Vitro Antineoplastic Drug Screen to assess whether sensitivity to any of the ..... II inhibitors showed the highest correlation (data not shown). This list contained ...
[CANCER RESEARCH56. 5211-5216. November IS, 19961
Enhanced Sensitivity to 1-@-D-Arabinofuranosylcytosine and Topoisomerase II Inhibitors in Tumor Cell Lines Harboring Activated ras Oncogenes' Han-Mo Koo, Anne Monks, Andrei Mikheev,2 Larry V. Rubinstein, Marcia Gray-Goodrich, Mary Jane McWilliams, W. Gregory Alvord, Herbert K. Oie, Adi F. Gazdar, Kenneth D. Paull, Helmut Zarbl,2 and George F. Vande Woude3 ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick. Maryland 21702-1201 IH-M. K., M. J. M., G. F. V. WI; Science Applications International Corporation-Frederick. National Cancer Institute-Frederick Cancer Research and Development Center, Frederick. Maryland 2! 702 (A. M.. M. G-G.J; Massachusetts Institute of Technology. Cambridge. Massachusetts 02139 [A. M.. H. ii: Division of Cancer Treatment. National Cancer Institute, NIH, Bethesda, Maryland 20892 IL V. R., H. K. 0., K. D. P.!: Data Management Services, Inc., National Cancer Institute-Frederick Cancer Research and Development Center. Frederick, Maryland 21 702 [W. G. A.J: and Simmons Cancer Center and Department of Pathology, Universiti of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235 [A. F. G./
ABSTRACT We used human tumor cell lines from the National Cancer Institute's In Vitro Antineoplastic Drug Screen to assess whether sensitivity to any of the —45,000compounds tested previously correlated with the presence of a ma oncogene.
Among
these cell lines, the mutations
in Ki-ras2
clustered
in
non-small cell lung and colon carcinoma subpanels, and five of the six leukemia lines contained mutations in either N-ras or Ki-ras2. These analyses revealed a striking correlation with i-(l-D-arabinofuranosylcy. tosine (Ara-C) and 2,2'-O-cyclocytidine sensitivity in the cell lines harbor ing ras mutations compared to the tumor lines with wild-type ras alleles. Strong correlations were also found with topoisomerase (topo) IJ inhibi tors, especially 3'-hydroxydaunorubicin and an olivacine derivative. These
differential
sensitivities
persisted
in an additional
22 non-small
cell
The NCI-ADS database presently contains the differential sensitiv ity patterns of the 60 tumor cell lines to over 45,000 compounds. The sensitivity
of each cell line to a given compound
can be displayed
as
a mean graph, based on the drug concentration that gives a 50% inhibition of cell growth (G150) in 2-day assay (20, 21). Individual patterns can be compared to the entire database using the COMPARE pattern recognition program (21). COMPARE searches for similarities in mean graph patterns and ranks the similarity between patterns by a PCC (2 1). COMPARE analyses of the NCI-ADS database has iden tified compounds with similar chemical structures and/or related biochemical
mechanisms
of action
(21—29) and, recently,
substrates
and inhibitors of the P-glycoprotein, which mediates MDR, have been identified
(30, 31).
lung carcinoma lines (ras mutations, n 12 and wild-type ras, n 10). Thus, the association with Ara-C sensitivity was greatest while topo II inhibitors showed a lower, but significant, correlation. These results sug
We used COMPARE to assess whether compounds present in the database displayed selective growth inhibition against the tumor cell
gest that the ras oncogene
lines with ras oncogenes.
may play a determinant
role in rendering
tumor
The compounds
identified
by COMPARE
were further tested against an independent set of tumor cell lines. Our
cells sensitive to deoxycytidine analogues and topo II inhibitors.
results
revealed
that the presence
of a ras oncogene
in the tumor cell
lines correlated with enhanced sensitivity to deoxycytidine analogues including Ara-C, 2,2'-O-cyclocytidine, and gemcitabine. Low but
INTRODUCTION Tumor progression is a multistep process that is driven by genetic instability (1, 2). Defects in specific genes like DNA repair genes or the frequently mutated tumor suppressor genes and activated onco genes have been implicated in the genetic instability of tumor cells (reviewed in Refs. 3—9).In addition, profound changes occur in checkpoint function as well as in the regulation of apoptosis (reviewed in Refs. 5—12).Cancer cells in general display decreased sensitivity to a wide variety of therapeutic agents (13—16),but we and others (7—10) have proposed that through compromising cell cycle checkpoint func tions, the oncogene and tumor suppressor gene of cancer cells may be responsible for their preferential sensitivity to certain antineoplastic agents. To begin to test this hypothesis, we determined whether the presence of mutations in ras alleles in the 60 human tumor cell lines of the NCI-ADS4 (17) correlated with enhanced sensitivity to any of the —45,000natural and synthetic compounds that have been tested in the screen. We chose to examine the ras alleles since ras oncogene activation is the most frequently occurring gain-of-function mutation detected in human tumors (18, 19).
significant correlation 3'-hydroxydaunorubicin
MATERIALS
was also shown to topo II inhibitors, and an olivacine derivative.
especially
AND METHODS
Compounds and Cell Lines. Compoundsused in this studywereobtained through
the Drug Synthesis
and Chemistry
Branch,
NC!. The human tumor cell
lines of NCI-ADS and culture conditions has been described previously (20). The tumor cell lines derived from NSCL carcinoma (32) were maintained and assayed
for drug sensitivity
in RPM!
supplemented
with 10% FBS and 2 mM
L-glutamine.
Sequence Determination of ras Mutations. GenomicDNAs wereisolated from the cell lines of NCI-ADS using a Cell Culture DNA kit (Qiagen). The exons
I and 2 of each
ras gene
were amplified
by PCR from the genomic
DNAs using the primer pairs specific for each exon/intron junction. The PCR amplifications Elmer)
were
performed
and were all preceded
in a GeneAmp
PCR
System
with one cycle of denaturation
9600
(Perkin
at 94°C for 3 mm
and finished with the incubation at 72°Cfor 3 mm. The PCR conditions used were 40 cycles of: 94°C(15 s), 48°C(15 s), 72°C(15 s with 1 s extension per cycle) for Ki-ras2
exon I and N-ras exon I ; 94°C ( 15 s), 52°C ( I 5 s), 72°C (15
Received 7/1/96; accepted 9/I 9/96.
5 with
The costs of publication of this article were defrayed in part by the payment of page
(15 s), 48°C(15 s), 70°C(25 s with I s extension per cycle) for N-ras exon 2; and 94°C(15 s), 56°C(15 s), 72°C(15 s with I s extension per cycle) for Ha-rasl exon 1. The products were then purified from a 6% polyacrylamide
charges. This article must therefore be hereby marked advertisement 18 U.S.C. Section 1734 solely to indicate this fact. I
Supported
in
part
by
the
National
Cancer
Institute,
Department
in accordance with of
Health
and
Human
Servies, under contract with ABL. 2 Present
address:
Fred
gel. DNA sequence
Hutchinson
Cancer
Research
Center,
I 124
Columbia
3 To whom
requests
for reprints
4 The
abbreviations
used
are:
should
be addressed,
at ABL-Basic
Research
per
cycle)
for
was determined
Ki-ras2
exon
2 and
for both strands
Ha-rasl
directly
exon
2; 94°C
from the purified
PCR products using the same primers as those for PCR amplification.
Street,
Cl-OlS. Seattle. Washington 98104. National Cancer Institute-Frederick B, Frederick, MD 21702-1201.
1 s extension
sequencing
Program,
Cancer Research and Development Center, P. 0. Box
reactions
were
carried
out
with
the
PRISM
Ready
DNA
Reaction
DyeDeoxy Terminator cycle sequencing kit (ABI), and the products were analyzed on an ABI 373A DNA sequencer. The DNA sequences of primers
NCI-ADS,
National
Cancer
Institute
In
Vitro
Antineo
used
plastic Drug Screen; G130 and Gl@0,50 and 30% inhibition of cell growth, respectively;
PCC, Pearson correlation coefficient; MDR, multidrug resistance; Ara-C, l-@3-o-arabino furanosylcytosine; topo, topoisomerase; NSCL, non-small cell lung; FBS, fetal bovine serum; DGGE. denaturing gradient gel electrophoresis; AML, acute myeloid leukemia.
for
PCR
amplification
and
sequencing
were:
KR15,
GGCCTGCT
GAAAATGACTGA and KRI3, GTCCTGCACCAGTAATATGCfor Ki-ras2 exon 1; KR25, CCAGACTGTG11TCTCCC'ITC and KR23, CACAAA GAAAGCCCTCCCCA for Ki-ras2 exon 2; NR15, GACTGAGTACAAACT
5211
ras AND SENSITIVITY TO Ara-C AND TOPO 11 INHIBITORS
GGTGGandNR13, GGGCCTCACCTCTATGGTG for N-ras exon 1; NR25, GGTGAAACCTG1TrGTTGGA
and
NR23,
ATACACAGAGGAAGCCT
Table 1 Activating mutations andcolon of ras genes in the cell lines of leukemia. NSCL cancer subpanels of the NCI-ADSMutation
TCG for N-ras exon 2; HR15, CAGGCCCCTGAGGAGCGATG and HR13, UCGTCCACAAAATGGTTCT
CAGGArFCCTACCGG
for Ha-rasl
exon
1; and HR25,
TCCTG
and HR23, GGTFCACCTGTACTGGTGGA
Cellline
Subpanel
for
Ha-rasl exon 2. DGGE. The PCR amplification of each exon from genomic DNA, purifi cation and end-labeling of the PCR products, heteroduplex formation, and DNA fragments by DGGE, one primer from each pair used for the direct DNA was linked
at its 5-end
to a 54-bp
long,
thermostable,
OC-rich
clamp sequence (33). Drug Sensitivity Assays, Data Calculation, and COMPARE Algorithm. The assay methodology, sensitivity data calculation, and the COMPARE algorithm have been described in detail previously (20, 21). Briefly, cellular response to a compound was evaluated using a sulforhodamine-B assay after 48 h continuous exposure of cells with a range of the compound concentration, and the sensitivity of each cell line was calculated for the compound concen tration yielding a GI50 (20). The mean graph pattern generated from the
NSCL cancer
Colon cancer
by exact distribution
methods
using STATXACT
software
Software Co.).
a The
allelic
genomic
DNA.
status
The
of
exon
I 2: 12:
A549/ATCC EKVX
mutKl2: AGT(Ser)WIWtKI:
HOP-62 HOP-92
mut>wtKl2: TGT(Cys)WIWtWIWtKl:
mut>WtKl2: TGT(Cys)WtWtK2: mutK61: CAT(His)wtWI
COLO-205 HCC2998 HCT-l 16
(CYTEL
I 2:
mut/wtN12: TGT(Cys)K I: mutlwtK GCT(Ala)N I : mutlwtN TGT(Cys)KI:
NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522
relative sensitivities of cell lines tested was used by the COMPARE pattern recognition program to calculate PCCs for all compounds in NCI-ADS data base (21). Statistical Analysis. The 1-sided Ps by Wilcoxon rank sum test were calculated
1: mutlwtK GAT(Asp)Nl: mut/wtNl2: GTF(Val)N2: mutN61: CTA(Leu)wtwtNl:
HL-60(TB) K562 MOLT-4 RPMI-8226 SR
DGGE procedure were described elsewhere (33). To facilitate separation of the sequencing
Dc.iciE°Sequence―K
CCRF-CEM
Leukemia
wt WI Kl: mut/Wt
WI Wt K13: GAC(Asp)
HCT- I5
K 1: mut/WI
K I 3: GAC(Asp)
HT-29
WI
wt
KM-12
WI
wt
SW620
Kl: mut
ras
genes
number
determined
containing
by
Kl2: G1T(Val)
DGGE
a mutation
is also
analysis
of
indicated:
PCR-amplified
K, Ki-ras2;
N,
N-ras; mut, allele With a mutation; Wt, wild-type allele; >, elevated DGGE signal for the
mutant allele compared to the wild-type signal.
RESULTS
b The
Determination of Activating Mutations of ras Genes in Tumor
Cell Lines of NCI-AD5. The mutation status of ras alleles in each
sequence
of
a mutation
independently
determined
by
direct
sequencing
of
PCR-amplified genomic DNA. The codon number harboring a mutation is indicated With the sequence and amino acid encoded. C A
silent
mutation
at
59th
codon
of
Ha-rasl
(GCC—+GCT)
was
also
found.
tumor cell line of NCI-ADS was determined from PCR-amplified
genomic DNA prepared from each cell line. Each PCR-amplified DNA sample was then analyzed for the presence of an activating mutation in the 12th, 13th and 61st codons of the Ki-, N- and Ha-ras alleles (1 8, 19) by direct DNA sequencing and DGGE. Among the 60 cell lines analyzed, 17 cell lines were found to contain activating mutations in at least one of the ras alleles. Most of the cell lines with activating mutations in ras genes clustered in three of the nine cancer-subpanels of the NCI-ADS; mutations in the Ki-ras2 allele were found in four cell lines of the NSCL cancer subpanel and in three cell lines of the colon cancer subpanel, whereas five of six leukemia lines contained mutations in either the N-ras or Ki-ras2 allele (Table 1). The additional mutant ras alleles were detected in cell lines of the remaining six cancer subpanels: N..rasArg6l in SK-MEL-2 (melano ma); K-ras@12 in OVCAR-5 (ovarian carcinoma); K@rasAst@3in SN12C (renal carcinoma); and K@rasA@@1@l3 in MDA-MB-231 and H@rasAI@@l2 in HS578T (breast carcinomas). The frequency of activat ing mutations in ras genes among the NCI-ADS cell lines were
comparablewith that found previouslyin other human tumors and tumor cell lines (18, 19). Compounds That Are Preferentially Active against Tumor Cell Lines with Activated ras Oncogenes. We used COMPARE to assess whether any compounds in the database of NCI-ADS displayed se lective growth inhibition of the tumor cell lines in the NSCL and colon carcinoma subpanels containing activated ras oncogenes. We initially focused on the 16 cell lines among which the distribution of mutant and wild-type ras allele-containing cell lines was approxi mately equal (Table 1), allowing an optimal analysis of the database using COMPARE. To establish the ras mean graph for this analysis, we assigned the cell lines with activated ras oncogenes a positive value to equate with preferential drug sensitivity, whereas those with wild-type ras alleles were given a negative value. The COMPARE analysis was performed to identify compounds in the database that: (a) were tested at least three times against all sixteen cell lines; (b) gave a PCC of >0.75 with the ras mean graph pattern (under these 5212
conditions, i.e., n = 16 cell lines, the probability of an observing correlation is 1 @-@-
carboquone
(PCC = 0.76). However, these compounds displayed poor correla 5213
1•@
T + $2 .1.@t!I53
(I, C
oncogenes and enhanced sensitivity to the topo II inhibitors, 3'hydroxydaunorubicin (47), and an olivacine derivative, NSC 659687 (Ref. 48; PCC = 0.82 and 0.8 1, respectively; Fig. 2). The range of response for these compounds was, however, narrower than for the deoxycytidine analogues (compare Fig. I with Fig. 2), but as with the deoxycytidine compounds, the same four ras oncogene-containing leukemia lines were more sensitive to the topo II inhibitors than wild-type K562 (Table 2). Positive correlations with the ras mutant NSCL and colon carci noma cell lines were also observed for a 3-deazauridine derivative compound (PCC = 0.77), the lipophilic antifolate trimetrexate (PCC
were
cells (20), and the
presence of oncogenic ras mutations in five of six cell lines in the subpanel (Table 1) introduced a bias to the mean graph. These leukemia lines, therefore, were not a suitable data set to generate a statistically relevant differential pattern for COMPARE analysis. Nev ertheless, consistent with the findings in NSCL and colon carcinoma cell lines, four of the five ras oncogene-containing leukemia lines (Table 1) were highly sensitive to the deoxycytidine analogues corn pared to K562 line with wild-type ras alleles (Table 2). In addition to the deoxycytidine analogues, the COMPARE analy sis also revealed
tions with leukemia lines in which the K562 line with wild-type ras alleles was generally more sensitive to the compounds than ras oncogene-containing lines (data not shown). The RPMI-8226 cells,
> .
a,
Cl)
.
io@—
(I, C a,
5$7
Cl)
•1
4 i@-@
10@@—
Mut
Wt
Mut
Wt
Fig. 2. Sensitivity of the NSCL and colon carcinoma cell lines of NCI-ADS to topo II inhibitors 3'-hydroxydaunorubicin (a) and olivacine derivative NSC 659687 (b). Symbols and cell line number are as described in the legend to Fig. 1.
ras AND SENSITIVITYTO Am-C AND TOPOII INHIBITORS Table 2 Sensitivities of leukemia lines of NCI-ADS to deoxycytidine analogues and topo Ii inhibitors Deoxycytidine M)Ara-CCCRF-CEM― analogueCell
line―Sensitivity
K562 Itt―2,2'O-cyclocytidineCCRF-CEM―RPMI-8226―5.01
l0@GemcitabineHL-6O(TB)―
lines
were
ordered
by
the
b Cell
lines
with
ras oncogenes.
level
RPMI-8226―
1.29 X l0―
K5628.91
1.41 x
CCRF-CEM― SR― HL-60(TB)― RPMI-8226―
.00 1.51 1.82 2.19 9.33
K5621
I .20 X
K562
1.00 X l0@NSC
CCRF-CEM―
X I0' 4.37 X ltt'
MOLT-4―
8.71x 10'
SR― RPMI-8226―
1.48 X l0@@ 2.51 X l0-@
K5622.51 a Cell
1.91 X 10' 3.47 X l0@3'-Hydroxy
RPMI-8226―1
HL-60(TB)―
of
sensitivity
(GI@0 X 10' 1.51 X l0@ 2.40 X l0@ 2.51 x l0@
X X X X X
10.8 10.8 l0@ l0@
.20 1.58 2.00 5.62 1.91
SR― MOLT@4b
line―Sensitivity MOLT-4― HL-6O(TB)― CCRF-CEM―
X 6.17 X 2.19 X 2.29 X
MOLT@4b HL-60(TB)― SRb
II inhibitorCell
(Gl,0 M)Topo
daunorubicinSRb
l0@ i07 l0@ iO-7 l0'
659687MOLT-4―
X X X x x
10' Itt' 10' 10' Itt'
2.75 X l0@ to
each
compound.
inhibitors compared to other ras oncogene-containing leukemia lines (Table 2), also displayed the relative resistance to antifolates and alkylating agents (data not shown). Tests of Am-C and Topo II Inhibitors on Additional NSCL
lc). The basis of correlation between ras oncogenes and enhanced sensitivity to deoxycytidine analogues is not yet understood. How ever, with an equimolar saturation dose of Ara-C (i.e., 10 nM), we observed more efficient apoptotic cell death in the ras oncogene
Tumor Cell Lines. To extend our findings with the cell lines of
containing
NCI-ADS, we tested an additional 22 NSCL cell lines for which the
Despite a large variety of well-characterized topo II inhibitors tested in NCI-ADS, significant correlations with the ras mutation pattern was only observed with 3'-hydroxydaunorubicin and the oh vacine derivative, NSC 659687. Further characterization of these topo II inhibitors revealed that 3'-hydroxydaunorubicin and the ohivacine derivative were superiorly capable of overcoming MDR.―These re
profileof ras mutationshad been determinedpreviously(32). These cell lines, 10 of which contained wild-type ras alleles, were treated with Ma-C, 3'-hydroxydaunorubicin, and the olivacine derivative, NSC 659687, as well as trimetrexate. The same assay conditions as in
NCI-ADS were used, except that these cell lines were assayed in growth medium with 10% FBS instead of the 5% FBS growth con ditions used in the NCI-ADS (20). The sensitivity to Ara-C was evaluated at a drug concentration that gave a 30% inhibition of cell growth (G130), since 50% growth inhibition was not achieved, even at 1 mM Ara-C for most of the cell lines (data not shown). The differ entials in drug sensitivity observed in these cell lines were less than in NCI-ADS cell lines (compare Figs. 1 and 2 with 3). However, the NSCL cell lines with mutant ras alleles again strongly associated with greater sensitivity to Ara-C (1-sided P = 0.01 by Wilcoxon rank sum test); the mean GI30 Ara-C concentrations for mutant and wild-type ras-containing cell lines were 1.35 X i07 M and 1.95 X lO_6 M, respectively (Fig. 3a). As in the NCI-ADS test, the differential re sponse to 3'-hydroxydaunorubicin and the olivacine derivative com pound was lower than with Ara-C and also showed a lower correlation (P = 0.04 for both compounds; Fig. 3b and c). DISCUSSION Our search for chemotherapeutic agents in the NCI-ADS database that are preferentially more effective at inhibiting the growth of tumor cells harboring activated ras oncogenes identified two different classes
of compounds,
deoxycytidine
analogues
and topo II inhibitors
(Figs. 1 and 2, and Table 2). The preferential activities of the com pounds against ras oncogene-containing tumor cells were further
tested using additional tumor cell lines derived from NSCL carcino
cells than in cells with wild-type
sults suggested
that the activation
ras alleles.5
of ras oncogenes
in tumor
cells
specifically enhanced the intrinsic sensitivity to topo II inhibitors, but this ras oncogene-associated
sensitization
could be unveiled
only with
MDR-escaping topo II inhibitors or in the absence of MDR in the target cells.6 We also performed COMPARE analysis using all 60 cell lines and found that, again, the deoxycytidine analogues and the topo II inhibitors showed the highest correlation (data not shown). This list contained many more topo II inhibitors than deoxycytidine analogues with the highest PCC of —0.55(data not shown). Although most of the tumor cell lines with ras oncogenes displayed highly enhanced sensitivity to deoxycytidine analogues, SW620 and HCT- 15 derived from colon carcinomas and one NSCL carcinoma line were less sensitive to deoxycytidine analogues (Figs. 1 and 3). However, these cell lines were quite sensitive to the topo II inhibitors (Figs. 2 and 3). The sensitivity
of these cells to the topo II inhibitors
may be related to the “enhancing― effect of topo II inhibitors when combined with a deoxycytidine analogue, as in the standard Ara-C/ daunorubicin combination regimen used in the treatment of acute leukemias (49, 50). Does the ras oncogene play a significant role in rendering tumor cells sensitive to deoxycytidine analogues and topo II inhibitors? In a study of 99 patients with AML, Neubauer et a!. (5 1) observed a relationship between the presence of a ras oncogene in the heukemic cells and improved overall survival of the patients in response to treatment primarily comprised of Ara-C and a topo II inhibitor. It was speculated that the presence of the ras oncogene accounted for the prolonged survival of patients, either by its association with a lower
mas (Fig. 3). Although Ara-C and related compounds have been the single most effective group of therapeutics for the treatment of acute leukemias (49), these compounds have been essentially inactive against solid tumors. Gemcitabine, however, a deoxycytidine ana 5 H-M. Koo, unpublished observation. logue showing some activity against solid tumors (40—46),displayed 6 H.-M. Koo, M. Gray-Goodrich, A. Vaigro-Wolff, M. J. McWilliams, preferential activity against the mutant ras cell lines like Ara-C (Fig. Monks, and G. F. Vande Woude, manuscript in preparation. 5214
K. D. Paull, A.
@
;@ ras AND SENSITIVITY TO Ma-C AND TOPO II INHIBITORS
a
b
_____
io-7— I
•
1.. .@
S
ii +
S
>.
S
io@
S S
10.8.
1 @-@-
C
@@-8
•
.T
10.6@
U, C
I
a,
Cl)
10.8_
S
@,
@:
@‘ ..
@5S
U) C
.@ C
a,
.1
a,
.
Cl)
S
Cl)
io@—
S
.
10@-
S S
.
T .Ã '+
S
.
10-s—
. .
10.6_
. S -—-3
10@-
Mut
Wt
Mut
Wt
Mut
Wt
Fig. 3. Sensitivity of additional NSCL carcinoma cell lines to: a, Ara-C: GI30 = 1.35 X l0@ M for Mut (•)and 1.95 X l0_6 M for Wt ( •); b, 3'-hydroxydaunorubicin:
GI50 9.77 x l0—@ MforMutand 1.86X l0_6 si for Wt;andc, olivacinederivativeNSC659687:GI50= 3.47X 10' MforMutand 1.26X l0@ Mfor Wt.Thecompoundswere tested at ten 10-fold dilutions. Mean drug concentrations conferring a 30% inhibition of cell growth for Ara-C and a 50% inhibition for the topo II inhibitors for each cell line were determined from three independent experiments. Mean sensitivities (+) for each group of cell lines are shown; bars, SE.
heukemic burden or by rendering the leukemic cells more sensitive to the chemotherapeutic agents used (5 1). In addition, consistent with a ras oncogene affecting the chemosensitivity of AML, 6 of 10 initially ras oncogene-positive AML patients, who had been treated with Ara-C and a topo II inhibitor, had only wild-type ras alleles in their leukemic cells at relapse, indicating that the ras oncogene-positive leukemic cells had been selectively eliminated (52). Furthermore, a cohort of ras oncogene-positive AML patients exhibited a significantly longer duration of complete remission when treated with progressively higher doses of Ara-C as consol idation therapy, whereas the postremission Ara-C dose intensifi cation did not significantly impact on complete remission duration of AML patients with wild-type ras alleles (53). These clinical observations (51—53) are consistent with our results illustrating that the tumor cell hines derived from heukemias and NSCL and colon carcinomas with activated ras oncogenes were preferentially more sensitive to deoxycytidine analogues and topo II inhibitors. In addition, these results suggest that the combination therapy used in AML treatment (51) may be more effective at killing leukemic cells containing ras oncogenes, through either additive or syner gistic mechanisms (49, 50). Our results combined with the AML clinical data reveal a signifi cant correlation between the ras oncogene status of tumor cells and their sensitivity to clinically relevant drugs. One application of these findings may be to determine the therapeutic efficacy of the deoxy cytidine analogues and topo II inhibitors on other tumors bearing frequent ras mutations, such as NSCL, colon and, notably, pancreatic carcinomas (18, 19); it should be noted that gemcitabine has been approved recently for the treatment of locally advanced pancreatic 5215
carcinoma (54), in which a large percentage of the tumors contain activated ras oncogenes (18, 19). If the relationship between cancer specific
genotypes
and antineophastic
drugs
provides
an explanation
for the preferential sensitivity of tumor cells to successful chemother apy protocols, it is hoped that such approaches would direct chemo therapy away from purely empirical approaches to strategies based on cancer-specific
genotypes.
ACKNOWLEDGMENTS We thank B. Chabner, M. Grever, G. Johnson, and E. Sausville for helpful discussion
and continued
support.
We thank M. Strobel,
K. Fukasawa,
I. Daar,
E. Harlow, and C. D. Bloomfield for critical reading of the manuscript. We also thank C. Rhoderick and M. Reed for preparation of the manuscript.
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