Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms

Watters, Dianne Josephine; Khanna, Kum Kum; Beamish, Heather; Birrell, Geoffrey W.; Spring, Kevin; Kedar, Padmini S.; Gatei, Magtouf; Stenzel, Deborah; Hobson, Karen; Kozlov, Sergei; Zhang, Ning; Farrell, Áine; Ramsay, Jonathan; Gatti, Richard A.; Lavin, Martin F.

doi:10.1038/sj.onc.1201037

Cited by 173 publications

(141 citation statements)

References 38 publications

(61 reference statements)

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“…Immunostaining using anti-ATM and anti-FLAG antibodies localized the recombinant ATM predominantly in the nucleus, and a minor fraction remaining in the cytoplasm. This localization is similar to that of endogenous ATM in various cell types, as recently reported by several laboratories (Brown et al, 1997;Chen and Lee, 1996;Lakin et al, 1996;Watters et al, 1997). The cytoplasmic fraction of ATM appears to be microsomal (Brown et al, 1997;Watters et al, 1997), and immunoelectronmicroscopy recently performed by Watters et al (1997) showed association of the protein with certain vesicular structures.…”

Section: Discussionsupporting

confidence: 85%

“…Endogenous ATM is predominantly nuclear (Brown et al, 1997;Chen and Lee, 1996;Lakin et al, 1996;Watters et al, 1997). Immunostaining using anti-ATM and anti-FLAG antibodies was used to localize the endogenous protein in immortalized normal fibroblasts, and the recombinant protein produced in AT22IJE-T cells.…”

Section: Localization Of Recombinant Atm By Immunostainingmentioning

confidence: 99%

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Recombinant ATM protein complements the cellular A-T phenotype

et al. 1997

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Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunode®ciency, cancer predisposition, genome instability and radiation sensitivity. The cellular phenotype of A-T points to defects in signal transduction pathways involved in activation of cell cycle checkpoints by free radical damage, and other pathways that mediate the transmission of speci®c mitogenic stimuli. The product of the responsible gene, ATM, belongs to a family of large proteins that contribute to maintaining genome stability and cell cycle progression in various organisms. A recombinant vector that stably expresses a full-length ATM protein is a valuable tool for its functional analysis. We constructed and cloned a recombinant, full-length open reading frame of ATM using a combination of vectors and hosts that overcame an inherent instability of this sequence. Recombinant ATM was stably expressed in insect cells using a baculovirus vector, albeit at a low level, and in human A-T cells using an episomal expression vector. An amino-terminal FLAG epitope added to the protein allowed highly speci®c detection of the recombinant molecule by immunoblotting, immunoprecipitation and immunostaining, and its isolation using immunoanity. Similar to endogenous ATM, the recombinant protein is located mainly in the nucleus, with low levels in the cytoplasm. Ectopic expression of ATM in A-T cells restored normal sensitivity to ionizing radiation and the radiomimetic drug neocarzinostatin, and a normal pattern of post-irradiation DNA synthesis, which represents an Sphase checkpoint. These observations indicate that the recombinant, epitope-tagged protein is functional. Introduction into this molecule of a known A-T missense mutation, Glu2904Gly, resulted in apparent instability of the protein and inability to complement the A-T phenotype. These ®ndings indicate that the physiological defects characteristic of A-T cells result from the absence of the ATM protein, and that this de®ciency can be corrected by ectopic expression of this protein.

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Section: Discussionsupporting

confidence: 85%

Section: Localization Of Recombinant Atm By Immunostainingmentioning

confidence: 99%

Recombinant ATM protein complements the cellular A-T phenotype

et al. 1997

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“…This is consistent with the observation of endogenous ATM protein in normal fibroblast cells detected with immunofluorescent histochemistry. 7 A prior report emphasized that the ATM was distributed within the cytoplasm of the Purkinje cells, but not within the nuclei. 49 This discrepancy may stem from the differences within species or from different techniques used to stain the cells.…”

Section: Discussionmentioning

confidence: 99%

“…Sequence analyses have revealed that the ATM gene product is a polypeptide around 350 kDa that localizes to the nucleus and within vesicular structures in the cytoplasm. [5][6][7][8] The exact role of the ATM protein is not yet known. However, based on the pleiotropic phenotype of AT and its close relationship to a family of phosphatidylinositol (PI) 3-kinases, it is linked to signal transduction, cell cycle control, DNA repair-related mechanisms, and resistance to oxidative stress.…”

Section: Introductionmentioning

confidence: 99%

Functional expression of ATM gene carried by HSV amplicon vector in vitro and in vivo

Shackelford

Manuszak

et al. 2003

Gene Ther

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Ataxia-telangiectasia (AT) is a human autosomal recessive disease with a pleiotropic phenotype characterized by cerebellar degeneration, immunodeficiency, premature aging, cancer predisposition, and radiation sensitivity. The gene mutated in AT, ATM (for AT-mutated), had been cloned and found to have ionizing radiation and oxidative stressinducible kinase activity. No treatment can stop the progression of the disease. In this study, the complete open-reading frame of ATM cDNA was cloned into a Herpes simplex virus type-1 (HSV-1) amplicon vector (pTO-ATM), and the transduction of cultured AT cells was demonstrated by immunohistochemistry and Western blot analysis. Functional gene expression was evaluated by cell colony-forming assays following exposure to oxidative stress. The survival of AT cells with ATM gene transduction was about 100% higher compared to nontransduced cells after t-butyl hydroperoxide treatments. Next, the normal ATM gene expression in different regions of the rat brain was studied. Immunohistochemistry staining demonstrated weak endogenous ATM protein expression in neurons of the caudate-putamen, with significantly higher levels of expression detected in neurons in other brain regions. Exogenous ATM gene expression from pTO-ATM after viral transduction in the caudateputamen of the adult rat was examined. At 3 days after injection of the pTO-ATM viral vector, abundant positive ATM staining of the neurons was found at the injection sites, in comparison to the controls. These data demonstrate that the relatively large ATM cDNA can be transduced and expressed in vitro and in vivo from an HSV amplicon viral vector. These data provide initial evidence that the replacement of the ATM gene into the cells of AT patients might be possible some day.

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“…29,30 Immunostaining using anti-ATM (3E8) and anti-FLAG (FLAG M2) antibodies was used to localize the FLAG-ATM protein produced 48 h postinfection with the HGC-ATM amplicon vector. The results (Figure 3) indicate that the vector-expressed FLAG-ATM was also localized in the nucleus.…”

Section: Localization Of Flag-atm By Immunofluorescencementioning

confidence: 99%