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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2482-2490
Flavopiridol Induces Apoptosis of Normal Lymphoid Cells, Causes
Immunosuppression, and Has Potent Antitumor Activity In Vivo
Against Human Leukemia and Lymphoma Xenografts
By
Francisco Arguello,
Mark Alexander,
Judith A. Sterry,
Gabriela Tudor,
Erik M. Smith,
Naina T. Kalavar,
John F. Greene Jr,
William Koss,
C. David Morgan,
Sherman F. Stinson,
Timothy J. Siford,
W.
Gregory Alvord,
Richard L. Klabansky, and
Edward A. Sausville
From the Laboratory of Drug Discovery Research and Development,
Developmental Therapeutics Program Division of Cancer Treatment and
Diagnosis, the Science Application International Corporation, and Data
Management Services, Inc, National Cancer Institute-Frederick Cancer
Research and Development Center, Frederick, MD; the Division of
Clinical Laboratories, University of Rochester Strong Memorial
Hospital, Rochester, NY; and the Department of Pathology and Immunology
Section, Scott & White Clinic, Temple, TX.
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ABSTRACT |
Flavopiridol is a novel semisynthetic flavone derivative of the
alkaloid rohitukine. Flavopiridol is known to inhibit potently the
activity of multiple cyclin-dependent kinases. We have assessed its
effects on normal and malignant cells in preclinical animal models of
localized and disseminated human hematopoietic neoplasms. Flavopiridol,
when administered as daily bolus intravenous (IV) injections, produced
selective apoptosis of cells in the thymus, spleen, and lymph nodes,
resulting in atrophy of these organs. With the exception of the
intestinal crypts, apoptosis or tissue damage was absent in all other
organs investigated (kidneys, liver, lungs, bone/bone marrow, muscle,
and heart). Flavopiridol had a marked apoptotic effect documented by
DNA nick-end labeling, or DNA agarose gels in xenografts of human
hematopoietic tumors HL-60, SUDHL-4, and Nalm/6. After treatment with
7.5 mg/kg flavopiridol bolus IV or intraperitoneal on each of 5 consecutive days, 11 out of 12 advanced stage subcutaneous (s.c.) human
HL-60 xenografts underwent complete regressions, and animals remained
disease-free several months after one course of flavopiridol treatment.
SUDHL-4 s.c. lymphomas treated with flavopiridol at 7.5 mg/kg bolus IV for 5 days underwent either major (two out of eight mice) or complete (four out of eight mice) regression, with two animals remaining disease-free for more than 60 days. The overall growth delay was 73.2%. The acquired immunodeficiency syndrome-associated lymphoma AS283 showed no significant response when flavopiridol was used in
advanced s.c. tumors, but when treatment was initiated in early stages,
there was a complete regression of the early tumors, and a significant
overall growth delay (>84%). When flavopiridol was used in severe
combined immunodeficient mice bearing disseminated human acute
lymphoblastic leukemia Nalm/6 cells, there was 15-day prolongation in
survival (P = .0089). We conclude that flavopiridol greatly
influences apoptosis in both normal and malignant hematopoietic tissues. This activity was manifested in our study as a potent antileukemia or antilymphoma effect in human tumor xenografts, which
was dose and schedule dependent. These findings provide compelling
evidence for the use of flavopiridol in human hematologic malignancies.
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INTRODUCTION |
FLAVOPIRIDOL, previously designated
Behringwerke L86-8275,1,2 is a flavone synthetically
derived from the plant alkaloid rohitukine isolated from the leaves and
stems of Amoora rohituka,3 and later from
Dysoxylum binectariferum.4 Both plants are
indigenous to India, where they are widely used in traditional
medicine.5-7 Initial studies documented that flavopiridol reversibly inhibited the in vitro growth of MDA468 human breast carcinoma cells, a phenomenon that was associated with arrest of cells
in G1 or G2 phases of the cell cycle.2 This drug was subsequently shown to inhibit potently cyclin-dependent kinases (CDKs),
a family of kinases which govern progression of cells through the cell
cycle.8,9 Induction of G2 arrest by flavopiridol appears to
be caused by both direct inhibition of CDK1 and interference with the
regulatory phosphorylations of CDK1.10,11 The arrest of
cells in G1 by flavopiridol can be related to inhibition of both CDK2
and CDK4.12 Cocrystallization studies using a
des-chloro-flavopiridol and CDK213 have shown that the
aromatic portion of the compound binds to the hydrophobic
adenine-binding pocket of the adenosine triphosphate (ATP) site of
CDK2. Studies summarized by Sedlacek et al14 have
documented that higher concentrations of flavopiridol than those
associated with inhibition of CDKs can also inhibit a variety of other
kinases.
The antitumor activity of flavopiridol has been evaluated in vivo by
Czech et al1 using a variety of human solid-tumor cell
lines xenografted in the subrenal capsule and/or subcutaneous (s.c.) space of athymic nude mice. Their studies have included seven
human carcinoma cell lines of the lung, five of the colon, four of the
ovary, two of the breast, one of kidney, and one of the stomach, as
well as one glioma and one melanoma tumor cell lines. None of the
evaluated tumors underwent complete regressions, but 14 out of 21 tumor
cell lines responded to flavopiridol treatment with an average growth
delay of about 35% to 45%.1 More recently, Drees et
al15 have shown sensitivity of both soft agar colonies and
tumors in vivo from prostate cancer cells. Initial clinical studies
have commenced with a 72-hour continuous infusion because of the need
for frequent dosing in animal models to show cytostatic effect.16
While surveying the effect of flavopiridol in vitro on different cell
types, Parker et al17 noted that certain hematopoietic cells were notably sensitive to apoptosis. To extend our experience with flavopiridol's antitumor activity and in an effort to improve the
bioactivity of flavopiridol against malignant tumors in vivo, we
examined the dose-limiting toxicity and antitumor effect of flavopiridol in murine hosts bearing hematopoietic human tumor xenografts. In this report, we document that flavopiridol, when administered as a daily intravenous (IV) bolus administration, produces
selective apoptosis of cells of the thymus, spleen, and lymph nodes,
and has marked antitumor activity in several localized or disseminated
human hematologic tumor xenografts in severe combined immunodeficient
(SCID) or nude mice.
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MATERIALS AND METHODS |
Drugs and treatment into animals.
Flavopiridol (NSC: 649890) was synthesized and supplied to us by
Behringwerke AG (Marburg, Germany). For use in laboratory animals,
flavopiridol was dissolved in a pyrogen-free, sterile 0.9% NaCl
solution (McGaw, Ontario, Canada) containing 1% or 5% dimethyl
sulfoxide (DMSO). The flavopiridol solution was sterilized by
filtration using a pyrogen-free 0.22µ sterile filter.
For daily injections, a freshly prepared solution was stored at 4°C.
At 4°C, flavopiridol tended to precipitate, thus before injection the
flavopiridol solution was warmed up at 37°C and vigorously agitated
in a vortex. Flavopiridol was administered as a bolus IV or
intraperitoneal (IP), or as a continuous infusion s.c. using osmotic
mini-pumps (ALZA, Palo Alto, CA), as previously described in
detail.18 The antibiotic cephalexin (Dista Products & Eli Lilly, Indianapolis, IN) was purchased as a pediatric oral suspension (125 mg/5 mL). Except for the animals treated by continuous infusion, all flavopiridol and control animals received 5 mL of cephalexin in 250 mL of drinking water, left at libitum, starting 24 hours before, during, and up to 48 hours after flavopiridol treatment.
Animals.
Female athymic nude NCr-nu/nu (Taconic Farm, Germantown, NY), SCID/NCr,
immunocompetent C57BL/6 NCr, and CD2F1 mice (National Cancer Institute
[NCI]-Animal Production Program, Frederick, MD), ages 6 to 14 weeks,
were used in our studies. Animals were maintained according to the
guidelines established by the National Institutes of Health.
Tumor cell lines and injection of tumor cells into animals.
The human promyelocytic leukemia HL-60, human B-cell follicular
lymphoma SUDHL-4, and acquired immunodeficiency syndrome (AIDS)-related human B-cell lymphoma AS283 were obtained from the Tumor Cell Repository of the Division of Cancer Treatment, Diagnosis and Centers,
NCI-Frederick Cancer Research and Development Center (Frederick, MD).
The human acute lymphoblastic leukemia Nalm/619 was the
generous gift of Dr Daniel H. Ryan (University of Rochester Medical
Center, Rochester, NY). All cell lines were grown in RPMI-1640 medium
with 10% fetal calf serum and L-glutamine, and the cells were
maintained using standard tissue culture conditions. For the production
of s.c. tumors, 1 × 107 cells in 0.3 mL of medium without
serum were inoculated in the right flank of mice. To produce
disseminated disease in SCID mice, Nalm/6 and AS283 cells were injected
IV in a dose of 5 × 106 or 1 × 107 cells in
0.1 mL of medium without serum, as previously described in
detail.19
Toxicologic studies.
To evaluate the effect of flavopiridol on the normal function of
diverse tissues or organs, blood chemistry analyses were performed with
the Abbot Vision System (Abbott Laboratories, Abbott Park, IL) and
T-Stat portable analyzer (Sensor Devises, Inc, Waukesha, WI). For white
cell counts, the red cells were lysed first by mixing 10 µL of blood
with 440 µL of a 2% solution of acetic acid. Leukocytes were counted
manually using a hemocytometer. Differential leukocyte counts were made
by using standard staining with Wright and Giemsa. For histological
examination of the bone marrow, the bones of the legs were decalcified,
as described previously.20
Assays of immunosuppression.
Human peripheral blood lymphocytes (PBL) were obtained from different
healthy donors by using standard density gradient centrifugation techniques. PBL were pretreated with different concentrations of
flavopiridol for 24 hours at 37°C, washed, and assayed in their ability to incorporate 3H-methyl thymidine21
after stimulation with a variety of mitogens, including
phytohemagglutinin-P (PHA-P), concanavalin A (CON-A), and pokeweed
mitogen (PWM), all acquired from Sigma Chemical Co (St Louis, MO). The
mouse anti-human CD3 monoclonal antibody (MoAb) (clone HIT3a;
Pharmingen, San Diego, CA) was used as a selective mitogen of T cells.
Flavopiridol-treated PBL were stimulated with serially diluted mitogens
in microtiter plate format for 72 hours, and triplicate samples for
each flavopiridol and mitogen concentration were pulsed with
3H-methyl thymidine (ICN Biomedicals, Inc, Costa Mesa, CA),
at 1 µCi/well for the final 20 hours of the 72-hour incubation
period. The degree of stimulation by mitogens in flavopiridol-treated and control cells was determined using standard scintillation counting.
Appropriate controls were included in all assays. These consisted of
untreated PBL (with and without mitogens) and the use of actinomycin D
which inhibits replicative responses of PBL.21,22
Pharmacokinetic studies.
Pharmacokinetic studies of flavopiridol were conducted after five daily
bolus IV injections of 5 mg/kg flavopiridol in male, CD2F1 mice. The
dose volume was 1 µL/g body weight. After the first, third and fifth
dose, groups of three mice were exsanguinated via the suborbital plexus
at close intervals from 2 minutes through 8 hours after injection of
flavopiridol. For 3-day continuous s.c. infusions, we used
osmotic minipumps to release flavopiridol at a rate of 0.9 mg/kg/h during three consecutive days. Blood was collected and
processed as above from groups of three mice, 24, 48, 72, and 96 hours
after pump implantation. Aliquots (50 µL) of each plasma sample were
prepared for assay by the addition of 50 µL of 0.0125 mol/L sodium
borate buffer (pH 8.0), followed by extraction with 7.5 mL of
t-butylmethyl ether. The organic phase was removed, evaporated to
dryness using a centrifugal vacuum concentrator (Jouan, Inc,
Winchester, VA). The residue was dissolved in 250 µL of mobile phase
(see below), and 200 µL were analyzed by high performance liquid
chromatography. The analytical system consisted of a Hewlett-Packard
(Palo Alto, CA) model 1050 pump, autosampler, and ultraviolet detector
with associated software for system control and spectrum analysis. The
system was equipped with a stainless steel 4.6 × 150 mm column
containing J'Sphere H-80 packing (YMC, Inc, Wilmington, NC).
Chromatography was effected with an isocratic eluent at 1.0 mL/min
using a mobile phase consisting of acetonitrile-0.05 mol/L, pH 3.0 ammonium formate buffer (25:75, vol/vol). Ultraviolet
detection at a wavelength of 267 nm was used. Plasma standard curves
consisting of 5 standards were prepared and processed identically to
samples. For pharmacokinetic analysis, plots of flavopiridol
concentration as a function of time were constructed using the
geometric mean of the plasma concentrations and the mean of the time
intervals for each time point.
Evaluation of antitumor response in animals with localized s.c.
tumors.
Subcutaneous tumors were measured with caliper, and the weight of the
animals recorded at least three times a week. Tumor weight was
estimated from caliper measurements of two perpendicular dimensions of
the tumor in millimeters using the
formula:
To
calculate the percent growth delay, the median tumor weight (MTW) for
the treated group was divided by the MTW of the control group on each
individual day of the experiment in which tumor weights were measured
during the course of the experiment. The results were then averaged and
multiplied by 100 to transform a proportion into a percent. The percent
growth delay was then computed with this
formula:
Complete
tumor regression, represented by the disappearance of an existing
measurable tumor in the animal, is reported as a ratio of the number of
complete regressions observed to the total number of animals in the
group. In animals injected with leukemia and lymphoma cells IV, in
which tumor growth is not amenable to direct observation, blood samples
were obtained at timed intervals to quantify total levels of lactic
dehydrogenase (LDH) and LDH isoenzymes, as described by Arguello
et al.19
Documentation of drug-induced apoptosis.
To document drug-induced apoptosis in situ, we used ApopTag In
Situ Apoptosis Kit (Oncor, Gaithersburg, MD) immunostaining on
paraffin sections according to the manufacturer's instructions, and
DNA was also extracted from frozen tissue by homogenization for 5 minutes at 37°C, followed by DNA isolation as described by
Distelhorst et al.23 Thirty micrograms of DNA was then
analyzed on a 0.8% agarose gel.
Statistical analysis.
The statistical test for growth delay was accomplished as follows. Each
treatment condition in a group of experiments was compared with its
appropriate control group by taking the ratio of the MTW of the treated
group to the MTW of the control group on a particular day, and
comparing over all days how the ratios of the treated to control median
tumor weights differ from 100%. Under the null hypothesis of no growth
delay, this random variable has the expectation of unity across the
days of the experiment. A one sample t-test was performed to
determine whether the percent growth delay was significant.
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RESULTS |
Effect of flavopiridol on normal cells and tissues.
Histological evaluation of different organs (heart, lung, kidney,
spleen, thymus, bone/bone marrow, and small intestines) of
immunocompetent C57BL/6 mice treated with the maximal tolerable dose
(MTD) in this mouse species (10 mg/kg bolus IV for 4 consecutive days)
showed a series of abnormalities when compared with control nontreated
animals. The spleen of flavopiridol-treated mice were smaller, with
markedly diminished numbers of lymphocytes in white pulp areas, and
follicular centers were completely absent (Fig 1a and
b). The thymus in the
treated animals also showed a marked depletion of lymphoid cells, and
the organ was about 10% to 20% the size of that of control animals by
the fourth day of flavopiridol treatment (Fig 1c and d). ApopTag
immunohistological analysis of the thymus and spleen from mice treated
with flavopiridol showed increased apoptosis of lymphoid cells in these
organs (Fig 1e through h). Peripheral lymph nodes in the intestines
(Peyer's patches) were significantly depleted of lymphoid cells and
the follicular centers were also absent, and some apoptotic epithelial cells were seen in the intestinal crypts (data not shown). These intestinal changes were identical to that described in patients with
severe immunodeficiencies, grade I acute graft versus host disease, or
T-cell defects.24,25 None of the vehicle-treated control
animals had these lesions.
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| Fig 1.
Effect of flavopiridol on normal cells and
tissues. (a) Histological section (10×) of the spleen of an untreated
immunocompetent C57BL/6 mouse, showing a rich population of lymphocytes
forming the germinal centers and marginal zone of the folliculi of the white pulp, surrounded by the blood-filled sinusoids of the red pulp.
(b) Histological section (10×) of the spleen of an immunocompetent mouse 96 hours after initiation of treatment with daily IV bolus injection of flavopiridol, showing a marked depletion of lymphocytes, and only remnants of the white pulp. (c) Histological section of the
thymus (10×) of a nontreated immunocompetent mouse showing the
densely populated cortex by lymphocytes, surrounding the medullary areas of the lobules. (d) Histological section of a thymus (10×) of a
flavopiridol-treated mouse showing an atrophic thymus, in which most
lymphoid areas have disappeared. (e) ApopTag immunohistochemistry of
the spleen (50×) of a nontreated mouse showing the rare presence of
apoptotic brown-stained cells. (f) ApopTag immunohistochemistry of the
spleen (50×) of a mouse 48 hours after initiation of treatment with
flavopiridol, showing multiple brown-stained apoptotic lymphocytes in
the follicular centers of the white pulp. (g) ApopTag immunostaining of
the thymus (10×) of a nontreated immunocompetent mouse showing the
lack of apoptosis in the lymphocyte-formed cortex. (h) ApopTag immunostaining of the thymus (10×) of a flavopiridol-treated mouse, showing a brown-stained, atrophic cortex caused by the death of thymocytes through apoptosis.
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A dose-related leukopenia was also observed. Peripheral leukocyte
counts in three immunocompetent C57BL/6 mice treated with 5 mg/kg IV
during 5 consecutive days developed a moderate leukopenia (average,
5,512/µL; n = 3; day 4), when compared with their
leukocyte counts before initiation of treatment (average, 12,599/µL).
When other C57BL/6 mice were treated with 10 mg/kg for three
consecutive days, the leukopenia was more pronounced (average,
2,054/µL; n = 3; day 3), as compared with leukocyte counts before
treatment (average, 9,506/µL). However, 24 hours after cessation of
treatment, there was a rebound in the number of leukocyte counts to
19,000/µL average (~50% neutrophils). Thus, persistent leukopenia
is not a feature of flavopiridol action in mice. The bone marrow
sections of femurs and tibias examined from two out of three
flavopiridol-treated mice were not significantly different from
controls, whereas one mouse showed depletion of both red and white cell
elements.
Despite extensive blood chemistry analyses to evaluate a variety of
physiological functions, no abnormalities were observed, even when
immunodeficient nude mice were treated with lethal doses of
flavopiridol at 10 mg/kg bolus IV daily for 3 or more consecutive days.
However, histological examination of some organs showed bacterial
colonies. This prompted us to examine immunocompetent C57BL/6 mice
treated with a high dose of flavopiridol bolus IV (10 mg/kg/d during 7 days) for evidence of sepsis, while we tested in another group of mice
whether the prophylactic use of a broad-spectrum antibiotic,
cephalexin, would prevent the suspected septicemia. We found that four
out of five mice treated with flavopiridol alone developed mixed
positive hemocultures with Escherichia coli, Staphylococcus
aureus, and S saprophyticus, whereas only one
out of five mice treated with flavopiridol plus cephalexin developed bacteremia by E coli. After that study, all animals treated
with flavopiridol and their respective control mice received cephalexin in drinking water starting 24 hours before initiation of treatment with
flavopiridol, and ending 48 hours after the last day of IV flavopiridol
treatment. The prophylactic use of cephalexin allowed us to increase
our previous MTD in nude mice of 5 mg/kg bolus IV during 5 consecutive
days to 7.5 mg/kg IV for 5 days.
Immunosuppressive effects of flavopiridol.
The prominent effect of flavopiridol on lymphoid cell elements in
normal, nontumored animals (Fig 1), raised the possibility that the
drug could cause defects in immune cell function in response to
antigenic stimulation. Thus, we assessed the potential for flavopiridol
to interdict mitogen-stimulated lymphocyte proliferation. Substantial
decreases in thymidine incorporation were observed in
flavopiridol-pretreated PBL incubated with T-specific mitogens, PHA and
CON-A, and CD3 MoAbs, as well as B-specific mitogen, PWM (Table
1). Table 1 also shows that this
suppressive effect is dose dependent. The threshold inhibitory
flavopiridol concentration was shown within the range of 100 to 250 nmol/L in PBL, in which cell viability remained above 80%. At these
concentrations, previous studies2 have shown little effect
on 3[H] thymidine incorporation into exponentially
growing cells until cell cycle arrest is established.
Antitumor effect of flavopiridol in immunodeficient nude and SCID
mice bearing localized (s.c.) human lymphohematopoietic tumors.
Preliminary studies in our lab had shown that 5 mg/kg bolus IV during 5 days, without antibiotics, was the MTD of flavopiridol in nude mice.
This MTD produced complete regressions of large s.c. HL-60 xenografts
(five out of five) which lasted for about 15 days in three animals, and
two remained disease-free for months (data not shown). Table
2 summarizes the treatment of several xenograft models
of mice bearing large s.c. HL-60 tumors with 7.5 mg/kg/d × 5 by IV
injection plus the prophylactic use of cephalexin. This treatment
resulted, except for one animal, in complete regressions. When the same
dose was given IP, all six tumors also underwent complete regressions.
All animals with complete regressions have remained tumor-free for more
than 90 days. On the other hand, s.c. SUDHL-4 lymphoma treated with
flavopiridol at 7.5 mg/kg bolus IV for 5 days plus antibiotic underwent
either 50% (two out of eight mice) or 100% (four out of eight mice)
regression, but only two animals remained disease-free for more than 60 days, with an overall growth delay of greater than 73.2%. The
AIDS-related AS283 lymphoma did not experience tumor regressions when
flavopiridol was used in large s.c. tumors, other than an evident
growth delay of 45.8%. However, when the treatment was implemented in
early stages (tumors ~2 to 4 mm in diameter), there was a complete
regression of the early tumors and a significant growth delay of
greater than 84%. In experiments not shown, we treated athymic mice
bearing human HCT15 colon and U251 glioma tumors with the same regimen which caused prominent responses in hematopoietic tumors. No tumor regressions were observed; however, there was a modest (37.8% to
44.7%) growth delay, concordant with prior experience with flavopiridol in solid tumor xenografts.
To mimic schedules used in initial clinical trials with flavopiridol,
in which the drug is administered by continuous infusion during 72 hours,16 we implanted s.c. 3-day continuous infusion osmotic pumps in mice bearing the flavopiridol-sensitive
cell line HL-60. We found that a 72-hour continuous infusion of
flavopiridol at doses as high as 0.9 mg/kg/h (61.4 mg/kg/72 h) had at
best very modest effects on the HL-60 tumors.
Flavopiridol-mediated apoptosis of tumor cells.
Recent in vitro studies have shown that flavopiridol has the ability to
induce apoptosis of human lymphoma cells.17 To examine whether apoptosis could be shown in tumors after treatment in vivo with
flavopiridol, we removed s.c. HL-60 tumors after treatment with
flavopiridol at 7.5 mg/kg IV every other day × 4. Immunohistochemistry staining for apoptotic cells in situ
showed by 96 hours evidence of extensive
apoptosis (Fig 2a through d), which was
first evident by 24 hours (data not shown). A DNA "ladder" was
observed in SUDHL-4 tumors 72 hours after initiation of treatment (Fig
2e). Of interest, AS283 tumors, which did not show evidence of
persistent regressions (only growth delays), did not show evidence of
extensive apoptosis by immunohistochemistry nor agarose gels, despite
the obvious reduction in tumor mass (data not shown).
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| Fig 2.
Apoptotic death of human promyelocytic leukemia HL-60
cells in vivo after flavopiridol therapy. (a) Histological section of a
s.c. HL-60 tumor (100×) removed from a nontreated control nude mouse,
showing a large number of predominantly viable and dividing malignant
blasts. (b) ApopTag immunostaining of the same HL-60 tumor as (a),
showing the sporadic presence of brown-stained cells indicative of rare
spontaneous apoptosis. (c) Histological section of an HL-60 tumor
(100×) 96 hours after initiation of treatment with daily IV bolus
injection of flavopiridol, showing multiple fragments of condensed
chromatin ("apoptotic bodies") indicative of cell death through
apoptosis. (d) ApopTag immunostaining of an HL-60 tumor 96 hours after
initiation of treatment with bolus IV flavopiridol. Multiple cells
densely stained brown indicate DNA fragmentation. (e) Agarose gel
analysis of DNA isolated from SUDHL-4 nontreated lymphoma (lane 2), and
DNA isolated from a tumor 72 hours after initiation of treatment with
flavopiridol (lane 3), which shows the typical "ladder" pattern
of internucleosomal cleavage of DNA into multiples of 180 to 250 bp.
Lane 1 includes DNA molecular mass markers.
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Antitumor effect of flavopiridol in SCID mice bearing disseminated
human lymphohematopoietic tumors.
Because localized s.c. tumors do not really reflect the systemic nature
of human leukemia, or advanced stages of lymphomas in humans, SCID mice
received human acute lymphoblastic leukemia (ALL) Nalm/6 cells, or
AS283 human lymphoma cells IV to produce systemic
disease.19 When flavopiridol was used in SCID mice bearing
disseminated human ALL Nalm/6 cells, at a dose of 7.5 mg/kg bolus IV
during five consecutive days from days 3 to 7 after tumor cell
injection, and repeated again at days 17 to 21, there was 15-day
prolongation in survival (P = .0089) (Fig
3a). Also, serum levels of total LDH in
nontreated mice increased progressively over time, reaching total LDH
levels as high as 43,000 U/L, whereas LDH in mice treated with
flavopiridol remained within the basal levels (~2,000 U/L) for almost
the same 30-day period. We have recently shown that serum levels of
total LDH, human-specific LDH isoenzymes, and NMP 41/7 are highly
reliable serum markers to monitor the progression of human leukemias in
SCID mice.19 An enhanced survival was also seen in SCID
mice bearing systemic AIDS-related lymphoma AS283. Whereas control
nontreated animals developed paralysis approximately 25 days after
injection of AS283 cells because of the involvement of the central
nervous system (meningeal infiltration), animals treated with
flavopiridol from days 3 to 7 after tumor cell injection had an
improvement in survival, as compared with controls
(P = .0027) (Fig 3b). The SUDHL-4 did not produce systemic
disease in SCID mice after IV injection of 107 cells/mouse,
and HL-60 produced diseases in a very unpredictable fashion; thus,
these cell lines could not be used as models of human systemic disease.
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| Fig 3.
Effect of flavopiridol therapy in systemic leukemia and
lymphoma xenografts. (a) Kaplan and Meier survival curve of SCID mice bearing systemic human acute lymphoblastic leukemia (Nalm/6) treated with flavopiridol 7.5 mg/kg IV every other day × 5 at days 3 to 7, and repeated again at days 17 to 21 posttransplantation of cells ( ).
Control mice ( ) received the vehicle 1% DMSO in NaCl. (b) Kaplan
and Meier survival curve of SCID mice bearing systemic human AS283
human lymphoma after one 5-day cycle of flavopiridol bolus IV therapy
from days 3 to 7 posttransplantation of cells (). Control mice
received the vehicle ().
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Pharmacokinetic studies.
To correlate the pharmacological behavior of flavopiridol with the
occurrence of drug-induced apoptosis, we studied nontumored mice
treated in an identical fashion to animals with s.c. or disseminated tumors. There was no significant difference in the plasma
concentration-time profiles obtained after 1, 3, or 5 daily injections
of 5 mg/kg flavopiridol, so the groups were combined for the purpose of
pharmacokinetic analysis. After IV injection of flavopiridol, plasma
concentrations declined in a biexponential manner from maximum levels
after 2 minutes of approximately 7 µmol/L to approximately 0.1 µmol/L after 8 hours (Fig 4). The
half-life for the initial phase was 18 minutes, and for the terminal
phase (biological half-life) was 158 minutes. A comparison of the
apparent volumes of distribution for the central compartment (1.4 L/kg)
and for the whole body (7.1 L/kg) suggest that flavopiridol is highly
distributed into the tissues. The plasma clearance was 31 mL/min/kg. In
our s.c. infusion studies, of the 12 plasma samples collected between
24 and 96 hours from mice receiving 0.9 mg/kg/h flavopiridol, the drug
was not detectable in four (2, 24-hour; 1, 48-hour; 1, 72-hour; and 1, 96-hour samples), suggesting possible pump failure. The remaining seven
samples had a mean flavopiridol concentration of 427 nmol/L (range, 175 to 907 nmol/L).
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| Fig 4.
Plasma concentrations of flavopiridol observed in
combined groups receiving 1, 3, and 5 daily IV injections of 5 mg/kg in mice. The experimental data points (), representing the geometric mean of the assayed plasma concentrations for each time point, and the
best-fit curve generated by nonlinear regression analysis, are shown.
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DISCUSSION |
We have found that flavopiridol has a marked proapoptotic effect on
normal lymphoid organs, such as spleen, thymus, and intestinal lymphoid
tissues, when administered into animals using a daily bolus IV or IP
schedule. With the exception of the intestinal crypt mucosa, there was
no evidence of apoptosis or tissue damage in several other nonlymphoid
organs studied. Similarly, good antitumor activity characterized as
tumor regression with cell apoptosis was observed in human tumors of
lymphohematopoietic origin. These include HL-60 and SUDHL-4 s.c.
xenografts, and Nalm/6 and AS283 disseminated disease models. This
effect correlated with exposure of the animals to transient plasma peak
concentrations of the drug of 7 to 9 µmol/L over each of five days.
Evidence of impaired immunologic response to several mitogens on the
part of flavopiridol-treated human lymphocytes has also been
documented.
The ability of a drug to selectively affect cells of a certain lineage
is determined by many factors, including selective binding/uptake of
the drug, increased or specific presence of the drug's molecular
target, and/or intrinsic metabolic and detoxifying systems
present in the cells. Flavonoid compounds have been previously shown to
interact with normal and malignant hematopoietic
cells,26-29 through apparent specific binding to nuclear
"receptors" referenced as type II estrogen binding sites (type II
EBS).30,31 Larocca et al27,28 found that all
blast cells from seven patients with ALL and 16 with acute myeloblastic
leukemia expressed variable amounts of type II EBS, ranging between
3,109 and 239,450 sites/cell. They also discovered that the growth of
these cells could be inhibited by compounds that interact with type II
EBS, mainly estrogens and plant flavonoids.26-29 The
relation of type II EBS as potential flavonoid receptors to protein
kinases may be of interest to consider. Although flavopiridol most
potently affects CDKs,10-12 at the concentrations achieved
here many other protein kinases, eg, protein kinase
C,14 may be affected. Other targets,
especially those with nucleotide binding sites, may also exist, but
these have yet to be defined.
The organs affected by flavopiridol, eg, spleen, lymph nodes, and
thymus, are formed predominantly by lymphoid cells in resting, G0 phase, unless they are stimulated by
mitogens.32-34 Thus, induction of apoptosis in these
populations indicate a capacity of the drug to affect noncyclin cells
of certain lineage. Similar observations were recently made by Bible
and Kaufmann,35 who found that confluent noncycling A549
human lung carcinoma, as well as cells A549 arrested in G1 after
aphidicolin treatment, could be killed with high doses of flavopiridol
(eg, >500 nmol/L). The ability of flavopiridol to kill nondividing
cells raises the following possibilities: (1) Flavopiridol at high
concentrations may have an additional molecular target(s), other than
CDKs. This possibility is reinforced by the capacity of the drug to
inhibit other kinases when used at concentrations greater than 1 µmol/L.14 (2) CDKs may have important unsuspected cell
functions in resting cells. For example, Perkins et al36
have recently shown that CDK is a component of the p300-associated
regulatory apparatus that regulates the transcriptional activation of
nuclear factor kappaB, a factor that is responsive to specific
cytokines and stress and is often activated in association with cell
damage and growth cell arrest in eukaryotes. Nevertheless, future
experiments must address the basis for flavopiridol-induced apoptosis
of normal and malignant hematopoietic cells.
We have also observed that apoptosis was readily apparent in some
tumors, eg, HL-60, SUDHL-4, or Nalm/6, whereas the partial tumor
regression of AS283 lymphoma tumors occurred without evidence of
apoptosis (DNA fragmentation). Similar observations were made in vitro
by Bible and Kaufmann.35 They found that
cultured A549 human lung carcinoma cells, in contrast to HL-60 cells,
at 72 hours after exposure to greater than 300 nmol/L flavopiridol died without the classic changes of apoptosis. However, they pointed out
that other well-established apoptotic agents, eg, topotecan and
etoposide, also failed to induce apoptosis of A549 cells. These studies
highlight the fact that after a cytocidal stimulus, the occurrence of
apoptosis seems to be determined not by universally applicable actions
of the drug, but by response properties of the cells, particularly
their ability to engage in "programmed cell death." Others have
pointed out that it may not be coincidence that chemotherapy in humans
has been successful in tumors which have risen from the types of cells
that can readily die by apoptosis, eg, hematopoietic and germ
cells.37 Flavopiridol may be uniquely suited to trigger
this process in certain cell types.
We found that continuous infusion of flavopiridol for 3 days resulted
in plasma levels (average, 427 nmol/L) that exceed the in vitro
IC50 reported for most human tumor cells tested (20 nmol/L to 200 nmol/L), including those in the NCI's 60-cell line in vitro screen panel.14 However, this concentration resulted in
very modest antitumor effect in animals bearing HL-60. The best
antitumor effect in xenografted animals was observed after daily bolus
IV or IP administration of flavopiridol that resulted in peak plasma levels of about 7 µmol/L, followed by a progressive decline to approximately 100 nmol/L in 8 hours. Thus, relatively short-lived, but
repetitive high plasma levels of flavopiridol in the µmol/L range
seem to be an effective way to produce the maximum antitumor effect
with flavopiridol. Thus, efforts to achieve this concentration versus
time relationship in clinical trials should be pursued. Current
clinical trials with flavopiridol have consistently achieved concentrations in the range of 500 to 1,000 nmol/L with a continuous infusion scheme.16 The apparent need for high levels of
flavopiridol in vivo, as compared with those required in vitro, may be
dependent on a variety of factors, including high concentrations of
competing ATP in vivo, binding proteins in blood and tissues, rapid
metabolism into inactive compounds, presence of pharmacological
barriers, among other factors.38
Our preliminary studies indicate that flavopiridol has the potential
for immunosuppressive activity. Human lymphocytes pretreated in vitro
with flavopiridol were unable to respond to well-established mitogens
(Con-A, PHA-P, PWM, and anti-CD3 antibodies), as evidenced in this
study by their inability to incorporate radioactive thymidine. Indeed,
the results of our preliminary immunologic studies suggest a broad
immunosuppressive effect on both T and B lymphocytes by flavopiridol.
The recently concluded Phase I trial of flavopiridol16 did
not suggest neutropenia as a noted side effect at the maximal tolerated
dose. However, that trial used continuous infusion and did not achieve
the transient high drug concentrations seen here. Our results raise the
possibility that flavopiridol may have immunosuppressive activity in
humans when administered in an IV bolus schedule.
The potential use of flavopiridol in the treatment of human hematologic
tumors is clear. Flavopiridol has the ability to produce cure of
animals bearing large HL-60 tumor masses. Also, SUDHL-4 or AIDS-related
lymphoma AS283 tumors underwent transient, but complete regressions,
and/or had a growth delay of greater than 70% after
flavopiridol treatment. Flavopiridol also produced a substantial and
statistically significant prolongation in survival of SCID mice bearing
disseminated AIDS-related lymphoma AS283 and human ALL Nalm/6 cells. We
believe that the results obtained in these preclinical studies using
athymic nude and SCID mice bearing human leukemia and lymphoma
xenografts may predict the therapeutic potential of flavopiridol in
humans with hematologic malignancies.
| |
NOTE ADDED IN PROOF |
Quantitation of lymphocytes in results from the recently concluded
Phase I trial in humans with a 72-hour infusion does suggest dose-related lymphopenia (A. Senderowicz, unpublished results), but not
neutropenia.
| |
FOOTNOTES |
Submitted July 23, 1997;
accepted November 17, 1997.
Address reprint requests to Edward A. Sausville, MD, PhD, Developmental
Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, EPN/843 MSC 7458, 6130 Executive Blvd,
Rockville, MD 20852.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors express their gratitude to Drs Adrian M. Senderowicz, Alan
D. Harmon, Alan J. Bitonti, Jennifer A. Dumont, and Barry M. Markaverich for their helpful discussions and suggestions during the
performance of this study. Our special thanks to Salvatore Gangliano
for his assistance in the measurement of total LDH.
| |
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August 1, 2003;
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T. Nakanishi, J. E. Karp, M. Tan, L. A. Doyle, T. Peters, W. Yang, D. Wei, and D. D. Ross
Quantitative Analysis of Breast Cancer Resistance Protein and Cellular Resistance to Flavopiridol in Acute Leukemia Patients
Clin. Cancer Res.,
August 1, 2003;
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C. T. Kouroukis, A. Belch, M. Crump, E. Eisenhauer, R. D. Gascoyne, R. Meyer, R. Lohmann, P. Lopez, J. Powers, R. Turner, et al.
Flavopiridol in Untreated or Relapsed Mantle-Cell Lymphoma: Results of a Phase II Study of the National Cancer Institute of Canada Clinical Trials Group
J. Clin. Oncol.,
May 1, 2003;
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[Abstract]
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J. Brown, J. O'Prey, and P.R. Harrison
Enhanced sensitivity of human oral tumours to the flavonol, morin, during cancer progression: involvement of the Akt and stress kinase pathways
Carcinogenesis,
February 1, 2003;
24(2):
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[Abstract]
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R. A. Messmann, C. D. Ullmann, T. Lahusen, A. Kalehua, J. Wasfy, G. Melillo, I. Ding, D. Headlee, W. D. Figg, E. A. Sausville, et al.
Flavopiridol-related Proinflammatory Syndrome Is Associated with Induction of Interleukin-6
Clin. Cancer Res.,
February 1, 2003;
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[Abstract]
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M. Alonso, C. Tamasdan, D. C. Miller, and E. W. Newcomb
Flavopiridol Induces Apoptosis in Glioma Cell Lines Independent of Retinoblastoma and p53 Tumor Suppressor Pathway Alterations by a Caspase-independent Pathway
Mol. Cancer Ther.,
February 1, 2003;
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[Abstract]
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N. R. Wall, D. S. O'Connor, J. Plescia, Y. Pommier, and D. C. Altieri
Suppression of Survivin Phosphorylation on Thr34 by Flavopiridol Enhances Tumor Cell Apoptosis
Cancer Res.,
January 1, 2003;
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[Abstract]
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Y. Ma, W. D. Cress, and E. B. Haura
Flavopiridol-induced Apoptosis Is Mediated through Up-Regulation of E2F1 and Repression of Mcl-1
Mol. Cancer Ther.,
January 1, 2003;
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[Abstract]
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L. M. Schang
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December 1, 2002;
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A. R. Tan, D. Headlee, R. Messmann, E. A. Sausville, S. G. Arbuck, A. J. Murgo, G. Melillo, S. Zhai, W. D. Figg, S. M. Swain, et al.
Phase I Clinical and Pharmacokinetic Study of Flavopiridol Administered as a Daily 1-Hour Infusion in Patients With Advanced Neoplasms
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[Abstract]
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C. Yu, G. Krystal, P. Dent, and S. Grant
Flavopiridol Potentiates STI571-induced Mitochondrial Damage and Apoptosis in BCR-ABL-positive Human Leukemia Cells
Clin. Cancer Res.,
September 1, 2002;
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[Abstract]
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A. M. Senderowicz
The Cell Cycle as a Target for Cancer Therapy: Basic and Clinical Findings with the Small Molecule Inhibitors Flavopiridol and UCN-01
Oncologist,
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[Abstract]
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L. M. Schang, A. Bantly, M. Knockaert, F. Shaheen, L. Meijer, M. H. Malim, N. S. Gray, and P. A. Schaffer
Pharmacological Cyclin-Dependent Kinase Inhibitors Inhibit Replication of Wild-Type and Drug-Resistant Strains of Herpes Simplex Virus and Human Immunodeficiency Virus Type 1 by Targeting Cellular, Not Viral, Proteins
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June 27, 2002;
76(15):
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[Abstract]
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Y. A. Elsayed and E. A. Sausville
Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins
Oncologist,
December 1, 2001;
6(6):
517 - 537.
[Abstract]
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B. Hagenauer, A. Salamon, T. Thalhammer, O. Kunert, E. Haslinger, P. Klingler, A. M. Senderowicz, E. A. Sausville, and W. Jäger
In Vitro Glucuronidation of the Cyclin-Dependent Kinase Inhibitor Flavopiridol by Rat and Human Liver Microsomes: Involvement of UDP-Glucuronosyltransferases 1A1 and 1A9
Drug Metab. Dispos.,
April 1, 2001;
29(4):
407 - 414.
[Abstract]
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R. W. Robey, W. Y. Medina-Pérez, K. Nishiyama, T. Lahusen, K. Miyake, T. Litman, A. M. Senderowicz, D. D. Ross, and S. E. Bates
Overexpression of the ATP-binding Cassette Half-Transporter, ABCG2 (MXR/BCRP/ABCP1), in Flavopiridol-resistant Human Breast Cancer Cells
Clin. Cancer Res.,
January 1, 2001;
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[Abstract]
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R. J. Klasa, A. F. List, and B. D. Cheson
Rational Approaches to Design of Therapeutics Targeting Molecular Markers
Hematology,
January 1, 2001;
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[Abstract]
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T. V. Achenbach, E. P. Slater, H. Brummerhop, T. Bach, and R. Müller
Inhibition of Cyclin-Dependent Kinase Activity and Induction of Apoptosis by Preussin in Human Tumor Cells
Antimicrob. Agents Chemother.,
October 1, 2000;
44(10):
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[Abstract]
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T. V. Achenbach, R. Müller, and E. P. Slater
Synergistic Antitumor Effect of Chemotherapy and Antisense-mediated Ablation of the Cell Cycle Inhibitor p27KIP-1
Clin. Cancer Res.,
August 1, 2000;
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[Abstract]
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S. Kitada, J. M. Zapata, M. Andreeff, and J. C. Reed
Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia
Blood,
July 15, 2000;
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[Abstract]
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K. C. Bible, R. H. Bible Jr., T. J. Kottke, P. A. Svingen, K. Xu, Y.-P. Pang, E. Hajdu, and S. H. Kaufmann
Flavopiridol Binds to Duplex DNA
Cancer Res.,
May 1, 2000;
60(9):
2419 - 2428.
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A. M. Senderowicz and E. A. Sausville
Preclinical and Clinical Development of Cyclin-Dependent Kinase Modulators
J Natl Cancer Inst,
March 1, 2000;
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376 - 387.
[Abstract]
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L. M. Leoni, E. Hamel, D. Genini, H. Shih, C. J. Carrera, H. B. Cottam, and D. A. Carson
Indanocine, a Microtubule-Binding Indanone and a Selective Inducer of Apoptosis in Multidrug-Resistant Cancer Cells
J Natl Cancer Inst,
February 2, 2000;
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217 - 224.
[Abstract]
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K. C. Bible, S. A. Boerner, K. Kirkland, K. L. Anderl, D. Bartelt Jr., P. A. Svingen, T. J. Kottke, Y. K. Lee, S. Eckdahl, P. G. Stalboerger, et al.
Characterization of an Ovarian Carcinoma Cell Line Resistant to Cisplatin and Flavopiridol
Clin. Cancer Res.,
February 1, 2000;
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661 - 670.
[Abstract]
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W. M. Stadler, N. J. Vogelzang, R. Amato, J. Sosman, D. Taber, D. Liebowitz, and E. E. Vokes
Flavopiridol, A Novel Cyclin-Dependent Kinase Inhibitor, in Metastatic Renal Cancer: A University of Chicago Phase II Consortium Study
J. Clin. Oncol.,
January 14, 2000;
18(2):
371 - 371.
[Abstract]
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Y. Li, M. Bhuiyan, S. Alhasan, A. M. Senderowicz, and F. H. Sarkar
Induction of Apoptosis and Inhibition of c-erbB-2 in Breast Cancer Cells by Flavopiridol
Clin. Cancer Res.,
January 1, 2000;
6(1):
223 - 229.
[Abstract]
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G. Melillo, E. A. Sausville, K. Cloud, T. Lahusen, L. Varesio, and A. M. Senderowicz
Flavopiridol, a Protein Kinase Inhibitor, Down-Regulates Hypoxic Induction of Vascular Endothelial Growth Factor Expression in Human Monocytes
Cancer Res.,
November 1, 1999;
59(21):
5433 - 5437.
[Abstract]
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G. I. Shapiro, D. A. Koestner, C. B. Matranga, and B. J. Rollins
Flavopiridol Induces Cell Cycle Arrest and p53-independent Apoptosis in Non-Small Cell Lung Cancer Cell Lines
Clin. Cancer Res.,
October 1, 1999;
5(10):
2925 - 2938.
[Abstract]
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M. Mutoh, F.-D. T. Lung, Y.-Q. Long, P. P. Roller, R. S. Sikorski, and P. M. O'Connor
A p21Waf1/Cip1 Carboxyl-terminal Peptide Exhibited Cyclin-dependent Kinase-inhibitory Activity and Cytotoxicity When Introduced into Human Cells
Cancer Res.,
July 1, 1999;
59(14):
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[Abstract]
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T. V. Achenbach, R. Muller, and E. P. Slater
Bcl-2 Independence of Flavopiridol-induced Apoptosis. MITOCHONDRIAL DEPOLARIZATION IN THE ABSENCE OF CYTOCHROME c RELEASE
J. Biol. Chem.,
October 6, 2000;
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Abstract
Introduction
Abstract
