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DRUG METABOLISM AND DISPOSITION
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:304–312, 2001
Vol. 29, No. 3
168/887824
Printed in U.S.A.
P450 ENZYME EXPRESSION PATTERNS IN THE NCI HUMAN TUMOR CELL LINE PANEL
LI J. YU,1 JOCELYN MATIAS, DOMINIC A. SCUDIERO, KAREN M. HITE, ANNE MONKS, EDWARD A. SAUSVILLE,
DAVID J. WAXMAN
AND
Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts (L.J.Y., J.M., D.J.W.); SAICFrederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland (D.A.S., K.M.H., A.M.); and
Developmental Therapeutics Program, National Cancer Institute, Bethesda, Maryland (E.A.S.)
(Received July 6, 2000; accepted October 26, 2000)
This paper is available online at http://dmd.aspetjournals.org
ABSTRACT:
Significant negative correlations between the patterns of P450dependent 7-benzyloxyresorufin metabolism activity and cell line
chemosensitivity were observed for 10 standard anticancer agents
(including 6 alkylating agents) and 55 investigational compounds,
suggesting a role for P450 metabolism in the inactivation of these
agents. Negative correlations between 7-ethoxycoumarin O-deethylation and cell line chemosensitivity to a group of topoisomerase inhibitors were also seen, again suggesting P450-dependent
drug inactivation. P450 enzyme profiling may thus aid in interpreting the patterns of drug sensitivity and resistance in the NCI tumor
cell panel, and may facilitate the identification of anticancer agents
whose activity can be altered via cytochrome P450 metabolism.
The cytochrome P450s are a superfamily of hemeprotein monooxygenases that catalyze the oxidative metabolism of a large number of
drugs, environmental carcinogens, as well as steroids and other endobiotics. Approximately 60 human cytochrome P450 genes are
known (Nelson et al., 1996; Nelson, 1999) and encode proteins that
exhibit major differences with respect to their catalytic specificities,
tissue-specific patterns of expression, and interindividual differences.
These differences result from genetic polymorphisms (IngelmanSundberg et al., 1999) and from the differential responsiveness of
P450s2 to the large number of foreign chemical inducers and endogenous regulators that control P450 gene expression (Waxman, 1999).
P450 enzymes are highly expressed in human liver and in certain
extrahepatic tissues and have been studied extensively with respect to
their roles in drug metabolism (Rendic and Di Carlo, 1997). Much less
is known about the profiles of P450 expression in primary human
tumor tissue and in cultured tumor cell lines, in part due to the very
low enzyme levels present (Smith et al., 1993; Huang et al., 1996;
Nakajima et al., 1996; Murray et al., 1999).
The expression of P450 enzymes in tumor tissue can have a major
impact on the responsiveness of tumors to cancer chemotherapeutic
drugs, owing to the central role that these enzymes play in the
metabolism of numerous clinically useful anticancer agents (LeBlanc
and Waxman, 1989). In the case of antitumor prodrugs, such as
cyclophosphamide and ifosfamide, P450 metabolism is essential for
therapeutic activity (Sladek, 1994). Indeed, the expression in human
tumor cells of specific P450 enzymes that activate these oxazaphosphorine prodrugs can greatly sensitize the cells to drug cytotoxicity
(Chase et al., 1998; Jounaidi et al., 1998). P450 enzymes can also
impact the pharmacokinetics and the therapeutic activity of other
classes of anticancer drugs, including those that are converted by P450
to metabolites that retain antitumor activity and those that are inactivated as a consequence of P450 metabolism (LeBlanc and Waxman,
1989; Kivisto et al., 1995).
During the past decade the U.S. National Cancer Institute (NCI) has
used a panel of 60 human tumor cell lines, chosen to represent nine
different tumor types, to carry out a large in vitro screening program
for novel anticancer agents (Boyd and Paull, 1995; Monks et al.,
1997). To date more than 60,000 compounds have been characterized
with respect to their antitumor activity using this primary screen,
These studies were supported in part by Contract SAIC 97-CX-50351A and by
National Institutes of Health Grant CA49248 (to D.J.W.). Support was provided in
whole or in part with Federal funds from the National Cancer Institute, National
Institutes of Health, under Contract N01-CO-56000.
Disclaimer: The content of this article does not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does mention of
trade names, commercial products, or organization imply endorsement by the
U.S. government.
1
Current address: Central Research Division, Pfizer Inc., Groton, CT.
2
Abbreviations used are: P450 or CYP, cytochrome P450; NCI, U.S. National
Cancer Institute; P450 reductase, NADPH-cytochrome P450 oxidoreductase;
7-ECOD, 7-ethoxycoumarin O-deethylase; 7-EROD, 7-ethoxyresorufin O-deethylase; 7-BROD, 7-benzyloxyresorufin O-debenzylase; PCC, Pearson correlation
coefficient.
Send reprint requests to: Dr. David J. Waxman, Dept. of Biology, Boston
University, 5 Cummington St., Boston, MA. E-mail: [email protected]
304
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Cytochrome P450 (P450) enzyme expression patterns were determined for a panel of 60 human tumor cell lines, representing nine
tumor tissue types, used by the National Cancer Institute (NCI)
Anticancer Drug Screening Program. All 60 tumor cell lines displayed significant P450 activity, as well as P450 reductase activity,
as determined using the general P450 substrate 7-benzyloxyresorufin. Cell line-specific P450 enzyme patterns were observed using three other P450 substrates, 7-ethoxycoumarin, coumarin, and 7-ethoxyresorufin, each of which was metabolized at a
low rate. Using a pattern-matching computer program, COMPARE,
correlative relationships were investigated between the arrays of
P450 activities and the patterns of cytotoxicity exhibited by a large
group of anticancer agents of proven or potential clinical utility.
305
P450 EXPRESSION IN NCI TUMOR CELL PANEL
Materials and Methods
Isolation of Microsomes from Tumor Cell Lines. The 60 human tumor
cell lines used in this study are described elsewhere (Boyd, 1989; Monks et al.,
1997) (see listing in Table 2, below). Cells at passage number ranging from 3
to 17 were harvested in mid-log growth phase. Microsomes were prepared
from frozen cell pellets in procedures carried out at 0 to 4°C. Cell pellets were
suspended in ice-cold 0.1 M KPi (pH 7.4) buffer containing 0.1 mM EDTA and
20% (v/v) glycerol. The samples were then homogenized, sonicated three
times (2–10 s/sonication at a moderate instrument setting), and centrifuged at
low speed (15 min at 8000g). The supernatant was then centrifuged for 1 h at
140,000g. Final microsomal pellets were resuspended in the above KPi/EDTA/
glycerol buffer to give a protein concentration ⱖ5 mg/ml with a yield that
ranged from 1 to 12 mg of microsomal protein per 5 ⫻ 108 cells. Microsomes
were stored in aliquots at ⫺80°C.
NADPH-Cytochrome P450 (Cytochrome c) Reductase Assay. P450 reductase activities were measured spectrophotometrically at 550 nm. Cytochrome c (Sigma Chemical Co., St. Louis, MO), reduced by P450 reductase in
the presence of NADPH, has a chromophore that absorbs visible light at 550
⫺1
nm with ⑀ ⫽ 21,000 M cm . Reactions were carried out in a cuvette
containing cytochrome c (42.7 ␮M), 0.3 M KPi buffer, pH 7.7, 25 ␮g of
microsomal protein, and 120 ␮M NADPH in a final volume of 1 ml. Reactions
were initiated by the addition of NADPH, and the change in A550 nm was
monitored at room temperature for 5 min. Data obtained were shown to reflect
initial reaction rates, and represent two to three replicate assays for each
sample.
7-ECOD Assay. Activity was assayed in 100 mM KPi buffer (pH 7.4), 20%
glycerol, 0.1 mM EDTA, with 1 mM 7-ethoxycoumarin (Aldrich Chemical
Co., Milwaukee, WI) and 200 ␮g of microsomal protein in a total volume of
200 ␮l. Reactions were initiated by adding NADPH to 1 mM. Reactions were
incubated for 1 h at 37°C with gentle shaking then terminated by adding 25 ␮l
of ice-cold 2 M HCl. The samples were then extracted twice with 450 ␮l of
chloroform. The chloroform layers were combined and then back-extracted
with 1 ml of 30 mM sodium borate, pH 9.2. The 7-hydroxycoumarin metabolite was determined fluorometrically (370-nm excitation wavelength, 450-nm
emission wavelength) in comparison to authentic 7-hydroxycoumarin standard
(Aldrich Chemical Co.). Data presented are based on two to three replicate
assays for each sample.
Coumarin 7-Hydroxylation Assay. Reaction mixtures contained reagents
and microsomes assayed in the same conditions and concentrations as described for the 7-ECOD assay, but using 1 mM coumarin (Sigma Chemical
Co.) in place of 7-ethoxycoumarin as the substrate. Enzyme incubation, extraction of the 7-hydroxycoumarin metabolite, and fluorometric analysis of
enzyme activity were also performed using the same conditions as the 7-ECOD
assay. Data shown generally reflect averages of duplicate assays for each
sample.
Alkoxyresorufin O-Dealkylation Assays. Reactions used to monitor
7-EROD and 7-BROD activities (total volume, 2.5 ml) were carried out in a
3-ml fluorometer cell containing a microstirring bar and 250 ␮g of microsomal
protein. Samples were mixed with 4 ␮M substrate (7-ethoxyresorufin or
7-benzyloxyresorufin, delivered using 10 ␮l of 1 mM stock solution in dimethyl sulfoxide; Molecular Probes, Eugene, OR) in 0.1 M KPi (pH 7.4) and
0.1 mM EDTA buffer at room temperature. Reactions were started by the
addition of NADPH to 250 ␮M. Formation of the fluorescent metabolite,
resorufin, from either 7-ethoxyresorufin or 7-benzyloxyresorufin was measured at room temperature over an 8-min period using a Shimadzu RF-1501
fluorescence spectrophotometer (Shimadzu, Kyoto, Japan). Fluorescence was
read at 550 nm (excitation) and 586 nm (emission). Activity values were
quantitated using the 7-hydroxylated standard, resorufin (Molecular Probes).
Data obtained were shown to correspond to initial reaction rates and generally
represent two to three assays for each sample.
Drug Screening and COMPARE Analysis. The COMPARE program
(Paull et al., 1989, 1995) has identified drugs with common mechanisms of
action. The molecular target version of this program (Lee et al., 1994; Monks
et al., 1997) was used here to analyze possible relationships between individual
P450 enzyme activities and the cell line sensitivity patterns of standard agents.
The standard agent database comprises 170 chemicals for which a considerable
amount of information is available in terms of preclinical and/or clinical
antitumor properties and presumed mechanism of action (Boyd and Paull,
1995; Paull et al., 1995). In addition, correlative analysis was undertaken
between target patterns and a database of approximately 3000 active investigational compounds, whose cytotoxicity or growth-inhibitory activity has been
confirmed in more than one series of in vitro cell line screenings. The relative
sensitivities of the panel of 60 cell lines to a given compound, at a concentration causing 50% growth inhibition, are represented as a mean-graph pattern
(Paull et al., 1989). In the molecular target version of the pattern recognition
program COMPARE (Lee et al., 1994; Alvarez et al., 1995), each target
measurement (e.g., P450 reductase or one of the measured P450 activities) is
represented in a manner similar to a mean-graph and used as a seed to derive
correlations between toxicity patterns in the various databases and the pattern
of expression of the P450 enzymes. The compounds that correlated to the
target pattern, either positively or negatively, were ranked by Pearson correlation coefficient (PCC) and p value. A positive PCC indicates that greater
activity of the target enzyme may be associated with increased cell sensitivity
to the drug. In contrast, a negative PCC implies that greater activity of the
target enzyme may confer cellular resistance to the given drug. To evaluate
compounds of possible interest, the uncorrected two-tail p value was set at
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generating a series of lead compounds for further investigation and
evaluation (Weinstein et al., 1997). Although, as noted above, cytochrome P450 enzymes contribute to the metabolism of a large number
of drug substrates and can have a large impact on a drug’s anticancer
activity, little is presently known about the P450 activity levels
present in the individual tumor cell lines that constitute the NCI panel.
P450 expression in tumor cells may lead to the localized production of
intracellular drug metabolites, and may thereby either increase or
decrease the cytotoxicity of test chemicals being evaluated. Characterization of P450 expression patterns within the NCI tumor cell line
panel may thus provide insight into some of the factors that govern the
cell line-specific and/or tumor type-dependent drug sensitivity and
drug resistance patterns seen in these cells. The potential relevance of
P450 expression in tumor cells with respect to effective anticancer
drug screening is supported by our observation of a dramatic enhancement of the in vitro and in vivo cytotoxic action of the oxazaphosphorine anticancer drug cyclophosphamide when tumor cells are
transduced to express the rat CYP2B1 gene (Chen and Waxman,
1995b) or its human P450 ortholog, CYP2B6 (Jounaidi et al., 1998).
In the present study, we sought to characterize the NCI human
tumor cell line panel with the following goals: 1) to determine
whether cytochrome P450 enzyme activities are expressed at a measurable level in the tumor cell lines constituting this panel; 2) to
provide initial information regarding the individual P450 activities
present in each of the cell lines; and 3) to search for any correlative
relationship between the arrays of P450 enzyme activities displayed
by the 60 cell lines and their patterns of chemosensitivity or chemoresistance toward ⬃3000 compounds that have been characterized in the
NCI in vitro screening program and shown to exhibit reproducible
cytotoxic activity. Ultimately, these studies may help ascertain
whether P450 enzyme expression patterns can aid in the interpretation
of drug sensitivity patterns that individual tumor cell lines exhibit
toward cytotoxic agents previously identified by the NCI drug screening program and whose mechanism of action is presently unknown.
The present studies complement recent investigations of the same NCI
tumor cell line panel that describe the expression of other enzymes
potentially relevant to anticancer drug metabolism. These include
glutathione S-transferases and enzymes of glutathione metabolism
(Tew et al., 1996), aldehyde dehydrogenases, which can contribute to
the inactivation of the activated metabolites of a number of cancer
chemotherapeutic agents including cyclophosphamide and ifosfamide
(Sreerama and Sladek, 1997), and DT-diaphorase and other enzymes
that catalyze bioreductive metabolism of a variety of quinones and
related chemicals (Fitzsimmons et al., 1996).
⫺1
306
YU ET AL.
FIG. 1. NADPH-P450 reductase activity of NCI human tumor cell line microsomes.
⬍0.0012 for the standard agents (n ⫽ 170) and at ⬍6.7E-05 for the investigational database (n ⫽ ⬃3000), which reflects the equivalent of p ⬍ 0.2 after
the Bonferroni adjustment for multiple comparisons. Using the assigned criteria, the probability of such occurrence from the selected database by random
chance would be approximately 20%.
Correlative analysis was also carried out between the target P450 enzyme
patterns and compounds classified according to each of six major clinical
mechanisms (van Osdol et al., 1994): alkylating agents (n ⫽ 35); “anti-DNA
agents” including compounds directly incorporated into DNA, polymerase
inhibitors, and ribonucleotide reductase inhibitors (n ⫽ 16); nucleotide synthesis inhibitors including antifolates and antimetabolites (n ⫽ 19); topoisomerase I inhibitors, all of which are camptothecin analogs (n ⫽ 23); topoisomerase II inhibitors (n ⫽ 16); and tubulin active antimitotic agents (n ⫽ 13).
Using compounds grouped within a single mechanism of action (van Osdol et
al., 1994) to produce a general pattern of response, each target was correlated
with these general mechanisms, and if the upper and lower limits of the 95%
confidence interval pass through zero, then there was no positive or negative
association suggested. However, a significant association may be suggested if
positive correlations have a lower limit of ⬎0.1 and negative confidence limits
have an upper confidence limit of ⬍⫺0.1.
Data presented in Figs. 1 through 5 are posted and can be accessed at the
web site http://dtp.nci.nih.gov/servlet/gcDisplaySearch?aliasStr⫽Waxman.
Results
NADPH-P450 Reductase Activity in the NCI Panel. P450 reductase activity was readily measured in all 60 NCI cell line microsomes
(Fig. 1). The mean specific activity for the panel of cell microsomes was
60.2 ⫾ 6.1 nmol/min/mg of protein (mean ⫾ S.E.), and the individual
values ranged from 5 to 294 nmol of cytochrome c reduced/min/mg of
protein. Because P450 reductase is an obligatory, and often rate-limiting,
enzymatic component of microsomal P450 metabolism, P450 reductase
levels are likely to be an important codeterminant of the P450 activity of
those tumor cell lines that express one or more cytochrome P450 proteins.
Analysis of P450 reductase activity levels on the basis of tumor cell type
did not reveal any significant associations between P450 reductase activity and tissue of origin (data not shown).
COMPARE analysis revealed that the P450 reductase activity pattern of the cell line panel correlated negatively with cell sensitivity
patterns to two established anticancer agents, L-asparaginase and
fludarabine phosphate ( p ⬍ 0.0012). In contrast, examination of the
database of 3000 investigational compounds revealed no correlations
that met the p value cutoff set for that group (see Materials and
Methods).
7-ECOD Metabolism by NCI Cell Line Microsomes. 7-Ethoxycoumarin O-deethylation is a sensitive microsomal reaction that allows detection of two human P450s that metabolize this substrate at a
high rate, CYP1A1 and CYP2E1. Several other human P450s metabolize 7-ethoxycoumarin at much lower rates (Table 1) (Waxman et al.,
1991). Forty-nine of the 60 NCI cell lines were active in the 7-ECOD
reaction (Fig. 2). 7-ECOD activity was below the limits of detection
(⬍0.15 pmol/min/mg) in the other 11 cell lines. The melanoma cell
line SK-MEL-2 and the ovarian cancer cell line OVCAR-4 were
particularly active, followed by the colon cell line KM12. Overall, the
highest average 7-ECOD activities were seen in the melanoma and
prostate tumor cell line groups, while the lowest activities were seen
in the central nervous system tumors and leukemia groups. However,
these trends did not reach statistical significance as a consequence of
heterogeneity of 7-ECOD activity within each tumor group.
COMPARE analysis revealed that 7-ECOD activity exhibited weak
negative associations with the cytotoxicity patterns of several established anticancer drugs, but none reached the defined level of statistical significance (see Materials and Methods). Examination of the
database of 3000 investigational compounds identified one compound, a benzopyran (NSC 669781) whose cytotoxicity toward the
cell panel showed a strong negative correlation with the pattern of
7-ECOD activity (PCC ⫽ ⫺0.54 and p value ⫽ 2.7E-05), suggesting
that this agent may be inactivated by CYP1A1 or CYP2E1, the two
most active catalysts of the 7-ECOD reaction.
Coumarin 7-Hydroxylase Activity in the NCI Panel. This reaction is actively catalyzed by CYP2A6, with CYP2B6 also catalyzing
this reaction, albeit at a much lower rate (Table 1) (Waxman et al.,
1991). Measurable coumarin 7-hydroxylase activity (ⱖ0.1 pmol/min/
mg) was observed in 43 of the 54 cell lines tested (Fig. 3). A colon
tumor cell line, HCT-15, showed highest activity (0.76 pmol of
7-hydroxycoumarin produced/min/mg), but no coumarin 7-hydroxylase activity was detected in several of the leukemia, lung, ovarian,
and renal tumor cell lines (Fig. 3). No statistically significant correlations between the coumarin 7-hydroxylase activity pattern and the
patterns of sensitivity to any of the established or investigational
agents were detected, indicating that none of these compounds are
metabolized by CYP2A6, or alternatively, that CYP2A6 does not
contribute to the cellular sensitivity or resistance of these compounds
at its relatively low level of expression in the tumor cell panel.
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Enzyme activities were measured by monitoring NADPH-dependent cytochrome c reduction as described under Materials and Methods. Data reflect mean ⫾ range
values for two to three replicate assays of each cell line using two independently prepared microsome aliquots. For many samples the error bars are too small to be seen.
NCI cell lines are numbered as shown in Table 2.
P450 EXPRESSION IN NCI TUMOR CELL PANEL
TABLE 1
P450 enzyme activities exhibited by a panel of human lymphocyte microsomes
containing cDNA-expressed human P450s
P450 enzyme activities were measured for a panel of 11 cDNA-expressed human P450s
obtained from GENTEST Corp. using the assay methods described under Materials and
Methods. Data obtained are summarized here in the form of a Table. “⫺” indicates the
measured activity was below the limit of detection and “(⫹)” indicates activity was very low,
but detectable. Higher activities are compared on a scale of “⫹” to “⫹⫹⫹⫹⫹”. Measured
turnover numbers (pmol/min/pmol of P450) for CYP1A1 were approximately 4 (7-ECOD), 1
(7-EROD), and 5 (7-BROD). Coumarin hydroxylase data shown here are primarily based on
Waxman et al. (1991).
7-ECOD
Coumarin
7-EROD
7-BROD
⫹⫹⫹⫹⫹
⫹
⫺
⫹
⫹
⫺
⫹
⫺
⫹⫹
⫹
⫺
⫺
⫺
⫺
⫹⫹⫹⫹⫹
⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹⫹⫹
⫹
(⫹)
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫹⫹⫹
(⫹)
⫹⫹
(⫹)
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
7-EROD Activity of the NCI Panel. This P450 reaction is actively
catalyzed by CYP1A1, with a lower but measurable activity exhibited
by CYP1A2 and -1B1, as demonstrated using cDNA-expressed human P450 enzymes (Table 1). A broad range of chemical carcinogens,
including several of the major chemical carcinogens found in cigarette
smoke and auto exhaust, can induce CYP1A1 in various tissues,
including lung. Although CYP1A1 is not commonly detected in
cultured cell lines in the absence of exposure to P450 inducers,
CYP1A1 activity has not been assayed in the NCI 60-cell line panel.
In this context, it is notable that eight of the nine NCI lung cancer cell
lines exhibited 7-EROD activity (Fig. 4). The highest 7-EROD activities (⬃3–5 pmol of resorufin produced/min/mg of protein) were
observed for the ovarian cancer line OVCAR-4, for the colon cancer
line KM12, and for two lung tumor cell lines, A549/ATCC and
NCI-H23. 7-EROD activity was below the limit of detection (⬃0.1
pmol/min/mg) for 19 of the 60 cell lines. Mean 7-EROD activity
values from the seven colon tumor cell lines were ⬃5-fold higher than
that from the eight renal and six leukemia cell lines. The absolute level
of 7-EROD activity in the panel was low, however, consistent with the
general finding that CYP1A1 expression is low in cells not exposed to
aromatic hydrocarbon inducers.
COMPARE analysis did not reveal any notable correlations between 7-EROD activity and anticancer drug sensitivity. One compound from the active database, the microtubule assembly inhibitor
norhomohalichondrin B (NSC 700368), was negatively associated
with 7-EROD activity.
7-BROD Activity Patterns in the NCI Cell Line Panel. The
7-BROD assay is a more general assay for cytochrome P450 enzymes
than the three other P450 enzyme assays used in this study. This assay
measures the activity of multiple human P450s, including CYP1A1,
-1B1, -2B6, -2C8, -2C9, -2C19, -2D6, and -3A4 (Table 1). All 60 of
the NCI tumor cell lines showed 7-BROD activity that was readily
measurable and well above background (Fig. 5), as verified in control
incubations containing bovine serum albumin in place of cell microsomes, or complete assay mixtures incubated without NADPH. Analysis of the 7-BROD activity data on the basis of the tissue of tumor
origin indicated that the lowest 7-BROD activities were present in the
six leukemia cell lines.
By COMPARE analysis, 7-BROD activity was negatively correlated with 10 standard anticancer agents ( p ⬍ 0.002), six of which are
classified as alkylating agents (Table 3). Moreover, COMPARE analysis using the investigational database identified 55 active compounds, all of which were negatively correlated with 7-BROD activity
(PCC ⱖ 0.49 and p ⬍ 6.7E-05). These negative correlations suggest
that increased 7-BROD activity may confer resistance to these agents,
perhaps via a P450-dependent metabolic inactivation reaction.
Overall Comparisons of P450 Activity Patterns. Table 2 presents
an overall comparison of the four microsomal P450 enzyme activities
and P450 reductase activity measured for the NCI tumor cell line
panel. Sixteen of the tumor cell lines gave good positive activities for
all four P450 activities, indicating that multiple P450 enzymes are
likely to be expressed in each of these cell lines. The ovarian cancer
cell line OVCAR-4 gave the highest overall P450 activity with the
four substrates tested. The high P450 activity of this cell line is not
solely a reflection of its high P450 reductase activity, as several other
cell lines in the panel displayed similarly high P450 reductase activity
but did not exhibit the consistently high P450 enzyme activities seen
in OVCAR-4.
Cell lines positive for 7-ECOD metabolism were not necessarily
positive for coumarin 7-hydroxylation [e.g., cell lines K-562, RPMI8226, HL60(TB)], consistent with the broader catalytic participation
of human P450s in the 7-ECOD reaction (cf. Table 1). Several strong
7-ECOD positive cell lines, such as the breast cell line HS-578T, the
melanoma cell line SK-MEL-28, and the prostate cell line PC-3, did
not show measurable 7-EROD activity (Table 2), suggesting that these
cell lines are functionally devoid of CYP1A activity. Several cell lines
(SNB-19, M14, NALME-3M, and 786 – 0) were negative for 7-ECOD
assay but showed substantial activities in the coumarin hydroxylase,
7-EROD, and 7-BROD assays. These findings demonstrate that
unique P450 expression patterns characterize each of the individual
tumor cell lines.
Comparison of the breast carcinoma cell line MCF7 with its doxorubicin (Adriamycin)-resistant subline MCF7/ADR-RES revealed a
decrease in P450 reductase activity, which has been seen earlier (Chen
and Waxman, 1995a). Also seen were decreases in 7-EROD and
7-BROD activity and an increase in 7-ECOD activity (Table 2).
Multiple changes in gene expression characterize MCF7/ADR-RES
cells, but these changes do not necessarily contribute to the cell’s
drug-resistant phenotype (Chen and Waxman, 1995a).
To further investigate which specific P450 enzymes are expressed
and at what relative levels, we used a panel of seven anti-human P450
antibodies (anti-CYP1A1, -1A2, -2A6, -2B6, -2C, -2E1, and -3A) to
analyze microsomes prepared from 11 of the tumor cell lines by
Western blotting using enhanced chemiluminescence detection. In all
cases, strong P450 form-specific signals were obtained when human
liver microsomes (20 ␮g of protein) or cDNA-expressed P450s (1–5
pmol) were used as positive controls on the blots. However, none of
the Western blots showed detectable P450 protein signals with the
tumor cell microsomes (50 –75 ␮g of protein) (data not shown). This
observation is in agreement with the very low level of corresponding
P450 mRNAs seen in the same tumor cell line panel by microarraybased expression profiling (Ross et al., 2000).
With the exception of 7-BROD activity, the P450 activity values
presently described for the NCI cell line panel are all very low when
compared with typical human liver microsomal P450 activities. For
example, the 7-ECOD activities of the most active NCI tumor cell line
microsomes correspond to only ⬃1% of the specific catalytic activity
of typical human liver microsomes assayed in our laboratory (Figs. 2
and 4). This may be reflected in the lack of compounds whose
cytotoxicity patterns correlate with these target activity patterns; furthermore, it is consistent with our inability to detect in these cells
P450 enzyme proteins by Western blotting, as noted above.
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CYP
1A1
1A2
1B1
2A6
2B6
2C19
2C8
2C9-Arg
2E1
3A4
4A11
307
308
YU ET AL.
FIG. 3. Coumarin 7-hydroxylase activity of NCI cell line microsomes.
Data reflect duplicate assays of each cell line, generally obtained using two independently prepared microsomal aliquots for each cell line. A bovine serum albumin
activity background value was subtracted from the activity of each microsomal sample to give the net specific activities shown. The low activity values shown here were
verified for select cell lines in (⫺)-NADPH control experiments. Error bars represent the range of specific activity values, and for many cell lines the error bars are too
small to be seen. Shown for comparison at the left is the high coumarin 7-hydroxylase activity of cDNA-expressed CYP2A6. *Samples exhibited mean activities below
the detection limit (⬃0.1 pmol/min/mg). 7-HC, 7-hydroxycoumarin.
Associations with Mechanisms of Action and Other Molecular
Targets. Correlative analyses were carried out to identify any possible
associations between the measured cell panel P450 activity patterns
and anticancer drugs grouped according to each of six major clinical
mechanisms of action (van Osdol et al., 1994). Potentially meaningful
negative correlations were found between 7-ECOD activity patterns
and a group of 23 camptothecin analog topoisomerase I inhibitors
(95% confidence limits from ⫺0.34 to ⫺0.22). The 7-ECOD activity
pattern was more weakly associated (negatively) with the topoisom-
erase II inhibitor group (95% confidence limits from ⫺0.25 to ⫺0.11).
The P450 catalysts of 7-ECOD activity might therefore confer resistance to compounds with topoisomerase I or II inhibition activities.
7-BROD activity was negatively correlated and therefore potentially
associated with resistance to topoisomerase I-inhibiting camptothecin
analogs (95% confidence limits from ⫺0.32 to ⫺0.20), anti-DNA
agents (95% confidence limits from ⫺0.32 to ⫺0.18), and alkylating
agents (95% confidence limits from ⫺0.30 to ⫺0.21). This latter
association was further emphasized by the significant association
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 3, 2017
FIG. 2. 7-ECOD activity of NCI cell line microsomes.
Data reflect two to three replicate assays for each cell line. Background activity values, determined using bovine serum albumin in place of microsomal protein, were
subtracted from each of the measured activities to give the indicated net specific activities. Error bars represent the range of specific activity values, and for many cell lines
are too small to be seen. Also shown for comparison (samples at left) are the 7-ECOD activities of human liver microsome samples HLS8 and HLS9 and the 7-ECOD
activity of an MCF7 cell line stably transfected with rat P450 2B1 (Chen et al., 1996). 7-HC, 7-hydroxycoumarin.
P450 EXPRESSION IN NCI TUMOR CELL PANEL
309
FIG. 5. 7-BROD activity of NCI cell line microsomes.
Data shown reflect mean ⫾ range for two to three assays of each cell line. Background activity values were measured for select cell line microsomes in assays carried
out without NADPH or using bovine serum albumin in place of microsomal protein. Error bars are too small to be seen for many of the cell lines.
between 7-BROD activity and six standard alkylating agents (Table
3), as noted above.
Finally, analyses were carried out to determine whether any of the
four sets of P450 activity measurements were coordinately expressed
with any of the molecular targets previously measured in the NCI cell
line panel for which data are presently available in the public domain.
These include various oncogenes, tumor suppressor genes, drug transporters, cytokines, cell cycle molecules, DNA repair enzymes, and
metabolic enzymes (Monks et al., 1997). No associations were found,
other than correlations between P450 reductase and 7-BROD activity
and between 7- EROD and 7-BROD activity ( p ⬍ 0.05). The former
correlation supports the conclusion that P450 reductase can be rate
limiting for P450-dependent metabolism in the NCI cell line panel.
Discussion
The ultimate goal of the NCI anticancer drug screening program is
to discover new anticancer agents by large-scale screening of libraries
of compounds for their ability to inhibit tumor cell growth in a
broad-based panel of human tumor cell lines. The cytotoxicity of
agents identified in these in vitro assays can be greatly affected by the
expression of one or more cellular components, such as transport
proteins or enzymes that can activate or deactivate the potential
anticancer drug (Weinstein et al., 1997). Characterization of the NCI
panel with respect to these “drug responsiveness” determinants may
help identify factors that determine the selectivity of a given agent for
certain tumor cell lines in the panel. These studies may also aid in the
discovery of new molecular targets and may facilitate the design of
new chemotherapeutic strategies based on the altered expression (either increased or decreased expression) of the drug response determinant. Efforts have been made in recent years to profile the expression
patterns of various drug responsiveness determinants in the NCI
panel, including aldehyde dehydrogenases (Sreerama and Sladek,
1997), various reductases (Fitzsimmons et al., 1996), glutathione-
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 3, 2017
FIG. 4. 7-EROD activity of NCI cell line microsomes.
Data shown are based on two to three replicate assays for each cell line, generally using two independently prepared microsomal aliquots for each cell line. Several of
the microsomal samples yielded negative slopes in the continuous 7-EROD fluorescent assay, resulting in calculated negative activity values (*). Error bars represent the
range of specific activity values, and for many cell lines the error bars are too small to be seen. Shown for comparison at the left are the 7-EROD activities of human liver
microsome sample HLS8 and cDNA-expressed human CYP1A2. Detection limit ⫽ 0.1 pmol/min/mg.
310
YU ET AL.
TABLE 2
Summary of P450 activities measured in the NCI cell line panel
Higher activities are compared on a scale of “⫹” to “⫹⫹⫹⫹⫹” as defined in Part B below. Numbers shown for each cell line are used in the figures to identify each cell line.
Cell Line
7-ECOD
Coumarin
7-EROD
7-BROD
⫹⫹⫹⫹
⫹
⫹
⫹⫹
⫹
⫹⫹
⫹
⫹
⫹
⫹
⫹
⫹⫹
⫺
⫹⫹
⫹
⫺
⫺
⫹⫹
⫺
⫹⫹
(⫹)
⫺
⫺
⫹
⫹⫹⫹
⫹
⫹
⫹
(⫹)
⫹
⫹⫹
⫹⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹
⫺
(⫹)
⫹
⫺
⫺
⫹
(⫹)
N.A.
⫹
⫹
(⫹)
(⫹)
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫺
⫹⫹⫹
(⫹)
⫹⫹
⫺
⫹
⫹⫹
⫹⫹
N.A.
⫹⫹⫹
(⫹)
(⫹)
⫺
⫹
⫹
⫹
⫺
⫺
⫹
⫺
⫹⫹
⫹⫹
⫺
(⫹)
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫹
⫺
⫹⫹⫹⫹
⫹
N.A.
(⫹)
⫹
⫹⫹⫹⫹⫹
⫹⫹
⫹
⫹
⫺
⫹⫹
⫹⫹
⫹
(⫹)
⫹⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
(⫹)
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹
(⫹)
⫹⫹
⫺
⫺
N.A.
(⫹)
⫹
⫹
⫹⫹⫹
⫹⫹
⫺
⫹
⫹
⫹
⫹⫹⫹
⫹
(⫹)
⫹⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹
⫹
⫹⫹
⫹⫹⫹
⫹
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫺
⫺
⫺
⫹⫹⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹
⫹
⫹⫹
(⫹)
⫹
⫹
⫹
⫹
⫺
⫹⫹
⫹
⫹
⫹
⫹
⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹⫹
⫹⫹⫹⫹⫹
⫹⫹⫹⫹
⫹⫹⫹⫹
⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹⫹⫹
⫺
⫹
⫹
⫺
(⫹)
⫹⫹⫹
N.A.
⫺
⫺
⫹⫹
⫹
⫹⫹⫹⫹⫹
⫺
⫹
⫺
⫹⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫹
⫹
⫺
⫹⫹⫹
⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫹
⫹⫹
⫹⫹⫹
⫺
⫹⫹
⫹⫹
⫹
⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫺
⫺
(⫹)
⫹⫹⫹
⫹
N.A.
⫹
⫹⫹
⫺
⫹
⫺
⫺
(⫹)
⫺
⫹
⫹⫹⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫹
⫹⫹⫹
⫹⫹
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Part A
Leukemia
1 K-562
2 MOLT-4
3 CCRF-CEM
4 RPMI-8226
5 HL-60(TB)
6 SR
CNS
7 SF-268
8 SF-295
9 SF-539
10 SNB-19
11 SNB-75
12 U261
Breast
13 BT-549
14 HS-578T
15 MCF7
16 MCF7/ADR-RES
17 MDA-MB-231/ATCC
18 MDA-MB-435
19 MDA-N
20 T-47D
Colon
21 COLO 205
22 HCC-2998
23 HCT-116
24 HCT-15
25 HT29
26 KM12
27 SW-620
Lung
28 A549/ATCC
29 EKVX
30 HOP-62
31 HOP-92
32 NCI-H322M
33 NCI-H226
34 NCI-H23
35 NCI-H460
36 NCI-H522
Melanoma
37 LOX-IMVI
38 M14
39 MALME-3M
40 SK-MEL-2
41 SK-MEL-28
42 SK-MEL-5
43 UACC-257
44 UACC-62
Ovarian
45 IGORV1
46 OVCAR-3
47 OVCAR-4
48 OVCAR-5
49 OVCAR-8
50 SK-OV-3
Prostate
51 DU-145
52 PC-3
Renal
53 786-0
54 A498
55 ACHN
56 CAKI-1
57 RXF-393
58 SN12C
59 TK-10
60 UO-31
P450
Reductase
311
P450 EXPRESSION IN NCI TUMOR CELL PANEL
TABLE 2
Continues
P450
Reductase
7-ECOD
Coumarin
nmol/min/mg
7-EROD
7-BROD
0.2–1
1–2.5
2.5–4
4–5
⬎5
4–20
20–30
30–40
40–50
⬎50
pmol/min/mg
Part B. Definition of Scales Used in Part A
⫹
5–40
⫹⫹
40–75
⫹⫹⫹
75–125
⫹⫹⫹⫹
125–250
⫹⫹⫹⫹⫹
⬎250
0.2–0.4
0.4–0.75
0.75–1.25
1.25–2.0
⬎2.0
0.1–0.2
0.2–0.3
0.3–0.5
0.5–0.7
⬎0.7
N.A., not assayed.
⫺, activity not readily detectable; (⫹), activity at limit of detection.
TABLE 3
Correlation between 7-BROD activity pattern and standard agent sensitivity patterns
NAME
8-Cl CYC AMP
Melphalan
Caracemide
Asaley
Uracil nitrogen mustard
Flurorodopan
Thioguanine
Carmethiozole
Fludarabine phosphate
Chlorambucil
MOA
A7
A7
A7
A7
DI
A7
PCC
p Value
⫺0.467
⫺0.440
⫺0.429
⫺0.427
⫺0.424
⫺0.424
⫺0.421
⫺0.416
⫺0.414
⫺0.413
0.00017
0.00043
0.00062
0.00067
0.00074
0.00075
0.00080
0.00095
0.00100
0.00103
MOA, mechanism of action, classified according to van Osdol et al. (1994) for those compounds whose MOA is known. A7, alkylation at N7 position of guanine; DI, agent incorporated into
DNA.
Library: Ling Springer/Style: Drug Metab Dispos
associated enzymes (Tew et al., 1996), the drug transporters mdr-1/
P-glycoprotein and multidrug resistance-associated protein (Alvarez
et al., 1995, 1998), the tumor suppressor gene p53 (O’Connor et al.,
1997), and the inhibitor of apoptosis protein family (Tamm et al.,
1998). These studies have led to several interesting findings. For
example, a high correlation was observed between drug sensitivity of
a series of compounds found in NCI’s open database of ⬃30,000
compounds and the P-glycoprotein-dependent mechanism of resistance (Alvarez et al., 1995). The validity of this approach was established in experiments demonstrating that the compounds identified in
this manner are, in fact, P-glycoprotein substrates (Alvarez et al.,
1995). Similarly, the correlation between transcript levels of ␥-glutamyl cysteine synthetase and certain standard agents has suggested
an association between the capacity of cells to synthesize glutathione
and their resistance to alkylating agents (Tew et al., 1996).
This study presents the first systematic investigation of human
P450 enzyme activity patterns in a large panel of human tumor cell
lines. These initial findings, with the support of future, more comprehensive studies using additional P450 form-selective enzyme assays
(Chang and Waxman, 1998) and more sensitive detection methods for
P450 expression (e.g., reverse transcriptase-polymerase chain reaction), may provide for a more complete understanding of the characteristic patterns of anticancer drug responsiveness exhibited by each
cell line. Such information may be helpful in the identification of lead
anticancer agents that are inactivated, or alternatively, are activated
via P450 metabolism. This concept is supported by the uniformly
negative correlation between cellular 7-BROD activity and each of 10
standard antitumor agents and 55 other confirmed active anticancer
drugs, which indicates a role for this P450 activity in determining
cellular resistance to these groups of agents. P450 activity can also
enhance chemosensitivity, as previously demonstrated with the human
tumor cell line MCF7/2B1, a derivative of MCF7 breast carcinoma
that expresses P450 form CYP2B1 at a relatively low level yet
exhibits significant cytotoxic sensitization to cyclophosphamide, both
in vitro and in a nude mouse xenograft model (Chen and Waxman,
1995b; Chen et al., 1996). Tumor cell lines engineered to express specific
P450s could potentially be very helpful if incorporated into the NCI
anticancer drug screening program in identifying novel anticancer prodrugs that undergo P450 metabolism but which would likely escape
detection using the current panel of human tumor cell lines.
Recently, Fitzsimmons et al. (1996) reported NADPH-P450 reductase activity in S9 supernatant fractions prepared from the same panel
of 60 NCI tumor cell lines examined in this study. The S9 fraction
P450 reductase activities reported in that study are severalfold lower
than the microsomal P450 reductase activities described in this report,
consistent with the expected enrichment of P450 reductase in the
microsomal fraction. In addition, the pattern of P450 reductase activities in this panel of NCI cell lines reported by Fitzsimmons et al.
(1996) is different from what we observed in our study. Although the
basis for this discrepancy is uncertain, it is possible that other interfering enzyme activities present in the S9 supernatant could be a
contributing factor. The pattern of P450 reductase activity seen in the
present study was reasonably well correlated with the toxicity pattern
of two standard agents (L-asparaginase and fludarabine phosphate),
which was not found in the study of Fitzsimmons et al. (1996).
The correlations between the arrays of P450 enzyme activities and
the patterns of toxicity of standard agents or database compounds
described in this report may aid in the design of further experiments
to evaluate new hypotheses regarding the role of P450 enzymes in
metabolism of select anticancer agents. Follow-up studies of the
metabolism of standard agents and database compounds selected by
the COMPARE algorithm using panels of cDNA-expressed human
P450 enzymes are likely to be informative in this regard. Similarly, it
will be of interest to further investigate three tumor cell lines that are
reported to be sensitive to either cyclophosphamide or ifosfamide,
namely, renal carcinoma cell line RXF-393 and nonsmall cell lung
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NSC
284751
8806
253272
167780
34462
73754
752
602668
312887
3088
312
YU ET AL.
References
Alexandre E, David P, Viollon C, Wolf P, Jaeck D, Azimzadeh A, Nicod L, Boudjema K and
Richert L (1990) Expression of cytochromes P450 2E1, 3A4 and 1A1/1A2 in growing and
confluent human HepG2 hepatoma cells: Effect of ethanol. Toxicol In Vitro 13:427– 435.
Alvarez M, Paull K, Monks A, Hose C, Lee JS, Weinstein J, Grever M, Bates S and Fojo T
(1995) Generation of a drug resistance profile by quantitation of mdr-1/P- glycoprotein in the
cell lines of the National Cancer Institute Anticancer Drug Screen. J Clin Invest 95:2205–
2214.
Alvarez M, Robey R, Sandor V, Nishiyama K, Matsumoto Y, Paull K, Bates S and Fojo T (1998)
Using the national cancer institute anticancer drug screen to assess the effect of MRP
expression on drug sensitivity profiles. Mol Pharmacol 54:802– 814.
Boyd MR (1989) Where do we go from here? Lippincott, Philadelphia.
Boyd MR and Paull KD (1995) Some practical considerations and applications of the National
Cancer Institute in vitro anticancer drug discovery screen. Drug Dev Res 34:91–109.
Chang TKH and Waxman DJ (1998) Catalytic assays for human cytochrome P450, in Methods
in Molecular Biology Series: Cytochrome P450 Protocols (Phillips IR and Shephard EA eds)
pp 95–102, Humana Press, Inc., Totowa, NJ.
Chang TKH, Weber GF, Crespi CL and Waxman DJ (1993) Differential activation of cyclophosphamide and ifosphamide by cytochromes P450 2B and 3A in human liver microsomes.
Cancer Res 53:5629 –5637.
Chase M, Chung RY and Chiocca EA (1998) An oncolytic viral mutant that delivers the CYP2B1
transgene and augments cyclophosphamide chemotherapy. Nat Biotechnol 16:444 – 448.
Chen G and Waxman DJ (1995a) Complete reversal by thaliblastine of 490-fold Adriamycin
resistance in multidrug-resistant (MDR) human breast cancer cells. Evidence that multiple
biochemical changes in MDR cells need not correspond to multiple functional determinants for
drug resistance. J Pharmacol Exp Ther 274:1271–1277.
Chen L and Waxman DJ (1995b) Intratumoral activation and enhanced chemotherapeutic effect
of oxazaphosphorines following cytochrome P450 gene transfer: Development of a combined
chemotherapy/cancer gene therapy strategy. Cancer Res 55:581–589.
Chen L, Waxman DJ, Chen D and Kufe DW (1996) Sensitization of human breast cancer cells
to cyclophosphamide and ifosfamide by transfer of a liver cytochrome P450 gene. Cancer Res
56:1331–1340.
Fitzsimmons SA, Workman P, Grever M, Paull K, Camalier R and Lewis AD (1996) Reductase
enzyme expression across the National Cancer Institute tumor cell line panel: Correlation with
sensitivity to mitomycin C and EO9. J Natl Cancer Inst 88:259 –269.
Huang Z, Fasco MJ, Figge HL, Keyomarsi K and Kaminsky LS (1996) Expression of cytochromes P450 in human breast tissue and tumors. Drug Metab Dispos 24:899 –905.
Ingelman-Sundberg M, Oscarson M and McLellan RA (1999) Polymorphic human cytochrome
P450 enzymes: An opportunity for individualized drug treatment. Trends Pharmacol Sci
20:342–349.
Jounaidi Y, Hecht JED and Waxman DJ (1998) Retroviral transfer of human cytochrome P450
genes for oxazaphosphorine-based cancer gene therapy. Cancer Res 58:4391– 4401.
Kivisto KT, Kroemer HK and Eichelbaum M (1995) The role of human cytochrome P450
enzymes in the metabolism of anticancer agents: Implications for drug interactions. Br J Clin
Pharmacol 40:523–530.
LeBlanc GA and Waxman DJ (1989) Interaction of anticancer drugs with hepatic monooxygenase enzymes. Drug Metab Rev 20:395– 439.
Lee JS, Paull K, Alvarez M, Hose C, Monks A, Grever M, Fojo AT and Bates SE (1994)
Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute
drug screen. Mol Pharmacol 46:627– 638.
Monks A, Scudiero DA, Johnson GS, Paull KD and Sausville EA (1997) The NCI anti-cancer
drug screen: A smart screen to identify effectors of novel targets. Anticancer Drug Des
12:533–541.
Murray GI, McFadyen MC, Mitchell RT, Cheung YL, Kerr AC and Melvin WT (1999)
Cytochrome P450 CYP3A in human renal cell cancer. Br J Cancer 79:1836 –1842.
Nakajima T, Wang RS, Nimura Y, Pin YM, He M, Vainio H, Murayama N, Aoyama T and Iida
F (1996) Expression of cytochrome P450s and glutathione S-transferases in human esophagus
with squamous-cell carcinomas. Carcinogenesis 17:1477–1481.
Nelson DR (1999) Cytochrome P450 and the individuality of species. Arch Biochem Biophys
369:1–10.
Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR,
Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC and Nebert DW (1996) Cytochrome P450
superfamily: Update on new sequences, gene mapping, accession numbers, and nomenclature.
Pharmacogenetics 6:1– 42.
O’Connor PM, Jackman J, Bae I, Myers TG, Fan S, Mutoh M, Scudiero DA, Monks A, Sausville
EA, Weinstein JN, Friend S, Fornace AJ Jr and Kohn KW (1997) Characterization of the p53
tumor suppressor pathway in cell lines of the National Cancer Institute anticancer drug screen
and correlations with the growth-inhibitory potency of 123 anticancer agents. Cancer Res
57:4285– 4300.
Paull KD, Hamel E and Malspeis L, eds (1995) Cancer Chemotherapeutic Agents. American
Chemical Society, Washington, D.C.
Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, Plowman J and Boyd
MR (1989) Display and analysis of patterns of differential activity of drugs against human
tumor cell lines: Development of mean graph and COMPARE algorithm. J Natl Cancer Inst
81:1088 –1092.
Rendic S and Di Carlo FJ (1997) Human cytochrome P450 enzymes: A status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29:413–580.
Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, Spellman P, Iyer V, Jeffrey SS, Van de Rijn
M, Waltham M, et al. (2000) Systematic variation in gene expression patterns in human cancer
cell lines. Nat Genet 24:227–235.
Roy P, Yu LJ, Crespi CL and Waxman DJ (1999) Development of a substrate-activity based
approach to identify the major human liver P450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P450 profiles.
Drug Metab Dispos 27:655– 666.
Sladek NE (1994) Metabolism and pharmacokinetic behavior of cyclophosphamide and related
oxazaphosphorines, in Anticancer Drugs: Reactive Metabolism and Interactions (Powers G
ed) pp 79 –156, Pergamon Press, Oxford, England.
Smith G, Harrison DJ, East N, Rae F, Wolf H and Wolf CR (1993) Regulation of cytochrome
P450 gene expression in human colon and breast tumour xenografts. Br J Cancer 68:57– 63.
Sreerama L and Sladek NE (1997) Class 1 and class 3 aldehyde dehydrogenase levels in the
human tumor cell lines currently used by the National Cancer Institute to screen for potentially
useful antitumor agents. Adv Exp Med Biol 414:81–94.
Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T and Reed JC (1998)
IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95),
Bax, caspases, and anticancer drugs. Cancer Res 58:5315–5320.
Tew KD, Monks A, Barone L, Rosser D, Akerman G, Montali JA, Wheatley JB and Schmidt DE
Jr (1996) Glutathione-associated enzymes in the human cell lines of the National Cancer
Institute Drug Screening Program. Mol Pharmacol 50:149 –159.
van Osdol WW, Myers TG, Paull KD, Kohn KW and Weinstein JN (1994) Use of the Kohonen
self-organizing map to study the mechanisms of action of chemotherapeutic agents. J Natl
Cancer Inst 86:1853–1859.
Waxman DJ (1999) P450 gene induction by structurally diverse xenochemicals: Central role of
nuclear receptors CAR, PXR, and PPAR. Arch Biochem Biophys 369:11–23.
Waxman DJ, Lapenson DP, Aoyama T, Gelboin HV, Gonzalez FJ and Korzekwa K (1991)
Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome
P450s. Arch Biochem Biophys 290:160 –166.
Weinstein JN, Myers TG, O’Connor PM, Friend SH, Fornace AJ Jr, Kohn KW, Fojo T, Bates
SE, Rubinstein LV, Anderson NL, Buolamwini JK, van Osdol WW, Monks AP, Scudiero DA,
Sausville EA, Zaharevitz DW, Bunow B, Viswanadhan VN, Johnson GS, Wittes RE and Paull
KD (1997) An information-intensive approach to the molecular pharmacology of cancer.
Science (Wash DC) 275:343–349.
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carcinoma cell lines NCI-H226 and NCI-H522, in view of the apparent requirement for P450 activity to manifest the latent cytotoxic
potential of these anticancer prodrugs (Sreerama and Sladek, 1997).
Conceivably, such studies may reveal the expression in these cells of
one or more oxazaphosphorine-activating human P450 enzymes
(Chang et al., 1993; Roy et al., 1999). Finally, caution should be
exercised when extrapolating the present findings to in vivo tumor
models. An earlier report (Smith et al., 1993) indicated that P450
expression patterns in human colon and breast tumor lines can change
in response to a variety of factors when the cells are grown as
xenografts in immunodeficient mice. In addition, the cellular profile
of P450 activities measured in tumor cell lines such as the NCI 60
panel may be very different from those present in vivo in the tumor
tissue of origin due to a variety of factors, including the variable loss
of expression of individual P450s that is often seen in cultured cells
(Alexandre et al., 1990). Cellular P450 activities can also be influenced by many factors including cell passage number, growth phase,
culture conditions, and origin of the tumor tissue used initially to
develop the cell line. Thus, while the P450 activity profiles described
in this study may not be representative of the corresponding parent
tumor tissue, they are nevertheless informative with respect to interpretation of the drug sensitivity patterns of the cell lines constituting
this anticancer drug screening panel.