Mouse Prostate Reconstitution Model Research Articles

Validation of transrectal ultrasonographic volumetry for orthotopic prostate tumours in mice.

Orthotopic human prostate tumour models in athymic nude mice are regarded as being most suitable for fundamental and pre-clinical research on prostate cancer. The anatomic localization of the tumour in the pelvis, however, provides little possibility for monitoring tumour growth or regression. To assess time-related changes in orthotopic tumour volume, we applied transrectal ultrasonography (TRUS) to the murine prostate. This technique has the advantages of allowing accurate monitoring of tumours during therapeutic manipulations and a reduction of animal use due to a reduction of sacrificing endpoints. To validate the TRUS method, the mouse prostate reconstitution model, RM-9, and the prostate-specific antigen (PSA) producing human prostate cancer xenograft PC-346 were used. Volumetric calliper measurements were performed with a 30 MHz ultrasound probe designed for intra-arterial use in humans. Tumour weight, determined at various time-points, was found to be closely related to actual tumour weight (R = 0.99) and, in the PC-346 model, to the level of PSA in the plasma. Furthermore, the interobserver variation for TRUS was low for tumours above 50 mg. Thus, TRUS for murine prostate tumours proves to be an accurate, reproducible and sensitive method.

Mouse prostate reconstitution model system: A series of in vivo and in vitro models for benign and malignant prostatic disease.

An elucidation of the complex, morphological and molecular changes that underlie benign and malignant prostatic disease will likely lead to improved methods of diagnosis and therapy for those disorders. To identify and understand the interrelation of the phenotypic and genetic changes inherent in these important diseases requires the development and use of in vivo and in vitro models that closely mimic specific aspects of the disease process. Once the suspected molecular underpinnings of prostatic disease are uncovered, in vivo and in vitro models will be required for further testing of the functional significance of specific genetic alterations as they are identified. In addition models of prostatic disease are necessary to evaluate novel therapeutic approaches. The mouse prostate reconstitution (MPR) model system was developed more than a decade ago with these specific needs in mind. Over the years, specific modifications of the MPR model have demonstrated its versatility and applicability for the study of benign and malignant prostatic disease, including metastatic progression. We discuss various modifications of the MPR model system made for its application to specific aspects of prostatic disease; the clinically relevant information that has been gleaned thus far from the use of this model system; and advances on the horizon for the expansion of its role in prostate research. The MPR model system has contributed substantially to the understanding and treatment of benign and malignant prostatic diseases. Additional modifications in this series of in vivo and in vitro models will likely lead to further advances.

Prostate cancer gene therapy: herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models.

Prostate cancer is the most common internal malignancy in men in the United States. Most cancers are diagnosed when they are locally advanced or metastatic and there is no effective treatment. In this study we evaluated the effectiveness of cytotoxic gene therapy in human PC-3 and DU145 prostate cancer cell lines and in a rodent cell line, RM-1, derived from the mouse prostate reconstitution model system. The cell lines were efficiently transduced in vitro by a replicative-defective recombinant adenovirus (ADV) carrying the herpes simplex virus thymidine kinase gene (HSV-tk). A virus titer-dependent sensitivity to ganciclovir (GCV) was observed. To determine a target therapeutic viral dose in vivo, subcutaneous tumors were generated by injection of RM-1 cells in syngeneic male hosts and injected with escalating doses of HSV-tk virus (5 x 10(7) to 1 x 10(9) pfu). The mice received GCV twice daily for 6 days and were sacrificed when tumor volumes exceeded 2.5 cm3 or when they appeared to be in distress. Because the two highest doses were equally as effective, further controlled studies were performed with the lower dose of 5 x 10(8) pfu with ADV/RSV-tk or a control virus containing the beta-galactosidase gene (ADV/RSV-beta-Gal) and treated with GCV or saline (PBS). The mean tumor volume in the treated animals was 16% that of control animals at 13 days. Histologically, treated tumors demonstrated necrosis and had a significantly higher apoptotic index. Survival data indicated that the treatment animals lived 7 days (21 in total) longer than the control animals, with 1 treatment animal being totally free of tumor. These results demonstrate that HSV-tk + GCV cytotoxic gene therapy can inhibit the growth of mouse and human prostate cancer cells in vitro and interrupt tumor growth of an aggressive mouse prostate cancer cell line in vivo.

Progression to androgen insensitivity in a novelin vitro mouse model for prostate cancer

We have shown previously that the ras and myc oncogenes can induce poorly differentiated mouse prostate carcinomas in vivo with high frequency (greater than 90%) using inbred C57BL/6 mice in the mouse prostate reconstitution model system. To study the androgen sensitivity of these carcinomas, we have developed an in vitro model system which includes a cell line from normal urogenital sinus epithelium (CUGE) and cell lines from three ras + myc transformed mouse prostate carcinomas (RM-9, RM-1, and RM-2). CUGE cells, as well as all prostate carcinoma cell lines, were positive for cytokeratin 18 mRNA and immunoreactive to cytokeratin-specific antiserum. Two out of three of the early passage carcinoma cell lines were clonal with respect to Zipras/myc 9 retrovirus integration as determined by Southern blot analysis. Whereas significant mitogenic effects of testosterone (10 nM) were not seen in CUGE cells grown in serum-free medium, under similar conditions approx. 2-fold increases in cell number were seen in all low passage prostate carcinoma cell lines. Also, in the presence of growth inhibitory levels of suramin (50 micrograms/ml), testosterone was capable of significant growth stimulation in the carcinoma cell lines. With further propagation from low passage [20-25 population doublings (PD)] to high passage (75-100 PD), all carcinoma cell lines demonstrated increased and similar growth rate in the presence and absence of testosterone. These cell lines maintained stable androgen receptor numbers and binding kinetics during the transition from testosterone-responsive growth to reduced responsivity over multiple passages in culture (> 150 PD). Overall, our studies indicate that the capacity to bind testosterone is stably maintained through the transition of the androgen-sensitive to insensitive phenotype and raise the possibility that androgen sensitivity can persist throughout progression but is masked by the acquisition of autocrine pathways.

Tissue and cell—cell interactions in prostate cancer progression

Prostate cancer is unique among human cancers in the wide discrepancy between the high prevalence of histologic changes recognizable as cancer (latent cancer) and the much lower prevalence of clinically recognizable disease (clinical cancer). Latent or incidental prostate cancer is often found at autopsy, affecting nearly 33% of all men older than 50 years, yet the number of new cases of clinically manifest prostate cancer represents only a small fraction of this latent disease. This discrepancy indicates that life-threatening prostate cancer results from progression of only a subset of the latent and clinically insignificant lesions and not the progression of all small tumors. Therefore, we must understand the underlying molecular and cellular determinants of prostate cancer progression. The role of tissue and cell-cell interactions in prostate cancer progression is complex. During early stages of progression, relatively normally organized stroma can suppress discrete steps in carcinogenesis. This phenomenon is exemplified by studies using the mouse prostate reconstitution (MPR) model system, where investigators showed that intrinsic properties of the mesenchyme can modulate the conversion of oncogene-initiated benign hyperplastic prostatic epithelium to the malignant phenotype. When the ras and myc oncogenes were introduced into both the mesenchymal and epithelial compartments of the microdissected urogenital sinus, poorly differentiated prostate cancer was produced at a high frequency (more than 90%) in inbred C57BL/6 mice. In contrast, benign prostatic hyperplasia converted to cancer at a low frequency (less than 10%) in BALB/c MPR mice under similar conditions. Heterologous MPR composed of BALB/c mesenchyme and C57BL/6 epithelium or vice versa showed that intrinsic properties of BALB/c mesenchyme can arrest the progression of ras + myc-initiated C57BL/6 epithelium from benign hyperplasia to cancer. Further progression of prostate cancer beyond its earliest forms of the malignant phenotype leads to increasingly disorganized stroma, where inappropriate stromal cell-cancer cell interactions can occur. Such interactions may include endothelial cell-cancer cell, lymphocyte-cancer cell, and neuron-cancer cell interactions. In addition to stromal cell-cancer cell interactions, the direct interaction of cancer cells with other cancer cells may lead to the establishment of important selection criteria whereby subsets of prostate cancer cells progress to metastatic disease. Understanding these various cell-cell interactions requires additional experimental studies that address the molecular and cellular basis of prostate cancer progression using appropriate model systems that contain the critical cell types. Recently we modified the MPR model system to involve the use of p53 knock-out urogenital sinus tissue as a target for oncogene-initiated prostate cancer. Using this protocol, we produced metastatic prostate cancer from normal cells in vivo. Importantly, the organ specificity of the metastatic disease closely resembles that in humans, and thus we can study genetic and phenotypic variability throughout the course of progression. In addition, because we use recombinant retroviruses to transduce the initiating oncogenes, we can track clonal progression and determine common progenitors of organ-specific metastatic disease.

Mesenchymal‐epithelial interactions and transforming growth factor‐β1 expression during normal and abnormal prostatic growth

Mesenchymal-epithelial interactions are associated with growth and morphogenesis of the prostate. We have detected three isoforms of transforming growth factor beta (TGF-beta) in the developing mouse prostate that may mediate some of these interactions. Separation of the fetal urogenital sinus (UGS) tissue into mesenchymal and epithelial components indicated that mRNA expression of TGF-beta 1, 2, and 3 was more abundant in the mesenchyme compared to the epithelium. Immunohistochemical analysis revealed accumulation of TGF-beta 1 in the mesenchyme surrounding ductules in the UGS and neonatal prostate. Further analysis of TGF-beta 1 localization in surgically removed adult human prostate tissues revealed more intense staining associated with regions of abnormal growth compared to normally appearing tissue. The percent of the total stained area with extracellular accumulation of TGF-beta 1 was 59% in prostate cancer, 26% in benign prostatic hyperplasia (BPH), and 8.6% in normal tissue. In additional immunohistochemical studies we observed that intracellular TGF-beta 1 was predominantly associated with the epithelial cells in prostate cancer (epithelial cells = 33.5% of the total stained area, stromal cells = 13.3%, and unstained = 53.2%), whereas in BPH intracellular TGF-beta 1 was predominantly associated with stromal cells (stromal cells = 32.2% of the total stained area, epithelial cells = 12.3%, and unstained = 55.5%). Although additional experimental and clinical studies are needed to better understand the relationships between TGF-beta 1 and abnormal prostatic growth, our observations thus far suggest a role for TGF-beta 1 in the development of benign and malignant growth abnormalities in the prostate. One approach to establishing the pathobiological significance of TGF-beta 1 accumulation in the prostate is by introducing and overexpressing the TGF-beta 1 cDNA in prostate tissue using the mouse prostate reconstitution model system. We discuss applicability of transgenic animal and organ reconstitution models for experimental studies concerning TGF-beta-induced prostate growth abnormalities.

DNA ploidy and clonal selection in ras + myc – Induced mouse prostate cancer

An important goal in prostate cancer research is to define specific molecular and cellular alterations that are associated with malignant progression. The mouse prostate reconstitution model is a relevant and useful system as it allows the study of early events in cancer progression under conditions where oncogene-initiated cells are surrounded by normal tissue. Using this model, activated ras and myc oncogenes are introduced into urogenital sinus cells via the recombinant retrovirus Zipras/myc 9. After 4 weeks' growth as subcapsular renal grafts, poorly differentiated carcinomas are produced in C57BL/6 mice. In this study we examined the temporal relationships between morphological alterations, growth, DNA ploidy status and clonal selection as determined by Southern blotting in ras + myc-initiated carcinomas. Nuclear image analysis demonstrated that the emergence of a cycling DNA tetraploid cell population strongly correlated with growth and histologic progression. These tightly linked events culminated in the outgrowth of mono- or oligoclonal cancer.

The frequency of apoptosis correlates with the prognosis of Gleason Grade 3 adenocarcinoma of the prostate.

Although localized carcinomas are predominantly moderately differentiated (Gleason Grade 3), they demonstrate markedly different rates of progression. Previously, the authors reported a correlation between apoptosis and the malignant characteristics of carcinoma in the mouse prostate reconstitution model system and between apoptosis and Gleason grade in the human tumor. This study was undertaken to determine whether the frequency of apoptosis correlates with prognosis and to compare the prognostic significance of the apoptotic index with other prognostic features in Gleason Grade 3 carcinomas. The apoptotic and mitotic indices of malignant and nonmalignant epithelium in 28 consecutive radical prostatectomy specimens were determined for a carcinomas composed entirely of Gleason sum 6 (primary Grade 3, secondary Grade 3) with a clinical stage T2 classification. Each patient was followed for 5-9 years (median 6, years). The indices, defined as the number of apoptotic and mitotic bodies in an H & E-stained section per 100 grids delineated by an ocular measuring field, were determined. The actuarial time to progression, defined as a sustained rise in the serum prostate specific antigen level greater than or equal to 0.4 ng/ml, was correlated with the apoptotic index, the mitotic index, tumor volume, and pathologic stage. Neither pathologic stage nor tumor volume differed significantly between the group of 19 patients (68%) with no progression and the other 9 whose tumor progressed. The median apoptotic index of the carcinomas was 0.87 (range, 0.12-3.91). For patients with a low apoptotic index (< 0.87), the actuarial progression rate at 5 years was 7% +/- 14% (+/- 2 SE) compared with 50% +/- 26% for those with a high apoptotic index (P < 0.007). Mitotic index had no prognostic significance. In carcinoma of the prostate, apoptosis can be recognized in standard H & E sections and quantitated by light microscopy. The apoptotic index may provide additional prognostic information in the predominant grade of early stage carcinoma. Cancer 1995; 75:522-9.

Transgenic models for the study of prostate cancer

Transgenic model systems provide tools for obtaining information that clarifies important relationships between genetic alterations and carcinogenesis. One such relationship is the induction of specific growth factor activities by dominantly acting oncogenes. Using a "transgenic organ" model referred to as mouse prostate reconstitution (MPR) under conditions where the ras and myc oncogenes were introduced using a recombinant retrovirus into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer (PC) was produced with high frequency (> 90%) in inbred C57BL/6 mice. Time-course studies using northern blotting and immunohistochemical analysis showed that the transition from benign to malignant status invariably was associated with the induction of elevated transforming growth factor-beta 1 (TGF-beta 1) expression. Additional immunohistochemical analysis of TGF-beta 1 in human PC and benign prostatic hyperplasia (BPH) showed that positive extracellular staining was significantly more extensive in PC compared with BPH. This differential staining pattern was evident in focal areas of PC adjacent to BPH. These findings in both the MPR model system and human PC suggest that elevated TGF-beta 1 expression is involved in the progression to malignancy and that its pattern of expression may become a useful marker of PC. Additional studies using transgenic animal models will continue to provide important clinically useful information about PC in man.

Alterations in mRNA levels for growth-related genes after transplantation into castrated hosts in oncogene-induced clonal mouse prostate carcinoma.

A clonal mouse prostate carcinoma was established by the introduction of the ras and myc oncogenes via the recombinant retrovirus Zipras/myc 9 using a mouse prostate reconstitution model system. A single-cell suspension derived from an early passage ras+myc-induced carcinoma was inoculated into the flanks of intact or castrated adult male C57BL/6 mice, and tumors were harvested 3 wk postinoculation for northern and Southern blotting. Tumor volume analysis showed that this carcinoma was not dependent on testicular androgens for growth. Southern blot analysis of virus-cell DNA junction fragments revealed that tumor cell populations recovered from both intact and castrated mice were progeny of the same virus-infected cell. Northern blotting showed that mRNA levels for the four growth-related genes transforming growth factor-beta 1 (TGF-beta 1), transforming growth factor-beta 3 (TGF-beta 3), tissue-type plasminogen activator (tPA), and c-myc were significantly elevated in clonal mouse prostate carcinomas grown in castrated hosts. In contrast, androgen receptor mRNA levels were significantly reduced under the same conditions. The response of TGF-beta 1, tPA, and c-myc mRNA levels in the carcinomas grown in castrated hosts was similar to that shown previously in normal rat ventral prostate. However, unlike normal rat ventral prostate after castration, increased numbers of apoptotic cells were not seen in the castrated group relative to the intact group at the time of analysis, indicating that the altered gene expression was not associated with cell death. In addition, testosterone-repressed prostate mRNA number 2 levels, shown previously to be elevated after castration in normal rat ventral prostate, were not increased in the androgen-deprived clonal mouse prostate carcinomas. Therefore, this early passage clonal ras+myc-induced prostate carcinoma demonstrates unique patterns of expression for a set of growth-related genes in an androgen-deprived environment.