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T Chaplin

Publications and source records attributed to T Chaplin.

At least 19 recordsLinked to original sources
PubMed2003

Histone acetylation-mediated regulation of genes in leukaemic cells.

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader-Willi syndrome.

Acetylation↗
PubMed2001

Identification and molecular characterisation of a CALM-AF10 fusion in acute megakaryoblastic leukaemia.

The t(10;11)(p13;q14-21) is a non-random translocation described in acute lymphoblastic and myeloid leukaemias. It results in the fusion of the gene CALM, which encodes a clathrin assembly protein, on 11q14 to the gene AF10, a putative transcription factor on 10p13. Here we describe for the first time, the occurrence of a CALM-AF10 fusion in a case of acute megakaryoblastic leukaemia. Fluorescence in situ hybridisation and reverse transcriptase polymerase chain reaction were used to confirm the presence of a CALM-AF10 fusion. A novel splice variant of CALM missing nt 1927-2091 was also detected. Though CALM is a cytoplasmic protein, the chimaeric fusion product is able to localise to both the nucleus and cytoplasm. Analysis of the fusion variants suggests, however, that the critical fusion product is likely to be cytoplasmic and contain the interactive leucine zipper of AF10.

Active Transport, Cell Nucleus↗
PubMed2001

The characterisation of the lymphoma cell line U937, using comparative genomic hybridisation and multi-plex FISH.

The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed.

Chromosome Aberrations↗
PubMed2001

Molecular analysis of the genomic inversion and insertion of AF10 into MLL suggests a single-step event.

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions.

Adult↗
PubMed1999

The cloning, mapping and expression of a novel gene, BRL, related to the AF10 leukaemia gene.

The MLL gene is reciprocally translocated with one of a number of different partner genes in a proportion of human acute leukaemias. The precise mechanism of oncogenic transformation is unclear since most of the partner genes encode unrelated proteins. However, two partner genes, AF10 and AF17 are related through the presence of a cysteine rich region and a leucine zipper. The identification of other proteins with these structures will aid our understanding of their role in normal and leukaemic cells. We report the cloning of a novel human gene (BRL) which encodes a protein containing a cysteine rich region related to that of AF10 and AF17 and is overall most closely related to the previously known protein BR140. BRL maps to chromosome 22q13 and shows high levels of expression in testis and several cell lines. The deduced protein sequence also contains a bromodomain, four potential LXXLL motifs and four predicted nuclear localization signals. A monoclonal antibody raised to a BRL peptide sequence confirmed its widespread expression as a 120 Kd protein and demonstrated localization to the nucleus within spermatocytes.

Amino Acid Motifs↗
PubMed1999

Therapy-related adult acute lymphoblastic leukemia with t(4;11)(q21; q23): MLL rearrangement, p53 mutation and multilineage involvement.

A diagnosis of pro-B acute lymphoblastic leukemia (ALL) with CD15+ was made in a 42-year-old woman, 12 months after the treatment of uterine adenocarcinoma by carboplatinum, anthracyclines, etoposide and radiotherapy. Molecular cytogenetic studies revealed a karyotype with multiple chromosome changes, including the t(4;11)(q21;q23) and a 17p-chromosome, with MLL disruption and 17p13/p53 gene deletion in 86% of the cells. A p53 exon 6 mutation was documented, resulting in p53 protein stabilization, with 20% of the cells reacting with the 1801 anti-p53 monoclonal antibody. Dual-color FISH using MLL and p53 probes was performed on peripheral blood smears, providing direct evidence of the involvement of the blast cells and of the granulocytic lineage. Only a partial, shortlasting response was obtained by induction treatment, confirming that a poor prognosis is associated with therapy-related ALL with the 4;11 translocation.

Adult↗
PubMed1998

Expression pattern and cellular distribution of the murine homologue of AF10.

We have cloned Af10, the murine homologue of the MLL partner gene AF10. The predicted open reading frame of Af10 contains 1069 aa which are 90% identical to those of AF10. Af10 contains an N-terminal cysteine-rich region with a LAP/PHD finger, a leucine zipper domain and a glutamine-rich region at the C-terminus, features also found in the human proteins AF10 and AF17. A single 5. 5-kb transcript was detected in murine tissues with the highest level of expression in the testes. A polyclonal antibody raised to the cysteine-rich region of AF10 was able to identify a double band of 140 kDa on Western analysis in mouse testicular extracts. After subcellular separation Af10 was identified in both the nuclear and cytoplasmic extracts, again as a double band of 140 kDa in size. In situ hybridisation studies were performed with sense and antisense digoxigenin-labelled oligonucleotides. High levels of expression were noted in postmeiotic germ cells, especially in spermatids from around stage VI to stage VIII. High levels of expression were also seen in the white matter of the cerebellum, extending into the granular layer. The expression in differentiated rather than in proliferating cells suggests that the role of Af10 may lie in the suppression of proliferation rather than in differentiation. Since the LAP/PHD finger domains are lost in the MLL-AF10 fusion, arguably such a function could be carried out by this domain.

Amino Acid Sequence↗
PubMed1995

The leukemia-associated-protein (LAP) domain, a cysteine-rich motif, is present in a wide range of proteins, including MLL, AF10, and MLLT6 proteins.

We have identified and further characterized a Caenorhabditis elegans gene, CEZF, that encodes a protein with substantial homology to the zinc finger and leucine zipper motifs of the human gene products AF10, MLLT6, and BR140. The first part of the zinc finger region of CEZF has strong similarity to the corresponding regions of AF10 (66%) and MLLT6 (64%) at the cDNA level. As this region is structurally different from previously described zinc finger motifs, sequence homology searches were done. Twenty-five other proteins with a similar motif were identified. Because the functional domain of this motif is potentially disrupted in leukemia-associated chromosomal translocations, we propose the name of leukemia-associated protein (LAP) finger. On the basis of these comparisons, the LAP domain consensus sequence is Cys1-Xaa1-2-Cys2-Xaa9-21-Cys3-Xaa2-4 -Cys4-Xaa4-5-His5-Xaa2-Cys6-Xaa12-46 - Cys7-Xaa2-Cys8, where subscripted numbers represent the number of amino acid residues. We review the evidence that this motif binds zinc, is the important DNA-binding domain in this group of regulatory proteins, and may be involved in leukemogenesis.

Amino Acid Sequence↗
PubMed1995

The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene.

The gene on chromosome 10 at band p12 (AF10), involved in the t(10;11) translocation in acute myeloid leukemia, has been identified and shown to contain conserved zinc finger and leucine zipper domains. These regions are highly homologous to the equivalent regions on AF17, the gene involved in the t(11;17) translocations. A series of adult, childhood, and infant leukemias with either simple or complex versions of the t(10;11) has been examined by Southern analysis and shown to involve rearrangement to the HRX locus. Reverse transcriptase-polymerase chain reaction from either bone marrow or peripheral blood cells showed that HRX sequence was fused to AF10 sequence in all 8 cases and subsequent sequence analysis showed an in-frame fusion between the HRX and AF10 sequence. A consistent feature of these fusions was the juxtaposition of the leucine dimerization motif of AF10 onto the NH2-terminal region of HRX. The published data suggest that a similar conclusion can be drawn about the t(11;17) translocation, implying a critical role for this motif in the chimaeric HRX protein.

Adult↗
PubMed1995

A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia.

A novel class of conserved transcription factors has been identified from the molecular cloning of AF10, the gene involved in the t(10;11)(p12;q23) translocation of acute myeloid leukemias. AF10 encodes a 109-kD protein of 1,027 amino acids and contains an N-terminal zinc finger region and a C-terminal leucine zipper. These structures have been found to be conserved in sequence and position in three other proteins, AF17, BR140, and a previously unrecognized Caenorhabditis elegans gene, provisionally named CEZF. The overall structure, level of sequence conservation, and expression pattern suggest that these genes encode a new class of transcription factors, some of which are targets for chromosomal translocation in acute leukemia.

Adaptor Proteins, Signal Transducing↗
PubMed1995

AF6 gene on chromosome band 6q27 maps distal to the minimal region of deletion in epithelial ovarian cancer.

Chromosome 6 has been shown to contain at band q27 a minimal region of deletion associated with epithelial ovarian cancers and AF6, a gene disrupted in acute myeloid leukemia with t(6;11)(q27;q23). Using rapid amplification of cDNA ends by polymerase chain reaction, the breakpoint in AF6 was confirmed and a cDNA clone identified. This clone was used as a probe to screen a chromosome 6 cosmid library, and a single cosmid C-109F0645 was isolated. By fluorescence in situ hybridization, C-109F0465 was found to map distal to the critically deleted region associated with ovarian malignancies. AF6 is therefore distinct from and lies telomeric to this region.

Acute Disease↗
PubMed1993

In situ hybridisation analysis of a homogeneously staining region at 11q23-24 in an acute myeloid leukaemia (M5) using yeast artificial chromosomes.

An example of a homogeneously staining region (hsr), occurring in an acute myeloid leukaemia (M5) on chromosome 11 in the region of bands q23-q24, has been analysed. In situ hybridisation using yeast artificial chromosome (YAC) DNA demonstrated that the amplification did not include the CD3 gene cluster and did not affect the human trithorax gene known to be disrupted by translocations at 11q23. In contrast, the amplification was shown to include the sequence D11S543 which has been previously mapped to chromosome band 11q24. High resolution analysis using confocal microscopy allowed the individual amplicons to be visualised, and it was shown that the hsr consisted of an 8-fold amplification of the region surrounding the probe D11S543. From previous estimates of human chromosome size it was possible to calculate that the hsr was composed of amplicons approximately 10 megabases in length. It was concluded that the region amplified did not extend as far as the translocation breakpoints occurring at 11q23 in acute leukaemias.

Aged↗
PubMed1993

Detection and significance of bcr-abl mRNA transcripts and fusion proteins in Philadelphia-positive adult acute lymphoblastic leukemia.

Blast cells from an unselected consecutive series of 84 adults presenting with acute lymphoblastic leukemia (ALL) to St Bartholomew's Hospital over a seven year period were tested prospectively by cytogenetic and retrospectively by RT-PCR analysis for the presence of the Ph translocation and bcr-abl mRNA. This combination gave an overall figure of 20.3% for bcr-abl-positive and/or Ph-positive ALL. The incidence of bcr-abl-positive/Ph-positive ALL was most common between the ages of 31 and 50 years, becoming less common after the age of 50. Eight out of ten bcr-abl-positive patients expressed the e1a2 mRNA transcript, the other two expressed the b3a2 and b2a3 transcripts respectively. Cells from all patients with bcr-abl mRNA transcripts expressed the appropriate p190 or p210 bcr-abl protein and all were Ph-positive.

Adolescent↗
PubMed1992

Chromosome 11q23 translocations in both infant and adult acute leukemias are detected by in situ hybridization with a yeast artificial chromosome.

The yeast artificial chromosome (YAC-13HH4), which spans a 440-kb region of DNA just distal to the CD3 locus on chromosome 11 at band q23, has been used to characterize a range of chromosomal translocations in acute leukemias from both adults and infants. In situ hybridization was performed on metaphase cells from bone marrow of 17 leukemias and two cell lines with a variety of chromosome 11q23 abnormalities. It was established that in infant leukemias the translocations t(11;19), t(4;11), and t(5;11) had occurred in the region defined by YAC 13HH4. Additionally, the translocations t(4;11), t(6;11), t(9;11), t(X;11), and t(10;11) in other leukemias were found to disrupt the same region of chromosome 11q23, although an exception was found in one t(6;11) translocation for which the breakpoint was distal to the YAC. One patient had a t(9;11) translocation in a therapy-related leukemia, suggesting that this class of etoposide-related malignancy has similar breakpoints to those occurring in de novo leukemias. An example of a lymphoma-derived translocation t(4;11) was shown to involve a deletion of the region defined by YAC 13HH4. A leukemia with a deletion on chromosome 11 (q23-q25) was also studied and it was shown that the YAC sequence was unaffected. It was concluded that, with a few exceptions, the translocations at 11q23 in a wide range of acute infant and adult leukemias occur in a common region and may result from a common underlying mechanism.

Acute Disease↗
PubMed1992

Molecular cloning and analysis of chromosome band 11q23 involved in leukaemia-associated translocations.

Three overlapping yeast artificial chromosomes (YACs) spanning a 780 kb region of DNA around the CD3 locus on chromosome 11 have been isolated and characterised. The individual cloned regions have been mapped by in situ hybridisation to chromosome band 11q23, and a restriction enzyme map of this region has been constructed. The positions of these clones in relation to a series of leukaemia-associated chromosomal translocations has also been determined. It was concluded that, although two clones lay entirely proximal to the breakpoints examined, the third clone (13HH4) encompassed the breakpoints for the translocations t(4;11), t(6;11), and t(9;11). The t(9;11) was observed in an acute myeloid leukaemia in a patient previously treated for an unrelated malignancy. It would thus appear that the breakpoints at chromosome band 11q23 occurring in therapy-related leukaemias are in the same region as those found in adult and childhood acute leukaemias and may result from a common underlying mechanism.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Establishment of a lymphoblastoid cell line, SD-1, expressing the p190 bcr-abl chimaeric protein.

A lymphoblastoid cell line (SD-1) has been established by Epstein-Barr virus immortalisation of Philadelphia chromosome positive acute lymphoblastic leukaemia (ALL) cells. Using newly derived anti-bcr monoclonal and anti-abl polyclonal antibodies it was demonstrated that both the original leukaemic cells and the derived cell line expressed the p190 form of the bcr-abl protein found in a proportion of cases of Philadelphia chromosome positive ALL. Interestingly, the leukaemia and the derived cell line each displayed different, clonal patterns of immunoglobulin gene rearrangements providing direct evidence that the t(9;22) translocation which results in the expression of the p190 bcr-abl protein must occur before immunoglobulin heavy chain gene rearrangement. In contrast to the leukaemia, which had multiple chromosome abnormalities in addition to the t(9;22), the cell line had the t(9;22) translocation as its sole abnormality. Although SD-1 cells were demonstrated to express continuously the p190 bcr-abl protein, they were unable to form colonies in soft agar and did not cause tumours in splenectomised nude mice. This cell line therefore represents an appropriate target cell line in which to examine the cooperativity of the p190 bcr-abl protein with other activated oncogene products.

Animals↗
PubMed1991

Normal c-abl gene protein--a nuclear component.

The subcellular distribution of the c-abl and bcr-abl gene products from KG1A and K562 cells has been studied by two different techniques. Firstly, physical disruption followed by subcellular fractionation was used to demonstrate that normal c-abl (p145) was recovered from the cytosol and the nuclear fractions of KG1A cells. In contrast, bcr-abl products were recovered exclusively from the cytosol fraction of K562 cells. Secondly, indirect immunofluorescence was used to localize c-abl protein to the cytoplasm, nuclear membrane and infrequently to the nucleus of KG1A cells and bcr-abl protein to only the cytoplasm of K562 cells. Thus both the approaches indicate that there is a component of normal c-abl products which appears to be nuclear and this is not reflected in the distribution of the bcr-abl 210 kDa protein, which remains cytosolic.

Animals↗