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Published on: September 20, 2013
Altered centrosomes in ataxia-telangiectasia cells and rapamycin-treated Chinese hamster cells
Stefania Bonatti1, Marcella Simili, Pier Alberto Benedetti
1CNR Institute of Clinical Physiology, Pisa, Italy.
Abstract:
Rapamycin induces chromosome malsegregation in mammalian cell lines and yeast. Previous studies indicate that the function impaired in ataxia-telangiectasia (A-T) patients is necessary for both the growth inhibition and the chromosome malsegregation induced by rapamycin, and that treating the non-tumorigenic Chinese hamster cell line CHEF/18 with rapamycin results in supernumerary centrosomes and multipolar spindles. In this paper we report that lymphoblastoid cell lines established from A-T patients as well as hamster A-T-like cells are more resistant to rapamycin than the respective normal cell lines. Two cell lines derived from Nijmegen Breakage Syndrome (NBS) patients, who have clinical symptoms similar to those of A-T but a different molecular defect, were not resistant to rapamycin. Both A-T lymphoblastoid cells and A-T-like fibroblasts had giant centrosomes formed by more than two areas of gamma-tubulin-reacting material. Such giant centrosomes were also observed in CHEF/18 cells after prolonged treatment with rapamycin. Formation of giant centrosomes, possibly due to the coalescence of supernumerary centrosomes, was associated with increased aneuploidy in treated cells. Expression analysis of cell-cycle regulatory genes in rapamycin-treated human lymphoblastoid cells indicated that rapamycin decreased the expression of the tumor suppressor gene GADD45. The levels of RB, p21 and p53 mRNA were also decreased, although to a lesser extent. As rapamycin is often used as an immunosuppressant in pediatric transplant patients, these data indicate that caution should be taken, especially when the drug is given for prolonged periods of time.
Insights
Rapamycin causes chromosome problems in cells, but cells from ataxia-telangiectasia (A-T) patients are resistant. This resistance is linked to giant centrosomes and potential risks with long-term rapamycin use in patients.
Area of Science:
- Cell Biology
- Genetics
- Pharmacology
Background:
- Rapamycin induces chromosome malsegregation and centrosome abnormalities in mammalian cells.
- The ataxia-telangiectasia (A-T) gene product is implicated in rapamycin's effects on cell growth and chromosome stability.
- Previous studies showed rapamycin causes supernumerary centrosomes and multipolar spindles in CHEF/18 cells.
Purpose of the Study:
- To investigate the role of the A-T gene in rapamycin-induced cellular responses.
- To determine if A-T and Nijmegen Breakage Syndrome (NBS) cells exhibit altered sensitivity to rapamycin.
- To examine the impact of rapamycin on centrosome structure and cell cycle gene expression.
Main Methods:
- Culturing and treating lymphoblastoid cell lines from A-T patients and normal individuals with rapamycin.
- Treating hamster A-T-like cells and CHEF/18 cells with rapamycin.
- Analyzing centrosome morphology using gamma-tubulin staining.
- Assessing gene expression levels of cell-cycle regulators (GADD45, RB, p21, p53) via mRNA analysis.
Main Results:
- A-T lymphoblastoid cells and A-T-like hamster cells showed increased resistance to rapamycin compared to normal cells.
- Nijmegen Breakage Syndrome (NBS) cells did not exhibit rapamycin resistance.
- Rapamycin treatment led to the formation of giant centrosomes in A-T cells and prolonged treatment in CHEF/18 cells, associated with increased aneuploidy.
- Rapamycin decreased the expression of GADD45, RB, p21, and p53 mRNA in human lymphoblastoid cells.
Conclusions:
- The A-T gene function is crucial for mediating rapamycin's effects on chromosome stability and centrosome duplication.
- Giant centrosome formation and subsequent aneuploidy are consequences of rapamycin treatment in sensitive cells.
- Reduced expression of key cell cycle regulatory genes, including tumor suppressors, by rapamycin warrants caution during prolonged use, especially in pediatric transplant patients.
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