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Laryngeal squamous cell carcinoma is a major medical problem worldwide. Although our understanding of genetic changes and their consequences in laryngeal cancer has opened new therapeutic pathways over the years, the diagnostic as well as treatment options still need to be improved. In our previous study, we identified CRKL (22q11) as a novel putative oncogene overexpressed and amplified in a subset of LSCC tumors and cell lines. Here we analyze to what extent CRKL DNA copy number gains correlate with the higher expression of CRKL protein by performing IHC staining of the respective protein in LSCC cell lines (n = 3) and primary tumors (n = 40). Moreover, the importance of CRKL gene in regard to proliferation and motility of LSCC cells was analyzed with the application of RNA interference (siRNA). Beside the physiological cytoplasmic expression, the analysis of LSCC tumor samples revealed also nuclear expression of CRKL protein in 10/40 (25%) cases, of which three (7.5%), presented moderate or strong nuclear expression. Similarly, we observed a shift towards aberrantly strong nuclear abundance of the CRKL protein in LSCC cell lines with gene copy number amplifications. Moreover, siRNA mediated silencing of CRKL gene in the cell lines showing its overexpression, significantly reduced proliferation (p < 0.01) as well as cell migration (p < 0.05) rates. Altogether, these results show that the aberrantly strong nuclear localization of CRKL is a seldom but recurrent phenomenon in LSCC resulting from the increased DNA copy number and overexpression of the gene. Moreover, functional analyses suggest that proliferation and migration of the tumor cells depend on CRKL expression. [ABSTRACT FROM AUTHOR]

Objectives: Aberrations in Wnt and Shh signaling pathways are related to the pathogenesis of head and neck carcinomas, and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of methylation of negative regulators of Wnt signaling: CXXC4, DACT2, HDPR1, and FBXW11 and Shh signaling: HHIP, PTCH1, SUFU, ZIC1, and ZIC4 and correlate it with clinicopathological features in this group of patients. Materials and methods: Methylation-specific PCR was used to detect gene promoter methylation, and real-time PCR was used to assess gene expression level. Results: The analysis of the occurrence of gene promoter methylation in head and neck carcinoma cell lines indicated that CXXC4, DACT2, HHIP, ZIC1, and ZIC4 are methylated in these tumors. These genes were further analyzed in tumor sections from oral and laryngeal cancer patients. Gene methylation rate was higher in laryngeal tumors. The methylation index in tumor samples correlated with the overall survival in a subgroup of oral cancer patients who died of the disease. Moreover, ZIC4 methylation correlated with lymph node involvement in oral cancer patients. Conclusions: Our findings corroborate that the activation of Wnt signaling in head and neck squamous cell carcinoma (HNSCC) is related to epigenetic silencing of its negative regulators. Moreover, the results indicate that the same mechanism of activation may operate in the case of Shh signaling. Clinical relevance: The methylation of ZIC4 may be considered a new prognostic marker in oral cavity and oropharyngeal tumors. Further investigations should determine the diagnostic significance of methylation of ZIC4, HHIP, and DACT2 in head and neck carcinomas. [ABSTRACT FROM AUTHOR]

Volatile anaesthetics such as halothane, isoflurane and others were expected to produce a health challenge for operation room personnel because of prolonged occupational exposure to anaesthetic gases. To estimate a molecular background of adverse health effects, a cohort of 100 exposed individuals was studied by the single-cell gene electrophoresis (comet assay) test. DNA lesions in lymphocytes of the exposed group did not differ significantly compared with nonexposed blood donors. Then, the exposed group was further divided according to job position. A highest level of DNA lesions was established in nurses but without significant difference compared with other groups. When a time period of exposure was taken into account, a tendency to cumulate DNA lesions was found only in the group of anaesthesiologists. A very weak genotoxic effect established in this study is discussed in relation to DNA repair, adaptative response and potential selfelimination of sensitive individuals. [ABSTRACT FROM AUTHOR]

The deregulation of Wnt signaling has recently emerged as one of the drivers of head and neck cancers. This is frequently related to the methylation of several antagonists of this pathway. This study aimed at the assessment of the profile of methylation of Wnt pathway antagonists and the determination of the prognostic value of the methylation of selected genes in oral carcinomas. The methylation of DACH1, DKK1, LKB1, PPP2R2B, RUNX3, SFRP2, and WIF-1 was analyzed in 16 oral squamous cell carcinoma cell lines using the methylation-specific polymerase chain reaction. The methylation of selected genes was further analyzed in tumor sections from 43 primary oral carcinoma patients. The analysis of oral carcinoma cell lines showed very frequent methylation of SFRP2 and WIF-1 and also a less frequent methylation of DACH1 and DKK1. On the other hand, RUNX3 was methylated only in one cell line, while LKB1 and PPP2R2B were not methylated in any of the cell lines . The biallelic methylation of DKK1 correlated with the low level of expression of this gene. Further evaluation of the methylation of DACH1, DKK1, and WIF1 in a clinical patient group confirmed the frequent methylation of WIF1 and intermediate or low frequency of methylation of DACH1 or DKK1, respectively. Importantly, the methylation of WIF-1 correlated with shorter survival in oral cancer patients. Overall, the methylation of the antagonists of Wnt pathway is frequently detected in oral squamous cell carcinomas. The methylation of WIF1 may be considered a prognostic marker in oral cancers. [ABSTRACT FROM AUTHOR]

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184-71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes ( CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies. [ABSTRACT FROM AUTHOR]

Patients with squamous cell carcinoma of the head and neck (HNSCC) aged less than 45 years are categorized as young adults (YA) and in opinion of many authors in comparison to older (typical) patients (OP) are characterized with more serious form of the disease and often lack the classical risk factors associated with the illness. Hence, there is a need of an exact clinical analysis and a search for additional causative factors. The purpose of this study was to estimate the role of genetic factors in the etiology of HNSCC in YA. Studies carried out on 60 patients of the study group (YA) and 72 older control patients were directed to: (1) a degree of spontaneous and induced chromosome breaks estimated by bleomycin test, (2) a degree of spontaneous and induced DNA damage and a potential of DNA repair determined by comet assay and (3) polymorphism of genes of glutathione transferases M1 and T1, responsible for detoxification of metabolites of carcinogens of tobacco smoke, studied by PCR-based genotyping. The level of chromosome breaks (spontaneous and induced), the level of DNA damage (spontaneous and induced), DNA repair potential and the distribution of polymorphic variants of GSTT1 gene are not significantly different in YA and in OP, which suggests that these factors do not appear the causative factors for HNSCC in young age. The significant risk factor of HNSCC in YA may be GSTM1 null genotype, which may cause the defective detoxification of metabolites of polycyclic aromatic hydrocarbons of tobacco smoke. [ABSTRACT FROM AUTHOR]