Culture of mouse prostatic epithelial cells from genetically engineered mice

BACKGROUND: The lack of appropriate prostate cancer models is a major problem for prostate cancer research. Progress has been made towards the development of better in vivo rodent genetic models for prostatic disease. However, an in vitro model is often preferred for the elucidation of cellular mechanisms involved in the disease. METHODS: We microdissected the four prostatic lobes from young male mice, harvested the epithelial components, and grew epithelial cells from these tissues. We maintained the growth of these cells in long-term and three-dimensional culture. RESULTS: We have reproducibly harvested and cultured for extended passages mouse prostatic epithelial cells (MPECs) from a variety of mouse genetic strains. These cells express luminal and basal epithelial markers as well as the androgen receptor. Additionally, MPECs form classic branching structures in a three-dimensional collagen matrix. CONCLUSIONS: We have developed a novel culture system to harvest and grow MPECs in long-term culture. These cells will serve as a useful in vitro complement to studies using mouse genetic models for prostatic disease.     

Growth of a spontaneous canine prostatic adenocarcinoma in vivo and in vitro: isolation and characterization of a neoplastic prostatic epithelial cell line, CPA 1

Neoplastic epithelium derived from a spontaneous canine prostatic adenocarcinoma has been maintained and grown in cell culture and as xenografts in athymic mice. An epithelial cell line (CPA 1) has been isolated from primary cultures and has been partially characterized in vitro. The growth of this cell line was not modified by either androgens or estrogens, and high-affinity receptors for these steroids could not be demonstrated in these cells. Xenografts were serially transplantable, with growth being similar in both sexes. Receptors for androgens and estrogens could not be detected in homogenates of xenografts or primary tumor. The histological appearances of serially transplanted tumors, and of xenografts generated by inoculation of the cell line (CPA 1) and several cloned substrains, were very similar to that of the primary tumor and were judged to be well differentiated. The characteristics of this neoplastic cell type have been compared with those of normal prostatic epithelium.     

Prostatic tumor cell plasticity involves cooperative interactions of distinct phenotypic subpopulations: role in vasculogenic mimicry

BACKGROUND: Tumor cell plasticity represents a significant clinical challenge in that the fate and function of tumor cells can be elusive until a tumor mass is evident. A remarkable example of plasticity is tumor cell vasculogenic mimicry, recently described in aggressive uveal and cutaneous melanoma, in addition to ovarian carcinoma, whereby tumor cells express endothelial-associated genes and form de novo vasculogenic-like networks in three-dimensional (3-D) culture. In the current investigation, we examined whether there is evidence for vasculogenic mimicry in heterogeneous prostatic neoplasms. METHODS: Dunning rat and human prostate cancer cell lines (comprised of epithelial- and fibroblastic-like tumor subpopulations) were tested for their ability to express selected endothelial-associated genes, laminin, the alpha6beta1 laminin-binding integrin, and for their potential to form perfusable tubular networks in 3-D culture. Simultaneous morphological analysis of tumor-lined channels in rat and human tumors was also performed. RESULTS: Green fluorescent protein labeling of prostatic clonal subpopulations revealed unique cooperative interactions of epithelial- and fibroblastic-like tumor cells in the formation of perfusable vasculogenic-like networks. Furthermore, while these cell lines were shown to express various vascular markers, prostatic tumor cell-lined channels were also detected in vivo in high grade tumors, and occurred in some cases in close proximity to conventional endothelial-lined vasculature. CONCLUSIONS: A multidisciplinary approach to assess vasculogenic mimicry by prostatic tumor cells has revealed supportive evidence that it occurs in invasive, heterogeneous prostate cancer cell lines, and circumstantially in aggressive rat and human tumors. These results reflect the plasticity of aggressive prostatic tumor cells and may provide new prognostic markers for clinical diagnosis and new therapeutic intervention strategies.     

前列腺癌与良性前列腺增生细胞株差异核基质蛋白的鉴定

目的:鉴定人前列腺癌雄激素依赖性细胞株LNCaP、前列腺癌雄激素非依赖性细胞株PC-3与良性前列腺增生细胞株BPH-1之间差异表达的核基质蛋白(nuclear matrix proteins,NMPs)。方法:常规制备各细胞株NMPs,应用双向凝胶电泳对其进行分离;对差异表达的蛋白质点行MALDI-TOF-MS/MS质谱分析,数据库搜索并鉴定。结果:成功获得了分辨率高、重复性好的不同细胞株NMPs双向凝胶电泳图谱;初步鉴定出包含酶、调节蛋白、RNA结合蛋白及各种因子在内的12个差异表达的蛋白质,在癌细胞株中3个NMPs表达上调,9个下调。结论:人前列腺癌细胞与良性前列腺增生细胞之间NMPs表达存在明显差异;初步筛选出12个差异NMPs,其表达水平及功能与疾病的联系需进一步研究。     

Relevance of bioreactors and whole tissue cultures for the translation of new therapies to humans

The purpose of this review is to provide a brief overview of bioreactor‐based culture systems as alternatives to conventional two‐ and three‐dimensional counterparts. The role, challenges, and future aspirations of bioreactors in the musculoskeletal field (e.g., cartilage, intervertebral disc, tendon, and bone) are discussed. Bioreactors, by recapitulating physiological processes, can be used effectively as part of the initial in vitro screening, reducing that way the number of animal required for preclinical assessment, complying with the 3R principles and, in most cases, allowing working with human tissues. The clinical significance of bioreactors is that, by providing more physiologically relevant conditions to customarily used two‐ and three‐dimensional cultures, they hold the potential to provide a testing platform that is more predictable of a whole tissue response, thereby facilitating the screening of treatments before the initiation of clinical trials. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:10–21, 2018.     

簇蛋白在前列腺上皮细胞凋亡中的表达和作用

目的探讨簇蛋白在前列腺上皮细胞凋亡过程中的表达和作用，及与转化生长因子β1（transforming growth factor β1，TGF-β1）的关系。方法取10只实验犬，分别于去势前及去势后近期（〈15d）和去势后远期（〉15d）取前列腺组织制成石蜡切片，通过免疫组织化学检测前列腺组织中簇蛋白的表达及其表达部位，同时检测TGF-β1的表达。结果去势后近期（〈15d）与去势前的簇蛋白表达有显著性的差异，去势后远期（〉15d）与去势前簇蛋白的表达没有显著性差异；TGF-β1的表达也是如此。结论簇蛋白的表达与前列腺上皮细胞凋亡有关，和TGF-β1表达存在一定的相关性。     

Studies of androgen and estrogen binding in normal canine prostatic tissue and in epithelial and stromal cell lines derived from the canine prostate

Androgen and estrogen binding to cytosol proteins was examined in preparations derived from canine prostatic tissue and from epithelial and fibroblastoid cell lines. Binding parameters were characterized by saturation analysis using a protamine sulfate precipitation procedure and by analysis of the mobility of steroid-binding moieties in sucrose density gradients. Androgen-binding studies demonstrated the presence of a single class of high-affinity binding component (kD = 1-2 × 10^?9 M), with specificity for 5α-dihydrotestosterone, in cytosols derived from tissue homogenates and both cell types. In estrogen-binding studies two discrete classes of binding component were characterized of high (kD = 5-10 × 10^?10 M) and moderate (kD = 1-2 × 10^?8 M) affinities. Both species were present in cytosol derived from all three sources. The high-affinity component displayed specificity for estradiol-17β, and the second component displayed some capacity for androgen binding. These findings are discussed with reference to the anomalous actions of some androgens in the canine prostate and in relation to the putative role of steroids in stromal-epithelial interrelationships.     

Biomodulation of 5-Fu cytotoxicity by folinic acid and its stereoisomers: in vitro experiments with different cell lines of prostatic cancer

The results of cytotoxic chemotherapy for advanced, hormone-escaped prostate cancer have been disappointing. Evaluation of the effect of new drugs or new combinations with already known ones is required. The antimetabolite 5-fluorouracil (5-FU) has been shown to be active in prostate cancer, acting via inhibition of thymidylate synthase, an essential enzyme in DNA de novo synthesis. Experiments with cell lines of different tumors have shown that 5-FU activity can be modulated by addition of the coenzyme tetrahydrofolic acid (folinic acid). We investigated the effect of folinic acid and its stereoisomers on 5-FU action in different cell lines of prostate cancer. It was found that addition of non-toxic folinic acid led to a two- to fourfold better antiproliferative effect of 5-FU. The unnatural 6R isomer, which is a compound of chemically synthesized folinic acid, inhibited the modulatory effect of the natural 6S isomer. Our results indicated that a combination of folinic acid and 5-FU may result in a better response of patients with hormone-resistant prostate cancer than of patients treated with 5-FU alone.     

Three‐dimensional printing in orthopaedic surgery: review of current and future applications

Three‐dimensional (3D) printing is a rapidly evolving technology with the potential for significant contributions to surgical practice. There are many current applications for 3D printing technology with future applications being explored. This technology has applications in preoperative planning, education, custom manufacturing (implants, prosthetics and surgical guides) and exciting potential for biological applications. This article reviews the current and future applications of 3D technology in orthopaedic surgery.     

Nitrosulindac (NCX 1102): a new nitric oxide-donating non-steroidal anti-inflammatory drug (NO-NSAID), inhibits proliferation and induces apoptosis in human prostatic epithelial cell lines

BACKGROUND: The aim of our study was to explore the anti-tumoral potential of the Nitric Oxide-Donating Non-Steroidal Anti-Inflammatory Drugs (NO-NSAID) NCX1102 (nitrosulindac), on three human prostatic epithelial cell lines at varying degree of transformation (PNT1A, LNCaP, and PC3). METHODS: Cytotoxicity, anti-proliferative effects, cell-cycle alterations, morphological changes, and apoptosis were investigated after treatment with nitrosulindac in comparison to the native molecule sulindac. Involvement of the polyamine pathway in the action of nitrosulindac was also examined. RESULTS: Nitrosulindac but not sulindac exerted a cytotoxic effect on all cell lines and an anti-proliferative effect on LNCaP and PC3 cells only. Nitrosulindac differentially altered the cell cycle, induced mitotic arrest and displayed a pro-apoptotic activity in all cell lines. Finally, the polyamine pathway does not seem to be involved in the mechanism of nitrosulindac action. CONCLUSIONS: Our results demonstrate the anti-proliferative and proapoptotic activity of nitrosulindac on prostate cancer cell lines and suggest its potential interest for new strategies in the management of prostate cancer.     

Stromal/epithelial interactions of murine prostatic cell lines in vivo: a model for benign prostatic hyperplasia and the effect of doxazosin on tissue size

BACKGROUND: One of the major constraints in elucidating the mechanisms involved in the etiology of benign prostatic hyperplasia (BPH) is the lack of suitable model systems that are readily manipulable in vitro and in vivo. To address this issue, we have used murine prostatic cell lines to establish a novel in vivo model for studying prostatic cell interactions. METHODS: Luminal, basal, and smooth muscle (SM) cell lines were inoculated alone or in combinations under the renal capsule of intact or castrated male mice, and the growth and composition of prostatic tissue in the absence or presence of doxazosin was determined. RESULTS: Both the luminal and basal cell lines reconstituted prostatic tissue if co-inoculated under the renal capsule with normal SM cells, whereas none of the lines formed significant tissue when inoculated alone. Luminal cells produced and secreted prostatic secretory products. The growth of prostatic tissue formed from co-inoculation of basal and SM cells was androgen responsive. In addition, a significant reduction in prostatic tissue was noted in animals treated with doxazosin. CONCLUSION: We have established an in vivo model that uses prostatic epithelial and SM cell lines for investigating cellular interactions between epithelial and SM cells that regulate prostatic growth and function. This model will be useful for delineating the mechanisms by which prostatic cells interact and in determining the efficacy of new approaches aimed at interfering with prostatic stromal/epithelial interactions that result in abnormal cellular proliferation.