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H Axelson

Publications and source records attributed to H Axelson.

At least 19 recordsLinked to original sources
PubMed2005

Effects of the histone deacetylase inhibitor valproic acid on Notch signalling in human neuroblastoma cells.

Neuroblastoma (NB), a sympathetically derived childhood tumour, shows characteristics of neuronal precursor cells, suggesting a halted differentiation process. We have previously shown that the Notch signalling cascade, a key player during normal neurogenesis, also might be involved in NB differentiation. Valproic acid (VPA), a well-tolerated antiepileptic drug, has been shown to induce differentiation and cell death of NB cells, possibly associated with its recently described HDAC inhibiting activity. Stimulation of NB cells with VPA led to increased cell death and phenotypic changes associated with differentiation, that is, neurite extension and upregulation of neuronal markers. VPA treatment also led to an activated Notch signalling cascade as shown by increased levels of intracellular Notch-1 and Hes-1, mimicking the initial phase of induced differentiation. These results reinforce that VPA potentially could be used in differentiation therapy of NB and that the effects in part could be a consequence of interference with the Notch signalling cascade.

Anticonvulsants↗
PubMed2001

Differences in early and late responses between neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y neuroblastoma cells.

Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/ trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.

Blotting, Northern↗
PubMed2000

Induced neuroblastoma cell differentiation, associated with transient HES-1 activity and reduced HASH-1 expression, is inhibited by Notch1.

Neuroblastoma is a childhood tumor that originates from the sympathetic nervous system. The tumor cells have embryonic features, presumably as a consequence of an impaired capacity to respond to signals and transcriptional control mechanisms operating during normal differentiation. Two basic helix-loop-helix transcription factors, human achaete-scute homologue-1 (HASH-1) and hairy/enhancer of split homologue-1 (HES-1), are crucial for proper development of some neuronal cells. Here, their potential roles during sympathetic differentiation of human neuroblastoma cells have been investigated. In all tested protocols for induction of differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cells, HASH-1 expression was rapidly decreased with a concomitant, often transient, increase in HES-1 expression. In gel mobility shift assays, using extracts from neuroblastoma cells, HES-1 bound to an oligonucleotide corresponding to a sequence in the HASH-1 promoter including the so-called N-box, suggesting that the transiently increased HES-1 activity in differentiating neuroblastoma cells is involved in down-regulation of HASH-1. Constitutive expression of the intracellular domain of Notch1, which activates the HES-1 promoter in SH-SY5Y cells, inhibited spontaneous and induced morphological differentiation of these neuroblastoma cells. Our data show that functional sympathetic neuronal differentiation of neuroblastoma cells is associated with transient activation of HES-1 and down-regulation of HASH-1 expression.

Basic Helix-Loop-Helix Proteins↗
PubMed2000

Abolished angiogenicity and tumorigenicity of Burkitt lymphoma by interleukin-10.

Because of its immunosuppressive properties, interleukin-10 (IL-10) is thought to play an important role in a number of human disease states, including inflammation, autoimmunity, and transplant rejection. In this study, we demonstrate that introduction of human or viral IL-10 genes into Burkitt's lymphoma cells markedly reduced their ability to grow as subcutaneous (sc) tumors in SCID mice. In vivo assays for angiogenesis revealed an inhibition of the angiogenic capacity of the IL-10-transfected lines. Recombinant human IL-10 abolished and viral IL-10 reduced vascular endothelial growth factor (VEGF)-165-induced neovascularization. Furthermore, IL-10 blocked the VEGF- and fibroblast growth factor (FGF)-2-induced proliferation of microvascular endothelial cells in vitro. The current observations suggest a direct role for IL-10 in the prevention of angiogenesis in human lymphoid malignancies.

Animals↗
PubMed2000

HASH-1 and E2-2 are expressed in human neuroblastoma cells and form a functional complex.

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homolog-1 (MASH-1 in mouse and HASH-1 in human) is essential for proper development of olfactory and most peripheral autonomic neurons, and for the formation of distinct neuronal circuits within the central nervous system. We have previously shown that HASH-1 is expressed in neuroblastoma tumors and cell lines, and in this study we have used the yeast two-hybrid system to isolate HASH-1 interacting proteins from a human neuroblastoma cDNA library. Two of the isolated clones contained cDNA from the E2-2 gene (also known as ITF2/SEF2-1). We show that E2-2 interacts with HASH-1 in both yeast and mammalian cells. The HASH-1/E2-2 complex binds an E-box (CACCTG) in vitro, and transactivates an E-box containing reporter construct in vivo. Furthermore, E2-2 seems to be one of the major HASH-1 interacting proteins in extracts from neuroblastoma cells. In conclusion, E2-2 forms a functional complex with HASH-1, and might therefore be involved in the development of specific parts of the central and peripheral nervous systems.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

Patched 2, located in 1p32-34, is not mutated in high stage neuroblastoma tumors.

Neuroblastoma is a childhood malignancy originating from cells of the sympathetic nervous system, exhibiting a marked diversity in outcome, with spontaneous regression at one end of the spectrum and severe disease and death at the other end. Features associated with frequent recurrence, a poor prognosis, and high tumor stage are loss of heterozygosity in the distal region of chromosome 1p and amplification of the N-myc gene. Patched 2 is a novel homologue to the tumor suppressor gene Patched 1, and has been mapped to 1p32-34, a part of chromosome 1 frequently deleted in high stage neuroblastoma tumors. RT-PCR analysis of 9 neuroblastoma cell lines showed expression of both Patched 1 and 2. We analyzed 14, mainly high stage, neuroblastoma tumors for mutations in the Patched 2 gene with denaturing HPLC using the Wave DNA fragment analysis system. In four tumor samples variations were detected within the coding sequence, and two of them gave rise to amino-acid substitutions. These variations were, however, also detected in normal DNA from the respective patients. We conclude that Patched 2 is expressed, but not frequently mutated, in high stage neuroblastomas and is therefore not likely to be involved in the genesis of this tumor.

Chromatography, High Pressure Liquid↗
PubMed1999

Selection of antibodies for intracellular function using a two-hybrid in vivo system.

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

Animals↗
PubMed1999

Human achaete-scute homologue 1 (HASH-1) is downregulated in differentiating neuroblastoma cells.

The mammalian achaete-scute homologue, MASH-1, is crucial for early development of the sympathetic nervous system and is transiently expressed in sympathetic neuroblasts during embryogenesis. Here we report that the human homologue (HASH-1) was expressed in all analyzed cell lines (6/6) derived from the sympathetic nervous system tumor neuroblastoma. The majority of small-cell lung carcinoma (4/5) cell lines tested expressed HASH-1, while other nonneuronal/non-neuroendocrine cell lines were negative. Induced differentiation of neuroblastoma cells resulted in HASH-1 downregulation. This occurred concomitant with induction of neurite outgrowth and expression of the neuronal marker genes GAP-43 and neuropeptide Y. Constitutive expression of exogenous HASH-1 did not alter the capacity of the neuroblastoma cells to differentiate in response to differentiation-inducing agents. It is concluded that moderate HASH-1 expression does not compromise the capacity of these cells to differentiate.

Basic Helix-Loop-Helix Proteins↗
PubMed1999

The basic helix-loop-helix transcription factor dHAND, a marker gene for the developing human sympathetic nervous system, is expressed in both high- and low-stage neuroblastomas.

Neuroblastoma is derived from the sympathetic nervous system and might arise as a result of impaired differentiation, retaining the neuroblastic tumor cells in the cell cycle. Thus, to understand the genesis of neuroblastoma, the study of mechanisms and genes regulating normal sympathetic development is of potential interest. The basic helix-loop-helix transcription factors human achaete-scute homolog-1 (HASH-1) and deciduum, heart, autonomic nervous system, and neural crest derivatives (dHAND) are expressed in the sympathetic nervous system of embryonic mice and chicken, with undetectable postnatal expression. By in situ hybridization technique, we show that dHAND was expressed by human sympathetic neuronal and extra-adrenal chromaffin cells throughout embryonic and fetal life, and was initially expressed in immature chromaffin cells of the adrenal gland. With overt chromaffin differentiation, dHAND was down-regulated. HASH-1, in contrast, was expressed in human sympathetic cells only at the earliest embryonic ages examined (Week 6.5 to 7). All examined neuroblastoma specimens (25/25) and all cell lines (5/5) had detectable dHAND mRNA levels. HASH-1 expression in tumor specimens was more restricted, although all cell lines (5/5) were HASH-1-positive. These results show that neuroblastoma tumors have retained embryonic features, suggesting that many neuroblastomas are blocked at an early stage of normal development when HASH-1 and dHAND are expressed. dHAND also appears to be a reliable and potentially useful clinical diagnostic marker for neuroblastoma, because expression was not dependent on tumor or differentiation stages and other pediatric tumors were dHAND-negative.

Animals↗
PubMed1997

Chromosomal translocations and leukaemia: a role for LMO2 in T cell acute leukaemia, in transcription and in erythropoiesis.

The LMO2 gene associated with T cell acute leukaemia has been used as an example of a gene activated by association with the T cell receptor genes after chromosomal translocations. The gene is shown to encode a LIM protein which is involved in protein interactions and during normal haematopoiesis is necessary for erythroid development. LMO2 has been shown to cause tumours when aberrantly expressed and to be able to heterodimerise with TAL1 to facilitate tumour development.

Adaptor Proteins, Signal Transducing↗
PubMed1997

LIM-only protein Lmo2 forms a protein complex with erythroid transcription factor GATA-1.

The LIM-only protein Lmo2, originally identified as an oncogenic protein in human T cell leukemia, is essential for erythropoiesis. A possible role for Lmo2 in transcription during erythropoiesis has been investigated. Direct interaction of Lmo2 was observed in vitro and in vivo with the zinc finger transcription factor GATA-1, as well as with the basic helix-loop-helix (bHLH) transcription factor Tall. By using mammalian two-hybrid analysis, E47/Tall/Lmo2/GATA-1 protein complex could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis. This data suggest that variations in amounts of complexes involving Lmo2, Tall, and GATA-1 could be important for erythroid differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1.

The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Functional diversity of LIM proteins: amino-terminal activation domains in the oncogenic proteins RBTN1 and RBTN2.

The RBTN1 and RBTN2 genes are activated by distinct translocations involving chromosome 11 in some T cell acute leukaemias. The RBTN proteins belong to the LIM family which comprises proteins with one, two or three cysteine-rich LIM domains, sometimes together with homeodomains or protein kinase domains. The RBTN1 and RBTN2 proteins comprise only tandem LIM domains. We report that RBTN1 and RBTN2 proteins are capable of supporting transcriptional transactivation of specific reporter genes in transfection assays. The results, using intact proteins or fusions with the homeodomain of the heterologous protein Isl-1, show that this transcriptional activation ability resides in the NH2-terminal parts of both proteins. The use of yeast assays with RBTN2 shows that RBTN2 forms homodimers and that the NH2-terminal 27 amino acids are sufficient to facilitate transcriptional transactivation. These data expand the functional diversity of the LIM-domain protein family and they augment the previously defined relationship between chromosomal translocations and transcriptional activation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The amino-terminal phosphorylation sites of C-MYC are frequently mutated in Burkitt's lymphoma lines but not in mouse plasmacytomas and rat immunocytomas.

We sequenced the region encoding the amino-terminal phosphorylation sites of C-MYC in the Ig/MYC translocation-carrying Burkitt lymphomas (BL), mouse plasmacytomas (MPC) and rat immunocytomas (RIC). Mutations affecting the Thr-58 codon or the immediate flanking region were found in seven of the 10 in vitro propagated BL lines. No mutations were found in any of the eight BL biopsies analysed. Germ-line sequences were also found in six in vivo and five in vitro passaged MPCs and in four in vivo transplanted RICs. These findings indicate that mutations in this region do not represent a general phenomena in Ig/MYC translocation-carrying tumours, but may confer growth advantage on BL cells under continuous in vitro propagation.

Animals↗
PubMed1994

Three exceptional IgH/myc-translocation-carrying rat immunocytomas have breakpoints 50 to 80 kb 5' of c-myc.

The spontaneously arising immunocytoma of the Louvain rat (RIC) carries a consistent chromosomal translocation between chromosomes 6 and 7. This translocation juxtaposes immunoglobulin heavy chain and c-myc sequences. In an earlier study on 14 RIC tumors, we found that the translocation breakpoint is located within 1.5 kb immediately upstream of c-myc in 10 of the tumors. Here we describe 3 exceptional tumors that had no rearrangement within 20 kb 5' of c-myc. Using pulsed-field gel electrophoresis we show that the translocation breakpoints in these tumors are located 50-80 kb 5' of c-myc and that c-myc rearranges to the 3' end of the IgH cluster.

Animals↗
PubMed1994

Juxtaposition of N-myc and Ig kappa through a reciprocal t(6;12) translocation in a mouse plasmacytoma.

Nearly all mouse plasmacytomas (MPCs) carry an Ig/myc translocation. Any one of the three Ig loci may participate, while the myc contribution has been limited to c-myc, excluding other members of the myc gene family. The same is true for the other two known Ig/myc translocation-carrying tumors, Burkitt's lymphoma and rat immunocytoma. It is believed that the Ig/myc juxtaposition plays a decisive, rate limiting role in the genesis of the three tumors, acting through the constitutive activation of myc that makes it refractory to normal regulation. Here we describe the molecular analysis of a unique t(6;12)(CI;B) translocation that we previously identified in an exceptional MPC that expressed N-myc but not c-myc. We now show that the translocation led to the juxtaposition of N-myc and Ig kappa. This is the first case of an Ig/myc-carrying tumor that involves N-myc rather than c-myc. These findings suggest that the translocation may already have occurred at the pro- or pre-B cell stage at which N-myc is open for transcription. According to this interpretation, constitutive activation of N-myc would suppress the expression of c-myc, but would not interfere with the differentiation of the pro-B cell into a fully mature plasma cell. Its tumorigenic influence would become manifest only at the time when the cell would normally be programmed to leave the cycling compartment, in connection with its terminal differentiation.

Animals↗
PubMed1994

Hypersomy of chromosome 15 with retrovirally rearranged c-myc, loss of germline c-myc and IgK/c-myc juxtaposition in a macrophage-monocytic tumour line.

From a lymphoid tumour induced by 7,12-dimethylbenz-[a]-anthracene (DMBA) + methyl-N-nitrose-N-urea (MNU) in an [AKR Rb(6.15) x CBAT6T6]F1 mouse, a macrophage- monocyte line (KT-10) was isolated. Following ethyl methanesulfonate (EMS) treatment, a bromodeoxyuridine (BUdR) resistant subline was selected. Serial propagation of this line in vitro in the presence of BUdR (28 months) with periodic cytogenetic and molecular examinations, has led to the definition of four successive stages. During stage I, the cells were trisomic for chromosome 15. They contained Rb(6.15) and Rb(del6.15) of AKR and T(14;15) of CBA origin. Southern blotting showed the presence of both germline (G) and rearranged (R) c-myc. At stage II, Rb(del6.15) has duplicated. Both Rb(6.15) and T(14;15) persisted together with G-myc and R-myc. In stage III, the CBA-derived T(14;15) was lost, in parallel with G-myc. At this stage, a Dic.In(6.15) was detected. One of its arms was cytogenetically identical with the long arm of In(6.15) in the variant IgK/myc translocations. This chromosome carried R-myc and IgK in juxtaposition, as indicated by comigration on pulsed field electrophoresis (PFGE). At stage IV, the R-myc carrying AKR-derived chromosome 15s were present in six copies. Possible relationships between the increasing R/G myc ratio and changed growth characteristics in vivo and in vitro are discussed.

9,10-Dimethyl-1,2-benzanthracene↗