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Biomedical subjects

M Hubank

Publications and source records attributed to M Hubank.

10 recordsLinked to original sources
PubMed2001

Analysis of gene transcription in cells lacking DNA-PK activity.

The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs(-/-) and DNA-PKcs(+/+) mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

3T3 Cells↗
PubMed1999

DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage.

Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.

Animals↗
PubMed1998

Identification of androgen-regulated genes in mouse kidney by representational difference analysis and random arbitrarily primed polymerase chain reaction.

The molecular nature of tissue-specific gene regulation by androgens has not been well defined, partly as a result of the variable expression and incomplete regulation of currently available gene models. We have therefore aimed to establish more informative models by identifying alternative genes whose expression is tightly and coordinately regulated by androgens. Female C57BL/6 mice were dosed with dihydrotestosterone- or sham-treated for 8 days, after which kidneys were removed and complementary DNA (cDNA) prepared. We then applied the subtractive hybridization techniques of random arbitrarily primed-PCR and PCR-coupled subtractive hybridization method of cDNA representational difference analysis to the isolated cDNA. In addition to well characterized androgen-regulated genes [e.g. KAP (kidney androgen-regulated protein)], we demonstrate the differential expression of six genes previously not known to be under androgen control. RNA levels of SA, Cytochrome P450 4B1, IL-6ST (interleukin-6 signal transducer), OATP (organic anion transporter), and a newly identified gene, MJAM, were up-regulated by androgen, while 16-alpha-hydroxylase was decreased. Expression of these transcripts was inhibited in dihydrotestosterone-treated females by flutamide and in males by castration, confirming their dependence on androgens. Although all the genes demonstrate tissue-specific regulation by androgen, SA showed both kidney specificity and absolute requirement for androgen for its expression. These newly identified androgen-regulated genes will constitute very useful models for studying the nature of tissue-specific gene regulation by androgens.

Androgen Antagonists↗
PubMed1994

Identifying differences in mRNA expression by representational difference analysis of cDNA.

Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, the PCR-coupled subtractive process of representational difference analysis (RDA) [Lisitsyn, N. et al. (1993) Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time taken by conventional means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes. Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a predicted homologue of the natural killer cell antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives. cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses, and examination of pathogen-host interactions.

Animals↗
PubMed1994

Expression of the excision repair gene, ERCC3 (excision repair cross-complementing), during mouse development.

Expression of the human ERCC3 (excision repair cross-complementing) gene in cells from patients with xeroderma pigmentosum (XP) group B (XP-B) corrects the defect in repair of UV light-induced DNA damage. XP-B is one of three groups of XP which exhibit the clinical symptoms of both XP and Cockayne's Syndrome (CS). CS and XP-B/CS patients develop severe neurological dysfunction during development. In order to explore the link between the defective gene and the neurological deficits in XP/CS, we have studied the expression of ERCC3 mRNA in developing mice by in situ hybridisation. ERCC3 was found to be ubiquitously expressed in cells from all regions and all developmental stages, from 9 day post-coitum embryo, to 15 day post-natal brain. In post-natal brain, regional differences in expression correlated with cell density and there was no evidence of cell specific or developmental alterations in levels of expression. These results indicate that the constitutively expressed gene does not perform a discrete developmental function. The neurological defects apparent in XP-B are likely to arise pleiotypically from the participation of ERCC3 in interactions with other elements involved in particular aspects of neurodevelopmental control. These results emphasise the developmental importance of genes whose primary functions are apparently unconnected with development.

Animals↗
PubMed1990

Nuclear tri-iodothyronine (T3) binding in neonatal rat brain suggests a direct glial requirement for T3 during development.

Tri-iodothyronine (T3) binding studies were performed on neuronal and glial nuclei prepared from developing rats brain by discontinuous sucrose gradient centrifugation. Maximum binding capacities (MBC) and dissociation constants (Kd) were obtained from Eadie-Hofstee plots of transformed data. An ontogenic study on nuclei prepared from whole brain revealed that on day 5 after birth, glial nuclear MBC was 1774 +/- 201 (S.E.M.) fmol/mg DNA compared with 974 +/- 117 fmol/mg DNA for the neurones (P less than 0.01). Although diminishing to 667 +/- 112 fmol/mg DNA by day 21, alterations in neuronal MBC over the neonatal period were not statistically significant, whereas glial MBC diminished steadily to 557 +/- 133 fmol/mg DNA in glial nuclei (P less than 0.05). Over the same period, a significant reduction in Kd was noted only in the glia, from 3.17 +/- 0.40 to 1.83 +/- 0.34 nmol/l (P less than 0.03). Ligand specificity of the receptor in both nuclear types on day 21 was tri-iodoacetic acid greater than T3 greater than thyroxine greater than 3,3',5'-T3, but this was less clearly demonstrated at day 5. Regional studies on days 15 and 21 demonstrated that for both neuronal and glial nuclei, receptors are concentrated in the cerebral cortex and diminish in a cranio-caudal direction. Cerebral glial MBC on day 21 was 2215 +/- 147 fmol/mg DNA, at this stage still exceeding the cerebral neuronal capacity of 1111 +/- 207 fmol/mg DNA. The results indicate that neonatal glia may respond directly to thyroid hormones via nuclear receptor binding, and that receptors are predominantly located in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effect of maternal hypothyroxinaemia during fetal life on the calmodulin-regulated phosphatase activity in the brain of the adult progeny in the rat.

Calmodulin-regulated phosphatase activity was measured in the brain of 2-month-old rats born from hypothyroid and normal dams, using a fluorometric enzyme assay developed for this purpose. Calmodulin content was measured in the same brain regions by radioimmunoassay. Significant differences between groups in weight and protein content, basal phosphatase and calmodulin-regulated phosphatase activity were found. The brain region most affected was the cerebellum, where basal and calmodulin-regulated phosphatase activities, and protein content were increased. The data point towards a lasting effect of maternal hypothyroxinaemia on the brain function of the progeny.

Animals↗
PubMed1987

Differences in nuclear triiodothyronine binding in rat brain cells suggest phylogenetic specialization of neuronal functions.

The central nervous system depends on thyroid hormones (TH) in regard to its development, maturation, and maintenance of normal functions. As there is much evidence to suggest that the effects of TH are mainly mediated through specific nuclear binding sites, we have studied the anatomical distribution of T3 nuclear receptors in different regions of adult rat brain, and the localization of receptors in the fractionated neuronal and glial nuclei of neocortex, paleocortex, and cerebellum. Purified nuclei from the various brain regions were prepared by ultracentrifugation in 2.2 M sucrose. Purified neuronal and glial fractions were obtained by discontinuous sucrose gradient centrifugation in 2.2 and 2.4 M sucrose. The washed nuclear fractions were used for T3 binding assay at 37 C for 30 min and the data analyzed by least squares nonlinear regression analysis. Nonfractionated nuclei from all regions studied were found to have similar dissociation constant (Kd) values (1.04-1.38 nM) and Eadie-Hofstee plots indicated the presence of an apparently ubiquitous single class of high affinity, low capacity binding sites. The increase in binding from cerebellum (54 +/- 24 fmol/mg DNA; mean +/- SE) to neocortex (666 +/- 89 fmol/mg DNA) showed a caudo-cranial pattern. In fractionated neuronal nuclei, the same trend was observed, only to a greater degree (1628 +/- 266, 994 +/- 76 and 212 +/- 29 fmol/mg DNA in neocortex, paleocortex, and cerebellum, respectively); the difference between corresponding values for glial nuclei of neocortex and paleocortex (357 +/- 139 and 250 +/- 92 fmol/mg DNA, respectively) was not statistically significant, and no specific T3 binding was found in cerebellar glial nuclei. These data suggest that TH may have an important role in neurons from phylogenetically newer regions, concerned with higher mental functions. The caudo-rostral distribution pattern may also indicate a gradient of TH actions in central nervous system regions.

Animals↗
PubMed1987

The effect of 3,5,3'-triiodothyronine on leucine uptake and incorporation into protein in cultured neurons and subcellular fractions of rat central nervous system.

Previous studies have shown that nuclear thyroid hormone receptors in rat brain are preferentially localized within neurons. These cells also synthesize protein at a high rate, and the aim of the present study was to investigate any relationship between these two characteristics. In this paper we have shown that T3 stimulates leucine uptake and incorporation into protein in primary cell cultures of neurons. Stimulation was apparent with concentrations of hormone as low as 1.25 nM and increased in a dose-dependent manner up to 10 nM T3. However, the rapidity of the effect (evident at 25 min, and significant at 40 min) suggests that protein synthesis is stimulated at the level of translation, rather than transcription. More detailed study with 5 nM T3, revealed that incorporation into both soluble (cytoplasmic) and insoluble (membrane-associated) protein fractions was stimulated to similar degrees, and therefore the effect on protein synthesis was general. Furthermore, T3-mediated stimulation of leucine uptake into neurons was completely abolished in the presence of the protein synthesis inhibitors, actinomycin D and cycloheximide, and therefore the effect on leucine uptake was attributed to an increased requirement for the amino acid in protein synthesis (pleiotrophic effect). Parallel studies conducted with synaptosomes and mitochondria isolated from the central nervous system of adult euthyroid animals revealed that 5 nM T3 was without effect on leucine uptake and incorporation into protein. Possible reasons for this lack of effect are discussed.

Animals↗