Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Targeting of cancer cells with monoclonal antibodies specific for C3b(i)

Purpose: The goal of this research is to determine the feasibility of an immunotherapeutic approach based on the use of monoclonal antibodies (mAb) to target complement activation fragments on opsonized cancer cells. Methods: We investigated whether treatment of LNCaP and C4-2 human prostate cancer cell lines with normal human serum would allow for deposition of sufficient amounts of the complement-activation protein C3b and its fragments [collectively referred to as C3b(i)] such that these proteins could serve as cancer-cell-associated antigens for targeting by mAb. Radioimmunoassays, flow cytometry, and magnetic purging with specific immunomagnetic beads were used for the analyses. Results: In vitro opsonization of human prostate cancer cells with normal human serum resulted in deposition of C3b(i) in sufficient quantity (approx. 100,000 molecules/cell) for the cells to be targeted in a variety of protocols. We found that ⁵¹Cr-labeled and C3b(i)-opsonized cancer cells could be specifically purged at high efficiency (95%–99%) using anti-C3b(i) mAb covalently coupled to magnetic beads. Flow-cytometry experiments indicated that most normal white cells were not removed under similar conditions. Opsonization of cancer cells with sera from men with prostate cancer led to lower levels of cell-associated IgM and, subsequently, lower amounts of C3b(i) deposited than in normal subjects. Prototype experiments suggested that this deficiency could be corrected by addition of IgM from normal donor plasma. Conclusion: mAb directed against complement-activation products may provide new opportunities to deliver diagnostic and therapeutic agents selectively to cancer cells and tumor deposits. These opportunities may include ex vivo purging of C3b(i)-opsonized cancer cells prior to autologous bone marrow or stem cell transplantation. Received: 17 February 2000 / Accepted: 1 August 2000     

Matched pairs of human prostate stromal cells display differential tropic effects on LNCaP prostate cancer cells

Prostate stromal cells may play binary roles in the process of prostate cancer development. As the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. However, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal clones. In this report, we describe the morphologic and behavioral characteristics of these cells and their effect on LNCaP prostate cancer cells in co-culture. Unlike LNCaP prostate cancer cells, the isolated prostate stromal clones are large fibroblast-like cells with a slow proliferation rate. Growth and survival of these clones are not affected by androgens. The stromal cells display high resistance to serum starvation, while cancer-associated stromal clones have differentiated survival ability. In co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. This work thus established a panel of valuable human prostate stromal cell lines, which could be used in co-culture to study the interaction between prostate cancer and prostate stromal cells.     

中国人HLA纯合细胞的筛选及纯合性鉴定

本文报告了我们实验室在中国人中筛选HLA纯合子的方法和筛选流程。用HLA血清学分型法,从上海129个近亲婚配家庭中筛选到HLA-AB纯合子23个(分布于17个家庭)。其中19个HLA-AB纯合子(分布于14个家庭)进一步做了家系MLC棋盘。最终证明,它们中14个细胞(分布于10个家庭)的HLA-D位点也纯合。     

Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: Evaluation of androgen-induced growth and psa expression

Summary A novel in vitro human prostate cancer model was established by using a coculture technique in which isolated human prostate fibroblasts were observed to grow as a mixed culture with isolated human prostate cancer cells (LNCaP) on microcarrier beads under microgravity-simulated conditions. This model appears to be promising and deserves further exploration because: (a) cocultured human prostate fibroblasts and cancer epithelial cells appear to undergo patterns of histogenesis similar to those observed in human prostate tumors and (b) unlike the conventional cell culture on plastic dishes, cocultured human prostate fibroblasts and LNCaP cells in microgravity-simulated conditions responded to the inductive signals of growth and differentiation from dihydrotestosterone in a manner similar to that observed in the in vivo condition. These results offer an opportunity to examine molecular mechanisms of cellular signaling in response to androgen stimulation during normal and aberrant human prostate development. The microgravity-simulated three-dimensional prostate epithelial cell culture with prostate fibroblasts can be further explored as an ideal in vitro model for the study of normal and neoplastic prostate development. This model could also be adopted as a drug screening program for the discovery of novel therapeutic agents in the treatment of human prostate cancer and benign hyperplastic growth.     

Tumor–stroma co-evolution in prostate cancer progression and metastasis

Cancer development is complex and involves several layers of interactions and pleotropic signaling mechanisms leading to progression. Cancer cells associate with resident stromal fibroblasts, smooth muscle cells, macrophages, endothelium, neurons and migrating cells at metastatic sites and phenotypically and genotypically activate them. These become an integral part of the cancer cell community through activated cell signaling mechanisms. During this process, the cancer cells and cells in the cancer microenvironment “co-evolve” in part due to oxidative stress, and acquire the ability to mimic other cell types (which can be termed osteomimicry, vasculomimicry, neuromimicry and stem cell mimicry), and undergo transition from epithelium to mesenchyme with definitive morphologic and behavioral modifications. In our laboratory, we demonstrated that prostate cancer cells co-evolve in their genotypic and phenotypic characters with stroma and acquire osteomimetic properties allowing them to proliferate and survive in the skeleton as bone metastasis. Several signaling interactions in the bone microenvironment, mediated by reactive oxygen species, soluble and membrane bound factors, such as superoxide, β2-microglobulin and RANKL have been described. Targeting the signaling pathways in the cancer-associated stromal microenvironment in combination with known conventional therapeutic modalities could have a synergistic effect on cancer treatment. Since cancer cells are constantly interacting and acquiring adaptive and survival changes primarily directed by their microenvironment, it is imperative to delineate these interactions and co-target both cancer and stroma to improve the treatment and overall survival of cancer patients.     

内皮祖细胞(EPCs)研究进展

组织工程血管以及组织工程化组织的血管化因目前内皮种子细胞扩增能力和生物活力的不足而受到限制。EPCs(内皮祖细胞)是内皮细胞的前体细胞。在胚胎期,内皮细胞系与造血细胞系来源于血岛内共同的祖先细胞;出生后,EPCs存在于骨髓,并可被转移至外周血,参与缺血组织的血管重建和血管的内膜化。因此EPCs有望成为今后组织工程内皮种子细胞的重要来源。