A gene-directed enzyme pro-drug therapy (GDEPT) based on purine nucleoside phosphorylase (PNP)

No abstract

A single course of GDEPT based on OAdV-delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune-competent TRAMP mice.

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.

No abstract

To develop animal models that represent the broad spectrum of human prostate cancer, we created transgenic mice with targeted prostate-specific expression of two genes (EcoRI and c-fos) implicated in the induction of genomic instability. Expression of the transgenes was restricted to prostate epithelial cells by coupling them to the tissuespecific, hormonally regulated probasin promoter (PB). The effects of transgene expression were examined histologically in prostate sections at time points taken from 4 to 24 months of age. The progressive presence of regions of mild-to-severe hyperplasia, low-and high-grade prostatic intra-epithelial neoplasia, and well-differentiated adenocarcinoma was observed in both PBEcoRI lines but no significant pathology was detected in the PBfos line. Prostate tissue of PBEcoRI mice was examined for expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 at multiple time points. Although p53 does not appear to be mutated, levels of PCNA and Ki67 are elevated and correlate with the severity of the prostatic lesions. Overall, pre-neoplastic and neoplastic stages represented in the PBEcoRI model showed similarity to corresponding early stages of the human disease. This genomic instability-based model will be used to study the mechanisms involved in the early stages of prostate carcinogenesis and to investigate the nature of subsequent events necessary for the progression to advanced disease.Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer death in American men but its etiology is poorly understood. One in five American males will eventually develop the disease but the high rate of clinical incidence is exceeded by the prevalence of prostate carcinomas undetected until autopsy (1,2). Although several Abbreviations: PB, probasin promoter; PCNA, proliferating cell nuclear antigen; PIN, prostatic intra-epithelial neoplasia; TRAMP, transgenic adenocarcinoma mouse prostate.© Oxford University Press 1623 genetic alterations have been documented (3), critical early molecular events involved in neoplastic initiation and progression from latent prostate cancer to the aggressive form of clinical disease remain poorly understood. Therefore, animal models that faithfully represent the broad spectrum of the human disease are required. A number of animal models for prostate cancer have been developed (4) but many lack a natural environment and characterization of early and late stages is not possible. The most successful mouse models to date utilize targeted expression of the SV40 early genes (T/t antigens) that function as oncoproteins, in part, by binding to and interfering with the p53 and/or Rb tumor suppressor proteins or by interfering with protein phosphatase 2A activity (5).In an attempt to create a model for early prostate cancer, we generated transgenic mice in which the restriction enzyme EcoRI or the proto-oncogene c-fos were expressed from a truncated rat probasin promoter. This promoter has been shown to express the SV40 early ...

This chapter describes procedural aspects for development of ribozymes in general, and specifically, that cleave mRNA to an essential cellular gene, the AC40 subunit of RNA pol I. Ribozyme design includes functional selection of binding sites followed by computer modeling. These ribozymes are being used in vectors that target expression to the prostate via tissue specific promoters (Voeks, Norris, and Clawson, 1998) and have demonstrated efficacy.

To develop animal models that represent the broad spectrum of human prostate cancer, we created transgenic mice with targeted prostate-specific expression of two genes (ECO:RI and c-fos) implicated in the induction of genomic instability. Expression of the transgenes was restricted to prostate epithelial cells by coupling them to the tissue-specific, hormonally regulated probasin promoter (PB). The effects of transgene expression were examined histologically in prostate sections at time points taken from 4 to 24 months of age. The progressive presence of regions of mild-to-severe hyperplasia, low- and high-grade prostatic intra-epithelial neoplasia, and well-differentiated adenocarcinoma was observed in both PBECO:RI lines but no significant pathology was detected in the PBfos line. Prostate tissue of PBECO:RI mice was examined for expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 at multiple time points. Although p53 does not appear to be mutated, levels of PCNA and Ki67 are elevated and correlate with the severity of the prostatic lesions. Overall, pre-neoplastic and neoplastic stages represented in the PBECO:RI model showed similarity to corresponding early stages of the human disease. This genomic instability-based model will be used to study the mechanisms involved in the early stages of prostate carcinogenesis and to investigate the nature of subsequent events necessary for the progression to advanced disease.