Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.

BACKGROUND: Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes. METHODS: Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and ß-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes. CONCLUSIONS: These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.

Recent advances in molecular biomarkers for diabetes mellitus: a systematic review.

CONTEXT: Diabetes is a growing global metabolic epidemic. Current research is focussing on exploring how the biological processes and clinical outcomes of diabetes are related and developing novel biomarkers to measure these relationships, as this can subsequently improve diagnostic, therapeutic and management capacity. OBJECTIVE: The objective of this study is to identify the most recent advances in molecular biomarkers of diabetes and directions that warrant further research. METHODS: Using a systematic search strategy, the MEDLINE, CINAHL and OVID MEDLINE databases were canvassed for articles that investigated molecular biomarkers for diabetes. Initial selections were made based on article title, whilst final inclusion was informed by a critical appraisal of the full text of each article. RESULTS: The systematic search returned 246 records, of which 113 were unique. Following screening, 29 records were included in the final review. Three main research strategies (the development of novel technologies, broad biomarker panels, and targeted approaches) identified a number of potential biomarkers for diabetes including miR-126, C-reactive protein, 2-aminoadipic acid and betatrophin. CONCLUSION: The most promising research avenue identified is the detection and quantification of micro RNA. Further, the utilisation of functionalised electrodes as a means to detect biomarker compounds also warrants attention.

Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line.

Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 106 cells, respectively. Additionally, several ß cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0-20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.

Concise review: Nanoparticles and cellular carriers-allies in cancer imaging and cellular gene therapy?

Ineffective treatment and poor patient management continue to plague the arena of clinical oncology. The crucial issues include inadequate treatment efficacy due to ineffective targeting of cancer deposits, systemic toxicities, suboptimal cancer detection and disease monitoring. This has led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable therapies. Mesenchymal stem cells (MSCs) have the intrinsic ability to "home" to growing tumors and are hypoimmunogenic. Therefore, these can be used as (a) "Trojan Horses" to deliver gene therapy directly into the tumors and (b) carriers of nanoparticles to allow cell tracking and simultaneous cancer detection. The camouflage of MSC carriers can potentially tackle the issues of safety, vector, and/or transgene immunogenicity as well as nanoparticle clearance and toxicity. The versatility of the nanotechnology platform could allow cellular tracking using single or multimodal imaging modalities. Toward that end, noninvasive magnetic resonance imaging (MRI) is fast becoming a clinical favorite, though there is scope for improvement in its accuracy and sensitivity. In that, use of superparamagnetic iron-oxide nanoparticles (SPION) as MRI contrast enhancers may be the best option for tracking therapeutic MSC. The prospects and consequences of synergistic approaches using MSC carriers, gene therapy, and SPION in developing cancer diagnostics and therapeutics are discussed.

Improved granulocyte colony-stimulating factor mobilization of hemopoietic progenitors using cytokine combinations in primates.

Peripheral blood stem cells (PBSCs), usually mobilized with granulocyte colony-stimulating factor (G-CSF) alone or in combination with chemotherapy, are the preferred source of cells for hemopoietic stem cell transplantation. Up to 25% of otherwise eligible transplant recipients fail to harvest adequate PBSCs. Therefore it is important to investigate existing and novel reagents to improve PBSC mobilization. Because of marked interindividual variation in humans, we developed a robust nonhuman primate model that allows the direct comparison of the efficacy of two PBSC mobilization regimens within the same animal. Using this model, we compared pegylated G-CSF (pegG-CSF) with standard G-CSF and compared the combination of G-CSF and pegylated megakaryocyte growth and development factor (pegMGDF) with G-CSF plus stem cell factor (SCF) by measuring the levels of CD34(+) cells, colony-forming cells (CFCs), and SCID repopulating cells (SRCs) before and after cytokine administration. Mobilization of CD34(+) cells, CFCs and SRCs using pegG-CSF achieved similar levels to those resulting from 5 days of standard G-CSF. The combination of G-CSF+pegMGDF mobilized progenitors to levels similar to G-CSF+SCF but greater than standard G-CSF for CD34(+) cells and CFC. This first direct comparison of PBSC mobilization in individual primates demonstrates that peg-G-CSF is equivalent to daily G-CSF and that the addition of pegMGDF to G-CSF improves mobilization. In light of the development of new thrombopoietin agonists, these data offer the potential for improved stem cell mobilization strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells.

Due to their ease of isolation, gene modification and tumor-homing properties, mesenchymal stem cells (MSCs) are an attractive cellular vehicle for the delivery of toxic suicide genes to a variety of cancers in pre-clinical models. In addition, the incorporation of suicide genes in stem cell-derived cell replacement therapies improves their safety profile by permitting graft destruction in the event of unexpected tumorigeneses or unwanted differentiation. Due to the functional requirement of ATP for the Firefly luciferase gene Luc2 to produce light, luciferase-based reporting of cytotoxicity can be engineered into potential cell therapies. Consequently, we nucleofected mammalian expression plasmids containing both the Luc2 and the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) genes for expression in murine MSCs to assess luciferase as a reporter of suicide gene cytotoxicity, and MSC as vehicles of suicide gene therapy. In vitro bioluminescence imaging (BLI) showed that following the addition of the non-toxic prodrug fluorocytosine (5-FC), CDUPRT-expressing MSCs displayed enhanced cytotoxicity in comparison to Luc2 reporter MSC controls. This study demonstrates the utility of luciferase as a reporter of CDUPRT-mediated cytotoxicity in murine MSC using BLI.

High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells.

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.

Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic ß-Cells.

Combinatorial gene and cell therapy as a means of generating surrogate ß-cells has been investigated for the treatment of type 1 diabetes (T1D) for a number of years with varying success. One of the limitations of current cell therapies for T1D is the inability to generate sufficient quantities of functional transplantable insulin-producing cells. Due to their impressive immunomodulatory properties, in addition to their ease of expansion and genetic modification ex vivo, mesenchymal stem cells (MSCs) are an attractive alternative source of adult stem cells for regenerative medicine. To overcome the aforementioned limitation of current therapies, we assessed the utility of ex vivo expanded bone marrow-derived murine MSCs for their persistence in immune-competent and immune-deficient animal models and their ability to differentiate into surrogate ß-cells. CD45-/Ly6+ murine MSCs were isolated from the bone marrow of nonobese diabetic (NOD) mice and nucleofected to express the bioluminescent protein, Firefly luciferase (Luc2). The persistence of a subcutaneous (s.c.) transplant of Luc2-expressing MSCs was assessed in immune-competent (NOD) (n = 4) and immune-deficient (NOD/Scid) (n = 4) animal models of diabetes. Luc2-expressing MSCs persisted for 2 and 12 weeks, respectively, in NOD and NOD/Scid mice. Ex vivo expanded MSCs were transduced with the HMD lentiviral vector (MOI = 10) to express furin-cleavable human insulin (INS-FUR) and murine NeuroD1 and Pdx1. This was followed by the characterization of pancreatic transdifferentiation via reverse transcriptase polymerase chain reaction (RT-PCR) and static and glucose-stimulated insulin secretion (GSIS). INS-FUR-expressing MSCs were assessed for their ability to reverse diabetes after transplantation into streptozotocin- (STZ-) diabetic NOD/Scid mice (n = 5). Transduced MSCs did not undergo pancreatic transdifferentiation, as determined by RT-PCR analyses, lacked glucose responsiveness, and upon transplantation did not reverse diabetes. The data suggest that ex vivo expanded MSCs lose their multipotent differentiation potential and may be more useful as gene therapy targets prior to expansion.

CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

Due to their ease of isolation, differentiation capabilities, and immunomodulatory properties, the therapeutic potential of mesenchymal stem cells (MSCs) has been assessed in numerous pre-clinical and clinical settings. Currently, whole pancreas or islet transplantation is the only cure for people with type 1 diabetes (T1D) and, due to the autoimmune nature of the disease, MSCs have been utilised either natively or transdifferentiated into insulin-producing cells (IPCs) as an alternative treatment. However, the initial success in pre-clinical animal models has not translated into successful clinical outcomes. Thus, this review will summarise the current state of MSC-derived therapies for the treatment of T1D in both the pre-clinical and clinical setting, in particular their use as an immunomodulatory therapy and targets for the generation of IPCs via gene modification. In this review, we highlight the limitations of current clinical trials of MSCs for the treatment of T1D, and suggest the novel clustered regularly interspaced short palindromic repeat (CRISPR) gene-editing technology and improved clinical trial design as strategies to translate pre-clinical success to the clinical setting.

Transcription-targeted gene therapy for androgen-independent prostate cancer.

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.

Derivation of MPR and TRAMP models of prostate cancer and prostate cancer metastasis for evaluation of therapeutic strategies.

Pre-clinical models of primary and metastatic prostate cancer are increasingly needed to evaluate efficacy of the new therapeutic strategies currently under investigation. The androgen-independent RM1 and androgen-dependent TR cell lines derived from transgenic mouse models of prostate cancer were examined in this regard. Following implantation in immune competent mice, the RM1 cell line was able to generate extremely fast growing s.c. and iprost tumors and metastatic lung lesions providing a time period of approximately 14-17 days from the time of tumor establishment to animal sacrifice to assess therapies. Implantation of TR cell lines resulted in more slowly growing s.c. and iprost tumors and metastatic lung lesions that exhibited highly variable incidence and growth. These models represent the best available means to evaluate therapeutics in primary and metastatic prostate cancer variants in an intact immune system.

Application of the transgenic adenocarcinoma mouse prostate (TRAMP) model for pre-clinical therapeutic studies.

BACKGROUND: The suitability of (C57BL/6 TRAMP x C57BL/6)F1 transgenic (TRAMP+) mice with well- to moderately-differentiated prostate cancer (PCa) was assessed for pre-clinical therapeutic studies. MATERIALS AND METHODS: TRAMP+ and TRAMP- mice were assessed for variability in genitourinary tract weight, seminal vesicle weight, prostate weight/volume and histopathology. Time-points included the reported ages of average tumour onset (approximately 25 weeks) and PCa-induced death (approximately 33 weeks). RESULTS: Seventy % of TRAMP+ mice aged 25-33 weeks had well- to moderately-differentiated PCa. At 25-28 weeks, the mean genitourinary tract weight was 2X greater and the mean prostate weight/volume was 1.5X more in TRAMP+ than in TRAMP- mice, respectively. Prostate weight/volume showed significant increases (p < 0.0001) by 2X and 3X, respectively by 31-33 weeks of age. CONCLUSION: The window for using the TRAMP model successfully for pre-clinical experimentation is 25-33 weeks provided that mice with poorly-differentiated PCa showing a large tumour burden are excluded.

Sphingosine kinase 1 isoform-specific interactions in breast cancer.

Sphingosine kinase 1 (SK1) is a signaling enzyme that catalyzes the formation of sphingosine-1-phosphate. Overexpression of SK1 is causally associated with breast cancer progression and resistance to therapy. SK1 inhibitors are currently being investigated as promising breast cancer therapies. Two major transcriptional isoforms, SK143 kDa and SK151 kDa, have been identified; however, the 51 kDa variant is predominant in breast cancer cells. No studies have investigated the protein-protein interactions of the 51 kDa isoform and whether the two SK1 isoforms differ significantly in their interactions. Seeking an understanding of the regulation and role of SK1, we used a triple-labeling stable isotope labeling by amino acids in cell culture-based approach to identify SK1-interacting proteins common and unique to both isoforms. Of approximately 850 quantified proteins in SK1 immunoprecipitates, a high-confidence list of 30 protein interactions with each SK1 isoform was generated via a meta-analysis of multiple experimental replicates. Many of the novel identified SK1 interaction partners such as supervillin, drebrin, and the myristoylated alanine-rich C-kinase substrate-related protein supported and highlighted previously implicated roles of SK1 in breast cancer cell migration, adhesion, and cytoskeletal remodeling. Of these interactions, several were found to be exclusive to the 43 kDa isoform of SK1, including the protein phosphatase 2A, a previously identified SK1-interacting protein. Other proteins such as allograft inflammatory factor 1-like protein, the latent-transforming growth factor ß-binding protein, and dipeptidyl peptidase 2 were found to associate exclusively with the 51 kDa isoform of SK1. In this report, we have identified common and isoform-specific SK1-interacting partners that provide insight into the molecular mechanisms that drive SK1-mediated oncogenicity.

p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-ß-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) show great promise for ex vivo gene and cell-mediated therapies. The immunophenotype and in vitro differentiation capacity of primary baboon MSCs was demonstrated to be near-identical to that observed in human MSCs. To optimize gene transfer efficiency, we compared the efficiency of serotypes 1, 2, 3, 4, 5, 6, and 8 of adeno-associated virus (AAV) vectors for their ability to mediate transduction of human and baboon MSCs. AAV serotype 2 vectors were the most efficient in transducing MSCs from humans and baboons. As a reference, human Ad293 cells were transduced with these seven AAV serotypes, and were found to have the highest transduction levels followed by baboon MSCs, and then human MSCs. The order of increasing transduction efficiency for the serotypes tested was similar for human and baboon MSCs, but was different for human Ad293 cells. The transduction efficiency of MSCs isolated from different individuals was comparable within the same species. We also demonstrated that baboon MSCs transduced with AAV serotype 2 vectors retain their potential to differentiate into adipocytes in vitro, and can incorporate into injured muscle tissue of NODSCID mice in vivo. We detected beta-galactosidase reporter gene expression in host muscle tissue for up to 9 weeks in this study, indicating engraftment of transduced baboon MSCs and sustained transgene expression in vivo.

Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice.

BACKGROUND: We aimed to evaluate the efficacy of gene-directed enzyme-prodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'. METHODS: Stably transformed andogen-independent mouse prostate cancer (PC) cells, RM1-CDUPRT, -GFP or GFP/LacZ cells were used. To assess the local bystander effects of CDUPRT-GDEPT, immunocompetent C57BL/6 mice implanted with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions intraprostatically were treated with 5FC. Pseudo-metastases in the lungs were established by a tail vein injection of untransfected RM1 cells. At necropsy, prostate weight/volume and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. RESULTS: CDUPRT expression in RM1-GFP/CDUPRT cells or tumors was confirmed by enzymic conversion of 5FC into 5FU, using HPLC. Treatment of mice bearing intraprostatic RM1-GFP/CDUPRT tumors with 5FC resulted in complete regression of the tumors. A 'local bystander effect' was seen, even though only 20% of the cells expressed CDUPRT. More importantly a significant reduction in pseudo-metastases of RM1 cells in lungs indicated a 'distant bystander effect'. Immunohistochemical evaluation of the treated tumors showed increased necrosis and apoptosis, with decreased tumor vascularity. There was also a significant increase in tumour-infiltration by macrophages, CD4+ T and natural killer cells. CONCLUSIONS: We conclude that CDUPRT-GDEPT significantly suppressed the aggressive growth of RM1 prostate tumors and lung pseudo-metastases via immune mechanisms involving necrosis and apoptosis.

Purine nucleoside phosphorylase and fludarabine phosphate gene-directed enzyme prodrug therapy suppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer.

Gene-directed enzyme prodrug therapy based on the E. coli purine nucleoside phosphorylase (PNP) gene produces efficient tumour cell killing. PNP converts adenosine analogs into toxic metabolites that diffuse across cell membranes to kill neighbouring untransduced cells (PNP-GDEPT). Interference with DNA, RNA and protein synthesis kills dividing and non-dividing cells, an important consideration for slow-growing prostate tumours. This study examined the impact of administering PNP-GDEPT into orthotopically grown RM1 prostate cancers (PCas) on the growth of lung pseudo-metastases of immunocompetent mice. C57BL/6 mice bearing orthotopic RM1 PCas received a single intraprostatic injection of OAdV220 (10(10) particles), a recombinant ovine atadenovirus containing the PNP gene controlled by the Rous Sarcoma virus promoter, followed by fludarabine phosphate (approximately 600 mg/m(2)/day) administered intraperitoneally (ip) once daily for 5 days. Pseudo-metastases were induced 2 days after intraprostatic vector administration by tail-vein injection of untransduced RM1 cells. Mice given PNP-GDEPT showed a significant reduction both in prostate volume (approximately 50%) and in lung colony counts (approximately 60%). Apoptosis was increased two-fold in GDEPT-treated prostates compared with controls (P < 0.01), but was absent in the lungs. Staining for proliferating cell nuclear antigen (PCNA) indicated that proliferation of both RM1 prostate tumours (P < 0.01) and lung colonies (P < 0.01) was significantly suppressed after GDEPT. Although prostate tumour immune cell infiltration did not differ significantly between treatments, immunostaining for Thy-1.2 (CD90) showed that GDEPT promoted Thy-1.2(+) cell infiltration into the prostate tumour site. This study showed that a single course of PNP-GDEPT significantly suppressed local PCa growth and reduced lung colony formation in the aggressive RM1 tumour model.

Gene-directed enzyme prodrug therapy for prostate cancer in a mouse model that imitates the development of human disease.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) based on the E. coli enzyme purine nucleoside phosphorylase (PNP) represents a new approach for treating slow growing tumours like prostate cancer (PCa). Expressed enzyme converts a systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine. Infected and neighbouring cells are killed by a bystander effect that results from the inhibition of DNA and RNA synthesis. METHODS: These studies were carried out using the transgenic adenocarcinoma of the prostate (TRAMP) model that mimics human PCa development and progression. Control TRAMP mice were injected intraprostatically with vector vehicle and thereafter intraperitoneally with saline or fludarabine phosphate ( approximately 600 mg/m(2)/day) once daily for 5 consecutive days. Treated mice received a single intraprostatic injection containing 10(10) particles of OAdV220, an ovine atadenovirus which expresses the E. coli PNP gene under the control of the Rous sarcoma virus promoter, followed by systemic fludarabine treatment. The weight of the genitourinary tract, seminal vesicles and the prostate as well as animal survival were monitored. Tumours were also analysed histologically. RESULTS: Preliminary studies showed that fludarabine alone caused no significant change in genitourinary (GU) tract weight in TRAMP mice. Animals injected with vector and prodrug showed a significant reduction (36-47%) in GU tract weight (ANOVA p = 0.0002) and a 35-50% reduction in seminal vesicle weight (ANOVA p = 0.0007). In particular, the target organ showed a significant 57% reduction in prostate weight (ANOVA p = 0.0007). PNP-GDEPT mice also showed a survival advantage over control mice. Histological analysis suggested that the cancer progression was slowed in GDEPT-treated animals. CONCLUSION: A single course of GDEPT based on OAdV-delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune-competent TRAMP mice.