Reconstituted Model Systems Research Articles

Adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy leads to systemic activity against spontaneous and induced metastasis in an orthotopic mouse model of prostate cancer.

It is critical to develop new therapies, such as gene therapy, which can impact on both local and metastatic prostate cancer progression. We have developed an orthotopic mouse model of metastatic prostate cancer using a cell line (RM-1) derived from the mouse prostate reconstitution (MPR) model system. This mouse model closely simulates the anatomical and biological milieu of the prostate and allows for realistic testing of experimental gene therapy protocols. Adenovirus (ADV)-mediated transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene in conjunction with ganciclovir (GCV) in this model led to significant suppression of growth and of spontaneous metastasis at 14 days post-tumor inoculation. Longer-term studies produced a significant survival advantage and a continued suppression of metastatic activity for treatment animals despite regrowth of the primary tumor. Challenge by injection of tumor cells into the tail vein following excision of treated and control s.c. primary tumors resulted in 40% reduction in lung colonization in the treatment group, indicating the possible production of systemic anti-metastatic activity following a single in situ treatment with ADV/HSV-tk + GCV in this model system.

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.

Transforming growth factor-beta localization during mouse prostate morphogenesis and in prostatic growth abnormalities.

Growth and morphogenesis of the prostate involves mesenchymal-epithelial interactions. Transforming growth factor-beta 1 (TGF-beta1) is one growth factor that may play a role in these paracrine interactions. We have localized TGF-beta1 by molecular and immunohistochemical analysis in the developing mouse prostate. Accumulations of TGF-beta1 protein were localized in the mesenchyme surrounding ductules in fetal and neonatal prostate. Previous studies in the mouse prostate reconstitution (MPR) model system have localized accumulations of TGF-beta1 to regions of oncogene-induced abnormalities. In surgically excised adult human prostate tissues, localized accumulations of TGF-beta1 are associated with prostate cancer and benign prostatic hyperplasia (BPH). Intracellular TGF-beta1 was more often associated with stromal cells in BPH and with neoplastic epithelial cells in prostate cancer. The production and accumulation of TGF-beta1 appears to involve interactions between mesenchymal and epithelial cells. Further experimental studies may clarify the relationships between TGF-beta1 and abnormal prostatic growth.

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.

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.

Genetic predisposition and mesenchymal-epithelial interactions in ras+myc-induced carcinogenesis in reconstituted mouse prostate.

Using a mouse prostate reconstitution (MPR) model system, strain-specific responses to the ras and myc oncogenes were investigated. When ras + myc were introduced into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer was produced at a high frequency (> 90%) in inbred C57BL/6 mice. In contrast, under similar conditions, inbred BALB/c MPRs formed benign prostatic hyperplasia that converted to cancer at a low frequency (< 10%). Restricting the oncogenes to the mesenchymal or epithelial compartments revealed that oncogene activities were more pronounced in the mesenchyme of C57BL/6 mice and resulted in elevated transforming growth factor-beta 1 expression along with a severe desmoplastic reaction. Heterologous MPRs composed of BALB/c mesenchyme and C57BL/6 epithelium or vice versa demonstrated that intrinsic properties of BALB/c mesenchyme can arrest the progression of ras + myc-initiated C57BL/6 epithelium from benign hyperplasia to malignant carcinoma.

Transforming growth factor beta 1 as a biomarker for prostate cancer.

Using the mouse prostate reconstitution (MPR) model system, under conditions where the ras and myc oncogenes are introduced via a recombinant retrovirus into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer is produced with high frequency (> 90%) using inbred C57BL/6 mice. Northern blotting and immunohistochemical analysis showed that the transition from benign prostatic hyperplasia (BPH) to prostate cancer is invariably associated with the induction of elevated transforming growth factor-beta 1 (TGF-beta 1) expression. Similar analysis of TGF-beta 1 in human BPH and prostate cancer is consistent with our MPR results and indicates that the accumulation of extracellular TGF-beta 1 is significantly more intense in prostate cancer compared to normal or benign prostate tissues. Interestingly, where benign pathologies are observed in the prostatic stroma in the presence of benign prostatic epithelium, extracellular TGF-beta 1 is seen predominantly in the stromal compartment. Experimental studies clearly demonstrate that mRNA levels of TGF-beta 1 and other growth related genes are regulated by androgens in prostate cancer cells. Overall, our results suggest that elevated TGF-beta 1 is involved in the development of prostate cancer. Direct determination of TGF-beta 1 levels and distribution as well as analysis of localized and systemic effects produced by TGF-beta 1 may serve as useful biomarkers for prostate cancer.

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.

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-γ-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.

Lipid—protein interactions in reconstituted model systems

The complexity of cell membranes requires complementary approaches to evaluating the structural role of lipids. The aim of this review is to summarize recent advances in the study of integral membrane protein—lipid interactions using reconstituted model systems.

Microtubule Dynamics and Organelle Transport in Reticulomyxa

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba,Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

Tracer diffusion in F-actin and Ficoll mixtures. Toward a model for cytoplasm

We have previously reported that self-diffusion of inert tracer particles in the cytoplasm of living Swiss 3T3 cells is hindered in a size-dependent manner (Luby-Phelps, K., D.L. Taylor, and F. Lanni. 1986. J. Cell Biol. 102:2015-2022; Luby-Phelps, K., P.E. Castle, D.L. Taylor, and F. Lanni. 1987. Proc Natl. Acad. Sci. USA. 84:4910-4913). Lacking a theory that completely explains our data, we are attempting to understand the molecular architecture responsible for this phenomenon by studying tracer diffusion in simple, reconstituted model systems. This report contains our findings on tracer diffusion in concentrated solutions of Ficoll 70 or Ficoll 400, in solutions of entangled F-actin filaments, and in solutions of entangled F-actin containing a background of concentrated Ficoll particles or concentrated bovine serum albumin (BSA). A series of size-fractionated fluorescein-Ficolls were used as tracer particles. By fluorescence recovery after photobleaching (FRAP), we obtained the mean diffusion coefficients in a dilute, aqueous reference phase (Do), the mean diffusion coefficients in the model matrices (D), and the mean hydrodynamic radii (RH) for selected tracer fractions. For each model matrix, the results were compared with similar data obtained from living cells. As in concentrated solutions of globular proteins (Luby-Phelps et al., 1987), D/Do was not significantly size-dependent in concentrated solutions of Ficoll 400 or Ficoll 70. In contrast, D/Do decreased monotonically with increasing RH in solutions of F-actin ranging in concentration from 1 to 12 mg/ml. This size dependence was most pronounced at higher F-actin concentrations. However, the shape of the curve and the extrapolated value of D/Do in the limit, RH----O did not closely resemble the cellular data for tracers in the same size range (3 less than RH less than 30 nm). In mixtures of F-actin and Ficoll or F-actin and BSA, D/Do was well approximated by D/Do for the same concentration of F-actin alone multiplied by D/Do for the same concentrations of Ficoll or BSA alone. Based on these results, it is possible to model the submicroscopic architecture of cytoplasm in living cells as a densely entangled filament network (perhaps made up of F-actin and other filamentous structures) interpenetrated by a fluid phase crowded with globular macromolecules, which in cytoplasm would be primarily proteins.

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate.

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.

A comparative evaluation of methods for isolation of RNA-directed DNA polymerase from cells in a reconstituted system

We have compared the relative merits of several procedures for the isolation of RNA-directed DNA polymerase (EC 2.7.7.7) from cells using a reconstituted model system consisting of a mixture of woolly monkey (simian) sarcoma virus and a cultured human lymphoblastoid cell line, NC-37. When the cell-virus mixture was gently disrupted and fractionated by differential centrifugation, most of the added polymerase was recovered associated with a particulate fraction obtained from the post-mitochondrial supernatant. Purification of the polymerase was best achieved starting from this fraction. The particulate fraction itself can be purified by gel filtration through a Sepharose 2 B column. This procedure did not significantly alter the composition of viral and cellular DNA polymerases. Wheras as little as 7.5 · 10 5 viral particles were sufficient for the detection of RNA-directed DNA polymerase activity, a minimum of about 10 11 particles were necessary for the isolation and unequivocal characterization of the enzyme from the cell-virus mixture by subcellular fractionation and chromatographic separation from cellular DNA polymerases. Purified RNA-directed DNA polymerase had the same primer-template characteristics, sedimentation properties, and immunological cross reactivity as the enzyme purified from density gradient-banded virions of simian sarcoma virus. Methods involving total extraction of the cell-virus mixture either by repeated freezing and thawing followed by detergent treatment or by Dounce homogenization and treatment with high salt and detergent failed to provide RNA-directed DNA polymerase free of cellular DNA polymerases. Because of this, low levels of cellular RNA-directed DNA polymerase may be missed when these approaches are used.