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J D Norton

Publications and source records attributed to J D Norton.

At least 55 records · Page 3Linked to original sources
PubMed1993

Multiple signaling pathways mediate anti-Ig and IL-4-induced early response gene expression in human tonsillar B cells.

We have analyzed the relationship between the signaling pathways coupled to surface immunoglobulin and interleukin (IL)-4 receptors in human B cells from the patterns of expression of a panel of phorbol ester-inducible early response genes (ERG) activated by anti-IgM and IL-4 stimulation in vitro. Anti-IgM stimulation led to the induction of all eleven ERG tested. Two of these, the proto-oncogene, c-fos and an anonymous ERG 1R20 were insensitive to protein kinase C (PKC) inhibition with the drug, staurosporine and retained inducibility after down-regulation of PKC activity by purging with phorbol ester. These observations are consistent with previous data showing anti-IgM signaling through both PKC-dependent and PKC-independent pathways. c-fos and 1R20 were also the only ERG inducible in response to IL-4 stimulation and whilst ionomycin induced only c-fos, dibutyryl cyclic adenosine monophosphate stimulation led to induction of both c-fos and 1R20. These observations lend support to a role for the adenylate cyclase pathway being important for coupling of IL-4-generated signals to B cells responses. None of the anti-IgM-responsive ERG was further induced when B cells were co-stimulated with a combination of anti-IgM and IL-4, suggesting that the signaling cascades from these two agents are integrated downstream of third messenger pathways to synergistically promote B cell proliferation.

Alkaloids↗
PubMed1993

Phorbol ester induction of early response gene expression in lymphocytic leukemia and normal human B-cells.

The spectrum of early response genes induced following phorbol ester (phorbol 12-myristate 13-acetate-PMA) stimulation of B-chronic lymphocytic leukemia cells was compared with that in normal tonsillar B-cells by using a panel of 20 gene probes. Of these, 2 (fos-B and Fra-1) were not expressed in either cell type; 11 displayed a comparable pattern and magnitude of induction in both the cell types, and one anonymous gene (5L3) that was inducible in leukemic cells was not detectably expressed in normal B-cells. Four further anonymous cDNAs (1R21, 10A, 1R19 and 3L11) detected transcripts that were constitutively expressed in normal B-cells with a concomitant reduction in inducibility compared with leukemic B-cells, whilst jun-B and jun-D, which were both inducible in normal B-cells, were constitutively expressed and only marginally PMA-inducible in leukemic B-cells. These data demonstrate clear differences in the PMA-activated early response gene regulatory pathways between normal and lymphocytic leukemia B-cells, which may reflect perturbations in signal transduction pathways that manifest in the differentiation arrest characteristic of these malignant B-cells.

B-Lymphocytes↗
PubMed1993

Immunoglobulin VH4 gene usage in B lymphoid leukaemias.

VH4 gene rearrangements occur in a similar proportion of cases of B lineage acute lymphoblastic leukaemia (ALL) and B chronic lymphocytic leukaemia (B CLL). However, there may be differences in the pattern of VH4 gene usage between these disorders as is the case for VH1 gene rearrangements. To examine this, we analysed the sequences of 24 PCR-amplified clonal VH4 gene rearrangements from a series of 15 cases of ALL and nine cases of CLL. Five distinct groups of genes were rearranged, three of which (represented by V2-1, V71-2/V71-4, V4.21) have been described in rearranged form in normal B lymphoid tissues. The most frequently rearranged gene was V4.21 which is strongly associated with autoimmune reactivity. V71-2, V71-4 and V2-1 were more frequently rearranged in CLL than ALL. The remaining two groups (represented by V4.33, V4.35) have not previously been described in rearranged form. One of these, V4.35, was seen only in ALL rearrangements. Both V4.35 and a VH1 gene, 20P3, which is also preferentially rearranged in ALL, are located at the 3' end of the VH locus. The location of these genes suggests that their rearrangement may be developmentally regulated in ALL. The findings in this study confirm restricted repertoires of IgH gene rearrangement in ALL and CLL. Characterization of IgH repertoires provides a means of correlating these transformed B cell populations with normal B cell developmental compartments. Moreover, the distinctive repertoires in ALL and CLL may reflect important differences in the ontogenic timing and microenvironmental milieu of tumourigenesis in these disorders.

Base Sequence↗
PubMed1993

A polymerase chain reaction-based method for the detection of trisomy 12 in B chronic lymphocytic leukaemia.

We report a polymerase chain reaction (PCR)-based method for detection of clonal trisomy 12 cell populations in B cell chronic lymphocytic leukaemia which is particularly applicable to retrospective analysis or archival material. The method utilizes three restriction enzyme site polymorphisms linked to the KRAS2 locus on chromosome 12 to monitor allele imbalance resulting from duplication of this chromosome. 21/24 unselected chronic lymphocytic leukaemia cases (88%) were informative for at least one polymorphism and of these, eight (33% of total cases) displayed allelic imbalance consistent with trisomy 12.

Base Sequence↗
PubMed1993

Early response gene signalling cascades activated by ionising radiation in primary human B cells.

We have used a panel of 13 protein kinase C-responsive immediate early gene probes to dissect the cellular signalling pathways activated by ionising gamma radiation in primary human B cells. Of these 13 genes, a delayed transient induction was observed for only 8: c-fos, c-jun, jun-B, jun-D, c-myc, ergI/krox 24 and two 'anonymous' genes, 3L3 and 19A. Expression of c-myc and c-fos mRNAs was paralleled by the appearance of their encoded proteins suggesting that these oncoproteins may couple radiation signalling to cellular responses. Of three protein kinase C-coupled transcription factors examined by gel retardation assay, (AP1, NF kappa B, EgrK/Krox24) only NF kappa B and, to a lesser extent, AP1 was stimulated in response to irradiation. These observations are not obviously compatible with a simple model invoking protein kinase C in radiation signalling in primary B cells and suggest that the pleiotropic effects of ionising radiation on this cell type are mediated through a distinct cellular signalling cascade.

B-Lymphocytes↗
PubMed1993

An immediate early human gene encodes an Id-like helix-loop-helix protein and is regulated by protein kinase C activation in diverse cell types.

Transcription factors characterized by the presence of a helix-loop-helix (HLH) domain play a central role in the regulation of cell growth/differentiation and tumorigenesis. We report here the cDNA sequence of a human early-response gene, designated HLH 1R21, encoding a 15-kDa HLH protein that lacks a basic, DNA-binding domain and which by a number of criteria appears to be the human homologue of mouse HLH 462. Like its murine counterpart, HLH 1R21 protein functions as an Id (inhibitor of DNA binding) transcription factor by inhibiting the binding of E2A-containing protein complexes to muscle creatine kinase E-box enhancer oligonucleotide in vitro. However HLH 1R21 does not inhibit the binding of HLH Max protein to a Max-binding oligonucleotide in vitro, indicating that it has limited promiscuity in its ability to antagonize the function of other HLH transcription factors. In addition, HLH 1R21 mRNA transcripts are regulated by phorbol ester treatment of a diverse range of human cell lines and, when overexpressed in mouse NIH3T3 cells, HLH 1R21 induces a morphologically transformed phenotype.

3T3 Cells↗
PubMed1992

Analysis of mutations in the Gs protein alpha subunit gene in human leukaemia.

Mutations at codons 201 and 227 that cause oncogenic activation of the gene encoding the Gs alpha subunit protein occur in a subset of human solid tumours. To determine whether such oncogenic mutations occur in leukaemia we have analysed DNA from leukaemic cells from 59 patients representing a spectrum of acute and chronic leukaemia types by direct nucleotide sequence analysis of polymerase chain reaction (PCR) amplified DNA. No mutations were detected at oncogenic hot spots represented by codons 201 or 227, nor in any other codons extending from nucleotide position 196-230 in the Gs alpha subunit gene. This strongly suggests that this region of the Gs alpha gene is highly nonpolymorphic and that oncogenic mutations in this gene are unlikely to play a significant role in the pathogenesis of leukaemia.

Amino Acid Sequence↗
PubMed1991

Evidence for protein kinase C--independent pathways mediating phorbol ester induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells.

The effects of phorbol esters on many cell types are known to be mediated through activation of the protein kinase C (PKC) signal transduction pathway. By using the specific inhibitor of this enzyme 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H7) we have assessed the role of PKC activation in phorbol ester (phorbol 12-myristate 13-acetate, PMA)-induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells (B-CLL) as a model of terminal differentiation of human B lymphocytes. H7 affected a dose-dependent inhibition of PMA-induced thymidine and uridine uptake with ID50 values of 41 microM and 32 microM, respectively. A comparable ID50 value (34 microM) was obtained for H7 inhibition of B-CLL PKC activity in a cell-free system. PMA-induced changes in cell morphology, expression of CD20, CD37 and FMC7 surface antigens together with increased secretion of immunoglobulin were variably abrogated by H7 suggesting that PKC activation is more important in B cell activation/DNA synthesis than in the differentiative response. Consistent with this, expression of a sizable proportion of PMA-inducible genes was not significantly affected by H7. These data are consistent with the existence of a PMA-activated, PKC-independent signal transduction pathway which may be important, though by itself apparently insufficient, for eliciting full terminal differentiation in B lymphocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Independent clonal origin of T- and B-cell clones in a composite lymphoma.

We present a detailed immunohistological and genotypic analysis of an unusual case in which a peripheral T-cell lymphoma, with features of Lennert's and angioimmunoblastic lymphoma, occurred after treatment of a low grade plasmacytoid lymphoma. By analysis of immunoglobulin and T-cell receptor genes, we show that the two diseases had an independent clonal origin at the level of lymphoid commitment. However, by employing a novel polymerase chain reaction-based technique for analysis of B-cell clonality, we show the persistence of a residual minor clonal B-cell population in the subsequent T-cell lymphoma. Only 2 previous cases of composite lymphoma involving B- and T-cell clones have been demonstrated by molecular analysis. This study underlines the immunophenotypic and genotypic heterogeneity of peripheral T-cell lymphomas and illustrates an unusual disease course in which a T-cell lymphoma has arisen in the context of, and perhaps as a consequence of, a B-cell lymphoma.

B-Lymphocytes↗
PubMed1991

Immunoglobulin gene 'fingerprinting': an approach to analysis of B lymphoid clonality in lymphoproliferative disorders.

Rearrangement of the immunoglobulin heavy chain (IgH) gene is widely exploited as a marker of B cell lineage and clonality in the pathology of lymphoproliferative disorders. We have developed a simple, polymerase chain reaction (PCR) based method for detecting IgH gene rearrangement which relies on the observation that by using a panel of PCR amplimers specific for each of the six heavy chain variable region families in conjunction with a common joining region amplimer, clonal rearrangement can be detected in over 90% of cases of B lymphoid malignancy. By using radiolabelled amplimers and exploiting the size heterogeneity resulting from independent IgH rearrangement events, we show that high resolution gel electrophoresis can be used to generate a 'fingerprint' representing the spectrum of B cell clonality in complex populations of B lymphocytes. The method effectively scans the entire IgH gene rearrangement repertoire and is capable of detecting rare clonal or oligoclonal B lymphoid cell populations. In normal bone marrow mononuclear cells, clonal IgH rearrangement could be readily detected at a sensitivity of 10(-3). We illustrate the application of the method in assessing the spectrum of B cell clonality occurring in an autoimmune condition. Hashimoto's thyroiditis, and in a malignant B cell disorder, chronic lymphocytic leukaemia. In addition, we explore the potential application of the technique in tracking minimal residual disease and for monitoring clonal evolution in acute lymphoblastic leukaemia.

B-Lymphocytes↗
PubMed1991

Preferential rearrangement of developmentally regulated immunoglobulin VH1 genes in human B-lineage leukaemias.

Immunoglobulin heavy chain (IgH) variable region (VH) genes are rearranged and expressed in a programmed manner during B-cell development. In common with foetal/pre-immune B-cells, malignant B-lymphoid populations preferentially use a restricted repertoire of developmentally regulated VH genes. By nucleotide sequence analysis of polymerase chain reaction amplified IgH genes, we have compared the repertoire of VH1 family genes that are rearranged in mature, CD5+ B chronic lymphocytic leukaemia (CLL) with that in immature, CD5-B-lineage acute lymphoblastic leukaemia (ALL). The results revealed a non-random pattern pf VH1 usage in which no single VH1 family member was common to each of these disease groups. The VH1 gene, 51P1, which underlies an auto-antibody associated cross-reactive idiotype, 'G6', frequently expressed on foetal B-cells, was preferentially rearranged in CLL (three of nine rearranged alleles). Another developmentally regulated VH1 gene, 20P3, accounted for more than half of the VH1 specific IgH gene rearrangements in ALL (five of nine VH1 alleles). Such developmentally restricted VH1 genes may distinguish discrete, although not necessarily exclusive, stages or compartments in B-lymphopoiesis from which each of these disease types arise.

B-Lymphocytes↗
PubMed1991

An improved method for detection of B-lymphoid clonality by polymerase chain reaction.

Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and joining (JH) region-specific primers. The simplest methods utilize a single VH primer specific for sequences present in most VH regions corresponding to the third framework region (FR3). An alternative approach is to use a panel of VH family-specific primers specific for the first framework regions (FR1). In the course of nucleotide sequence analysis of IgH gene rearrangements amplified using a VH FR1 primer panel, these authors previously observed 3' VH region deletion and/or base mis-matches sufficient to prevent efficient priming from the VH FR3 primer target sequence in a significant minority of cases of B-lineage malignancy. An improved PCR method has therefore been developed by using a panel of seven VH FR1 family-specific primers incorporated in a single reaction. By using this method clonal IgH gene rearrangement is detected in 15 of 16 cases of B-lineage malignancy. Significantly, this series included four cases of B-lymphoma in which previous attempts to detect PCR clonal IgH gene rearrangements using a VH FR3 primer were unsuccessful. In two of these cases, nucleotide sequence analysis of the amplified DNA showed that failure to prime with the VH FR3 primer was likely to be attributable to insufficient homology with the target sequence. The use of the approach described in this paper should significantly improve the reliability of detection of B-lymphoid clonality by PCR.

Base Sequence↗
PubMed1991

Immunoglobulin heavy chain gene fingerprinting reveals widespread oligoclonality in B-lineage acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) of B-cell lineage typically arises as a monoclonal expansion of committed B-lymphocyte precursors that are arrested at an immature stage of differentiation. From Southern hybridization analysis of immunoglobulin heavy chain (IgH) genes in leukaemic blasts, the occurrence of a sizeable minority of patients displaying multiple (greater than two) rearranged heavy chain alleles has been widely reported. In at least some patients these data are consistent with the presence of oligoclonal populations of precursor B-cells. We have used a more sensitive, polymerase chain reaction based immunoglobulin gene 'fingerprinting' approach in the analysis of B-cell clonality in eight patients with common ALL which were apparently monoclonal on the basis of Southern blot analysis of their IgH genes. The results revealed an oligoclonal pattern of IgH gene rearrangement in half of the patients analysed, implying that oligoclonality at the level of B-cell commitment, as defined by IgH gene rearrangement, is much more widespread in this disease than has previously been recognized.

Alleles↗
PubMed1990

Cell-type-specific early response gene expression during plasmacytoid differentiation of human B lymphocytic leukemia cells.

Monoclonal B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) can be induced to undergo plasmacytoid differentiation in vitro by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). By differential screening of a cDNA library derived from cells treated with phorbol ester we have isolated and characterised a set of early response genes (ERGs) displaying rapid transient up-regulation of expression in response to PMA. Cross-hybridisation studies showed that PMA probably induces the expression of over one hundred distinct genes, implying an ERG complexity comparable to that activated by mitogenic stimulation of fibroblasts and normal T lymphocytes. Of 13 genes analysed in detail, most were induced by PMA without a requirement for de novo protein synthesis, whilst nuclear run-on analysis showed that at least some of the more abundant classes of ERG were up-regulated through transcriptional mechanisms. In a proliferating variant B-CLL population, few differences in ERG expression were seen, suggesting that these genes are part of a gene regulatory pathway coupled to the differentiative rather than the proliferative response of B-CLL cells. However, studies in a range of cell types revealed a surprisingly diverse pattern of PMA-induced expression where most ERGs were relatively B-CLL-specific. This implies an extreme diversity of gene regulatory pathways activated in the primary response by phorbol ester generally and suggests that the onset of PMA-induced plasmacytoid differentiation of B-CLL cells is preceded by activation of a complex gene regulatory program that is largely unique to this maturation-arrested B cell.

B-Lymphocytes↗
PubMed1990

Coupling of early response gene expression to distinct regulatory pathways during alpha-interferon and phorbol ester-induced plasmacytoid differentiation of B chronic lymphocytic leukaemia cells.

Phorbol esters (phorbol 12-myristate 13-acetate; PMA) and alpha-interferon (alpha-IFN) act through divergent signal transduction pathways to induce B chronic lymphocytic leukaemia cells (B-CLL) to undergo plasmacytoid differentiation in vitro. By using a panel of PMA-inducible early response gene probes we show that these two different effectors are coupled to second messenger pathways that do not converge on a common gene regulatory programme in differentiation of B-CLL cells. Moreover, using the calcium ionophore, A23187, four distinct regulatory classes of early response gene could be defined implying that multiple regulatory pathways may mediate the process of terminal differentiation in B lymphocytes.

B-Lymphocytes↗
PubMed1990

Independent regulation of mouse VL30 retrotransposon expression in response to serum and oncogenic cell transformation.

The nucleotide sequence of the long terminal repeats (LTRs) of retrovirus-transmissible mouse VL30 cDNA clones, NVL-1 and NVL-2 were determined and compared with that of the prototype NVL-3. Both shared the typical U3 R U5 structure together with unusual features of redundancy in the tRNAgly primer binding site and adjacent inverted repeat. NVL-1 and NVL-2 LTRs were almost identical and differed from the NVL-3 LTR in the U3 domain harbouring transcriptional regulatory determinants. S1 nuclease analysis of cellular and virus-encapsidated RNA suggested that NVL-1/2 and NVL-3 elements retrotranspose with comparable efficiency but that in contrast to transformation-regulated VL30 expression which affects all types of NVL element, only NVL-1/2 elements were found to be serum responsive. Both modes of VL30 regulation were found to be coupled through protein kinase C-independent pathways. Expression of N-ras transactivated U3 enhancer determinants in all classes of LTR. However the same region of NVL-1/2 LTR did not confer serum responsiveness implying that cis regulatory determinants of VL30 elements mediating growth factor responsiveness are at least in part dissociable from those responsible for cell transformation-regulated expression.

Base Sequence↗