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Pyrazoloacridine Is Active in Multidrug-resistant Neuroblastoma Cell Lines
with Nonfunctional p53
Nino Keshelava, Denice Tsao-Wei and C.
Patrick Reynolds
Division of Hematology-Oncology, Children’s Hospital Los
Angeles, and Departments of Pediatrics [N. K., C. P. R.*], Pathology [C. P. R.],
and Preventive Medicine [D. T-W.], University of Southern California Keck School
of Medicine, Los Angeles, California 90033. *To whom requests for reprints should be addressed, at Division
of Hematology-Oncology, MS# 57, Children’s Hospital Los Angeles,
4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: (323) 669-5646;
Fax: (323) 664-9226/9455; E-mail: preynolds@chla.usc.edu.
Purpose. The purpose of this study was to determine the activity of
pyrazoloacridine (PZA) in neuroblastomas that have acquired high-level
resistance to multiple drugs (not associated with multidrug
resistance-associated protein or P-glycoprotein) during therapy,
including those with loss of p53 function.
Experimental Design. We determined the activity of PZA in 12 drug-sensitive
and 10 multidrug-resistant (MDR) neuroblastoma cell lines.
Six of the
MDR cell lines lacked functional p53.
Drug cytotoxicity was measured
using the DIMSCAN fluorescence/digital imaging microscopy assay with
a 4-log dynamic range.
Results. LC90 (i.e., the drug concentration that was
lethal for 90% of the cell population) values ranged from 0.01 to 1.1
µM for the drug-sensitive cell lines, from 0.8 to
2.4 µM for the MDR cell lines with functional
p53, and from 0.9 to 2.1 µM for the MDR cell
lines that lacked functional p53.
To confirm that PZA cytotoxicity is
independent of p53 function, we compared two neuroblastoma cell lines
in which p53 function was abrogated via human papilloma virus-16 E6
transduction (which mediates increased degradation of p53) to the
mock-transduced (LXSN) controls.
LC90 values for human
papilloma virus-16 E6 clones (abrogated p53) ranged from 0.2 to 2.04
µM, whereas LC90 values for LXSN
controls (functional p53) were 0.1 and 0.5 µM.
PZA was active with 72-h in vitro exposures against p53-nonfunctional
MDR cells at drug levels (2–3 µM) obtained
for shorter periods (1–3 h) in Phase I and II clinical trials.
Washout experiments showed that 3 µM PZA
achieved 0.5–1 log of cell kill with 1–3-h exposures versus
3 logs at 24 h.
Conclusions. These data suggest that expanding the time of PZA systemic
exposure, which may be clinically tolerable with hematopoietic stem
cell support, should be tested in clinical trials.
Prolonged systemic
exposure to PZA with hematopoietic stem cell support may be effective
against recurrent neuroblastomas that have failed conventional
chemotherapeutic regimens, including those neuroblastomas with loss
of p53 function.
© 2003 American
Association for Cancer Research
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