+, CD5+) or
with CD11c (CD20+, CD5+, CD11c+) or
CD22 (CD20+, CD5+, CD22+) were
evaluated.
Western Blot Analysis.
The patient’s isolated lymphocytes were washed with PBS, resuspended
in Triton X-100 lysis buffer, and handled as described previously
(7)
. For Western analysis, 20 µg from each sample were
separated on a 12% SDS-polyacrylamide gel and transferred to
nitrocellulose membranes. The membranes were blocked and then incubated
in 1:1000 concentration of Bax, Bcl-2 (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA), and PARP (Travigen, Gaithersburg, MD)
antibodies. The membranes were exposed to horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology,
Inc.). The proteins were visualized using the enhanced
chemiluminescence reagents (Amersham International Ltd.,
Buckinghamshire, United Kingdom).
dCK.
Patient lymphocytes were analyzed at the pre-Bryo and post-Bryo time
points for dCK activity using 2-CdA as a substrate, as described
previously (7)
. Cells were harvested, washed once with
PBS, and resuspended at a concentration of 10 x 106
cells/ml in dCK sonication buffer consisting of 10 mM
Tris-HCl (pH 7.6), 2 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, and 10% glycerol. Samples were stored
at -70°C until assayed.
Cytosolic 5'-NT Activity.
Isolated patient lymphocytes were washed once with PBS and resuspended
at a concentration of 10 x 106 cells/ml in 5'-NT
sonication buffer consisting of 20 mM imidazole-HCl (pH
7.0), 20 mM MgCl2, 0.1 mM EGTA, and
0.1 mM phenylmethylsulfonyl fluoride. Samples were stored
at -70°C until assayed as described previously (7)
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Results are presented as a ratio of the dCK activity (expressed as
pmol/mg protein/min):5'-NT activity (expressed as pmol inosine
monophosphate dephosphorylated/mg protein/min).
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RESULTS |
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. At the post-Bryo time point,
nondifferentiated cells were reduced to 7%, and the differentiated
cells were increased to 93%. At post-2CdA, nondifferentiated cells
were 53%, and differentiated cells were 47%. During the second and
the third cycle, we did not observe a marked increase of differentiated
cells as seen after the first cycle. However, the percentage of
differentiated cells remained increased compared to the pre-Bryo level
of the first cycle.
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. At the post-Bryo time point,
CD22-positive cells were increased to 93%, whereas the percentage of
double-positive cells for CD11c and CD22 remained the same. At the
post-2CdA time point, CD22-positive cells were 92%, and
double-positive cells for CD11c and CD22 were 5%. During the second
and third cycle, CD22-positive cells remained elevated from 76% to
90%, and the double-positive cells for CD11c and CD22 were elevated
from 6% to 8%.
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. Before the onset of treatment, the total WBC count and
lymphocyte count were 60,500 and 37,100/µl, respectively. After the
first cycle, the total WBC count was reduced to 26,000/µl and
remained decreased for up to 2 months after the third cycle. Lymphocyte
count was also reduced to 5,940/µl after first cycle and gradually
declined thereafter. According to the National Cancer Institute Working
Group criteria (8)
, the patient achieved partial
remission. The hemoglobin, neutrophil, and platelet counts were
maintained above 11 gm/dl, 1.5 x 109/liter, and
100 x 109/liter, respectively, and the patient was
free of hymphadenopathy and hepatosplenomegaly. Unfortunately, although
there was a significant reduction in lymphocyte count in the peripheral
blood, bone marrow examination showed more than 30% lymphocytes, which
precluded a designation of complete remission. The patient’s absolute
lymphocyte count increased to >10 x 109/liter 2
months after the discontinuation of therapy.
Western Blot Analysis.
The gradual increase in the Bax:Bcl-2 ratio from 0.90 in first cycle
to 7.5 in second cycle and 14.3 in the third cycle was primarily due to
the reduction of Bcl-2 expression and an increase in Bax expression
(Fig. 2)
. In all three cycles, there was
a relative reduction of Bcl-2 expression compared to the internal
control glyceraldehyde-3-phosphate dehydrogenase as determined by
densitometric analysis of the Western blots. At the post-Bryo time
point of three cycles, the relative expression of Bcl-2 was 1.94, 0.18,
and 0.09, respectively. In the first cycle, PARP (116 kDa) was
significantly reduced post-Bryo when compared to that recorded in the
pre-Bryo. The profile of PARP in the second cycle did not appear to
change except for further change of the 116-kDa band to 85-kDa band by
post-2CdA. In the third cycle, post-Bryo, PARP cleavage was complete,
with no detectable 116- or 85-kDa bands. Post-2CdA showed significant
cleavage of the PARP 116-kDa to 85-kDa bands.
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. Post-2CdA was excluded because
dCK uses 2-CdA as its substrate. In the first, second, and third cycle,
at post-Bryo, the dCK:5'-NT ratio was higher than the pre-Bryo ratio,
but there were no statistical differences between the means.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bryo-1 has been reported to induce differentiation of CLL cells of B-cell origin to a HC stage in vitro (2) . We have previously documented that Bryo-1-treated CLL cells exhibit increased sensitivity to 2-CdA, a drug active in treating de novo HCL but not active in fludarabine-resistant CLL. In WSU-CLL cells in vitro as well as the xenograft model in SCID mice, the efficacy of 2-CdA was enhanced when the cells were first exposed to Bryo-1 (6 , 7) . Administration of Bryo-1 followed by 2-CdA appeared to be sequence-dependent because neither concurrent nor reverse sequential use of the two agents was more effective than either agent alone (6) .
Bryo-1 is a potent modulator of PKC, which elicits a wide range of biological effects including in vitro and in vivo antineoplastic activity, induction of differentiation, pleotropic immunoenhancing effects on both T and B lymphocytes, activation of polymorphonuclear leukocytes, and enhancement of the production and function of hemopoietic growth factors (9) . Bryo-1-induced translocation of PKC to the plasma membrane leads to the phosphorylation of specific protein substrates and eventually induces down-regulation of PKC (10) . In patients, activation of PKC activity is increased within first couple of hours after treatment with Bryo-1 and is then subsequently down-regulated by 24 h on (11) .
We have demonstrated here that Bryo-1 infusion for 72 h induced
differentiation of the CLL cells (Fig. 1)
, as evidenced by the presence
of cell membrane processes and increased nuclear:cytoplasmic ratio.
By using flow cytometry, we further showed that differentiated cells
bear HC-associated surface markers CD22 and CD11c (Table 1)
.
Differentiation was most evident after the first cycle of Bryo-1
treatment. Despite subsequent administration of Bryo-1, we did not
observe a marked change in cell differentiation in the second and third
cycle compared to that found in the first cycle, possibly because of an
accumulation of Bryo-1-resistant CLL cells over time. These
observations confirm that Bryo-1 induces differentiation in peripheral
blood lymphocytes in vivo similar to that demonstrated using
the WSU-CLL cell line in vitro and in vivo using
a SCID mouse xenograft model.
Among the purine analogues, 2-CdA has the ability to induce apoptosis
in nondividing lymphocytes at concentrations that spare other cell
types (12)
. For this reason, 2-CdA is used in the
treatment of indolent lymphoid malignancies, including HCL and
low-grade lymphoma. The cytotoxicity of 2-CdA depends mainly on its
selective and progressive phosphorylation by dCK and accumulation of
its 5'-triphosphate metabolite (2-CdATP) in lymphocytes. 2-CdAMP can be
dephosphorylated back to the nucleoside by a cytosolic 5'-NT. Hence,
both the in vitro sensitivity of cultured CLL cells and the
in vivo response of patients with CLL to 2-CdA correlate
with the high ratio of dCK:5'-NT (12, 13, 14)
. Bryo-1
increases the sensitivity to 2-CdA by increasing the dCK:5'-NT ratio
(7)
and by increasing intracellular incorporation of 2-CdA
(13)
. The phosphorylated metabolite of 2-CdA, 2-CdATP, is
a potent inhibitor of ribonucleotide reductase and DNA polymerase 
,
and these two enzymes are responsible for DNA synthesis and repair in
actively dividing cells (15
, 16)
. Inhibition of the repair
mechanism can lead to DNA fragmentation, which is characteristic of
apoptosis in dividing cells.
PARP cleavage is an accepted indicator for the initiation of apoptosis.
PARP is a 116-kDa nuclear protein that maintains DNA repair pathways.
Once it is cleaved into 85- and 22-kDa fragments, the DNA repair
mechanism is impaired, which ultimately leads the cells to undergo
apoptosis. Hence, the presence of the 85-kDa band is an indicator for
PARP cleavage. In peripheral mononuclear cells, the sequential
treatment with Bryo-1 and 2-CdA led to an increase in the Bax:Bcl-2
ratio as well as evidence of PARP cleavage (Fig. 2)
. This indicates
that the signal transduction pathways leading to apoptosis have been
initiated in these peripheral blood lymphocytes. In vitro,
studies have shown that the activation of caspase 3 precedes PARP
cleavage and is easily detected by Western blot analysis
(17)
. We did not observe caspase 3 or cleavage in the
peripheral blood lymphocytes isolated from this patient (data not
shown). The significant reduction of total WBC count and lymphocyte
count during the treatment and 2 months after the termination of
treatment provides evidence for the effectiveness of sequential
treatment of Bryo-1 and 2-CdA in eliminating malignant CLL cells by
differentiating the cells to HC and inducing apoptosis.
In this patient, the dCK:5'-NT ratio was elevated after 3 days of infusion of Bryo-1. This suggests that the Bryo 1-induced elevation of the dCK:5'-NT ratio may lead to increased sensitivity of peripheral blood lymphocytes of this patient to 2-CdA. The increased sensitivity to 2-CdA of the peripheral blood lymphocytes induced by Bryo-1 is reflected by the elevated dCK:5'-NT ratio at the post-Bryo time point. Hence, to further understand the efficacy of 2-CdA, peripheral blood lymphocytes of the patient should be analyzed for dCK and 5'-NT activity 1 day or 2 days after the initiation of the 5-day infusion of 2-CdA.
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FOOTNOTES |
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1 Supported by NIH Grant CA79837, Grant 6323-99
from the Leukemia Society of America, and a grant from OrthoBiotech
Company. Flow cytometry was done in the Molecular and Cellular Imaging
and Analytical Cytometry Core Facility of Karmanos Cancer Institute and
Wayne State University (P30-CA22453-20). 
2 To whom requests for reprints should be
addressed, at Wayne State University School of Medicine, Lande
Building, Room 317, 550 East Canfield, Detroit, MI 48201.
3 The abbreviations used are: CLL, chronic
lymphocytic leukemia; Bryo-1, bryostatin 1; HC, hairy cell; HCL, HC
leukemia; 2-CdA, 2-chlorodeoxyadenosine; dCK, deoxycytidine kinase;
5'-NT, 5'-nucleotidase; PKC, protein kinase C; PARP, poly(ADP)ribose
polymerase.
Received 9/29/99; revised 1/17/00; accepted 1/18/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß. J. Biol. Chem., 265: 4033-4040, 1990.This article has been cited by other articles:
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  T. E. Battle and D. A. Frank STAT1 mediates differentiation of chronic lymphocytic leukemia cells in response to Bryostatin 1 Blood, October 15, 2003; 102(8): 3016 - 3024. [Abstract] [Full Text] [PDF]
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