Affiliations of authors: L. R. Kelland, S. Y. Sharp, P. M. Rogers, P. Workman, Cancer Research Campaign Centre for Cancer Therapeutics, The Institute of Cancer Research, Surrey, U.K.; T. G. Myers, Developmental Therapeutics Program, Information Technology Branch, National Cancer Institute, Bethesda, MD.
Correspondence to: Paul Workman, Ph.D., Cancer Research Campaign Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Rd., Sutton, Surrey SM2 5NG, U.K. (e-mail-paulw{at}icr.ac.uk).
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ABSTRACT |
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INTRODUCTION |
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DT-diaphorase, an obligate two-electron-reducing enzyme [reduced nicotinamide-adenine dinucleotide (phosphate) : quinone oxidoreductase; EC 1.6.99.2], catalyzes the reduction of various quinones (18). As a result, cells rich in DT-diaphorase are especially sensitive to quinone-containing bioreductive anticancer agents, such as mitomycin C and the indoloquinone EO9, which act as prodrugs for activation to toxic forms by DT-diaphorase (19-21). Some tumor types (notably, colon and non-small-cell lung cancers) have been shown to contain relatively high levels of DT-diaphorase (22-26). Thus, these cancers may be particularly suitable for treatments that use a DT-diaphorase prodrug approach. Although previous studies (27) have shown that geldanamycin is a substrate for DT-diaphorase, a cell line derived from human colorectal cancer and expressing DT-diaphorase did not appear to be particulary sensitive to geldanamycin. However, it is not known whether cells expressing high levels of DT-diaphorase show altered sensitivity to 17AAG.
The primary aim of this study was to investigate whether DT-diaphorase activity has a role in the sensitivity of human tumor cells to 17AAG. Initially, sensitivity to 17AAG was determined by use of the CRC/Institute of Cancer Research (ICR) panel of 15 human colorectal and 11 ovarian carcinoma cell lines, including some resistant to classical agents. Comparative data were obtained in selected lines for the 17-amino metabolite and the additional Hsp90-binding agents geldanamycin and radicicol. The correlation between sensitivity and DT-diaphorase activity seen in a subset of the CRC/ICR panel (selected to span the range of sensitivity to 17AAG) was then examined and confirmed with data from the NCI panel of 60 human tumor cell lines (28). This led to the hypothesis that high DT-diaphorase expression was a major factor in determining cellular sensitivity to 17AAG but not to geldanamycin or radicicol. To provide further conclusive data, sensitivity to 17AAG was determined in a newly established isogenic pair of cell lines that differ only in the expression of the active NQO1 gene. This pair is composed of the human colon BE line [which contains a disabling point mutation in the NQO1 gene encoding DT-diaphorase (29)] and a subline stably transfected with the NQO1 gene and expressing high levels of functional DT-diaphorase. Finally, evidence that the Hsp90 inhibitory mechanism was retained by 17AAG in colon cell lines expressing high and low levels of DT-diaphorase was obtained by immunoblot analysis of Raf-1, mutant p53, Hsp70, and Hsp90 proteins. The results suggest that determination of patients' NQO1 genotype and of tumor DT-diaphorase activity should be included in the clinical evaluation of 17AAG because variations in these characteristics could affect the toxicity and efficacy of the drug.
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MATERIALS AND METHODS |
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We used panels of human colon and ovarian cell lines. We obtained cell lines from commercial cell culture collections or derived them in-house as described previously (30). In some cases, we used sublines derived from a particular parent line with acquired drug resistance to cisplatin (CH1cisR and A2780cisR ovarian lines) or to doxorubicin (CH1doxR and an SKOV-3 subline stably overexpressing the multidrug-resistance protein MRP1) (30-32). All lines were grown as monolayers in Dulbecco's modified Eagle medium containing 10% fetal calf serum, 2 mM glutamine, and 0.5 µg/mL hydrocortisone in 6% CO2/94% air. All lines were free of Mycoplasma contamination.
Drugs and Chemicals
Geldanamycin, 17AAG, and 17-amino,17-demethoxygeldanamycin were supplied by E. Sausville (NCI). The remaining drugs (herbimycin, radicicol, streptonigrin, and dicoumarol) and chemicals were obtained from Sigma Chemical Co. (Poole, U.K.).
Growth Inhibition Studies
We used the sulforhodamine B assay as described previously (30-32) for growth inhibition studies. Briefly, we seeded tumor cells into 96-well microtiter plates, allowed the cells to attach overnight, and then added the drug to quadruplicate wells as indicated. Unless otherwise indicated, we exposed cells to a drug for 4 days. Thereafter, the cell number in treated versus control wells was estimated after treatment with 10% trichloroacetic acid and staining with 0.4% sulforhodamine B in 1% acetic acid. The IC50 was calculated as the drug concentration that inhibits cell growth by 50% compared with control growth.
Stable Transfection of the NQO1 Gene Into the BE Human Colon Carcinoma Cell Line
BE cells contain a point mutation in the NQO1 gene and thus have no functional DT-diaphorase enzyme activity (29). We used the bicistronic expression vector pEFIRES-P (33) to express the NQO1 gene in BE cells, Lipofectamine (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) for transfection, and puromycin (0.5 µg/mL) for selection. Resulting clones were screened for DT-diaphorase enzyme activity or protein by an enzyme assay or immunoblotting, respectively (see below). Full details of the vector construction and the biologic properties of the stable transfectants will be published elsewhere (Sharp SY, Kelland LR, Valenti MR, Brunton LA, Hobbs S, Workman P: unpublished results). The stable transfectants, designated BE-F397 clone 2 and BE-F397 clone 5, were used in these studies.
DT-Diaphorase Assay
To determine whether 17AAG was a good substrate for DT-diaphorase, we used the standard cytochrome c assay, as described previously for the bioreductive indoloquinone EO9 (34) and geldanamycin (27), but replaced menadione with 17AAG as the substrate and intermediate electron acceptor. We assayed extracts of the human colon cell line HT29 or purified human DT-diaphorase protein (from J. Skelly, ICR). For preparation of cell extracts, 2 x 107 cells were trypsinized, washed twice in ice-cold phosphate-buffered saline (PBS), and centrifuged (MSE Centaur I; 1100 rpm for 5 minutes at room temperature). The cell pellet then was resuspended in 0.5-1 mL of lysis buffer (PBS containing 1% Triton X-114 and 500 µM phenylmethylsulfonyl fluoride) and left on ice for 30 minutes. After centrifugation (MSE Microcentrifuge; 12 000 rpm for 5 minutes at room temperature), the supernatant was used for protein determination and the enzyme assay. Results obtained for 17AAG were compared with those for geldanamycin, EO9, and streptonigrin, an excellent substrate for DT-diaphorase (35). For all drugs, the difference in reduction of the menadione substrate in the absence and presence of dicoumarol (100 µM), a standard inhibitor of DT-diaphorase, was determined (27).
Immunoblotting
This analysis was performed as described previously (30-32). Briefly, 5 x 106 cells were trypsinized, washed with PBS, and lysed in 100 µL of lysis buffer at 4 °C for 1 hour. Lysis buffer contained 10 mL of 150 mM NaCl-50 mM Tris-HCl (pH 7.5), 500 µL of 20 mM phenylmethylsulfonyl fluoride, 2 µL of aprotinin (10 mg/mL, stock solution), 2 µL of leupeptin (10 mg/mL, stock solution), 100 µL of 10 mM sodium orthovanadate, 100 µL of Nonidet P-40, and 100 µL of 20% sodium dodecyl sulfate (SDS). Lysates were centrifuged (MSE Microcentrifuge; 12 000 rpm for 15 minutes at 4 °C), and the resulting protein extracts were separated (50 µg/lane) by SDS-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose filters. Antibodies to Hsp90 and Hsp70 were obtained from StressGen (Victoria, Canada), and antibodies to Raf-1 and p53 (DO1) were from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody to the rat DT-diaphorase (which cross-reacts with human diaphorase) was supplied by R. Knox (previously at CRC/ICR, now at Enzacta Ltd., Salisbury, U.K.). Antibody binding was identified with horseradish peroxidase-labeled secondary antibodies combined with enhanced chemiluminescence reagents (Amersham, Buckinghamshire, U.K.) and autoradiography.
In Vivo Effects
BE vector control cells and BE-F397 clone 2 cells were established as subcutaneous xenografts by injection of 5 x 106 cells into the flanks of adult female athymic nude (nu/nu) mice. The antitumor effect of 17AAG was determined in mice bearing comparably sized tumors (6-8 mm in diameter) derived from these cells. Animals were randomly assigned to receive vehicle alone (five or six mice) or 17AAG (five animals; dose schedule = 80 mg/kg per day in 10% dimethyl sulfoxide and 90% egg phospholipid by intraperitoneal injection on days 1-4 and days 7-11). Before this clinical formulation was available, 17AAG was administered to mice bearing HT29 xenografts in 10% dimethyl sulfoxide-0.05% Tween 20-90% NaCl, with a dose schedule of 80 mg/kg per day on days 0-3 and days 6-10. This dose and schedule were derived from previously performed experiments [NCI drug data file on 17AAG and (11)].
Tumor size was determined twice weekly by caliper measurements, and tumor volumes were
calculated (volume = [a x b2 x ]/6,
where a and b are orthogonal tumor diameters). Tumor volumes were then
expressed as a percentage of the volume at the start of treatment (relative tumor volume). The
effect of the drug was determined by the growth delay, i.e., the difference in days required for the
volume of tumors in control and treated animals to double. All procedures involving animals were
performed within the guidelines set out by the Institute's Animal Ethics Committee and the
United Kingdom Coordinating Committee for Cancer Research's ad hoc
Committee on the Welfare of Animals in Experimental Neoplasia (36).
Statistical Analyses
Where indicated, errors are presented as standard deviation (n 3). Correlation tests and
linear regression analyses were computed with SAS JMP (SAS Institute, Cary, NC). We assessed
correlations with a Spearman calculation for the CRC/ICR panel and with a Pearson calculation
for the NCI panel. Although the Spearman statistic is technically more robust, the Pearson
statistic was used for correlations in the NCI panel for historic continuity. The likelihood test for
linear model comparison was performed with S-Plus (Mathsoft, Seattle, WA). All P
values are two-sided.
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RESULTS |
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The in vitro growth inhibition properties of geldanamycin,
17AAG, and radicicol against panels of human colon (15 lines) and
ovarian (11 lines) carcinoma cell lines are shown in Table 1,
A. The IC50 value for
17-amino,17-demethoxygeldanamycin, the major metabolite of 17AAG, is
also included for some lines. In most cell lines, all four compounds
potently inhibited growth, with IC50 values of less than 2.5
µM. Notably, one ovarian cell line (the 41M line) was
relatively resistant (IC50 >2.5 µM) to all
four Hsp90-interactive compounds. On average, geldanamycin was the most
potent agent (mean IC50 = 50.1 nM), with similar
values obtained for 17-amino,17-demethoxygeldanamycin (mean
IC50 = 47 nM in a subset of nine cell lines). 17AAG
showed intermediate potency (mean IC50 = 220.4 nM),
and the least potent agent was radicicol (mean IC50 =
587.4 nM).
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The in vitro potencies of geldanamycin, 17AAG, and
radicicol have also been evaluated in various anticancer drug-resistant
sublines. These lines possess acquired resistance to cisplatin (cisR
lines) or to doxorubicin through overexpression of P-glycoprotein (doxR
line) or of MRP1 (SKOV-3 S2) (Table 1, B). Although little
cross-resistance to geldanamycin was observed in the
cisplatin-resistant cell lines, geldanamycin was markedly less potent
in the P-glycoprotein-overexpressing cell lines and in the
MRP1-overexpressing cell lines than in the parent lines, suggesting
that geldanamycin is a substrate for these multidrug-resistant efflux
proteins. The picture is rather less clear for 17AAG because the
parental CH1 ovarian cell line is relatively resistant to 17AAG,
although there is at least a 2.5-fold cross-resistance to 17AAG in
CH1doxR. The level of cross-resistance for geldanamycin and 17AAG was
similar in the MRP-overexpressing ovarian line. Like geldanamycin,
17AAG retains full activity in the cisplatin-resistant lines.
Growth Inhibition and DT-Diaphorase Enzyme Activity
Because geldanamycin and 17AAG are quinone-based compounds and BE
cells have a disabling point mutation in the NQO1 gene (29),
the lack of DT-diaphorase activity in these cells could be involved in
their surprisingly high relative resistance to 17AAG and low relative
resistance to geldanamycin. To explore this possibility, we measured
DT-diaphorase enzyme activity and IC50 values for
geldanamycin, 17AAG, and radicicol in 11 cell lines (selected from
those shown in Table 1), with a broad spectrum of responses to these
compounds (Fig. 3
). A statistically significant
negative Spearman correlation was apparent for 17AAG (r =
-.81; P = .002). Cells with marginal DT-diaphorase levels
were relatively resistant to 17AAG, but there was no statistically
significant correlation between sensitivity to geldanamycin or
radicicol and DT-diaphorase levels (P = .33 and .76,
respectively). Thus, we have identified the potential for a causal link
between expression of DT-diaphorase and sensitivity to 17AAG, but not
geldanamycin, in the CRC/ICR panel of colorectal and ovarian cell lines.
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Activity in Isogenic BE Colon Cell Lines That Contain or Lack the Active NQO1 Gene
To more directly investigate the role of DT-diaphorase in mediating the cytotoxicity of 17AAG, we stably transfected the BE cell line with the NQO1 gene encoding DT-diaphorase. As shown by immunoblotting, the resulting BE-F397 clone 2 and the naturally high DT-diaphorase-containing colon line HT29 possess similar levels of DT-diaphorase protein (unpublished results). Enzyme activity data supported the immunoblotting observations. Values (measured as the dicoumarol-inhibitable reduction of menadione and expressed as micromoles of cytochrome c reduced per minute per milligram of protein) are as follows: BE vector control cells, unmeasurable activity (<0.002); BE-F397 clone 2, 1.4 ± 0.5; BE-F397 clone 5, 1.3 ± 0.2; and HT29, 0.94 ± 0.2. This HT29 activity is similar to the activity obtained previously with the same assay (27). Functional validation of the model was provided by the observation that introduction of the DT-diaphorase gene into BE cells substantially enhanced the potency of streptonigrin, an excellent DT-diaphorase substrate and bioreductive agent. The degree of potentiation correlated with DT-diaphorase levels and activity (117-fold potentiation in BE-F397 clone 5 and 142-fold potentiation in BE-F397 clone 2). Further details will be published elsewhere.
Dose-response curves for geldanamycin and 17AAG in BE vector control cells and BE-F397
clone 2 are shown in Fig. 4, A. Although the two lines showed similar
sensitivity to geldanamycin, BE vector control cells lacking DT-diaphorase were markedly less
sensitive to 17AAG. The degrees of potentiation (in terms of IC50 values) for
geldanamycin, 17AAG, 17-amino,17-demethoxygeldanamycin, radicicol, and herbimycin observed
when DT-diaphorase was introduced into the BE colon cell line are shown in Fig. 4,
B. Notably, a 32-fold potentiation was observed with 17AAG, whereas a less than
threefold potentiation was observed for all other compounds evaluated. In a second test of the
effect of DT-diaphorase on the growth inhibitory properties of these compounds (Fig. 4,
B), HT29 colon cells (naturally high in DT-diaphorase activity) were
compared with BE parent cells (no measurable DT-diaphorase activity). Results generally
mirrored those results observed with the isogenic-transfected pair of BE lines, with only 17AAG,
of the Hsp90 inhibitors tested, showing a marked DT-diaphorase-mediated differential effect
(87-fold potentiation). It is of interest in this pair of lines that HT29 cells had a strikingly greater
sensitivity to radicicol than did BE cells, an effect not seen with the isogenic BE cell line pair.
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Having demonstrated a potentially important role for DT-diaphorase
in cellular sensitivity to 17AAG, we used a menadione substrate
replacement assay as described previously (27,34) to determine
the ability of this agent, geldanamycin, and
17-amino,17-demethoxygeldanamycin to act as substrates for purified
human DT-diaphorase (Table 2). Streptonigrin (35), an
excellent substrate for DT-diaphorase, was also included in the
comparison. We found that 17AAG was a reasonable substrate for
DT-diaphorase, but it is not appreciably better than geldanamycin or
17-amino,17-demethoxygeldanamycin. This is perhaps surprising in view
of the cellular data. The DT-diaphorase-mediated reduction rate was
similar for all three analogues, each at a substrate concentration of
10 µM. At 50 µM, 17AAG and
17-amino,17-demethoxygeldanamycin gave twofold to threefold higher
rates than geldanamycin, and the difference was even greater at 100
µM. Geldanamycin at 100 µM resulted in
substrate inhibition, which was not observed with the other two
analogues at 100 µM. The latter two concentrations,
however, are much higher than the pharmacologically relevant range. It
also should be noted that all three of the ansamycin analogues gave
reaction rates that were substantially lower than rates observed for
streptonigrin (Table 2
). With the structurally
distinct Hsp90 inhibitor radicicol, which lacks a quinone moiety, no
reduction was observed.
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To determine whether the mode of action of 17AAG was the same in
cells expressing low and high levels of DT-diaphorase and to guide the
choice of molecular pharmacodynamic markers in the imminent clinical
trial, we measured the levels of Raf-1, mutant p53, Hsp90, and Hsp70
proteins in vector control cells and transfected BE cells treated with
17AAG (or geldanamycin). Levels of these proteins 6 and 24 hours after
the addition of equitoxic (continuous exposure to 5x and 10x
IC50) or equimolar (0.15 and 0.3 µM)
geldanamycin or 17AAG are shown in Fig. 5. No change
in Hsp90 protein levels was observed. A similar marked reduction,
especially at 24 hours, was observed for Raf-1 and p53 proteins in the
BE vector control cells and BE-F397 clone 2 cells at equitoxic
concentrations. By contrast, an increase in Hsp70 levels was observed.
For geldanamycin or 17AAG at equimolar concentrations (0.15 or 0.3
µM), no change in any of the four proteins was observed in
the BE vector control cells expressing low levels of DT-diaphorase,
consistent with their cellular resistance at these concentrations.
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We determined the effect of 17AAG on the response of the BE vector
control cells and BE-F397 cells when grown subcutaneously as solid
tumor xenografts in nude mice. 17AAG was administered at the maximum
tolerated dose of 80 mg/kg per day intraperitoneally on days 0-4 and
days 7-11, a schedule that is active on sensitive xenografts [NCI
drug data file on 17AAG and (11)]. The xenograft tumor grown
from the transfected BE-F397 cells (Fig. 6, B) was
more sensitive than the BE vector control cells (Fig. 6,
A). The growth
delays, calculated from the time required to reach twice the treatment
volume, were 11.4 days for the BE-F397 xenograft and 5.8 days for the
vector control. For the HT29 xenograft (and a similar schedule of 80
mg/kg per day intraperitoneally on days 0-3 and days 6-10), a growth
delay of 16.6 days was observed (Fig. 6,
C). Experiments (not shown)
confirmed that the differences in DT-diaphorase expression seen in
vitro were maintained in the xenograft (data not shown). Thus, the
HT29 line with a naturally high level of DT-diaphorase and also the
transfected BE-F397 line were more sensitive in vivo than the
BE vector control cells that have a low level of DT-diaphorase activity.
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DISCUSSION |
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The correlation seen between expression of DT-diaphorase activity and sensitivity to 17AAG but not to geldanamycin or radicicol shows that the effect is not generic across all Hsp90 inhibitors or, indeed, across all benzoquinone ansamycins. The precise mechanism by which high levels of DT-diaphorase in tumor cells result in sensitivity to 17AAG is not clear. The observation that DT-diaphorase activity affects tumor cell sensitivity to 17AAG but not to geldanamycin or 17-amino,17-demethoxygeldanamycin is not explicable in terms of their respective behavior as substrates for the purified human enzyme. Although we have demonstrated that 17AAG is a reasonable substrate for human DT-diaphorase, it was not appreciably better than geldanamycin or 17-amino,17-demethoxygeldanamycin, particularly at more relevant drug concentrations. Only at the markedly suprapharmacologic concentrations of 50 and 100 µM was 17AAG reduced at a statistically significantly faster rate than geldanamycin. For 17-amino,17-demethoxygeldanamycin, there was no appreciable difference in rate compared with geldanamycin.
Given the close structural similarity of 17AAG, 17-amino,17-demethoxygeldanamycin, and
geldanamycin (Fig. 1), it is clear that it is the allyl substitution on the
amino group at position 17 that is responsible for the DT-diaphorase effect. Preliminary results
with a range of 17AAG analogues are consistent with this observation. We hypothesize that the
behavior of the reduction product of 17AAG must differ from the reduction products derived
from geldanamycin analogues with other substituents.
The xenograft experiment confirmed that DT-diaphorase-transfected BE-F397 cells were more sensitive than BE vector control cells in a solid tumor in vivo. The naturally high DT-diaphorase-containing HT29 xenograft was also more sensitive than the BE vector control xenograft. Dose-response data were not generated in these experiments. However, it seems likely that the differences seen in the in vivo xenografts were not as large as those observed in the same lines in vitro. One factor that would tend to decrease the contribution of DT-diaphorase levels in the xenograft experiments is the metabolism of 17AAG to the 17-amino derivative, which is the major metabolite in the mouse (13). This could be important because we show in this article that sensitivity to the 17-amino metabolite is not affected by DT-diaphorase. Formation of the 17-amino metabolite is catalyzed by cytochrome P450, specifically CYP3A4 in human microsomes (13). Thus, we propose that the sensitivity of a given patient's tumor to 17AAG may be affected by the balance between DT-diaphorase and CYP3A4 metabolism. Consequently, we urge that both enzymes (or surrogates thereof) be monitored in the clinical studies that are now under way with 17 AAG.
We determined that 17AAG was operating through the Hsp90 protein to stimulate degradation of the oncogenic client proteins Raf-1 and mutant p53 by use of 17AAG at equitoxic and equimolar concentrations and cells expressing high and low levels of DT-diaphorase. The depletion of client proteins reported previously for both 17AAG and geldanamycin (4-6,9) was seen in cells expressing high and low levels of DT-diaphorase. At equitoxic concentrations of 17AAG or geldanamycin (5x and 10x IC50) in the isogenic BE cell lines after 6 hours and, especially, after 24 hours of drug exposure, there was a similar and marked reduction in Raf-1 and mutant p53 proteins. At the fixed concentrations of 0.15 or 0.3 µM 17AAG, which inhibited growth of wild-type NQO1-transfected cells but not BE vector control cells, there was no reduction in Raf-1 or p53 protein in cells with low levels of DT-diaphorase, whereas depletion was seen in the cells with high levels of DT-diaphorase that did respond to these concentrations. Thus, target activity was maintained in the presence of the respective active concentrations of 17AAG, independent of the expression of DT-diaphorase. This rules out the possibility that different target mechanisms operate in cells expressing low and high levels of DT-diaphorase. Rather, DT-diaphorase expression increases the potency of 17AAG via client protein depletion.
In contrast to effects reported in melanoma xenografts after administration of 17AAG (11), no difference in the levels of Hsp90 was observed in our experiments. Hsp70 levels, however, were increased, consistent with the removal of Hsp90-induced transcriptional repression of Hsp70 when Hsp90 is inhibited (38). Again, this effect was seen at equitoxic concentrations of 17AAG in both high and low DT-diaphorase lines, consistent with retention of the Hsp90-binding mechanism.
The high constitutive expression of p53 in BE cells suggests a mutant p53 genotype. Effects on mutant p53 were consistent with cell cycle effects of geldanamycin reported in cell lines expressing wild-type or mutant p53 (39). In our own studies on the A2780 human ovarian carcinoma cell line (wild-type for p53) and a subline stably transfected with the viral p53-inactivating gene HPVE6 (40), we found no difference in sensitivity to geldanamycin or 17AAG. Overall, the results indicate that p53 status is unlikely to influence sensitivity to 17AAG.
In summary, although uncertainties remain regarding the precise mechanism involved, our results clearly show that expression of DT-diaphorase can influence a tumor's sensitivity to 17AAG. It is also possible that NQO1 expression could affect toxicity of 17AAG toward normal tissues. There are obvious implications for the clinical evaluation of 17AAG as an anticancer agent because 5%-20% of the population (depending on ethnicity) is homozygous for the genetic polymorphism used in this study, the DT-diaphorase-disabling point mutation in the NQO1 gene present in the BE colon cell line (41). In addition, the expression of DT-diaphorase in human tumors is very variable (25,26), as it is in the cell lines studied herein and elsewhere (22-24). We suggest that, in addition to measuring degradation of oncogenic client proteins and/or an increase in Hsp70 after treatment with 17AAG as potential markers of activity and therapeutic response, NQO1/DT-diaphorase genotype, CYP3A4 status, and also tumor DT-diaphorase levels should be determined. In particular, we propose that these measurements may provide useful indicators of efficacy and/or toxicity and should be considered for the phase I clinical trials of 17AAG that have recently begun under the auspices of the NCI and CRC.
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NOTES |
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We thank Dr. E. Sausville at the NCI for helpful discussion and the supply of geldanamycin and derivatives, Drs. A. Monks and N. Scudeiro at the NCI for providing NCI cell screen data, M. Valenti and L. Brunton for technical assistance with the xenograft experiment. and Dr. M. Walton for discussion of data.
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Manuscript received April 17, 1999; revised September 10, 1999; accepted September 17, 1999.
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