Alan J Mighell | PubMed Health

PubMed2006

Phenotypic changes associated with DYNACTIN-2 (DCTN2) over expression characterise SJSA-1 osteosarcoma cells.

DYNACTIN-2 (DCTN2) localises to chromosome 12q13-q15, a region prone to stable amplification in several cancers. Transient DCTN2 overexpression has a significant impact on cellular phenotype primarily due to disruption of the DYNEIN-dynactin motor. Changes reported include alterations of microtubule-directed movement of molecular (e.g. TP53) and organelle (e.g. Golgi) cargoes towards the nucleus, centrosome biology, cellular movement and mitosis with a potential predisposition to mitotic block and polyploidy. These changes would be expected to be of relevance to carcinogenesis. To investigate this, we report the first study of DCTN2 genomic amplification and sustained DCTN2 overexpression in cancer cells. QFMPCR was employed to characterise the extent of chromosome 12q13-q15 amplicons in SJSA-1, SJRH30, U373MG and CCF-STTG1 cancer cells. DCTN2 amplification was present in SJSA-1, U373MG and SJRH30 cells, yet was incomplete at the 5'-end in SJRH30 cells. Only SJSA-1 cells were characterised by DCTN2 overexpression on Western blot analyses. Microscopy studies distinguished SJSA-1 cells by greater DCTN2 immunofluorescence and diminished centrosome and 58K protein Golgi-marker focus compared to SJRH30 cells. Indirect evidence derived from the published work of others indicated that TP53 transport into the nucleus was unimpaired. Furthermore, we observed that SJSA-1 cells were easy to propagate. In conclusion, persistent DCTN2 overexpression can be tolerated in SJSA-1 cancer cells despite phenotypic abnormalities predicted from transient overexpression studies. This preliminary study does not support a major role for DCTN2 overexpression in carcinogenesis, although further studies would be necessary to confirm this.

Adenocarcinoma↗

PubMed2003

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

BACKGROUND: Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. RESULTS: We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. CONCLUSION: Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Benzothiazoles↗

PubMed2002

Identification of microcephalin, a protein implicated in determining the size of the human brain.

Primary microcephaly (MIM 251200) is an autosomal recessive neurodevelopmental condition in which there is a global reduction in cerebral cortex volume, to a size comparable with that of early hominids. We previously mapped the MCPH1 locus, for primary microcephaly, to chromosome 8p23, and here we report that a gene within this interval, encoding a BRCA1 C-terminal domain-containing protein, is mutated in MCPH1 families sharing an ancestral 8p23 haplotype. This gene, microcephalin, is expressed in the developing cerebral cortex of the fetal brain. Further study of this and related genes may provide important new insights into neocortical development and evolution.

Adolescent↗

PubMed2002

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development.

We report the initial characterization of mOb1 (Odd homeoBox 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.

Amino Acid Sequence↗

PubMed2002

Expression of mOb1, a novel atypical 73 amino acid K50-homeodomain protein, during mouse development.

We report the initial characterization of mOb1 (Odd homeoBox 1), which encodes an atypical 73 amino acid K50-homeodomain protein localised in the cytoplasm and absent from nuclei during mouse development. Conserved orthologues were present in man, rat, cow, pig and chicken, but not in fish, amphibians or invertebrates. Temporo-spatial patterns of mOb1 transcript and mOb1 protein expression were coincident in developing mouse embryos. Cardiac expression was first observed at E8.25 in linear heart tube myocardium and briefly in both horns of the sinus venosus. Myocardial expression peaked at E13.5, where after it diminished and was not detectable above background by adulthood. At no stage was expression observed in endocardium, endocardial cushion tissue, the coronary arteries or great vessels. At E13.5 and E15.5, mOb1 expression broadened to include skeletal muscle, stratified epithelium (upper aerodigestive tract and skin), epithelium of developing airways, vibrissae, midbrain/hindbrain junction, meninges, mesenchymal cellular condensations that preceded cartilage formation and chondrocytes.

Amino Acid Sequence↗