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Control experiments established that the IC50 value for
formaldehyde was in excess of 200 µM with MCF-7 and MCF-
7/ADR cells. Additional control experiments established that
3 h incubation of cells with 1% DMSO in growth media did
not alter the survival of MCF-7 or MCF-7/ADR cells.
Toxicity of Hyd r olyzed Ep id oxofor m . The following
stock solutions of Epidoxoform in DMSO were prepared: 1
mM, 500 µM, 100 µM, 50 µM, 10 µM, 5 µM, 1 µM, and 100
nM. These were then diluted 10× with autoclaved Millipore
water. The resulting solutions in 90% water/10% DMSO (v/
v) were incubated at 37 °C for 20 h. A 20 µL aliquot of each
solution containing hydrolyzed Epidoxoform was then added
to each of the appropriate wells of a 96-well plate each
containing 1000 cells in 180 µL of RPMI 1640 media. At this
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Ack n ow led gm en t. We are grateful to the National
Cancer Institute of the NIH (Grant CA24665) and the
American Cancer Society (RPG-98-110-01-ROG) for
financial assistance. D.J .T. thanks the Division of
Medicinal Chemistry of the American Chemical Society
and Wyeth-Ayerst for a predoctoral fellowship. We
thank Pharmacia-Upjohn (Milan, Italy) and Gensia-
Sicor (Milan, Italy) for samples of epidoxorubicin. We
particularly thank Dr. Vincent Ursino of Vinchem
(Clatham, NJ ) and Dr. Antonino Suarato (Pharmacia)
for facilitating the receipt of samples from Gensia-Sicor
and Pharmcia, respectively. We thank Dr. William W.
Wells for MCF-7/ADR cells, Dr. David Brook for help
with the kinetic analysis using Mathematica, and Dr.
Deborah Wuttke for help with the NMR experiments
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Su p p or tin g In for m a tion Ava ila ble: Supporting Infor-
mation Available: 1H NMR spectra for epidoxorubicin and
Epidoxoform in DCCl3, derivation of the kinetic and equilib-
rium expressions for the hydrolysis at pH 7.3, kinetic analysis
of the hydrolysis of Epidoxoform at pH 6, schematic diagrams
for proposed drug-DNA adducts, and dose/response curves
and raw data for cell experiments (9 pages). Ordering
information is given on any current masthead page.
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