title: Nuclear bud formation: a novel manifestation of Zidovudine genotoxicity.
abstract: Normal diploid somatic mammalian cell division generates 2 daughter cells as a result of a strict and well-controlled mitotic process. However, some defects during the progression of that process could generate an unbalanced distribution of chromosomes, aneuploidy and eventually, a malignant phenotype. Previous observations using a transgenic mouse model with diminished DNA repair capacity revealed the presence of nuclear buds (NBs) induced in vitro by the nucleoside analog zidovudine (Retrovir(R), 3'-azido-3'-deoxythymidine, AZT). Here we used bone marrow mesenchymal cells, taken from mice with the Xpa(-/-)Trp53(+/-) genotype, that were cultured and exposed to 0 and 100 muM AZT for 24 hours. Fixed and denatured cells were processed by fluorescence in situ hybridization (FISH) with whole chromosome painting probes used to identify chromosomes in cells growing on glass chamber slides (2 probes/slide). A variety of sizes and shapes of NBs were observed. Some NBs had a large connection with the main nucleus (>(1/4) of the NB diameter), others hada smaller connection (<(1/4) of the NB diameter), some were circular and positioned close to the nucleus, while some resided in the cytoplasm separated from the nucleus or connected by a thin chromatin strand. We had hypothesized that NBs would progress in the process of budding until separation occurred, but this was not proven by time-lapse photography studies performed for 20 hours. From 1,126 cells scored in the unexposed cultures, 10.39 % of cells carried NBs, while from 1,108 cells scored in the AZT-exposed cultures 29.16% of cells carried NBs (p = 0.001). In AZT-exposed cells there were a total of 322 NBs scored; 46.6% or 150 NBs contained positive signals for one or both probes used, while 53% or 172 NBs had no probe signal. In addition, FISH analysis showed no preferential localization of any chromosome within the NBs. Among the NBs that carried no probe signal, the presence of positive signals with inversion of DAPI imaging demonstrated centromeric content. It has been hypothesized that NBs occur as a result of expulsion of amplified DNA from the main nucleus; however, this data demonstrates that NBs may contain any chromosome, suggesting that NBs do not consist of just amplified DNA.
authors.affiliation: Cytogenetics and Microscopy Core, National Human Genome Research Institute, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dutra
authors.forename: E
authors.initials: E
authors.lastname: Pak
authors.forename: S
authors.initials: S
authors.lastname: Wincovitch
authors.forename: K
authors.initials: K
authors.lastname: John
authors.forename: M C
authors.initials: MC
authors.lastname: Poirier
authors.forename: O A
authors.initials: OA
authors.lastname: Olivero
mesh_terms: D000818:Animals; D019380:Anti-HIV Agents / Q000633:toxicity*; D002467:Cell Nucleus / Q000187:drug effects*; D051379:Mice; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics; D015215:Zidovudine / Q000633:toxicity*
chemical_list: D019380:Anti-HIV Agents; D016159:Tumor Suppressor Protein p53; D051760:Xeroderma Pigmentosum Group A Protein; C497315:Xpa protein, mouse; D015215:Zidovudine
references.citation: Hum Genet. 1988 Nov;80(3):224-34
references.pmid: 3192212
references.citation: Mutat Res. 2004 Jul 13;551(1-2):167-80
references.pmid: 15225591
references.citation: Mutat Res. 2007 Apr 1;617(1-2):33-45
references.pmid: 17250856
references.citation: Genes Chromosomes Cancer. 1994 Apr;9(4):292-5
references.pmid: 7519052
references.citation: Genes Chromosomes Cancer. 1994 Jun;10(2):85-94
references.pmid: 7520271
references.citation: J Cell Biol. 1999 Jan 11;144(1):11-20
references.pmid: 9885240
references.citation: Nature. 1999 Nov 11;402(6758):187-91
references.pmid: 10647011
references.citation: Environ Mol Mutagen. 2007 Apr-May;48(3-4):215-23
references.pmid: 16395695
references.citation: Mutat Res. 2000 Mar 14;448(1):81-90
references.pmid: 10751625
references.citation: Toxicol Pathol. 2001;29 Suppl:109-16
references.pmid: 11695546
references.citation: Nat Protoc. 2007;2(5):1084-104
references.pmid: 17546000
references.citation: Mutat Res. 2009 Jun 1;665(1-2):67-74
references.pmid: 19427513
references.citation: Environ Mol Mutagen. 2009 Oct;50(8):718-24
references.pmid: 19562754
references.citation: J Cell Biol. 1998 Mar 23;140(6):1307-20
references.pmid: 9508765
references.citation: Proc Natl Acad Sci U S A. 1986 May;83(9):2934-8
references.pmid: 3458254