Abstract: BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.
Abstract: Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.
Abstract: Neuroblastoma is a heterogeneous neural crest-derived embryonic childhood neoplasm that is the second most common solid tumor found in children. Despite recent advances in combined therapy, the overall survival of patients with high-stage disease has not improved in the last decades. Treatment failure is in part attributed to multidrug resistance. To address the mechanisms involved in the development of multidrug resistance, we have generated two doxorubicin-resistant neuroblastoma cell lines (IGRN-91R and LAN-1R). These cells were shown to overexpress the MDR1 gene coding for the P-glycoprotein and were resistant to other MDR1- and non-MDR1-substrate drugs. Indeed, the MDR1 inhibitor verapamil only partially restored sensitivity to drugs, confirming that P-glycoprotein-mediated drug efflux was not responsible for 100% resistance. High-resolution and array-based comparative genomic hybridization analyses revealed the presence of an amplicon in the 7q21 region as the unique genomic alteration common to both doxorubicin-resistant cell lines. In addition to the MDR1 locus, this large amplified region is likely to harbor additional genes potentially involved in the development of drug resistance. This study represents the first molecular cytogenetic and genomic approach to identifying genomic regions involved in the multidrug-resistant phenotype of neuroblastoma. These results could lead to the identification of relevant target genes for the development of new therapeutic modalities.
Abstract: Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.
Abstract: BACKGROUND: Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. METHODS: NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. RESULTS: Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL-receptors or TRAIL is not affected by sub-toxic doses of HDACIs. CONCLUSION: HDACIs were shown to activate the mitochondrial pathway and to sensitise NB cells to TRAIL by enhancing the amplitude of the apoptotic cascade and by restoring an apoptosis-prone ratio of pro- to anti-apoptotic proteins. Combining HDACIs and TRAIL could therefore represent a weakly toxic and promising strategy to target TRAIL-resistant tumours such as neuroblastomas.
Abstract: Human neuroblastoma (NB) is the second most frequent solid tumor of childhood and represents a highly heterogeneous disease at clinical and biologic levels. Little progress has been made to improve the poor prognosis of patients with high-stage NB. Tumor progression and metastatic dissemination still represent major obstacles to the successful treatment of advanced stage disease. In order to develop and evaluate new, targeted, therapeutic strategies, fully defined and biologically relevant in vivo models of NB are strongly needed. We have developed an orthotopic model of metastatic human NB in the nude mouse, using 2 well-characterized NB cell lines. Tumor growth, vascular properties and metastatic patterns were investigated using a sensitive and newly developed in vivo echographic technology in addition to immunohistochemistry and PCR analyses. Results show that implantation of low numbers of NB cells directly into the adrenal gland of nude mice resulted in rapid and homogeneous tumor growth without tumor morbidity. Nude mice were shown to rapidly develop highly vascularized adrenal tumors that selectively metastasized to the liver and bone marrow. In addition, the newly formed mouse vessels in orthotopic but not in heterotopic tumors, were found to express the highly angiogenic alphavbeta3 integrin marker, indicating the development of a truly malignant neovasculature in orthotopic conditions only. This observation confirms the impact of the regional microenvironment on tumor biology and suggests the existence of cross-talk with the tumor cells. In conclusion, such model faithfully reproduces the growth, vascular and metastatic patterns as observed in patients. It therefore represents a powerful and biologically relevant tool to improve our understanding of the biology of NB and to develop and assess new antiangiogenic and metastasis-targeted therapies.
Abstract: Standard cytomorphological examination of bone marrow (BM) aspirates does not appear to be sensitive enough to detect single neuroblastoma cells. The SIOPEN Neuroblastoma Bone Marrow Committee developed a sensitive and reproducible anti-GD2 immunocytochemical assay and introduced morphological and immunocytological criteria for the interpretation of results. Fixed cytospins were incubated with a commercially available anti-GD2 monoclonal antibody and an APAAP kit. Cells fulfilling all morphological and immunocytological criteria were called criteria-positive cells (CPCs). Not convincingly interpretable cells fulfilled some, but not all, criteria, and negative cells displayed only exclusion criteria. The genetic profile of doubtful cells was checked by fluorescence in situ hybridization. Ideally, 3 x 10(6) cells were analyzed to reach a 95% probability of detecting one tumor cell in 1 x 10(6) mononuclear cells. Four quality control rounds were organized to validate the method. A total of 111 quality control samples were analyzed. Two main improvements were achieved: in discordant cases, the range between the lowest and highest reported result was reduced by half, and discordant results were only found in samples with less than 10 CPCs per 1 x 10(6). This article describes the first internationally standardized protocol to detect and quantify rare neuroblastoma cells by immunocytochemistry. This method is an indispensable tool for multicenter studies evaluating the clinical significance of minimal residual disease in neuroblastoma.
Abstract: Neuroblastoma (NB) is a childhood neoplasm which heterogeneous behavior can be explained by differential regulation of apoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces rapid apoptosis in most tumor cells and thus represents a promising anticancer agent. We have reported silencing of caspase-8 expression in highly malignant NB cells as a possible mechanism of resistance to TRAIL-induced apoptosis. To explore the particular contribution of caspase-8 in such resistance, retroviral-mediated stable caspase-8 expression was induced in the IGR-N91 cells. As a result, sensitivity to TRAIL was fully restored in the caspase-8-complemented cells. TRAIL-induced cell death could be further enhanced by cotreatment of IGR-N91-C8 and SH-EP cells with cycloheximide or subtoxic concentrations of chemotherapeutic drugs in a caspase-dependent manner. Sensitization to TRAIL involved enhanced death receptor DR5 expression, activation of Bid and the complete caspases cascade. Interestingly, combined treatments also enhanced the cleavage-mediated inactivation of antiapoptotic molecules, XIAP, Bcl-x(L) and RIP.Our results show that restoration of active caspase-8 expression in a caspase-8-deficient NB cell line is necessary and sufficient to fully restore TRAIL sensitivity. Moreover, the synergistic effect of drugs and TRAIL results from activation of the caspase cascade via a mitochondrial pathway-mediated amplification loop and from the inactivation of apoptosis inhibitors.
Abstract: PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. Materials and METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.
Abstract: The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.
Abstract: Human neuroblastoma (NB) is a highly heterogeneous childhood cancer secreting a high level of vascular endothelial growth factor (VEGF). Its vascularization has been clearly correlated with metastatic progression and poor outcome. Thus, molecules that target the vascular endothelium are regarded as new therapeutics of clinical interest. Angiostatin, an internal fragment of plasminogen containing the first four kringle structures, has been described as a powerful angiogenic inhibitor. We used a recombinant adenovirus encoding the human angiostatin kringle 1-3 directly fused to human serum albumin HSA (AdK3-HSA). Coupling to HSA has been previously shown to increase the in vivo half-life of this angiostatic factor, and to lead to tumor growth inhibition in the MDA-MB-231 carcinoma model. For the assessment of antiangiogenic gene therapy in the human NB IGR-N835 tumor model, 5 x 10(9) PFU of AdK3-HSA were intravenously injected in tumor-bearing athymic mice presenting either of the following experimental settings: early stage, established, and minimal residual tumors. No delay in tumor growth was observed in animals treated with AdK3-HSA as compared to those treated with the empty virus AdCO1. In early-stage tumors, kinetics of tumor occurrence and tumor growth were similar in AdK3-HSA- and AdCO1-treated animals. K3-HSA was found to be expressed at high levels (the mean value for the three experiments being 19.4+/-15.9 microg/ml) in the circulation of all animals up to 21-35 days after virus injection. In addition, IGR-N835 tumors were found to be highly vascularized and to release high amounts of angiogenic factors, in particular VEGF (665+/-370 pg/mg total protein). Thus, in spite of high circulating levels, K3-HSA may be unable to displace the NB proangiogenic switch. In this regard, a more promising target to inhibit NB angiogenesis seems to be the VEGF/VEGFR system.
Abstract: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumor cells, a process sometimes potentiated by chemotherapeutic drugs or cycloheximide (CHX). Childhood neuroblastoma (NB) is a clinically and biologically heterogeneous neoplasm whose behavior can be explained by differential regulation of apoptosis. The non-invasive S-type NB cell lines are sensitive to TRAIL, whereas the invasive N-type NB cell lines are resistant. We have reported the silencing of caspase-8 expression in N-type cells as a possible mechanism of death receptor-mediated resistance to apoptosis in NB. The recently observed deregulation of caspase-10 in these cells prompted us to investigate the particular contribution of caspase-8 silencing in the resistance to TRAIL in N-type cells. Stable caspase-8 expression was therefore restored in the IGR-N91 cell line by retroviral infection. The IGR-N91-C8 cells became sensitive to TRAIL-mediated apoptosis, whereas the control vector-infected IGR-N91-M cells remained resistant. Interestingly, the apoptotic response to TRAIL was enhanced by co-treatment of SH-EP and IGR-N91-C8 cells with CHX or with sub-toxic concentration of doxorubicin (DOX) in a caspase-dependent manner, as cells could be protected from death by specific caspase-8 or pan-caspase inhibitors. CHX or DOX was shown to enhance TRAIL-induced caspase-8 activation and loss of mitochondrial transmembrane potential. In conclusion, restoration of active caspase-8 expression in caspase-8- and caspase-10-deficient IGR-N-91 cell line is necessary and sufficient to fully restore TRAIL-mediated cell death. Moreover, DOX and CHX were able to sensitize NB cell lines to TRAIL-induced apoptosis in a caspase-8-dependent manner by engaging death receptor and mitochondrial signaling pathways.
Abstract: Death induced by doxorubicin (dox) in neuroblastoma (NB) cells was originally thought to occur via the Fas pathway, however since studies suggest that caspase-8 expression is silenced in most high stage NB tumors, it is more probable that dox-induced death occurs via a different mechanism. Caspase-8 silenced N-type invasive NB cell lines LAN-1 and IMR-32 were investigated for their sensitivity to dox, and compared to S-type noninvasive SH-EP NB cells expressing caspase-8. All cell lines had similar sensitivities to dox, independently of caspase-8 expression. Dox induced caspase-3, -7, -8 and -9 and Bid cleavage in S-type cells and death was blocked by caspase inhibitors but not by oxygen radical scavenger BHA. In contrast, dox-induced death in N-type cells was caspase-independent and was inhibited by BHA. Dox induced a drop in mitochondrial membrane permeability in all cell lines. Dox-induced death in S-type cells gave rise to apoptotic nuclei, whereas in N-type cells nuclei were non-apoptotic in morphology. Transfection of SH-EP cells with a dominant negative FADD mutant inhibited TRAIL-induced death, but had no effect on dox-induced apoptosis. These results suggest that S-type cells undergo apoptosis after dox treatment independently of death receptors, whereas N-type cells are killed by a caspase-independent mechanism.
Abstract: CD44 is a polymorphic transmembrane glycoprotein that exists as multiple isoforms resulting from alternative splicing and posttranslational modifications. Enhanced expression of CD44 has been correlated to the tumorigenicity and metastatic behavior in different malignant tumors. In contrast, human neuroblastomas exhibit an inverse correlation between CD44 expression and tumor malignancy. To determine whether there is a CD44 silencing in sympatho-adrenal precursors as a possible explanation for the down-regulation of CD44 in neuroblastomas, the expression of standard CD44H and v6, v7, v7v8, or v10 isoforms was analyzed by immunohistochemistry in human adrenal glands of 14- to 20-week-old gestational age fetuses. All of the fetal neuroblasts localized in the adrenal gland parenchyma and migrating from the sympathetic nerve chain into the fetal adrenal cortex strongly expressed CD44H but none of the CD44 isoforms could be detected in these cells. In contrast, a strong expression of CD44v7 and v6 was detected in the fetal adrenal cells. These results indicate that, as for many other cell types, the CD44H expressed by fetal neuroblasts may contribute to controlling their migration into the adrenal medulla and that the down-regulation of CD44H in neuroblastoma cells should be explained by mechanisms other than the fetal suppression of CD44H expression in their normal counterparts.
Abstract: BACKGROUND: CD44 represents a heterogeneous group of surface glycoproteins involved in cell-cell and cell-matrix interactions. CD44H is the major receptor for hyaluronate, and most if not all CD44H known functions are attributed to its ability to recognize hyaluronate. We have previously demonstrated a lack of CD44 expression in high stages and NMYC-amplified tumors and further have shown that NMYC-amplified cell lines either did not express CD44 at all or expressed a nonfunctional receptor. On the other hand, nonamplified cells constitutively expressed an active receptor, suggesting that absence of CD44-mediated hy aluronate binding could be related to increased malignancy in human neuroblastoma. PROCEDURE: In the present study we have compared the glycosylated structure of CD44 expressed by NMYC amplified vs. nonamplified cell lines in relation to their adhesive properties for hyaluronate. These adhesive properties were measured after modifications of the carbohydrate structure with enzymes and inhibitors of N- or O-linked glycosylation. RESULTS AND CONCLUSIONS: Our results indicate that increased sialylation, defective N-linked glycosylation, and substitution of the CD44 glycoprotein with keratan sulfate glycosaminoglycan might include modifications observed on neuroblastoma cells that could account for the inability of the receptor to bind hyaluronate.
Abstract: BACKGROUND AND PROCEDURE: To determine the possible role of Fas/FasL system in the particularly heterogeneous behaviour of neuroblastoma (NB), we have measured the functional expression of Fas and its ligand, FasL, in primary neuroblastoma samples and cell lines by immunohistochemistry and flow cytometry. RESULTS: Our results reveal that while Fas expression is associated with low stage and more mature tumors, heterogeneous FasL expression was mostly detected in high stage tumors, with our apparent correlation to MYCN amplification. Flow cytometric analysis of cell lines demonstrated a high expression of Fas in epithelial-type, HLA class I positive cell lines, which was lost upon activation with phorbol esters. In contrast, Fas ligand was detected in only a small subset of cell lines. CONCLUSIONS: In some cell lines, cytotoxic assays revealed the ability of NB-associated Fas receptor to transduce an apoptotic signal upon triggering. The pattern of functional Fas/FasL expression in tumours and cell lines suggests that this system may be involved in the evasion of highly malignant neuroblastoma cells to host immune response.
Abstract: Human neuroblastoma (NB) is a highly heterogeneous childhood cancer that is aggressively malignant or can undergo spontaneous regression that may involve apoptosis. NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Noninvasive S-type cell lines (NB cell lines of substrate adherent phenotype) are highly sensitive to TRAIL, whereas invasive N-type cell lines (NB cell lines of neuronal phenotype) are resistant. Whereas both S- and N-type cell lines express TRAIL-R2, FADD, and caspase-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a malignant stage IV NB tumor when compared with a benign ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage NB tumors. Caspase-8 expression can be induced by demethylation with 5-aza-2'deoxycytidine, which enhances sensitivity to TRAIL. Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.
Abstract: Background and Procedures NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective apoptotic ligand TRAIL. Noninvasive S-type cell lines are highly sensitive to TRAIL, whereas invasive N-type cell lines are resistant. RESULTS: Although both S- and N-type cell lines express TRAIL-R2, FADD, and caspases-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a stage IV NB tumor when compared to a ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage tumors, and expression can be induced by demethylation. CONCLUSIONS: Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.
Abstract: Highly malignant neuroblastoma tumors with MYCN amplification have been shown to downregulate the expression of the CD44 adhesion receptor. We have previously shown that MYCN amplified neuroblastoma cell lines either lack CD44 expression or express a nonfunctional, nonhyaluronic acid-binding CD44 receptor. By analysis of cells with manipulated expression of either CD44 or MYCN, we demonstrate that transfection of cells with a CD44 full-length cDNA construct produced a functional receptor in single copy MYCN cells and a nonfunctional CD44 receptor in MYCN amplified cells, similar to the CD44 receptor expressed by cells with enforced MYCN. Analysis of the in vivo growth properties of the transfectants revealed that the restoration of a functional CD44 receptor in nonamplified cells resulted in the suppression of in vivo cell growth, therefore linking the MYCN-related lack of hyaluronic acid-binding function of CD44 to the highly tumorigenic properties of a subset of neuroblastoma cells.
Abstract: TRKA expression was evaluated on 122 untreated neuroblastomas by immunohistochemistry using an antibody with predetermined specificity. This procedure is simple and reliable for protein detection at cellular level in a routine clinical setting. Fourteen tumours were classified as benign ganglioneuroma with a restricted expression of TRKA on ganglion cells; these patients were excluded from the following analysis. A total of 108 tumours were classified as neuroblastoma or ganglioneuroblastoma; 74 expressed TRKA protein, which strongly correlated with low stage, absence of N-MYC amplification, age (<1 year), CD44 expression and favourable clinical outcome. In a univariate analysis including tumour stage, age, histology, N-MYC amplification, CD44 and TRKA expression, all parameters had significant prognostic value. The absence of TRKA expression on CD44-positive or N-MYC non-amplified tumours permits the characterization of a subgroup of patients with intermediate prognosis. However, in a multivariate analysis taking into consideration the prognostic factors mentioned above, CD44 and tumour stage were the only independent prognostic factors for the prediction of patients' event-free survival.
Abstract: CD44 represents a heterogeneous group of surface glycoproteins, involved in cell-cell and cell-matrix interactions. CD44 is the major receptor for hyaluronate (HA), a component of cell matrices, and most of CD44 known functions are attributed to its ability to recognize HA. We have recently shown that although a majority of human neuroblastomas (NBs), a childhood cancer, express high levels of CD44H, high stages and tumors with amplification of the NMYC proto-oncogene fail to express CD44. Lack of CD44 expression is strongly associated with the presence of NMYC amplification and has been further shown to represent a new feature for predicting risk of disease progression and dissemination. In the present study, we have investigated the role of CD44 expressed by NB cell lines and the possible relationship among the presence of NMYC amplification, functional expression of CD44 receptor, and tumorigenic properties of NB cells. A panel of cell lines with variable NMYC amplification and/or overexpression, as well as clonal and stable NMYC-transfected NB cells, were analyzed for CD44 expression and ability to bind HA. Our results confirmed previous observations that in NB cell lines lack of CD44 is not always related to the presence of NMYC amplification, with a number of cell lines or transfectants with both CD44 expression and NMYC amplification. However, the ability of the CD44 receptor to bind immobilized hyaluronan was restricted to CD44H+ cell lines without NMYC amplification (SH-EP and ACN). The HA-binding function was CD44 dependent and could be specifically blocked by an anti-CD44 antibody. No induction of functional HA binding was obtained with NMYC-amplified cell lines or NMYC transfectants, despite an induced increase of CD44 expression upon differentiation or after tentative activation of the receptor with phorbol esters. Inhibition of N-linked glycosylation with tunicamycin resulted in decreased HA binding of cells bearing an active CD44 receptor. We conclude that NMYC-amplified NB cell lines either do not express CD44 at all or express a nonfunctional receptor, whereas nonamplified cells constitutively express an active receptor. The lack of functional HA binding in NB cells might be partly due to incomplete N-glycosylation. The involvement of NMYC in the regulation of N-linked glycosylation can be suspected.
Abstract: This multicentric analysis of tumours obtained from 140 patients with neuroblastoma confirms that the lack of CD44 expression is a highly significant factor of poor prognosis and, as previously published in multivariate analysis of the four factors, i.e. MYCN amplification, CD44 expression, age and tumour stage, CD44 expression and tumour stage were the only independent prognostic factors of event-free survival (Combaret et al., J Clin Oncol 1996, 14, 25-34). Furthermore, CD44 analysis affords significant prognostic discrimination in subgroups of patients with or without MYCN amplified tumours, both in low-stage neuroblastomas and high-grade neuroblastomas. In the subgroup of patients with low-stage neuroblastoma and the stage 4 subgroup, CD44 was the only independent prognostic factor for the prediction of event-free survival in a multivariate analysis. In conclusion, CD44 is one of the most powerful factors for predicting clinical outcome in neuroblastoma at the time of initial staging.
Abstract: PURPOSE: In contrast to other human tumors, a repression of the cell-surface glycoprotein CD44 on neuroblastoma is a marker of aggressiveness that usually correlates to N-myc amplification. We thus compared the prognostic value of both markers in the initial staging of 121 children treated for neuroblastoma in collaborative institutions. METHODS: Frozen samples were analyzed by a rapid and well-standardized technique of immunostaining with monoclonal antibodies (MoAbs) against epitopes in the CD44 constant region. RESULTS: In this retrospective series, CD44 was expressed on 102 specimens and strongly correlated with favorable tumor stages and histology, younger age, and normal N-myc copy numbers. In univariate analysis, CD44 expression and normal N-myc were the most powerful markers of favorable clinical outcome (P < 10(-6) and chi 2 = 65.40 and P < 10(-6) and chi 2 = 42.56, respectively), but analysis of CD44 affords significant prognostic discrimination in subgroups of patients with or without N-myc-amplified tumors. In the subgroup of stage IV neuroblastomas, CD44 was the only significant prognostic marker (P < .02, chi 2 = 5.76), whereas N-myc status was not discriminant. In multivariate analysis of five factors, ie, N-myc amplification, CD44 expression, age, tumor stage, and histology, the only independent prognostic factors of event-free survival were CD44 expression and tumor stage. CONCLUSION: The analysis of CD44 cell-surface expression must be recommended as an additional biologic marker in the initial staging of the disease.
Abstract: Metastasis in children with neuroblastoma (NB) is a poor prognostic factor despite intensive therapy. In the near future, stem cell factor (SCF) is likely to be used clinically to accelerate bone marrow (BM) recovery after high-dose chemotherapy in patients with advanced NB. The high frequency of BM metastases in NB could be secondary to BM-derived human growth factors (HGF) modulating the adhesion, secondary growth (or both) of circulating metastatic NB cells. To test this hypothesis, we studied the in vitro effects on NB cell lines grown in chemically defined medium of SCF, interleukin (IL)-1 beta, IL-3, IL-6, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta) used alone or in combination. The antigenic expression of NB-associated cell adhesion molecules (CAM) HLA class 1, intercellular CAM-1, neural-CAM and CD44 were assayed by monoclonal antibodies and flow cytometry, and DNA synthesis by 3H-thymidine uptake. The expression of CAM was not modulated by SCF or other HGFs. An increase in thymidine uptake was induced by bFGF alone in IMR-32 cells, while SCF and other HGFs had no notable effect. Our results indicate that SCF and other BM-derived HGFs are unlikely to have a generalised effect on the expression of adhesion molecules by NB cells or proliferation. The clinical administration of recombinant human SCF to children with NB should be safe.
Abstract: The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment and tumour metastasis. A large family of variants or isoforms, generated by alternative splicing of a single gene, has been reported to be involved in the malignant process, by conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumour characterised by an aggressive and metastatic behaviour in advanced stages, with amplification of the MYCN protooncogene. In this report, we show that the CD44 standard molecule was highly expressed in the majority of tumours of stages 1-3, in all stage 4s and ganglioneuromas, but only in a subset of stage 4 tumours. A lack of CD44 expression was observed in all MYCN amplified stage 4 tumours, thus demonstrating a highly significant inverse relationship between MYCN amplification and CD44 expression in neuroblastoma. In addition, the expression of 4 different CD44 isoforms was measured on all specimens and was always found to be negative. Using neuroblastoma cell lines and MYCN expressing transfectants, we show that CD44 expression by neuroblastoma cell lines is not directly related to MYCN amplification, but is associated to the stage of differentiation or lineage, and to the tumorigenic properties of the cells. In addition, CD44 expression can be upmodulated parallel to differentiation or maturation as induced by retinoic acid, bromodeoxyuridine or phorbol ester. In contrast, cytokines such as IFN gamma, TNF alpha, or growth factors such as bFGF, SCF and TGF beta were ineffective in modulating CD44 expression.
Abstract: We have attempted to distinguish in human neuroblastoma between the effects of mycN on differentiation and its potential to promote malignant progression. Others have observed out-growth of autocrine cells with evidence of an advanced malignant phenotype in a mycN-transfected clonal cell line derived from the single-copy mycN neuroblastoma, SK-N-SH. We have now transfected the parental cell line with the same mycN expression vector and selected 5 clones characterized by unique and stable chromosomal integration sites and variable exogenous copy numbers. mycN gene expression was variable in the different clones and correlated roughly with the copy number of transfected mycN genes. Clones with minimal levels of mycN gene expression had a neuroblastic phenotype and low numbers of surface HLA class-I molecules. Clones with high levels of mycN expression had a Schwann/glial-like phenotype with higher surface HLA class-I display without imbalance of expression of specific loci and accelerated growth. Two such clones were capable of anchorage-independent growth in the absence of serum, and acquired tumorigenic properties. Our results show that exogenous mycN expression can be associated with a differentiation of neuroblastoma cells along the Schwann/glial pathway and can induce accelerated and autonomous growth.
Abstract: The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis.
Abstract: The expression of cellular adhesion molecules (CAM) involved in cell adhesion and immune recognition was measured on neuroblastoma tissue samples, on a neuroblastoma (NB) cell line, SK-N-SH, and on 3 phenotypically different variants, SH-SY5Y, SH-EP, SH-IN, representing neuronal, Schwannian/glial or intermediate NB-cell types. Immunohistochemical analysis of CAM expression by NB and related tumors at different stages of differentiation revealed a co-expression of several CAM (ICAM-1/CD54, LFA-3, VLA-2 and HLA-ABC) associated with low stages and more highly differentiated NB tumors and peripheral neuroepitheliomas (PN). In contrast, N-CAM was uniformly expressed on all NB tumors. Flow cytometric analysis of CAM surface expression by SK-N-SH and variant cells revealed highly variable phenotypes. Expression of ICAM-1, LFA-3, VLA-2 and HLA-ABC molecules was associated with the epithelial cell type represented by the SH-EP variant. In contrast, low expression of these molecules and high expression of N-CAM was associated with the neuronal SH-SY5Y cells. Exposure of the NB cells to differentiation inducers (retinoic acid, 5'-bromodeoxyuridine and phorbol esters) and cytokines (tau-interferon, alpha-tumor necrosis factor) resulted in a variable up-regulation of the expression of all CAMs, except N-CAM, regardless of the type of differentiation induced. In an attempt to establish a link between the pattern of expression of CAM on NB cells and their susceptibility to natural killer (NK) or lymphokine-activated killer (LAK) cell lysis, the analysis revealed that NB cells expressing CAM and a differentiated phenotype were less susceptible to NK lysis, but no difference in the sensitivity of the NB cell types to LAK effectors was observed. Treatment of NB target cells with cytokines or PMA decreased their susceptibility to NK and LAK lysis, while induction of differentiation with RA or BUdR resulted in no changes in the sensitivity to NK and LAK lysis. In conclusion, expression of HLA-ABC and several co-regulated CAMs was shown to be associated with a differentiated phenotype in NB, with an overall decreased sensitivity to NK/LAK effector cells.
Abstract: The surface antigenic profile of 10 surgically removed uveal melanoma lesions and 5 conjunctival melanomas was analyzed with a panel of 22 monoclonal antibodies (mAbs) raised against membrane bound cutaneous melanoma-associated antigens (MAA). In addition these lesions were tested for their reactivity with mAbs against MHC class I and II molecules, CD7 (Pan-T) and CD10 (CALLA). The anti-MAA mAbs can be divided into two major groups: first those mAbs detecting markers expressed by the majority of uveal melanomas such as NKI-Beteb, NKI/C3, G7E2, M-2-2-4, Mel-14, G7A5, AMF6, AMF7, Pal M1, Pal M2, Me14/D12. The staining intensity for these mAbs was rather high, ranging in intensity between 70 and 100%. The second group of antibodies includes mAbs detecting markers not or very poorly expressed on ocular melanomas. The anti-ICAM-1 mAb P358 did not react with any of the lesions tested and mAb Muc18 and Muc54 only with one and two out of 15 lesions, respectively. The majority of spindle lesions and mixed type lesions and half of the epitheloid type lesions expressed HLA class I molecules, while HLA class II molecules were found on half of the spindle and epitheloid type lesions and on a small number of mixed cell type lesions. All spindle lesions were found to express the CD10 (CALLA) molecule and less than half of the other type of lesions were stained with an anti CD10 mAb. The melanoma associated ganglioside GD3 was mainly expressed on epitheloid type lesions while GD2 was predominantly expressed on mixed type lesions. In essence, the overall surface phenotype of the uveal melanoma lesions tested, as defined by the panel of mAbs used, differs markedly from the surface phenotype of cutaneous melanoma lesions defined by a very similar antibody panel.
Abstract: Neuroblastoma cell lines and tumors are characterized by low HLA class I expression. The majority of neuroblastoma cell lines and a high percentage of disseminated tumors display amplification of the nuclear protooncogene N-myc. An inverse correlation between HLA class I expression and N-myc amplification and overexpression has been recently described in neuroblastomas (NBs). In this study we have shown that cytokines (recombinant gamma-interferon, recombinant alpha-tumor necrosis factor), differentiation agents (dibutyryl cyclic AMP, phorbol myristate acetate) and growth factors (nerve growth factor, epithelial growth factor) were able to influence the growth rate and surface expression of HLA class I molecules as well as of a tumor-associated antigen on 2 representative NB cell lines. Induced decreased growth rate in NB cells was not always related to decreased N-myc expression. Analysis at the mRNA level revealed that both N-myc and HLA class I RNA steady-state levels could be modulated by several substances, including recombinant gamma-interferon, phorbol myristate acetate, dibutyryl cyclic AMP, and epithelial growth factor and were not necessarily linked. An inverse correlation between N-myc and HLA mRNA levels was observed only after exposure of NB cells to recombinant alpha-tumor necrosis factor. We conclude that N-myc and HLA class I RNA steady-state levels can be modulated independently and suggest that they are not necessarily inversely regulated.
Abstract: Three monoclonal antibodies (IgG2) have been produced from hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice hyperimmunized with gamma-interferon-treated neuroblastoma cells. The 3 MAbs, 7A4, 2A6 and IG8, detected an antigen present on neuroblastoma tumors and cell lines, but also on some neuro-ectoderm-derived tissues and cells. All 3 clones were shown to react with an epitope of the di-sialo-ganglioside GD2 molecules highly expressed by some neuro-ectoderm-derived tumors, mainly neuroblastoma. Whereas MAb IG8 specificity was restricted to GD2 and its o-acylated form, MAb 2A6 and 7A4 were also able to detect GD3 at high concentration of antibody as shown by TLC analysis and immunodetection. The 3 MAbs were able to lyse 100% neuroblastoma cells in the presence of rabbit or human complement. Direct binding assays with 125I-labelled MAbs showed that MAb 7A4 might be a good candidate for in vivo immunolocalization experiments. The high proportion of anti-GD2 MAbs obtained by our fusion and the increased binding of anti-GD2 MAbs on gamma-IFN-treated neuroblastoma cells suggests a modulation of the exposure and an increase in the immunogenicity of GD2 induced by gamma-IFN.
Abstract: A panel of 8 new Mabs have been produced against neuroblastoma cells (LAN-1) previously treated with IFN-gamma. All selected Mabs from 2 different fusions have been shown to detect epitopes on the GD2 ganglioside molecules highly expressed on all cells of neural crest origin including neuroblastoma, glioblastoma and melanoma. Our results imply that modulation of GD2 exposure on NB cells is dependent on culture conditions and moreover that IFN-gamma increases the surface expression of GD2 and thereby enhances their immunogenicity.
Abstract: To evaluate the feasibility and clinical usefulness of immunocytochemical detection of bone marrow metastases in neuroblastoma, we studied bone marrow samples from patients undergoing intensive therapy, followed in the majority of cases by autologous bone marrow rescue. Two monoclonal antibodies were used in an indirect immunoenzymatic assay to test 384 samples collected from multiple bone marrow sites during 79 staging procedures in 48 patients. Of 578 immunocytochemical tests, 59 (10%) yielded non-evaluable results. Analysis by individual bone marrow sites showed an agreement between cytological and immunocytochemical examinations in 276 of 309 (89%) evaluable tests with 5 A7 and in 179 of 210 (85%) with UJ 13 A. Infiltration by neuroblastoma cells was reported in 9% of samples by cytology, in 6% by immunochemistry with 5 A7 and in 16% with 13 A. Analysis of results by staging demonstrated agreement between cytological examination and immunocytochemical detection with both monoclonal antibodies in 60 of 75 (80%) evaluable stagings. Bone marrow metastasis was detected by cytology in 22% of stagings, by immunochemistry with 5 A7 in 23%, with UJ 13 A in 25%. Detailed analysis of discordant results revealed that they were related partly to bone marrow sampling variability associated with focal and minimal metastasis of neuroblastoma cells. These data suggest the clinical usefulness of immunocytochemical detection as a complementary test to cytological examination for accurate evaluation of bone marrow infiltration in patients with disseminated neuroblastoma.
Abstract: Testing with a panel of 26 monoclonal antibodies (MAbs) showed the antigenic profile of 13 human neuroblastoma cell lines to be characterized by a generally poor antigenic expression; therefore, Interferon-gamma (IFN-gamma), dibutyryl cyclic-AMP and retinoic acid were used to analyse the modulation of surface antigenic expression during differentiation. Treatment of neuroblastoma cell lines with IFN-gamma resulted mainly in induction or increase of class-I MHC antigenic expression. Induction of class-II MHC antigens was obtained on only one neuroblastoma cell line out of 13, thus representing an exceptional event. An increase in some other antigens expressed by neuroblastoma cell lines was also observed. In contrast, and in addition to morphological maturation, treatment of these cell lines with the differentiation inducer dibutyryl-cyclic-AMP (dbc-AMP), resulted in general down-modulation of antigenic expression, particularly of neuroblastoma-associated 5A7 or Leu7 antigens. Retinoic acid treatment had no significant effect on MHC antigens, but it decreased expression of 5A7 and Leu7 antigens, and markedly increased the expression of the melanoma-associated antigen Me14-D12. The similarity between the antigenic profile of in vitro differentiated neuroblastoma cells and that of mature ganglioneuroma cells suggests that compounds like cyclic-AMP or retinoic acid are excellent tools for further investigations of the mechanisms of neuroblastoma differentiation and might have important clinical applications.
Abstract: A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.
Abstract: HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.
Abstract: HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.
Abstract: cDNA clones encoding different human Ia antigen beta chains were isolated by use of a complementation-expression assay in Xenopus oocytes. The assay was based on two previous findings. First, oocytes injected with mRNA from a human B-cell line express HLA-DR antigen. The three intracellular DR chains are assembled in oocytes and can be immunoprecipitated with anti-DR monoclonal antibodies. Second, we have isolated cDNA clones encoding DR alpha and intermediate chains. In order to identify beta-chain cDNA clones, mRNA was hybrid-selected with pools of cDNA clones, mixed with mRNA for the alpha and intermediate chains, and injected into oocytes. We isolated two distinct clones that could select DR beta-chain mRNA as demonstrated by assembly of the translation product with DR alpha chains and immunoprecipitation with DR-specific monoclonal antibodies. One clone is specific for a beta chain of the DR locus. The other clone, much weaker in its ability to select DR mRNA, encodes another Ia-like beta chain. Full-length cDNA clones corresponding to the DR and Ia-like beta chains were isolated and compared. Cross-hybridization was detectable in the coding regions but not in the 3' untranslated regions. Distinct RNAs homologous to the DR and the Ia-like beta-chain clones were present in B cells but were undetectable in three T-cell lines.
Abstract: The expression at the cell surface level of two previously defined human Ia antigen subsets, NG1 and NG2, was studied. Two monoclonal hybridoma antibodies, D1-12 (anti-NG1) and D4-22 (anti-NG2), labeled with distinct fluorochromes, were used as probes. The results indicate that NG1 and NG2 molecules are simultaneously expressed on the same cell, and moreover, that they are identifiable as spatially separate structures. In addition, when a series of 6 other anti-Ia monoclonal antibodies previously described were analyzed in a checkerboard test of binding inhibition at the cell surface level, they were found to closely mimic the reactivity pattern of either D1-12 or D4-22 antibody, suggesting that NG1 and NG2 Ia subsets express independent clusters of highly immunogenic epitopes.
Abstract: The expression on several established human glioma cell lines of two well-defined differentiation antigens, HLA-DR and the common acute lymphoblastic leukemia antigen (CALLA) has been demonstrated. Rabbit anti-CALLA antiserum and monoclonal anti-Ia antibodies specifically lysed glioma cells in the presence of complement. Absorption of anti-CALLA antiserum and anti-Ia antibodies by glioma cells abolished their cytotoxicity against blasts isolated from a common acute lymphoblastic leukemia. Immunoprecipitation of solubilized glioma cells by monoclonal anti-Ia antibodies revealed two polypeptide chains of 28 and 33 kDa, whereas the anti-CALLA antiserum precipitated a single polypeptide chain of 100 kDa.
Abstract: HLA-DR antigens, the human equivalent of mouse Ia antigens, are multimeric surface glycoproteins characterized by a high degree of allelic polymorphism. They are expressed specifically on macrophages and lymphocytes and they play a key role in the regulation of the immune response. We have investigated this complex genetic system by a direct study of the genes involved through molecular cloning. This paper deals with the cloning, in plasmids, of full-length cDNA sequences for the HLA-DR alpha chain from the human B-cell line Raji. The approach relies on a translation assay of mRNA injected into frog oocytes and recognition of translation products by polyclonal and monoclonal antibodies. After enrichment of specific mRNA and cloning of cDNA, plasmid clones were analyzed by hybridization-selection of mRNA and translation in oocytes. A clone was identified and used to screen a cDNA library from which several full-length HLA-DR alpha chain plasmids were isolated. DNA sequence determination of one such clone confirmed its identity and also established the amino acid sequence of the NH2-terminal signal sequence of HLA-DR alpha chains. The translation product of HLA-DR alpha chain mRNA purified by hybridization-selection gives a single alpha chain spot on two-dimensional gels, whereas the alpha chain released from the alpha/beta HLA-DR complex gives about seven distinct spots. Finally, the results of analysis of genomic DNA by Southern blotting are compatible with the existence of a single nonpolymorphic alpha chain gene and indicate extensive cross-hybridization with a homologous gene in mouse DNA.
Abstract: Two distinct subsets of human Ia molecules, called NG1 and NG2, present in all individuals irrespective of their HLA-DR phenotype, which were previously defined by their reactivity with two monoclonal hybridoma antibodies, D1--12 and D4--22, were analyzed by two-dimensional peptide mapping techniques. Results show that, in the Ia molecular pool from a single individual, small beta subunits of the NG1 and NG2 subsets display significant differences from each other. In addition, beta subunits of the same subset from two different allotype Ia molecular pools are also different from each other, thus indicating that NG1 and NG2 subsets carry polymorphic specificities. Moreover, large alpha chains of NG1 and NG2 subsets are different from each other; however, no significant differences are observed in alpha chains of the same subset when different allotype Ia pools are analyzed. The possible genetic implications of these findings are discussed.
Abstract: An antiserum was produced by immunization of rabbits with membrane preparations of a human lymphoblastoid B cell line, Daudi. After absorption with a human endometrial carcinoma cell line, this antiserum appeared to be specific for antigen(s) present on adult and fetal thymocytes as well as on tonsillar lymphocytes but absent, or present in very small amounts, on normal or phytohemagglutinin- (PHA) stimulated peripheral blood lymphocytes (PBL). When T and B cell-enriched fractions from tonsillar lymphocytes were tested with the anti-Daudi serum, the reactivity was equally distributed in each population. Among 13 human lymphoblastoid cell lines tested, reactivity was demonstrated on three out of four T cell lines, and on four out of nine B cell lines. The positive reacting B cell lines were derived from two African and two American Burkitt lymphomas. The antigen(s) described does not seem to be related either to human Ia-type antigens or to Epstein-Barr virus-associated antigens because these antigens are not present on fetal or adult thymocytes. Reciprocal absorption experiments indicate that this anti-Daudi serum detects the same antigenic structures present on certain subpopulations of T and B lymphocytes.
Abstract: An antiserum was obtained by immunization of rabbits with human thymocyte membrane fractions. After appropriate absorptions, the antiserum was shown to detect specifically a population of T-cells. When tested by complement-mediated cytotoxicity the antiserum lysed 95% of thymocytes, 65% of normal PBL and 45% of tonsillar lymphocytes. It was also cytotoxic for three different T-cell lines (MOLT-4, CCRF-CEM and CCRF-HSB-2). When peripheral or tonsillar lymphocytes were separated into populations enriched in B- and T-cells, the percentage of cells lysed by the antiserum correlated well with the proportion of E-rosetting cells. Treatment of PBL with the antiserum and complement resulted in an increase of SmIg-positive B-cells in the residual cell fraction, which could no longer form E-rosettes. Treatment of PBL with the antiserum alone completely inhibited the E-rosette formation. The cytotoxic index on PBL from patients with various lymphoid disorders always correlated with the proportion of T-cells as assessed by E-rosette formation. Finally, the absorptive capacity of thymocytes for the antiserum was ten times higher as compared to that of PBL or tonsil cells.
