CD10-positive stromal cells in gastric carcinoma: correlation with invasion and metastasis

BACKGROUND: CD10 is a cell surface metalloproteinase expressed by a variety of normal cell types, including lymphoid precursor cells, germinal center B lymphocytes and some epithelial cells. Although accumulating data indicate that CD10 expression by stromal cells is involved in colorectal carcinogenesis and it is a novel prognostic factor in breast carcinoma, CD10-positive stromal cells and their correlation with invasion and metastasis have not been studied in gastric carcinoma. The aim of this study was to immunohistochemically investigate the rate of CD10 production in the stromal cells in our gastric carcinoma collection and clarify its correlation with invasion and metastasis. METHODS: One hundred and sixteen cases of gastric carcinoma were analyzed immunohistochemically using a monoclonal CD10 antibody (clone 56C6). RESULTS: The expression of CD10 by stromal cells was significantly higher in the primary gastric carcinomas than in normal and dysplasia mucosas (P = 0.014). More frequent expression of CD10 by stromal cells was detected in differentiated carcinoma than in undifferentiated carcinoma (P < 0.001). CD10 expression by the stromal cells was associated with depth of invasion and lymph node metastasis (P < 0.05). Stromal CD10 expression was lower in gastric carcinoma without vessel invasion than in those with vessel invasion (P = 0.001). However, no association was observed between stromal CD10 expression and TNM stage. In differentiated carcinoma, stromal CD10 expression was associated with the depth of invasion, lymph node metastasis, vessel invasion and TNM stage (P < 0.01). CONCLUSION: These results indicate that stromal cells expressing CD10 may play an important role in gastric carcinogenesis. CD10 expression by stromal cells seems to promote invasion and metastasis of differentiated gastric carcinoma.     

CD34-positive Early Stages of Human T-Cell Differentiation

Thymus, the main organ for T lymphopoiesis, requires a permanent influx of progenitors from bone marrow (BM) or fetal liver. An essential question relating to early T-cell development is the identification of the progenitor population which actually homes to the thymus. Recent findings have shown that human multipotent progenitor/stem cells expressing CD34 have the capacity to differentiate into T cells when introduced into a thymic environment. More mature CD34+ bone marrow cells coexpressing CD7 and having a poor myeloid differentiation capacity can also efficiently differentiate into T cells in vitro. These lymphoid committed precursors might be the true thymic repopulating cells. In the thymus, cells with a similar CD34+7+ phenotype include the most primitive thymocyte precursors. CD34+ thymocytes have no myeloid differentiation potential, but may include precursors for natural killer (NK) cells. Interleukin-7 (IL7) is a potent in vitro growth factor for CD34+ thymocytes. Whereas current data do not support a crucial role for IL2, patients with IL2 receptor y chain (IL2Ry) deficiency lack T- and NK cells. The recent demonstration that IL2Ry is part of the receptor for IL7 strongly suggests that this cytokine plays an essential role in in vivo T lymphocyte and NK development.     

Quantitative evaluation of lysozyme- and CD68-positive Kupffer cells in diethylnitrosamine-induced hepatocellular carcinomas in monkeys

Quantitative analysis of lysozyme- and CD68-positive Kupffercells was carried out in connection with diethylnitrosamine-inducedhepatocarcinogenesis in non-human primates. The number of Kupffercells/mm2 was determined in 28 cases of hepatocellular carcinoma(HCC) and seven age-matched controls. The Kupffer cell counts(mean ± SEM) gradually decreased in the following order,irrespective of the histochemical markers (lysozyme or CD 68)used: healthy control liver (101.7 ± 13.5 and 103.2 ±11.9 respectively), non-cirrhotic and non-neoplastic host liver(54.3 ± 13.6 and 50.5 ± 15.4), cirrhotic hostliver (26.2 ± 8.2 and 27.2 ± 3.3), HCC tissue(20.7 ± 4.4 and 19.3 ± 4.1) and metastatic fociin the lung (9.8 ± 1.8 and 9.7 ± 2.8). The differencebetween the normal liver and the non-neoplastic, non-cirrhoticportions of the HCC-bearing liver was significant (P < 0.05).A highly significant difference was found between the numberof Kupffer cells found in healthy control or non-neoplasticliver and those found in HCC nodules (P < 0.0001 and P <0.0005 respectively). The results obtained by hematoxylin andeosin staining and lysozyme/CD68 immunohistochemistry were highlysimilar, indicating that this decrease was attributable primarilyto numeric loss of Kupffer cells. The results suggest that thereduction in the number of Kupffer cells in HCC is a constantfeature of hepatocarcinogenesis not only in rodent models, butalso in non-human primates.     

CD34阳性及阴性成年人急性T淋巴细胞白血病临床分析

目的 分析CD34阳性及阴性成年急性T淋巴细胞白血病(T-ALL)患者的临床特点及预后,探讨CD34表达对T-ALL预后的价值.方法 回顾性分析郑州大学第一附属医院血液科2012年1月至2015年7月收治的75例初治成年T-ALL患者.根据CD34的表达情况,分为CD34阳性组与阴性组,对两组患者的临床特点及预后进行比较.结果 75例初治成年T-ALL患者中,CD34阳性组24例(32.0％),CD34阴性组51例(68.0％).CD34阳性组与阴性组在性别、年龄、肝脾大、淋巴结肿大、血小板减少、白细胞增高、染色体异常、4周完全缓解(CR)率、中枢神经系统白血病(CNSL)方面,差异均无统计学意义(均P＞ 0.05);初诊时CD34阳性组血红蛋白(Hb) ＜90g/L、伴髓系抗原表达者的比例高于阴性组,差异均有统计学意义(x 2=5.888,P=0.015;x 2=10.758,P=0.001).18例选择造血干细胞移植(HSCT),57例未选择HSCT.在未选择HSCT的患者中,CD34阳性组中位生存期为5个月,阴性组为32个月,两者差异有统计学意义(x2=9.172,P=0.002).结论 成年T-ALL患者CD34表达与Hb＜ 90 g/L及髓系抗原表达有关;未选择HSCT患者的CD34表达可能与预后呈负相关.     

Dietary flavonoids induce MLL translocations in primary human CD34+ cells

Genetic abnormalities leading to infant leukemias already occur during fetal development and often involve rearrangements of the mixed-lineage leukemia (MLL) gene. These rearrangements resemble the aberrations observed in therapy-related leukemias following treatment with topoisomerase II (topoII)-inhibiting agents such as etoposide. Since flavonoids are potent topoII inhibitors, we examined the role of three widely consumed dietary flavonoids (quercetin, genistein and kaempferol) on the development of MLL rearrangements in primary human CD34(+) cells. Using the neutral Comet assay, we demonstrated a dose-dependent double-strand break (DSB) formation after exposure to flavonoids. An incorrect repair of these DSBs resulted in chromosomal translocations that co-localized with those identified in infant leukemias. Most of these translocations were formed by microhomology-mediated end joining. Moreover, in all but one translocation, SINE/Alu or LINE/L1 repetitive elements were present in at least one side of the breakpoint junction. Beside MLL translocations, fluorescence in situ hybridization analysis demonstrated monosomy or trisomy of MLL in 8-10% of the quercetin-exposed CD34(+) cells. Our study demonstrates that biologically relevant concentrations of flavonoids can induce MLL abnormalities in primary hematopoietic progenitor cells. This is particularly alarming knowing that the differences in metabolism and excretion rate between mother and fetus can lead to a higher flavonoid concentration on the fetal side. Therefore, it is important to raise public awareness and set guidelines for marketing flavonoid supplements to reduce the risk of infant leukemias.     

Cell kinetics of CD34-positive hematopoietic cells following chemotherapy plus colony-stimulating factors in advanced breast cancer

Bone-marrow (BM) hematopoietic precursors are recruited into proliferative activity when colony-stimulating factors (CSF) are sequenced with chemotherapy (CT). Previous studies suggested that further CT can be safely administered only when the increased proliferative activity of these cells has subsided, because most cytostatic drugs selectively damage cycling cells. The safest interval between CSF discontinuation and the start of the next CT course needs to be ascertained in vivo. Thirty patients with advanced breast cancer were treated with an intensified FEC regimen, planned at 21-day intervals, sequenced with granulocyte-macrophage (GM)-CSF (15 patients) or granulocyte (G)-CSF (15 patients). Using flow cytometry (FCM) we evaluated the proliferation kinetics of CD34⁺ BM hematopoietic progenitors before CT + CSF and at different times after CSF administration was stopped. FEC + GM- and FEC + G-CSF sequences both induced a rapid and sustained increase in the percentage of BM myeloid precursors (BMMP%) and in the cycling status of CD34+ BM cells. However, while the BMMP% remained elevated in both cases after CSF were stopped, the enhanced proliferative activity of CD34+ cells decreased more rapidly after GM- than after G-CSF. Using FCM, CD34⁺ BM-derived hematopoietic presursor cell kinetics is readily evaluated in the clinical setting. The administration of CSF following CT increases both the proliferative activity of CD34+ BM cells and the BMMP%. After CSF were discontinued a kinetic refractoriness of hematopoietic progenitors was more evident after GM-CSF than after G-CSF. These data may be of value in designing clinical trials to avoid cytostatic damage to the BM hematopoietic stem-cell compartment. © 1995 Wiley-Liss, Inc.     

Ajoene, a garlic-derived natural compound, enhances chemotherapy-induced apoptosis in human myeloid leukaemia CD34-positive resistant cells

The reputation of garlic as an effective remedy for tumours extends back to the Egyptian Codex Ebers of 1550 BC. Several garlic compounds, including allicin and its corresponding sulfide, inhibit the proliferation of several human malignant cells. Ajoene is a garlic-derived compound produced most efficiently from pure allicin and has the advantage of a greater chemical stability than allicin. Recently, ajoene was shown to inhibit proliferation and induce apoptosis of human leukaemia CD34-negative cells including HL-60, U937, HEL and OCIM-I. More significantly, ajoene was shown to induce 30% apoptosis in myeloblasts from a chronic myeloid leukaemia patient in blastic crisis. Acute myeloid leukaemia (AML) is a heterogeneous malignant disease in which disease progression at the level of CD34-positive cells has a major impact on resistance to chemotherapy and relapse. MATERIALS AND METHODS: The aim of the present study was to investigate the effect of ajoene on changes in the expression of apoptosis-related proteins: bcl-2 and caspase-3, induced by two principal drugs used in treatment of AML, cytarabine and fludarabine, in KGI human myeloid leukaemia CD34-positive-resistant cells. Both quantitative ELISA measurement of bcl-2 and colourimetric measurement of active caspase-3 were used. RESULTS: Quantitative ELISA measurement of bcl-2 (units per million cells) showed treatment of KG1-resistant leukaemia cells with 40 microM ajoene alone to significantly reduce the bcl-2-expression from 239.5 +/- 1.5 in control cultures to only 22.0 +/- 4.0 in ajoene-treated cultures. Fludarabine had significantly more inhibitory effect on bcl-2-expression than cytarabine in KGI-resistant myeloid leukaemia cells. Ajoene significantly enhanced the inhibitory effect of the two chemotherapeutic drugs, cytarabine and fludarabine, on bcl-2-expression in KGI cells. Bcl-2-expression could not be detected in fludarabine + ajoene-treated cultures. The Western blot of bcl-2-expression in KGI control and treated cells confirmed the quantitative ELISA measurements. Quantitative measurement of activated caspase-3 (pg per million cells) showed the two drugs, cytarabine and fludarabine, significantly increased the activated caspase-3 level in KGI myeloid leukaemia cells. CONCLUSION: The addition of ajoene enhanced the activation of caspase-3 in both cytarabine- and fludarabine-treated KGI cells. In conclusion, the present results suggest a potential role for the combination of ajoene with fludarabine-based chemotherapy in the treatment of refractory and/or relapsed AML patients. Further studies are warranted to evaluate a similar enhancing effect for ajoene in blast cells from AML patients in primary cultures.     

IKAROS isoform 6 enhances BCR-ABL1-mediated proliferation of human CD34+ hematopoietic cells on stromal cells

The BCR-ABL1 induces chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Recent studies revealed high ratios of loss of the IKZF1 gene which encodes IKAROS in BCR-ABL1+ ALL and lymphoblastic crisis (LBC) of CML. However, little is known about the cooperativity between the aberrant IKAROS and BCR-ABL1 in primary human hematopoietic cells. We investigated the effects of expression of BCR-ABL1 and/or IK6, a natural dominant negative isoform of IKAROS, on proliferation and differentiation of human CD34+ cord blood cells with or without human bone marrow-derived stromal cells which support early B cell differentiation. Cell proliferation was remarkably enhanced by co-expression of BCR-ABL1 and IK6, with reduced expression of glycophorin A and increased expression of CD41, especially on stromal cells, compared with expression of BCR-ABL1 alone that resulted in expansion of erythroid progenitors. Interestingly, p190BCR-ABL1 showed higher dependency on stromal cells to stimulate cell growth with IK6, than p210BCR-ABL1. Furthermore, the cooperation was found to depend on direct cell adhesive interaction of hematopoietic progenitors with stromal cells. These findings suggest that IK6 and BCR-ABL1 synergistically contribute to leukemogenesis in human bone marrow stromal microenvironment, and may provide a clue to elucidate the mechanisms of leukemogenesis of Ph+ ALL and CML-LBC.     

Hematopoietic stem cells can be CD34+ or CD34-.

Until recently, it was thought that the most primitive HSC have a fixed phenotype within a hierarchical differentiation system, and that changes in engraftment and renewal potential occur in a stepwise fashion linked with differentiation. In this review, we summarize the data from several different species and different animal models of hematopoietic stem cell function. Taking into account all of the published data, it becomes clear that the hematopoietic stem cell compartment contains more than one phenotypically identifiable population capable of self-renewal and long term pluripotent engraftment. It is clear that some stem cells express CD34, and others do not. The exact phenotypic progression between these cells needs to be further defined, because different in vivo and ex vivo manipulations may shift the stem cells from one phenotype to another, and this can complicate interpretation of experimental transplant data.     

CD7 and CD56-positive primary effusion lymphoma in a human immunodeficiency virus-negative host

Primary effusion lymphoma is an entity with distinctive features. The majority of cases are diagnosed in patients infected with human immunodeficiency virus. We report a case of pleural-based primary effusion lymphoma in an elderly patient negative for human immunodeficiency virus. By flow cytometry, lymphoma cells expressed CD7, CD38, CD45, CD56, HLA-DR, and kappa surface light chains. A monoclonal rearrangement of the immunoglobulin heavy chain and the presence of human herpesvirus 8 genome were detected. Our case lacked CD30 or CD138 with expression of surface light chains. There was strong expression of CD7 and CD56. These findings are unusual or unique in primary effusion lymphoma. Our report suggests that aberrant expression of T cell and natural killer cell markers can be seen in primary effusion lymphoma.     

Circulating CD34+, CD133+, and vascular endothelial growth factor receptor 2-positive endothelial progenitor cells in myelofibrosis with myeloid metaplasia.

PURPOSE: Endothelial progenitor cells (EPCs) are present in circulation and contribute to vasculogenesis in adults. We measured the number of circulating EPCs in patients with myelofibrosis with myeloid metaplasia (MMM), and we examined the relationship between the number of EPCs and severity of the MMM disease process. PATIENTS AND METHODS: The number of EPCs was measured by assaying the CD34+CD133+ vascular endothelial growth factor receptor 2 (VEGFR2) -positive cell phenotype in 110 MMM patients, 16 patients with other Philadelphia-negative chronic myeloproliferative disorders (Ph-negative CMPDs), and 14 healthy participants. In four MMM patients, the capacity of selected CD34+ cells to form endothelial colonies (CFU-End) in vitro was tested. RESULTS: CD34+, CD133+, and VEGFR2-positive EPCs were detectable in unselected peripheral-blood cells of 50.9% MMM patients, 37.5% control patients, and 21% healthy participants. Patients with MMM had a median of 0.26% EPCs, significantly higher than that in healthy controls (median, 0%) and in patients with other Ph-negative CMPDs (median, 0.1%). In 14.5% of MMM patients, the numbers of EPCs were greater than the highest value found in patients with other Ph-negative CMPDs. CD34+ selected cells produced colony-forming unit-endothelial (CFU-End), which were vascular endothelial (VE) -cadherin positive, CD31+, von Willebrand factor positive, and CD45-. In MMM patients, the larger the number of EPCs, the smaller the number of circulating immature myeloid cells and circulating CD45+CD34+ hematopoietic progenitor cells. Increased numbers of EPCs were associated with younger age and a diagnosis of prefibrotic MMM. CONCLUSION: Circulating EPCs are elevated in MMM patients in the early stage of the disease. Heightened mobilization of EPCs may represent an important mechanism for development of neoangiogenesis in MMM.     

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to contain CD34⁺ natural suppressor cells that inhibit the activity of intratumoral T-cells. The present study evaluated whether GM-CSF production and the presence of CD34⁺ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer-related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM-CSF and for CD34⁺ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4-fold the levels of GM-CSF and had approximately 2.5-fold the number of CD34⁺ cells as did cancers of patients that remained disease-free. In a second method of analysis, the prognostic significance of high vs. low GM-CSF and CD34⁺ cell values was evaluated. These analyses showed that patients whose cancers produced high GM-CSF levels or had a high CD34⁺ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM-CSF or had a low CD34⁺ cell content remained disease-free (16% and 19%, respectively). Our results indicate that the presence of CD34⁺ cells in GM-CSF-producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34⁺ cells in GM-CSF-producing HNSCC leads to increased tumor recurrence or metastasis.Int. J. Cancer 74:69–74. © 1997 Wiley-Liss, Inc.     

The CD9 molecule on stromal cells

Numerous functions have been attributed to CD9 and other members of the transmembrane 4 (TM4) superfamily. CD9 is thought to be involved in cell proliferation, differentiation, motility and survival. It may also function as part of toxin and virus receptor complexes. Although much remains to be learned about molecular mechanisms, the molecule associates with several integrins, small G proteins, MHC class II molecules and other TM4 superfamily proteins on a given cell surface membrane. Here, we briefly discuss the CD9 displayed on stromal cells that support hematopoiesis and the potential importance of this molecule to osteoclast differentiation.     

Ex-vivo expansion and maturation of CD34-positive hematopoietic progenitors optimization of culture conditions

The aim of this study was to look for an ex vivo culture system for clinical application. We evaluated the ex vivo expansion of peripheral blood CD34+ cells in gas-permeable bags and whether or not an exogenous protein source would be required in these kind of cultures. We also evaluated maturation of the cells during culture. Cells were cultured for 15 days in medium supplemented with SCF, G-CSF, IL3 and IL6. The bags supported the expansion of hematopoietic cells in a similar manner to small scale flasks system: (a) the expansion means of total nucleated cells on day +5 were 12.5-fold for bag versus 5-fold for flask, on day +10 were 44.12-fold for bag versus 41-fold for flask and on day +15 were 67.7-fold for bag versus 84.2-fold for flask, (b) the peak values of CFU-GM were reached on day +10 (9.2-fold for bag vs. 12-fold for flask), and (c) maximal expansion of CD15+/CD11b- population occurred on day +10 (517.5-fold for bag vs. 2959.2-fold for flask). So, we did not find any advantages by culturing further than day +10. We subsequently investigated the use of serum-free medium. The study showed better results when we used medium supplemented with autologous plasma versus serum-free system. In summary, these data described a strategy of culture clinically feasible and safe, using gas-permeable bags, and the kinetics and differentiation of neutrophils and neutrophil precursors from selected CD34+ cells in liquid cultures. Ex vivo expansion of this population might result in earlier engraftment as compared with that for selected stem cells alone.     

Frequent gene dosage alterations in stromal cells of epithelial ovarian carcinomas

Stromal cells are an active and integral part of epithelial neoplasms. We have previously observed allelic imbalance on chromosome 3p21 in both stromal and epithelial cells of ovarian tumors. This study was designed to explore gene dosage alterations throughout human chromosomes from stromal and epithelial cells of epithelial ovarian carcinomas. Thirteen stromal and 24 epithelial samples, microdissected from epithelial ovarian carcinomas, were analyzed using multiplex ligation-dependent probe amplification technique. Analysis covered 110 cancer related genes. Frequent genetic alterations were detected both in the stroma and epithelium of ovarian carcinomas. The mean number of altered genes per tumor was 10.8 in stroma and 23.6 in epithelium. In the stroma, the mean number of gains was 6.6 and of losses 4.2 and in the epithelium 13.7 and 9.9. The high number of changes associated with advanced tumor stage (p = 0.035) and death due to ovarian cancer (p = 0.032). The most frequent alteration was the deletion of the deleted in colorectal carcinoma (DCC) on chromosome 18q21.3 in 62% of samples. Loss of DCC was related to endometrioid subtype (p = 0.033). Large chromosomal aberrations were detected on the basis of alterations in adjacent genes. Most importantly, 38 genes showed similar genetic alterations (gain-gain or loss-loss) in stromal and epithelial compartments of 11 tumor pairs. Thus, frequent genetic alterations in stromal cells of epithelial ovarian carcinomas resembled those of malignant epithelial cells and may indicate a common precursor cell type. Epithelial-mesenchymal transition may generate transformed cancer cells and modify the tumor microenvironment with distinct properties.     

CD164--a novel sialomucin on CD34+ cells

Hematopoiesis in adult bone marrow is a tightly regulated process involving interactions between cytokine and adhesion receptors on hematopoietic progenitor cells and their cognate ligands in the immediate microenvironment. These interactions control hematopoietic stem cell self-renewal, quiescence, commitment and migration. Recently, sialomucins have assumed some importance in hematopoiesis, with six of these receptors, CD34, PSGL-1, CD43, PCLP, CD45RA and CD164, having been identified on primitive hematopoietic precursor cells and/or their associated stromal/endothelial elements. This article reviews the cloning, expression and function of the recently identified sialomucin, CD164, which is highly expressed by primitive hematopoietic progenitor cells. The CD164 receptor is implicated in mediating or regulating hematopoietic precursor cell adhesion to stroma, and may serve as a potent negative regulator of hematopoietic progenitor cell proliferation.     

乳腺间质CD34和平滑肌肌动蛋白免疫组化检测在乳腺癌诊断中应用

目的 :评价免疫组化检测乳腺间质纤维细胞CD34和平滑肌肌动蛋白 (SMA)表达在乳腺癌诊断中价值。方法 :6 7例乳腺活检组织进行CD34和SMA免疫组化检测 ,其中乳腺癌 4 2例 ,乳腺良性病变 2 5例。观察各类乳腺病变间质的CD34和SMA表达状况 ,并分析CD34和SMA表达的相关性。结果 :2 5例良性病变中CD34表达 10 0 % (2 5 / 2 5 ) ,SMA表达 4 8% (12 / 2 5 )。 34例浸润性乳腺癌 (不包括 8例单纯导管内癌 )CD34全部缺乏表达 (0 /34) ,SMA表达 10 0 % (34/ 34)。CD34和SMA表达在乳腺浸润性癌和乳腺良性病变中表达的差异均具有显著性 (P<0 .0 1和P <0 .0 1)。导管内癌 (单纯导管内癌 8例和伴有浸润性癌的导管内癌成分 13例 )导管周围间质的纤维细胞CD34和SMA表达不均一 ,呈现为不同程度丢失CD34表达和获得SMA表达 ,并且CD34和SMA的表达呈负相关 (r= 0 .931,P <0 .0 1)。结论 :本研究结果表明乳腺间质纤维细胞CD34表达缺乏是浸润性乳腺癌间质改变的一项敏感指标。导管内癌管周间质的纤维细胞CD34和SMA表达不均一体现了从原位癌向浸润性癌发展的过程 ,因此 ,导管内癌管周间质丢失CD34 纤维细胞同时出现SMA 肌纤维母细胞反应可能是癌细胞向间质浸润的前兆。     

c-myc protein kinetics during growth and differentiation of CD34 (MY10)-positive blast cells from normal human marrow

We have used flow cytometry to quantitate nuclear c-myc protein, at each phase of the cell cycle, during in-vitro differentiation of CD34-positive stem cells isolated from normal human bone marrow by the monoclonal antibody, MY10. Mean c-myc protein levels in CD34-positive cells, consisting of greater than 70% blasts, are lower than a marrow fraction containing myeloid cells of intermediate maturation, but have an invariant proportional relationship, with regard to nuclear mass, over the cell cycle. The majority of these primitive cells are non-cycling, as revealed by DNA content. Under our assay conditions, nuclear c-myc protein distribution over the cell cycle did not change as these progenitors entered a proliferative phase in culture. In cultures containing factors supporting myeloid maturation, mean G0/G1 p62c-myc levels initially decline, then rise above starting values as promyelocytes and myelocytes differentiate from CD34-positive cells, and as proliferation begins. With further myeloid maturation, and while cell numbers are increasing, c-myc protein continues to increase. C-myc protein kinetics differ in cultures in which macrophages, rather than myeloid cells, predominate. These data indicate that a complex relationship exists between c-myc gene expression and proliferation, maturation and lineage in haemopoietic cells, and lend support to the notion that early down regulation may be causally associated with the differentiation process.     

CD40配体对转CD40 cDNA的人肺癌细胞功能调节的研究

目的 研究CD40配体(CD40L,CD154)对CD40阴性肺癌细胞系转染CD40 cDNA后(即CD40高表达细胞系)的调节作用,评价CD40作为治疗靶抗原的潜能。方法 通过流式细胞术筛选CD40阴性细胞系,即GLC-82细胞。以3D5细胞为模板,以pCDNA3为载体,通过基因克隆技术制备CD40 cDNA并转染到GLC-82细胞中,建立CD40高表达细胞系,即GLC-82／CD40。在细胞培养液中加入0．1μg／ml CD40L,通过流式细胞术和MTT试验检测CD40L对细胞表型、细胞生长、细胞周期及凋亡的影响。结果 Western蛋白印迹检测、限制性酶切电泳、DNA测序和流式细胞仪测定均证明了CD40 cDNA转染成功,转染后细胞的CD40表达率高达95．9％。CD40L与CD40作用后,使GLC-82／CD40细胞系上MHC-1、ICAM-1和Fas分子的表达增强,EGFR表达减弱。细胞生长受到抑制,第5天受抑最明显,受抑率为30％,但无周期特异性变化。CD40L停用48h后,各种指标的变化有所恢复。CD40L对原有高表达CD40的Calu-3肺癌细胞上述指标的影响较GLC-82／CD40更明显,而对不表达CD40的GLC82细胞无明显影响。所有肺癌细胞系均未出现细胞凋亡。结论 CD40L对转染CD40 cDNA后CD40高表达肺癌细胞系具有免疫导向治疗的潜能。