Pharmaceutical Research, Vol. 20, No. 7, July 2003 (© 2003)
`
`Research Paper
`
`Synergy between
`3ⴕ-Azido-3ⴕ-deoxythymidine and
`Paclitaxel in Human Pharynx
`FaDu Cells
`
`Jeffrey S. Johnston,1 Andrew Johnson,1 Yuebo Gan,1
`M. Guillaume Wientjes,1,2 and Jessie L.-S. Au1,2,3
`
`Received December 8, 2002; accepted March 17, 2003
`
`Purpose. We recently demonstrated simultaneous targeting of telo-
`mere and telomerase as a novel cancer therapeutic approach, and that
`telomerase inhibitors such as 3⬘-azido-3⬘-deoxythymidine (AZT) sig-
`nificantly enhanced the antitumor activity of paclitaxel, which causes
`telomere erosion, in telomerase-positive human pharynx FaDu tu-
`mors in vitro and in vivo (1). The present study evaluated the synergy
`between AZT and paclitaxel to identify optimal combinations for
`future clinical evaluation.
`Methods. FaDu cells were incubated with or without AZT for 24 h
`and then treated with AZT with or without paclitaxel for an addi-
`tional 48 h. Under these conditions, single agent paclitaxel produced
`a 60% maximum reduction of cell number (IC50 was 7.3 nM), and
`single agent AZT produced a 97% reduction (IC50 was 5.6 ␮M).
`Synergy was evaluated using fixed-concentration and fixed-ratio
`methods, and data were analyzed by the combination index method.
`Results. The results indicate a concentration-dependent synergy be-
`tween the two drugs; the synergy was higher for combinations con-
`taining greater paclitaxel-to-AZT concentration ratios and increased
`with the level of drug effect. For example, in combinations containing
`1 ␮M AZT, synergy was 1.3-fold at the 20% effect level and 3.1-fold
`at the 60% effect level. Because the major antitumor activity, deter-
`mined by comparing the posttreatment cell number to the pretreat-
`ment cell number, was antiproliferation at the 20% effect level and
`cell kill at the 60% effect level, our results suggest that AZT mainly
`enhances the cell kill effect of paclitaxel.
`Conclusion. In summary, the present study demonstrates a synergis-
`tic interaction between paclitaxel and AZT and supports a combina-
`tion using a low and nontoxic AZT dose in combination with a thera-
`peutically active dose of paclitaxel.
`
`KEY WORDS: paclitaxel; AZT; synergy; telomere; telomerase.
`
`INTRODUCTION
`
`Telomeres are specific DNA structures at the ends of
`chromosomes that protect chromosomes from end-to-end fu-
`sion, maintain chromosome integrity, reversibly repress tran-
`scription of neighboring genes, and play a role in chromo-
`some positioning in the nucleus (1). Because of the inability of
`DNA polymerases to replicate the 3⬘ end of chromosomes,
`telomeres are shortened by 50 to 200 bp per cell division in
`
`1 College of Pharmacy, The Ohio State University, Columbus, Ohio
`43210.
`2 James Cancer Hospital and Solove Research Institute, The Ohio
`State University, Columbus, Ohio 43210.
`3 To whom correspondence should be addressed. (e-mail: au.1@
`osu.edu)
`ABBREVIATIONS: AZT, 3⬘-azido-3⬘-deoxythymidine; AZTTP,
`AZT triphosphate; CI, combination index; IC, inhibitory drug con-
`centration; SRB, sulforhodamine B.
`
`normal somatic cells. Loss of telomeres to below a threshold
`value is believed to induce senescence. Telomerase is a ribo-
`nucleoprotein DNA polymerase that synthesizes telomeric
`repeats de novo and is involved in multiple cellular processes,
`including cell differentiation, proliferation, inhibition of ap-
`optosis, tumorigenesis, and possibly DNA repair and drug
`resistance (3–6). Telomerase is present in nearly all immortal
`cell lines, germ-line cells, stem cells, and about 90% of human
`tumors, but seldom in normal somatic cells (7).
`The selective expression of telomerase in tumor cells
`makes telomerase an attractive therapeutic target. However,
`telomerase inhibition results in cytotoxicity only after a sig-
`nificant lag time. For example, telomerase inhibitors resulted
`in cytotoxicity in HeLa cells after 23 to 26 cell doublings (8).
`The long lag time may also allow for activation of the telo-
`merase-independent alternative mechanism of telomere
`maintenance (9). These concerns limit the therapeutic poten-
`tial of telomerase inhibitors.
`We recently demonstrated simultaneous targeting of
`telomere and telomerase to be a novel approach to targeting
`cancer cells that use telomerase for telomere maintenance.
`We further showed that telomerase inhibitors [i.e., antisense
`to the RNA component of telomerase and 3⬘-azido-3⬘-
`deoxythymidine (AZT)] significantly enhanced the antitumor
`activity of paclitaxel, which causes telomere erosion, in telom-
`erase-positive human pharynx FaDu tumors in vitro and in
`vivo (1). The present study evaluated the concentration-
`dependent synergy between AZT and paclitaxel, to identify
`synergistic combinations for future clinical evaluation.
`
`MATERIALS AND METHODS
`
`Chemicals and Reagents
`
`Paclitaxel was a gift from the Bristol-Myers Squibb Co.
`(Wallingford, CT) and the National Cancer Institute
`(Bethesda, MD). AZT was supplied by the National Cancer
`Institute (Bethesda, MD). Gentamicin was purchased from
`Solo Pak Laboratories (Franklin Park, IL), and other cell
`culture supplies from GIBCO Laboratories (Grand Island,
`NY).
`
`Cell Culture
`
`FaDu cells were purchased from American Type Culture
`Collection (ATCC, Manassas, VA) and maintained in mini-
`mum essential medium. Culture medium was supplemented
`with 9% heat-inactivated fetal bovine serum, 2 mM L-
`glutamine, 90 ␮g/ml gentamicin, and 90 ␮g/ml sodium cefo-
`taxamine. Cells were incubated with complete medium at
`37°C in a humidified atmosphere of 5% CO2 in air. For ex-
`periments, cells were harvested from preconfluent cultures
`after two rinses with versene and a 10-min incubation with
`trypsin. The harvested cells were resuspended in fresh me-
`dium. Cells were seeded in 96-well microtiter plates and al-
`lowed to attach for 20 to 24 h.
`
`Drug Treatment
`
`Stock solutions of paclitaxel were prepared in 100%
`ethanol at a concentration of 1 mM and stored at −70°C.
`
`957
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`Stock solutions of AZT were prepared in double-distilled
`sterile water at a concentration of 10 mM.
`Drug treatment was initiated after cells were allowed to
`attach to the growth surface. On the day of experiments, the
`culture medium was removed and replaced with drug-
`containing medium. Treatment with AZT was initiated 24 h
`before paclitaxel treatment to allow for the conversion of
`AZT to AZTTP, which is the active metabolite that inhibits
`telomerase (10). Cells were then treated with paclitaxel, with
`or without AZT, for an additional 48 h. Control cells were
`processed similarly but without drugs.
`
`Drug Activity Evaluation
`
`The remaining cell number after drug treatment was
`measured using the sulforhodamine B (SRB) assay, which
`stains for cellular protein and represents the overall drug ef-
`fect, i.e., the combination of cytostatic and cytotoxic effects
`(11). In brief, cells (2,000 cells per well) were seeded onto
`96-well plates. At the end of drug treatment, cells were fixed
`with 0.2 ml 10% trichloroacetic acid at 4°C for 1 h, rinsed with
`distilled water, allowed to air dry, stained with SRB (0.05 ml
`of 0.4%), rinsed with 1% glacial acetic acid, and again allowed
`to air dry. Tris-HCl (0.2 ml, 10 mM) was then added to each
`well to dissolve the SRB, which was measured by absorbance
`at 490 nm using an EL 340 microplate biokinetics reader (Bio-
`Tek Instruments, Inc., Winooski, VT).
`
`Evaluation of Interaction
`
`The nature of the drug interaction was evaluated using
`two methods. The first method used fixed concentrations of
`AZT together with increasing concentrations of paclitaxel,
`i.e., the fixed-concentration method. The advantage of this
`method is that it yields the conventional sigmoidal concentra-
`tion–effect curves showing increases in effect as a function of
`increasing paclitaxel concentration and provides a measure of
`the enhancement of the paclitaxel activity at a fixed AZT
`concentration. The latter facilitates the selection of the AZT
`dose to be used during in vivo studies. However, the experi-
`mental design of the fixed concentration ratio is such that
`only limited AZT concentrations can be studied.
`The second method used fixed concentration ratios of
`paclitaxel and the telomerase inhibitor, i.e., the fixed-ratio
`method. The advantage of this method is that it enables the
`measurement of the nature of the interaction between pacli-
`taxel and AZT at much broader concentration ranges as com-
`pared to the first method. An additional advantage is that it
`allows the identification of the optimal ratios of the two drugs
`that give the maximal synergy. For this method, cells were
`treated with solutions containing both drugs at 5 to 320% of
`their respective initial concentrations. The initial concentra-
`tions were 7 nM for paclitaxel and 5 ␮M for AZT, which were
`approximately their respective IC50 values. The concentration
`ratio of paclitaxel to AZT was kept at four fixed ratios (80:20,
`60:40, 40:60, and 20:80).
`
`Synergy Determination
`
`The nature of the interaction between paclitaxel and
`AZT was analyzed using the combination index method (12).
`Concentration–effect curves for single agents and their com-
`binations were used to determine the amount of each agent,
`
`either alone or in combination, needed to achieve a given
`level of effect. The combination index (CI) was calculated as
`follows.
`
`CI = ICA,B
`+ ICB,A
`ICB
`ICA
`ICA and ICB are the concentrations of agents A and B
`needed to produce a given level of cytotoxicity when used
`alone, whereas ICA,B and ICB,A are the concentrations
`needed to produce the same effect when used in combination.
`A CI value of 1 indicates additive interaction, values less than
`1 indicate synergistic action, and values greater than 1 indi-
`cate antagonistic interaction (13).
`
`RESULTS
`
`Cytotoxicity of Paclitaxel and AZT: Results of
`Fixed-Concentration Method
`
`Cells were treated with paclitaxel, with or without AZT,
`for 48 h. For the combination treatment, cells were also pre-
`treated with AZT for 24 h. Table I summarizes the IC50 val-
`ues.
`
`Treatment with single agent paclitaxel for 48 h resulted
`in a maximum cell number reduction of about 60% (Fig. 1).
`This effect was reached at 100 nM paclitaxel and was not
`increased by increasing the drug concentration another 10-
`fold. The remaining cell numbers after treatment with 1, 5, 10,
`50, 100, and 500 nM paclitaxel were 122 ± 1, 102 ± 4, 93 ± 2,
`71 ± 3, 66 ± 8, and 66 ± 3% of the pretreatment cell number,
`respectively (mean ± SD of three experiments). Hence, the
`antitumor activity reflected inhibition of proliferation at ⱕ10
`nM paclitaxel and cell kill at higher concentrations. Because
`
`Table I. Synergy between Paclitaxel and AZT: Results of Fixed-
`Amount Method
`
`AZT (␮M)a
`
`Paclitaxel IC50 or
`IC70 (nM)
`
`Combination
`index
`
`Synergy,
`fold
`
`50% effect
`0.0
`1.0
`5.0
`10.0
`70% effect
`0.0
`1.0
`5.0
`10.0
`
`7.34 ± 3.18
`4.92 ± 2.77
`2.23 ± 1.48
`0.78 ± 0.43
`
`>1,000
`160 ± 10.4
`10.5 ± 5.9
`5.10 ± 3.0
`
`Not applicable Not applicable
`0.54 ± 0.15b
`1.94 ± 0.53
`0.73 ± 0.02b
`1.37 ± 0.04
`NDc
`NDc
`
`Not applicable Not applicable
`0.11 ± 0.02b
`9.14 ± 1.50
`0.39 ± 0.01b
`2.34 ± 0.36
`0.78 ± 0.01b
`1.50 ± 0.39
`
`Note: Synergy between paclitaxel and AZT was determined using the
`fixed-concentration method. Cells were treated with paclitaxel solu-
`tions (0–1,000 nM) and fixed concentrations of AZT (0, 1, 5, and 10
`␮M). Drug activity was measured by the SRB assay. The results were
`analyzed for the nature of interaction between paclitaxel and AZT as
`described in Materials and Methods. A combination index of less
`than 1 indicates synergy, and its inverse value indicates the extent of
`synergy. Results at two effect levels, i.e., 50% and 70%, are shown.
`Mean ± SD of three experiments, with six replicates per experiment.
`a IC50 and IC70 values of single agent AZT were 5.82 ± 1.10 ␮M and
`11.22 ± 1.50 ␮M.
`b p < 0.05 compared to 1.
`c Cannot be determined because single-agent AZT produced greater
`than 50% cytotoxicity.
`
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`Synergy between Paclitaxel and AZT in FaDu Cells
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`959
`
`Fig. 1. Cytotoxicity of paclitaxel and/or AZT: results of fixed-
`concentration method. Cells were treated with paclitaxel, with or
`without AZT, for 48 h. For the combination treatment, cells were also
`pretreated with AZT for 24 h. Drug effect was measured as reduction
`of number of FaDu cells attached to growth surface. Note that the
`results have been normalized for the 10, 50, and 70% cytotoxicity
`derived from single agent AZT at 1, 5, and 10 ␮M (i.e., the effect of
`the combination was expressed as a percentage of the value of AZT-
`treated sample). Results of a representative experiment are shown
`(six replicates per experiment). The IC50 values are shown in Table I.
`Symbols: 䊉, paclitaxel alone; 䊊, AZT alone (inset); 䊏, paclitaxel plus
`1 ␮M AZT; 䉱, paclitaxel plus 5 ␮M AZT; 䉲, paclitaxel plus 10 ␮M
`AZT.
`
`the IC50 of paclitaxel was about 10 nM, the major drug effect
`at concentrations below its IC50 was antiproliferation,
`whereas cell kill became more prominent at higher concen-
`trations.
`Treatment with single agent AZT for 72 h resulted in a
`97% maximum cell number reduction at 100 ␮M AZT (Fig. 1,
`inset). The synergy studies used AZT concentrations ranging
`from 0.25 to 100 ␮M. At these AZT concentrations, the cell
`number reduction ranged from <1 to 97%.
`Figure 1 also shows the results of the paclitaxel and AZT
`combinations using the fixed-concentration method. Note
`that the results have been normalized for the cytotoxicity
`derived from single agent AZT (i.e., the effect of the combi-
`nation was expressed as a percentage of the value of the
`AZT-treated sample). Cotreatment with AZT significantly
`increased paclitaxel activity. First, there was an increase in the
`maximum effect, resulting in nearly complete reduction of cell
`number in the combinations containing 5 or 10 ␮M AZT.
`Second, the concentration–effect curve of paclitaxel (after
`corrected for AZT activity) was shifted to the left, and the
`IC50 of paclitaxel was decreased by 1.5- to 9.4-fold (Table I)
`by 1 to 10 ␮M AZT (p < 0.05, Student’s t test).
`
`Cytotoxicity of Paclitaxel and AZT: Results of
`Fixed-Ratio Method
`
`Cells were treated as described for the fixed-
`concentration method, except that paclitaxel and AZT were
`applied at preselected ratios of concentrations (see Methods).
`The results of single agent AZT or paclitaxel, used at the
`same concentrations as in the combinations, are shown for
`comparison (Fig. 2). Consistent with the results of the fixed-
`concentration method, AZT enhanced paclitaxel cytotoxicity
`such that the concentration–effect curves were shifted to the
`left. Furthermore, there was an increase in the maximum ef-
`
`Fig. 2. Cytotoxicity of paclitaxel and/or AZT: results of fixed-ratio
`method. Cells were treated with solutions containing both drugs at 5
`to 320% of their respective initial concentrations (7 nM for paclitaxel
`and 5 ␮M for AZT), which were approximately their respective IC50
`values. The concentrations of paclitaxel to AZT were kept at four
`fixed ratios: 䊉, 80:20; 䊊, 60:40; 䉱, 40:60; 䉭, 20:80.
`
`fect, resulting in nearly complete reduction of cell number in
`all paclitaxel/AZT combinations.
`
`Synergy Determination
`
`Figure 3 shows the combination index analysis of the
`results of the fixed concentration method at the three AZT
`concentrations (1, 5, or 10 ␮M). Table I summarizes the CI
`values and extents of synergy. The CI values were below 1.0,
`indicating synergy between paclitaxel and AZT. The extent of
`synergy (equal to the inverse value of combination index) was
`inversely related to the AZT concentrations and ranged from
`9.0-fold at 1 ␮M to 1.3-fold at 10 ␮M AZT. The results further
`show greater extents of synergy at higher drug effect levels.
`For example, for combinations containing 1 ␮M AZT, the
`extent of synergy was between 1.3- and 1.8-fold at 10 to 50%
`drug effect levels and much greater at higher drug effect lev-
`els (9.0-fold at the 70% effect level). Note that because 10 ␮M
`AZT resulted in greater than 60% cell growth inhibition and
`because paclitaxel, at the highest concentration of 1,000 nM,
`produced ⱕ70% maximum inhibition, the only CI that could
`
`Fig. 3. Analysis of synergy between paclitaxel and AZT: results of
`fixed-concentration method. Results depicted in Fig.1 were analyzed
`by the combination index method as described in Methods. A com-
`bination index of <1 indicates synergy. Symbols: 䊏, paclitaxel plus 1
`␮M AZT; 䉱, paclitaxel plus 5 ␮M AZT; 䉲, paclitaxel plus 10 ␮M
`AZT. Data represent the average of the means from three experi-
`ments. Bar, 1 SD. (A) Relationship between combination index and
`drug effect level. A 50% drug effect level equals to 50% reduction in
`cell number compared to the untreated control. (B) Relationship
`between combination index and AZT concentration, at 70% drug
`effect level.
`
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`be calculated was at the 70% effect level for combinations
`containing 10 ␮M AZT.
`Figure 3B shows a plot of CI values vs. AZT levels ob-
`tained from the fixed concentration method. This result indi-
`cates an inverse relationship between the extent of synergy
`and AZT concentration, with a 9.1-fold synergy at 1 ␮M AZT
`and 1.5-fold synergy at 10 ␮M AZT (Table I).
`Figure 4 and Table II show the results of the combination
`index analysis for the fixed-ratio studies. Consistent with the
`results of the fixed-concentration method, the extent of syn-
`ergy was greater at higher drug effect levels (Fig. 4A). For
`example, the combination indices were indistinguishable from
`1.0 at effect levels ⱕ40% but were significantly less than 1.0 at
`higher effect levels for each of the four ratios examined. Also
`consistent with the fixed-concentration method, the extent of
`synergy increased with the paclitaxel-to-AZT ratios in the
`combinations. The maximal synergy of about 3.3-fold was
`achieved at the 60% effect level with a paclitaxel:AZT ratio
`of 80:20 (Fig. 4B and Table II).
`
`DISCUSSION
`
`Results of the present study indicate a concentration-
`dependent synergy between paclitaxel and AZT. The extent
`of synergy was greatest for combinations containing higher
`paclitaxel concentrations and/or greater paclitaxel-to-AZT
`concentration ratios and increased with the level of drug ef-
`fect. For example, in combinations containing 1 ␮M AZT,
`synergy was 1.3-fold at the 20% effect level and 3.1-fold at the
`60% effect level. These data, because the major antitumor
`activity of paclitaxel was antiproliferation at the 20% effect
`level and cell kill at the 60% effect levels, suggest that AZT
`mainly enhances the cell kill effect of paclitaxel. This is con-
`sistent with our previous finding, established using a different
`method to measure apoptosis (i.e., release of DNA–histone
`complex from the nucleus), that AZT enhances the pacli-
`taxel-induced apoptosis, presumably by increasing the cell
`fraction with damaged telomere (1). There are multiple stud-
`ies suggesting a relationship between telomere instability/
`erosion and apoptosis (8,14–20). For example, apoptosis in
`human HeLa 293 and MW451 cells induced by hydroxyl radi-
`
`Fig. 4. Analysis of synergy between paclitaxel and AZT: results of
`fixed-ratio method. Results depicted in Fig. 1 were analyzed by the
`combination index method as described in Methods. A combination
`index of <1 indicates synergy. Symbols for the different paclitaxel-
`to-AZT concentration ratios are: 䊉, 80:20; 䊊, 60:40; 䉱, 40:60; 䉭,
`20:80. Data represent the average of the means from three experi-
`ments. Bar, 1 SD. (A) Relationship between combination index and
`drug effect level. A 50% drug effect level equals 50% reduction in cell
`number compared to the untreated control. (B) Relationship be-
`tween combination index and the paclitaxel-to-AZT ratios at the
`60% drug effect level.
`
`Table II. Synergy between Paclitaxel and AZT: Results of Fixed
`Ratio Method
`
`Paclitaxel:
`AZT
`ratio
`
`Paclitaxel
`concentration
`(nM)a
`
`AZT
`concentration
`(␮M)b
`
`Combination
`index
`
`Synergy,
`fold
`
`20% effect
`80:20
`60:40
`40:60
`20:80
`50% effect
`80:20
`60:40
`40:60
`20:80
`
`0.84 ± 0.30
`0.94 ± 0.40
`1.12 ± 0.27
`1.20 ± 0.18
`
`5.10 ± 1.67
`3.84 ± 1.30
`2.35 ± 0.78
`1.27 ± 0.45
`
`1.18 ± 0.98
`1.02 ± 0.82
`0.88 ± 0.47
`0.53 ± 0.14
`
`0.91 ± 0.30
`1.83 ± 0.62
`2.51 ± 0.84
`3.62 ± 1.29
`
`0.84 ± 0.30
`0.94 ± 0.40
`1.12 ± 0.27
`1.20 ± 0.18
`
`0.60 ± 0.12c
`0.65 ± 0.12c
`0.64 ± 0.09c
`0.73 ± 0.15c
`
`1.19 ± 0.39
`1.07 ± 0.69
`0.89 ± 0.26
`0.85 ± 0.12
`
`1.70 ± 0.30
`1.57 ± 0.28
`1.59 ± 0.20
`1.41 ± 0.31
`
`Note: Synergy between paclitaxel and AZT in human FaDu cells was
`determined using the fixed-ratio method. The paclitaxel-to-AZT con-
`centration ratios were kept constant for each concentration–response
`curve. Cells were treated with solutions containing both drugs at 5 to
`320% of their respective initial concentrations (7 nM for paclitaxel
`and 5 ␮M for AZT). Drug activity was measured by the SRB assay.
`The results were analyzed for the nature of interaction between
`paclitaxel and AZT as described in Materials and Methods. A com-
`bination index less than 1 indicates synergy, and its inverse value
`indicates the extent of synergy. Results at two effect levels, i.e., 20%
`and 50%, are shown. Mean ± SD of three experiments, with four
`replicates per experiment.
`a IC20 and IC50 values of paclitaxel were 1.46 ± 0.78 nM and 11.6 ±
`1.89 nM, respectively.
`b IC20 and IC50 values of AZT were 2.22 ± 0.92 ␮M and 5.95 ± 1.89
`␮M, respectively.
`c p < 0.05 compared to 1.0 (additivity).
`
`cals showed telomere erosion without caspase activation (21),
`and elongation of telomeres in human fibroblast IDH4 and
`prostate DU145 cells resulted in higher resistance to apopto-
`sis induced by serum depletion (17). Telomere erosion by
`paclitaxel is associated with cell death (21). Further studies
`are needed to determine the mechanisms of telomere erosion
`by paclitaxel and the role of telomere erosion in paclitaxel-
`induced apoptosis.
`The mechanism of the lessened synergy between pacli-
`taxel and AZT at higher AZT concentrations (i.e., >5 ␮M) is
`not clear. AZT has multiple pharmacologic actions including
`inhibition of reverse transcriptase, the reverse transcriptase
`component of human telomerase, integrase, DNA polymer-
`ase ␥, and thymidine kinase and is preferentially incorporated
`into telomeric DNA and Z-DNA-containing regions of Chi-
`nese hamster ovary cells (22–27). AZT at concentrations
`above 3 ␮M increased the percentage of cells in S-phase and
`prolonged the cell doubling time (28–30). We previously
`showed, using fluorescence in situ hybridization analysis, that
`paclitaxel selectively shortened telomeres in M-phase cells
`but not interphase cells (1). Hence, blockade of cells in the
`S-phase at high AZT concentrations may diminish the telo-
`mere erosion effect of paclitaxel and diminishes the synergy.
`Further studies are needed to test this hypothesis.
`In summary, the present study demonstrates a synergistic
`interaction between paclitaxel and AZT and supports the use
`of a combination of a low and nontoxic AZT dose and a
`therapeutically active dose of paclitaxel.
`
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`
`ACKNOWLEDGMENTS
`
`This study was supported in part by a research grant
`R01CA77091 from the National Cancer Institute, NIH,
`DHHS.
`
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