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Cell cycle arrest of human gingival fibroblasts and periodontal ligament cells by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin
Umeå University, Faculty of Medicine, Odontology, Oral Cell Biology. Umeå University, Faculty of Medicine, Odontology, Oral Microbiology.
Umeå University, Faculty of Medicine, Odontology, Periodontology.
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2004 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, Vol. 112, no 10, 674-685 p.Article in journal (Refereed) Published
Abstract [en]

The cytolethal distending toxin (Cdt) is produced by several Gram-negative bacterial species and causes growth arrest and morphological alterations in mammalian cells. Actinobacillus actinomycetemcomitans, which is involved in the pathogenesis of localized aggressive periodontitis, also produces a Cdt that affects periodontal connective tissue cells. The aim of this study was to investigate in which phase of the cell cycle these cells are arrested and enlarged when challenged with A. actinomycetemcomitans, and to evaluate the involvement of its Cdt. Human gingival fibroblasts and periodontal ligament cells were challenged with A. actinomycetemcomitans extract, or with purified Cdt, and cell cycle analysis was performed by propidium iodide staining and flow cytometry. Cells exposed to an A. actinomycetemcomitans wild-type strain, or to purified Cdt, were arrested in both G1 and G2/M phases, and appeared enlarged compared to the corresponding controls. The cellular enlargement occurred in both G1 and G2/M arrested cells. In contrast, cells exposed to an A. actinomycetemcomitans cdt-knockout mutant strain showed cell cycle phase distribution and size similar to the controls. In conclusion, A. actinomycetemcomitans causes a combined G1 and G2/M growth arrest and enlargement in periodontal connective tissue cells, which is attributed to its Cdt.

Place, publisher, year, edition, pages
2004. Vol. 112, no 10, 674-685 p.
Identifiers
URN: urn:nbn:se:umu:diva-6487DOI: 10.1111/j.1600-0463.2004.apm1121006.xPubMedID: 15601319OAI: oai:DiVA.org:umu-6487DiVA: diva2:146156
Available from: 2007-12-13 Created: 2007-12-13 Last updated: 2009-12-08Bibliographically approved
In thesis
1. Cellular and molecular responses of periodontal connective tissue cells to Actinobacillus actinomycetemcomitans cytolethal distending toxin
Open this publication in new window or tab >>Cellular and molecular responses of periodontal connective tissue cells to Actinobacillus actinomycetemcomitans cytolethal distending toxin
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt.

A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations.

The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement.

In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.

Place, publisher, year, edition, pages
Umeå: Odontologi, 2004. 49 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 88
Keyword
Medical sciences, Actinobacillus actinomycetemcomitans, cytolethal distending toxin, periodontal connective tissue cells, localized aggressive periodontitis, growth arrest, bone resorption, receptor activator of NF-κB ligand, osteoprotegerin, pro-inflammatory cytokines, MEDICIN OCH VÅRD
National Category
Medical and Health Sciences
Research subject
Odontology
Identifiers
urn:nbn:se:umu:diva-345 (URN)91-7305-746-0 (ISBN)
Public defence
2004-12-10, Sal 933, 3A, Norrlands Universitessjukhus, Umeå, 13:00 (English)
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Available from: 2004-11-01 Created: 2004-11-01 Last updated: 2009-08-24Bibliographically approved

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