Cell line modeling to study biomarker panel in prostate cancer.

BACKGROUND: African-American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS: We assembled a PCa cell line model that included currently available African-American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS: The dysregulation of the multiplex biomarker panel in primary African-American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African-American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African-Americans and Caucasians as a prelude to future translational studies. CONCLUSION: We have characterized a novel in vitro cell line model that could be used to study the biological basis of disparity in PCa between African-Americans and Caucasians.

Novel In Vivo model for combinatorial fluorescence labeling in mouse prostate.

BACKGROUND: The epithelial layer of prostate glands contains several types of cells, including luminal and basal cells. Yet there is paucity of animal models to study the cellular origin of normal or neoplastic development in the prostate to facilitate the treatment of heterogenous prostate diseases by targeting individual cell lineages. METHODS: We developed a mouse model that expresses different types of fluorescent proteins (XFPs) specifically in prostatic cells. Using an in vivo stochastic fluorescent protein combinatorial strategy, XFP signals were expressed specifically in prostate of Protein Kinase D1 (PKD1) knock-out, K-Ras(G) (12) (D) knock-in, and Phosphatase and tensin homolog (PTEN) and PKD1 double knock-out mice under the control of PB-Cre promoter. RESULTS: In vivo XFP signals were observed in prostate of PKD1 knock-out, K-Ras(G) (12) (D) knock-in, and PTEN PKD1 double knock-out mice, which developed normal, hyperplastic, and neoplastic prostate, respectively. The patchy expression pattern of XFPs in neoplasia tissue indicated the clonal origin of cancer cells in the prostate. CONCLUSIONS: The transgenic mouse models demonstrate combinatorial fluorescent protein expression in normal and cancerous prostatic tissues. This novel prostate-specific fluorescent labeled mouse model, which we named Prorainbow, could be useful in studying benign and malignant pathology of prostate.

Novel 3D co-culture model for epithelial-stromal cells interaction in prostate cancer.

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial-stromal interactions including paracrine function, yet an ideal three-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model in double-layered alginate hydrogel microspheres, incorporating prostate cancer epithelial and stromal cells in separate compartments of the microspheres. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate cancer. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also demonstrated that the amount of sE-cad is influenced by epithelial-stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction.

Cell therapy with human renal cell cultures containing erythropoietin-positive cells improves chronic kidney injury.

New therapeutic strategies for chronic kidney disease (CKD) are necessary to offset the rising incidence of CKD and donor shortage. Erythropoietin (EPO), a cytokine produced by fibroblast-like cells in the kidney, has recently emerged as a renoprotective factor with anti-inflammatory, antioxidant properties. This study (a) determined whether human renal cultures (human primary kidney cells [hPKC]) can be enriched in EPO-positive cells (hPKC(F+)) by using magnetic-bead sorting; (b) characterized hPKC(F+) following cell separation; and (c) established that intrarenal delivery of enriched hPKC(F+) cells would be more beneficial in treatment of renal injury, inflammation, and oxidative stress than unsorted hPKC cultures in a chronic kidney injury model. Fluorescence-activated cell sorting analysis revealed higher expression of EPO (36%) and CD73 (27%) in hPKC(F+) as compared with hPKC. After induction of renal injury, intrarenal delivery of hPKC(F+) or hPKC significantly reduced serum creatinine, interstitial fibrosis in the medulla, and abundance of CD68-positive cells in the cortex and medulla (p < .05). However, only hPKC(F+) attenuated interstitial fibrosis in the renal cortex and decreased urinary albumin (3.5-fold) and urinary tubular injury marker kidney injury molecule 1 (16-fold). hPKC(F+) also significantly reduced levels of renal cortical monocyte chemotactic protein 1 (1.8-fold) and oxidative DNA marker 8-hydroxy-deoxyguanosine (8-OHdG) (2.4-fold). After 12 weeks, we detected few injected cells, which were localized mostly to the cortical interstitium. Although cell therapy with either hPKC(F+) or hPKC improved renal function, the hPKC(F+) subpopulation provides greater renoprotection, perhaps through attenuation of inflammation and oxidative stress. We conclude that hPKC(F+) may be used as components of cell-based therapies for degenerative kidney diseases.

Controlled regulation of erythropoietin by primary cultured renal cells for renal failure induced anemia.

PURPOSE: Renal failure induced anemia develops as a result of inadequate production of erythropoietin, which is the primary regulator of red blood cell production. We previously noted that culture expanded primary renal cells stably express erythropoietin and suggested that these cells may be used as a potential treatment for renal failure induced anemia. We investigated whether these cells are able to regulate erythropoietin expression in a controlled manner under different oxygen and environmental conditions. MATERIALS AND METHODS: Primary rat renal cells were exposed to different hypoxic (0.1% to 1% O(2)) and normoxic environments. Erythropoietin expression was assessed using reverse transcriptase-polymerase chain reaction. Erythropoietin production was measured in culture medium using Meso Scale Discovery® assays. Results were plotted to compare different levels of production to the control. RESULTS: Cultured renal cells expressed high levels of erythropoietin under hypoxia for up to 24 hours with a gradual decrease thereafter. However, erythropoietin expression was decreased when cells were switched from a hypoxic to a normoxic environment within the initial 24 hours. This indicated that cultured renal cells have the capacity to sense environmental oxygen tension and regulate erythropoietin expression accordingly. In addition, erythropoietin release in medium followed a pattern similar to that of gene expression under normoxic and hypoxic conditions. CONCLUSIONS: These findings indicate that primary renal cells have the ability to regulate erythropoietin gene expression and release through environment dependent mechanisms. This also suggests that with further study the possibility exists of developing these cells as a potential method to treat renal failure induced anemia.

TRAIL and interferon-alpha act synergistically to induce renal cell carcinoma apoptosis.

PURPOSE: Despite modern targeted therapy metastatic renal cell carcinoma remains a deadly disease. Interferon-alpha (Calbiochem(R)) is currently used to treat this condition, mainly combined with the targeted anti-vascular endothelial growth factor antibody bevacizumab. TRAIL (Apo2 ligand/tumor necrosis factor related apoptosis inducing ligand) (Calbiochem) is a novel antineoplastic agent now in early phase clinical trials. Interferon-alpha and TRAIL can act synergistically to kill cancer cells but to our knowledge this has never been tested in the context of renal cell carcinoma. We hypothesized that TRAIL and interferon-alpha could synergistically induce apoptosis in renal cell carcinoma cells. MATERIALS AND METHODS: We treated renal cell carcinoma cell lines with recombinant TRAIL and/or interferon-alpha. Viability and apoptosis were assessed by MTS assay, flow cytometry and Western blot. Synergy was confirmed by isobologram. Interferon-alpha induced changes in renal cell carcinoma cell signaling were assessed by Western blot, flow cytometry and enzyme-linked immunosorbent assay. RESULTS: TRAIL and interferon-alpha acted synergistically to increase apoptotic cell death in renal cell carcinoma cells. Interferon-alpha treatment altered the ability of cells to activate extracellular signal-regulated kinase while inhibiting extracellular signal-regulated kinase with UO126 abrogated TRAIL and interferon-alpha apoptotic synergy. Interferon-alpha did not induce changes in TRAIL or death receptor expression, or change other known mediators of the intrinsic and extrinsic apoptotic cascade in the cells. CONCLUSIONS: TRAIL plus interferon-alpha synergistically induces apoptosis in renal cell carcinoma cells, which is due at least in part to interferon-alpha mediated changes in extracellular signal-regulated kinase activation. TRAIL and interferon-alpha combination therapy may be a novel approach to advanced renal cell carcinoma that warrants further testing in vivo.

Erythropoietin producing cells for potential cell therapy.

INTRODUCTION: Anemia is an inevitable outcome of chronic renal failure due to the kidney's decreased ability to produce erythropoietin (EPO). We examined the feasibility of isolating and expanding EPO-producing cells for cell-based therapy. MATERIALS AND METHODS: Renal cells from 7- to 10-day-old mice were culture-expanded. The cells at each subculture stage were characterized for EPO expression, using immunocytochemistry, FACS, and Western Blot analysis, with EPO-specific antibodies. To assess the levels of EPO expression, cells incubated under normoxic and hypoxic conditions were analyzed. RESULTS: Immunocytochemical analysis of the cultured renal cells expressed EPO at each subculture stage (P1-P3). Western Blot analysis of the detergent-solubilized cell extracts detected EPO (34 kDa) protein in the kidney cells of all passages tested. CONCLUSION: These results demonstrate that EPO-producing renal cells can be grown and expanded in culture. The cells stably expressed EPO at multiple subculture stages and they are able to form tissue in vivo. This study shows that EPO-producing cells may be used as a potential treatment option for anemia caused by chronic renal failure.