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Multiple myeloma is the second most frequent hematological cancer after lymphoma and remains an incurable disease. The pervasive support provided by the bone marrow microenvironment to myeloma cells is crucial for their survival. Here, an unbiased assessment of receptor tyrosine kinases overexpressed in myeloma identified ROR2, a receptor for the WNT noncanonical pathway, as highly expressed in myeloma cells. Its ligand, WNT5A is the most abundant growth factor in the bone marrow of myeloma patients. ROR2 mediates myeloma cells interactions with the surrounding bone marrow and its depletion resulted in detachment of myeloma cells from their niche in an in vivo model, triggering apoptosis and thus markedly delaying disease progression. Using in vitro and ex vivo 3D-culture systems, ROR2 was shown to exert a pivotal role in the adhesion of cancer cells to the microenvironment. Genomic studies revealed that the pathways mostly deregulated by ROR2 overexpression were PI3K/AKT and mTOR. Treatment of cells with specific PI3K inhibitors already used in the clinic reduced myeloma cell adhesion to the bone marrow. Together, our findings support the view that ROR2 and its downstream targets represent a novel therapeutic strategy for the large subgroup of MM patients whose cancer cells show ROR2 overexpression.

In diffuse large B-cell lymphoma (DLBCL), the clinical and biological significance of concordant and discordant bone marrow (BM) involvement have not been well investigated. We evaluated 712 de novo DLBCL patients with front-line rituximab-containing treatment, including 263 patients with positive and 449 with negative BM status. Compared with negative BM disease, concordant BM adversely impacted overall and progression-free survival, independent of the International Prognostic Index (IPI) and cell-of-origin classification. Once BM is concordantly involved, poor prognosis was not associated with the extent of BM involvement. Conversely, patients with discordant BM showed favorable overall survival similar to stage I–II DLBCL. A BM-adjusted IPI, using three parameters: concordant BM involvement, age >60 years, and performance status >1, improves the risk stratification for DLBCL with positive BM. Intensive immunochemotherapy seemingly rendered survival benefit for patients with concordant BM, as did rituximab maintenance for the discordant BM group. Frequently revealing adverse clinical and molecular characteristics, patients with concordant BM demonstrated gene expression signatures relevant to tumor cell proliferation, migration and immune escape. In conclusion, clinical and biological heterogeneity is seen in DLBCL with positive BM but concordant BM involvement represents a distinct subset with unfavorable gene signatures, high-risk clinicopathologic features and poor prognosis.

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novoDLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1deletions and PRDM1mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients.

Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a unique subtype of DLBCL. The impact of rituximab on survival and patterns of treatment failure in PT-DLBCL patient remain controversial. We analyzed the clinical and biological feature of 280 PT-DLBCL cases, 64% of which were treated with rituximab-containing regimens. Although most (95%) patients achieved complete remission, a continuous risk of relapse was observed. Rituximab significantly reduced the cumulative risk of relapse (P=0.022) and improved both progression-free survival (PFS, P=0.012) and overall survival (OS, P=0.027) of PT-DLBCL patients (5-year PFS, 56% vs 36%; 5-year OS, 68% vs 48%). Central nervous system and contralateral testis were the most common sites of relapse, but other extranodal and nodal sites of relapse were also observed. Most cases of PT-DLBCL had a non-germinal center B-cell like (84%) immunophenotype and an activated B-cell like (86%) gene expression profile (GEP) subtype. The distinctive GEP signature of primary testicular lymphoma was relevant to tumor cell proliferation, dysregulated expression of adhesion molecules and immune response, likely accounting for the poor outcome. Accordingly, forkhead box P1 transcription factor (FOXP1) and T-cell leukemia/lymphoma 1 (TCL1) oncogenic activation were confirmed and predicted a significant trend of poor survival. This study provides valuable observations for better understanding of both clinical and biological features in PT-DLBCL patients.

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.

The immunosuppressive activity of rapamycin (RAPA) and its efficacy as an anti-rejection agent in organ transplantation have been ascribed principally to its anti-proliferative effects on T cells, while the activity on monocytes is partially unknown. In vitro, RAPA reduced monocyte survival by inducing a caspase-independent cell death. RAPA-induced monocyte cell death (RAPA-CD) was impeded by activation of granulocyte macrophage-colony stimulating factor family receptors or toll-like receptor 4, and by exposure to inflammatory cytokines. In vivo, in patients who received RAPA monotherapy as part of pre-conditioning for islet transplantation, RAPA affected survival of myeloid lineage cells. In the peripheral blood, CD33+ and CD14+ cells decreased, whereas lymphocytes appeared unaffected. In the bone marrow, myeloid precursors such as CD15+ and CD15+/CD16+ were selectively and significantly decreased, but no major cytotoxic effects were observed. The RAPA-CD suggests a dependence of monocytes on mammalian target of RAPA pathways for nutrient usage, and this feature implies that RAPA could be selectively useful as a treatment to reduce monocytes or myeloid cells in conditions where these cells negatively affect patient, suggesting a potential anti-inflammatory action of this drug.

The immunosuppressive activity of rapamycin (RAPA) and its efficacy as an anti-rejection agent in organ transplantation have been ascribed principally to its anti-proliferative effects on T cells, while the activity on monocytes is partially unknown. In vitro, RAPA reduced monocyte survival by inducing a caspase-independent cell death. RAPA-induced monocyte cell death (RAPA-CD) was impeded by activation of granulocyte macrophage-colony stimulating factor family receptors or toll-like receptor 4, and by exposure to inflammatory cytokines. In vivo, in patients who received RAPA monotherapy as part of pre-conditioning for islet transplantation, RAPA affected survival of myeloid lineage cells. In the peripheral blood, CD33+and CD14+cells decreased, whereas lymphocytes appeared unaffected. In the bone marrow, myeloid precursors such as CD15+and CD15+/CD16+were selectively and significantly decreased, but no major cytotoxic effects were observed. The RAPA-CD suggests a dependence of monocytes on mammalian target of RAPA pathways for nutrient usage, and this feature implies that RAPA could be selectively useful as a treatment to reduce monocytes or myeloid cells in conditions where these cells negatively affect patient, suggesting a potential anti-inflammatory action of this drug.

The authors assessed MATILDE chemotherapy followed by response-tailored radiation therapy in 41 patients aged 70 years or younger with primary CNS lymphoma in a Phase II trial. With response rates of 76% after MATILDE and 83% after chemotherapy with or without radiation therapy, this was an active strategy, particularly in low- to intermediate-risk patients (International Extranodal Lymphoma Study Group IELSG score). Myelosuppression was the dose-limiting toxicity, with 9.5% of lethal complications. After a median follow-up of 49 months, a plateau in the survival curve (5-year overall survival: 41 ± 7%) was obtained.

To characterize the therapeutic variables correlated to outcome in 370 patients with primary CNS lymphoma.

Intravascular lymphomatosis (IL) is a rare entity only recently included in lymphoma classification, whose main feature is the exclusive or predominant growth of neoplastic cells within blood vessels. The vast majority of the patients affected by IL belong to the 7th or 8th decade of life and present with skin rash or CNS diffuse necrotic or demyelinating lesions. Case report. SS, a 13-year-old girl, was admitted to a Neurosurgery Unit because of endocranic hypertension, where, after CT and MRI documenting a IV ventricle 3 cm diameter tumor, she was submitted to complete tumor excision: extemporary diagnosis was suggestive of medulloblastoma. When referred to us she had persistent fever with normal blood and spinal fluid cultures. Whole CNS MRI did not give evidence of residual or metastatic disease while CSF cytology showed only pleiocytosis. Treatment was started according to our ongoing protocol for medulloblastoma with pre-radiation chemotherapy. Before delivering radiotherapy (RT), upon review of histologic specimens, the definitive diagnosis of IL B-phenotype was made. The girl was re-admitted and, after a complete re-staging, chemotherapy was intensified according to our schedule for high-grade B-cell lymphoma and CNS was irradiated up to a total dose of 25 Gy. She remained alive in continuous complete remission at 21 months after diagnosis. The case here reported is unique for age, tumor presentation, and, so far, favourable outcome, in spite of the delayed histological diagnosis.

Intravascular Lymphomatosis (IL) is a rare and usually aggressive form of non-Hodgkin's lymphoma characterized by the growth of neoplastic cells within vascular lumina that usually presents with skin or central nervous system (CNS) involvement. The mechanism(s) for the selective intravascular growth of this neoplasm remain(s) unexplained. We now report clinical and immunohistologic data on surgical material from 6 cases of IL; in 4 of 6 cases, autopsies were performed. Our IL cases shared the following features: (1) B-cell lineage; (2) lack of skin involvement at presentation; (3) aggressive behavior; and (4) lack of extravascular lymphomatous masses; in addition, 1 case had an associated gastric low-grade MALT lymphoma. We studied by immunohistochemistry formalin-fixed, paraffin-embedded sections with monoclonal antibodies to molecules known to be involved in lymphocyte and endothelial adhesion phenomena, that is, CD29 (@b"1 integrin subunit), CD43 (leukosialin), CD44 (H-CAM), CD54 (ICAM-1), embryonal N-CAM (e-NCAM), and EMA (episialin). In all cases, the surfaces of IL aggregates reacted for CD44 but were consistently negative for CD29; also absent was CD54. Conversely, the integrity of the endothelial cells was underscored by their even reactivity for CD29, CD44, and CD54. Given that CD29 is currently regarded as critical for lymphocyte trafficking in general and for transvascular migration in particular, and CD54 is also involved in transvascular lymphocyte migration, we conclude that their consistent absence in IL may contribute to its intravascular and disseminated distribution pattern. The rather frequent association of IL with various conventional lymphomas is known; yet, one of our cases appears to be the first report of IL associated with a low-grade MALT lymphoma.

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.

1.The effects of retinoic acid,?-interferon, cytosine arabinoside, nerve growth factor, tumor necrosis factor, and 12-O-tetradecanoylphorbol 13-acetate on the human neuroblastoma cell line, LAN-5, were studied. Intracellular levels of acetylcholinesterase, neuron-specific enolase, catecholamines and related neurotransmitters, vasointestinal peptide, and substance P were evaluated after induction.2.Cell morphology was strongly affected by retinoic acid,?-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. The main effects of retinoic acid and?-interferon were the loosening of cell clusters and the extension of long neurites; cytosine arabinoside induced cell body swelling and marked neuritogenesis. Following 12-O-tetradecanoylphorbol 13-acetate treatment, the cells became small, round, and neuritic. Conversely, modifications induced by nerve growth factor and tumor necrosis factor were mild. Cell proliferation rate was reduced by retinoic acid,?-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate, while nerve growth factor and tumor necrosis factor were devoid of effects.3.Acetylcholinesterase activity was significantly stimulated by retinoic acid and by?-interferon. Neuron-specific enolase activity was unaffected by all treatments except 12-O-tetradecanoylphorbol 13-acetate, which enhanced it by 1.6-fold.4.The cellular catecholamine and related metabolite content was lowered by retinoic acid and?-interferon, while cytosine arabinoside and, even more, 12-O-tetradecanoylphorbol 13-acetate showed a stimulatory activity on their intracellular accumulation.5.Finally, the cell-associated vasointestinal peptide level was strikingly increased by?-interferon and, to a lesser extent, by retinoic acid, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate.6.It is concluded that the most relevant biochemical changes associated with LAN-5 cells differentiation involve the repertoire of neurotransmitters and neuropeptides. These events vary in quality and in quantity, likely due to the pattern complexity of gene expression triggered by each inducer in determining the diversity of neuronal phenotypes.

Summary 1.The effects of gamma-interferon (γ-IFN), retinoic acid (RA), and cytosine arabinoside (ARA-C) on the growth, morphology, and phenotype of the human neuroblastoma (NB) cell lines, LAN-1 and GI-ME-N, have been extensively tested.2.RA,γ-IFN, and ARA-C induced a dose-dependent morphological differentiation and growth inhibition, without affecting cell viability. Cells exposed to 10−6M RA or 1000 U/mlγ-IFN significantly decreased their growth rate within the first 24 and 48 hr of culture, respectively. Cells became smaller and polygonal and sprouted long cellular processes with varicosites along their courses. In contrast, ARA-C-differentiated cells were larger and flattened, with few elongated dendritic processes.3.Analysis of membrane and cytoskeletal markers by immunofluorescence and Western blot showed several changes in NB-specific antigen expression after 5 days of treatment with all inducing agents. Analysis of labeled phosphatidylinositol metabolites from prelabeled cells showed, within 1 min of treatment with RA, a rapid decrease in inositol 1,4,5-trisphosphate and of 1,2-diacylglicerol levels. No changes in inositol phospholipid metabolism were observed inγ-IFN- or ARA-C-treated cells.4.We conclude that RA-induced decrease in phosphatidylinositol (PI) hydrolysis is not likely to be a consequence of the acquisition of a different phenotype, as its changes precede the acquisition of neuronal markers. In addition,γ-IFN and ARA-C, both inducing a mature phenotype, did not affect PI hydrolysis.5.Decreased PI hydrolysis seems to be sufficient, although not necessary, to commit NB cells to neuronal differentiation. Analysis of molecular mechanisms associated with NB cell differentiation may be helpful to clarify the potential of various biological agents in affecting the development of the neural cell.

Although neuronal cells are a major target of phorbol ester action, the activity of the various protein kinase C (PKC) isoenzymes have not been studied in detail in human neuroblasts. Differentiation of the LAN‐5 human neuroblastoma cell line by interferon‐γ (IFN‐γ) is accompanied by a twofold increase in PKC activity. Since PKC is a multigene family, we investigated which isoforms were expressed in control and differentiated cells, and which of these isoenzymes is involved in neuronal differentiation. We found that: (1) PKC activity is higher in differentiated than in undifferentiated cells; (2) RT‐PCR analysis showed the expression of mRNA for PKCα, ‐γ, ‐δ ‐ε and‐ζ and the absence of mRNA for β in untreated LAN‐5 cells; (3) Western blot evaluation with PKC isoform‐specific antibodies showed the same pattern of PKC expression in non‐differentiated cells; (4) Expression of PKCε mRNA was significantly enhanced by IFN‐γ‐induced differentiation, while the other isoforms were not affected; (5) Differentiation of LAN‐5 cells with IFN‐γ or retinoic acid induced overexpression of the PKCε protein, while inhibition of cell proliferation by fetal calf serum starvation was without effect. These findings suggest that expression of PKCε isoform is tightly coupled with neuronal differentiation and may play a role in the maintenance of the differentiated state.

Although neuronal cells are a major target of phorbol ester action, the activity of the various protein kinase C (PKC) isoenzymes have not been studied in detail in human neuroblasts. Differentiation of the LAN-5 human neuroblastoma cell line by interferon-γ (IFN-γ) is accompanied by a twofold increase in PKC activity. Since PKC is a multigene family, we investigated which isoforms were expressed in control and differentiated cells, and which of these isoenzymes is involved in neuronal differentiation. We found that: (1) PKC activity is higher in differentiated than in undifferentiated cells; (2) RT-PCR analysis showed the expression of mRNA for PKCα, -γ, -δ -ε and-ζ and the absence of mRNA for β in untreated LAN-5 cells; (3) Western blot evaluation with PKC isoform-specific antibodies showed the same pattern of PKC expression in non-differentiated cells; (4) Expression of PKCε mRNA was significantly enhanced by IFN-γ-induced differentiation, while the other isoforms were not affected; (5) Differentiation of LAN-5 cells with IFN-γ or retinoic acid induced overexpression of the PKCε protein, while inhibition of cell proliferation by fetal calf serum starvation was without effect. These findings suggest that expression of PKCε isoform is tightly coupled with neuronal differentiation and may play a role in the maintenance of the differentiated state.

A peculiar human cell line (GI-ME-N) derived from the metastatic bone marrow of a 2-yr-old patient with stage IV neuroblastoma (NB) was extensively characterized. Cell-type-specific markers, tumorigenicity in nude mice, morphology, cytogenetics, and amplificationexpression of the N-myc gene were evaluated. All metaphases presented the typical 1p deletion. Surface markers specific for NB cells, vimentin, and neurofilament proteins were all clearly detectable with immunofluorescence andor western blot procedures. Moreover, it was found that GI-ME-N cells did not express N-myc oncogene or HLA class 1 antigens, and were not classified as peripheral neuroectodermal tumor cells. However, extremely short latency and survival times, comparable to peripheral neuroectodermal tumor cells, were observed in nude mice grafted with GI-ME-N. In addition, no correlations were. observed in tumorigenicity of N-myc amplified (IMR32) versusunamplified (SK-N-SH GI-ME-N) human NB cell lines in nude mice. We conclude that N-myc amplification expression do not correlate with the aggressiveness of human NB in athymic animals, which is not always explained by the peripheral neuroectodermal tumor cell nature of the malignant cells, either.

Two human neuroblastoma cell lines, LAN‐5 and GI‐CA‐N, have been analyzed for their capability to adhere to different extracellular matrix (ECM) components. The GI‐CA‐N cells adhered to all the tested substrates: laminin (LN), type I and type IV collagen (Coll I, Coll IV), vitronectin (VN), and fibronectin (FN). Conversely LAN‐5 cells weakly attached to FN and VN, whilst adhesion on LN and Coll I and IV was strong and induced a rapid elongation of cell processes. By means of RT‐PCR and immunoprecipitation we showed that the integrin pattern of these two lines was different and could explain their diversity in adhesion capability. Both cell lines express a large amount of the β1integrin subunit, associated with different α chains, probably responsible for their adhesion to some ECM proteins. After treatment of LAN‐5 cells with biological differentiating agents, such as γ‐interferon, alone or in combination with tumour necrosis factor‐α (TNF‐α), or retinoic acid, the levels of α1β1, α2β1, and α3β1integrin expression were enhanced, while the amount of α, remained constant. In contrast, treatment of LAN‐5 cells with TNF‐α, that did not induce any maturation, or starvation in 2% foetal calf serum, that inhibited cell proliferation without affecting neural differentiation, did not induce any change in the integrin assessment. Messenger‐RNAs for the two α6isoforms, A and B, were present in both cell lines. However, in LAN‐5 cells, the protein product was neither detectable nor inducible by differentiation. Our results confirm the specific modulation of the α1β1integrin expression in human neuronal development, and show, for the first time, the involvement of α2β1and α3β1, heterodimers in this maturational process.

ABSTRACT: A peculiar human cell line (GI-ME-N) derived from the metastatic bone marrow of a 2-yr-old patient with stage IV neuroblastoma (NB) was extensively characterized. Cell-type-specific markers, tumorigenicity in nude mice, morphology, cytogenetics, and amplification/expression of the N-myc gene were evaluated. All metaphases presented the typical 1p deletion. Surface markers specific for NB cells, vimentin, and neurofilament proteins were all clearly detectable with immunofluorescence and/or western blot procedures. Moreover, it was found that GI-ME-N cells did not express N-myc oncogene or HLA class 1 antigens, and were not classified as peripheral neuroectodermal tumor cells. However, extremely short latency and survival times, comparable to peripheral neuroectodermal tumor cells, were observed in nude mice grafted with GI-ME-N. In addition, no correlations were. observed in tumorigenicity of N-myc amplified (IMR32) versus unamplified (SK-N-SH GI-ME-N) human NB cell lines in nude mice. We conclude that N-myc amplification/expression do not correlate with the aggressiveness of human NB in athymic animals, which is not always explained by the peripheral neuroectodermal tumor cell nature of the malignant cells, either.

The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN-1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and γ-interferon (γ-IFN). Analysis of labelled phosphatidylinositol metabolises from prelabelled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol within 1 min of induction of LAN-1 cell dilfferentiation by RA, while no changes were observed in γ-IFN-treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA-treated LAN-1 cells and suggest that phosphoinositide-derived metabolises may not constitute general regulators of cellular differentiation.

Several T-cell functions are controlled by the regulatory peptide interleukin 2 (IL-2). Binding of IL-2 with specific receptors has been well documented, but the molecular mechanism by which IL-2/IL-2 receptor interaction is transduced is not known. We have found that treatment of IL-2-dependent T-cell lines with IL-2 is followed by a rapid stimulation of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H]inositol. Increased incorporation of the metabolic precursor into phosphatidylinositol and phosphatidylinositol 4-monophosphate, together with the appearance of radiolabeled phosphatidylinositol 4,5-bisphosphate, occurred within minutes of treatment with IL-2 of factor-dependent CT6 cells. Analysis of labeled water-soluble compounds from prelabeled cells indicated a rapid (within 1 min) stimulation of inositol phospholipid hydrolysis following IL-2 treatment. Increased recovery of [3H] inositol phosphates and appearance of [3H]inositol trisphosphate were observed after treatment with IL-2 of CT6 cells, as well as of a second IL-2-dependent cell line, CTB6. These findings suggests that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain IL-2 signals are transduced.

The precise cellular origin and the pathogenetic mechanism(s) leading to the neoplastic transformation of anaplastic large cell lymphoma (ALCL) and the Reed-Sternberg cell of Hodgkin's disease (HD) remains largely uncertain. Classical cytogenetic analysis has shown a unique translocation involving bands 2p23 and 5q35 bands in a variable number of ALCLs. It has been recently shown that the nucleophosmin/B23 (NPM) gene (5q35) and a novel anaplastic lymphoma kinase (ALK; 2p23) are the fused genes of t(2;5). To investigate the presence and the precise frequency of NPM-ALK gene products among ALCL and HD cases, a large and well-characterized panel of ALCL (n = 49) and HD (n = 72) cases was studied using multiple strategies including reverse transcriptase- polymerase chain reaction (RT-PCR), Southern blot analysis, and immunohistochemistry. Overall, 6 (3 T and 3 null) of 49 ALCL and 3 (2 nodular sclerosis and 1 mixed cellularity) of 72 HD showed the presence of NPM-ALK transcripts by RT-PCR. NPM-ALK gene rearrangements were detected in all RT-PCR, NPM-ALK-positive ALCL by Southern blot analysis. Furthermore, in all the available cases we were able to show the presence of ALK-related protein using a specific polyclonal antiserum recognizing the cytoplasmic domain of ALK by immunohistochemistry. Our data show that NPM-ALK gene transcripts are identified in a subpopulation of ALCL, almost exclusively in T or null cell in origin, and in rare cases of HD. These findings show that some HD may be closely related to ALCL, giving us new insights on the pathogenesis and possibly biologic evolution of HD.

Interferon-gamma (IFN-gamma) is a potent growth-inhibitory cytokine also endowed with differentiating activity on neural cells. Binding of IFN-gamma to its high-affinity receptor induces a rapid and transient activation of phospholipase A2 (PLA2). The mechanism coupling the IFN-gamma receptor (IFN-gamma-R) to PLA2 activation is not clearly defined, and no information is available on this mechanism in neuroblast cells. We have tested the hypothesis that GTP-binding proteins (G-proteins) may couple the IFN-gamma-R to PLA2 in the human neuroblastoma (NB) cell line LAN-5. Incubation of NB cells with IFN-gamma resulted in a rapid increase in [3H]arachidonic acid (AA) release, and this effect was blocked by pretreatment with anti-IFN-gamma antibodies. IFN-gamma-stimulated AA release was still observed in permeabilized cells that were blocked by pretreatment with anti-IFN-gamma-R antibodies. Exposure of permeabilized LAN-5 cells to guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, induced a dose-dependent release of [3H]AA. A non-specific nucleotide effect was excluded, since similar stimulatory effects on AA mobilization were not observed by GTP, ATP, CTP, ADP and GDP. IFN-gamma-stimulated AA release was completely blocked by the guanine nucleotide analogue that inhibits G-protein function, guanosine 5′-[beta-thio]diphosphate (GDP[S]). A role for G-proteins in IFN-gamma-R coupling to PLA2 was further supported by the inhibition of IFN-gamma-induced [3H]AA release by treatment of permeabilized cells with pertussis toxin and with the antiserum against the common alpha-subunits of G-proteins. To determine a possible contribution to AA mobilization by the phospholipase C and diacyglycerol lipase pathway or by protein kinase C activation, the effects of neomycin, a phospholipase C inhibitor, and PMA (phorbol 12-myristate 13-acetate), a direct activator of protein kinase C, were investigated. Neither neomycin nor PMA affected either basal or IFN-gamma-stimulated AA release. Ca2+ concentration, which has been shown to regulate the activity of some PLA2s, does not appear to play an important role in the regulation of the IFN-gamma-stimulated PLA2 activity, since incubating permeabilized cells in different concentrations of Ca2+ induced AA release without affecting the IFN-gamma response. Altogether, these findings suggest the existence of IFN-gamma-R, which couples a Ca(2+)-independent PLA2 activation via pertussis-toxin-sensitive G-proteins.

DOCSPER—A Synthetic Lipid Fit for In Vivo ApplicationDOCSPER [1,3-Dioleoyloxy-2-(N5-carbamoyl-spermine)-propane] is a cationic amphiphile consisting of a hydrophobic 1,3 dioleylglycerol moiety and threefold positively charged spermine head group (1). We optimised the 5-step-synthesis of the lipospermine and after up-scaling we have obtained sufficient amounts to initiate preclinical investigations. DOCSPER was tested for its ability to transfect eukaryotic cells in vitro. It has proven to possess high transfection efficiency in comparison to commercially available liposomal transfection agents. Furthermore, DOCSPER was extensively tested in several in vivo studies (23). These studies revealed a high transfection efficiency, whereas very low toxicity levels were detected. Thus, the results clearly indicate that the cationic lipid DOCSPER is a reliable, low-risk system for broad applications in gene therapy.Groth D. et al. Int J Pharm 1998; 162:143–157.Nikol S. et al. Int J Angiol 2000; 9:87–95.Armeanu S. et al. Mol Ther 2000; 1(4):366–375.

Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic LipoplexesEarlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (1). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled.The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60 of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90 of the particles was in the blood. Currently, we compare the encapsulation efficiency, stability and targetability of the CCL with stabilized antisense lipid particles (SALP) (2), while also the biological activity of these carriers is addressed. In conclusion our results demonstrate that antisense ODN can be targeted very efficiently to EC in vivo, employing plasma-stable CCL, surface modified with negatively charged albumin.ReferencesStuart DD, Allen TM. BBA 2000; 1463:219–229.Semple S. et al. BBA 2001; 1510: 152–166.

The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN‐1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and γ‐interferon (γ‐IFN). Analysis of labelled phosphatidylinositol metabolises from prelabelled cells indicated a rapid decrease of inositol 1,4,5‐trisphosphate and 1,2‐diacylglycerol within 1 min of induction of LAN‐1 cell dilfferentiation by RA, while no changes were observed in γ‐IFN‐treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA‐treated LAN‐1 cells and suggest that phosphoinositide‐derived metabolises may not constitute general regulators of cellular differentiation.

Iodine labeled metaiodobenzylguanidine (MIBG) is a radiopharmaceutical employed for both diagnosis and metabolic radiotherapy of neuroblastoma (NB). Resistance to the radiotherapeutic effects of MIBG is common, due to lack of MIBG accumulation by NB cells. MIBG enters competent cells via the noradrenaline transporter; this function requires a relative cellular maturation and is missing in most NB cell lines. In vitro differentiation of NB cells can be achieved with γ-interferon (γ-IFN) and other agents. We have verified that γ-IFN-induced differentiation of NB cells is specifically associated with an increase in their ability to incorporate MIBG. This phenomenon is due to enhancement of MIBG transporter activity, according to pharmacological sensitivity and semiquantitative PCR-based analysis of specific MIBG transporter mRNA. New therapeutic strategies, based on both differentiation therapy and targeted radiotherapy of NB can so be devised.

In order to define some distinctive criteria of differentiation, we studied the effects of retinoic acid (RA), ?-interferon (?-IFN), cytosine arabinoside (ARA-C), nerve growth factor (NGF), tumor necrosis factor (TNF), and 12-0-tetradecanoylphorbol-13 acetate (TPA) on the human neuroblastoma cell line, LAN-5. Intracellular levels of acetylcholinesterase, neuron specific enolase, catecholamines, and related neurotransmitters were evaluated after 8 days of treatment with various inducers. Our data indicate that the most striking biochemical changes accompanying LAN-5 cell differentiation are a shift from a catecholaminergic to a cholinergic phenotype, and/or a drastic increase in the catecholamines levels. Moreover, small changes may be independent of thein vitro maturation. Thus, biochemical phenotyping, more than morphological evaluation, is a fruitful approach to monitor neuronal cell differentiation.

In the last few years we have been able to produce a large body of evidences that ARA-C is capable of inducing neuronal differentiation of human neuroblasts in vitro. Three different human neuroblastoma (NB) cell lines have been tested with ARA-C, LAN-1, LAN-5 and Gl-ME-N. ARA-C actually modifies both surface and cytoskelcton markers' expression while inducing substained morphologic differentiation of all three NB cell lines. Specifically, ARA-C induces concentration, dependent growth inhibition, neurite sprouting, and neurofilaments expression in Gl-ME-N cells, while decreasing the level of membrane antigens specific for neuroblast cells. The maximum effect was obtained using a dose as low as 0.1 mgml. Similar modifications were recorded inducing bolh LAN-1 and LAN-5 human NB cells with ARA-C. The fact that ARA-C treatment of human NB cells started one day after plating with no subsequent decrease in cell viability in the cultures, strongly suggests that its effects were not the result of selection of more differentiated cells by differences in plating efficiency or ARA-C cytotoxicity. This conclusion was supported by time-lapse pholomycroscopic observations, which clearly showed individual ARA-C treated NB cells undergoing morphologic differentiation.The results rise the suspicious that the interpretation of ARA-C mechanisms of action on P19 carcinoembrional cells is overscmplicistic. We actually do not thing thai “…one can sclectively enrich for neurons by treating the induced culture with the antimetabolyte ARA-C” as suggested by Chung JJM et al. (Cell 64, 189-200, 1991).In fact, as previously demonstrated by our group, ARA-C is not onlv capable of arrest the overgrowing of non-neuronal cells, but is actively capable of pushing neuroblast cells along the neuronal differentiation pathway.