Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors

Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes [TEMs]) (1) are a distinct hematopoietic lineage of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin.     

Role of glycosylphosphatidylinositol-specific phospholipase D in the homing of umbilical cord blood, mobilized peripheral blood and bone marrow-derived hematopoietic stem/progenitor cells

Recent studies suggested that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) correlated with tumor malignancy and prognosis of certain tumors. As hematopoietic stem/progenitor cells (HS/PC) homing was similar to tumor invasion and metastasis in some mechanisms, which arose our intrests in whether GPI-PLD contribution to the homing of HS/PC. In this study, CD34⁺ cells from umbilical cord blood (UCB), mobilized peripheral blood (MPB), and bone marrow (BM) were assayed for their differences in adhesion, migration, respectively. The expression of GPI-anchored proteins (CD48, CD90) on the cells were analyzed by flow cytometry. Semi-quantitive RT-PCR was used to detect GPI-PLD expression in the three different CD34⁺ cells. The results showed that GPI-PLD had no effect on the adhesion of CD34⁺ cells. While, spontaneous and SDF-1 induced migration of UCB and MPB, but not BM CD34⁺ cells were decreased after 1,10-phenanthroline (an inhibitor of GPI-PLD) pretreatment. Furthermore, we found little difference in GPI-anchored adhesion molecules (CD48, CD90) expression between untreated and pretreated CD34⁺ cells. GPI-PLD mRNA was low expressed in MPB and undetected in UCB and BM CD34⁺ cells. Our results suggested that GPI-PLD probably had no contribution to HS/PC homing, which may due to its low or no expression in UCB, BM and MPB CD34⁺ cells.     

Human monoclonal antibody detects a cell surface antigen expressed on hematopoietic malignant cells of lymphoid lineage

An antigen with a molecular weight of 150 kilodaltons expressed on certain leukemia and lymphoma cells was recognized by a human monoclonal antibody (3H12), which had been established by the fusion of lymphocytes from a small cell lung cancer patient with a mouse myeloma cell line (P3U1). Peripheral blood mononuclear cells from 3 out of 4 cases with lymphoid crisis of chronic myelogenous leukemia (CML) were positively stained by 3H12, while cells from 5 cases with myeloid crisis of CML did not react to this antibody. The antibody did not show any reactivity to cells from the chronic phase of CML, other types of leukemias or normal hematopoietic cells. We further examined 29 cell lines of hematopoietic origin and found that 2 undifferentiated cells (BV-173 and K-562) reacted to the 3H12 antibody. In addition, we found that 3 out of 6 Burkitt lymphoma cells (DAUDI, RAJI and HR1K) reacted to 3H12. Taken together, these results suggest that the antigen recognized by 3H12 is a differentiation-associated antigen expressed on immature lymphoid cells, and could potentially be a reliable cell lineage marker.     

正常供者外周血造血干细胞动员效果及影响因素

 目的　探讨粒细胞集落刺激因子（G-CSF）动员正常供者外周血造血干细胞的效果、毒副作用及影响因素。方法　对59名异基因造血干细胞正常供者采用G-CSF 皮下注射3 ~ 5 d,使用COBE Spectra血细胞分离机采集外周血干细胞,流式细胞术检测采集物中CD+34细胞数。结果　所有供者第一次采集的单个核细胞（MNC）及CD+34细胞量平均值分别为4.4（1.12～13.06）×108／kg供者体重及3.78（1.14～12.92）×106／kg供者体重。患者不良反应轻微。男性供者、年龄小于45岁者及采集前白细胞计数高者采集所得CD+34细胞数较高。结论　G-CSF作为正常供者动员剂安全有效。患者性别、年龄及采集前白细胞计数可作为预测因素。     

Aberrant promotor methylation in MDS hematopoietic cells during in vitro lineage specific differentiation is differently associated with DNMT isoforms

Aberrant promoter methylation may contribute to the hematopoietic disturbances in myelodysplastic syndromes (MDS). To explore a possible mechanism, we therefore analyzed expression of DNA methyltransferase (DNMT) subtypes kinetics and aberrant promoter methylation of key regulatory genes during MDS hematopoiesis. An in vitro model of MDS lineage-specific hematopoiesis was generated by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low-risk: RA/n=6, RARS/n=3; high-risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Promoter methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite-treated genomic DNA. Expression of DNMT1, DNMT3a and DNMT3b was analyzed and correlated with gene promoter methylation for each lineage at different time points. DNMT expression (all isoforms) was increased during thrombopoiesis whereas elevated DNMT1 level were seen during erythropoiesis. Associations between aberrant promoter methylation and DNMT expression were found in high-risk MDS for all lineages and during erythropoiesis. Hypermethylation of p15, p16, p73, survivin, CHK2, RARb and DAPK were associated with elevated DNMT isoform expression. No general overexpression of DNMT subtype was detected during MDS hematopoiesis. However a negative association of DNMT3a and 3b expression with MDS disease risk (IPSS) could be observed. Our data indicate that all mammalian DNMT isoforms may be involved in the aberrantly methylated phenotype in MDS but seem also to be essential for the differentiation of normal hematopoietic stem cells. In particular elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in MDS.     

Role of different normal hematopoietic regulatory proteins in the differentiation of myeloid leukemic cells

There are 4 different normal myeloid hematopoietic cell growth-inducing proteins MGI-1 (CSF or IL-3) that induce normal precursor cells to multiply and form clones containing only macrophages (MGI-1M = M-CSF = CSF-1), only granulocytes (MGI-1G = G-CSF), both granulocytes and macrophages (MGI-1GM = GM-CSF), or granulocytes, macrophages, eosinophils, mast cells, megakaryocytes and erythroid cells (interleukin-3) (IL-3). There is another type of normal myeloid regulatory protein (MGI-2) with no MGI-1 (CSF or IL-3) activity which can induce differentiation of normal myeloid precursors and certain clones of myeloid leukemic cells. The present results with MGI-2 and pure recombinant MGI-1G, MGI-1GM and IL-3 have shown that different clones of myeloid leukemic cells can be induced to differentiate by different hematopoietic regulatory proteins. One type of leukemic clone is induced to differentiate to mature cells only by MGI-2 and is partially differentiated by MGI-1G, a second type is differentiated only by MGI-1GM or IL-3, and other workers have found a third type that is differentiated only by MGI-1G. The presence of surface receptors does not necessarily make leukemic cells differentiation-competent for these hematopoietic regulatory proteins. All 4 types of MGI-1 (CSF or IL-3) induce endogenous synthesis of MGI-2 in normal myeloid precursor cells. It is suggested that, in addition to their potential therapeutic effect on the development of normal hematopoietic cells, MGI-2, MGI-1G, MGI-1GM and IL-3 all have the potential for differentiation-directed therapy of leukemia in leukemic cells that can be differentiated by one of these normal hematopoietic regulatory proteins.     

Retrotransposons as mutagens in the induction of growth autonomy in hematopoietic cells

Factor-independent mutants of hematopoietic cells, especially of multipotent cells, are valuable tools to identify genes that regulate stem cell proliferation and differentiation and thus may be important in leukemogenesis. Factor-independent mutants from both myeloid precursor and hematopoietic stem cell lines were isolated. The frequency of such mutants in a given cell population was one to two orders of magnitude lower for the multipotent cell line FDC-Pmix (3.6 x 10(-9)) than for the myeloid precursors, FDC-P1-M (1.7 x 10(-8)) and D35 (2.2 x 10(-7)). Analysis of these mutants revealed several mechanisms by which growth autonomy was obtained, either with or without direct contribution of growth factor gene activation. The molecular basis of spontaneous activation of the Multi-CSF (Interleukin3) gene was determined and compared to activation of the GM-CSF gene in a previous study. Multi-CSF gene activation in both precursor and stem cells was caused by the insertion of an intracisternal A particle (IAP) provirus. In two independent mutants of the D35 cell line, activation of the Multi-CSF or the GM-CSF gene was caused by almost identical IAPs with a 99% homology in the U3 and R region of the long terminal repeat. This result demonstrates that only one class of IAPs, or perhaps a single provirus, is involved in transposition and gene activation in a particular cell line. A unique example of anti-sense promotion from an IAP provirus in one Multi-CSF mutant underlines the versatility of these elements as natural insertional mutagens.     

血管抑制素对血管内皮细胞系ECV-304的抑制作用

 目的　观察血管抑制素 (Angiostatin)对血管内皮细胞系ECV 3 0 4的抑制作用。 方法　利用MTT法分析结果 ,实验分为 :1 .将血管抑制素与细胞同时加入 ;2 .先接种细胞 ,培养 2 4h后再加血管抑制素。结果　血管抑制素对ECV 3 0 4的抑制作用呈典型的量效关系。结论　血管抑制素对血管内皮细胞有显著的抑制作用。     

Adenovirus as a gene therapy vector for hematopoietic cells

Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.     

Oxaliplatin-DNA adduct formation in white blood cells of cancer patients

In this study, we investigated the kinetics of oxaliplatin-DNA adduct formation in white blood cells of cancer patients in relation to efficacy as well as oxaliplatin-associated neurotoxicity. Thirty-seven patients with various solid tumours received 130 mg m(-2) oxaliplatin as a 2-h infusion. Oxaliplatin-DNA adduct levels were measured in the first cycle using adsorptive stripping voltammetry. Platinum concentrations were measured in ultrafiltrate and plasma using a validated flameless atomic absorption spectrometry method. DNA adduct levels showed a characteristic time course, but were not correlated to platinum pharmacokinetics and varied considerably among individuals. In patients showing tumour response, adduct levels after 24 and 48 h were significantly higher than in nonresponders. Oxaliplatin-induced neurotoxicity was more pronounced but was not significantly different in patients with high adduct levels. The potential of oxaliplatin-DNA adduct measurements as pharmacodynamic end point should be further investigated in future trials.     

Peritumoral blood and lymphatic vessel invasion as a prognostic indicator in stage-I breast-carcinoma

Between 1978 and 1986 we treated 318 consecutive patients with stage I breast cancer. Median follow-up time is 8.5 years. In all cases the invasion of peritumoral lymphatic (LVI) and blood vessels (BVI) was studied by the hematoxylin-eosin staining method. Different ten-year survival (86% vs 67%) and disease-free survival (82% vs 61%) probabilities were found in patients with or without LVI. BVI showed low sensitivity and specificity, therefore vessel invasion was reassessed on 190 specimens with the immunoperoxidase technique using the Ulex Europeus Type 1 lectine. A significant correlation between the vessel staining intensity and the presence of intraluminal metastases was found. With this method a better sensitivity was obtained, but it was associated with a loss of specificity and prognostic significance.     

Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture

Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.     

Significance of blood vessel leakiness in cancer

Despite major advances in the field of tumor angiogenesis, relatively little attention has been paid to the permeability of blood vessels in tumors. The leakiness of tumor vessels is well documented in experimental tumor models and in human cancer, but the mechanism is poorly understood, as are the implications to the rate of cancer growth, predisposition to metastasis, and delivery of macromolecular therapeutics to tumor cells. Sixteen experts in the fields of cancer biology and vascular biology gathered at the William Guy Forbeck "Focus on the Future" Conference to discuss this topic. The meeting was the first of its kind focused on the significance of blood vessel leakiness in tumors. The participants discussed the cellular basis of tumor vessel leakiness, endothelial barrier function of blood vessels, monitoring tumor vessel leakiness, mediators of endothelial leakiness, consequences of tumor vessel leakiness, genomic analysis of vascular targets, targeting drugs to tumor vessels, and therapeutic manipulation of tumor vessels. The group concluded that a more complete understanding of the basic biology of tumor vessels will be necessary to fully appreciate the consequences of vessel leakiness in cancer. New research tools such as intravital measurements of tumor blood flow and vessel leakiness, in vivo phage display, magnetic resonance imaging, and use of selective angiogenesis inhibitors will contribute to this understanding.     

Reduced blood vessel formation and tumor growth in alpha5-integrin-negative teratocarcinomas and embryoid bodies.

Embryonic stem (ES) cells-wild-type, heterozygous, or null for alpha5-integrin-were injected ectopically into syngeneic mice to develop teratocarcinomas. alpha5-null-derived teratocarcinomas were significantly smaller than the wild-type or alpha5 heterozygous tumors. Histological analysis revealed the presence of tissues derived from all three germ layers, in all tumors. However, alpha5-null teratocarcinomas displayed less undifferentiated tissue than did the controls. Decreased proliferation and increased apoptosis were observed in the undifferentiated areas of the alpha5-null teratocarcinomas. The expression of extracellular matrix proteins, fibronectin and tenascin-C, and the basement membrane components, laminin, entactin/nidogen, and collagen IV, was similar in the different tumors, although the deposition of these molecules was more disorganized in alpha5-null teratocarcinomas. The absence of alpha5-integrin in the various tissues of the alpha5-null tumors was confirmed by immunohistochemistry. Many vessels, but not all, stained positively for alpha5-integrin, showing that they were host derived. Analysis of the area occupied by vessels revealed, on average, an 8-fold decrease in alpha5-null teratocarcinomas compared with control tumors. Staining for smooth muscle alpha-actin showed that pericytes and smooth muscle cells were recruited around the vessels in all tumors, suggesting similar vessel differentiation. Deposition of EIIIA and EIIIB and fibronectin around the vessels was observed in all tumors. The fact that some, although few, alpha5-integrin-negative vessels existed in alpha5-null tumors indicated that alpha5-/- ES cells could differentiate into endothelial cells. Endothelial cell differentiation and vessel formation were analyzed also in vitro. alpha5-null ES cells were differentiated into embryoid bodies, although they were delayed in growth and attachment. Differentiation into endothelial cells was achieved, but the organization into a complex vasculature was delayed compared with controls. We conclude that alpha5beta1-integrin plays a significant role in vessel formation both in ES cell cultures and in teratocarcinomas. Reduced vascularization likely contributed to the reduced proliferation and increased apoptosis observed in alpha5-null teratocarcinomas.     

MCS+血细胞分离机在自体外周血造血干细胞采集中的应用

目的探讨MCS+血细胞分离机在外周血干细胞采集中的使用和故障处理。方法总结我科自2002年1月至2004年12月共收治81例患者,采用MCS+血细胞分离机进行165次外周血干细胞采集,分析采集成功率、不良反应发生率和故障处理等情况。结果81例病人中90%为使用G-CSF后3~4天开始采集。68例患者进行股静脉插管采集,占84.0%;13例经肘正中静脉穿刺采集,占16.0%。采集过程最长6.3小时,最短4.2小时,平均4.8小时。按照累计采集物CD34+细胞总数>2.0×106/kg为采集成功标准,81例患者中78例需要连续2次采集,另有3例患者3次连续采集。单次采集处理血量为7326±627.3m l,采集物为87±18.4m l。81例患者165次采集中有10次出现ACD-A过敏反应(6.1%)。采集中1例患者出现终产品袋出口处被血凝块堵塞,2例患者出现插管血流不畅,血流速度低于20m l/m in,机器无法进行正常运转,故障发生率1.82%,上述情况经及时处理,未造成严重不良反应,均顺利完成采集。结论采用MCS+血细胞分离机进行外周血干细胞采集方法可靠,平均2次采集可达到临床治疗要求。     

In vitro studies on the invasion of blood vessel wall by choriocarcinoma cells

The development of metastases represents the lethal event in the clinical course of most neoplastic diseases. The complex mechanism responsible for the spread of cancer are governed by interactions between tumour cells and the host tissues. Choriocarcinoma cells and cultured blood vessels were used to study the interaction between tumour and endothelial cells. Seeding of tumour cells onto blood vessel wall caused retraction of endothelial cells leading to the exposure of underlying tissue. Subsequently, the tumour cells penetrated the sub-endothelial connective tissue and the internal elastic lamina.     

Mitochondria as a target for inducing death of malignant hematopoietic cells

Mitochondria plays a central role in apoptotic cell death. The intermembrane space of mitochondria contains a number of soluble molecules whose release from the organelle to the cytosol or the nucleus induces cell death. Thus, molecules that directly trigger mitochondria membrane permeabilisation are efficient cytotoxic drugs. Mitochondria is one of the cellular targets for commonly used epipodophyllotoxins, adenine deoxynucleoside analogs and taxanes as well as recently developped agents such as the pentacyclic triterpene betulinic acid and the lymphotoxic agent FTY720. Most informations on anthracyclines point to the mitochondrial membrane as the main target of cardiotoxicity. Mitochondria is also a target for arsenite trioxide, an old cytotoxic agent recently used for treating acute promyelocytic leukemia, lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid developped as a chemosensitizer, the retinoic acid receptor gamma activator CD437 and nitric oxide (NO). Recently, cytotoxic drugs have been specifically designed to directly affect the mitochondrial function. These include the positively charged alpha-helical peptides, which are attracted to and disrupt the negatively charged mitochondrial membrane, thus inducing mammalian cell apoptosis when targeted intracellularly. Various strategies have been proposed also to directly inhibit Bcl-2 and related anti-apoptotic proteins, including antisense oligonucleotides (e.g. Genasense, currently tested in phase III trials), small molecules that mimic the BH3 dimerization domain of these proteins and kinase inhibitors. Ligands of the mitochondrial benzodiazepine receptor such as the isoquinolone carboxamide derivative PK11195 also overcome the membrane-stabilizing effect of Bcl-2, whereas the adenosine nucleotide translocator (ANT) and the mitochondrial DNA are two other potential cellular targets for cytotoxic agents. Potentially, new compounds directly targeting the mitochondria may be useful in treating hematological malignancies. The challenge is now to selectively target these mitochondria permeabilizing agents to malignant cells. This review briefly summarizes the role of the mitochondria in cell death and describes these various strategies for targeting the mitochondria to induce apoptosis.