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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 672-682
Cytosine Deaminase Adenoviral Vector and 5-Fluorocytosine
Selectively Reduce Breast Cancer Cells 1 Million-Fold When They
Contaminate Hematopoietic Cells: A Potential Purging Method for
Autologous Transplantation
By
F. Garcia-Sanchez,
G. Pizzorno,
S.Q. Fu,
T. Nanakorn,
D.S. Krause,
J. Liang,
E. Adams,
J.J. Leffert,
L.H. Yin,
M.R. Cooperberg,
E. Hanania,
W.L. Wang,
J.H. Won,
X.Y. Peng,
R. Cote,
R. Brown,
B. Burtness,
R. Giles,
R. Crystal, and
A.B. Deisseroth
From the Department of Internal Medicine, Section of Medical
Oncology, and the Department of Laboratory Medicine, Yale University
School of Medicine, New Haven, CT; the Division of Pulmonary and
Critical Care Medicine, The New York Hospital-Cornell Medical Center,
New York, NY; the M.D. Anderson Cancer Center, Houston, TX; Systemix
Inc, Palo Alto, CA; the Department of Pathology, University of Southern
California School of Medicine, Los Angeles, CA; and Quality Biological,
Inc, Gaithersburg, MD.
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ABSTRACT |
Ad.CMV-CD is a replication incompetent adenoviral vector carrying a
cytomegalovirus (CMV)-driven transcription unit of the cytosine
deaminase (CD) gene. The CD transcription unit in this vector catalyzes
the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU).
This adenoviral vector prodrug activation system has been proposed for
use in selectively sensitizing breast cancer cells, which may
contaminate collections of autologous stem cells products from breast
cancer patients, to the toxic effects of 5-FC, without damaging the
reconstitutive capability of the normal hematopoietic cells. This
system could conceivably kill even the nondividing breast cancer cells,
because the levels of 5-FU generated by this system are 10 to 30 times
that associated with systemic administration of 5-FU. The incorporation
of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA
processing and protein synthesis so that even nondividing cells die of
protein starvation. To test if the CD adenoviral vector sensitizes
breast cancer cells to 5-FC, we exposed primary explants of normal
human mammary epithelial cells (HMECs) and the established breast
cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial
cells to the effects of 48 hours of exposure to 5-FC. We next tested
the selectivity of this system for BCC. When peripheral blood
mononuclear cells (PBMCs), collected from cancer patients during the
recovery phase from conventional dose chemotherapy-induced
myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in
serum-free conditions, little or no detectable conversion of 5-FC into
5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In
contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when
MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for 48 hours. All of the BCC lines tested were
shown to be sensitive to infection by adenoviral vectors when exposed
to a recombinant adenoviral vector containing the reporter gene
betagalactosidase (Ad.CMV- gal). In contrast, less than 1% of the
CD34-selected cells and their more immature subsets, such as the
CD34+CD38 or
CD34+CD33 subpopulations, were positive
for infection by the Ad.CMV-gal vector, as judged by
fluorescence-activated cell sorting (FACS) analysis, when
exposed to the adenoviral vector under conditions that did not commit
the early hematopoietic precursor cells to maturation. When artificial
mixtures of hematopoietic cells and BCCs were exposed for 90 minutes to
the Ad.CMV-CD vector and to 5-FC for 10 days or more, a greater than 1 million fold reduction in the number of BCCs, as measured by
colony-limiting dilution assays, was observed. To test if the
conditions were damaging for the hematopoietic reconstituting cells,
marrow cells collected from 5-FU-treated male donor mice were
incubated with the cytosine deaminase adenoviral vector and then
exposed to 5-FC either for 4 days in vitro before transplantation or
for 14 days immediately after transplantation in vivo. There was no
significant decrease in the reconstituting capability of the male
marrow cells, as measured by their persistence in female irradiated
recipients for up to 6 months after transplantation. These observations
suggest that adenovirus-mediated gene transfer of the Escherichia
coli cytosine deaminase gene followed by exposure to the nontoxic
pro-drug 5-FC may be a potential strategy to selectively reduce the
level of contaminating BCCs in collections of hematopoietic cells used for autografts in breast cancer patients.
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INTRODUCTION |
THE DISEASE-FREE SURVIVAL of
poor prognosis newly resected or advanced disease breast cancer
patients1 and those with metastatic disease2
can be improved by the delivery of intensive combination chemotherapy
treatments followed by hematopoietic rescue with autologous stem cell
transplantation. Approximately 8,000 of such treatments are performed
every year in the United States.1
Currently, breast cancer patients with gross contamination of the bone
marrow by tumor cells are usually excluded from such treatments.
Recently, a study based on polymerase chain reaction (PCR) analysis for
cytokeratin 19 (CK-19), a marker for metastatic circulating cells, has
shown that the majority of advanced disease breast cancer patients
already have cancer cells contaminating their bone marrow.3
The presence of a positive PCR assay for CK-19 in these patients is
associated with a decrease in disease-free interval after
transplantation3 and an increased probability of relapse
from 19% to greater than 80%.
These data have led to an increased interest in procedures that can
selectively remove breast cancer cells (BCCs) from autologous hematopoietic cells to be used for transplant, without damaging the
hematopoietic reconstituting cells. Methods applied to this problem
include monoclonal antibodies (MoAbs) targeted to breast cancer-associated cell surface markers,4 breast cancer
specific MoAbs conjugated to toxins,5 in vitro incubation
with cytotoxic drugs,6 lectin agglutination,7
phototherapy,8 and biological modifiers.9
Unfortunately, many of these systems for eradicating BCCs also lead to
the destruction of clonogenic progenitor cells and thereby cause a
prolongation of neutropenia after intensive therapy and
transplantation. No combination of MoAbs using positive selection of
normal hematopoietic cells and negative selection of BCCs has yet to
become commercially available for use in reducing the level of
neoplastic cells in autologous hematopoietic stem cells.
One promising strategy for removing BCCs from autologous bone marrow or
peripheral blood mononuclear cells (PBMCs) is to selectively infect the
BCCs with replication-incompetent adenoviral vectors that carry genes
that encode for pro-drug activation enzymes. We have studied the
feasibility of using a pro-drug activation approach for the eradication
of neoplastic epithelial breast cells that contaminate collections of
autologous hematopoietic cells. Specifically, we used a
replication-incompetent adenoviral vector to selectively introduce the
bacterial chemotherapy sensitization gene, cytosine deaminase (CD),
into BCCs ex vivo, without introducing this gene into the normal early
hematopoietic reconstituting cells.
CD converts the innocuous antibiotic pro-drug 5-fluorocytosine (5-FC)
into the cytotoxic chemotherapeutic agent 5-fluorouracil (5-FU),
thereby sensitizing the BCCs to 5-FC. The adenoviral vector is one of
the most attractive candidates for the delivery of chemosensitization genetic elements, because it can infect epithelial neoplastic cells at
high frequency, although it does not efficiently infect the very early
normal hematopoietic stem cells,11,12 unless they are
induced to mature to later stages of differentiation by exposure to
serum and hematopoietic growth factors, such as interleukin-3
(IL-3).13,14 5-FU generated by the CD adenoviral vector and
5-FC is converted into its phosphorylated metabolites, a prerequisite
for incorporation into DNA and RNA.12 If the level of 5-FU
becomes high enough (>30 µmol/L), as is the case with cells that
contain the CD gene, the incorporation of 5-FU into RNA is sufficiently
high to disrupt protein synthesis with consequent death of even
nondividing cells.15-18 Thus, adenoviral vectors containing
the bacterial CD gene could be potentially useful for ex vivo and in
vivo killing of even nondividing BCCs and can therefore be considered
for human bone marrow purging. Results demonstrating the feasibility of
this approach are the subject of this report.
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MATERIALS AND METHODS |
Cells and cell culture.
The BCC lines MCF-7, MDA-MB-453, MDA-MB-436, MDA-MB-231, MDA-468, T47D,
and BT-20 (obtained from American Type Culture Collection [ATCC],
Rockville, MD) were grown in DMEM-F12 (GIBCO-BRL,
Gaithersburg, MD) containing 10% heat-inactivated fetal bovine serum
(FBS; HyClone Laboratories Inc, Logan, UT). The transformed human
kidney cell line 293 (obtained from ATCC) was propagated in Dulbecco's
modified Eagle's medium (DMEM; GIBCO-BRL) containing 10%
heat-inactivated FBS. Human mammary epithelial cells (HMECs), derived
from an explant of normal breast epithelium, were obtained from
Clonetics Corp (San Diego, CA) and grown according to the
manufacturer's specifications. These cells were maintained in a 5%
CO2/95% air humidified atmosphere. Mobilized peripheral
blood (MPB) cells were obtained from cancer patients undergoing
therapeutic autologous stem cell harvest. Mononuclear cells (MNCs) were
isolated by density centrifugation over Ficoll (Histopaque-1077; Sigma
Chemical Co, St Louis, MO) at 500g for 30 minutes and washed
twice with Medium 199/HBSS (JRH Biosciences, Lenexa, KS).
Chemicals.
5-FC and 5-FU were purchased from Sigma Chemical Co and
6-[3H]5-Fluorocytosine (4.1 Ci/mmol) was purchased from
Moravek Biochemicals Inc (Brea, CA).
Drug cytotoxicity assays.
To test for drug sensitivity, 1 × 104 MCF-7
breast cancer cells, MDA-MD-453 breast cancer cells, or primary normal
breast epithelial cells (HMECs) were plated in triplicate in tissue
culture medium supplemented with 5-FC or 5-FU (0 to 10 mmol/L) in
96-well flat-bottom tissue culture plates (Corning Inc, New York, NY).
Drugs were added approximately 24 hours after seeding. After 5 to 6 days in culture, the effect on cell proliferation was assessed using a
fluorimetric/colorimetric assay (Alamar Blue Assay; Alamar Biosciences Inc, Campbell, CA).
Drug cytotoxicity assays after exposure to the CD adenoviral vector.
To test for adenoviral induced sensitization to 5-FC, 1 × 104 MCF-7 cells, MDA-453 cells, or HMECs were infected with
the Ad.CMV-CD at different multiplicities of infection (MOI; 10, 50, and 100) in serum-free medium for 90 minutes. After washing, cells were then plated in triplicate into 96-well flat-bottom tissue culture plates. Different concentrations of 5-FC were added to the cells after
infection and, after 5 days, the number of cells remaining was
determined as described above.
In vitro testing of the selectivity of Ad.CMV-CD-mediated
sensitization of BCCs to 5-FC.
Peripheral blood stem cells, MCF-7 BCCs, and mixtures of the two cell
populations (a total of 2.5 × 106 cells) were
incubated for 48 hours in 25-cm2 flasks containing 10 mL of
medium with 500 µmol/L 5-FC and 20 µCi of [6-3H]5-FC
(Moravek Biochemicals, Brea, CA). A 1-mL aliquot of the medium was
sampled and mixed with an equal amount of ice-cold methanol,
centrifuged at 7,500g for 5 minutes, and stored at
 20°C before analysis. The conversion from 5-FC to 5-FU was
evaluated by high-performance liquid chromatography (HPLC)
using a Microsorb C18 reverse phase column (25 cm × 4.6 mm
internal diameter; Rainin, Inc, Woburn, MA) eluted with 50 mmol/L potassium phosphate monobasic (KH2PO4),
pH 3.0, at room temperature. Fractions were collected at 1-minute
intervals and radioactivity was counted after the addition of
scintillation fluid.
To evaluate drug metabolism and incorporation, MCF-7 BCCs were
incubated with 500 µmol/L 5-FC containing 40 µCi of
[6-3H]5-FC for 48 hours after infection with 100 MOI of
Ad.CMV-CD or with 5 µmol/L 5-FU in the presence of 25 µCi
[6-3H]5-FU for 24 hours at 37°C and then washed twice
with ice-cold phosphate-buffered saline (PBS), and the mono-layer was
treated with 0.5 mL of 5% trichloroacetic acid (TCA). The pellet was
washed twice with 5% TCA and the incorporated radioactivity was
determined after digestion with NCS tissue solubilizer (Amersham,
Arlington Heights, IL). To analyze the fluorinated metabolites, TCA
cell extracts were neutralized with Freon/trioctylamine extraction and
separated on a Spherisorb SAX HPLC column (25 cm × 4.6 mm i.d.)
at room temperature.14 The column was eluted with a sodium phosphate (pH 3.3) gradient from 0.02 to 0.3 mol/L for 40 minutes at
0.7 mL/min. The effluent was collected in 1-mL fractions and radioactivity was determined. Unlabeled standards were added to each
aliquot assay to positively locate the fluoronucleotides.
Recombinant adenovirus.
A replication-incompetent recombinant adenoviral vector obtained from
the laboratory of Dr Ron Crystal (Cornell Medical School, New York,
NY) that contained the cytosine deaminase gene (Ad.CMV-CD) in a cytomegalovirus (CMV)-driven transcription unit was used in this
series of experiments.19 In this vector, a portion of the
E1a and E1b gene region of human adenovirus serotype 5 had been
replaced by the bacterial cytosine deaminase gene under the transcriptional control of the human CMV promoter as described previously.19 A similar adenoviral vector (Ad.CMV-gal)
was engineered in our laboratory in which a -galactosidase
transcription unit was inserted into the E1a and E1b
region.20 Recombinant adenovirus was purified by a cesium
chloride (CsCl) gradient density centrifugation. The final viral band
was diluted 1:1 with sterile glycerol and stored at 70°C.
The number of adenovirus particles in viral stocks was determined by
limiting dilution and plaque formation of 293 cells exposed to various
dilutions of the vector (plaque-forming units [pfu]). Absence of
replication-competent virus was confirmed by the limiting dilution and
plaque formation assays in HeLa cells exposed to the vector.
Fluorescein di--D-galactopyranoside (FDG) flow cytometry analysis
of infectivity of the -galactosidase adenoviral vector for BCCs
versus hematopoietic cells.
Mobilized peripheral blood or enriched CD34+ cells were
exposed for 30 minutes, 90 minutes, or 24 hours to the Ad.CMV-gal virus (100 MOI) in serum-free medium (Iscove's modified Dulbecco's medium [IMDM]; GIBCO-BRL), washed twice with PBS, and
incubated for 24 hours in 24-well flat-bottom plates with IMDM
containing 5% heat-inactivated FBS in the absence of growth factors.
Twenty-four hours after exposure to Ad.CMV-gal, the cells were
washed once with PBS and prepared for flow cytometry. Cells were
stained with CD34 phycoerythrin (PE)-conjugated MoAb alone (HPCA-2;
Becton Dickinson, San Jose, CA) or in combination with CD38
Cy5-PE-conjugated or CD33 Cy5-PE-conjugated MoAbs (Caltag
Laboratories, Burlingame, CA).
After 30 minutes of incubation in the dark at 4°C, cells were
washed with PBS containing 1% bovine serum albumin and stained with
fluorescein di--D-galactopyranoside (Molecular Probes, Inc, Eugene,
OR), as previously described.21,22 A 2 mmol/L solution of
FDG (substrate) in 98:1:1 H2O/DMSO/ethanol was mixed with
an equal volume (50 µL) of cell suspension that was prewarmed to 37°C. After 1 minute of incubation at 37°C, an equal volume (50 µL) of ice-cold 2× strength PBS was added.
The samples were maintained at 4°C for 3 to 4 hours before analysis
by flow cytometry. Cells were treated with Chloroquine to suppress
endogenous -galactosidase activity. Viability studies were performed
using propidium iodine. Flow cytometry was performed with a FACStar
flow cytometer (Becton Dickinson). The frequency of FDG+
cells for each particular immunophenotype was calculated as follows: Frequency (%) = (number of a particular immunophenotype that was FDG+ among vector exposed cells) (number of a
particular immunophenotype that was FDG+ among cells not
exposed to vector)/(number of a particular immunophenotype among
vector-exposed cells).
BCCs were harvested from monolayer cultures with 0.5 mmol/L EGTA
[ethylene glycol-bis(-aminoethyl
ether)-N,N,N ,N-tetraacetic acid, pH 7.0] and exposed in
suspension to the adenoviral vectors for 90 minutes or 24 hours, as
specified. After vector exposure, cells were washed twice with PBS and
seeded in tissue culture medium. Twenty-four hours after vector
exposure, the cells were trypsinized, washed twice in PBS, and stained
with FDG.
Integrin analysis.
MoAbs used to detect the human  V3
[LM609] and V5 [P1F6] integrins were
purchased from Chemicon International (Tamecula, CA). BCC lines and
CD34+ cells were stained with each MoAb using standard
procedures.23,24 Flow cytometry was performed using a
FACStar flow cytometer (Becton Dickinson).
Analysis of the CD adenoviral vector sensitization of clonogenic
cells in BCC lines or hematopoietic cells to 5-FC.
Methods used for this assay have been described in detail previously by
our laboratory.25 To determine the proliferative capacity
of hematopoietic cells and BCCs, mixtures composed of CD34+
cells and MCF-7 cells were exposed in suspension for 90 minutes to the
Ad.CMV-CD vector in duplicate assays, washed twice in PBS, and left in
culture for a 2-day period in the presence or absence of 5-FC. To
determine the proliferative capacity of the hematopoietic cells,
granulocyte-macrophage colony-forming unit (CFU-GM) cells were assayed
as follows25: 2 to 3 × 103
CD34+ selected cells were cultured in 1 mL of 0.9%
methylcellulose in IMDM, 30% fetal bovine serum, 1% bovine serum
albumin, 104 mol/L 2-mercaptoethanol, 2 mmol/L
L-glutamine, 5% serum containing phytohemoagglutinin leukocyte
conditioned medium (PHA-LCM), and 3 U/mL of recombinant human
erythropoietin (MethoCult, StemCell Technologies Inc, Vancouver,
British Columbia, Canada). Cells were cultured in 35-mm plates and
incubated for 14 days at 37°C in a humidified atmosphere containing
5% CO2 in air. Colonies consisting of 50 cells or more
were counted as CFU-GM. A limiting dilution assay was used to determine
the effects of the Ad.CMV-CD vector on BCCs. At the end of the 2-day
incubation period, all cells were collected and washed twice in PBS. A
total of 104 cells were serially diluted 10-fold, and each
dilution was plated into a 96-well flat-bottom microtiter plate that
contained 200 µL medium DMEM (DMEM-F12, 10% FBS) per well and
incubated for 14 days. The clonogenic growth of surviving tumor cells
was evaluated by phase-contrast microscopy, scoring the number of wells
with at least 1 colony containing 20 or more cells.
Evaluation of the efficacy of the 5-FC/CD adenoviral vector system
under conditions that simulate in vitro purging of BCCs from
collections of autologous hematopoietic cells.
MCF-7 BCCs (9×107 cells) and 7 × 109 HL60 cells were mixed in a 50 mL volume of PBS and
exposed to the cytosine deaminase adenoviral vector (in a ratio of
pfu/total cells of 400/1) for 90 minutes. The cells were then diluted
and plated overnight in 150-cm2 tissue culture flasks. An
aliquot of these cells was subjected to serial 10-fold dilutions up to
a 1/106 fold dilution. The cells were plated in
150-cm2 flasks in up to 1/103 dilutions, in
75-cm2 flasks in up to 1/104 dilutions, and in
25-cm2 flasks at higher dilutions. The first dilution was
less than 10-fold so that the final 10-fold dilution would produce 1 colony equivalent (20 cells). These cells were then incubated
overnight, rinsed free of nonadherent cells, and incubated in medium
supplemented with 500 µmol/L 5-FC for 14 days. The colonies were
subsequently counted after staining with methylene blue.
Evaluation of the safety of the 5-FC CD adenoviral vector system for
hematopoietic reconstituting cells in a mouse transplantation model.
Hematopoietic cells were harvested from the femurs of Balb/C male donor
mice 48 hours after treatment with 150 mg/kg of 5-FU. The hematopoietic
cells were exposed to the adenoviral vector carrying the cytosine
deaminase gene for 90 minutes and rinsed. Between 1 and 2 million of
the cells were then transplanted into female recipient mice exposed to
725 to 750 cGy of total body irradiation.25 In some cases,
the marrow cells were exposed to 5-FC by either incubating them in
QBSF58 serum-free medium (Quality Biologicals Inc, Gaithersburg, MD)
supplemented with 100 ng/mL of stem cell factor (Amgen Inc, Thousand
Oaks, CA) for 96 hours in suspension culture in the absence of serum at
37°C, with or without supplementation with 500 µmol/L 5-FC before
transplantation, or alternatively exposed in vivo after transplantation
to 5-FC (peak serum concentrations in the 500 µmol/L range) for 14 days in vivo immediately after the transplantation into the female recipients.
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RESULTS |
Time course of expression of adenoviral transgenes after infection.
MCF-7 cells were exposed to the -galactosidase adenoviral vector for
90 minutes, after which the cells were incubated in complete tissue
culture medium and assayed for -galactosidase activity by enzymatic
analysis of extracts of cells at 1, 2, 4, 6, 8, 10, and 12 days after
infection. As shown in Fig 1, the expression of the transgene quickly reached its maximum and persisted at that level for 4 to 5 days before starting to decrease.
-Galactosidase activity was still detectable well beyond 10 days.
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| Fig 1.
Time course of expression of -galactosidase gene in
MCF-7 BCCs. BCCs were exposed to the -galactosidase adenoviral
vector for 90 minutes, incubated for 24 hours in complete medium, and then tested for expression of the transgene by an enzymatic assay on
cell lysates at the indicated times (days) after the infection.
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Comparative cytotoxicity effect of 5-FC and 5-FU in normal breast
epithelium and BCC lines in the absence of Ad.CMV-CD.
We evaluated the antiproliferative effect of 5-FC or 5-FU at different
concentrations of fluoropyrimidines (ranging between 0 and 10 mmol/L),
in the absence of Ad.CMV-CD vector, on several BCC lines and on primary
HMECs, as shown in Table 1. The results of
these experiments showed that 5-FC is 1,000-fold less potent as an
inhibitor than 5-FU for HMECs and 10,000-fold less toxic for the two
BCC lines than 5-FU.
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Table 1.
Differential Cell Growth Inhibition (IC50)
of 5-FC and 5-FU in Primary Normal HMECs and in BCC Lines, MCF-7 and
MDA-MB-453, Before or After Incubation With the Ad.CMV-CD Vector
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Increased sensitivity of HMECs and BCCs to 5-FC after exposure to
Ad.CMV-CD.
The BCCs were exposed to the Ad.CMV-CD vector for a short period of
time (90 minutes) in serum-free conditions and then incubated for 2 days in 5-FC. As shown by the data presented in Table 1, after exposure
of the cell lines to the CD adenoviral vector, they became sensitive to
concentrations of 5-FC that were nontoxic before exposure to the virus
(Table 1). More importantly, even the slowly dividing primary explants
of normal breast cells (HMECs) showed growth inhibition between 85%
and 100% at concentrations of 5-FC in the range of 100 to 1,000 µmol/L after exposure to the Ad.CMV-CD vector.
These data clearly indicate that the cytosine deaminase vector
(Ad.CMV-CD) can efficiently increase the sensitivity of BCC lines or
primary explants of normal breast epithelial cells to 5-FC between 100- and 1,000-fold. We also studied the toxicity of 5-FC to the BCCs after
they were exposed to varying ratios of the cytosine deaminase
adenoviral vector (Table 1). These experiments suggest that even very
low ratios of the CD adenoviral vector/cell can generate significant
toxicity. These results may underestimate the true potential of the
5-FC/CD adenoviral system, because the cells were exposed to 5-FC for
only 2 days before analysis. As will be shown below, longer incubation
in 5-FC increases cell kill.
Study of the selectivity of infectivity of BCCs versus hematopoietic
cells by Ad.CMV-CD.
The conversion of 5-FC into 5-FU 48 hours after 90 minutes of exposure
in serum-free medium to the AD.CMV-CD vector was evaluated as a measure
of the infectivity of the adenoviral vector for PBMCs compared with the
BCC line MCF-7 (Fig 2). When PBMCs were
incubated in 500 µmol/L of 5-FC (2.5 µmol total 5-FC present in the
medium) after exposure of the cells to the Ad.CMV-CD vector (100 MOI), almost no detectable conversion of 5-FC to 5-FU occurred in the hematopoietic cells (0.018 µmol). In contrast, when the MCF-7 BCC
line was exposed to the Ad.CMV-CD vector (100 MOI) for 90 minutes (or
for 24 hours) and then incubated an additional 48 hours in 5-FC, almost
70% of 5-FC was converted into 5-FU (1.74 µmol). Importantly, the
adenoviral vector showed infectivity for BCCs even when they were
diluted in a 1,000-fold excess of PBMCs, leading to the conversion of
6.4% (0.16 µmol) of 5-FC into 5-FU (Fig 2). The concentration of
5-FU generated in the medium by a culture of 100% MCF-7 cells exposed
to 500 µmol/L 5-FC reached a 5-FU concentration of 347 µmol/L, a
concentration well above that necessary to kill most of the established
cancer cell lines.
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| Fig 2.
Conversion of 5-FC into 5-FU in PBSCs, MCF-7 BCCs, and an
0.1% mixture of MCF-7 cells in PBSCs after exposure to
Ad.CMV-CD adenoviral vector. Cells (2.5 × 106)
were exposed for 90 minutes to 100 MOI of Ad.CMV-CD and then incubated
for 48 hours in a 25-cm2 flask with 10 mL of medium
containing 500 µmol/L 5-FC and 40 µCi of [6-3H]5-FC.
One milliliter of medium was collected after 24 hours, mixed with an
equal amount of ice-cold methanol, and analyzed by HPLC as described in
the Materials and Methods. The numbers above each histogram column
indicate the total amount of drug in micromoles (either 5-FC or 5-FU)
per flask.
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We then studied the incorporation of 5-FU into RNA and DNA of MCF-7
cells treated at the known IC50 concentration of the
fluoropyrimidine for this adenocarcinoma breast cell line
(Fig 3). The IC50 concentration for the MCF-7 breast adenocarcinoma cells was 5 µmol/L of 5-FU generating 15 pmol/106 cells of 5-FU nucleotides with 17 pmol/106 cells of the fluoropyrimidine incorporated into
the nucleic acids (RNA and DNA) during a 24-hour period. In contrast,
MCF-7 cells exposed to the gene therapy system of Ad.CMV-CD (100 MOI)
for 90 minutes followed by 48 hours of incubation in 500 µmol/L 5-FC were able to generate a 320-fold excess of fluorinated nucleotides (FUXP) compared with the same cell line at the IC50
concentration of 5-FU, with an incorporation of 170 pmol/106 cells into the nucleic acids, a 10-fold gain in
5-FU incorporation with the vector system over direct treatment with
5-FU (Fig 3). Even when 0.1% of MCF-7 cells were mixed with a
population of PBMCs, exposed to Ad.CMV-CD and 50 µmol/L of 5-FC, the
presence of 2,500 breast cancer cells (0.1% of the total cells) was
able to generate a concentration of 32 µmol/L 5-FU (see Fig 2), well above the 5 µmol/L that is the IC50 value for the MCF-7
cell line for 72 hours of exposure. This is substantially below the
IC50 for hematopoietic stem cells, which is 650 µmol/L of
5-FU.26
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| Fig 3.
Comparative metabolism and nucleic acids incorporation of
5-FC and 5-FU in MCF-7 BCCs exposed to the 5-FC/Ad.CMV-CD gene therapy system and 5-FU. MCF-7 cells were incubated for 90 minutes with 100 MOI
of Ad.CMV-CD followed by 48 hours of exposure to 500 µmol/L 5-FC with
20 µCi of [6-3H]5-FC or for 24 hours with 5 µmol/L
5-FU in the presence of 25 µCi of [6-3H]5-FU. The
conversion of 5-FC into 5-FU was determined after the collection of 1 mL of medium by HPLC as described in the Materials and Methods.
Formation of fluorinated nucleotides (FUXP) was determined in the
neutralized TCA cell extract by HPLC analysis using a Spherisorb SAX
column eluted with a gradient of sodium phosphate, pH 3.3. The
incorporation into nucleic acids was determined in the TCA pellets
after digestion with NCS tissue solubilizer. The numbers above the
histograms represent the micromoles of 5-FU or FUXP measured.
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BCCs are more sensitive to adenoviral infection than
CD34+ cells.
We directly tested the infectivity of several BCC lines (MDA-MB-468,
MDA-MB-436, MDA-MB-453, MDA-MB-231, and T-47D) using the adenoviral
vector Ad.CMV-Gal (100 MOI) after 90 minutes of exposure and an
additional 24 hours of incubation in a fluorescein isothiocyanate
(FITC)-conjugated substrate (FDG) for the -galactosidase reaction.
More than 90% of the cells in each of the breast cancer populations studied were infected by the adenoviral vector
(Fig 4), as measured by increased
fluorescence, because the presence of the -galactosidase enzyme
would generate higher levels of fluorescein (Fig 4).
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| Fig 4.
Infectivity of different human BCC lines by the
-galactosidase Ad.CMV adenoviral vector. BCCs were exposed in
suspension for 90 minutes (red line) or 24 hours (blue line) to 100 MOI
of the Ad.CMV-gal vector. Twenty-four hours later, the cells were washed with PBS, stained with FDG, and analyzed by flow cytometry as
described in the Materials and Methods. The profile defined by the
black line is the control in the absence of virus. The red and the
blue profiles are from samples exposed to the vector for
90 minutes and 24 hours, respectively.
|
|
The cells were exposed to the Ad.CMV-gal vector for 90 minutes (red
line) or 24 hours (blue line). There was no difference in
the fluorescence intensity between the cells exposed to the vector for
90 minutes or for 24 hours of exposure, demonstrating that a long-term
exposure to the vector is not necessary to reach a high frequency of
infection in BCC lines. However, the extent of infection, either at 90 minutes or 24 hours, is different for each cell line, ranging between a
0.5-log to a 2-log increase in peak fluorescence intensity.
We used the same method to determine the infectivity of early
hematopoietic cells by the Ad.CMV-gal vector (100 MOI) at 30 minutes, 90 minutes, and 24 hours of exposure. We specifically focused
on CD34+ cells and the
CD34+CD38 and
CD34+CD33 subsets that represent earlier
stages of hematopoietic differentiation. The results of these
experiments show that less than 1% of the CD34+,
CD34+CD38, and
CD34+CD33 hematopoietic cells are
infected by the Ad.CMV-gal vector, even at 24 hours of vector
exposure, under our experimental conditions, in which no growth factors
are added. Other investigators11,12 have reported similar
low frequencies of infection of early hematopoietic precursor cells by
the adenoviral vector. If the hematopoietic cells are incubated in
growth factors capable of inducing maturation of the early
hematopoietic cells, the infectivity by the adenoviral vector
increases.13,14
Patterns of integrin expression may explain differences in
infectivity of the adenoviral vector observed among BCC lines.
It has been proposed that the integrins
V3 and V5
play an important role as coreceptors for adenovirus
infection.23,24 To test this hypothesis, we studied the
level of expression of these integrins in the BCC lines (MDA-MB-453,
T47D, MDA-MB-231, MCF-7, MDA-MB-468, BT-20, and MDA-MB-436) and in
CD34+ hematopoietic cells. The results of these experiments
are shown in Table 2. There was a very low
level of expression for V3 in
CD34+ cells (4.4%). A low level of
V3 integrin was seen in all of the BCC
lines (see Table 2) except for the MDA-MB-231 cell line, which was
42.5% positive. In contrast, a substantial but variable level of
expression of V5 was detected in all of
the BCCs tested (see Table 2). In contrast, the
V5 integrin receptor was not detectable
in the vast majority CD34+ cells. This integrin has been
reported to play a key role in adenoviral vector uptake and release
from the post-uptake endosome. BCCs have a 4- to 27-fold higher level
of V5 than CD34+ cells, as
shown in Table 2. The level of adenoviral infectivity in the BCC lines,
as measured by the expression of -galactosidase from the
-galactosidase adenoviral vector, appeared to be related to the
presence of the V5 integrin (see Table
2).
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Table 2.
Correlation Between Integrin Expression in Different
Cell Types, Infectivity by CD Adenoviral Vector as Measured by
Adenoviral Vector -Galactosidase Activity at 72 Hours After
Infection, and Functional Expression of the Adenoviral Vector Cytosine
Deaminase Gene as Measured by Conversion of 5-FC to 5-FU During a
24-Hour Period at 72 Hours After Infection With CD Adenoviral
Vector
|
|
The pattern of expression of integrins on the BCCs in marrow samples
from breast cancer patients was also evaluated by Richard Cote. He
found that all the BCCs metastatic to the bone marrow were positive for
the V3 integrins (data not shown), as has been previously reported in much larger numbers of
patients.27 He also studied the infectivity of primary BCCs
present in marrow of advanced breast cancer patients and found them to
be infectable with the -galactosidase adenoviral vector (data not
shown).
Correlation between integrin expression, infectivity, and 5-FC
conversion in BCC lines.
We have followed the expression of -galactosidase, as a measure of
cell infectivity, over a 72-hour period in BCC lines expressing different levels of V5 after incubation
with the Ad.CMV-gal adenoviral vector. As shown in Table 2, the
fluorescence intensity level of FDG, indicating -galactosidase
activity, was 12- to 15-fold higher in the T47D and MCF-7 cell lines
that presented high levels (90% to 96%) of
V5 integrin expression, as compared with
MB-MDA-453 cells that showed only a 15.8% presence of
V5. Similarly, the conversion of 5-FC
into 5-FU was twofold to threefold higher in the T47D and MCF-7 cell
lines with high V5 expression, than with
the MDA-MB-453 cell line, which showed low levels of the
V5 integrin, after exposure to the
Ad.CMV-CD vector and incubation with 500 µmol/L 5-FC for a 24-hour
period starting 48 hours after exposure to the adenoviral vector (Table
2). These results further confirm the crucial role of integrins in the
uptake of the adenoviral vector and ultimately in the expression of the transgene.
Sensitivity of mixtures of BCCs and hematopoietic progenitor cells to
treatment with Ad.CMV-CD plus 5-FC.
We used a methylcellulose assay for measuring the effect of the 5-FC/CD
adenoviral system on the clonogenic progenitor hematopoietic cells and
a limiting dilution colony formation assay to evaluate the growth of
colonies from cultures of MCF-7 BCCs after exposure to the CD
adenoviral vector and 5-FC. We prepared mixtures of BCCs in a
1,000-fold excess of normal human PBMCs and plated the cells (10,000 cells/well for each condition: virus alone, 5-FC alone, and virus plus
5-FC) in microwell plates as described in the Materials and Methods. We
exposed the cells to 5-FC (100 µmol/L) for 2 days in the absence of
the Ad.CMV-CD vector and with both 5-FC (100 µmol/L) and Ad.CMV-CD
vector (100 MOI). The data from these experiments indicate that vector
exposure for 90 minutes, followed by incubation in 5-FC for 48 hours,
decreased the proliferative capacity of clonogenic BCCs, even when the
BCCs were in a 1,000-fold excess of normal PBMCs. In this low-density
culture system, no viable colonies of MCF-7 were seen after incubation
to the adenoviral vector plus 5-FC (data not shown). This may again be
an underestimation of the true potential of this system, because the
target cells were incubated in 5-FC for only 2 days. As shown below,
longer periods of incubation of virally exposed cells in 5-FC result in
greater levels of cell kill.
The toxicity of the purging system to clonogenic hematopoietic cells
was also tested using a methylcellulose colony assay. We measured the
number of colonies generated by PBMCs exposed for 90 minutes to the
Ad.CMV-CD vector (100 MOI) and then cultured in suspension in the
presence of 10 µmol/L and 50 µmol/L 5-FC for 48 hours, when plated
in methylcellulose medium. No significant decrease in the numbers of
CFU-GM in colonies was detected even when the myeloid cells were mixed
with 0.1% of BCCs and then exposed to the Ad.CMV-CD and 5-FC. The data
presented in Table 3 show that the early
hematopoietic precrusor cells, which give rise to the hematopoietic
clonogenic progenitor cells, are resistant to the 5-FC and Ad.CMV-CD
vector treatment, even when exposed in the presence of low percentage
of BCCs.
Testing of the potential efficacy of 5-FC/CD adenoviral vector
purging of BCC from mixtures with hematopoietic cells.
A mixture of 2 × 106 MCF-7 cells in a 100-fold excess
of HL60 cells was exposed to the CD adenoviral vector (400 MOI) for 90 minutes; the cells were then subjected to 10-fold serial dilutions and
plated overnight. The nonadherent cells were rinsed away, and the cells
were incubated in 500 µmol/L 5-FC for 14 days. In a second
experiment, we made a mixture of 9 × 107 MCF-7 cells
and 7 × 109 HL60 hematopoietic cells, exposed the
cells to the cytosine deaminase vector (200 MOI) for 90 minutes, plated
the cells directly or after serial dilution by factors of 10 up to
1/1,000,000 and then incubated overnight, rinsed off the nonadherent
cells, and incubated the cells for 14 days in 500 µmol/L
5-FC. Colony counts at the end of this period, even in
flask that were inoculated with 107 MCF-7 cells, showed no
colonies detectable even in the undiluted cultures, in which the cells
have been exposed to the cytosine deaminase vector and incubated in
5-FC (Table 4). This shows that the
vector/5-FC system can decrease the level of BCCs by more than 1 million fold when present in a mixture with hematopoietic cells.
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Table 4.
MCF-7 Clonogenic Cells After Exposure of a Mixture of
9 × 107 MCF-7 Cells in a 100-Fold Excess of
HL60 Cells to the CD Adenoviral Vector (200 MOI) for 90 Minutes
|
|
Testing of the safety of the 5-FC/CD adenoviral vector system through
use of an in vivo engraftment transplantation assay after exposure to
Ad.CMV-CD and 5-FC.
A potential adverse effect of all ex vivo purging procedures is the
toxicity to the 1/10,000 hematopoietic early precursor cells that are
responsible for hematopoietic reconstitution after transplantation into
lethally irradiated recipients. We have already demonstrated the lack
of toxicity of the adenoviral infection, Ad.CMV-CD, and 5-FC treatment
to the hematopoietic progenitor cells measured by CFU-GM
methylcellulose assay after exposure to the vector and 5-FC in vitro
(see Table 4). To test the effect of the 5-FC/CD adenoviral system on
the viability of the more primitive reconstituting hematopoietic cells,
we used a murine bone marrow transplantation model.
We exposed marrow cells collected from 5-FU-treated male donor mice to
the cytosine deaminase adenoviral vector for 90 minutes. We then
incubated the marrow cells for 4 days in serum-free medium (QBSF 58 medium) supplemented with 100 ng/mL stem cell factor and 500 µmol/L
5-FC. One to two million of these cells were then used in the
transplant into lethally irradiated female mice. Sixty-eight percent of
the mice survived the transplant. As shown in
Fig 5, most of the deaths occurred before
day 10 after transplantation. Deaths that occur before 10 days after
transplantation are usually ascribed to infection or radiation
toxicity, rather than to failure to engraft. Thus, most of the
hematopoietic reconstituting cells survived the in vitro incubation
with 5-FC after exposure to the cytosine deaminase adenoviral vector.
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| Fig 5.
Kaplan-Meier analysis of survival. Group 1: 22 mice
received 1 to 2 million fresh marrow cells. Group 2: 21 mice
transplanted with 1 to 2 million marrow cells cultured in QBSF58
serum-free medium for 4 days, as was group 3, but not exposed to the CD
adenoviral vector/5-FC. Group 3: 19 mice transplanted with 1 to 2 million marrow cells exposed to the CD adenoviral vector followed by
5-FC. Survival for each group was estimated by the Kaplan-Meier model. Log-rank test was used to check for equality of survival functions. Sixty-two mice were studied. Follow-up was 83.9 ± 43.4 days
(mean ± SD), with a range of 4 to 145 days. Survival functions:
Results are given as the mean ± SE, with the 95% confidence interval
in parentheses. Group 1: 22 mice were transplanted and 19 survived (from 3 different experiments). Three died at day 6. Survival function:
86.4 ± 7.3 (63.4 to 95.4). Group 2: 21 mice were transplanted, 19 survived and 2 died at days 6 and 8 (2 different experiments). Survival: 90.5 ± 6.4 (67 to 97.5). Group 3: 19 mice were
transplanted. Six died at days 4 (1 mouse), 6 (2 mice), 9 (2 mice), and 18 (1 mouse) (4 different experiments total). Survival: 68.4 ± 10.7 (42.8 to 84.4). Log-rank test: P = .16. There was no
difference in survival among these 3 groups.
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|
Because of the data presented in Table 4, in which a 1 million fold
reduction of the BCC in an excess of hematopoietic cells was achieved
by incubation of the cytosine deaminase vector exposed cells to 10 or
more days of 5-FC incubation, we also used the mouse model to test
whether CD adenoviral-infected cells, exposed to 14 days of 5-FC
immediately after infusion, would maintain the reconstituting
capability. One to two million of these cells were used in the
transplant into lethally irradiated female recipient mice. As shown by
the data in Table 5, the survival of the
mice undergoing transplantation with vector-exposed cells with or
without the posttransplantation 14-day infusion of 5-FC, at
intraperitoneal doses of 5-FC that generate 500 µmol/L peak serum
concentrations, was not significantly different from the survival of
mice undergoing transplantation with vector-exposed cells without 5-FC
treatment (Table 5).
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Table 5.
Transplantation of Marrow Cells Exposed Before
Transplantation to Cytosine Deaminase Adenoviral Vector Followed by
Posttransplantation In Vivo Exposure to 14 Days of 5-FC
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|
Individual posttransplantation white blood cell counts (WBC) and
differential counts performed in all animals in each group (transplants
with cells exposed to vector followed by 4 days of in vitro 5-FC
pretransplantation incubation or 14 days of posttransplantation in vivo
5-FC exposure) showed no differences in leukocytes, lymphocytes, platelets, or hematocrit volumes among the different transplant groups
when determined at 2 or 6 months after transplantation (data not
shown). These data suggest that the grafts are stable in all of the
treatment groups.
To measure specifically whether the male donor cells that were exposed
before transplantation in vitro to the cytosine deaminase adenoviral
vector followed by 4 days of pretransplantation in vitro exposure to
5-FC or exposed to the vector before transplantation followed by 14 days of posttransplantation 5-FC exposure could stably repopulate the
female recipient lethally irradiated mice, lethally irradiated female
recipient mice transplanted with Ad.CMV-CD-exposed cells exposed to
5-FC either before or after transplantation were killed 1 to 6 months
after transplantation. Then, G-banding cytogenetic analysis for the Y
chromosome was performed on the bone marrow cells from these animals.
We performed this analysis both for the animals transplanted with
vector-exposed cells that either were incubated in vitro for 4 days in
QBSF58 serum-free medium supplemented with 500 µmol/L 5-FC (2 animals
at 1 to 2 months after transplantation) or were exposed after
transplantation in vivo to 5-FC for 14 days (4 animals at 6 months
after transplantation).
Because all transplants involved the infusion of male donor cells into
lethally irradiated female recipients, we were able to use cytogenetics
to test stable and complete engraftment of the Ad.CMV-CD and
5-FC-treated cells. All of the metaphase spreads studied for each of 2 female recipient animals 1 and 2 months after transplantation showed
100% of the cells as donor male cells. In addition, greater than 99%
of the metaphases studied at 6 months in both groups (4 days of in
vitro 5-FC exposure in QBSF58 serum-free medium before transplantation
or 14 days of posttransplantation 5-FC in vivo exposure) showed a male
karyotype. These results demonstrate that the engrafting capability of
the male reconstituting hematopoietic cells was not affected by the
pretransplantation Ad.CMV-CD exposure followed by either 4 days of
pretransplantation 5-FC in vitro or 14 days of posttransplantation in
vivo 5-FC administration. All of these data show that a sufficient
number of the hematopoietic reconstituting cells survived the
5-FC/adenoviral vector exposure to generate complete and stable
engraftment in lethally irradiated female recipients.
| |
DISCUSSION |
Retroviral marking studies in autologous transplantation used to treat
both hematopoietic and solid tumor neoplasms have shown that the
presence of neoplastic cells in the infused hematopoietic cells used to
restore marrow function after transplantation can contribute to relapse
after transplantation.28-30 Fields et al3 recently reported that patients infused with autologous hematopoietic cells that are free of contaminating BCCs by PCR assay have a longer
disease-free interval than do those patients infused with autologous
cells that are positive by PCR assay from BCCs. We therefore decided to
test if a replication incompetent adenoviral vector could be used to
selectively deliver a chemotherapy sensitization gene, cytosine
deaminase, to BCCs without altering the hematopoietic cells. The goal
was to sensitize the BCCs to drugs that are nontoxic for the
hematopoietic cells.
We chose for this purpose a bacterial gene, cytosine deaminase, that
catalyzes the conversion from 5-FC into 5-FU. We showed that there were
three types of differences between BCCs and normal immature
hematopoietic reconstituting cells that protect the hematopoietic cells
and sensitize the BCCs using this system. We first tested whether
hematopoietic cells and BCCs show differences to infection by the
adenoviral vector. -Galactosidase assays on breast cancer and
CD34-selected cells exposed for 24 hours to an adenoviral vector
carrying the Lac-Z gene showed that less than 1% of the CD34-selected
early hematopoietic cells were infected by the vector, whereas up to
100% of each of 6 of the BCC lines tested scored positive for
infection in the -galactosidase assay. We also used the cytosine
deaminase adenoviral vector to show that conversion of 5-FC to 5-FU, as
measured by HPLC, in CD34-selected hematopoietic cells exposed to the
cytosine deaminase vector was at the undetectable level, whereas
greater than 70% conversion was seen in the MCF-7 BCC line after
exposure to the cytosine deaminase vector. Thus, this first difference
demonstrated definitively that the CD34-selected cell and its
CD34+CD33 and
CD34+CD38 subsets exposed to the vector
under nondifferentiating conditions were not infectable by the
adenoviral vector, whereas the BCC were infectable.
Direct assay of the CD34-selected cells for the presence of the
V3 and V5
integrins that have been reported to bind the penton proteins of the
adenoviral vector,23,24 a step necessary for uptake of the
vector and release of adenoviral vectors from the postuptake endosome
(which is necessary for high levels of expression of the vector
transgenes), were absent from the surface of the early hematopoietic
cells. Other workers have shown that the adenoviral vector does not
bind efficiently to these hematopoietic cells, presumably due to the
absence of the receptor for the fibrillar protein of the vector that
mediates vector binding. Our data and those of others25
have shown that both the BCC lines and the BCCs metastatic to the
marrow have the requisite integrin receptors for adenoviral uptake and
are infectable by these vectors.
The conditions of the vector exposure and in vitro purging conditions
(serum-free medium [QBSF58] and the absence of late-acting growth
factors) were designed to minimize the tendency of the hematopoietic
cells to differentiate. Neering et al13 have shown that,
during differentiation, the early hematopoietic cells become infectable
by the adenoviral vector. Studies in which the adenoviral vector has
been mixed with CD34-selected hematopoietic cells in the presence of
serum and hematopoietic growth factors13,14 that induce
differentiation have shown higher levels of infectability than in our
studies or those of others11,12 in which the conditions of
exposure to the vector (serum-free medium, no growth factors added) did
not induce differentiation of the hematopoietic precursor cells. Thus,
it is imperative to use conditions during the 5-FC in vitro incubation
after exposure of the hematopoietic cells to the adenoviral vector that
do not induce the marrow cells to differentiate to maintain the
specificity of infection.
A second difference between BCCs and normal immature hematopoietic
cells is the increased ability of BCCs to convert 5-FU into
phosphorylated metabolites, a necessary step for 5-FU to exert toxicity
to a cell through inhibition of thymidylate synthase or by
incorporation into nucleic acids. We found that the levels of
orotate-phosphoribosyl transferase, a critical enzyme that catalyzes
the conversion of 5-FU to 5-FUMP, is 50- to 100-fold lower in
CD34-selected immature normal hematopoietic cells than in neoplastic
epithelial cells (data not shown). This second difference between
epithelial neoplastic cells and normal immature hematopoietic cells in
the capability to metabolize 5-FU so that it will be incorporated into
DNA and RNA provides a second level of protection for the early normal
hematopoietic cells from 5-FU.
Finally, a third difference between normal immature hematopoietic cells
and neoplastic epithelial cells, which protects the immature
hematopoietic cells from the toxicity of the cytosine deaminase
adenoviral vector/5-FC treatment, is the greater intrinsic resistance
of the hematopoietic stem cell to 5-FU. 5-FU exposure is used as a
method for fractionating the more sensitive mature hematopoietic cells
from the early reconstituting cells for transplantation in mouse models
and for isolating human stem cells,26 because the early
hematopoietic cells are so resistant to the 5-FU. In contrast, 5-FU is
used in the therapy of breast cancer because BCCs are sensitive to
5-FU.
Our direct measurements show that 100% of the BCCs are sensitive to
5-FU at levels (5 µmol/L) that are less than the threshold at which
early hematopoietic cells show toxicity from 5-FU (500 µmol/L). This
is very important, because 5-FU may diffuse out of the epithelial
neoplastic cells before phosphorylation and could potentially destroy
the hematopoietic cells not initially infected by the cytosine
deaminase vector. The resistance of the immature hematopoietic cells is
at least 100-fold greater than that of the BCCs.
Direct analysis of mixtures of BCCs and CD34-selected hematopoietic
cells in our studies, using assays for clonogenic BCCs and
hematopoietic cells, show that the precursors for the clonogenic progenitor hematopoietic cells are not sensitive to exposure to the
5-FC and cytosine deaminase vector system, whereas a 1 million fold
reduction in clonogenic BCC can be produced by the cytosine deaminase
vector/5-FC system. In addition, our experiments show that even very
slowly growing primary explants of normal breast epithelial cells are
sensitive to the effects of the 5-FC and cytosine deaminase adenoviral
vector gene therapy method. These results suggest that the nondividing
BCCs will be destroyed as well as the dividing BCCs by this treatment.
Because a major fraction of BCCs that contaminate collections of
hematopoietic cells used for transplantation are nondividing, the
mechanism of cell death is probably due to the incorporation of 5-FU
into RNA at a level that inhibits protein synthesis.15,16
The ultimate test for the selectivity of the 5-FC/cytosine deaminase
adenoviral vector treatment (toxicity for BCCs and the absence of
significant toxicity for the early reconstituting hematopoietic cells)
is to test for engraftment of the 5-FC/CD adenoviral vector-treated hematopoietic cells in lethally irradiated recipient mice. Our transplantation data in Balb/C mice show stability of engraftment (for
up to 6 months) and complete engraftment by the male donor cells
exposed to the adenoviral vector and 5-FC (either before transplantation or after transplantation) after transplantation into
female recipient mice as determined by cytogenetic analysis and blood
counts. A study of the data in Table 5 and Fig 5 suggests that the
14-day in vivo exposure to the 5-FC may be safer in terms of the
reconstitutive capacity of the marrow cells used for transplant than is
the 4 days of pretransplantation in vitro 5-FC exposure. Clearly, there
is an advantage of increased levels ( >1 million-fold) of BCC kill
with the posttransplantation 14-day in vivo exposure to 5-FC over the
pretransplantation 4-day exposure to 5-FC, due to the longer time of
5-FC exposure. Thus, in a clinical trial already approved at Yale, we
will be proposing to use a pretransplantation exposure to the Ad.CMV-CD
vector and a posttransplantation in vivo exposure to the 5-FC (for 14 days).
Other investigators have reported studies with adenoviral vectors
carrying the P53 gene to correct cells in which mutation in P53 in the
BCCs has occurred.11 Induction of cell death directly occurred in only a fraction of the primary BCCs in these studies. Another disadvantage of this latter P53 system is that it will be
ineffective for BCCs in which the P53 mutation exerts a
transdominant-negative effect on P53 function.
The BCL-X short transcription unit has also been
introduced into BCCs in an attempt to directly eliminate them for bone
marrow purging purpose.12 We do not yet know whether this
system has adverse effects on the early hematopoietic cells. In
addition, this vector was only partially suppressive to established BCC lines. Moreover, we do not know what the effect of such a vector would
have on slow or nondividing breast cancer cells that are primary
explants rather than established rapidly proliferating BCC lines.
Another pro-drug activation system based on the herpes virus thymidine
(HVTK) is limited by the fact that the sensitization to ganciclovir
generated by incorporation of the HVTK gene into BCCs occurs only if
the cells are in S-phase.31 The cytosine deaminase
adenoviral vector system we have used in the studies in this report has
an obvious advantage, because it is potentially toxic for both dividing
and nondividing breast cancer cells. The very high intracellular levels
of 5-FU generated in cells infected with the cytosine deaminase
adenoviral vector probably results in concentrations of 5-FU necessary
for a sufficient incorporation of 5-FU into RNA to kill even
nondividing cells.15,16
Another advantage of the cytosine deaminase/5-FC system is that the
level of exposure to 5-FU generated in the BCCs is much higher than
that which can safely be achieved by infusion of 5-FU in the systemic
circulation.15-19 Thus, the vector/5-FC system provides a
unique therapeutic advantage over the direct systemic delivery of 5-FU.
The cytosine deaminase vector used in our studies has already been
approved for use in human cancer gene therapy trials conducted by
Ronald Crystal (Cornell Medical School, New York,
NY).19 Thus, the availability of this vector,
the prevalence of patients with detectable contamination of their
marrow and peripheral blood with BCCs, and the selectivity of the
5-FC/cytosine deaminase adenoviral vector system shown in our studies
for the BCCs suggest that this method may be useful in the future for improving the overall outcome of autotransplants in advanced breast cancer patients. These findings indicate that the cytosine deaminase vector used to sensitize BCCs to 5-FC, which is nontoxic for
hematopoietic cells up to 650 µmol/L, is a method of potential
utility for in vitro purging of autologous cells used for
transplantation in breast cancer patients.
| |
FOOTNOTES |
Submitted April 14, 1997;
accepted March 5, 1998.
A.B.D. received support from the Anderson Chair for
Cancer Treatment and Research and the George and Barbara Bush Leukemia Research Fund at the M.D. Anderson Cancer Center, the Ensign
Professorship of Medicine at the Yale University School of Medicine,
the Hull Development Fund at the Yale Cancer Center, and the Susan B. Komen Fund for Breast Cancer Research. F.G.-S. was
supported by a Fellowship from the Spanish Ministry of Education.
Address reprint requests to A.B. Deisseroth, MD, PhD, Gene Therapy
Program, Yale Cancer Center, and Medical Oncology Section, Yale
University School of Medicine, WWW 221, 333 Cedar St, New Haven, CT
06520-8032; e-mail: deisseroab{at}maspo2.mas.yale.edu.
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 acknowledge Drs Manuel Ramirez and Wing Leung from Johns
Hopkins University (Baltimore, MD) for the statistical evaluation of
the animal data. Many thanks to Rocco Carbone (Yale Cancer Center FACS
Shared Resource Core) for expert flow cytogenetic analysis. Thanks to
Ron Brown of Quality Biological, Inc for a gift of QBSF58 medium.
| |
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Abstract
Introduction
Abstract