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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
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.
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.
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 672-682
© 1998 by the American Society of Hematology.
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