CD43, but not P-selectin glycoprotein ligand-1, functions as an E-selectin counter-receptor in human pre-B-cell leukemia NALL-1

B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is characterized by a high rate of tissue infiltration. The mechanism of BCP-ALL cell extravasation is not fully understood. In the present study, we have investigated the major carrier of carbohydrate selectin ligands in the BCP-ALL cell line NALL-1 and its possible role in the extravascular infiltration of the leukemic cells. B-precursor ALL cell lines and clinical samples from patients with BCP-ALL essentially exhibited positive flow cytometric reactivity with E-selectin, and the reactivity was significantly diminished by O-sialoglycoprotein endopeptidase treatment in NALL-1 cells. B-precursor ALL cell lines adhered well to E-selectin but only very weakly to P-selectin with low-shear-force cell adhesion assay. Although BCP-ALL cell lines did not express the well-known core protein P-selectin glycoprotein ligand-1 (PSGL-1), a major proportion of the carbohydrate selectin ligand was carried by a sialomucin, CD43, in NALL-1 cells. Most clinical samples from patients with BCP-ALL exhibited a PSGL-1(neg/low)/CD43(high) phenotype. NALL-1 cells rolled well on E-selectin, but knockdown of CD43 on NALL-1 cells resulted in reduced rolling activity on E-selectin. In addition, the CD43 knockdown NALL-1 cells showed decreased tissue engraftment compared with the control cells when introduced into gamma-irradiated immunodeficient mice. These results strongly suggest that CD43 but not PSGL-1 plays an important role in the extravascular infiltration of NALL-1 cells and that the degree of tissue engraftment of B-precursor ALL cells may be controlled by manipulating CD43 expression.     

CREB antisense oligonucleotides induce non-apoptotic cell death in proliferating leukemia cells, but not normal hematopoietic cells, by a bizarre non-antisense mechanism.

We report that antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) against cyclic AMP response element-binding protein (CREB) induce the death of human leukemia cell lines including HL-60, Kasumi-1 and K562, OCI-AML1a and also primary leukemia cells isolated from patients with acute myelocytic leukemia and chronic myelocytic leukemia in blastic crisis. In contrast, normal human bone marrow CD34+ cells and normal peripheral blood lymphocytes were resistant to the antisense-mediated cell death. We found that antisense-treated HL-60 cells had prominent nuclear fragmentations but lacked apoptotic features including internucleosomal DNA cleavage and TUNEL positivity. Cell cycle analysis demonstrated a remarkable reduction in G1 phase population along with a mild accumulation of S phase and good preservation of G2/M phase, indicating cells died at G2/M without cycling into G1 phase. None of the sense-sequenced PS-ODNs induced cell death. Further, neither the expression nor the message of CREB protein was reduced by antisense treatment, indicating that cell death was mediated by a non-antisense mechanism. On the other hand, no consensus oligonucleotide sequence for cell death induction was detected. Rather, we found a good correlation between the melting temperatures and the anti-proliferative activities of the oligonucleotides. Thus, CREB antisense PS-ODNs selectively induce a non-apoptotic cell death in leukemic cells by an unknown hybridization-dependent mechanism.     

Expression of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) genes in blasts of infant acute lymphoblastic leukemia with organ involvement

The mRNA contents of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of MMP (TIMP)-1 and TIMP-2 in leukemia cells from 33 infants with acute lymphoblastic leukemia (ALL) were quantified at initial presentation, and the correlation between their expression and patient clinical characteristics was examined. The mRNA contents of MMP-2 and MMP-9 were not associated with any patient characteristics. Positive correlation was found between hepatosplenomegaly and the MMP-2/TIMP-1 and MMP-2/TIMP-2 ratios (p=0.005 and 0.009) and between CNS involvement and the MMP-2/TIMP-2 ratio (p=0.012). The results suggest that MMP/TIMP balance is closely related to the infiltration of leukemia cells into extramedullary organs.     

Liposome-incorporated Grb2 antisense oligodeoxynucleotide increases the survival of mice bearing bcr-abl-positive leukemia xenografts

We previously demonstrated that liposome-incorporated antisense oligodeoxynucleotide specific for the grb2 mRNA (L-Grb2) inhibited Grb2 protein expression and the proliferation of bcr-abl-positive leukemia cell lines. To determine whether L-Grb2 has the potential of being a therapeutic modality against bcr-abl-positive leukemia, we studied the tissue distribution of L-Grb2 in normal mice before studying its effects in mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 was widely distributed in the body. The highest tissue concentrations of L-Grb2 were found in the spleen and liver, which are the organs where the tumor mass of bcr-abl-positive leukemia is mainly found. At 4 h post-injection, the amount of L-Grb2 detected per g of tissue was 64 microg in spleen and 50 microg in liver. Intravenous injection of bcr-abl-positive 32D mouse leukemia cells into radiated NOD/scid mice caused a lethal leukemia syndrome; we determined whether L-Grb2 could prolong the survival of mice bearing such xenografts. One day after leukemia cell inoculation, mice received twice weekly intravenous injections of L-Grb2. At an injection dose of 15 mg of L-Grb2 per kg of mouse body weight, 80% of mice treated with L-Grb2 survived to 48 days (end of study) whereas 0% of mice treated with the same dose of liposomal control oligonucleotide survived; the mean survival duration of these groups was 44 and 20 days, respectively. Our data indicate that L-Grb2 prolonged the survival of mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 may be used as a novel cancer therapeutic modality.     

Suppression of a squamous cell carcinoma (SCC)-related serpin, SCC antigen, inhibits tumor growth with increased intratumor infiltration of natural killer cells

Squamous cell carcinoma (SCC) antigen (SCCA), a member of the ovalbumin serine proteinase inhibitor family, serves as a circulating marker of squamous cell carcinoma (SC). One of the SCCAs, SCCA1, has been suggested to play a role in the attenuation of apoptosis in vitro and in the augmentation of tumor growth in vivo. In the present study, the infection of a SCC cell line (SKG IIIa) with recombinant retrovirus that expressed the antisense SCCA mRNA suppressed expression of SCCA in vitro. Local administration of this retrovirus into tumors by inoculation in nude mice suppressed tumor growth. Treatment of tumor tissue in vivo is also associated with increased numbers of apoptotic tumor cells and large mononuclear cells in the tumor. To test the possible role of SCCA in the infiltration of large mononuclear cells, we analyzed the effect of SCCA1 on migration of natural killer (NK) cells induced by monocyte-chemoattractant protein-1 in vitro. SCCA1 suppressed migration of NK cells completely, and this inhibitory effect was lost by mutation of the reactive site loop of SCCA1. These results suggest that antisense SCCA may suppress the growth of SCC in vivo not only by the augmentation of intracellular apoptosis but also by the increased infiltration of NK cells into the tumor.     

红色荧光蛋白标记的小鼠白血病模型的建立

 目的　利用红色荧光蛋白（DsRed）标记的小鼠淋巴瘤EL4细胞株,建立荧光标记的小鼠白血病模型,并对模型进行分析和鉴定。方法　将EL4／DsRed细胞以低剂量（5×102／只）、中剂量（5×103／只）、高剂量（2.5×104／只）经尾静脉注入经清髓照射的C57BL／6小鼠体内,同时接种骨髓细胞5×106／只,采用流式细胞术（FCM）、反转录聚合酶链反应（RT-PCR）、组织病理等方法鉴定小鼠成模情况。结果　C57BL／6小鼠接种不同剂量EL4／DsRed细胞后白血病发病率达100 %,移植后第2周FCM示受鼠肝、脾、骨髓和外周血中有大量EL4／DsRed细胞;各组组织器官病理检查呈现出不同程度的肿瘤细胞浸润。结论　用DsRed标记的EL4细胞以5×102／只植入C57BL／6小鼠,即可成功建立荧光标记的小鼠白血病模型,可为白血病发病机制、微小残留病检测等研究提供有价值的动物模型。     

WT1 in acute leukemia, chronic myelogenous leukemia and myelodysplastic syndrome: therapeutic potential of WT1 targeted therapies.

Among clinicians, initial awareness of the Wilms' tumor gene was limited mostly to pediatric oncologists. Almost a decade ago, overexpression of Wilms' tumor 1 (WT1) was observed in adult acute leukemia. Subsequent studies indicated that WT1 overexpression occurs in most cases of acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS). Limited tissue expression of WT1 in adults suggests that WT1 can be a target for leukemia/MDS therapy. WT1 expression in stem/progenitor cells remains unsettled. However, lack of progenitor cell suppression by WT1 antisense or WT1-specific cytotoxic T cells provide some assurance that WT1 expression in progenitor cells is minimal or absent. Immunotherapy-based WT1 approaches are furthest along in preclinical development. WT1-specific cytotoxic lymphocytes can be generated from normals and leukemic patients. In mice, WT1 vaccines elicit specific immune responses without evidence of tissue damage. In this paper, we review studies validating the immunogenicity of WT1 and propose that leukemia and MDS may be a good clinical model to test the efficacy of a WT1 vaccine.     

T cells in the human metastatic melanoma microenvironment express site‐specific homing receptors and retention integrins

T‐cell infiltration into the metastatic melanoma microenvironment (MME) correlates with improved patient survival. However, diffuse infiltration into tumor occurs in only 8% of melanoma metastases. Little is known about mechanisms governing T‐cell infiltration into human melanoma metastases or about how those mechanisms may be altered therapeutically. We hypothesized that T cells in the MME would be enriched for chemokine receptors CCR4, CCR5, CXCR3 and homing receptors relevant to the tissue site. Viably cryopreserved single cell suspensions from nineteen melanoma metastases representing three metastatic sites (tumor‐infiltrated lymph node, skin and small bowel) were evaluated by multiparameter flow cytometry and compared to benign lymph nodes and peripheral blood mononuclear cells from patients with Stage IIB–IV melanoma. T cells in the melanoma metastases contained large effector memory populations, high proportions of activated, moderately differentiated cells and few regulatory T cells. Site‐specific homing was suggested in bowel, with high expression of CCR9. We neither encounter the anticipated enrichment of integrin α4β7 in bowel, cutaneous leukocyte antigen (CLA) in skin, nor integrin α4β1 or receptor CXCR3 in metastatic sites. Retention integrins αEβ7, α1β1 and α2β1 were significantly elevated in metastases. These data suggest limited tissue site‐specific homing to human melanoma metastases, but a significant role for retention integrins in maintaining intratumoral T cells. Our findings also raise the possibility that T‐cell homing, infiltration, and retention in melanoma metastases may be increased by increasing expression of ligands for CLA, α4β1 and CXCR3 on intratumoral endothelium.     

Expression of the high-affinity selectin glycan ligand C2-O-sLeX by colon carcinoma cells

The selectin family of adhesion proteins directs leukocytes in the blood to lymphoid organs and sites of inflammation, and is also thought to be involved in the dissemination of carcinomas expressing sialylated Lewis glycan structures, such as sialyl-Lewis X (sLeX). The expression of core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) by leukocytes allows for the biosynthesis of core 2 O-glycans that when terminated by sLeX can serve as high-affinity selectin glycan ligands. In particular, the sLeX-modified core 2 O-glycan structure C2-O-sLeX has been directly demonstrated to confer significantly higher affinity selectin binding than sLeX. We have recently described the reactivity of the mAb CHO-131, which is dependent on the enzymes alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase, and C2GnT, and specifically recognizes the glycan structure C2-O-sLeX. Here we examined a defined pair of colon carcinoma cell lines that are distinct in their capacity to bind E-selectin, as demonstrated by shear flow assays involving whole blood and shear stresses that occur in the microvasculature. CHO-131 demonstrated reactivity with such cancer cells, but only with the cell line that avidly attached to E-selectin. Hence, we demonstrate for the first time the detection of C2-O-sLeX on colon carcinoma cells, which, as with leukocytes, may be directly relevant to the expression of high affinity glycan ligands for the selectins.     

Survivin反义RNA真核表达载体的构建及鉴定

目的 为了特异封闭白血病细胞survivin的表达，抑制其功能，本实验构建了survivin反义核酸载体并导入白血病细胞系中。方法 应用RT—PCR获得survivin的cDNA片段，反向插入pcDNA3质粒载体中；经限制性酶切和测序鉴定所构建的反义核酸是否正确；采用电转染方法将重组体导入HL—60细胞中；RT—PCR技术检测转染细胞survivin表达的变化。结果 经限制性酶切和测序鉴定证明survivin反义核酸已成功构建；RT—PCR产物电泳结果显示，与转染前细胞、空质粒转染细胞相比，转染survivin反义核酸的细胞survivin mRNA水平明显降低。结论 本实验已成功建立了survivin反义核酸真核表达载体，而且在白血病细胞系中发挥了特异封闭作用，为进一步研究survivin反义核酸在白血病治疗中的作用提供了实验基础。     

Enhanced invasiveness of drug‐resistant acute myeloid leukemia cells through increased expression of matrix metalloproteinase‐2

The acquired drug resistance as well as extramedullary tissue infiltration of leukemic cells is a major obstacle in leukemia treatment. Excessive egress of leukemia cell blasts results in invasion into various organs or tissues, which is facilitated by the catalytic activities of matrix metalloproteinases (MMPs). However, the migration of chemoresistant leukemia cells remains unclear. Here, we generated drug‐resistant variants of the human acute myeloid leukemia cell line (AML‐2/WT) by stepwise exposure to anticancer drugs and evaluated the level of MMP‐2 in the drug‐resistant variants, along with their invasiveness. Each of the drug‐resistant cell variants demonstrated predominant increases in the expression and gelatinolytic activity of MMP‐2 as well as in invasiveness, which were significantly suppressed by both a MMP‐2 inhibitor and a blocking antibody. Knockdown experiments using MMP‐2 short hairpin RNA also indicated that its upregulation was strongly associated with the cells' increased invasive properties. Importantly, elevated levels of MMP‐2 activity and invasiveness were observed in ex vivo mononuclear cell of bone marrow from patients with poor responses to chemotherapy. These findings suggest that advanced malignancy due to acquired drug resistance is responsible for the progressive invasiveness of leukemia cells via MMP‐2. © 2009 UICC     

THP-1 monocytic leukemia cells express Fas ligand constitutively and kill Fas-positive Jurkat cells

Normal human monocytes express Fas and are susceptible to Fas ligand (FasL)-induced apoptosis. Because the myeloid leukemia cell lines HL-60 and THP-1 can be differentiated into functional monocytes and macrophages, we studied their expression of Fas and Fas ligand (FasL) to determine whether there were differentiation-associated changes in these proteins. THP-1, HL-60 and HCW-2, both before and after treatment with PMA, expressed high levels of Fas ligand (FasL), but did not express Fas. The FasL expressed by THP-1 cells was functional as measured by their ability to kill Jurkat T-cells by apoptosis. The THP-1 Fas gene appears to be silent, because bacterial lipopolysaccharide (LPS) induced Fas expression in fully differentiated THP-1 cells. Our results suggest that FasL expression by leukemia cells may account in part for the pathophysiology of myeloid leukemia, and that PMA-differentiated THP-1 cells, while possessing many of the functional properties of normal macrophages, are abnormal with respect to a major apoptotic pathway.     

Recruited monocytic myeloid‐derived suppressor cells promote the arrest of tumor cells in the premetastatic niche through an IL‐1β‐mediated increase in E‐selectin expression

The tumor premetastatic niche initiated by primary tumors is constructed by multiple molecular factors and cellular components and provides permissive condition that allows circulating tumor cells to successfully metastasize. Myeloid‐derived suppressor cells (MDSCs), a population of immature cells in pathological conditions, play a critical role in the formation of the premetastatic niche. However, few researches are focused on the function of monocytic MDSCs (mo‐MDSCs), a subtype of MDSCs, in the construction of the niche. Here, we show that the number of mo‐MDSCs is significantly increased in the premetastatic lungs of tumor‐bearing mice, thus promoting tumor cell arrest and metastasis. Before the arrival of tumor cells, the lung‐recruited mo‐MDSCs produced IL‐1β, thereby increasing E‐selectin expression and promoting tumor cell arrest on endothelial cells. Depletion of mo‐MDSCs in the premetastatic lungs decreased IL‐1β production, resulting in reduced E‐selectin expression. In addition, compared with alveolar macrophages and interstitial macrophages, mo‐MDSCs were the major source of IL‐1β expression in the premetastatic lungs. Cytokine array analyses and transwell experiments revealed that CCL12 recruits mo‐MDSCs to premetastatic lungs. CCL12 knockdown in tumor‐bearing mice significantly decreased mo‐MDSC infiltration into the premetastatic lungs, leading to reduced E‐selectin expression. Overall, the permissive conditions produced by the infiltrated mo‐MDSCs correlated with increased tumor cell arrest and metastasis. These results reveal a novel role of mo‐MDSCs in constructing the premetastatic niche. Thus, inhibition of mo‐MDSCs infiltration may change the premetastatic niche to normal condition and attenuate tumor metastasis.     

P—选择素及其配体与肿瘤转移关系的研究

P-选择素是选择素这类粘附分子家族中的一员,它可以介导各种白细胞和某些恶性肿瘤细胞的粘附,P-选择素通常可以在被激活的血小板和上皮细胞表面出现,它一般是贮存在细胞内的颗粒中,受到生理或病理刺激后数分钟内就会运动到细胞表面,已被鉴定的P-选择素的糖蛋白配体有PSGL-1和CD24两种,P-选择素配体可以在多种人的癌细胞和癌细胞系中发现,并发现可能在肿瘤转移中起着重要作用。     

Suppression of proline-directed protein kinase F(A) expression potentiates erythroid differentiation of human myeloid leukemia cells

BACKGROUND: Initial clinic studies revealed that the overexpression of proline-directed protein kinase F(A) (PDPK F(A)) is associated conversely with various stages of tumor tissue differentiation. However, the role of overexpressed PDPK F(A) in tumor cell differentiation remains unknown and needs to be established. In this report, the authors explore the potential role of PDPK F(A) in cellular differentiation by investigating the effects of partial inhibition of this kinase on erythroid differentiation of chronic myeloid leukemia cells (K562). METHODS: PDPK F(A) antisense expression vector and its specific antibody were developed successfully. Two stable, transfected antisense clones of human myeloid leukemia cells were subcloned that expressed approximately 80% and approximately 50% of the total PDPK F(A) existing in control-transfected clones, as determined by both immunoprecipitate activity assay and immunoblot analysis. In sharp contrast, the PDPK F(A) antisense clones expressed no significant suppression of any other related PDPK members' expression, demonstrating the specificity of these two antisense clones. RESULTS: The antisense clones proportionally induced spontaneous erythroid differentiation up to approximately 30% of the total K562 cells. Moreover, antisense suppression of PDPK F(A) expression appeared to potentiate sodium butyrate/hemin-induced erythroid differentiation of K562 cells to a more complete stage compared with the control. CONCLUSIONS: The results demonstrate that specific antisense suppression of overexpressed PDPK F(A) in human myeloid leukemia cells is sufficient to potentiate both spontaneous and drug-induced erythroid differentiation, indicating that PDPK F(A) is an important negative regulator in controlling the erythroid differentiation of human myeloid leukemia cells.     

Adult T cell leukemia-like disease experimentally induced in rabbits

An HTLV-I-transformed T cell line, obtained from the peripheral blood of a virus-infected (B/J X Chbb:HM) F1 rabbit, was able to kill syngeneic newborn rabbits within 7 days, when inoculated intraperitoneally at a dose of 1 X 10(8) cells. Inoculation of 1 X 10(7) cells killed or rendered moribund 50% of inoculated animals, while surviving animals exhibited cell-mediated cytotoxic activities against the transformed cells. The peripheral blood leukocyte counts increased in all surviving animals, in association with appearance of abnormal lymphocytes with convoluted or lobulated nuclei. Pathological examination of animals that died one week post-inoculation revealed no tumors in the abdominal cavity, but accumulation of ascites containing abnormal lymphocytes. Histological examination showed leukemic infiltration in the liver, lungs, spleen and mesenteric lymph nodes. The same cell line was also able to kill syngeneic adult rabbits in 8-10 days when inoculated intravenously, but not intraperitoneally, at a dose of 1 X 10(8) cells. Leukemic infiltration was observed in the major organs of these animals. Adult animals which were already virus carriers were resistant to this lethal inoculation. This rabbit ATL-like disease may prove to be useful as an experimental model for acute adult T cell leukemia.