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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3804-3816
By
From the Division of Hematology-Oncology, Walter Reed Army Medical
Center, Washington, DC; the Department of Medicine, Uniformed Services
University of Health Sciences, Bethesda, MD; the Division of
Hematologic Malignancies, Johns Hopkins Oncology Center, Baltimore, MD;
Biotechnologies, Ltd, Laurel, MD; the Department of Laboratory Medicine
and Pathology, University of Minnesota, Minneapolis, MN; and the
Developmental Therapeutics Program, National Cancer Institute,
Bethesda, MD.
Flavopiridol has been reported to induce apoptosis in lymphoid cell
lines via downregulation of bcl-2. The in vitro activity of
flavopiridol against human chronic lymphocytic leukemia (CLL) cells and
potential mechanisms of action for inducing cytotoxicity were studied.
The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a
flavopiridol concentration of 1.15 µmol/L (95% confidence interval
[CI] ±0.31), 0.18 µmol/L (95% CI
±0.04), and 0.16 µmol/L (95% CI ±0.04), respectively. Loss of
viability in human CLL cells correlated with early induction of
apoptosis. Exposure of CLL cells to 0.18 µmol/L of flavopiridol
resulted in both decreased expression of p53 protein and cleavage of
the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes
in bcl-2 protein expression alterations. We evaluated flavopiridol's
dependence on intact p53 by exposing splenocytes from wild-type
(p53+/+) and p53 null (p53
B-CELL CHRONIC lymphocytic leukemia (CLL)
is the most common leukemia in the Western hemisphere, with
approximately 10,000 new cases diagnosed each year.1
Relative to other forms of leukemia, the overall prognosis of CLL is
good, with even the most advanced stage patients having a median
survival of 3 years.2 However, unlike most of the other
forms of acute and chronic leukemia, substantial therapeutic progress
has not been made over the past 40 years in either prolonging survival
or introducing curative therapy. The addition of fludarabine as initial
therapy for symptomatic CLL patients has led to a higher rate of
complete responses (27% v 3%) and duration of
progression-free survival (33 v 17 months) as compared with
previously used alkylator-based therapies.3 Although
attaining a complete clinical response after therapy is the initial
step toward improving survival in CLL, the majority of patients either
do not attain complete remission or fail to respond to fludarabine.
Furthermore, all patients with CLL treated with fludarabine eventually
relapse, making its role as a single agent purely palliative.
Therefore, identifying new agents with novel mechanisms of action that
complement fludarabine's cytotoxicity and abrogate the resistance
induced by intrinsic CLL drug-resistance factors will be necessary if
further advances in the therapy of this disease are to be realized.
Several biologic factors, including abnormal p53 function,
overexpression of bcl-2, and incubation of tumor cells with
interleukin-4 (IL-4), have been associated with either in vitro or in
vivo drug resistance in CLL.4-12 Of these, the most
extensively studied, uniformly predictive factor for poor response to
therapy and inferior survival in CLL patients is aberrant p53 function,
as characterized by point mutations or chromosome 17p13
deletions.4-7 Indeed, virtually no responses to either
alkylator or purine analog therapy have been documented in multiple
single institution case series for those CLL patients with abnormal p53
function.4-7 Introduction of a therapeutic agent that has
the ability to overcome the drug resistance associated with p53
mutation in CLL would potentially be a major advance for the treatment
of the disease. We describe here that flavopiridol, a novel synthetic
flavone currently entering phase II trials, demonstrates marked in
vitro cytotoxicity toward human CLL cells and may circumvent either
IL-4-induced resistance or that incurred by a p53 mutation.
Patients, Cell Separation, and Culture Conditions
Treatment of Mice and Harvested Cells
Viability Assays Viability assays of isolated mononuclear cells from CLL patients were performed using the MTT assay. Cells (1 × 106 per well) in a volume of 100 µL were placed in a 96-well flat-bottom plate and the test drug (100 µL per well at 2× final concentration) or medium alone was added to the plates. All human experiments were performed in quadruplicate. Cells were incubated for fixed times (4 hours, 24 hours, and 96 hours). After the incubation, 50 µL of MTT (Sigma Chemical Co, St Louis, MO) at a concentration of 2 mg/mL was added to each well. Cells were incubated for 24 hours and centrifuged at 300g for 5 minutes, and 150 µL of supernatant was removed. A total of 200 µL of protamine sulfate (Sigma) in phosphate-buffered saline (PBS) at 25 mg/mL was added to each well. Centrifugation was repeated, followed by the replacement of 200 µL of protamine sulfate. Plates were centrifuged and 200 µL of protamine sulfate was removed. Plates were then allowed to air dry. The precipitated MTT formazan was solubilized with 150 µL of dimethyl sulfoxide (DMSO) with constant agitation for 4 hours. After this procedure, the optical density at 540 nm was obtained using an Anthos Reader 2001 (Anthos Labtec Inc, Frederick, MD) with a Biolise-Windows program. Cell viability was expressed as the ratio of absorption between drugged cells/control sample.
Apoptosis Assays After 4 and 24 hours of incubation with 0.18 µmol/L flavopiridol in supplemented RPMI and 10% FBS, apoptosis studies were performed using the following techniques.TdT/PI.
Cells (5 × 105) were added to cold 1% buffered
formaldehyde (10% Formadehyde [methanol free, ultrapure grade;
Polysciences, Inc, Warrington, PA], prepared in Dulbecco's
phosphate-buffered saline without CaCl2 and
MgCl2 [PBS]) for 15 minutes on ice. Cells were then
washed with PBS and resuspended in 70% methanol and stored at
Annexin/PI. After incubation with 0.18 µmol/L of flavopiridol or medium for 4 or 24 hours, 5 × 105 cells were washed with PBS and then resuspended in binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 150 mmol/L NaCl 5 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L CaCl2) containing 2 µL of Annexin V-FITC stock (BioWhittaker, Inc, Walkersville, MD) and 10 µL of 20 µg/mL PI (Sigma). After incubation for 10 minutes at room temperature in a light-protected area, the specimens were quantified by flow cytometry on a FACScan (Becton Dickinson). Protein Extraction and Western Blot Analysis The bcl-2, bax, p53, p27, and CPP32 protein expressions were analyzed by Western blot after incubation in either medium or two concentrations of flavopiridol (0.18 and 0.33 µmol/L) for 1, 4, and 24 hours. Whole-cell lysates were prepared by pelleting 1.25 × 108 PBS-washed mononuclear cells in a microcentrifuge, aspirating the supernatant, and adding 0.5 mL of cold lysis buffer, as described previously.14 This cell suspension was incubated at 4°C for 40 minutes with constant agitation and then centrifuged for 15 minutes at 14,000 rpm at 4°C. The supernatant was recovered, alliquoted, and frozen at 80°C.
Polymerase Chain Reaction (PCR) of Murine Splenocytes The genotypes of the mice were verified (1) by lification of the p53 exon 6/intron 6/exon 7 fragment in the p53 wild-type mouse and demonstration of the lack of product in the p53 knock out mouse and (2) by amplification of a neo/p53 exon 7 hybrid product in the p53 knock-out mouse and the lack of hybrid product in the p53 wild-type mouse. The amplification methods used have been previously described.16
Fluorescent In Situ Hybridization for 17p13 Deletions From peripheral blood that had been collected in EDTA collection tubes, smears were prepared from the patient 28a described in Table 2 and Fig 3 and air-dried at ambient temperatures for 1 to 7 days. Before hybridization, the slides were denatured in 70% formamine/2× SCC (0.3 mol/L sodium chloride in 0.03 mol/L citric acid, pH 7.0) at 74°C ± 1°C for 5 minutes, followed by serial dehydration in ethanol. The slides were then drained and maintained on a 45°C to 50°C slide warmer for approximately 2 minutes during the time of probe denaturation. One microliter of the probe LSI p53 (17p13.1), labeled with Spectrum Orange (Vysis, Downers Grove, IL), was mixed with 2 µL purified H2O and 7 µL of Vysis' LSI Hybridization Buffer. The probe mixture was next denatured in a 74°C ± 1°C water bath for 5 minutes. The denatured probe mixture was applied to the denatured specimen target (~10 µL probe mixture for a 22 mm × 22 mm target area). A glass coverslip was applied to the target area and sealed with rubber cement. Hybridization was performed in a humidified chamber at 37°C for 14 to 18 hours. Posthybridization washes consisted of a 2-minute wash in 4× SSC/0.3% NP-40 at 74°C ± 1°C followed by 1 minute in 2× SSC/0.1% NP-40 (Sigma) at room temperature. The slides were then air-dried in the dark, counter-stained with Vysis' DAPI in Antifade, cover-slipped, and examined with a Zeiss Standard Fluorescent Microscope (Olympus Optical Co, Tokyo, Japan) equipped with epifluorescence optics and dual bandpass for DAPI/Spectrum Orange. One hundred to 200 cells were counted, and the percentages of 0, 1, or 2 signals were determined. A diagnosis of monoallelic deletion of p53 was established when the percentage of cells with 1 signal exceeded the controls' mean by greater than 3 standard deviations. The same procedure outlined above was applied to 16 blood smears that had been obtained from patients who had undergone bone marrow biopsy for staging of breast cancer and who had no evidence of metastatic breast cancer or other malignancy in the blood or bone marrow. For these 16 cases, the mean and standard deviation of the percentage of cells with 1 hybridization signal (±3 SD) was 10.3% (±20.3%). Patient 28a had 35% of cells showing 1 hybridization signal and 65% demonstrating 2 hybridization signals.Statistics Groups of data were compared using paired or nonpaired Student's t-tests (two-sided) as appropriate. Nonparametric data were analyzed using the Wilcoxan signed-rank test. JMP Statistics software (SAS Institute, Trumbull, CT) or Quatropro software (Novell Inc, Orem, UT) was used to perform these analyses.
Flavopiridol Is Cytotoxic Toward CLL Cells With an Optimal Exposure Period of 24 Hours Peripheral mononuclear cells from 11 consecutive patients with CLL studied in the laboratory (UPN 1a, 2a, 3a, 5a, 6a, 7a, 9a, 10a, 1b, 2b, 3b, and 4b; see Table 2 for clinical data) were exposed to either medium or varying (0.01, 0.033, 0.1, 0.33, 1, 3.3, 10, 33, and 100 µmol/L) concentrations of flavopiridol. Cells were incubated either in drug or medium as follows: 4 hours and then developed; 4 hours and then washed of drug and developed after in vitro incubation in fresh medium for 4 days; 24 hours and then developed; 24 hours and then washed of drug and developed after in vitro incubation in fresh medium for 4 days; and developed after 4 days of continuous exposure to the agent. No loss of viability was noted immediately after 4 hours of flavopiridol exposure (data not shown). The viability of CLL cells at each other time point and concentration are depicted in Table 1. All of the patients with CLL demonstrated in vitro response to flavopiridol, including 5 patients with fludarabine-refractory CLL. The average concentration of flavopiridol required to produce 50% cytotoxicity (LC50) was 1.15 µmol/L (range, 0.52 to 1.93 µmol/L; 95% confidence interval [CI] ±0.31 µmol/L) after 4 hours of exposure to this agent followed by incubation in fresh medium until 4 days. After 24 hours of exposure to flavopiridol with immediate addition of MTT or incubation in fresh medium until 4 days, the LC50 decreased to 0.18 µmol/L (range, 0.09 to 0.33 µmol/L; 95% CI ±0.04 µmol/L) and 0.19 µmol/L (range, 0.1 to 0.26 µmol/L; 95% CI ±0.04 µmol/L), respectively. After continuous 96-hour exposure to flavopiridol, the LC50 declined to 0.16 µmol/L (range, 0.04 to 0.24 µmol/L; 95% CI ±0.05 µmol/L). Comparing these time points, the LC50 was significantly lower for both the 24-hour (P = .02) and the 96-hour (P = .02) flavopiridol exposures as compared with the 4-hour incubation. The LC50 was not significantly different (P = .57) between CLL cells exposed to flavopiridol for 24 versus 96 hours. Therefore, these data support a 24-hour treatment administration schedule for phase II studies of flavopiridol in patients with B-CLL.
Flavopiridol Is Equivalently Cytotoxic to Untreated and Previously Treated B-CLL Because in vitro drug resistance is more frequently observed in previously treated patients, we next assessed if the degree of flavopiridol cytotoxicity varied by pretreatment status. A cohort of 27 patients with either untreated (n = 16) or previously treated (n = 11) CLL were identified from our institutions. The clinical history of these patients at the time of phlebotomy is described in Table 2. Of the previously treated CLL patients, 5 had fludarabine refractory disease. Mononuclear cells isolated from these patients were exposed to varying (0.01, 0.033, 0.1, 0.33, 1, 3.3, 10, 33, and 100 µmol/L) concentrations of flavopiridol for 4 days. Patients with untreated B-CLL had a mean LC50 of 0.12 (range, 0.02 to 0.22 µmol/L; 95% CI ±0.03 µmol/L) after 4 days of exposure to flavopiridol. A cohort of previously treated B-CLL had a similar mean LC50 of 0.15 µmol/L (range, 0.03 to 0.24 µmol/L; 95% CI ±0.05 µmol/L) after 4 days of exposure to flavopiridol that was not significantly different (P = .77) from the untreated patients. Even for the 6 patients who were clinically fludarabine refractory, the LC50 was 0.17 µmol/L (range, 0.09 to 0.21 µmol/L; 95% CI ±0.05 µmol/L), suggesting that in vitro sensitivity to flavopiridol is not altered by prior treatment and more advanced disease.
Flavopiridol Is Also Cytotoxic Toward Normal Mononuclear Cells To assess the tumor selectivity of flavopiridol against CLL cells, we exposed mononuclear cell isolates from 9 healthy volunteers to varying (0.01, 0.033, 0.1, 0.33, 1, 3.3, 10, 33, and 100 µmol/L) concentrations of flavopiridol. Cells were incubated under identical conditions to those of CLL cells, as described previously. No cytotoxicity was observed immediately after 4 hours of incubation. The LC50 after 4 hours of agent exposure followed by incubation in fresh medium until 4 days was 1.21 µmol/L (range, 0.60 to 1.90 µmol/L; 95% CI ±0.61 µmol/L) for normal cells. After 24 hours of exposure followed by incubation for 96 hours or a continuous 96-hour exposure to flavopiridol, the LC50 declined to 0.24 µmol/L (range, 0.15 to 0.27 µmol/L; 95% CI ±0.03 µmol/L) and 0.18 µmol/L (range, 0.02 to 0.21 µmol/L; 95% CI ±0.05 µmol/L), respectively. As shown in Fig 1, the LC50 was not significantly different (P = .57) between the mononuclear cells obtained either from volunteers or patients with CLL exposed to flavopiridol for 24 versus 96 hours, respectively. In fact, comparison of the therapeutic index between B-CLL cells and normal mononuclear cells demonstrated no significant difference at 4 (P = .16), 24 (P = .70), and 96 (P = .82) hours, respectively.
Flavopiridol Induces Apoptosis in CLL Cells In an attempt to characterize if the cytotoxicity induced by flavopiridol was due to apoptosis, mononuclear cells from CLL patients (n = 7) were incubated for either 4 or 24 hours in medium or 0.18 µmol/L of flavopiridol. Unlike fludarabine, which induces apoptosis only after 1 day of exposure (data not shown), flavopiridol produced apoptosis as early as 4 hours postincubation. Indeed, the mean spontaneous apoptosis rate in medium incubated CLL cells at 4 hours was 12.8% (range, 4.4% to 17.7%; 95% CI ±3.6%), which increased significantly (P = .01) to 25.0% (range, 11.8% to 37%; 95% CI ±6.5%) with flavopiridol exposure. At 24 hours, the mean spontaneous apoptosis rate in medium was 20% (range, 10.2% to 34.1%; 95% CIs ±7.8%) that increased significantly (P = .02) to 73.4% (range, 60.7% to 87.4%; 95% CI ±11.0%). A representative tunnel assay of CLL cells (patient UPN 9a) incubated in medium or 0.18 µmol/L of flavopiridol assessing apoptosis concurrently with cycle status is shown in Fig 2. Noteworthy is the fact that flavopiridol appears to be inducing apoptosis in G0-1 arrested cells. Because necrotic cells sometimes show uptake of TdT, a separate assessment of apoptosis was performed using annexin-V and propidium iodine. Figure 3 demonstrates a representative annexin-V/PI assay with CLL cells (patient UPN 28a) incubated in medium or 0.18 µmol/L of flavopiridol for 4 and 24 hours. This demonstrates a definitive population of cells with the altered annexin-V phospholipid observed with apoptosis, but lacking PI staining. Similar apoptosis was observed with the tunnel assay outlined above (data not shown). These data support the observation of others that flavopiridol is inducing cytotoxicity at least in part through the pathway of apoptosis.
Flavopiridol Cytotoxicity Does Not Correlate With Changes in bcl-2 or bax Protein Expression To determine if flavopiridol was inducing early apoptosis either through decreased expression of bcl-2 or increased bax protein, we incubated mononuclear cells from CLL patients with 0.18 µmol/L flavopiridol or medium with subsequent assessment of bcl-2 protein (n = 7) or bax (n = 3) protein expression at 4 and 24 hours. Figure 4A depicts bcl-2 expression in 3 patient samples at the 0.18 µmol/L flavopiridol concentration, demonstrating minimal change in bcl-2 protein expression as compared with medium. Quantification of protein banding by densitometry in these seven experiments demonstrated similar or slightly increased expression of bcl-2 expression with 0.18 µmol/L of flavopiridol as compared with medium at 4 and 24 hours, respectively. Mononuclear cells from 2 additional CLL patients were incubated with a higher concentration of flavopiridol (0.33 µmol/L), which induced a high rate of apoptosis (85% and 88%) as measured by the TdT/PI at 1 day, but similarly failed to demonstrate a change in bcl-2 protein expression. The protein expression of bax was also not altered with 0.18 µmol/L flavopiridol incubation as compared with medium-matched control (data not shown). These data suggest that flavopiridol-induced apoptosis in human CLL cells does not occur as a consequence of bcl-2 protein modulation.
Flavopiridol Cytotoxicity Does Not Correlate With Changes in Expression of p27 It has recently been preliminarily identified that the cell cycle inhibitor p27 is variably overexpressed in CLL lymphocytes, is induced by IL-4 incubation, and may have a role in suppressing apoptosis. Because p27 binds to CDK2 and flavopiridol inhibits this kinase, we sought to determine if flavopiridol was modulating expression of this protein. We incubated mononuclear cells from 3 CLL patients in medium or flavopiridol (0.18 or 0.33 µmol/L), which induced a high rate of apoptosis as measured by the TdT/PI at 1 day, but failed to demonstrate a change in p27 protein expression, as shown in Fig 4B.IL-4 Induces Resistance to F-ara-A, But Not Flavopiridol In vitro incubation of B-CLL cells with IL-4 has been demonstrated to induce drug resistance toward chlorambucil and prednisone. We sought to determine if another agent currently used in the treatment of CLL (ie, F-ara-a) and flavopiridol were affected by IL-4 incubation. Cells from 6 patients with B-CLL were exposed to varying concentrations (0.01, 0.033, 0.1, 0.33, 1, 3.3, 10, 33, and 100 µmol/L) of F-ara-a and flavopiridol with or without 10 ng/mL of IL-4. As demonstrated in Fig 5A, incubation with IL-4 for 4 days increased the LC50 of F-ara-a in 5 of 6 CLL cell samples tested from a mean of 11.6 µmol/L (range, 2.2 to 29.6 µmol/L; 95% CI ±9.4 µmol/L) to 29.3 µmol/L (range, 2.89 to 74.0 µmol/L; 95% CI ±24.6 µmol/L). The magnitude of this change was not equivalent between patients, with only 3 having significant increases over the respective controls. In contrast, as shown in Fig 5B for the same 6 CLL patients, the LC50 of cells exposed in medium and flavopiridol was 0.23 µmol/L (range, 0.2 to 0.3 µmol/L; 95% CI ±0.03 µmol/L) versus 0.24 µmol/L (range, 0.2 to 0.28 µmol/L; 95% CI ±0.02 µmol/L) for medium and IL-4 and flavopiridol. These data suggest that IL-4 incubation does not increase the in vitro drug resistance to human CLL cells exposed to flavopiridol.
Flavopiridol Causes a Decrease in p53 Protein Expression The normal CLL cellular response to DNA damage induced by alkylator and/or purine analog exposure is an increase of p53 protein expression. To determine if flavopiridol was inducing apoptosis via a similar pathway, we incubated mononuclear CLL cells from 5 patients with 0.18 µmol/L of flavopiridol or medium with subsequent assessment of p53 protein expression at 1, 4, and 24 hours. Figure 6A depicts a representative Western blot demonstrating a decrease in p53 protein expression with flavopiridol exposure at 4 and 24 hours as compared with medium-only control samples. Figure 6B depicts a reprobing experiment with actin, demonstrating equivalent protein loading among each of the samples. Fast Green Staining demonstrated similar results (data not shown).
Flavopiridol Induces Apoptosis Independent of p53 Status The observation that p53 protein expression was actually decreased in response to flavopiridol exposure suggested that this agent might be operating in a unique, p53-independent fashion. To test this hypothesis, we explored whether the observed effects of flavopiridol were dependent on intact p53. Splenocytes from 4 wild-type and 3 typed p53 null type mice were exposed to varying (0.01, 0.1, 0.33, and 1 µmol/L) concentrations of flavopiridol. The absolute molecular status of the wild-type and p53 null type mice were secondarily confirmed by identifying the presence or absence of the p53 gene and neogene as previously described16,17 and is shown in Fig 7. The cytotoxicity as assayed by trypan blue after 24 hours of incubation at each concentration of flavopiridol is summarized in Fig 8. Viability of the p53 null (mean, 81%; 95% CI ±4.3%) and wild-type (mean, 90%; 95% CI ±2%) mice splenocytes after 24 hours of incubation in medium was similar. A noticeable decline in splenocyte viability was noted with incremental increases in flavopiridol concentrations without preferential cytotoxicity to p53+/+ as compared with the p53 /
splenocytes. This nonpreferential toxicity was also observed in the
thymocytes (data not shown). In contrast to this, the viability of the
p53/ splenocytes (89%) was affected
minimally by 500 cGy of irradiation as compared with almost complete
loss of viability (22%) in the p53+/+ splenocytes.
Incubation with F-ara-a at varying concentrations yielded a result
similar to that observed after irradiation. After 24 hours of
incubation with F-ara-a, the p53/ splenocytes
had a LC50 of greater than 100 µmol/L, contrasting with
that of p53+/+ cells in which the LC50 was only
3.95 µmol/L. The possible clinical relevance of these murine
experiments to B-CLL is exemplified by the in vitro response of UPN28a
to flavopiridol. This patient had a 17p13 deletion, as shown in
Fig 9. In vitro drug incubation with
fludarabine and flavopiridol demonstrated a LC50 of greater than 100 µmol/L for F-ara-a and 0.22 µmol/L for flavopiridol. This
is further demonstrated by Fig 3 illustrating marked apoptosis in CLL
cells from this same patient after 24 hours of 0.18 µmol/L of
flavopiridol exposure.
Flavopiridol Exposure Results in Activation of Caspase 3 The protein caspase 3 exists in zymogen form, is overexpressed in B-CLL, and when cleaved to its 17- to 20-kD and 10-kD fragments cleaves the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) that commits the cell to apoptosis. Because caspase is activated at a point in the apoptotic pathway distal to p53, we sought to determine if flavopiridol incubation resulted in cleavage of this protein. We incubated mononuclear CLL cells from 3 patients with 0.18 µmol/L of flavopiridol or medium with subsequent assessment of caspase 3 protein expression and its fragments at 1, 4, and 24 hours. Figure 10A depicts a representative Western blot demonstrating a decrease in the zymogen caspase-3 32-kD protein. Detection of the subsequent appearance of the caspase-3 protein cleavage 20-kD fragment at 24 hours of flavopiridol exposure as compared with medium control is demonstrated in Fig 10B.
Whereas Senderowicz et al17 preliminarily reported the observation of in vitro apoptosis in a single patient with human B-CLL, this manuscript evaluates the effect of flavopiridol in multiple patients with this disease. Furthermore, we show that this agent has marked activity against B-cell CLL, irrespective of previous treatment, purine analog refractoriness, the presence of a 17p13 deletion, or IL-4 incubation. In a recently completed phase I study administering flavopiridol as a 72-hour infusion, a median plasma concentration of 0.44 µmol/L was attained at the recommended phase II starting dose.18 At this concentration, hypotension and diarrhea were noted, but not myelosuppression. These attainable plasma drug levels and dose-limiting toxicities have been noted by a second group.19 This mean attainable concentration of flavopiridol represents 2.4 times the LC50 of human B-cell CLL cells in vitro. The apoptosis/cytotoxicity observed with flavopiridol in CLL cells was observed early at 4 to 24 hours as compared with fludarabine, in which significant apoptosis/cytotoxicity is not noted before 4 days. In contrast, the maximal flavopiridol drug effect at this low, clinically attainable concentration requires 24 hours of continuous exposure. Incubation with flavopiridol beyond 24 hours produced an insignificant change in the LC50 of CLL cells.
Submitted September 18, 1997;
accepted July 7, 1998.
Address reprint requests to John C. Byrd, MD, Hematology-Oncology Service, ward 78, Walter Reed Army Medical Center, Washington, DC 20307.
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A. J. Johnson, D. M. Lucas, N. Muthusamy, L. L. Smith, R. B. Edwards, M. D. De Lay, C. M. Croce, M. R. Grever, and J. C. Byrd Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia Blood, August 15, 2006; 108(4): 1334 - 1338. [Abstract] [Full Text] [PDF]
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 E. B. Sambol, G. Ambrosini, R. C. Geha, P. T. Kennealey, P. DeCarolis, R. O'Connor, Y. V. Wu, M. Motwani, J.-H. Chen, G. K. Schwartz, et al. Flavopiridol Targets c-KIT Transcription and Induces Apoptosis in Gastrointestinal Stromal Tumor Cells Cancer Res., June 1, 2006; 66(11): 5858 - 5866. [Abstract] [Full Text] [PDF]
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J. A. Whitlock, M. Krailo, J. M. Reid, S. L. Ruben, M. M. Ames, W. Owen, and G. Reaman Phase I Clinical and Pharmacokinetic Study of Flavopiridol in Children With Refractory Solid Tumors: A Children's Oncology Group Study J. Clin. Oncol., December 20, 2005; 23(36): 9179 - 9186. [Abstract] [Full Text] [PDF]
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G. K. Schwartz and M. A. Shah Targeting the Cell Cycle: A New Approach to Cancer Therapy J. Clin. Oncol., December 20, 2005; 23(36): 9408 - 9421. [Abstract] [Full Text] [PDF]
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K. C. Bible, J. L. Lensing, S. A. Nelson, Y. K. Lee, J. M. Reid, M. M. Ames, C. R. Isham, J. Piens, S. L. Rubin, J. Rubin, et al. Phase 1 Trial of Flavopiridol Combined with Cisplatin or Carboplatin in Patients with Advanced Malignancies with the Assessment of Pharmacokinetic and Pharmacodynamic End Points Clin. Cancer Res., August 15, 2005; 11(16): 5935 - 5941. [Abstract] [Full Text] [PDF]
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T. E. Battle, J. Arbiser, and D. A. Frank The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells Blood, July 15, 2005; 106(2): 690 - 697. [Abstract] [Full Text] [PDF]
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G. K. Schwartz Development of Cell Cycle Active Drugs for the Treatment of Gastrointestinal Cancers: A New Approach to Cancer Therapy J. Clin. Oncol., July 10, 2005; 23(20): 4499 - 4508. [Abstract] [Full Text] [PDF]
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J. R. Brown Chronic Lymphocytic Leukemia: A Niche for Flavopiridol? Clin. Cancer Res., June 1, 2005; 11(11): 3971 - 3973. [Full Text] [PDF]
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J. C. Byrd, B. L. Peterson, J. Gabrilove, O. M. Odenike, M. R. Grever, K. Rai, R. A. Larson, and the Cancer and Leukemia Group B Treatment of Relapsed Chronic Lymphocytic Leukemia by 72-Hour Continuous Infusion or 1-Hour Bolus Infusion of Flavopiridol: Results from Cancer and Leukemia Group B Study 19805 Clin. Cancer Res., June 1, 2005; 11(11): 4176 - 4181. [Abstract] [Full Text] [PDF]
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A. J. Johnson, L. L. Smith, J. Zhu, N. A. Heerema, S. Jefferson, A. Mone, M. Grever, C.-S. Chen, and J. C. Byrd A novel celecoxib derivative, OSU03012, induces cytotoxicity in primary CLL cells and transformed B-cell lymphoma cell line via a caspase- and Bcl-2-independent mechanism Blood, March 15, 2005; 105(6): 2504 - 2509. [Abstract] [Full Text] [PDF]
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M. Puppo, S. Pastorino, G. Melillo, A. Pezzolo, L. Varesio, and M. C. Bosco Induction of Apoptosis by Flavopiridol in Human Neuroblastoma Cells Is Enhanced under Hypoxia and Associated With N-myc Proto-oncogene Down-Regulation Clin. Cancer Res., December 15, 2004; 10(24): 8704 - 8719. [Abstract] [Full Text] [PDF]
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L. J. Rush, A. Raval, P. Funchain, A. J. Johnson, L. Smith, D. M. Lucas, M. Bembea, T.-H. Liu, N. A. Heerema, L. Rassenti, et al. Epigenetic Profiling in Chronic Lymphocytic Leukemia Reveals Novel Methylation Targets Cancer Res., April 1, 2004; 64(7): 2424 - 2433. [Abstract] [Full Text] [PDF]
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A. P. Mone, P. Huang, H. Pelicano, C. M. Cheney, J. M. Green, J. Y. Tso, A. J. Johnson, S. Jefferson, T. S. Lin, and J. C. Byrd Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells Blood, March 1, 2004; 103(5): 1846 - 1854. [Abstract] [Full Text] [PDF]
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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; 9(9): 3320 - 3328. [Abstract] [Full Text] [PDF]
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J. L. Aron, M. R. Parthun, G. Marcucci, S. Kitada, A. P. Mone, M. E. Davis, T. Shen, T. Murphy, J. Wickham, C. Kanakry, et al. Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8-mediated apoptosis and down-regulation of c-FLIP protein Blood, July 15, 2003; 102(2): 652 - 658. [Abstract] [Full Text] [PDF]
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U. Raju, E. Nakata, K. A. Mason, K. K. Ang, and L. Milas Flavopiridol, a Cyclin-dependent Kinase Inhibitor, Enhances Radiosensitivity of Ovarian Carcinoma Cells Cancer Res., June 15, 2003; 63(12): 3263 - 3267. [Abstract] [Full Text] [PDF]
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J. C. Byrd, D. M. Lucas, A. P. Mone, J. B. Kitner, J. J. Drabick, and M. R. Grever KRN5500: a novel therapeutic agent with in vitro activity against human B-cell chronic lymphocytic leukemia cells mediates cytotoxicity via the intrinsic pathway of apoptosis Blood, June 1, 2003; 101(11): 4547 - 4550. [Abstract] [Full Text] [PDF]
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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; 9(2): 562 - 570. [Abstract] [Full Text] [PDF]
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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; 2(2): 139 - 150. [Abstract] [Full Text] [PDF]
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S. Wittmann, P. Bali, S. Donapaty, R. Nimmanapalli, F. Guo, H. Yamaguchi, M. Huang, R. Jove, H. G. Wang, and K. Bhalla Flavopiridol Down-Regulates Antiapoptotic Proteins and Sensitizes Human Breast Cancer Cells to Epothilone B-induced Apoptosis Cancer Res., January 1, 2003; 63(1): 93 - 99. [Abstract] [Full Text] [PDF]
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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; 2(1): 73 - 81. [Abstract] [Full Text] [PDF]
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J. E. Karp, D. D. Ross, W. Yang, M. L. Tidwell, Y. Wei, J. Greer, D. L. Mann, T. Nakanishi, J. J. Wright, and A. D. Colevas Timed Sequential Therapy of Acute Leukemia with Flavopiridol: In Vitro Model for a Phase I Clinical Trial Clin. Cancer Res., January 1, 2003; 9(1): 307 - 315. [Abstract] [Full Text] [PDF]
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I. Gojo, B. Zhang, and R. G. Fenton The Cyclin-dependent Kinase Inhibitor Flavopiridol Induces Apoptosis in Multiple Myeloma Cells through Transcriptional Repression and Down-Regulation of Mcl-1 Clin. Cancer Res., November 1, 2002; 8(11): 3527 - 3538. [Abstract] [Full Text] [PDF]
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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 J. Clin. Oncol., October 1, 2002; 20(19): 4074 - 4082. [Abstract] [Full Text] [PDF]
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G. K. Dy and A. A. Adjei Novel Targets for Lung Cancer Therapy: Part II J. Clin. Oncol., July 1, 2002; 20(13): 3016 - 3028. [Abstract] [Full Text] [PDF]
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D. F. Dukers, C. J. L. M. Meijer, R. L. ten Berge, W. Vos, G. J. Ossenkoppele, and J. J. Oudejans High numbers of active caspase 3-positive Reed-Sternberg cells in pretreatment biopsy specimens of patients with Hodgkin disease predict favorable clinical outcome Blood, June 17, 2002; 100(1): 36 - 42. [Abstract] [Full Text] [PDF]
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C. M. Barrett, F. L. Lewis, J. B. Roaten, T. W. Sweatman, M. Israel, J. L. Cleveland, and L. Lothstein Novel Extranuclear-targeted Anthracyclines Override the Antiapoptotic Functions of Bcl-2 and Target Protein Kinase C Pathways to Induce Apoptosis Mol. Cancer Ther., May 1, 2002; 1(7): 469 - 481. [Abstract] [Full Text] [PDF]
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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. [Abstract] [Full Text]
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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; 6(2): 661 - 670. [Abstract] [Full Text]
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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] [Full Text]
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J. C. Byrd, C. Shinn, R. Ravi, C. R. Willis, J. K. Waselenko, I. W. Flinn, N. A. Dawson, and M. R. Grever Depsipeptide (FR901228): A Novel Therapeutic Agent With Selective, In Vitro Activity Against Human B-Cell Chronic Lymphocytic Leukemia Cells Blood, August 15, 1999; 94(4): 1401 - 1408. [Abstract] [Full Text] [PDF]
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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; 275(41): 32089 - 32097. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
)
and exon 7 reverse primers (W3
) Human CLL cells (n = 11 patients); (
) normal mononuclear cells (n = 9 patients).
Cytotoxicity is expressed as the LC50 (drug concentration
required to reduce viability by 50%).
)
or presence (
) of IL-4 (10 ng/mL). After 4 days of incubation, the
viability was assessed using the MTT test and is expressed for each
patient as the LC50 (drug concentration required to reduce
viability by 50%). (B) The in vitro effect of IL-4 incubation on in
vitro drug resistance to flavopiridol in human CLL cells. The cells
were obtained from CLL patients after obtaining informed consent,
isolated, and cultured at 1 × 106/mL in medium (control)
and varying concentrations of flavopiridol (0.01 to 100 µmol/L) in
the absence (
) of IL-4 (10 ng/mL). After 4 days of
incubation, the viability was assessed using the MTT test and is
expressed for each patient as the LC50 (drug concentration
required to reduce viability by 50%).
Family protease CPP32 (caspase-3) in non-Hodgkin's lymphomas, chronic lymphocytic leukemias and reactive lymph nodes.
Blood
89:3817, 1997