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Vol. 2, 123-129, February 2003     Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research

17AAG: Low Target Binding Affinity and Potent Cell Activity—Finding an Explanation1

Gabriela Chiosis2, Henri Huezo, Neal Rosen, Edward Mimnaugh, Luke Whitesell and Len Neckers

Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [G. C., H. H., N. R.]; Cell and Cancer Biology Branch, National Cancer Institute, NIH, Rockville, Maryland 20850 [E. M., L. N.]; and Steele Memorial Children’s Research Center, University of Arizona, Tucson, Arizona 85724 [L. W.]

2 To whom requests for reprints should be addressed, at Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 271, New York, NY 10021. Phone: (212) 639-8929; Fax: (212) 717-3627; E-mail: chiosisg{at}mskcc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ansamycin geldanamycin (GM) and its derivative, 17AAG, now in early clinical trials in cancer patients, have potent activity against several cancer cells at low nanomolar concentrations. The main target of these drugs is the molecular chaperone heat shock protein 90. Contrary to the high antitumor potency, the affinity of these drugs for the chaperone was determined to be ~1 µM. We propose that this difference can partly be explained by the physicochemical characteristics of the ansamycins. GM and 17AAG accumulate in cells, producing higher intracellular concentrations than expected. We conclude that although apparent activity for ansamycins can be seen at low nanomolar concentration, their real activity correlates with the heat shock protein 90 binding affinity and is in the low micromolar concentration range. We suggest that in the clinic, micromolar concentrations of 17AAG must accumulate in the tumor cells to achieve antitumor effects in patients comparable with ones achieved in tissue culture settings.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GM3 and HA (Fig. 1) are ansamycin antibiotics isolated from Streptomyces hygroscopicus on the basis of their ability to revert the malignant phenotype of v-src transformed fibroblasts (1, 2). The ansamycins were found to be active against many cancer cell lines inducing growth arrest and differentiation (3–5). Addition of these compounds to cells induces the proteasomal degradation of a subset of proteins involved in signal transduction (i.e., steroid receptors, Raf-1 kinase, and certain transmembrane tyrosine kinases; Refs. 3, 4). In addition, the molecules cause the selective degradation of certain mutated proteins known to be involved in cancer such as v-Src, Bcr-Abl, and p53 but have little or no effect on their normal counterparts (6–11). Cancer cells treated with ansamycins usually arrest in G1, followed by morphological and functional differentiation (12, 13). However, some cell lines undergo G2-M arrest (12, 14). Despite their cellular potency, the introduction of these compounds to the clinic was prevented by their high liver toxicity and/or cellular instability (6, 15). A derivative of GM, 17-AAG, was found to have similar cellular effects but lower hepatotoxicity than the parent compound and is currently in early clinical trials in cancer patients (4) at five institutions.4



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  		Fig. 1. Representative Hsp90 inhibitors.

 
The main target of these drugs was demonstrated to be the Hsp90 family of chaperones (16, 17). GM, HA, and 17AAG bind to a conserved pocket in the NH2-terminal portion of Hsp90. The endogenous ligands for the pocket are ATP and ADP. These nucleotides function as a switch in determining the conformation of the chaperone (18, 19). The binding of ansamycins to Hsp90 prevents binding of ATP and has profound effects on the composition of Hsp90-containing multimolecular chaperone complexes and on signaling proteins, which depend on the chaperone (20–25). Ansamycins’ affinity for the isolated Hsp90 protein have been determined by several methods to be in the low micromolar range (26–28). When tested against several tumor cell lines, these compounds were found to inhibit growth with IC50s ranging from 2 nM to >40 µM. The low nanomolar values obtained are at odds with the determined affinity of these drugs for Hsp90. The inconsistency has been observed but never addressed in the literature.

In search of drug-like small molecule inhibitors of Hsp90, we developed simple soluble compounds that could help in elucidating the potency issue for GM and the related series.

The X-ray structure of GM, radicicol (RD), and ADP bound to the NH2-terminal pocket of Hsp90 {alpha} has been determined (16, 17, 27). We studied the shape, volume, and mode of interaction of these ligands when inside the pocket and designed the novel Hsp90 inhibitor PU3 (Fig. 1; Ref. 29). PU3 exhibited a relative binding affinity of 15–20 µM for Hsp90 and elicited qualitative effects on cellular protein expression, proliferation, and differentiation very similar to those induced by 17AAG (29). Synthesis of a library of compounds based on the PU3 skeleton produced a compound, PU24FCl (Fig. 1), that exhibited a relative affinity for Hsp90 comparable with 17AAG (30, 31). Addition of PU24FCl to cancer cells induced the growth arrest and differentiation of several cancer cell lines at concentrations ranging between 1 and 5 µM, values consistent with the binding affinity for the chaperone, however, 10–100-fold greater than 17AAG (Ref. 31 and unpublished data).

Here we show that in tissue culture experiments, when the molar amount of ansamycins is kept constant but the volume of the medium is increased, no activity change is observed. In contrast, when such an experiment is repeated with the purine derivatives, PU3 and PU24FCl, activity diminishes with concentration. We find that upon addition to aqueous tissue culture media, ansamycins rapidly accumulate into cells and are mostly depleted from the bulk media. Thus, addition of 10 nM GM to cells can lead to an intracellular drug concentration that is in the micromolar range.

Our experiments suggest that the real activity of GM and 17AAG is, contrary to the observed apparent values, in the low micromolar range and consistent with the previously reported affinity of these compounds for Hsp90.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
GM and 17AAG were obtained from the Developmental Therapeutics Program/National Cancer Institute. The synthesis of PU3, PU29FCl, and PU24FCl was described elsewhere (29–31). MCF-7 cells and PC3-M were obtained from the American Type Culture Collection (Manassas, VA) and maintained in 1:1 mixture of DMEM:Ham’s F-12 supplemented with 2 mM glutamine, 50 units/ml penicillin, 50 units/ml streptomycin, and 5% heat-inactivated fetal bovine serum (Gemini Bioproducts) and incubated at 37°C in 5% CO2. Proliferating prostate epithelial cells seeded in 96-well plates were purchased from Clonetics no. CC-0088 (Walkersville, MD).

Growth Arrest Studies.
Growth inhibition studies were performed using the sulforhodamine B assay as described previously (5, 32). Stock cultures were grown in T-175 flasks containing 30 ml of DMEM (HG, F-12, nonessential amino acids, and penicillin and streptomycin), with glutamine, and 10% fetal bovine serum. Cells were dissociated with 0.05% trypsin and 0.02% EDTA in PBS without calcium and magnesium. Experimental cultures were plated in microtiter plates (Nunc) in the indicated volumes of growth medium at a density of 1000 MCF-7 cells/well. One column of wells was left without cells to serve as the blank control. Cells were allowed to attach overnight. The following day, growth medium having drug or DMSO at twice the desired initial concentration was added to the plate in triplicate and was serially diluted at a 1:1 ratio in the microtiter plate. After 72 h of growth, 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 is calculated as the drug concentration that inhibits cell growth by 50% compared with control growth.

Proliferating prostate epithelial cells seeded in 96-well plates were purchased from Clonetics no. CC-0088. Upon receipt, cells were placed in a humidified incubator at 37°C in 5% CO2 and allowed to equilibrate for 3 h. Media were removed by suction and replaced with fresh media provided by the manufacturer. Cells were then treated with drugs or DMSO for 72 h and the IC50s determined as described above.

Production of Tritiated GM.
Biosynthetic radiolabeling of GM was achieved using [methyl-3H]methionine (84.8 Ci/mmol, NEN#NET-061X) and a modification of previously reported methods (33). Briefly, fermentation broth cultures (50 ml) of S. hygroscopicus var. geldanus var. nova (National Cancer Institute, Natural Products Branch UC5208) were supplemented with tritiated methionine (10 µCi/ml) 2 days after inoculation. Incubation with agitation at room temperature was continued for an additional 48 h. Culture supernatants were collected by centrifugation and extracted three times with chloroform. The pooled extracts were applied to a silica gel column (bed volume = 1 ml; Sigma), and the column was washed with chloroform followed by 1% methanol in chloroform. GM was eluted using 5% methanol in chloroform, and the yellow drug containing fractions were collected, combined, and subjected to a second silica gel purification step. Chemical purity was estimated at >95% by silica gel thin layer chromatography using authentic GM as a standard. Essentially, all of the radioactivity detected by autoradiography of thin layer chromatography plates comigrated with the yellow GM spots. Aliquots of the purified, radiolabeled GA solution were counted by liquid scintillation spectroscopy and converted to dpm, and the UV spectrum (200–400 nm) of the purified GA was also recorded. Using the molar extinction coefficient of 19,300 for the GA peak at 306 nm, the concentration of [3H]GA was calculated to be 2.34 mM with a specific radioactivity of 9.02 Curies/mol.

Cellular Uptake and Localization of GM.
Two million PC3-M cells were added to 25 cm2 flasks and allowed to attach and begin growing overnight. The next day the medium was removed by aspiration and replaced with 2 ml of DMEM without serum to prevent GM binding to serum proteins. Radioactive GM was diluted with cold GM and added to the cells to produce a final concentration of 2 µM (equivalent to 4 nmol of GM) and 38,000 dpm/ml radioactivity. The cells were then incubated at 37°C under standard cell culture conditions for 0, 2, 6, 10, 20, 30, 60, and 90 min. At each time point, the medium was removed by aspiration, and the cells were washed twice with ice-cold PBS. After removal of the PBS, 0.5 ml of 1 N NaOH were added to each flask to digest the cells. The NaOH was then neutralized with 0.5 ml of 1 N HCl, and 100 µl of 2 M Tris (pH 7.5) buffer were added to adjust the pH. Cell-associated radioactivity was measured in aliquots by liquid scintillation counting; the protein concentrations were determined by the bicinchoninic acid (BCA) microplate method (34). The amount of incorporated GM was determined using the following approximations: trypsinized tissue culture PC3-M cells have a diameter of 10–20 µm, thus a radius of 5–10 µm. This value incorporated in the formula V = 4/3 x {pi}r3 calculates their volume at 525-4200 x 10-12 cm3. Therefore, the 2.5–3 million cells would occupy a volume of 1.5–12.6 µl. A cell-associated radioactivity of 14,000 dpm/flask indicates a cellular content of 0.75 nmol of GM. Considering the volume occupied by the cells (approximate average of 7 µl), it was calculated that the cell concentration of GM would be 108 µM. Radioactivity of the media was measured to be an average of 25,000 dpm/ml. This value translates into a concentration of GM in media of 1.3 µM.

The entire experiment, with the exception of the first two time points (2 and 6 min) and with the addition of a 120-min time point, was repeated in a 6-well plate format with essentially the same profile of drug uptake.

Cell Fractionation Experiment.
A 15-cm plate of exponentially growing PC-3M cells was exposed to 2 µM GM at 33,800 dpm/ml in serum-free medium for 30 min. The cells were rinsed twice in cold PBS, scraped, and lysed in ice-cold water using a Dounce homogenizer with 10 strokes. The homogenate was diluted with an equal volume of 0.5 M sucrose-100 mM Tris (pH 7.4) before differential centrifugation.

Mass Spectrum Analysis.
A total of 5 x 104 MCF7 cells/well was seeded into a 12-well plate and allowed to attach overnight and grow to 80–90% confluency. The medium was removed by aspiration and replaced with fresh solution. PU3 (100 µM) or DMSO was added to the wells. Wells without cells but with media and drug served as positive controls. Wells with medium only served as the negative controls. Twenty-four h later, the drug was extracted from media by adding 400 µl of chloroform to 1 ml of media from drug-treated cells, DMSO-treated cells, or blank wells. The drug was extracted from cells by removing the medium and then adding 250 µl of isopropanol to the cell monolayer, followed by vigorously mixing the cell monolayer with the isopropanol. The drug was extracted in 100 µl of chloroform. Equal aliquots of the organic extracts were analyzed by low-resolution mass spectra recorded in the positive ion mode under electron-spray ionization. The concentration of drug present was determined by comparing the intensity of the recorded peaks to an external standard of PU3.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cellular Growth Inhibition by GM, 17AAG, and the Synthetic Molecules PU3 and PU24FCl.
MCF7 cells were treated in parallel in a 96-well plate with various concentrations of 17AAG and volumes of media added to the same number of cells. Addition of 17AAG to the MCF-7 cell line resulted in growth inhibition that was proportional to the molar amount and not the concentration of added drug. First we varied the volume of the media added to the cells but kept the number of added moles of 17AAG constant (8.75 pmol; Fig. 1A). Contrary to the expected higher inhibitory efficacy in the wells that contained less volume (higher drug concentration), we observed identical inhibition of growth. When the growth inhibitory effect of 17AAG was determined by serially diluting 25 pmol of drug in 50 µl of media (500 nM initial concentration), the value coincided with the number determined when 25 pmol of drug were serially diluted in 100 µl of media (250 nM initial concentration; Fig. 2B). On the contrary, in the wells in which the concentrations were kept identical by adjusting the number of moles in the corresponding volumes, we observed a paradoxical increase of drug efficacy. The IC50 of 17AAG when the drug was measured by serially diluting 25 pmol in 50 µl (500 nM initial concentration; curve 1, Fig. 2C) was determined to be almost twice the value determined by serially diluting 50 pmol in 100 µl (500 nM initial concentration; curve 2, Fig. 2C). An identical behavior was determined for GM (Fig. 2D).



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  		Fig. 2. MCF-7 cells were treated with: (A) and (B) different concentrations of 17AAG obtained by adding the same molar amount of drug to different volumes of media and (C) same concentrations of 17AAG obtained by serially diluting 25 pmol of 17AAG in 50 µl of media (curve 1, C) and 50 pmol of 17AAG in 100 µl of media (curve 2, C) for 72 h as described in "Materials and Methods." D, MCF-7 cells were treated with GM. Curve 1 was obtained by serial dilution of 25 pmol of GM in 50 µl of media (500 nM) and curve 2 by serial dilution of 150 pmol of GM in 300 µl of media (500 nM). All experiments were conducted in triplicate wells.

 
Both PU3 and PU24FCl exhibit favorable M logPs (2.2 and 2.4, respectively) and are much more soluble in water compared with ansamycins. The inhibitory effect of PU24FCl was determined by serially diluting the drug in a 96-well plate at a volume of 50 µl of media. PU24FCl inhibits the MCF-7 breast cancer cell line with an IC50 of 2.5–3 µM. At 10 µM, the inhibition was maximum (Fig. 3A, curve 1). The amount of drug corresponding to this concentration (500 pmol) was the starting point of another experiment in which increasing volumes of media were added to wells containing 500 pmol of PU24FCl added to same number of cells (decreasing concentration). As expected, the curve obtained by plotting the resulting growth inhibition values completely overlaps with curve 1 (Fig. 3A, curve 2). This behavior is mirrored by PU3; the drug activity is proportional to its concentration regardless of the volume of medium used in the assay (Fig. 3B).



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  		Fig. 3. MCF-7 cells were treated with PU24FCl. A, curve 1 represents the inhibitory effect of different concentrations of drug compared with control in an experiment conducted in 50 µl of media/well in a 96-well plate. The amount of drug corresponding to maximum percentage of growth arrest in curve 1 (10 µM) was kept constant and added to wells that contained increasing volumes of media. The observed inhibitory effects are plotted in curve 2. B, MCF-7 cells were treated with PU3. Growth inhibition is proportional to concentration not the molar amount of drug. Experiments were conducted in a 48-well plate for 72 h as described in "Materials and Methods."

 
It is noteworthy that 50% growth inhibition of MCF-7 cells was observed with PU3 at 50 µM (29), a value rendering this compound a 25–30-fold weaker inhibitor than PU24FCl. This ratio was also observed in the relative affinities for Hsp90 (15–20 versus 0.55 µM). In general, the potency of our purine-scaffold Hsp90 inhibitors was found to be linearly proportional to their measured affinity for the Hsp90 pocket (data not shown), suggesting that these synthetic compounds affect the cancer cells primarily through their Hsp90 inhibitory effect.

Cellular Uptake of GM and PU3.
To give a quantitative aspect to our observations, cells were treated with radiolabeled GM to determine the amount of accumulated drug. Cells were treated with tritiated GM for various time intervals and the cell-associated radioactivity was measured (Fig. 4A). Although the concentration of added GM in the incubation media was calculated to be 2 µM, the value determined inside the cell corresponded to an approximated average concentration of 100 µM. Radioactivity of the media also was measured and translated to a concentration of GM of 1.3 µM. These results indicate that under the experimental conditions used, there is a rapid 80-fold accumulation of drug in cells over that of the medium.



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  		Fig. 4. A, PC3-M cells were treated with tritiated GM as described in "Materials and Methods." Cell-accumulated GM was determined at several time points by measuring the radioactivity of the cellular mass. B and C, MCF-7 cells were treated with PU3 as described in "Materials and Methods." Drug present in media (C) and accumulated within cells (B) was extracted in chloroform and the concentration of drug in both extracts determined by mass spectrum analysis.

 
Most of the radiolabeled GM accumulated into the cytosol (91%), whereas only small fractions were found in other compartments of the cell (1.6% nucleus, 4.5% mitochondria, 2.5% microsomes).

When cells were treated with 100 µM PU3, it was demonstrated by mass spectroscopy that more drug was in the media (Fig. 4C) than in the cell extract (Fig. 4B). The concentration of the drug partitioned in the media was four to five times higher than in the cells. The experiment was repeated with PU24FCl (10 µM). Cells were treated with PU24FCl in serum containing and serum-free media for 15 min, 60 min, 6 h, and 24 h and the extracts analyzed by mass spectra. The drug was detected in all extracts, with higher amounts in the media (data not shown).

Effect of 17AAG and the Synthetic Hsp90 Inhibitors on Primary Epithelial Prostate Cells.
Accumulation of ansamycins in cells could be a characteristic of the tumor cells and/or a restricted tissue culture phenomenon. In tissue culture, we observe comparable activities against tumor and normal cells for GM and 17AAG. Primary prostate epithelial cells were treated with 17AAG or the synthetic inhibitors (Fig. 5). Growth inhibition was observed with PU24FCl, PU29FCl, and PU3 (estimated 35, 40, and 280 µM, respectively) at concentrations an order of magnitude higher than necessary for the inhibition of prostate tumor cells tested (1–5 µM for PU24FCl and PU29FCl and 45–50 µM for PU3; data not shown). 17AAG, however, did not differentiate between the two cell types. Inhibition of the primary epithelial cells was observed at an IC50 = 30 nM with 17AAG, a value that is equivalent to IC50s obtained for several prostate cancer cell lines (25–45 nM; Ref. 35). This observation would suggest a constant, nonspecific accumulation of GM and 17AAG into cells conflicting with the existent in vivo data. In prostate cancer xenografts, 17AAG inhibited tumor growth at doses that were nontoxic to the animals (35). Although high levels of 17AAG were observed immediately after administration in both normal tissue and tumors, the drug was biexponentially cleared from normal tissue while persisting for >24 h in tumors (36). These data may imply that unselective accumulation of GM in tissue culture is a peculiarity of the drug in these settings and not a characteristic of Hsp90 inhibitors.



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  		Fig. 5. Primary epithelial prostate cells were treated with drugs or vehicle for 72 h as described in "Materials and Methods." PU29FCl is another synthetic Hsp90 inhibitor that has a binding affinity for the chaperone similar to PU24FCl.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
First, these findings may explain much of the variability in IC50s reported for ansamycins in the literature. In light of our data, the number of the cells plated, the volume of the media and the size of the plates (i.e., 96- versus 8-well plate) would greatly influence the resulting IC50s. In the future, we recommend that investigators report, in addition to plate type, number of cells and drug concentrations, the total amount of drug, and media volume when they publish work on ansamycins.

On the basis of the work presented, it is generally suggested that when IC50s and phenotypic changes noticed in the cell studies are much lower than the observed binding constant to target or the IC50 for an enzymatic process or protein-protein interaction that one investigates whether concentration of drug has occurred in the cell.

The question raises as what is to be defined as pharmacologically relevant when studying the effect of GM or 17AAG (or any other similarly behaving compound) on a certain tumor line. Although the apparent concentration of added ansamycins in tissue culture is in the nanomolar range, because of their accumulation into cells, the real concentration at which effects are seen is in the low micromolar range.

The cellular concentration phenomena observed in tissue culture seen for GM and 17AAG has potential clinical implications. One of the requirements for a clinically successful cancer drug is to achieve active concentrations at the site of its action, the tumor cell. 17AAG is now in Phase I clinical trial and maximum peak serum levels of 2–3 µM (37–39) or 16 µM (40), depending on the schedule and dosing used, and prolonged levels of 100–200 nM are obtained without significant toxicity. Although typical tumor concentrations achieved in Phase I studies are not known, it is clear that minimally micromolar levels of 17AAG must be achieved to produce effects comparable with those observed in tissue culture studies. Monitoring of pharmacodynamic markers in tumor biopsy specimens is a difficult but seemingly unavoidable task in the 17AAG clinical trials (40, 41).

Although GM is equally toxic to cancer and normal cells in tissue culture, in vivo it was shown to selectively accumulate in cancer cells and to be effective against tumors at concentrations that are nontoxic to animals (35, 36, 42–45). The observed lack of selectivity in tissue culture is not because of Hsp90 inhibition. The synthetic Hsp90 inhibitors, PU3 and PU24FCl, are at least one order of magnitude more active in inhibiting the growth of tumor versus normal cells. It is important to differentiate between unselective accumulation into cells in tissue culture because of physicochemical peculiarities of a compound in those settings and selective accumulation into cancer versus normal cells because of different biological characteristics of these cells.

Cancer cells may be more sensitive to interference with Hsp90 chaperone function than normal cells. Hsp90 overexpression has been shown in multiple tumor types and as a function of oncogenic transformation (46–50). There is growing evidence that the Hsp90 chaperone plays a necessary role in maintaining transformation, and it could have several functions in this regard. Cancer cells grow in an environment of hypoxia, low pH, and low nutrient concentration. They also rapidly adapt to or are selected to become resistant to radiation and cytotoxic chemotherapy. The general role of Hsp90 in maintaining protein stability under stress may be necessary for cell viability under these conditions. Secondly, cancer cells harbor many mutated oncogenic proteins. Some of these are gain-of-function mutations that are required for the transformed phenotype. Hsp90 may be required for maintaining the folded, functionally active conformation of these proteins. Thirdly, activation of signaling pathways mediated by steroid receptors, Raf-1 and other Hsp90 targets, is necessary for the growth and survival of many tumors.

Although chronic high levels of Hsp90 inhibitors could be toxic to normal cells, (as the tissue culture experiments with 17AAG suggest), the in vivo data indicate that there is a window of activity where the drug has antitumor activity with minimal side effects to the mouse.

The observations presented in this article raise several important issues that extend beyond the particular case of Hsp90 and ansamycins and may explain the behavior of many agents known to accumulate into cells. Indeed, such behavior has been observed in the case of taxanes and epothilones. Although the concentration of epothilones required for microtubule polymerization was determined to be in the micromolar range, with apparent Kis of 0.4 µM (51), profound inhibition of cell proliferation was obtained in the lower nanomolar range. Epothilones, as well as paclitaxel, were found to accumulate several hundred-fold inside cells compared with external medium concentrations (52, 53).

The authors advise that the physical and chemical characteristics of a drug should not be overlooked when studying its biological function as this may result in confusing and conflicting data.

In summary, we propose that the apparent nanomolar activity seen for ansamycins in tissue culture is because of their accumulation in cells and that their real activity correlates with the Hsp90 binding affinity and is in the low micromolar concentration range.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Susan G. Komen Foundation Grant 2000-201, NIH-National Cancer Institute Grant 1U01CA91178-01, and a generous donation from the Taub Foundation. Back

3 The abbreviations used are: GM, geldanamycin; HA, herbimycin; Hsp, heat shock protein. Back

4 Institutions at which Phase I clinical trial with 17AAG is ongoing: United States, National Cancer Institute; Memorial Sloan-Kettering Cancer Center; Mayo Clinic; University of Pittsburgh; and United Kingdom, Royal Marsden Hospital. Back

Received 4/26/02; revised 6/27/02; accepted 11/ 7/02.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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