Study On STK15 Gene Abnormality And Centrosomal Amplification, Chromosomal Instability In Laryngeal Carcionma

Chromosomal abnormality which can usually be caused by abnormal mitosis has been considered the feature of tumor cells. The centrosome, the major microtubule organizing center regulates cell division through forming bipolar mitotic spindles and plays an essential role in the maintenance of chromosomal stability. Therefore, the chromosomal abnormality in tumor cells may correlate much with centrosomal abnormality. Now many centrosomal abnormalities have been found in quite a few kinds of tumors among which amplification is one of the most common abnormalities. STK15 gene associating with centrosome can induce its amplification, chromosomal instability and cell transformation.Laryngeal carcinoma is a kind of head and neck cancer with high occurrence in northeast of China. In our study semi - quantitative RT- PCR and PCR - SSCP were used to test the expressional level and mutation of STK15 gene in 50 cases of laryngeal carcinoma and human laryngeal squamous cell cancer cell line Hep - 2; Meanwhile Hep - 2 cell line was used as an example to detect centrosomal amplification by immunofluorescence and chromosomal instability by routine and high- resolution G - banding and fluorescent in situ hybridization ( FISH). This will provide the evidence of how centrosomal amplification and chromosomal instability induced by STK15 gene abnormalityfunctions in laryngeal carcinoma, so as to probe its occurrence mechanism and provide scientific basis for early diagnosis and therapy.Materials and Methods1. Materials1. 1 Human laryngeal squamous cell carcinoma tissue, human laryngeal squamous cell cancer cell line Hep - 2; normal human embryonic lung fibroblast WI - 38 cell line1. 2 Reagents for RT - PCR and PCR - SSCP1. 3 Monoclonal anti - y - tubulin antibody derived from a mouse with GTU - 88 hybridoma; FITC conjugated goat anti - mouse IgG and other relevant reagents1.4 Reagents for G - banding and FISH2. Methods2.1 STK15 gene mRNA expressional level was tested in 50 cases of laryngeal carcinoma with paired normal tissue as control and Hep -2 ceUs with WI -38 cells as control by RT - PCR.2. 2 Mutation of STK15 gene exon 6 and exon 7 in the same tissues and cells was detected by PCR- SSCP.2.3 Immunofluorescent antibodies were applied to test centroso-mal amplification in Hep - 2 cell line as an example.2. 4 Karyotype analysis in Hep -2 cell line was carried out u-sing routine and high resolute G - banding ; The number of chromosome 20 was analyzed by FISH.Results1. In the 50 cases of laryngeal carcinoma, there were 34(68% ) cases whose expressions of STK15 gene in tumor were higher than paired normal tissues and the expression of STK15 gene in Hep - 2 cell line was higher than that in WI - 38 cell line. The tumor group and contrast one had significant difference by statistic analysis.2. No mutation was found in exon 6 and 7 of STK15 gene in the above tissues and cells.3. Apparent centrosomal amplification was found in Hep - 2 cell line: The number of centrosome changed from 1 to? and Hep - 2 cells with amplified centrosomes (that is more than 2 in one cell) were 11-23% which were much higher than those in WI -38 cell line.4. (1) Karyotype analysis showed that chromosomal instability in Hep - 2 cell line was common: The chromosomal number changed form 43 to 84 and chromosomal model ranged from 69 to 74. The structural aberration included translocation, deletion and isochromo-some and was represented by 13 marker chromosomes. (2) The result of FISH showed apparent polysomy of chromosome 20.Discussion1. STK15 gene abnormality in tumorsSTK15 gene has been verified as an oncogene and it regulates the maturation of centrosome, formation of spindle and accurate segregation of chromosomes. Its overexpression can lead to centrosomal amplification, chromosomal instability and cell transformation. The ex-pressional level of this gene was tested in 50 cases of laryngeal carcinoma and Hep -2 cell line and STK15 gene ov...