Gonadotropin-releasing hormone receptor–targeted gene ... · Gene expression–directed prodrug therapy is a two step therapeutic approach for cancer gene therapy. In the first

Gonadotropin-releasing hormone receptor–targetedgene therapy of gynecologic cancers

Carsten Grundker, Abdohamid Huschmand Nia,and Gunter Emons

Department of Gynecology and Obstetrics, Georg-August-University, Gottingen, Germany

AbstractThe majority of ovarian, endometrial, and breast cancersexpress gonadotropin-releasing hormone (GnRH) recep-tors. Apart from reproductive organs (ovaries, fallopiantubes, and uterus) that are normally removed duringsurgical therapy of ovarian or endometrial cancer,pituitary gonadotrophs also express GnRH receptors.The signal transduction pathway in tumor cells isbasically different from the classic GnRH receptor signaltransduction, which is known to operate in the pituitarygonadotrophs and can therefore be considered tumorspecific. Other organs and hematopoetic stem cells donot express GnRH receptors. We have recently shownspecific activation of nucleus factor KB in ovarian,endometrial, and breast cancers after treatment withGnRH agonists. Based on this tumor-specific signalingpathway and the distribution pattern of GnRH receptors,we have developed and successfully tested a genetherapy concept by using a GnRH analogue as aninducer for the transcription of a therapeutic gene incell culture and in nude mice. [Mol Cancer Ther2005;4(2):225–31]

IntroductionMore than 80% of ovarian and endometrial cancers and>50% of breast cancers express gonadotropin-releasinghormone receptors (GnRH-R; ref. 1). Apart from pituitarycells and reproductive organs (ovaries, fallopian tubes, anduterus) that are normally removed during surgical therapyof ovarian or endometrial cancer, other organs andhematopoetic stem cells do not express GnRH-R (2). TheGnRH-R is therefore a promising target for a tumor-specificgene therapy.

In addition to its unique distribution pattern, the GnRH-R signal transduction pathway in tumor cells is differentfrom the classic pathways via phospholipase C or proteinkinase C, known from pituitary cells (1). In tumor cells,GnRH analogues rather interfere with the mitogenic signaltransduction pathway of growth factor receptors andrelated oncogene products associated with tyrosine kinaseactivity (3). A comparable mechanism has been describedin human prostate cancer cells (4). These findings indicatethe existence of a tumor-specific signal transductionpathway for GnRH in GnRH-R-positive cancers.

We have recently shown activation of the nucleus factornB (NFnB) in ovarian, endometrial and breast cancers aftertreatment with GnRH agonist triptorelin (5). This wouldmake triptorelin an ideal candidate to induce the expres-sion of a therapeutic gene fused to a NFnB-enhancersequence in GnRH-R-positive tumors. In this study, wehave evaluated this novel gene therapy concept based ontissue-specific GnRH-R expression pattern and its signalingpathway characteristic for these tumors.

Gene expression–directed prodrug therapy is a two steptherapeutic approach for cancer gene therapy. In the firststep, a therapeutic gene is delivered into the tumor andbrought to expression. In a second step, a prodrug is givenand is selectively activated in transfected cells by theexpressed gene product. The first gene-directed enzymeprodrug therapy system described was the thymidine kinasegene of the herpes simplex virus thymidine kinase (HSV-TK)in combination with the prodrug ganciclovir (6). A largenumber of experiments have been done with this system,in different types of tumors and initial studies in animalmodels were very promising. This encouraged investigatorsto move into clinical trials where poor results have beenobtained thus far. The current state of preclinical researchand the results of the clinical trials as reviewed by Fillat et al.(7) show that in the last 15 years a large effort has been madewith numerous different strategies to enhance HSV-TK/ganciclovir efficacy in cellular and in vivo models.

To show the proof of principle of our gene therapyconcept, we decided to use HSV-TK/ganciclovir as it isa well known and well established therapeutic system.

Materials andMethodsAnimalsFemale athymic (nude) mice (CD1 nu/nu), 6 to 8 weeks

old on arrival, were obtained from Charles River (Sulzfeld,Germany). The mice were housed in sterile cages in atemperature-controlled room with 12-hour light/12-hourdark schedule and were fed autoclaved chow and waterad libidum . All experiments were done according to theGerman ethical guidelines and the German laws forprotection of animals.

Received 7/20/04; revised 11/23/04; accepted 12/1/04.

Grant support: Faculty of Medicine, Georg-August-University, Gottingen,Germany.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Requests for reprints: Carsten Grundker, Department of Gynecology andObstetrics, Robert-Koch-Street 40, D-37099 Gottingen, Germany.Phone: 49-0-551-399810; Fax: 49-0-551-399811.E-mail: [email protected]

Copyright C 2005 American Association for Cancer Research.

Molecular Cancer Therapeutics 225

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Cell Lines and Culture ConditionsThe human endometrial cancer cell lines Ishikawa,

HEC-1A, and Hec-1B and the ovarian cancer cell linesEFO-21, OVCAR-3, and SK-OV-3 were obtained fromAmerican Type Culture Collection (Manassas, VA) or thesources detailed previously (8). aT3-1, a human GnRH-R-positive mouse gonadotrope cell line, was kindly providedby Dr. P.M. Mellon (Department of Reproductive Medicine,University of California, San Diego, CA). 3T3 is a GnRH-R-negative human fibroblast cell line obtained fromAmerican Type Culture Collection. The cell lines weremaintained in MEM medium (Life Technologies, Berlin,Germany) supplemented with 2.2 g NaHCO3/L (Bio-chrom, Berlin, Germany), 100 units/mL penicillin (LifeTechnologies), 100 Ag/mL streptomycin (Life Technolo-gies), 10% (v/v) FCS (Life Technologies), and 40 IUinsulin/L (Hoechst, Frankfurt, Germany) at 37jC inhumidified 5% CO2. aT3-1 was maintained in RPMI1640 (Life Technologies).

Plasmids andTransfectionpNFnB-SEAP (Clontech, Palo Alto, CA) is designed to

monitor the activation of NFnB and NFnB-mediated signaltransduction pathways using the reporter gene secretedalkaline phosphatase (SEAP). pNFnB-SEAP contains fourtandem copies of the nB4 enhancer fused to the HSV-TKpromoter (Fig. 1). pTK-SEAP (Clontech) was used as anegative control to determine the background signalsassociated with the culture medium. The enhancerlesspTK-SEAP contains HSV-TK promoter upstream of theSEAP coding sequence.

pNFnB-LacZ was constructed in our laboratory byinsertion of LacZ gene (h-galactosidase) from pIND-LacZ(Invitrogen BV, Groningen, the Netherlands) into HindIII-XbaI site of pNFnB-SEAP after removal of the SEAP gene(Fig. 1A). pNFnB-TK was constructed in our laboratory byinsertion of HSV-TK gene from pNGVL1-TK (NationalGene Vector Laboratory, University of Michigan, AnnArbor, MI) into HindIII-XbaI site of pNFnB-SEAP afterremoval of the SEAP gene. pNFnB-TK contains fourtandem copies of the nB4 enhancer fused to the HSV-TKpromoter followed by the HSV-TK gene as therapeutic gene(Fig. 1A).

Transfections were done with Superfect liposomereagents (Qiagen, Hilden, Germany) according to manu-facturer’s instructions.

GnRH Agonist Triptorelin-Induced NFnnB ActivationIn vitro

Cells were grown to f50% confluence on 30-mm plates.Transfections with pNFnB-SEAP (see above) were doneusing the Superfect liposome reagents and following themanufacturer’s instructions (Qiagen) and cells werecultured for 96 hours in the absence of FCS and phenolred with or without 100 nmol/L GnRH agonist triptorelin.Every 24 hours, 100 AL of the media were taken andanalyzed for SEAP activity. Chemiluminescence detectionof SEAP activity was done according the manufacturersinstructions (Clontech) using a plate fluorometer (Bert-hold, Bad Wildbach, Germany).

GnRH Agonist Triptorelin-Induced NFnnB ActivationIn vivo

Tumors were initiated by i.p. injection of 1 � 107 cancercells with Matrigel (Collaborative Biomedical Products,Bedford, MA). After 3 to 4 weeks, all animals had developedsolid tumors of about 115 mm3 (HEC-1B) or 80 mm3

(OVCAR-3) and the treatment was initiated. Each groupwas composed of 10 animals. The in vivo experimentswere done as follows: 50 AL pNFnB-LacZ (0.5 Ag/AL) plusSuperfect reagent (50 AL) were injected i.p. Half of eachgroup were injected i.v. with triptorelin (300 nmol/20 g).After 24 hours, the mice were sacrificed. Tumor tissueswere collected for h-galactosidase staining. All organs wereremoved, cleaned, and also stained for h-galactosidaseactivity. Stainings for h-galctosidase activity were doneaccording to manufacturer’s (Promega, Madison, WI)

Figure 1. A, NFnB-SEAP, NFnB-LacZ, and NFnB-TK contain fourtandem copies of the nB4 enhancer fused to the HSV-TK promoterfollowed by SEAP or LacZ (h-galactosidase) reporter gene or the HSV-TKgene as therapeutic gene. pNFnB-LacZ was constructed by insertion ofLac-Z gene into HindIII-Xba I site of pNFnB-SEAP after removal of theSEAP gene. pNFnB-TK was constructed by insertion of HSV-TK geneinto HindIII-Xba I site of pNFnB-SEAP after removal of the SEAP gene. B,GnRH-induced activation of NFnB in GnRH-R-positive endometrial,ovarian, and breast cancer cells. Activated GnRH-R induces NFnBactivation and nuclear translocation of activated NFnB. Activated NFnBnow couples to nB DNA binding sites and induces cellular mechanisms(1 ). Based on this mechanism, we have developed a gene therapy, whichspecifically kills targeted tumor cells, expressing GnRH-R (2 ). ActivatedNFnB couples to the nB DNA binding site of the pNFnB-TK plasmid andinduces expression of the therapeutic enzyme HSV-TK. HSV-TK nowcatalyzes the activation of the prodrug ganciclovir to an active drugwhich kills the tumor cells.

GnRH Receptor–Targeted Gene Therapy226




instructions for in situ staining of cells growing asmonolayer. Tumor tissue and organs were washed withPBS and kept in fresh 0.25% glutaraldehyde (Sigma, St.Louis, MO)/PBS solution for 30 minutes before they werewashed with PBS and stained over night at 37jC in stainingsolution. This solution contains in 30 mL PBS: 8mgspermidine (Sigma), 60 AL of 1 mol/L MgCl2, 2% NP40,300 AL of 1% potasiumdeoxycholat, 150 AL of 0.5 mol/LK3Fe(CN)6�3H2O, 150 AL of 0.5 mol/L K3Fe(CN)6 (Sigma),and 15 mg 5-bromo-4-chloro-3-indolyl-h-D-galactopyrano-side (Stratagene, La Jolla, CA).

GnRH AgonistTriptorelin-Induced Activation of HSV-TK In vitro

Cells were grown to f70% confluence on six-wellplates and transfected with pNFnB-TK (see above) or forcontrol with pNGVL1-TK (see above) which contains aconstitutively active HSV-TK. Transfections were doneusing the Superfect liposome reagents and following themanufacturer’s instructions (Qiagen). After 24 hours, thecells were treated with or without GnRH agonist tripto-relin (100 nmol/L) followed by a treatment with orwithout ganciclovir (15 Amol/L). After 72 hours, viablecells were stained.

GnRH AgonistTriptorelin-Induced Activation of HSV-TK In vivo

Tumors were initiated by i.p. or s.c. injection of 1 � 107

cancer cells with Matrigel (Collaborative BiomedicalProducts). After 3 to 4 weeks all animals had developedsolid tumors of about 115 mm3 (HEC-1B) or 80 mm3

(OVCAR-3) and the treatment was initiated. Each groupwas composed of 10 animals. The in vivo experimentswere done as follows: 50 AL pNFnB-TK (0.5 Ag/AL) plusSuperfect reagent (50 AL) were injected either i.p. or directlyinto the s.c. tumors. Transfection was repeated every fourthday. Half of each group were injected i.v. with triptorelin(15 nmol/g every fourth day). The pNFnB-TK-transfectedmice were treated with ganciclovir (20 Ag/g every fourthday). Tumor volumes were measured every 2 days. Themice were killed after 20 days.

Statistical AnalysisAll data are expressed as the mean F SE. The data from

the time course experiments in nude mice were tested forsignificant differences by one-way ANOVA followed byStudent-Newman-Keuls test for comparison of individualgroups, after a Bartlett test had shown that variances werehomogenous. Differences were considered statisticallysignificant at a P < 0.05.

ResultsBasic Mechanisms of the GnRH-R-Targeted Gene

TherapyA GnRH agonist (e.g., triptorelin) induces activation of

NFnB in GnRH-R-positive cancer cells (Fig. 1B). ActivatedNFnB now couples to the nB DNA binding sites of thepNFnB-TK plasmid and induces expression of the thera-peutic enzyme HSV-TK. Through this the prodrug ganci-clovir is activated.

GnRH Agonist Triptorelin-Induced NFKB ActivationIn vitro

To examine GnRH agonist triptorelin-induced activa-tion of NFnB, we transiently transfected GnRH-R-positiveEFO-21 ovarian cancer cells with a reporter vectorcontaining a nB4 cis-acting DNA sequence (responseelement) and the SEAP reporter gene or the pTK-SEAPvector as a negative control. SEAP activity was detectedusing a chemiluminescence assay. During culture of thetransfected EFO-21 GnRH agonist triptorelin treatmentresulted in a 5.4-fold (Fig. 2A) increase of NFnB-inducedSEAP expression (P < 0.001). To show that our genetherapy concept based on signal transduction pathway ofGnRH is specific for GnRH-R-positive tumors, we hadto show that pNFnB-activation is observed neither inGnRH-R-negative cells nor in GnRH-R-positive nonmalig-nant cells. GnRH-R-negative SK-OV-3 ovarian cancer cells

Figure 2. NFnB-induced SEAP expression of GnRH-R-positive EFO-21ovarian cancer cells (A), GnRH-R-negative SK-OV-3 ovarian cancer cells(B), GnRH-R-negative 3T3 fibroblast cell line (C), and GnRH-R-positiveaT3-1 pituitary cell line (D) transfected with pNFnB-SEAP with or without(100 nmol/L GnRH agonist triptorelin treatment. After treatment withtriptorelin, a significant (P < 0.001) increase of SEAP expression isobserved in the EFO-21 cancer cell line only (A). Column 1, SEAP controlplasmid without triptorelin treatment; column 2, SEAP control plasmidwith triptorelin treatment; column 3, pNFnB-SEAP without triptorelintreatment; column 4, pNFnB-SEAP with triptorelin treatment.





were transfected with pNFnB-SEAP and treated with orwithout triptorelin. The SEAP-activity remained unchangedat a low level after triptorelin treatment (Fig. 2B). In pNFnB-SEAP transfected GnRH-R-negative 3T3 fibroblast cells NFnBactivity remained unchanged at a low level after triptorelintreatment (Fig. 2C). The same experiment was done with theaT3-1 gonadotrope cells which express GnRH-R. In aT3-1cells, NFnB-induced SEAP expression was not affected bytriptorelin (Fig. 2D). These data indicate that GnRH agonisttriptorelin-induced NFnB activation is restricted to cancercells and requires the presence of GnRH-R (Fig. 2A).

GnRH Agonist Triptorelin-Induced NFKB ActivationIn vivo

To examine GnRH agonist triptorelin-induced activationof NFnB in vivo , mice bearing endometrial or ovariantumors i.p. were transiently transfected with a reportervector containing a nB4 response element and the LacZreporter gene. Triptorelin-induced NFnB activity within thetumor is shown as h-galactosidase activity (Fig. 3). NFnBwas induced by triptorelin in the tumor only (Fig. 4A).Apart from the reproductive organs ovary (Fig. 4B) anduterus (Fig. 4C) that are normally removed during surgicaltherapy, other organs (Fig. 4D–K) did not show triptorelin-induced NFnB activity in vivo . The blue staining onstomach (Fig. 4H) are tumor cells grown on the surface ofthe organ, not the organ itself.

GnRH AgonistTriptorelin-Induced Activation of HSV-TK In vitro

To examine GnRH agonist triptorelin-induced activationof NFnB and specific killing of GnRH-R-positive tumorcells in vitro , we used the therapeutic HSV-TK gene underthe control of four copies of the nB enhancer fused to theHSV-TK promoter. Using HSV-TK as therapeutic gene,ganciclovir as prodrug, and GnRH agonist triptorelin asinductor we could show the specific killing of NFnB-TKtransfected GnRH-R-positive cancer cells in vitro (Fig. 5,right). Without triptorelin treatment, the NFnB-TK trans-fected cells could not be killed by ganciclovir (Fig. 5,

middle). Using a vector with a constitutively active HSV-TKgene (pNGVL1-TK) incubation with ganciclovir resulted incell death with or without triptorelin treatment (Fig. 5, left).On GnRH-R-negative cells, not shown here, no killingeffects were observed. Because of the different GnRHreceptor signal transduction, no toxic effects wereobserved in GnRH-R-positive pituitary cells.

GnRHAgonistTriptorelin-Induced Activation of HSV-TK In vivo

To show the proof-of-principle of our gene therapyconcept in vivo , mice bearing endometrial or ovariantumors i.p. or s.c. were transiently transfected withNFnB-TK (Fig. 1) and treated with GnRH agonist triptor-elin and the prodrug ganciclovir. Transfection and treat-ment were repeated every fourth day. Every second day,the tumor volume (s.c. tumors) or development of ascitisindicated as increase of body weight (i.p. tumors) wasmeasured. The increase of the tumor volume of the micereceiving gene therapy with s.c. endometrial or ovariancancer was significantly lower (P < 0.05) than with thecontrol animals (Fig. 6B). Development of ascitis indicatedas increase of body weight of the mice receiving gene therapywith i.p. endometrial or ovarian cancer was significantlylower (P < 0.01) than that of control animals (Fig. 6A). Toxicside effects were not observed. To further exclude side effects,a long-time experiment using two animals without tumorwas conducted. The mice were transiently transfected withNFnB-TK and treated with GnRH agonist triptorelin and theprodrug ganciclovir for 8 weeks. No effects were observed.

If the tumor growth had exceeded the limitations accord-ing the ethical guidelines the mouse had to be killed (c). Onemouse in the gene therapy group bearing a s.c. endometrialtumor died 20 days after the beginning of the treatment (b).

DiscussionIn this study, we show the proof of principle of a targetedHSV-TK suicide gene therapy which uses both tissue

Figure 3. Transcription factorNFnB can be activated via GnRH-Rand up-regulate target genes. Usingthe reporter gene LacZ, the conceptof the gene therapy via GnRH-R-dependent tumor-specific signaltransduction was successfully testedin nude mice bearing Hec-1A endo-metrial tumors (i.p.). Transfectionwith pNFnB-LacZ (i.p.). Treatmentwith GnRH agonist triptorelin (i.v.).X-Gal staining after tumor replace-ment. Experiments using OVCAR-3human ovarian tumor cells gaveidentical results. Representative ofthree separate experiments. All ex-perimental groups consisted of fiveanimals.





specific expression of GnRH-R and tumor-specific signaltransduction of this receptor.

TK is an enzyme that specifically functions in the salvageof thymidine for DNA synthesis. Although human TK ispresent in normal cells, the viral form can selectivelyactivate antiviral agents such as ganciclovir which isthereafter toxic for mammalian cells. Selective expressionof viral TK in cancer cells could permit effective treatmentwith ganciclovir. Application of drug sensitization with theHSV-TK/ganciclovir system in solid tumors was pioneeredby Mootlen et al. and has been used in clinical trials ofdifferent tumors including cancers of the uterus, the ovary,

and the breast (9–12). Although the mechanisms involvedin tumor regression remain to be fully elucidated, it is likelythat HSV-TK gene transfer to tumor cells results in thephosphorylation of ganciclovir to toxic nucleotide precur-sors, which in turn disrupt DNA synthesis in cellsexpressing the transgene, as well as not transfectedneighboring tumor cells through cell-to-cell transfer oftoxic metabolites (bystander effect; ref. 13). One of theunique features of the tumor destruction in the HSV-TKsystem is the observation that not all the tumor cells mustcontain the inserted gene to be killed by ganciclovir (9).Ural et al. (14) found that in situations in which only 10%of the cells were transfected, tumor cell proliferationdecreased 70% compared with the control. This bystandereffect is important in the gene therapy, because none ofthe thus far available gene transfer techniques is able totransfer a therapeutic construct into every single cell of atumor. The bystander effect has been reported in gyneco-logic cancers as well (15–19). Recent studies from Kunishigeet al. using lipofectin-mediated gene transfer of HSV-TK toa human cervical adenocarcinoma cell line and a humanendometrial adenocarcinoma cell line showed sensitivity toganciclovir and presented evidence of the bystander effectin both cell lines in vitro and in vivo (17). Theseinvestigators observed tumor reduction when mixtures ofHSV-TK-negative cells with >20% HSV-TK-positive cellswere injected into SCID mice (17).

In the setting of ovarian cancer, the HSV-TK has beenused for gene therapy. HSV-TK-based gene therapyprotocols have relied on the fact that 90% of ovariancancers are confined to the peritoneum, even at relapse.This closed compartment permits uncomplicated i.p.delivery of concentrated liposomal and viral vectors foreffective transduction of plasmids containing HSV-TK.Efficacy of this approach is further magnified by thebystander effect (20). Incorporation of the viral enzyme by10% of tumor cells yields a 70% cell kill in some studieswith xenografts, obviating the need to target 100% of the

Figure 4. Due to the tissue specificexpression and signaling of GnRH-R,GnRH agonist triptorelin-induced acti-vation of NFnB is limited to thereproductive tissues and their carcino-mas. Hec-1A endometrium carcinomagrown i.p. (A), ovary (B), uterus (C),brain (D), lung (E), heart (F), kidney(G), stomach (H), intestine (I), liver(K). Transfection with pNFnB-LacZ(i.p.) with (left ) or without (right )treatment with GnRH agonist triptor-elin (i.v.). X-Gal staining after tumor/organ replacement. Experiments usingOVCAR-3 human ovarian tumor cellsgave identical results. Representativeof three separate experiments. Allexperimental groups consisted of fiveanimals.

Figure 5. Selective killing of GnRH-R-positive EFO-21 ovarian cancercells in vitro . Using the gene therapy vector pNFnB-TK containing HSV-TKas therapeutic gene under the control of four copies of the nB enhancerfused to the HSV-TK promoter, the prodrug ganciclovir and GnRH agonisttriptorelin as inductor the specific killing of GnRH-R-positive cancer cellscould be shown (right ). Without triptorelin treatment, the cells could notbe killed by ganciclovir (middle). Using a vector with a constitutive activeHSV-TK (pNGVL1-TK), treatment with ganciclovir resulted in cell deathwith or without triptorelin treatment (left ). Representative of threeseparate experiments. Experiments using GnRH-R-positive endometrialcancer cell lines Ishikawa, HEC-1A, and Hec-1B and GnRH-R-positiveovarian cancer cell line OVCAR-3 gave identical results.





tumor cells (20). Several trials have been completed (21–23). In phase I reports, 38% of patients with recurrentdisease have experienced stabilization of tumor growth(23). Toxicities have been tolerable.

However, if such a gene therapy could be madetumor specific, this would increase effectiveness andreduce side effects. In our study, we could show thatGnRH agonist-induced activation of a reporter gene viaNFnB enhancer sequences was restricted to GnRH-R-positive human cancer cell lines in vitro . Using thereporter gene LacZ , the concept of gene therapy viaGnRH-R-dependent tumor-specific signal transductionpathway was successfully tested in nude mice bearing

xenografts of human ovarian or endometrial cancers.Only the GnRH-R-positive tumors as well as thereproductive organs that are normally removed duringsurgical therapy showed GnRH-induced activation ofNFnB. All other organs did not show any activation ofNFnB. Using HSV-TK as therapeutic gene, ganciclovir asprodrug and GnRH agonist triptorelin as inducer wecould show specific killing of GnRH-R-positive cancercells in vitro . No effects were observed on GnRH-R-negative cells. Because of the facts that the GnRH signaltransduction in pituitary gonadotrophs is different to thatacting in reproductive tumors and in breast cancer cells(1) and in addition that GnRH do not induce NFnBactivation in pituitary gonadotrophs, the therapeutic genecould not be activated in pituitary gonadotrophs andtherefore no toxic effects could as well be observed in theGnRH-R-positive pituitary gonadotrophs in vitro . Tofurther exclude side effects in vivo a long timeexperiment (8 weeks) using two animals withouttumor was conducted. No effects were observed.

The proof-of-principle could be shown in vivo in nudemice bearing endometrial or ovarian cancers s.c. and i.p.The tumor volume of the mice receiving gene therapy wassignificantly lower as compared with control animals. Toxicside effects were not observed.

Until now, for practical reasons, we have only usedliposomal transfections, which might be adequate for i.p.tumors, such as ovarian cancer, where tumor spreadlimited to the peritoneal surface is common. Therefore,our nude mouse model with i.p. xenografts of humanovarian cancers was adequate for the evaluation of ourgene therapy concept for ovarian cancer. If successfullyoptimized, this strategy could be directly taken to clinicaldevelopment for the treatment of ovarian cancer. Becausemodern liposomal techniques are not toxic and havenearly the same efficacy as viral systems, the use of anonviral method should be preferred in the therapy ofovarian cancer. Using i.p. xenografts of human endome-trial cancer cells, this model was used to test the principalapplicability of our approach for the gene therapy of thiscancer. As this tumor mainly form metastases outside theperitoneal cavity, the development of a viral transfectionstrategy will be necessary.

In summary, the proof-of-principle of the GnRH-R-targeted gene therapy could be shown in vitro andin vivo on tumor-bearing nude mice. The tumor volumein mice receiving gene therapy was significantly lowercompared with control animals. Toxic side effects werenot observed.

Acknowledgments

We thank Ferring Pharmaceuticals (Copenhagen, Denmark) for the gift ofthe GnRH agonist triptorelin and Renate Dietrich for excellent technicalassistance.

References

1. Grundker C, Gunthert AR,Westphalen S, EmonsG. Biology of GnRH systemsin human gynecological cancers. Eur J Endocrinol 2002;146:1–14.

Figure 6. GnRH agonist triptorelin-induced activation of HSV-TK in vivo .Development of ascitis indicated as increase of bodyweight of mice with ani.p.A,Hec-1B endometrial tumor.B, increase of tumor volume of mice withs.c. Hec-1B endometrial cancer. *, Day of treatment. c, Tumor growth hadexceededthelimitationsaccordingtheethicalguidelinesandthemousehadtobekilled.b,Onemousedied.Point, meanof three independent experiments;bars, FSE. All experimental groups consisted of five animals. ExperimentsusingOVCAR-3 ovarian cancer xenografts gave comparable results.





2. Grundker C, Volker P, Griesinger F, et al. Antitumor effects ofcytotoxic LHRH analog AN-152 on human endometrial and ovariancancers xenografted into nude mice. Am J Obstet Gynecol 2002;187:528–37.

3. Emons G, Muller V, Ortmann O, et al. Luteinizing hormone-releasinghormone agonist triptorelin antagonizes signal transduction and mitogenicactivity of epidermal growth factor in human ovarian and endometrialcancer cell lines. Int J Oncol 1996;9:1129–37.

4. Moretti RM, Marelli MM, Dondi D, et al. Luteinizing hormone-releasinghormone agonists interfere with the stimulatory actions of epidermalgrowth factor in human prostatic cancer cell lines, LNCaP and DU 145.J Clin Endocrinol Metab 1996;81:3930–7.

5. Grundker C, Schulz K, Gunthert AR, Emons G. Luteinizing hormone-releasinghormone induces nuclear factor nB-activation and inhibits apoptosis in ovariancancer cells. J Clin Endocrinol Metab 2000;85:3815–20.

6. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM.In vivo gene transfer with retroviral vector-producer cells for treatment ofexperimental brain tumors. Science 1992;256:1550–2.

7. Fillat C, Carrio M, Cascante A, Sangro B. Suicide gene therapymediated by the herpes simplex virus thymidine kinase gene/ganciclovir system: fifteen years of application. Curr Gene Ther2003;3:13–26.

8. Emons G, Ortmann O, Schulz KD, Schally AV. Growth-inhibitoryactions of analogues of luteinizing hormone releasing hormone on tumorcells. Trends Endocrinol Metab 1997;8:355–62.

9. Moolten FL. Tumor chemosensitivity conferred by inserted herpesthymidine kinase genes: paradigm for a prospective cancer controlstrategy. Cancer Res 1986;46:5276–81.

10. Moolten FL,Wells JM. Curability of tumors bearing herpes thymidine kinasegenes transferred by retroviral vectors. J Natl Cancer Inst 1990;82:297–300.

11. Kwong YL, Chen SH, Kosai K, Finegold MJ, Woo SLC. Adenoviral-mediated suicide gene therapy for hepatic metastasis o breast cancer.Cancer Gene Ther 1996;3:339–44.

12. Qian C, Bilbao R, Bruna O, Prieto J. Induction of sensitivity toganciclovir in human hepatocellular carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase.Hepatology 1995;22:118–23.

13. Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidencethat metabolic cooperation is responsible foe the bystander effectobserved with HSVtk retroviral gene therapy. Hum Gene Ther1993;4:725 – 31.

14. Ural AU, Takebe N, Adhikari D, et al. Gene therapy for endometrialcarcinoma with the herpes simplex thymidine kinase gene. Gynecol Oncol2000;76:305–10.

15. Tong XV, Block A, Chen SH, Woo SLC, Kieback DG. Adenovirus-mediated thymidine kinase gene transduction in human epithelial ovariancancer cell lines followed by exposure to ganciclovir. Anticancer Res1996;16:1611–8.

16. Rosenfield ME, Wang M, Siegal GD, et al. Adenoviral-mediateddelivery of herpes simplex virus thymidine kinase results in tumorreduction and prolonged survival in a SCID mouse model of humanovarian carcinoma. J Mol Med 1996;74:455–62.

17. Kunishige I, Samejima Y, Shiki Y, et al. Suicide gene therapy forhuman uterine adenocarcinoma cells using herpes simplex virus thymidinekinase. Gynecol Oncol 1999;72:16–25.

18. Kim J, Hwang ES, Kim JS, You EH, Lee SH. Intraperitoneal genetherapy with adenoviral-mediated p53 tumor suppressor gene for ovariancancer model in nude mice. Cancer Gene Ther 1999;6:172–8.

19. Tong XW, Kieback DG, Ramesh R, Freeman SH. Molecular aspects ofovarian cancer. Is gene therapy the solution? Hematol Oncol Clin North Am1999;13:109–33.

20. Wolf JK, Jenkins AD. Gene therapy for ovarian cancer (review). Int JOncol 2002;21:461–8.

21. Robinson W, Adams J, Marrogi A, Freeman S. Vaccine therapy forovarian cancer using herpes simplex virus-thymidine kinase (HSV-TK)suicide gene transfer technique: a phase I study. Gene Ther Mol Biol1998;2:31–40.

22. Hasenburg A, Tong XW, Fischer A, et al. Adenovirus-mediatedthymidine kinase gene therapy in combination with topotecan for patientswith recurrent ovarian cancer: 2.5-year follow-up. Gynecol Oncol 2001;83:549–54.

23. Alvarez RD, Gomez-Navarro J, Wang M, et al. Adenoviral-mediatedsuicide gene therapy for ovarian cancer. Mol Ther 2000;2:524–30.





2005;4:225-232. Mol Cancer Ther Carsten Gründker, Abdohamid Huschmand Nia and Günter Emons therapy of gynecologic cancers

targeted gene−Gonadotropin-releasing hormone receptor

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