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.
Subject(s)
Ataxia Telangiectasia/genetics , Centrosome/drug effects , Centrosome/ultrastructure , Chromosome Aberrations/chemically induced , Nijmegen Breakage Syndrome/genetics , Sirolimus/pharmacology , Animals , Ataxia Telangiectasia/pathology , Cell Cycle , Cell Line , Centrosome/metabolism , Chromosomes/ultrastructure , Cricetinae , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Dose-Response Relationship, Drug , Drug Resistance , Fibroblasts/metabolism , Gene Expression Regulation , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocytes/metabolism , Metaphase , Micronucleus Tests , Microscopy, Confocal , Models, Statistical , Phenotype , Retinoblastoma Protein/metabolism , Spindle Apparatus/drug effects , Time Factors , Tumor Suppressor Protein p53/metabolism , GADD45 ProteinsABSTRACT
No single biomarker can predict the risk for malignant transformation of precancerous lesions of the head and neck. Micronucleus frequency, nuclear p53 accumulation and mitotic index were determined in proliferating basal cells using paraffin-embedded specimens from normal, dysplastic and malignant tissues. p53 accumulation was detected by immunohistochemistry using pAb 1081 and pAb 240 antibodies. Micronuclei were scored in the same cell population and classified for the presence/ absence of p53 accumulation in the main nucleus. Fifty-three carcinomas and 15 precancerous lesions were studied. Both micronuclei and p53 accumulation were found in precancerous lesions, suggesting that they are early events in head and neck squamous cell carcinoma progression. The two biomarkers were not related to each other: indeed, micronucleus frequency was higher in p53-negative than in p53-positive cells. Three patients with precancerous lesions later developed carcinomas; all three cases showed high frequencies of both micronuclei and cells accumulating p53 protein.
