The effects of human chorionic gonadotrophin (hCG) and its subunits on in vitro bladder cancer cell growth have been assessed using the a tetrazolium salt reduction assay (MTT). Intact hCG, alpha-hCG and beta-core hCG all had no effect on cell growth, while beta-hCG increased MTT reduction in all four bladder cancer lines tested. The magnitude of beta-hCG stimulation was maximal in the T24 line, which does not itself produce beta-hCG and appeared to be correspondingly lower in beta-hCG-secreting lines. The addition of antibodies to beta-hCG inhibited MTT reduction among high secretors but failed to inhibit MTT reduction in non-beta-hCG producers. These results are consistent with the poor prognosis associated with beta-hCG expression by bladder tumours in vivo and suggest an autocrine/paracrine stimulation of tumour growth by endogenously produced beta-hCG

Chard, T.

Gillott, D.

Iles, R.

English

Middlesex University Research Repository

British Journal of Cancer (1996) 73, 323-326© 1996 Stockton Press All rights reserved 0007-0920/96 $12.00 %The effects of beta-human chorionic gonadotrophin on the in vitro growth ofbladder cancer cell linesDJ Gillott, RK Iles and T ChardAcademic Unit of Reproductive Physiology, Obstetrics and Gynaecology, St. Bartholomew's Hospital, West Smithfield, LondonECIA 7BE, UK.Summary The effects of human chorionic gonadotrophin (hCG) and its subunits on in vitro bladder cancercell growth have been assessed using the a tetrazolium salt reduction assay (MTT). Intact hCG, a-hCG and f,-core hCG all had no effect on cell growth, while fl-hCG increased MTT reduction in all four bladder cancerlines tested. The magnitude of ,B-hCG stimulation was maximal in the T24 line, which does not itself produceP-hCG and appeared to be correspondingly lower in ,B-hCG-secreting lines. The addition of antibodies to f,-hCG inhibited MTT reduction among high secretors but failed to inhibit MTT reduction in non-fl-hCGproducers. These results are consistent with the poor prognosis associated with ,B-hCG expression by bladdertumours in vivo and suggest an autocrine/paracrine stimulation of tumour growth by endogenously produced f,-hCG.Keywords: beta-human chorionic gonadotrophin; cysteine knot; autocrine/paracrine; growth factor; bladdercancer; MTT assayHuman chorionic gonadotrophin (hCG) is a member of thefamily of glycoprotein hormones, including follicle-stimula-tion hormone (FSH), luteinising hormone (LH) and thyroid-stimulating hormone (TSH), all of which are heterodimericand share a common a-subunit. Each hormone has a uniqueP-subunit, although the f-chain of hCG exhibits 81%homology with that of LH and may have arisen from theLH gene following duplication and readthrough in the 3'direction (Fiddes and Talmadge, 1984). In primates andequids, CG originates from the conceptus and rescues thecorpus luteum by binding to an LH receptor (Yoshimi et al.,1969; Braunstein et al., 1976; Bolton et al., 1980). The lone f-subunit cannot bind to the LH receptor, the intactheterodimer being required for both binding and activation(Pierce and Parsons, 1981).The ectopic production of fl-hCG by a proportion ofbladder cancers has been reported by several authors(Rodenburg et al., 1985; Dexeus et al., 1986, Iles et al.,1987) and cannot be accounted for simply by geneduplication or rearrangement. Consequently, it is likely thatthe control mechanisms governing fl-hCG expression areabnormal in secreting tumours (Iles et al., 1988). As a clinicalmarker fl-hCG may have some value for monitoring intherapy (Marcillac et al., 1993); high levels have beenassociated with both radio-resistance and metastases(Martin et al., 1989), but current opinion holds that thesecretion is an epiphenomenon of little clinical significance(Jacobsen et al., 1990; Smith et al., 1994). However, a recentstudy carried out in this unit showed that 50% of T2/T3patients had elevated urinary fl-hCG (>3.74 IU mmol- 'creatinine), and 90% of these went on to develop metastases,while only 3% of the non-expressing group developedmetastatic disease. In addition, survival curves for thesepatients, when divided into P-hCG expressors and non-expressors, show approximately 10% and 60% survivalrespectively at 17 months, suggesting a valuable prognosticfunction (Iles et al., 1996).We have also shown that primary cultures of normalurothelium can secrete fl-hCG (Iles et al., 1990). TheCorrespondence: RK Iles, Williamson Laboratory for MolecularOncology, Joint Academic Units of Obstetric Gynaecology andReproductive Physiology, St. Bartholomew's Hospital MedicalCollege, West Smithfield, London EC17A 7BE, UKReceived 27 March 1995; revised 21 July 1995; accepted 11September 1995production of fl-hCG by normal urothelium, together witha complete absence of intact hCG, suggests the possibility ofa hitherto unknown biological role for this molecule. Wehave investigated this idea further by examining the effects offl-hCG on the growth of various bladder cancer cell lines invitro.Materials and methodsCell linesThe f-human chorionic gonadotrophin-secreting bladdercancer cell lines RTI 12, SCaBER and 5637 (Iles et al.,1987) were obtained from the American Type CultureCollection (Rockville, MD, USA). The non-secreting bladdercancer line T24 (Bubenik et al., 1973) was obtained from DrJ Masters of the Institute of Urology, London, UK.Hormones and antibodiesIntact hCG along with free a- and fl-subunits were derivedfrom NIH preparation CR123 obtained from Dr DianaBlithe (Endocrine Branch, The National Institute of ChildHealth and Human Development, NIH, Bethesda, MD,USA). Drs RE Canfield and S Birken of Columbia Universityhave purified all the CR series of hCG reference standards,i.e. intact hCG and free cx- and fl-subunits, for the NIH andthe WHO (Birken et al., 1990). These have been distributedworldwide and have been used as the First InternationalReference Preparation of hCG and free subunits forimmunoassay and the Third International standard forintact hCG bioassay. Here we used the preparation labelled123 of the CR series, which was first released in 1977. Thepurity of these preparations is well recognised. However,several degradation products of hCG metabolism haverecently been recognised, the ultimate step being theformation of urinary f-core. Intermediate steps include thenicking of the f-subunit at residues 45-50. The extent ofsuch nicking varies with each preparation and CR123 hasbeen estimated to contain 10-16% nicked fl-chains (Birkenet al., 1991). The in-house preparation of highly purified fi-core used in these experiments has been previously describedby Lee et al. (1991).Sheep antisera 750 and 752 were prepared in-house,following immunisation with free fl-hCG isolated frompregnancy urine. These antisera were found to react withonly the free f-subunit of hCG but not intact hCG, LH,fi-hCG growth factor actiitr_ DJ Gillott et al324FSH, TSH or the free a-subunit and the hCG urinarymetabolite f-core. (Iles et al. unpublished data). Thesepolyclonal sheep antisera are now commercially availablefrom Polyclonal Antibodies, Llyndysal Dyfed, Wales, UK). Acontrol antisera of normal (non-immune) sheep sera wasobtained from Polyclonal Antibodies.MTT assayThe 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide (MTT) assay (Mosman, 1983) was used to estimate cellnumber following exposure to intact hCG, free a- and f-subunits, urinary metabolite fl-core and to anti-,B-hCGantisera.Microtitre plates (Coming 96-flat-bottomed wells) wereseeded with 200 pl of 0.1 x 106 ml-' cell suspension (20 000cells per well) in RPMI + 10% fetal calf serum (FCS) for 24h before the replacement of media by fresh culture mediacontaining test materials (0-500 ng ml-' ligands or 1:1000dilution of antisera). Plates were then incubated for a further36 h, followed by 1 h incubation in fresh medium, thenaddition of 20 ul millipore filtered MTT (5 mg ml-' in PBS).After 4 h incubation all fluid was removed and 100 pl ofacidified isopropanol (containing 0.04 M HCI) was added toeach well and maintained at room temperature for 15 min toallow formazan crystals to dissolve. Absorbance measure-ments were carried out at 570 nm against a 630 nm blank; atleast three replicates of each treatment were included. Resultswere expressed as optical density as a per cent of untreatedcontrols.ResultsStimulation by hCG and fragmentsIntact hCG, a-hCG and fl-core had no effect on the growthof any of the cell lines, while 25 ng ml-' ,B-hCG produced a152% increase in MTT reduction by T24 cells; (Figure 1,Table I). 5637, SCaBER and RT112 were stimulated to alesser extent increasing MTT reduction by 132, 112 and116% of controls respectively (Figure 2, Table II).Effect of anti-#-subunit antiseraConcurrent addition of specific antisera with the fl-hCG-containing media eliminated the stimulatory effect of the fi-hCG on the responding cell lines. This was best illustratedwhen antisera was concurrently added to escalating doses (0-50 ng ml-') of free fl-hCG in cultures of the T24 cell line(which does not secrete its own fl-hCG). The dose-responsecurve to the P-hCG was obliterated with co-addition of theanti-f-subunit antisera and MTT reduction matched that ofcontrols (Figure 3). This was not seen when non-immunecontrol sheep serum (NSS) was used (data not shown).E0--MCr0) _c- 4_0._0"00.0.+oTable I Dose-effect of hCG and subunits on T24 cell growth asmeasured by MTT reduction relative to untreated controlsIntact hCG cx-hCG fl-hCG #-coreLigand (per cent (per cent (per cent (per cent(ng ml-1) of control) of control) of control) of control)0 100.0±4.2 100.0+ 10.0 100.0± 14.1 100.0+ 7.31.57 90.9 ± 5.2 108.1 +4.2 118.0+4.5 95.9+ 5.23.13 87.8±2.9 112.9±8.5 123.2+3.1 94.2±6.96.25 86.5+ 3.7 105.4+ 5.7 130.4+9.7 98.9+ 3.212.50 84.4+4.1 102.6+6.3 139.5 ± 7.5 91.2+ 8.725.00 90.4 + 17.5 104.2 + 7.7 154.0 ± 10.2 99.7 + 9.90- cf)0(00.-*a~0 L00.00 5 10 15 20 25 30p-hCG (ng ml-1)Figure 2 A comparison of the dose-dependent growth responseof four epithelial bladder cancer cell lines (T24, SCaBER, RT1 12and 5637) to fl-hCG.Table H Dose -effect of f,-hCG on cell growth of ,B-hCG-expressing (SCaBER, RTI 12 and 5637) and non-expressing (T24)cell lines as measured by MTT reduction relative to untreatedcontrolsT24 5637 SCaBER RT112fl-hCG (per cent (per cent (per cent (per cent(ng ml-') of control) of control) of control) of control)0 100.0+14.1 100.0+7.3 100.0+15.4 100.0+5.71.57 118.0+4.5 102.8 +6.8 98.6+2.7 93.7 +4.73.13 123.2 +3.1 106.7 +9.2 102.4+4.6 94.7+ 5.96.25 130.4+9.7 107.6+6.5 99.0+2.6 99.2+9.012.50 139.5 + 7.5 116.2+9.4 101.7 +4.8 100.8 ± 7.325.00 154.0+10.2 132.0+7.0 111.8+7.7 115.5+ 16.6Antisera to free f-subunit at a 1:1000 dilution added tocell lines SCaBER and 5637 (lines that synthesised andsecreted their own fl-hCG) lowered MTT reduction to 60%and 70% of control levels respectively. However, the control1Ec 10oco1.- I0-0C 44O)L0-.--004L -0 1.20iCG0 5 10 15 20 25 30Ligand (ng mF1)Figure 1 Dose-dependent effects of hCG, free a- and fl-subunitsand metabolite fl-core on the growth of the T24 cell line asmeasured by tetrazolium salt reduction expressed as a percentageof untreated control values.60501401301201100090800.- F~~~~~~PhCG7 ,B~~~~~~~-hCG+_ ~~~~~~~~anti-pi-hCG-~~~~~.,0 0.2 0.4 0.6 0.8 1.0Relative antibody dilutionFigure 3 Abolition of the dose-dependent fl-hCG growthstimulation of T24 cells by the co-addition of anti-fl-hCGantibodies to the cultures.fl-hCG growth factor actvtxDJ Gillott et a!325NSS at the same dilution did not alter MTT reductionrelative to controls. Furthermore, the T24 cell line (whichdoes not secrete f-hCG) was unaffected by the addition ofthe fl-subunit antisera (Figure 4).DiscussionThe observation that 70% of established bladder cancer celllines and normal urothelial cells established as finite lifespancultures secrete variable quantities of,-hCG prompted ourinvestigation into a putative biological activity of free fl-hCG(Iles, 1991). The MTT assay clearly demonstrates an increasein cell numbers following fl-hCG treatment but no such effectwith intact hCG or oc-hCG (Figure 1). It is interesting to notethat f-core had no effect either. This metabolite, which has ashortened carboxy terminus, fewer carbohydrate residues andseveral nicks in the amino acid chain (Birken et al., 1988) isfound excreted in the urine. The absence of an effect with thismolecule provides further evidence for the specificity of thegrowth effect observed with the free fl-subunit.The effect of exogenous f-hCG on responding cell lineswas inhibited in a dose-dependent manner by antisera to fi-hCG (Figure 3). This suggests a highly specific type ofinteraction, possibly mediated by a receptor, as normal sheepserum produced no diminution of growth response in any ofthe lines. The growth response was highest in the T24 cellline, which does not itself secrete fl-hCG, while highersecretors exhibited less growth stimulation. This implies thatfl-hCG producers may be self-stimulating populations in vitro(or indeed, in vivo), high level producers being incapable offurther stimulation by exogenous fl-hCG. Alternatively, theremight be more than one subpopulation of urothelial cells,with secretors and responders in varying proportions thatdetermine overall production or response rates. The recentlyelucidated three-dimensional structure of f-hCG (Lapthorn etal., 1994) includes at its centre a distinctive arrangement ofprotein chain folds that is stabilised by six disulphide bondsand known as the cysteine knot motif. This motif is found inat least three growth factors: nerve cell growth factor (NGF),transforming growth factor (TGF-fl2) and platelet-derivedgrowth factor (PDGF-BB). These molecules are able to bindto their receptors as homodimers, a possibility that couldexist with fl-hCG and should be investigated further.x 120co0) 110E10000C 90~8O7060a- 50T24 5637 SCaBERCell lineFigure 4 The effect of 1:1000 dilution anti-fl-hCG antibodies inmedia on cell growth, as measured by tetrazolium salt reduction,for fl-hCG secreting cell lines 5637 and SCaBER and non-expressing cell line T24 compared with untreated controls andcells exposed to normal (non-immune) sheep sera (NSS) at 1:1000dilution. O, Control; f3, NSS(1:1000); M, anti-free ,B-hCG.When no exogenous /3-hCG was added, antisera against /3-hCG considerably inhibited growth in the high-producinglines such as SCaBER, with little or no growth inhibitionperceived in the non-secreting T24 line (Figure 4). Thisprovides further evidence for the validity and specificity ofthe observed effects of the subunit itself and constitutesimportant new evidence for an autocrine/paracrine effect off-hCG in urothelial cell carcinomas, while suggesting apossible mechanism for some of the poor prognosticassociations with fl-hCG that have been reported.In conclusion, fl-hCG (but not intact hCG, oa-hCG or 1l-core) is able to specifically increase cell growth in bladderepithelial bladder cancer lines, this effect is mediated by aspecific interaction that is obliterated by anti-f,-hCG serum.These findings could be explained if it is postulated that thefree f-subunit acts as a growth factor.AcknowledgementsThis study was supported by grants from the Paul Balint Trust andThe John Ellerman Foundation.ReferencesBIRKEN S, ARMSTRONG EG, KOLKS MAG, COLE LA, AGOSTO GM,KRICHEVSKY A, VAITUKAITIS JL AND CANFIELD RE. (1988).Structure of human chorionic gonadotrophin fl-subunit corefragment from pregnancy urine. Endocrinology, 123, 572- 583.BIRKEN S, KRICHEVSKY A, O'CONNOR J, LUSTBADER J ANDCANFIELD RE. (1990). Chemistry and immunochemistry ofhCG,its subunits and fragments. In Glycoprotein Hormones,Serono Symposia, USA, Chin WW, Boime I (eds). pp. 45-61Raven Press: New York.BIRKEN S, GAWINOWICZ MA, KARDANA A AND COLE LA. (1991).The heterogeneity of human chorionic gonadotropin (hCG). II.Characteristics and origins of nicks in hCG reference standards.Endocrinology, 129, 1551-1558.BOLTON RA, COULAM CBAND RYAN RJ. (1980). Specific binding ofhuman chorionic gonadotrophin to human corpora lutea in themenstrual cycle. Obstet. Gynecol., 56, 336-338.BRAUNSTIEN GD, RASOR J, ADLER D, DANZER H AND WADE MW.(1976). Serum human chorionic gonadotrophin levels throughoutnormal pregnancy. Am. J. Obstet. Gynecol., 126, 678-681.BUBENIK J, BARESOVA M, VIKLICKY V, JAKOUBKOVA J, SAINER-OVA H AND DONNER J. (1973). Established cell line of urinarybladder carcinoma (T24) containing tumour-specific antigens. Int.J. Cancer., 11, 765-773.DEXUS F, LOGOTHETIS C, HOSSAN E AND SAMUELS ML. (1986).Carcinoembryonic antigen and beta-human chorionic gonado-trophin as serum markers for advanced urothelial malignancies.J. Urol., 136, 403 - 407.FIDDES JC AND TALMADGE K. (1984). Structure, expression, andevolution of the genes for the human glycoprotein hormones. Rec.Prog. Horm. Res., 40, 43.ILES RK. (1991). Beta human chorionic gonadotrophin productionby bladder tumour cells: Incidence, molecular analysis andclinical significance. PhD Thesis, UK: University of London.ILES RK, OLIVER RTD, KITAU M, WALKER C AND CHARD T.(1987). In vitro secretion of human chorionic gonadotrophin bybladder tumour cells. Br. J. Cancer, 55, 623 - 626.ILES RK, CZEPULKOWSKI BH, YOUNG BD AND CHARD T. (1988).Amplification or rearrangement of the fl-human chorionicgonadotrophin human LH gene cluster is not responsible forthe ectopic production of ,BhCG bladder tumour cells. J. Mol.Endocrinol., 2, 113 -117.ILES RK, PURKIS PE, WHITEHEAD PC, OLIVER RTD, LEIGH I ANDCHARD T. (1990). Expression of beta human chorionicgonadotrophin by non-trophoblastic non-endocrine 'normal'and malignant epithelial cells. Br. J. Cancer, 61, 663-666.ILES RK, PERSAD R, SHARMA KB, DICKINSON, SMITH P ANDCHARD T. (1996). Urinary concentration of human chorionicgonadotrophin and its fragments as a prognostic marker inbladder cancer. Br. J. Urol. (in press).JACOBSEN AB, NESLAND JM, FOSSA SD AND PETTERSEN EO.(1990). Human chorionic gonadotrophin, neuron specific enolaseand deoxyribonucleic acid flow cytometry in patients with highgrade bladder cancer. J. Urol., 143, 706- 709.LAPTHORN AJ, HARRIS DC, LITTLEJOHN A, LUSTBADER JW,CANFIELD RE, MACHIN KJ, MORGAN FJ AND ISAACS NW.(1994). Crystal structure of human chorionic gonadotrophin.Nature, 369, 455-461.f,-hCG growth factor activityDJ Gillott et at326LEE CL, ILES RK, SHEPHERD JH, HUDSON CN AND CHARD T.(1991). 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The effects of beta-human chorionic gonadotrophin on the in vitro growth of bladder cancer cell lines

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The effects of beta-human chorionic gonadotrophin on the in vitro growth of bladder cancer cell lines

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