Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos.

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase.

We purified CALLA from human kidney and isolated a cDNA clone reactive with two oligonucleotide probes corresponding to two distinct peptides. The amino acid sequence translated from the CALLA cDNA revealed 100% identity with that of human neutral endopeptidase (NEP, enkephalinase). The distribution of CALLA antigen and NEP in normal tissues are similar.

Age-dependent endothelial nitric oxide synthase uncoupling in pulmonary arteries of endoglin heterozygous mice.

Endoglin is a TGF-beta superfamily receptor critical for endothelial cell function. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia type I (HHT1), and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng(+/-)) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng(+/-) mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng(+/-) vs. control vessels. The vasodilatory responses were inhibited by l-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O(2) species (ROS) in adult Eng(+/-) vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng(+/-) lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng(+/-) mice. Our data suggest that eNOS-dependent H(2)O(2) generation in Eng(+/-) lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations.

Screening for pulmonary and cerebral arteriovenous malformations in children with hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterised by vascular dysplasia complicated by visceral arteriovenous malformations (AVMs). To date, the diagnostic yield of screening procedures for pulmonary and cerebral AVMs in children with definite or potential HHT is not well defined. The aim of the present study was to prospectively evaluate the diagnostic yield of a screening protocol for pulmonary and cerebral AVMs in children with either a definite or potential HHT diagnosis. All children referred for evaluation for HHT between 1996 and 2008 were included in the present analysis. Screening tests for AVMs included chest computed tomography and brain magnetic resonance imaging. 61 children with a definite clinical and/or genetic diagnosis of HHT were asymptomatic for visceral AVMs at their first baseline assessment (mean+/-SD age 8.7+/-4.7 yrs; range 0-17.0 yrs). Of these, 15 (25%) had pulmonary and/or cerebral AVMs diagnosed on initial screening tests. Pulmonary AVMs predominated in paediatric HHT patients (14 out of 15 patients) and were found in eight children aged <10 yrs. 55 children had a potential HHT diagnosis as they fulfilled only one or two HHT clinical diagnostic criteria and did not have a confirmatory genetic diagnosis (age 10.9+/-4.8 yrs; range 0-17.9 yrs). None of these children had pulmonary or cerebral AVMs on initial screening tests. The present data suggest that children with a definite HHT diagnosis have a high frequency of pulmonary AVMs even when clinically asymptomatic. In contrast, no AVMs were observed in children not fulfilling HHT diagnostic criteria. Genetic testing appears to be useful in defining an at-risk group for pulmonary AVMs in childhood.

Endoglin modulates cellular responses to TGF-beta 1.

Endoglin is a homodimeric membrane glycoprotein which can bind the beta 1 and beta 3 isoforms of transforming growth factor-beta (TGF-beta). We reported previously that endoglin is upregulated during monocyte differentiation. We have now observed that TGF-beta itself can stimulate the expression of endoglin in cultured human monocytes and in the U-937 monocytic line. To study the functional role of endoglin, stable transfectants of U-937 cells were generated which overexpress L- or S- endoglin isoforms, differing in their cytoplasmic domain. Inhibition of cellular proliferation and downregulation of c-myc mRNA which are normally induced by TGF-beta 1 in U-937 cells were totally abrogated in L-endoglin transfectants and much reduced in the S-endoglin transfectants. Inhibition of proliferation by TGF-beta 2 was not altered in the transfectants, in agreement with the isoform specificity of endoglin. Additional responses of U-937 cells to TGF-beta 1, including stimulation of fibronectin synthesis, cellular adhesion, platelet/endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation, and homotypic aggregation were also inhibited in the endoglin transfectants. However, modulation of integrin and PECAM-1 levels and stimulation of mRNA levels for TGF-beta 1 and its receptors R-I, R-II, and betaglycan occurred normally in the endoglin transfectants. No changes in total ligand binding were observed in L-endoglin transfectants relative to mock, while a 1.5-fold increase was seen in S-endoglin transfectants. The degradation rate of the ligand was the same in all transfectants. Elucidating the mechanism by which endoglin modulates several cellular responses to TGF-beta 1 without interfering with ligand binding or degradation should increase our understanding of the complex pathways which mediate the effects of this factor.

A model of human acute lymphoblastic leukemia in immune-deficient SCID mice.

A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies.

Differentially androgen-modulated genes in ovarian epithelial cells from BRCA mutation carriers and control patients predict ovarian cancer survival and disease progression.

Epidemiological studies have implicated androgens in the etiology and progression of epithelial ovarian cancer. We previously reported that some androgen responses were dysregulated in malignant ovarian epithelial cells relative to control, non-malignant ovarian surface epithelial (OSE) cells. Moreover, dysregulated androgen responses were observed in OSE cells derived from patients with germline BRCA-1 or -2 mutations (OSEb), which account for the majority of familial ovarian cancer predisposition, and such altered responses may be involved in ovarian carcinogenesis or progression. In the present study, gene expression profiling using cDNA microarrays identified 17 genes differentially expressed in response to continuous androgen exposure in OSEb cells and ovarian cancer cells as compared to OSE cells derived from control patients. A subset of these differentially affected genes was selected and verified by quantitative real-time reverse transcription-polymerase chain reaction. Six of the gene products mapped to the OPHID protein-protein interaction database, and five were networked within two interacting partners. Basic leucine zipper transcription factor 2 (BACH2) and acetylcholinesterase (ACHE), which were upregulated by androgen in OSEb cells relative to OSE cells, were further investigated using an ovarian cancer tissue microarray from a separate set of 149 clinical samples. Both cytoplasmic ACHE and BACH2 immunostaining were significantly increased in ovarian cancer relative to benign cases. High levels of cytoplasmic ACHE staining correlated with decreased survival, whereas nuclear BACH2 staining correlated with decreased time to disease recurrence. The finding that products of genes differentially responsive to androgen in OSEb cells may predict survival and disease progression supports a role for altered androgen effects in ovarian cancer. In addition to BACH2 and ACHE, this study highlights a set of potentially functionally related genes for further investigation in ovarian cancer.

A murine model of hereditary hemorrhagic telangiectasia.

Endoglin (CD105), an accessory protein of the TGF-beta receptor superfamily, is highly expressed on endothelial cells. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is associated with mutations in the Endoglin gene, leading to haploinsufficiency. To generate a disease model and ascertain the role of endoglin in development, we generated mice lacking 1 or both copies of the gene. Endoglin null embryos die at gestational day 10.0-10.5 due to defects in vessel and heart development. Vessel formation appears normal until hemorrhage occurs in yolk sacs and embryos. The primitive vascular plexus of the yolk sac fails to mature into defined vessels, and vascular channels dilate and rupture. Internal bleeding is seen in the peritoneal cavity, implying fragile vessels. Heart development is arrested at day 9.0, and the atrioventricular canal endocardium fails to undergo mesenchymal transformation and cushion-tissue formation. These data suggest that endoglin is critical for both angiogenesis and heart valve formation. Some heterozygotes, either with an inbred 129/Ola or mixed C57BL/6-129/Ola background, show signs of HHT, such as telangiectases or recurrent nosebleeds. In this murine model of HHT, it appears that epigenetic factors and modifier genes, some of which are present in 129/Ola, contribute to disease heterogeneity.

Common acute lymphoblastic leukemia antigen expressed on leukemia and melanoma cell lines has neutral endopeptidase activity.

We have previously reported that the amino acid sequence of the common acute lymphoblastic leukemia antigen (CALLA, CD10) translated from a normal human kidney cDNA clone is identical to that of neutral endopeptidase (NEP, EC 3.4.24.11). In this study, we show that by flow cytometry, a monoclonal antibody (135A3) produced against rabbit NEP reacted selectively with leukemia and melanoma cell lines expressing CALLA on their surface. A glycoprotein of apparent Mr 100,000 was immunoprecipitated from surface labeled NALM-1 leukemia or Mel-1477 melanoma cells with monoclonal antibodies to NEP (135A3) or CALLA (44C10). mRNAs hybridizing to a NEP-specific probe were present in CALLA+ leukemia and melanoma cell lines, but absent from CALLA- lines. NEP enzymatic activity was detected on intact cells from CALLA+ lines, but not CALLA- lines. The activity was blocked by two selective inhibitors of NEP, thiorphan and phosphoramidon. CALLA antigen purified from the NALM-6 leukemic cell line by affinity to 44C10-IgG Sepharose retained a peptidase activity that was completely blocked by thiorphan and phosphoramidon. Thus the CALLA antigen present at the surface of leukemia and melanoma cell lines is an enzymatically active neutral endopeptidase.

Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative.

Endoglin (CD105), a component of the TGF-beta 1 receptor complex, is the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We have identified a novel endoglin splice site mutation, leading to an in-frame deletion of exon 3, in a new-born from a family with HHT. Expression of normal and mutant endoglin proteins was analyzed in umbilical vein endothelial cells from this baby and in activated monocytes from the affected father. In both samples, only normal dimeric endoglin (160 kD) was observed at the cell surface, at 50% of control levels. Despite an intact transmembrane region, mutant protein was only detectable by metabolic labeling, as an intracellular homodimer of 130 kD. In monocytes from three clinically affected HHT1 patients, with known mutations creating premature stop codons in exons 8 and 10, surface endoglin was also reduced by half and no mutant was detected. Overexpression into COS-1 cells of endoglin cDNA truncated in exons 7 and 11, revealed their intracellular expression, inability to be secreted and to form heterodimers at the cell surface. These results indicate that mutated forms of endoglin are transiently expressed intracellularly and not likely to act as dominant negative proteins, as proposed previously. A reduction in the level of functional endoglin is thus involved in the generation of HHT1, and associated arteriovenous malformations.

Hereditary haemorrhagic telangiectasia: current views on genetics and mechanisms of disease.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterised by epistaxis, telangiectases, and multiorgan vascular dysplasia. The two major types of disease, HHT1 and HHT2, are caused by mutations in the ENG (endoglin) and ACVRL1 genes, respectively. The corresponding endoglin and ALK-1 proteins are specific endothelial receptors of the transforming growth factor beta superfamily essential for maintaining vascular integrity. Many mutations have been identified in ENG and ACVRL1 genes and support the haploinsufficiency model for HHT. Two more genes have recently been implicated in HHT: MADH4 mutated in a combined syndrome of juvenile polyposis and HHT (JPHT), and an unidentified HHT3 gene linked to chromosome 5. Current knowledge on the genetics of HHT is summarised, including the pathways that link the genes responsible for HHT and the potential mechanisms underlying the pathogenesis of the disease.

Hereditary haemorrhagic telangiectasia: mutation detection, test sensitivity and novel mutations.

BACKGROUND: Hereditary haemorrhagic telangiectasia (HHT) is a genetic disorder present in 1 in 8000 people and associated with arteriovenous malformations. Genetic testing can identify individuals at risk of developing the disease and is a useful diagnostic tool. OBJECTIVE: To present a strategy for mutation detection in families clinically diagnosed with HHT. METHODS: An optimised strategy for detecting mutations that predispose to HHT is presented. The strategy includes quantitative multiplex polymerase chain reaction, sequence analysis, RNA analysis, validation of missense mutations by amino acid conservation analysis for the ENG (endoglin) and ACVRL1 (ALK1) genes, and analysis of an ACVRL1 protein structural model. If no causative ENG or ACVRL1 mutation is found, proband samples are referred for sequence analysis of MADH4 (associated with a combined syndrome of juvenile polyposis and HHT). RESULTS: Data obtained over the past eight years were summarised and 16 novel mutations described. Mutations were identified in 155 of 194 families with a confirmed clinical diagnosis (80% sensitivity). Of 155 mutations identified, 94 were in ENG (61%), 58 in ACVRL1 (37%), and three in MADH4 (2%). CONCLUSIONS: For most missense variants of ENG and ACVRL1 reported to date, study of amino acid conservation showed good concordance between prediction of altered protein function and disease occurrence. The 39 families (20%) yet to be resolved may carry ENG, ACVRL1, or MADH4 mutations too complex or difficult to detect, or mutations in genes yet to be identified.

Response of non-T, non-B acute lymphocytic leukemia cells to phorbol ester.

Non-T, Non-B acute lymphocytic leukemia cells were cultured in vitro with or without the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a potential modulator of differentiation. The eight cases studied were representative of non-T, non-B acute lymphocytic leukemia (ALL) cells and expressed amounts of la antigens varying from 0.9 X 10(5) to 7.1 X 10(5) molecules/cell; these levels were measured in a cellular radioimmunoassay with 21w4 monoclonal antibody directed at a monomorphic human la determinant. With all cases, TPA caused a significant increase in the level of la. Cultures with TPA expressed 4.3 times the amount of la found on fresh ALL cells, and a correlation was observed (r = 0.92) between the level of la following culture with TPA and that found on fresh ALL cells. A 25% increase in the modal volume of ALL cells was also caused by TPA. There was no detectable induction of surface or cytoplasmic immunoglobulin and no change in the expression of the common ALL antigen. Inhibition of [3H]thymidine incorporation and stimulation of 14C-labeled amino acid incorporation were observed in the presence of TPA, suggesting that the increase in la level occurs concurrently with an increase in protein synthesis induced by phorbol ester. Following culture with TPA, a substantial increase in the ability of the ALL cells to stimulate in a mixed-lymphocyte reaction was obtained. These results suggest that ALL cells, like other cell types, are susceptible to the effects of TPA and respond by changes in cell volume, surface antigen expression, and mixed-lymphocyte reaction stimulating capacity.

Down-regulation of transforming growth factor beta receptors by androgen in ovarian cancer cells.

Steroid hormones have been implicated in the etiology and/or progression of epithelial ovarian cancer. As ovarian surface epithelial cells are growth inhibited by transforming growth factor beta (TGF-beta), we tested whether steroid hormones could regulate the expression of TGF-beta1 or its receptors in ovarian cancer cells, as assessed by quantitative reverse transcription-PCR. Treatment of ovarian cancer HEY cells with 500 nM 5alpha-dihydrotestosterone (DHT), but not estradiol-17beta or progesterone, for 60 h down-regulated the expression of mRNA for TGF-beta receptors I and II (TbetaR-I and TbetaR-II), betaglycan, and endoglin but had no effect on TGF-beta1 mRNA levels. Androgen receptor (AR) mRNA expression in HEY cells was compared to other ovarian cancer cell lines. OVCAR-3 cells expressed AR mRNA levels similar to that of androgen-responsive LNCaP prostate cancer cells, whereas SKOV-3 and HEY cells expressed only 3 and 0.01%, respectively. Western blot analysis and saturation binding assays confirmed the expression of AR protein in these three cell lines, but at the limit of detection in SKOV-3 and HEY cells. Treatment of SKOV-3 and HEY cells for 24 h with 1-50 nM DHT resulted in a dose-dependent down-regulation of TbetaR-II mRNA. The AR antagonist hydroxyflutamide did not reverse the effect of DHT on SKOV-3 cells but by itself down-regulated TbetaR-II mRNA. This apparent androgen-mimetic action of hydroxyflutamide and the ability of SKOV-3 and HEY cells to respond to DHT may be due to their expression of AR-associating protein 70, an AR co-activator reported to amplify AR transactivation and to result in agonist activity of AR antagonists. DHT was able to reverse TGF-beta1 growth-inhibitory action in SKOV-3 cells and in a primary culture of ovarian cancer cells derived from ascites. Thus, androgens may promote ovarian cancer progression in part by decreasing TGF-beta receptor levels, thereby allowing ovarian cancer cells to escape TGF-beta1 growth inhibition.

Endoglin is overexpressed after arterial injury and is required for transforming growth factor-beta-induced inhibition of smooth muscle cell migration.

Endoglin is a homodimeric membrane glycoprotein primarily expressed on endothelial cells. In association with transforming growth factor (TGF)-ss receptors I and II, it can bind TGF-beta1 and -beta3 and form a functional receptor complex. There is increasing evidence that endoglin can modulate the cellular response to TGF-beta, a factor implicated in vascular lesion formation in human and experimental models. The purpose of this study was to analyze the expression of endoglin in normal and balloon-injured porcine coronary arteries and in normal and atherosclerotic human coronary arteries and to determine its ability to mediate the effects of TGF-beta on the migration of vascular smooth muscle cells (SMCs). In normal porcine coronary arteries, endoglin was of low abundance and was found primarily on endothelial cells and adventitial fibroblasts, as well as on a minority of medial SMCs. On days 3, 7, and 14 after angioplasty, endoglin was present not only on endothelial cells but also on adventitial myofibroblasts and medial SMCs of porcine coronary arteries. By day 28, few or no cells expressed endoglin. In situ hybridization revealed that endoglin mRNA expression appeared to be highest in endothelial cells on days 3, 7, and 14 days after injury and absent thereafter. With a second balloon injury, a similar pattern of endoglin protein and mRNA expression was observed. In human vascular tissue, endoglin immunolabeling was higher in endarterectomy specimens removed from diseased coronary arteries than in normal internal mammary arteries. In vitro, antisense oligonucleotides to endoglin decreased its expression and antagonized the TGF-beta-mediated inhibition of human and porcine SMC migration. In summary, upregulation of endoglin occurs during arterial repair and in established atherosclerotic plaques and may be required for modulation of SMC migration by TGF-beta.

The induction of CD10 on a pre-B leukemia cell line occurs with progression of the disease in scid mice.

We have previously demonstrated the engraftment and dissemination of human pre-B acute lymphoblastic leukemia (ALL) cells into scid mice. In the current study, the temporal pattern of infiltration of a CD10- pre-B leukemia line (G2) in various murine tissues and the progression of the disease in the whole animal were monitored by quantifying human CD44 mRNA expression by the polymerase chain reaction (PCR). Irradiated scid mice were injected intravenously with 10(6) G2 cells and killed 3 days to 10 weeks later. After 2 weeks, leukemic cells were found mostly in bone marrow, but also in lung. At 6 to 7 weeks, spleen and lung contained 30% human RNA, while peripheral blood, liver, and kidney contained 2-3%. Infiltration to brain and thymus was observed at 8-9 weeks. In terms of the whole animal, spleen and liver were the major sites of tumor burden. The induction of CD10 expression was previously observed in transplanted CD10- G2 leukemic cells recovered from scid thymus at 10-12 weeks, which corresponds to the terminal stage of disease. In this study, the CD10 expression on the leukemic cells was monitored at earlier time points by flow cytometry and quantitative PCR. Induction of CD10 was first observed in bone marrow, spleen, peripheral blood, and liver at 6-7 weeks (10-fold), at the time of the onset of dissemination of the leukemia. Despite the presence of 30% human RNA in lung at 6-7 weeks, CD10 induction was not significant in that site before 10 weeks. Increased levels of CD10 were seen in all tissues between 8 and 10 weeks; the highest levels were observed in leukemic cells proliferating in thymus (113-fold) and in those found in circulation. These findings suggest that initial induction of CD10 occurs in hematopoietic tissues at the time of rapid proliferation of the leukemic cells and their infiltration of several tissues. At later time points, the increase in CD10 expression is seen on the leukemic cells found in all peripheral organs suggesting an association with disease progression.

Tumor formation by a human pre-B leukemia cell line in scid mice is enhanced by matrigel and is associated with induction of CD10 expression.

We have previously demonstrated the engraftment of human pre-B acute lymphoblastic leukemia (ALL) cells injected intravenously into irradiated scid mice. We now report on the ability of the reconstituted extracellular matrix, Matrigel, to promote the formation of subcutaneous tumors in non-irradiated scid mice by a CD10- pre-B ALL cell line termed G2. Lymphatic tumors infiltrating the dermis were seen in all eight mice sacrificed 10-13 weeks after the co-injection of G2 cells and Matrigel but in only 2/8 mice injected with leukemic cells alone. Infiltration of bone marrow, spleen, thymus, lung and liver was observed earlier and was more extensive in the Matrigel-treated group. The tumor cells derived from Matrigel-treated mice could be passaged in vitro and their colony-forming ability was higher than that of the original G2 line. When re-injected intravenously into non-irradiated scid mice, the tumor cells invaded the thymus earlier than did the G2 cells. The expression of CD10/neutral endopeptidase was induced at high levels in all tumors, in Matrigel or non Matrigel-treated animals. This up-regulation was transient as the tumor variants grown in vitro or in vivo lost expression of CD10. However, 6-8 weeks later, induction of CD10 was observed on both tumor variants and parental G2 cells growing in the thymus and at a lower level on cells in bone marrow and spleen. Culturing G2 cells in vitro at high density or in the presence of documented growth-promoting cytokines such as IL-3, IL-6, IL-7, and GM-CSF did not stimulate the expression of CD10 mRNA. The induction of this surface endopeptidase was thus associated with growth of leukemic cells in the specific microenvironments provided by the lymphoid tumors and the thymus in scid mice. The function of CD10 might be related to the hydrolysis of peptides which are critical in regulating interactions between adjacent pre-B cells, the stromal microenvironment and the transduction of growth and/or differentiation signals.

Homotypic aggregation of pre-B leukemic cell lines by antibodies to VLA integrins correlates with their expression of CD9.

The molecules effecting adhesion of acute lymphoblastic leukemia (ALL) cells are not well defined. We investigated the expression of very late activation (VLA) integrins in five human leukemic cell lines of pre-B cell phenotype. VLA-4 was found to be the dominant integrin in all five, three possessed VLA-5, and one VLA-6. None had VLA-2, or VLA-3. Since certain anti-VLA-4 monoclonal antibodies (mAb) have been reported to induce homotypic aggregation of T and B lymphocytes we investigated the possibility that VLA-4 might be involved in aggregation of pre-B cells. mAb 44H6 (anti-VLA-alpha 4), and 4B4 (anti-VLA-beta 1) induced strong aggregation which was not blocked by the anti-FC gamma IIR mAb IV.3. However, aggregation was effected in only three of the five lines suggesting the involvement of molecules other than VLA-4. The level of expression of CD9, but not that of CD11a, CD18, CD19, CD44, or CD54, was found to correlate with the level of aggregation. Of mAb directed to CD9, CD19, CD44, endoglin, and HLA-DR only mAb to CD9 induced aggregation. Admixture of mAb ALB6 (anti-CD9) and mAb 44H6 neither potentiated nor inhibited the response indicating a common effector mechanism. We suggest that the level of CD9 may determine the level of VLA-regulated adhesion, and therefore the adhesive phenotype of leukemic pre-B cells.

Lymphoid blast crisis of B-lineage phenotype with monosomy 7 in a patient with juvenile chronic myelogenous leukemia (JCML).

We studied a patient with juvenile chronic myelogenous leukemia (JCML) whose terminal course was characterized by transformation to acute lymphoblastic leukemia. Karyotypic studies identified monosomy 7 in leukemic myelomonocytic marrow cells during the chronic phase and in the lymphoblasts during the transformation phase. Our ability to sustain the transformed lymphoblasts in culture allowed us to characterize them further. CD19, HLA-DR, and CD10 were present, consistent with a pre-B acute lymphoblastic leukemia phenotype. CD14 (My-4) and CD13 (My-7) were negative. Rearrangement of immunoglobulin heavy- and light-chain genes identified monoclonal populations of cells of the B lineage. This case provides further evidence that JCML is a clonal disease of pluripotent stem-cell origin.

Endoglin is expressed on a subpopulation of immature erythroid cells of normal human bone marrow.

A monoclonal antibody (1G2) was raised by immunization of a Balb/c mouse with the leukemic blasts from a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). Sequential immunoprecipitation of the protein from human umbilical vein endothelial cells with 1G2 and the endoglin-specific monoclonal antibody 44G4 indicated that both antibodies react with the same molecule, a homodimer of molecular mass 180,000. This protein was first identified on acute lymphoblastic leukemia and was shown to be primarily associated with endothelial cells. In addition, 1G2 and 44G4 identified the same subpopulation of human bone marrow mononuclear cells (BMMNC), as established by two colour immunofluorescence analysis. By cell sorting and colony assays it could be demonstrated that endoglin is not expressed on hemopoietic precursor cells (CFU-G, CFU-GM, CFU-GEMM, BFU-E). May-Grünwald-Giemsa staining of sorted BMMNC revealed that 1G2 recognized immature proerythroblasts and double-fluorescence analysis showed that endoglin is present on a subset of glycophorin A-positive BMMNC. 1G2 was not reactive on bone marrow B-cells (CD19, CD20), T-cells (CD3, CD7), natural killer cells (CD56), myeloid cells (CD13, CD14, CD15, CD33), and on CD34-positive cells. Endoglin contains an arginine-glycine-aspartic acid sequence, a feature generally associated with extracellular matrix proteins which interact with integrins. It is suggested that proerythroblasts may utilize endoglin to interact with integrins in cell-cell adhesion events in the stromal or hemopoietic compartment of the bone marrow.