INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3)POTENTIATES PACLITAXEL-INDUCED APOPTOSIS IN HUMAN BREASTCANCER CELLSC.A. FOWLER*, C.M. PERKS, P.V. NEWCOMB, P.B. SAVAGE, J.R. FARNDON and J.M.P. HOLLY

Division of Surgery, Department of Hospital Medicine, Bristol Royal Infirmary, Bristol, United Kingdom

Variability in response to chemotherapy is poorly under-stood. Paclitaxel-induced apoptosis was assessed in humanHs578T breast cancer cells, using the MTT assay, cell count-ing, morphological features and flow cytometry. Pre-dosingcells with non-glycosylated insulin-like growth factor bindingprotein-3 (ngIGFBP-3) had no effect on the cells per se butaccentuated paclitaxel-induced apoptosis. The apoptoticpathway was further examined by measuring caspase-3 ac-tivity in cell lysates at time points over 48 hr after dosing withpaclitaxel. Activity increased significantly, and Western im-munoblots for caspase-3 in conditioned media showed thatthe inactive precursor decreased after incubation with pacli-taxel. Endogenous production of IGFBP-3 by the cells afterincubation with paclitaxel was evaluated using Western li-gand blotting, specific IGFBP-3 immunoblotting and radio-immunoassay. Paclitaxel increased endogenous IGFBP-3,which was further increased if the cells had been pre-dosedwith ngIGFBP-3. These findings suggest that IGFBP-3 may bean important modulator of paclitaxel-induced apoptosis. Int.J. Cancer 88:448–453, 2000.© 2000 Wiley-Liss, Inc.

The response to chemotherapy varies depending on the tumourand host characteristics. The reasons for this are not always clear.Understanding what controls sensitivity to drug action is importantif we are to unravel these variable responses and subsequentlyimprove treatment and prognosis.

The ability to induce programmed cell death, or apoptosis, intumour cells is crucial to the success of many treatments. Apopto-sis can be initiated in response to a range of physiological triggersactivating different signalling pathways. Central to apoptotic path-ways is a family of proteases, the caspases. They are present asinactive pro-enzymes until a signal initiates activation, starting acascade event. They mediate highly specific proteolytic cleavageevents that degrade intracellular proteins vital for cell survival,resulting in apoptosis (Enariet al., 1996).

Cytotoxic drugs (Meynet al.,1994) and radiation (Meynet al.,1993) can initiate apoptosis, hence, their use in the treatment ofcancer. Paclitaxel is a cytotoxic drug, derived from the yew tree,which affects spindle formation (Rowinskyet al.,1992). It causesapoptosis in a number of cell lines and is thought to actvia tumournecrosis factor-a (TNF-a) and subsequent ceramide generation(Bogdan and Ding, 1992; Kimet al., 1991). Ceramide acts as asecond messenger in one signalling pathway that activates apopto-sis. This action can be mimicked using synthetic ceramide ana-logues. The amount of apoptosis initiated by ceramide can besignificantly increased by the addition of non-glycosylated insulin-like growth factor binding protein-3 (ngIGFBP-3) in an IGF-independent manner (Gillet al., 1997).

The IGFBPs bind specifically to the IGFs (IGF-I and IGF-II)and alter their availability and action. IGFs are potent mitogens fora number of different cell types, including human breast cancercells (Quinnet al., 1996). The main carrier of the IGFs in humanserum is IGFBP-3. Interest in IGFBP-3 has grown since it has beensuggested that IGFBP-3 may also have an inhibitory role indepen-dent of IGF-I. IGFBP-3 fragments bind to chick embryo fibroblastcell surface and inhibit cell growth (Lalouet al., 1996). Trans-forming growth factor-b (TGF-b) causes cell growth inhibition inhuman breast cancer cells, and this effect is mediated by enhancedexpression of IGFBP-3 (Ohet al.,1995). IGFBP-3 inhibits mono-

layer growth in Hs578T human breast cancer cells (Ohet al.,1993a), and this action may be through specific IGFBP-3 bindingsites on the cell surface (Ohet al., 1993b).

The Hs578T breast cancer line is ideal for studying the IGF-independent effects of IGFBP-3 as it lacks a functional IGFreceptor and is therefore non-responsive to the IGFs in terms ofproliferation and cell survival (De Leonet al., 1992). We haveexamined whether IGFBP-3 can alter paclitaxel-induced activationof caspase-3 and apoptosis in Hs578T cells.

MATERIAL AND METHODS

Recombinant human ngIGFBP-3 was a kind gift from Dr C.Maack (Celtrix, Palo Alto, CA). Caspase-3 antibody was pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA). Pacli-taxel and all other chemicals were purchased from Sigma (Poole,UK). Tissue culture plastics were obtained from Nunc–GIBCOLife Technologies (Paisley, UK).

Cell culturesThe human breast cancer cell line Hs578T was purchased from

ECACC (Porton Down, UK) and grown in a humidified 5% CO2atmosphere at 37°C. Cells were maintained in DMEM with glu-tamax-1 supplemented with 10% FCS, penicillin (5,000 IU/ml)and streptomycin (5 mg/ml), referred to hereafter as growth media(GM). Experiments were performed on cells in serum-free HEPESDMEM and Ham’s nutrient mix F-12 with sodium bicarbonate(0.12%) (SFM), BSA (0.2 mg/ml) and transferrin (0.01 mg/ml)and supplemented as before.

MTT assayThe MTT assay was used as a crude measure of cell viability.

MTT is a yellow tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which is converted by metaboli-cally active cells into a coloured water-insoluble formazan salt.Cells were seeded at 53 104/ml (150 ml GM/well) in 96-wellplates and grown for 24 hr. GM was replaced with SFM, BSA (0.2mg/ml) and transferrin (0.01 mg/ml) (100ml/well) for 24 hr priorto dosing. Dosing was as described under Results for appropriateincubation times. Following this, MTT reagent (7.5 mg/ml in PBS)was added to the cells (10ml/well) and the cultures were incubatedfor 30 min at 37°C. The reaction was stopped by addition ofacidified triton buffer [0.1 M HCl, 10 % (v/v) Triton X-100; 50ml/well], and the tetrazolium crystals were dissolved by mixing ona Titertek plate shaker for 20 min at room temperature. Sampleswere measured on a Bio-Rad (Richmond, CA) 450 plate reader ata test wavelength of 595 nm and a reference wavelength of 650nm. Results represent the mean6 SEM of 5 wells from 1 exper-iment that was representative of experiments repeated at least 3times.

*Correspondence to: Division of Surgery, Department of Hospital Med-icine, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK. Fax:101179282736. E-mail: ClareAFowler@cs.com

Received 13 July 1999; Revised 22 March 2000; Accepted 19 April2000

Int. J. Cancer:88, 448–453 (2000)© 2000 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

Flow cytometryFlow cytometry was used to quantify the amount of apoptosis in

any given sample. In fixed apoptotic cells, fragmented DNA waswashed out, leaving a lower DNA content, and the cells, therefore,appeared as a pre-G1 peak on a DNA cell cycle histogram. Cellswere seeded at 0.43 106/T25 flask and grown in GM for 24 hr.Cells were dosed in triplicate as described in Results. Floating cellsfrom the supernatant and the PBS wash were collected and addedto the trypsinised cells from each flask. Cell viability was mea-sured in 50ml aliquots of cells added to 50ml Trypan blue andloaded onto a haemocytometer, and the percentage of dead cellsper sample was calculated. Each sample of cells was fixed in 70%ethanol for a minimum of 24 hr prior to analysis by flow cytom-etry. Fixed cells were pelleted (5000g for 5 min) and washed 3times with PBS (5000g for 5 min). Supernatant was removed, andcells were resuspended in reaction buffer (propidium iodide, 0.05mg/ml; sodium citrate, 0.1%; RNase A, 0.02 mg/ml; NP-40, 0.3%;pH 8.3) and incubated at 4°C for 30 min prior to measurement ona FACSCalibur flow cytometer (Becton Dickinson, Crowley, UK)by an argon laser at 488 nm for excitation. Data were analysedusing Cell Quest software (Becton Dickinson, Crowley, Oxford).

Morphological assessmentTo assess whether paclitaxel-induced cell death showed the

classical morphological features associated with apoptosis, fixedand stained cell aliquots were examined under oil immersion at amagnification of3100.

Caspase-3 activity measurementA caspase-3 assay kit (PharMingen, Crowley, UK) was used to

measure activity. Active caspase-3 cleaves the fluorogenic sub-strate Ac-DEVD-AMC after the aspartic acid (D) and before theAMC group. AMC becomes fluorescent when it is cleaved fromthe peptide by active caspase-3. In brief, cells were seeded at 0.43106/T25 flask and grown in GM for 24 hr. Cells were dosed intriplicate as described in Results. Cell lysates were prepared atvarious time points and frozen at –20°C. In a 96-well plate, 200mlof reaction buffer (HEPES), 5ml of reconstituted caspase-3 flu-orogenic substrate (Ac-DEVD-AMC) and 50ml of cell lysate wereadded to each well. The reaction mixture was incubated at 37°C for1 hr. Fluorescence was measured using a spectrofluorometer (Flu-orolite 1000; Dynex, Billinghurst, UK).

Western ligand blotting and immunoblottingConditioned medium was collected from the T25 flasks used for

flow cytometry and cell viability, after the cells had been spun out.The remaining solution was concentrated 10-fold using Millipore(Watford, UK) Ultrafree-MC filter units. The pattern of IGFBPssecreted by the cells in response to various treatments and subse-quent immunoblotting of the membranes for IGFBP-3 was deter-mined using the methods described previously (Coulsonet al.,1991; Hugheset al., 1995). Briefly, samples were separated by12% SDS-PAGE and the proteins then transferred onto a nylonmembrane and probed with a mixture of [125I]IGF-I and [125I]IGF-II. The membrane was washed, and bands were revealed byautoradiography. For immunoblotting, nylon membranes were in-cubated with a specific polyclonal antibody for IGFBP-3 (SCH-2/6at 1:15,000) overnight at room temperature. Following the removalof excess unbound antibody, an anti-rabbit antibody (1:10,000) forIGFBP-3 conjugated to peroxidase was added for 1 hr. Binding ofthe peroxidase was visualised using enhanced chemiluminescenceaccording to the manufacturer’s instructions (Amersham, Ayles-bury, UK).

To assess caspase-3, cells were grown in T25 and treated asabove. Cells were lysed on ice for 10 min (1 ml; 10 mM Tris-HCl,5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50mM sodium fluoride, 100mM sodium orthovanadate, 1% Triton, 1mM phenylmethylsulphonyl fluoride; pH 7.6). A protein assay wasthen performed on the lysates for normalisation of protein levelsbetween samples. Proteins were separated, and immunoblotting

was performed as above, using a specific polyclonal antibody forcaspase-3 (1:133) overnight and an anti-goat antibody (1:4,000)for caspase-3 detection.

RadioimmunoassayLevels of IGFBP-3 in conditioned media were measured using

radioimmunoassay as previously described (Yatemanet al.,1993).

Statistical analysisData were analysed using the Microsoft (Redmond, WA) Excel

version 5.0 software package. Significant effects were determinedusing Student’st-test. A statistically significant difference wasconsidered to be present atp , 0.05.

RESULTS

Effect of paclitaxel on Hs578T cellsInitially, the effect of paclitaxel on Hs578T cells was investi-

gated using the MTT assay, which measured the percentage ofmetabolically active cells remaining after incubation with pacli-taxel (25 to 1,000 nM) after 24 and 48 hr. There was a dose-dependent decrease in metabolically active cells seen more clearlyat 48 hr (Fig. 1a). Trypan blue was used to establish whether thisdecrease in metabolic activity was due to cell death. A dose-dependent increase in the percentage of dead cells was found (Fig.1b); i.e., cells treated with 500 nM paclitaxel showed a 5.5-foldincrease in cell death when compared with controls (p , 0.01).

Flow cytometry showed a dose-dependent increase in the per-centage of cells in the pre-G1 phase (Fig. 1c). Even the lowest doseof paclitaxel (25 nM) caused a significant increase (p , 0.01) inthe percentage of apoptotic cells when compared with untreatedcells.

Figure 2a shows an untreated cell and Figure 2b,c shows cellstreated with paclitaxel (75 nM), which demonstrate the classicalfeatures of apoptosis including plasma membrane blebbing, cyto-plasmic condensation, nuclear fragmentation and cell fragmenta-tion into apoptotic bodies.

IGFBP-3 accentuates the effect of paclitaxel on Hs578T cellsSince ngIGFBP-3 accentuates the apoptotic effect of ceramide

on Hs578T cells (Gillet al.,1997), it was necessary to see whetherngIGFBP-3 could interact with the effect of paclitaxel on thesecells. Cells were pre-incubated with ngIGFBP-3 (500 ng/ml) for 24hr before dosing the cells with paclitaxel (75 nM) and ngIGFBP-3for 48 hr. The percentage of dead cells was assessed using Trypanblue uptake (Fig. 3a). ngIGFBP-3 did not have an effect, aspreviously observed (Ohet al.,1993a), but the effect of paclitaxelwas significantly enhanced when the cells were pre-incubated withngIGFBP-3 (p , 0.05). Flow cytometry confirmed that the accen-tuation of cell death by ngIGFBP-3 was associated with a specificincrease in apoptosis (Fig. 3b).

Caspase-3 activity is increased by paclitaxelCell lysates were prepared from Hs578T cells untreated or

treated with paclitaxel (75 nM). Lysates were collected at set timepoints from 30 min to 48 hr after dosing. Figure 4a shows asignificant increase in caspase-3 activity in treated cells comparedwith controls (p , 0.01) at 48 hr. To examine whether this increasein caspase-3 activity was associated with a loss of caspase-3pro-enzyme (32K), Western immunoblotting of lysates was per-formed (Fig. 4b). There was a significant decrease in the caspase-3pro-enzyme in paclitaxel-treated cells (p , 0.001) and, interest-ingly, a small (1.2-fold) increase when cells were incubated withngIGFBP-3 alone. When pre-incubation with ngIGFBP-3 wasfollowed by dosing with paclitaxel, there was again a loss ofcaspase-3 pro-enzyme compared with ngIGFBP-3 alone (Fig. 4c).

Hs578T cells treated with paclitaxel releaseendogenous IGFBP-3

The effect of paclitaxel on levels of endogenously producedIGFBP-3 was studied (Fig. 5a). Hs578T cells secreted IGFBP-3,

449IGFBP-3 ALTERS PACLITAXEL-INDUCED APOPTOSIS

shown as a band at 46 kDa (Fig. 5, lane a). Treatment withpaclitaxel (25 to 500 nM) increased IGFBP-3 levels in conditionedmedia (Fig. 5, lanes b–d). To confirm that the protein seen on theligand blot was IGFBP-3, a Western immunoblot for IGFBP-3 wasperformed. Figure 5b is a representative immunoblot showing anincrease in endogenous IGFBP-3 when cells were treated with

paclitaxel compared with untreated cells; no proteolysis was ob-served. Endogenous production of IGFBP-3 was also assessed byradioimmunoassay. Figure 5c shows a significant increase (p,0.01) in the amount of IGFBP-3, from 10.8 ng/ml in the condi-tioned media of control cells to 17.9 ng/ml in cells treated with 25nM paclitaxel; this does not appear to be dose-related.

FIGURE 1 – Effect of paclitaxel on Hs578T cells.(a) Effect ofincreasing doses of paclitaxel (25 to 1,000 nM) on the metabolicactivity of Hs578T cells after 24 (solid squares) or 48 (open squares)hr; in whichp , 0.05 to 0.001 at doses of 100 to 1,000 nM paclitaxelat 24 hr andp , 0.01 to 0.001 at 75 to 1,000 nM paclitaxel at 48 hr.MTT activity was assayed as described in Material and Methods.Results represent the mean6 SEM of 5 wells from 1 experiment thatwas representative of experiments repeated at least 3 times.(b) Pac-litaxel causes dose-dependent cell death.(c) Paclitaxel induces adose-dependent increase in apoptosis. Cells incubated with paclitaxelfor 48 hr were prepared for flow cytometry as described in Materialand Methods. The graph shows the percentage of cells in the pre-G1phase of the cell cycle for each dose of paclitaxel (25, 75 and 500 nM).Results represent the mean6 SEM for each paclitaxel dose performedin triplicate at the same time and are representative of experimentsrepeated at least 3 times (*p , 0.05, **p , 0.01, ***p , 0.001).

FIGURE 2 – Morphological features of apoptosis in Hs578T cellstreated with paclitaxel. Hs578T cells either untreated(a) or treated(b,c) with 75 nM paclitaxel were cytospun and treated with Wright’sstain, which stains the nuclei dark blue and the cytoplasm orange.Untreated cells(a) exhibit a large nucleus and faintly stained cyto-plasm. Treated cells show the classical features of apoptosis seeninitially (b) and more markedly(c).

450 FOWLER ET AL.

Cells were then pre-incubated with ngIGFBP-3 (500 ng/ml) for24 hr before treatment with paclitaxel (75 nM), to test whether theobserved increase in apoptosis was accompanied by an increase inendogenously produced IGFBP-3. Figure 6a shows a representa-tive Western immunoblot. Cells treated with paclitaxel (lane b)showed an increase in endogenous IGFBP-3 compared with un-treated cells (lane a). Cells pre-incubated with ngIGFBP-3 (lane d)showed a further increase in endogenous IGFBP-3, again with noproteolysis. These observations were confirmed by the radioim-munoassay results (Fig. 6b).

DISCUSSION

The IGFs are important mitogenic polypeptides that are in-volved in normal and malignant cellular proliferation. The role ofthe IGF-I receptor and its ligands, IGF-I and IGF-II, in modulatingapoptosis has been demonstrated in a variety of experimentalmodels. The IGFs protect cells from apoptosis, and cell lines thatover-express the IGF-I receptor are less susceptible to apoptosis(Resnicoffet al.,1995). In patients with breast cancer, circulatingIGF-I levels are elevated (Peyratet al.,1993) and the stromal cellssurrounding the tumour express IGF-I (Peyrat and Bonneterre,

1992), suggesting that IGF-I may modulate apoptosis in breastcancer cells.

The IGFBPs are a critical component of the IGF system. Itwas first thought that they were present primarily as carrierproteins for the IGFs, modulating availability and regulatingbiological action. Evidence now suggests that they also havetheir own inherent actions independent of the IGFs. In Chinesehamster ovary cells, IGFBP-1 specifically binds to thea5b1integrin receptor, where, independently of IGF, it stimulatescell migration (Joneset al.,1993). The growth-inhibitory effectof TGF-b is associated with a rise in endogenous IGFBP-3,which can be blocked with an IGFBP-3 anti-sense oligode-oxynucleotide (Ohet al., 1995). Andress and Birnbaum (1992)showed that osteoblast-derived IGFBP-5 stimulated mitogene-sis in an IGF-independent manner.

FIGURE 3 – Pre-incubation of Hs578T cells with ngIGFBP-3 beforedosing with paclitaxel enhanced cell death(a) and accentuated apo-ptosis(b). Cells were plated in T25 flasks and allowed to settle for 24hr before changing to SFM for 24 hr.(a) Percentage of dead cells aftereach treatment assessed by Trypan blue counting.(b) Percentage ofcells in the pre-G1 phase of the cell cycle, as measured by flowcytometry. Results represent the mean6 SEM of 3 experiments, eachdone in triplicate (*p , 0.05, ***p , 0.001).

FIGURE 4 – (a) Cell lysates of Hs578T cells show an increase incaspase-3 activity when treated with paclitaxel (75 nM). Cells lysateswere prepared from untreated cells (solid squares) and treated cells(open squares) after dosing with paclitaxel from 30 min to 48 hr.Lysates were plated in 96-well plates, and caspase-3 activity wasmeasured as described under Material and Methods. Results representthe mean6 SEM of each treatment in triplicate simultaneously and arerepresentative of experiments repeated at least 3 times (**p , 0.01).(b) Cell lysates of Hs578T cells show a marked decrease in inactivecaspase-3 precursor when treated with paclitaxel. Cell lysates fromuntreated cells (lane a) and cells treated for 48 hr with 75 nM paclitaxel(lane b) or 500 ng/ml ngIGFBP-3 alone (lane c) or cells pre-incubatedwith ngIGFBP-3 for 24 hr before dosing with paclitaxel for 48 hr (laned) were collected and samples separated by gel electrophoresis. Mem-branes were prepared and probed as described in Material and Meth-ods, to assess the presence of inactive caspase-3 precursor (32K); arepresentative immunoblot is shown.(c) Mean6 SEM optical densityreadings from immunoblots performed at least 3 times (***p , 0.001).

451IGFBP-3 ALTERS PACLITAXEL-INDUCED APOPTOSIS

Pivotal to the normal development of tissues and organs is thehomeostatic mechanism apoptosis. It is a tightly regulated processwith characteristic biochemical and morphological features occur-ring in a wide variety of tissues (Kerret al., 1972). Apoptosis isinduced in response to both physiological (e.g., post-lactationalbreast) and pathological stimuli. Many factors modulate apoptosis,including extracellular signals such as growth factors and intracel-lular signals such as p53, Bcl-2 and Bax. Many cytotoxic agentsused in cancer therapy can induce apoptosis in tumours and normaltissues, and this correlates with the efficacy of the agents (Milasetal., 1994). If apoptosis could be increased in the tumours, it maybe possible to enhance the efficacy of such cytotoxic agents. Drugresistance of tumours, however, may be due to insensitivity to theinduction of apoptosis.

Our study shows that the cytotoxic agent paclitaxel inducesapoptosis of Hs578T human breast cancer cells, as seen morpho-logically and quantified using flow cytometry. Pre-incubating thecells with ngIGFBP-3 before addition of paclitaxel caused a sig-nificant increase in the amount of apoptosis, while ngIGFBP-3alone had no effect. As the Hs578T cells are non-responsive to theIGFs, we conclude that ngIGFBP-3 interacts with the apoptoticpathway initiated by paclitaxel in an IGF-independent manner. Wehave also shown that associated with paclitaxel-induced apoptosisthere is an increase in the endogenous production of IGFBP-3 seenon Western ligand blots and confirmed with IGFBP-3 immuno-blotting and radioimmunoassay. When the cells were pre-incu-

bated with ngIGFBP-3, further enhancement of endogenous IG-FBP-3 production was observed. We speculate that since IGFBP-3accentuates paclitaxel-induced apoptosis, it may prime the cells topotentiate the apoptotic trigger.

Gill et al.(1997) showed that ngIGFBP-3 enhanced the effect ofa synthetic ceramide analogue. Paclitaxel has been reported to actvia TNF-a, which is associated with endogenous production ofceramide, and these results are consistent with IGFBP-3 interact-ing with this pathway. Once the signalling pathway is activated,the caspases play an important role in apoptosis and caspase 3activation is seen. This activation is concomitant with a loss of thecaspase-3 pro-enzyme (32K). When cells are incubated with ngIG-FBP-3 alone, there is a small increase in the pro-enzyme, the exactimplications of which are not known; however, this may be, asabove, ngIGFBP-3 priming the cells and decreasing their thresholdfor subsequent apoptotic triggers.

IGF and IGFBP-3 are the most prevalent growth factor andbinding protein, respectively, in plasma, and the balance betweenthem may be important in maintaining tissue homeostasis. Epide-miological evidence supports this view as high IGF-I levels havebeen associated with a 7-fold increased risk of breast cancer whenadjusted for serum levels of IGFBP-3 (Hankinsonet al., 1998).Shifting the balance may have important consequences on diseaseprocesses; likewise, it may alter the efficacy of certain drug treat-ments. Paclitaxel is effective in the treatment of some tumours; thismay in part be due to its ability to alter endogenous levels ofIGFBP-3. It mediates the growth-inhibitory effects of many dif-ferent anti-proliferative agents. If the endogenous IGFBP-3 sub-sequently increases the amount of caspase-3 pro-enzyme, this maybe associated with increased apoptosis. Alternatively, altering theamount of IGFBP-3 may shift the IGF–IGFBP-3 balance, whichmay in turn affect the efficacy of paclitaxel. These observationscould have important implications for future cancer treatments.

FIGURE 5 – Conditioned media from Hs578T cells treated with pac-litaxel shows increased endogenous IGFBP-3:(a) ligand blot, (b)Western immunoblot,(c) radioimmunoassay. Conditioned mediumfrom untreated cells and cells treated for 48 hr with paclitaxel (25, 75and 500 nM) was concentrated, and samples were separated by gelelectrophoresis. Membranes were prepared and probed as described inMaterial and Methods, to assess the production of endogenous IG-FBP-3. (a,b) Representative ligand and Western blots, respectively:lane a, untreated cells; lane b, 25 nM paclitaxel; lane c, 75 nMpaclitaxel; lane d, 500 nM paclitaxel.(c) Amount of endogenousIGFBP-3 as measured by radioimmunoassay in the conditioned me-dium of untreated and treated cells. Results are means6 SEM of 6readings from 2 experiments (**p , 0.01).

FIGURE 6 – Conditioned media from Hs578T cells pre-incubatedwith ngIGFBP-3 before treatment with paclitaxel show increasedendogenous IGFBP-3:(a) Western immunoblot,(b) radioimmunoas-say. Conditioned medium from untreated cells (lane a), cells treatedwith 75 nM paclitaxel (lane b) or 500 ng/ml ngIGFBP-3 (lane c) aloneand from cells pre-incubated with ngIGFBP-3 for 24 hr before dosingwith paclitaxel for 48 hr (lane d) was concentrated and separated by gelelectrophoresis as described in Material and Methods.(a) Represen-tative immunoblot.(b) Amount of endogenous IGFBP-3 as measuredby radioimmunoassay in the conditioned medium of untreated andtreated cells. Results are means6 SEM of 6 readings from 2 exper-iments (*p , 0.05, **p , 0.01).

452 FOWLER ET AL.

REFERENCES

ANDRESS, D.L. and BIRNBAUM, R.S., Human osteoblast-derived insulin-likegrowth factor (IGF) binding protein-5 stimulates osteoblast mitogenesisand potentiates IGF action.J. biol. Chem.,267,22467–22472 (1992).BOGDAN, C. and DING, A., Taxol, a microtubule-stabilising antineoplasticagent, induces expression of tumor necrosis factor alpha and interleukin-1in macrophages.J. Leukocyte Biol.,52, 119–121 (1992).COULSON, V.J., WASS, J.A., ABDULLA , A.F., COTERILL, A.M. and HOLLY,J.M., Insulin-like growth factor binding proteins (IGFBPs) in acromegaly.Growth Regul.,1, 119–124 (1991).DE LEON, D.D., WILSON, D.M., POWERS, M. and ROSENFELD, R.G., Effectsof insulin-like growth factors (IGFs) and IGF receptor antibodies on theproliferation of human breast cancer cells.Growth Factors,6, 327–336(1992).ENARI, M., TALANIAN , R.V., WONG, W.W. and NAGATA, S., Sequentialactivation of ICE-like and CPP32-like proteases during Fas-mediated ap-optosis.Nature (Lond.),380,723–726 (1996).GILL , Z.P., PERKS, C.M., NEWCOMB, P.V. and HOLLY, J.M., Insulin-likegrowth factor-binding protein (IGFBP-3) predisposes breast cancer cells toprogrammed cell death in a non-IGF-dependent manner.J. biol. Chem.,272,25602–25607 (1997).HANKINSON, S.E., WILLETT, W.C., COLDITZ, G.A., HUNTER, D.J., MICHAUD,D.S., DEROO, B., ROSNER, B., SPEIZER, F.E. and POLLAK , M., Circulatingconcentrations of insulin-like growth factor-I and risk of breast cancer [seecomments].Lancet,351,1393–1396 (1998).HUGHES, S.C., JOHNSON, M.R., HEINRICH, G. and HOLLY, J.M., Couldabnormalities in insulin-like growth factors and their binding proteinsduring pregnancy result in gestational diabetes?J. Endocrinol.,147,517–524 (1995).JONES, J.I., GOCKERMAN, A., BUSBY, W.H., JR., WRIGHT, G. and CLEM-MONS, D.R., Insulin-like growth factor binding protein 1 stimulates cellmigration and binds to the alpha 5 beta 1 integrin by means of itsArg-Gly-Asp sequence.Proc. nat. Acad. Sci. (Wash.),90, 10553–10557(1993).KERR, J.F., WYLLIE , A.H. and CURRIE, A.R., Apoptosis: a basic biologicalphenomenon with wide-ranging implications in tissue kinetics.Brit. J.Cancer,26, 239–257 (1972).KIM, M.Y., LINARDIC, C., OBEID, L. and HANNUN, Y., Identification ofsphingomyelin turnover as an effector mechanism for the action of tumornecrosis factor alpha and gamma-interferon. Specific role in cell differen-tiation. J. biol. Chem.,266,484–489 (1991).LALOU, C., LASSARRE, C. and BINOUX, M., A proteolytic fragment ofinsulin-like growth factor (IGF) binding protein-3 that fails to bind IGFsinhibits the mitogenic effects of IGF- I and insulin.Endocrinology,137,3206–3212 (1996).

MEYN, R.E., STEPHENS, L.C., ANG, K.K., HUNTER, N.R., BROCK, W.A.,MILAS, L. and PETERS, L.J., Heterogeneity in the development of apoptosisin irradiated murine tumours of different histologies.Int. J. radiat. Biol.,64, 583–591 (1993).

MEYN, R.E., STEPHENS, L.C., HUNTER, N.R. and MILAS, L., Induction ofapoptosis in murine tumors by cyclophosphamide.Cancer Chemother.Pharmacol.,33, 410–414 (1994).

MILAS, L., STEPHENS, L.C. and MEYN, R.E., Relation of apoptosis to cancertherapy.In Vivo, 8, 665–673 (1994).

OH, Y., MULLER, H.L., LAMSON, G. and ROSENFELD, R.G., Insulin-likegrowth factor (IGF)-independent action of IGF-binding protein-3 inHs578T human breast cancer cells. Cell surface binding and growth inhi-bition. J. biol. Chem.,268,14964–14971 (1993a).

OH, Y., MULLER, H.L., NG, L. and ROSENFELD, R.G., Transforming growthfactor-beta–induced cell growth inhibition in human breast cancer cells ismediated through insulin-like growth factor-binding protein-3 action.J.biol. Chem.,270,13589–13592 (1995).

OH, Y., MULLER, H.L., PHAM, H. and ROSENFELD, R.G., Demonstration ofreceptors for insulin-like growth factor binding protein-3 on Hs578Thuman breast cancer cells.J. biol. Chem.,268,26045–26048 (1993b).

PEYRAT, J.P. and BONNETERRE, J., Type 1 IGF receptor in human breastdiseases.Breast Cancer Res. Treat.,22, 59–67 (1992).

PEYRAT, J.P., BONNETERRE, J., HECQUET, B., VENNIN, P., LOUCHEZ, M.M.,FOURNIER, C., LEFEBVRE, J. and DEMAILLE , A., Plasma insulin-like growthfactor-1 (IGF-1) concentrations in human breast cancer.Europ. J. Cancer,4, 492–497 (1993).

QUINN, K.A., TRESTON, A.M., UNSWORTH, E.J., MILLER, M.J., VOS, M.,GRIMLEY, C., BATTEY, J., MULSHINE, J.L. and CUTTITTA, F., Insulin-likegrowth factor expression in human cancer cell lines.J. biol. Chem.,271,11477–11483 (1996).

RESNICOFF, M., ABRAHAM, D., YUTANAWIBOONCHAI, W., ROTMAN, H.L.,KAJSTURA, J., RUBIN, R., ZOLTICK, P. and BASERGA, R., The insulin-likegrowth factor I receptor protects tumor cells from apoptosisin vivo. CancerRes.,55, 2463–2469 (1995).

ROWINSKY, E.K., ONETTO, N., CANETTA, R.M. and ARBUCK, S.G., Taxol:the first of the taxanes, an important new class of antitumor agents.Semin.Oncol.,19, 646–662 (1992).

YATEMAN, M.E., CLAFFEY, D.C., CWYFAN HUGHES, S.C., FROST, V.J.,WASS, J.A. and HOLLY, J.M., Cytokines modulate the sensitivity of humanfibroblasts to stimulation with insulin-like growth factor-I (IGF-I) byaltering endogenous IGF-binding protein production.J. Endocrinol.,137,151–159 (1993).

453IGFBP-3 ALTERS PACLITAXEL-INDUCED APOPTOSIS