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. 2016 Aug 8;11(8):e0160901.
doi: 10.1371/journal.pone.0160901. eCollection 2016.

Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

Worapong Singchat  1 , Ekarat Hitakomate  2 , Budsaba Rerkarmnuaychoke  3 , Aorarat Suntronpong  1 , Beiyuan Fu  4 , Winai Bodhisuwan  5 , Surin Peyachoknagul  1   6 , Fengtang Yang  4 , Sittichai Koontongkaew  2 , Kornsorn Srikulnath  1   6
Affiliations

Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

Worapong Singchat et al. PLoS One. .

Abstract

Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence in situ hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (SIX3), 11p15.5 (H19), and 11q21q22.3 (MAML2, PGR, TRPC6, and MMP family) regions, and deletion of 9p23 (PTPRD) and 16q23.1 (WWOX) regions were identified in HN31 and HN12. Interestingly, partial loss of PTPRD (9p23) and WWOX (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of PTPRD, with no detectable expression of the WWOX gene. This suggests that the scarcity of PTPRD and WWOX genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. DAPI stained- and M-FISH (multiplex fluorescence in situ hybridization) karyotypes of four head and neck squamous cell carcinoma cell lines.
DAPI stained and M-FISH karyotype of a representative metaphase indicates for HN30 (a and b), HN31(c and d), HN4 (e and f), and HN12 (g and h). Scale bars represent 10 μm.
Fig 2
Fig 2. Graphical representation of two-dimensional unsupervised hierarchical clustering of chromosomal aberrations in four head and neck squamous cell carcinoma cell lines (10 metaphases per cell line).
Each column refers to a metaphase in each cell line, and each row to type of chromosomal abnormality. Red indicates the presence of each abnormality. Black indicates the absence of each abnormality.
Fig 3
Fig 3. C-banded metaphase spread of four head and neck squamous cell carcinoma cell lines.
C-banded metaphase spread of HN30, HN31, HN4, and HN12 are shown in (a, b, c, and d). Arrows indicate chromosome 9, and arrowheads indicate chromosomes 1. Scale bars represent 10 μm.
Fig 4
Fig 4. Amplification of SIX3 and H19, and deletion of PTPRD and WWOX in four head and neck squamous cell carcinoma cell lines.
The X-axis represents the normalize log2 ratio fluorescence intensity thresholds -0.9 (loss) and 0.53 (gain), while the Y-axis represents the ideogram of human chromosome. Arrows indicate amplification of SIX3 (2p21) and H19 (11p15.5), and deletion of PTPRD (9p23) and WWOX (16q23.1) in HN31 and HN12 cell lines.
Fig 5
Fig 5. mRNA expression of candidate genes in four head and neck squamous cell carcinoma cell lines and a human mucoepidermoid pulmonary carcinoma NCI as compared with a non-tumorigenic human skin keratinocyte cell line HaCaT by real time quantitative reverse transcription PCR.
The X-axis indicates relative expression level to HaCaT, and the Y-axis indicates expression level of each cell line. The level of mRNA expression of SIX3, H19, PTPRD, and WWOX are shown in (a, b, c, and d). Data were expressed as mean ± standard deviation. The levels of statistical significance were represented as * for P ≤ 0.05, ** for P ≤ 0.01, and *** for P ≤ 0.001. N/A indicates expression not detectable.
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