Chromosome Abnormalities, Centrosome Amplification, And Telomere Dysfunction In Head And Neck Squamous Cell Carcinomas

Chapter 1. Non-random chromosome abnormalities in head and neck squamous carcinomasObjectives To detect and analyze chromosomal abnormalities in head and neck squamous carcinomas (HNSCC). Methods 18 cell lines and 3 normal tissues of HNSCC were cultured, and harvest when in metaphase. G-banding of chromosomes was with Wright's stain. Chromosomal changes were characterized with karyotypes according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). Results 1. Loss of chromosome material occurred more often than gain；Chromosomal imbalances detected in HNSCC cell lines included loss of 2q, 3p, 4, 8p, 9, 13, 14, 15, 17, 18, 21, and 22; and gain of 3q, 7, 8q, 11q13, and 20. 2. Most of chromosomal breakpoints were located to the centromeric regions. Conclusion Multiple unbalanced chromosomal structural rearrangements and numerical changes in HNSCC led to activation and amplification of Oncogenes, and inactivation and lost of Tumor suppressor genes; which contribute to the development of HNSCC.Chapter 2 Centrosome amplification in heand and neck squamous cell carcinomasObjectives To study the underlying mutational mechanisms of chromosomal evolution in head and neck squamous cell carcinomas, centrosome expression and metaphase morphology were investigated in 8 HNSCC cell lines. Methods Cell cultures were harvest and stained with haematoxylin and eosin, to analyze cellular metaphase morphology. Centrosomes were detected by anti-γtubulin antibodies using the technique of immunofluorescence hybridization. Epithelial cell cultures from 6 normal head and neck tissues were used as normal control in the experiment. Results Metaphase multipolar mitoses and centrosome amplification were found in all HNSCC cell lines at the average frequencies of 9.9±4.8% and 6.7±3.1%separately. Also there was a significant positive correlation between them, r=0.857,P＜0.01. However, the normal controls seldom showed metaphase multipolar mitoses and centrosome amplification, with the average frequencies of 0.8±0.9% and 1.2±1.4% separately. The frequencies of HNSCC cell lines were greatly higher than those of normal controls, P＜0.01. Conclusions Supernumeray centrosomes caused multipolar cell division and contribute to gross chromosomal aberrations of HNSCC.Chapter 3. Telomeric dysfunction and chromosomal breakage-fusion- bridge cycle in head and neck squamous cell carcinomasObjectives To explore the mechanisms of chromosome aberrations, we studied the chromosome telomeres expression and anaphase morphologies in HNSCC cell lines. Methods Cell cultures of 8 HNSCC cell lines were stained with haematoxylin and eosin, to analyze mitotic cells with anaphase bridge morphology. Chrosomal telomeric TTAGGG repeats were detected by in situ hybridization with fluorescein-conjugated (CCCTAA)3 peptide nucleic acid probes. Epithelial cell cultures from 6 normal head and neck tissues were used as normal controls. Results All HNSCC cell lines showed TTAGGG-negative chromosome ends with the mean number of 12.8±5.4 per cell; and presented anaphase bridges at the frequency of 22.7±9.9 %. Moreover, the frequency of anaphase bridges was positively correlated to the mean number of negative telomeric ends, r=0.797, P＜0.01.On the other hand, the normal controls scarcely present the two events, with the average numbers of 0.3±0.3 and 1.6±1.4 %. The mean numbers of HNSCC cell lines were significantly higher than those of normal controls, P＜0.005. Conclusions Shortening of telomeric repeats disrupted normal mitotic process by triggering chromosomal BFB cycles, thus led to the development of complex chromosome aberrations and genomic imbalances.