Suicide Gene Therapy For Cancer Research Articles

Suicide gene therapy of gallbladder cancer using a cancer-selectively replicating adenovirus carrrying uracil phosphoribosyltransferase (UPRT) gene

Suicide gene therapy of gallbladder cancer using a cancer-selectively replicating adenovirus carrrying uracil phosphoribosyltransferase (UPRT) gene

Heat-directed suicide gene therapy for breast cancer.

Adjuvant hyperthermia can improve treatment outcome for locally recurrent breast cancer (LRBC). Previously, we demonstrated that infection of human breast cancer cells with a recombinant adenovirus expressing beta-galactosidase from the human hsp70b gene promoter (Ad.70b.betagal) results in 50- to 800-fold increases in reporter gene expression following heat treatment (30 minutes at 43 degrees C). Here, we describe a heat-directed suicide gene therapy strategy based on an adenoviral vector (Ad.70b.CDTK) in which expression of the dual prodrug-activating E. coli cytosine deaminase/herpes simplex virus thymidine kinase (CDTK) fusion gene is under the control of the hsp70b promoter. Treatment of T47D and MCF-7 breast cancer cells with mild hyperthermia (43 degrees C/30 minutes) and prodrugs (100 microg/ml 5-fluorocytosine and 10 microg/ml ganciclovir) following infection with Ad.70b.CDTK (10-100 PFU/cell) resulted in 30- to 60-fold decreases in clonogenic survival relative to control cultures treated with heat or prodrugs alone. Clonogenic survival declined even further (up to 240-fold) following heat treatment at 41.5 degrees C for 120 minutes. A decreased clonogenic survival was accompanied by tumor cell apoptosis. These results demonstrate that this combined treatment strategy can be highly effective against heat- and radiation-resistant breast tumor cells and supports the continued development of heat-directed CDTK suicide gene therapy strategies for LRBC.

An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice

The most serious problem in current gene therapy is discrepancies between experimental data and actual clinical outcomes, which may be due to insufficient analyses and/or inappropriate animal models. We have explored suicide gene therapy by using various clinically relevant animal models and doubt the clinical use of maximal suicide gene expression, which has been generally recommended. To explore this subject further, we studied what expression level of suicide gene and what promoter led to the maximal clinical benefit in the case of hepatic metastatic cancer in mice. Therapeutic and adverse side effects of 4 adenoviral vectors that express herpes simplex virus thymidine kinase (HSV-tk) under different promoters were scrupulously investigated in 2 mouse models of hepatic metastasis of gastric cancer that possess clinical characteristics. Surprisingly, increases in HSV-tk expression beyond a certain point, achieved by the Rous sarcoma virus long terminal repeat promoter, not only enhanced the adverse side effects of lethal hepatotoxicity and ganciclovir-independent cytotoxicity but also failed to further increase therapeutic potential. Moreover, the carcinoembryonic antigen (CEA) tumor-specific promoter, the therapeutic potential of which had been underestimated, was much more useful—even in the case of low CEA-producing cancer—than had been previously reported. In conclusion, the optimal therapeutic expression level of a suicide gene is a novel concept and a crucial factor for successful cancer gene therapy. The present results, which contradict those of previous studies, alert researchers about possible problems with ongoing and future clinical trials that lack this concept. (H EPATOLOGY 2003;37:155-163.)

Transcriptionally targeted retroviral vector for combined suicide and immunomodulating gene therapy of thyroid cancer.

Gene therapy may be an effective approach to thyroid carcinoma refractory to conventional treatment. A transcriptionally targeted retroviral vector for gene therapy of thyroid carcinomas was generated replacing the viral enhancer with the enhancer sequence of the human thyroglobulin (TG) gene, yielding a chimeric long-terminal repeat. The TG enhancer was used to drive the expression of either a reporter gene (beta-galactosidase) or two therapeutic genes, i.e. the prodrug-activating enzyme thymidine kinase of herpes simplex virus (HSV-TK) and human IL-2, separated by an internal ribosome entry site. The corresponding vector having an unmodified long-terminal repeat was used as control. The targeted vector allowed selective transgene expression and cell killing in differentiated thyroid tumor cells but not in anaplastic thyroid carcinoma cells and nonthyroid cells, as demonstrated by quantitative RT-PCR and cytotoxicity assays. Nude mice injected with tumor cells underwent near complete or complete regression of tumors transduced with the control vector after ganciclovir treatment. On the other hand, infection with the thyroid-specific vector led to regression only of TG-expressing tumors. In addition, tumors expressing human IL-2 showed significant growth retardation, compared with nontransduced tumors while exhibiting signs of necrosis and presence of an inflammatory infiltrate. However, HSV-TK/IL-2 plus ganciclovir was significantly more efficient than HSV-TK/IL-2 alone in eradicating tumor masses. Our results indicate that replacement of viral enhancer with TG enhancer confers selectivity of transgene expression in thyroid cells. Thus, the combined thyroid-specific expression of two therapeutic genes (cytokine and suicide genes), although a safe tumor-targeted treatment, would allow an increased anticancer effect.

Development of gene therapy using prostate-specific membrane antigen promoter/enhancer with Cre Recombinase/LoxP system for prostate cancer cells under androgen ablation condition.

To enhance the efficacy of suicide gene therapy for prostate cancer under androgen deprivation, we designed a promoter system that consists of the prostate‐specific membrane antigen (PSMA) promoter/enhancer (PEPM) and Cre‐loxP DNA recombination system. We constructed two kinds of plasmids. One plasmid contains a Cre recombinase (Cre) under the control of PEPM and the other expresses CMV‐lox‐luciferase/herpes simplex virus thymidine kinase (TK). In PSMA‐positive LNCaP cells, the promoter activity of the PEPM‐Cre plus CMV‐lox‐luciferase demonstrated 800‐fold greater activity compared with that of the PSMA promoter alone. However, no enhancement of the promoter activity was observed in the PSMA‐negative cells. Furthermore, in contrast to prostate specific antigen promoter/enhancer (PP), the promoter activity of PEPM did not decrease when the LNCaP cells were cultured in charcoal‐stripped fetal bovine serum (CFBS). In an in vitro gene therapy model with LNCaP cells, the cell growth inhibition in the presence of ganciclovir (GCV) was more evident in the cells transfected with the PEPM‐Cre plus CMV‐lox‐TK than in the cells with the PP‐TK, and the difference in efficacy between the two plasmids was more remarkable when the cells were maintained in CFBS medium. The therapeutic effect of PEPM‐Cre plus CMV‐lox‐TK was also observed in xenografted LNCaP cells on nude mice when the plasmids were directly injected into tumors and GCV was administered intraperitoneally. These findings indicate that the combination of the PSMA promoter/enhancer and the Cre‐loxP system can enhance the PSMA promoter activity even under androgen ablation conditions and can exert its anti‐tumor effect both in vitro and in vivo.

Gene therapy for prostate cancer using the cytosine deaminase/uracil phosphoribosyltransferase suicide system.

Cytosine deaminase (CD) activates prodrug 5-FC to 5-FU and is used for suicide gene therapy (the CD/5-FC system). E. coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that directly converts 5-FU into 5-fluorouridine monophosphate and improves the antitumoral effect of 5-FU. This study demonstrates the effectiveness of transduction of the UPRT gene in addition to CD/5-FC cancer suicide gene therapy. We investigated a combined suicide gene transduction therapy for human hormone independent prostate cancer cell line DU145 using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD+UPRT/5-FC system). Cells transfected with AdCA-UPRT showed approximately 57 times lower IC50 to 5-FU compared with those transfected with AdCA-LacZ. Furthermore, cells transfected with AdCA-CD and AdCA-UPRT proved to be more sensitive to 5-FC compared with those transfected with AdCA-CD. Intratumoral injection of AdCA-CD and AdCA-UPRT drastically suppressed the growth of tumors which had generated from DU145 cells inoculated into athymic (nude) mice compared with those injected with AdCA-LacZ or AdCA-LacZ and AdCA-CD. These results suggest that the CD+UPRT/5-FC system could be a powerful factor in human prostate cancer suicide gene therapy.

Current progress in suicide gene therapy for cancer.

Standard chemotherapeutic agents and ionizing radiation destroy dividing cells. Because tumor cells divide more rapidly than normal cells, there is a therapeutic index in which damage to the cancer cells is maximized while keeping the toxicity to the normal host cells acceptable. Suicide gene therapy strives to deliver genes to the cancer cells, which convert nontoxic prodrugs into active chemotherapeutic agents. With this strategy, the systemically administered prodrug is converted to the active chemotherapeutic agent only in cancer cells, thereby allowing a maximal therapeutic effect while limiting systemic toxicity. A literature search was conducted using the MEDLINE database from 1990 to 2001 to identify articles related to suicide gene therapy for cancer. A number of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene. Various vectors, including liposomes, retroviruses, and adenoviruses, have been used to transfer these suicide genes to tumor cells. These strategies have been effective in cell culture experiments, laboratory animals, and some early clinical trials. Advances in tissue- and cell-specific delivery of suicide genes using specific promoters will improve the clinical utility of suicide gene therapy.

Targeted Gene Therapy of Ovarian Cancer Using an Ovarian-Specific Promoter

Objectives. The “suicide” gene therapy of cancer using promoters such as cytomegalovirus could cause severe toxicity to normal tissues due to a lack of specificity of prodrug activation. Therefore, we investigated gene therapy of ovarian cancer using ovarian-specific promoter (OSP1) to limit the synthesis of the prodrug activating enzyme HSVtk to ovarian cancer cells.Methods. The HSVtk expressing plasmid pOSP1–HSVtk was created and transfected into an ovarian cancer cell line OVCAR3. The ganciclovir (GCV) sensitivity of the stable transfectants was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Tissue specificity of this promoter was evaluated by comparing the sensitivity to GCV between ovarian and nonovarian cancer cell lines after they were transfected with pOSP1–HSVtk. One transfectant sensitive to GCV was implanted intraperitoneally to immunocompromised mice which were treated subsequently with GCV. Furthermore, this ovarian cancer survival model was used to evaluate the in vivo efficacy of cationic lipid mediated pOSP1–HSVtk gene delivery followed by GCV treatment.Results. Stable transfectants of OVCAR3 cells bearing OSP1–HSVtk became more sensitive to GCV treatment compared to the parental cell line and vector transfected OVCAR3 cell line. OSP1–HSVtk could specifically sensitize the OVCAR3 ovarian cancer cell line to GCV. SCID mice transplanted with the OVCAR3 transfectant and treated with GCV survived longer than the mice without GCV treatment (P = 0.032). In vivo gene delivery mediated by a cationic lipid (GL67) followed by GCV treatment yielded a longer survival in the OVCAR3 survival model (P = 0.016).Conclusions. The OSP1 promoter can selectively direct suicide gene therapy of ovarian cancer and the in vivo efficacy is improved by using a cationic lipid GL67 as delivery vehicle as opposed to the direct injection of plasmid.

Combined suicide gene therapy and oncolytic viral therapy for prostate cancer

Combined suicide gene therapy and oncolytic viral therapy for prostate cancer

Bystander Effects of Nucleoside Analogs Phosphorylated in the Cytosol or Mitochondria

The efficiency of nucleoside kinase suicide gene therapy for cancer is highly dependent on “bystander” cell killing, i.e., the transfer of cytotoxic phosphorylated nucleoside analogs to cells adjacent to those expressing the suicide enzyme. We have recently studied the possible use of mitochondrial nucleoside kinases as suicide genes. In the present study, we investigated if nucleoside analogs phosphorylated in the mitochondrial matrix cause bystander killing. We used deoxycytidine kinase-deficient Chinese hamster ovary cells reconstituted with deoxycytidine kinase targeted to either the cytosol or mitochondria matrix and determined the bystander cell killing when these cells were incubated with the nucleoside analogs 1-β-d-arabinofuranosylcytosine and 2′,2′-difluorodeoxycytidine. A bystander effect occurred when nucleoside analogs were phosphorylated in the cytosol, but not when these compounds were phosphorylated in the mitochondria. These findings suggest that nucleoside kinases targeted to the mitochondrial matrix have limited use in suicide gene therapy when efficient bystander cell killing is required.

Suicide Gene Therapy of Cancer

Suicide Gene Therapy of Cancer

Necrotic, rather than apoptotic, cell death caused by cytochrome P450-activated ifosfamide.

Feline kidney cells were transfected with a vector overexpressing cytochrome P450 2B1 (CYP2B1). Transfected cells acquired a new specific biochemical activity, which could be demonstrated by a rapid CYP2B1 detection assay and showed selective sensitivity to the antitumorigenic prodrug ifosfamide (IFO). Further, the cell-killing effect was also mediated on nonmodified cells like feline kidney cells, mouse lymphoma, and human pancreatic cells in the vicinity of the CYP2B1-expressing cells due to the diffusible nature of the activated IFO metabolites. One of these, phosphoramide mustard, causes interstrand DNA cross-linking and it has been thought that the inability to repair this damage results in apoptosis. Surprisingly, our results clearly demonstrate a necrotic mechanism of IFO-induced cell death. This may have important implications for the activation of the immune system during CYP2B1/IFO suicide gene therapy of cancer.

In vivo sensitization of ovarian tumors to chemotherapy by expression of E. coli purine nucleoside phosphorylase in a small fraction of cells.

This report examines a major barrier to suicide gene therapy in cancer and other diseases: namely, bystander cell killing. Existing vectors for in vivo gene delivery are inefficient and often transduce or transfect less than 1% of target cells. The E. coli PNP gene brings about cellular necrosis under conditions when 1 in 100 to 1 in 1000 cells express the gene product in vitro. In vivo bystander killing at or near this magnitude has not been reported previously. In the present experiments, transfection of cells with the E. coli PNP gene controlled by a SV40 promoter resulted in 30 nmol 6-methyl purine deoxyriboside (MeP-dR) converted per milligram tumor cell extract per hour (or conversion units (CU)). This level of expression led to elimination of entire populations of tumor cells in vitro after treatment with MeP-dR. Much earlier killing was observed using a tat transactivated E. coli PNP vector (approximately seven-fold higher activity, 230 CU). In vivo effects on tumor growth were next examined. Human ovarian tumors transfected with E. coli PNP were excised 5 days after i.p. implantation from the peritoneal cavities of mice in order to determine both E. coli PNP enzymatic activity and the fraction of cells expressing the gene. PNP activity at 5 days after gene transfer was approximately 170 CU and was expressed in approximately 0.1% of the tumor cells as judged by in situ hybridization. The expression of E. coli PNP at this level produced a 30% increase in life span (P < 0.001) and 49% reduction in tumor size (P < 0.005) after MeP-dR treatment, as compared with control tumors. Our observations lead to the conclusion that pronounced bystander killing by E. coli PNP is conferred in vivo, and that vectors capable of transgene expression in as few as one in 1000 cells can produce substantial antitumor effects if expression on a per cell basis is very high.

CL1-GFP: AN ANDROGEN INDEPENDENT METASTATIC TUMOR MODEL FOR PROSTATE CANCER

The mechanisms responsible for tumor progression to androgen independence in prostate cancer (CaP) remain unknown. To characterize these changes and provide a basis for rational therapeutic strategies for advanced CaP, an in vivo model from a highly aggressive androgen independent CaP cell line with distinct cellular and molecular properties was developed. An aggressive androgen-independent cell line designated CL1 was derived from a slow-growing, and androgen-dependent, parental LNCaP cell line through in-vitro androgen-deprivation and selection. CL1 was stably transfected with a green fluorescence protein gene (CL1-GFP) and orthotopically injected into SCID mice. The pathologic behavior, histology, and molecular determinants of CL1 tumor and metastases were determined and characterized by standard light and fluorescent microscopy, and quantitative RT-PCR analysis. CL1 is an anaplastic prostate cancer cell line which demonstrates extensive local invasion and metastases to various organs that can be visualized via GFP expression. When compared with parental LNCaP cells, RT-PCR analysis of the tumor revealed an over-expression of EGFR, b-FGF, VEGF, TGF-beta, IL-8, IL-6, and bcl-2 and a down regulated expression of the p53, E-cadherin and PTEN. In contrast to LNCaP cells, CL1 tumors express lower levels of androgen receptor and barely detectable PSA mRNA. CL1-GFP represents an aggressive androgen-independent CaP tumor model derived through androgen deprivation whose pathologic development and molecular properties in animals resembles the clinical characteristics of hormone refractory prostate cancer (HRPC). Metastatic sites of CL1-GFP can be visualized with fluorescence microscopy offering a unique therapeutic model for the evaluation of drug sensitivity and other therapeutic modalities.

Gene therapy of rat medullary thyroid cancer by naked nitric oxide synthase II DNA injection

Background Nitric oxide (NO), produced by NO synthase II (NOS II), is the main mediator of the tumoricidal action of activated macrophages. In the present study we examined the potential of the NOS II gene as a suicide gene for medullary thyroid cancer (MTC) therapy. Methods We orthotopically transplanted rMTC 6–23 cells into the inbred strain of Wag/Rij rats and constructed a plasmid carrying the NOS II gene under the control of the cytomegalovirus (CMV) promoter. Results Successive injections of tumor cells (Day 0) and naked DNA (Day 2) caused strong inhibition of tumor growth (50%, p<0.05). Plasmid injection into established tumors (14-day tumors) resulted in the development of large cavities due to tumor cell destruction, with a significant reduction in tumor tissue volume (35%, p<0.05). Adjacent quiescent tissues were unaffected. Cell death occurred by apoptosis as demonstrated by specific labeling. Macrophages and CD4+ lymphocytes were recruited in the treated tumors. However, tumor-specific T lymphocytes were undetectable in the spleen of treated rats. In control experiments using Lac Z as a reporter gene, expression of β-galactosidase was detected in only 1% of the tumor cells. Conclusions Despite a low gene transfer efficiency, NOS II plasmid produced a strong anti-tumor action resulting from its marked ‘bystander’ effect mainly due to NO production and diffusion. Therefore the NOS II gene appears to be a promising suicide gene therapy of human cancer. Copyright © 2000 John Wiley & Sons, Ltd.

In vivo adenovirus-mediated suicide gene therapy of orthotopic bladder cancer.

These studies were undertaken to determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase (ADV/RSV-tk) and the prodrug ganciclovir (GCV) in an orthotopic murine bladder cancer model. We utilized a replication-defective adenoviral construct containing the beta-galactosidase gene as a control and the herpes simplex virus thymidine kinase gene as the therapeutic vector under the transcription control of the Rous sarcoma virus long terminal repeat promoter. Intravesically created, orthotopic bladder tumors were established in syngeneic C3H/He female mice. India ink injection and beta-galactosidase studies were performed to determine if transurethral administration, direct tumor injection, or the combination was the most efficient route of virus administration. Optimal dosing of ADV/RSV-tk was determined by direct tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate but similar controlled experiments. Growth curve studies demonstrated reliable tumor formation by 14 days. Direct transvesical tumor injection resulted in the best distribution and intratumor gene expression as measured by X-gal staining. Dose-ranging experiments demonstrated an optimal viral dose of 5 x 10(8) plaque-forming units and a greater than twofold reduction in tumor growth for the animals treated with ADV/RSV-tk compared to controls. Efficacy studies demonstrated a greater than threefold reduction in tumor growth. No clinical or gross pathologic toxicity was detected. Long-term survival results suggested a survival benefit for the treatment animals compared to controls. We conclude that ADV/RSV-tk in combination with GCV provides effective therapy for orthotopic murine bladder cancer by significantly inhibiting tumor growth with limited toxicity to the host. These data provide further support for testing this suicide gene therapy strategy in human Phase I trials.

Efficacy of herpes simplex virus thymidine kinase in combination with cytokine gene therapy in an experimental metastatic breast cancer model.

Immunotherapy in combination with suicide gene therapy for breast cancer was tested using a metastatic animal model. Subcutaneous injection of the nonimmunogenic breast cancer cell line 4T1 in BALB/C mice gave rise to tumors in 100% of mice with both micrometastases and macrometastases in the lung. We used the herpes simplex virus thymidine kinase (HSV-TK) gene along with the cytokine genes granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) to determine their effect on tumor regression and inhibition of lung metastasis. Adenoviral (AV) vectors carrying these transgenes, in separate constructs, were used in this study. Intratumoral administration of AV-TK followed by 10 days of ganciclovir treatment resulted in a delay in tumor growth and, in some cases, in a low to moderate reduction in tumor volume. Inclusion of either GM-CSF or IL-2 gene with HSV-TK resulted in a slightly greater reduction in tumor volume, although these data were not significantly different from those obtained for TK treatment alone. However, when both cytokine genes were combined with TK, a substantial reduction in tumor growth was observed compared with HSV-TK alone (P < .02). Tumor weight data also exhibited superior efficacy of TK/GM-CSF/IL-2 treatment when compared with animals treated with TK gene only (P < .01). More importantly, TK/GM-CSF/IL-2 combination gene therapy induced a significant reduction in lung metastasis compared with any other treatment groups in the 4T1 model (P < .001 between TK GM-CSF/IL-2 versus TK only). Surgical excision of primary tumors after TK/GM-CSF/IL-2 plus ganciclovir treatment resulted in anti-metastatic activity that was similar to that observed for animals in which no surgery was performed. Survival analysis showed a significant difference between animals treated with AV-TK/GM-CSF/IL-2 and animals treated with TK only at 35 days after virus injection (P < .01). Immunophenotypic data suggest infiltration of lymphocytes within the tumor microenvironment in TK- and cytokine gene-treated animals. Thus, the overall data presented here demonstrate that TK gene therapy in combination with GM-CSF and IL-2 gene-mediated immunotherapy strategies have important implications in the treatment of breast cancer.

Suicide gene therapy for urogenital cancer: current outcome and prospects.

Viral-mediated transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene has been demonstrated by several investigators to confer sensitivity to nucleoside analogs such as ganciclovir (GCV) in a variety of tumor cells including brain, prostate, bladder, kidney, ovary, head and neck, lung, pancreas, and liver cancers. Fourteen suicide gene clinical protocols using adenovirus vectors have been conducted, including four in prostate cancer. Two additional protocols for prostate cancer are in preparation in Japan and the Netherlands. A study conducted at Baylor College of Medicine was the first to demonstrate the safety of HSV-tk plus GCV therapy for human prostate cancer and the anticancer activity of gene therapy in this disease. However, it is still in the early stage of its development, with a number of problems to be overcome. Systemic delivery, specific introduction, and specific expression of the target gene are the major issues to be managed in order to establish a clinically relevant treatment strategy.

Effective suicide gene therapy in vivo by EBV-based plasmid vector coupled with polyamidoamine dendrimer.

This study demonstrates in vivo effectiveness of a nonviral vector system, Epstein-Barr virus (EBV)-based plasmid vector coupled with polyamidoamine (PAMAM) dendrimer (EBV/polyplex), in suicide gene therapy of cancer. The EBV-based vector is a plasmid vector containing EBV nuclear antigen 1 (EBNA1) gene and oriP from EBV genome. HSV-1 tk gene was transferred into Ewing's sarcoma cell lines, A4573 and KP-EWS-YI, by using an EBV-based plasmid vector, pSES.Tk, or a conventional plasmid vector, pS.Tk. Cells transfected with pSES.Tk/dendrimer showed approximately 100 times lower ID50 to ganciclovir (GCV) compared with those transfected with pS. Tk/dendrimer. Intratumoral injection of pSES.Tk/dendrimer but not pS. Tk/dendrimer drastically suppressed the growth of tumors which had generated from A4573 or Huh7 hepatocellular carcinoma (HCC) cells inoculated into severe combined immunodeficiency (SCID) mice. The treatment with pSES.Tk/dendrimer also resulted in significant prolongation of survival of the mice implanted with A4573. These results suggest that the EBV/polyplex system could be useful for in vivo suicide gene therapy of cancer. Gene Therapy (2000) 7, 53-60.

Combined suicide gene therapy and radiotherapy for prostate cancer: A phase I study

Combined suicide gene therapy and radiotherapy for prostate cancer: A phase I study