Expression of hematopoietic growth factor receptors on early hematopoietic precursors: detection and regulation

Since the original isolation of colony-stimulating factors from human serum, conditioned medium of murine or human cell lines, or freshly isolated human mononuclear cells, a revolutionary explosion of ideas has occurred in our understanding of molecular controls of the hematopoietic stem cell self-renewal and differentiation. With the availability of techniques of molecular cloning in the early 1 980s, the first hematopoietically activated cytokines led to molecular clones expressed in bacteria, yeast, or mammalian cellular systems. There then followed a development of techniques leading to the molecular cloning and expression of many hematopoietic growth factors and their receptors, as well as the primary, secondary, and tertiary molecules in signal transduction into activation of specific genes for differentiation or self-renewal. The clinical use of these factors in the diagnosis, treatment, and incorporation into new cell therapies for a variety of diseases is a subject of current interest.     

Expression of human tenascin in synovitis and its regulation by interleukin-1.

OBJECTIVE. Tenascin is an extracellular matrix glycoprotein with effects on cell adhesion, cell migration, and lymphocyte activation. We proposed to identify the expression of human tenascin messenger RNA (mRNA) and protein in inflammatory synovitis and in normal synovium, and to identify potential regulatory cytokines. METHODS. Immunohistochemistry and in situ hybridization were used to identify the expression of tenascin in synovium. Northern blot analysis of RNA and both immunoblot analysis and enzyme-linked immunosorbent assay of proteins were used to identify tenascin in synovial cell cultures. RESULTS. Tenascin was found along the synovial lining layer and in perivascular areas of normal synovium. In inflammatory synovitis, tenascin protein and mRNA expression were shown to be increased in the synovial lining layer, in perivascular areas, in lymphoid aggregates, and in areas of fibrosis. Interleukin-1, a major mediator of tissue injury in inflammatory synovitis, induced tenascin expression and deposition in primary synovial fibroblast cultures. CONCLUSION. Tenascin mRNA and protein are increased in inflammatory synovitis, and interleukin-1 is an inducer of tenascin in synovial fibroblasts. This identifies a new pathway by which interleukin-1 alters the extracellular matrix composition in synovitis. Since tenascin has effects on lymphocyte activation and cell adhesion, the induction of tenascin in inflammatory synovitis may play a role in the pathophysiology of arthritis.     

Expression of BRCA1 gene in ewe mammary epithelial cells during pregnancy: regulation by growth hormone and steroid hormones.

OBJECTIVE: Steroid hormones (estradiol and progesterone) in association with prolactin and growth hormone are involved in lobulo alveolar development of the mammary gland during pregnancy. We hypothesized that the BRCA1 gene may be induced by these different hormones. METHODS AND RESULTS: In this study, we have demonstrated by Northern blot and in situ hybridization, that the expression of ovine (o) BRCA1 mRNA in mammary epithelial cells increased dramatically during a short period in the second half of pregnancy (days 70 to 112) and decreased at the end of pregnancy. The increase in oBRCA1 mRNA expression is concomitant with rapid lobulo alveolar growth. Using an in vivo protocol to artificially induce mammary gland development, we demonstrated by the real-time RT-PCR method that growth hormone in association with estrogen, progesterone and hydrocortisone induces an increase of BRCA1 mRNA expression in the ewe mammary gland. Moreover, we showed that estradiol and progesterone induce oBRCA1 expression in primary cultures of ewe mammary gland. CONCLUSIONS: These results suggest that BRCA1 is a potential regulator of the effects of steroid hormones and growth hormone in the induction of mammary epithelial cell proliferation.     

Expression of the alpha 1-proteinase inhibitor gene in human monocytes and macrophages.

Expression of the alpha 1-proteinase inhibitor (alpha 1PI) gene was studied in human mononuclear cells. Using RNA blot and dot hybridization, alpha 1PI mRNA was detected in human peripheral blood monocytes, bronchoalveolar and breast milk macrophages, but not in B or T lymphocytes. Using incorporation of a radiolabeled amino acid precursor, synthesis and secretion of alpha 1PI were demonstrated in human monocytes and macrophages, but not in lymphocytes. In addition, alpha 1PI was secreted in functionally active form as shown by complexing with serine proteases. Biosynthesis of alpha 1PI by mononuclear phagocytes was greatest during the first 24 hr in culture and progressively decreased over the next 10 days. The reduction in alpha 1PI biosynthesis in vitro involved a mechanism acting at the pretranslational level as alpha 1PI mRNA content also progressively declined over 10 days in culture. The ease of sampling human monocytes and macrophages now permits examination of the biochemical defect in homozygous PiZ and PiS alpha 1PI deficiencies and study of the functional significance of locally produced alpha 1PI in normal tissues and sites of injury or inflammation.     

Expression of human tenascin in synovitis and its regulation by interleukin-1

Objective. Tenascin is an extracellular matrix glycoprotein with effects on cell adhesion, cell migration, and lymphocyte activation. We proposed to identify the expression of human tenascin messenger RNA (mRNA) and protein in inflammatory synovitis and in normal synovium, and to identify potential regulatory cytokines. Methods. Immunohistochemistry and in situ hybridization were used to identify the expression of tenascin in synovium. Northern blot analysis of RNA and both immunoblot analysis and enzyme-linked immunosorbent assay of proteins were used to identify tenascin in synovial cell cultures. Results. Tenascin was found along the synovial lining layer and in perivascular areas of normal synovium. In inflammatory synovitis, tenascin protein and mRNA expression were shown to be increased in the synovial lining layer, in perivascular areas, in lymphoid aggregates, and in areas of fibrosis. Interleukin-1, a major mediator of tissue injury in inflammatory synovitis, induced tenascin expression and deposition in primary synovial fibroblast cultures. Conclusion. Tenascin mRNA and protein are increased in inflammatory synovitis, and interleukin-1 is an inducer of tenascin in synovial fibroblasts. This identifies a new pathway by which interleukin-1 alters the extracellular matrix composition in synovitis. Since tenascin has effects on lymphocyte activation and cell adhesion, the induction of tenascin in inflammatory synovitis may play a role in the pathophysiology of arthritis.     

Notch1与Jagged1蛋白在大肠腺癌组织中的表达及临床病理意义

目的探讨Notch1和Jagged1蛋白在大肠腺癌发生发展的作用。方法采用免疫组织化学PV-9000法和Western blotting法检测78份大肠腺癌组织(大肠癌组),40份大肠腺瘤组织(腺瘤组)及40份正常大肠组织(距大肠腺癌组织远端5㎝,正常组)Notch1与Jagged1蛋白表达情况,分析其与大肠癌临床病理参数的关系。结果大肠癌组Notch1、Jagged1蛋白阳性率明显高于腺瘤组及正常组,P<0.05;Notch1和Jagged1蛋白表达强度与大肠癌组织学分级、Dukes分期和淋巴结转移密切相关,P均<0.05。大肠癌组Notch1与Jagged1蛋白表达关系呈正相关(r=0.407,P<0.01)。结论 Notch1和Jagged1蛋白在大肠腺癌的发生与发展中起重要作用。     

Density-dependent regulation of growth of BSC-1 cells in cell culture: control of growth by serum factors.

BSC-1 cells grow slowly, to high cell density, in medium with 0.1% calf serum. An increase in the serum concentration increases both the growth rate of the cells and the final cell density. The serum can be replaced to some extent by epidermal growth factor (EGF). Initiation of DNA synthesis in BSC-1 cells that have spread into a "wound" in a crowded cell layer requires the addition of a trace of serum or EGF, if the cells have previously been deprived of serum. The binding of 125I-labeled EGF to low-density and high-density BSC-1 cells has been studied. Binding is faster to low-density cells. Cells at low cell density also bind much more EGF per cell than cells at high cell density. The fraction of bound 125I-labeled EGF that is present on the cell surface as intact EGF is larger at low than at high cell density. The results indicate that the number of available EGF receptors per cell decreases drastically as the cell density increases. It is suggested that a decrease in the number of available EGF receptor sites per cell, and the accompanying decrease in sensitivity of the cells to EGF, contributes to density-dependent regulation of growth of these cells.     

Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells. Possible role in the hematopoietic microenvironment

In the hematopoietic microenvironment, bone marrow endothelial cells may play an important role in trafficking and maintenance of progenitor and stem cells due to adhesive interactions and paracrine secretion of hematopoietic growth factors. However, it is unknown whether progenitors in turn modulate endothelial proliferation and function.We analyzed mRNA expression (Northern blot) and release of vascular endothelial growth factor-A (VEGF-A), which specifically acts on endothelial cells, by cytokine-stimulated peripheral blood-derived CD34+ hematopoietic progenitor cells.While unstimulated CD34+ cells expressed VEGF-A mRNA weakly without cytokine release in vitro, incubation for 24 hours with a single cytokine (e.g., kit ligand [KL]) resulted in increased VEGF-A mRNA expression and significant secretion of VEGF-A into the supernatant. The amount of VEGF released was substantially augmented by incubation with a combination of cytokines (e.g., KL, IL-3, GM-CSF, G-CSF), or by exposure to hematopoietic cytokines for a longer time period. In addition, we show that VEGF induced the release of hematopoietic growth factors (GM-CSF) by bone marrow endothelial cells and that in vitro stromal cell-derived factor-1 (SDF-1) driven transendothelial progenitor cell migration was increased by the presence of VEGF, which might be due to pore formation (increased endothelial fenestration).In vivo, release of VEGF by progenitor cells may result in a paracrine loop supporting proliferation of both endothelium and progenitors and may facilitate transendothelial migration during cytokine-induced progenitor cell mobilization.     

Role of hematopoietic growth factors/flt3 ligand in expansion and regulation of dendritic cells

Dendritic cells (DCs) are hematopoietic cells that initiate immune responses by presenting antigen to T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a primary growth factor for DCs in vitro, but recently it was recognized that other factors including flt3 ligand (FL) and G-CSF expand various DC subsets in vivo. DCs undergo a complex series of maturation and activation steps after they acquire antigen and before they can activate resting T cells. In addition, they must traffic to T-cell-rich areas of lymph nodes (LN) to achieve this. Each of these steps is tightly regulated, and in the last year progress has been made in identifying some of the key molecules involved in each of these steps. This progress will further the efforts underway to develop DCs as vaccine adjuvants.     

Accelerated cell-cycling of hematopoietic progenitor cells by growth factors

Recent advances in molecular biology have led to the identification of hematopoietic growth factors that support and influence the proliferation of hematopoietic progenitor cells in vitro and in vivo. Although these factors have been extensively studied, little is known of their role in the regulation of cell-cycling of hematopoietic progenitors, especially in the early stage of hematopoiesis. In the present study, we examined the effects of early acting growth factors on proliferative kinetics of hematopoietic progenitors by monitoring the number of cells in individual developing colonies, using an in vitro clonal assay. Interleukin-11 (IL-11) or steel factor (SF), alone or in combination, shortened the time for the size of IL-3-dependent colonies to double. Consecutive replating experiments provided evidence for direct action of growth factors on the growth rate of hematopoietic progenitor cells. Shortening of the time for the total cell number in the colonies to double was due to a reduction in time for each single cell within the respective colonies to become two daughter cells, and there was no alteration in the incidence of cells with a proliferative capacity. Cell-cycle analysis demonstrated that IL-11 has the potential to induce a shortened time for cell-cycle of hematopoietic progenitor cells without affecting distribution of each fraction of the cell- cycle, whereas SF has the potential to reduce cell-cycle time mainly by decreasing the time required for hematopoietic progenitor cells to go through the G1 phase. These results suggest that growth factors may modulate cell-cycling of hematopoietic progenitor cells.     

Expression and secretion of type beta transforming growth factor by activated human macrophages.

Alveolar macrophages activated with concanavalin A and peripheral blood monocytes activated with lipopolysaccharide secrete type beta transforming growth factor (TGF-beta). There is minimal TGF-beta secretion in unactivated monocytes, even though TGF-beta mRNA is expressed in these cells at a level similar to that in activated, lipopolysaccharide-treated cultures. U937 lymphoma cells, which have monocytic characteristics, also express mRNA for TGF-beta. Freshly isolated monocytes, both control and lipopolysaccharide-treated, secrete an acid-labile binding protein that inhibits TGF-beta action. We conclude the following: (i) that expression of TGF-beta mRNA is unrelated to monocyte activation, (ii) that secretion of TGF-beta is induced by monocyte activation, and (iii) that cosecretion of TGF-beta and its monocyte/macrophage-derived binding protein may modulate growth factor action. In contrast, monocytic expression of other growth factor genes, such as the B chain of platelet-derived growth factor, is not constitutive and requires activation.     

Role of hematopoietic growth factors in angiogenesis

In early ontogeny, hematopoiesis is closely associated with angiogenesis. This article reviews recent studies of the effect of hematopoietic growth factors on several endothelial cell functions together with recent findings about angiogenesis and antiangiogenic therapies in hematopoietic malignancies such as leukemia, lymphoma and myeloma.     

锌指蛋白2在胰腺癌中的表达

目的对锌指蛋白2(HZF2)在胰腺癌中的表达及意义进行初步的探讨。方法收集20例手术病理证实的胰腺癌标本,应用RT-PCR检测锌指蛋白基因HZF2 mRNA的表达,并分析该表达与胰腺癌临床病理特征的关系。结果胰腺癌组织中HZF2 mRNA表达值平均为143.1±12.2,较相应癌旁组织表达值82.2±7.6高1.40~2.34倍(P<0.01)。HZF2 mRNA的表达与TNM分期呈正相关,而与性别、肿瘤部位、肿瘤大小、分化程度无关。结论锌指蛋白2在胰腺癌中高表达,在胰腺癌的发生发展中可能起一定作用。