Neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYPT), and chromogranin A (CGA) are immunohistochemical markers for diagnosing lung neuroendocrine tumors (LNETs). However, the precise expression mechanisms have not been studied in enough detail. The purpose of the present study is to define the molecular mechanisms of NCAM1, SYPT, and CGA gene expressions, using cultivated lung cancer cells and focusing upon NeuroD1 (ND1), achaete-scute homolog-like 1 (ASCL1), and known transcription factors, repressor element 1 (RE1)-silencing transcription factor (REST) and c-AMP responsive element-binding protein (CREB). Promoter assays, chromatin immunoprecipitation, and transfection experiments revealed that ND1 activated NCAM1, that ASCL1 weakly upregulated SYPT expression, and that CGA expression was not regulated by ND1 or ASCL1. REST expression was restricted in non-small cell lung cancer (NSCLC) cells, and knockdown of REST could cause as much SYPT expression as in SCLC cells and weak CGA expression in NSCLC cells. However, CGA gene upregulation via CREB activation was not found in REST-lacking NSCLC cells, indicating the requirement of some additional mechanism for sufficient expression. These results suggest that NCAM1, SYPT and CGA expressions are differently regulated by neuroendocrine phenotype-specific transcription factors and provide a reason why NCAM1 and SYPT are frequently expressed in LNETs, irrespective of malignancy grade.
Mechanisms of endocrine secretory granule (SG) formation in thyroid C cells and medullary thyroid cancer (MTC) cells have not been fully elucidated. Here we directly demonstrated that PROX1, a developmental homeobox gene, is transcriptionally involved in SG formation in MTC, which is derived from C cells. Analyses using gene expression databases on web sites revealed that, among thyroid cancer cells, MTC cells specifically and highly express PROX1 as well as several SG-forming molecule genes. Immunohistochemical analyses showed that in vivo MTC and C cells expressed PROX1, although follicular thyroid cancer and papillary thyroid cancer cells, normal follicular cells did not. Knockdown of PROX1 in an MTC cells reduced SGs detected by electron microscopy, and decreased expression of SG-related genes (chromogranin A, chromogranin B, secretogranin II, secretogranin III, synaptophysin, and carboxypeptidase E). Conversely, the introduction of a PROX1 transgene into a papillary thyroid cancer and anaplastic thyroid cancer cells induced the expression of SG-related genes. Reporter assays using the promoter sequence of chromogranin A showed that PROX1 activates the chromogranin A gene in addition to the known regulatory mechanisms, which are mediated via the cAMP response element binding protein and the repressor element 1-silencing transcription factor. Furthermore, chromatin immunoprecipitation-PCR assays demonstrated that PROX1 binds to the transcriptional regulatory element of the chromogranin A gene. In conclusion, PROX1 is an important regulator of endocrine SG formation in MTC cells.
Abbreviations:PTC, papillary thyroid carcinoma; SNP, single nucleotide polymorphism; TERT, telomerase reverse transcriptase CorrespondencePapillary thyroid carcinoma (PTC) is the most common thyroid malignancy. Point mutations in the telomerase reverse transcriptase (TERT) promoter, C228T and C250T and oncogene BRAF V600E have been investigated as risk factors for PTC.However, little research has been done on the single nucleotide polymorphism rs2853669 in the TERT promoter in PTC. This study aimed to clarify the clinicopathological significance of rs2853669 in Japanese patients with PTC. The genetic frequencies of rs2853669, C228T, C250T and BRAF V600E were investigated in 58 patients with PTC and compared with the clinicopathological parameters of PTC. rs2853669, C228T, C250T and BRAF V600E were found in 58.6%, 17.2%, 5.2% and 37.0% of the PTC patients, respectively. PTC with rs2853669 and C228T were associated only with tumor sizes larger than 2.0 cm (P < 0.05). Furthermore, the coexistence of rs2853669 and C228T was strongly associated with tumor size (P < 0.01), with an odds ratio of 6.4 (P < 0.05). We showed that rs2853669, as well as C228T, may be a risk factor for the aggressiveness of PTC, and the coexistence of these mutations might represent greater risk.
Follicular thyroid neoplasm is a common tumor, and consists of follicular thyroid adenoma (FTA) and carcinoma (FTC). The mechanisms of tumor development of FTA and FTC are not well-understood. Single-nucleotide polymorphisms (SNPs) and point mutations in the telomerase reverse transcriptase (TERT) promoter have been associated with tumor development of many cancers. In order to clarify the significance of TERT promoter SNPs and mutations, including rs2853669 (−245T>C), C228T, and C250T, we analyzed 59 FTA patients and 19 FTC patients. Rs2853669 was found in 67.8% (40/59) and 57.9% (11/19) of FTAs and FTCs, respectively, and homozygous rs2853669 (CC) was more frequently found in FTC than in FTA. Furthermore, in FTA, rs2853669 was significantly associated with tumor size greater than 2.0 cm (P < 0.05).C228T was found in 5.1% and 36.8% of FTAs and FTCs, respectively. Frequencies of rs2853669 or/and C228T mutation were 71.2% in FTAs and 73.7%, in FTCs, and were significantly associated with larger tumor sizes in FTAs (P < 0.05). Rs2853669 is considered to be associated with tumor development in FTA and FTC.C228T, C250T, follicular thyroid adenoma, follicular thyroid carcinoma, follicular thyroid neoplasms, point mutation, rs2853669, single-nucleotide polymorphism, TERT promoter
<b><i>Background/Aim:</i></b> The telomerase reverse transcriptase (<i>TERT</i>) promoter has a regulatory single nucleotide polymorphism (rSNP), rs2853669, and occasionally shows point mutations C228T and C250T. Although C228T and C250T have been well examined to increase <i>TERT</i> promoter activity and are known as risk factors for thyroid carcinoma, the significance of rs2853669 has not been well investigated. This study aimed to clarify the influence of rs2853669 on <i>TERT</i> promoter activity in thyroid carcinoma cells. <b><i>Materials:</i></b> Seven of 8 examined thyroid cell lines had rs2853669, 5 had C228T, and 1 had C250T. <b><i>Results:</i></b> Three papillary thyroid carcinoma cell lines, harboring both rs2853669 and C228T, showed higher <i>TERT</i> mRNA expression on real-time PCR than the other cell lines. Anaplastic thyroid carcinoma cell lines, in contrast, showed variable <i>TERT</i> mRNA expression depending on the combination of rs2853669, C228T, and C250T. Luciferase assays, performed to compare the influences of rs2853669, C228T, and C250T on <i>TERT</i> promoter activity in thyroid carcinoma, showed that rs2853669, as well as C228T, increased the promoter activity, and the combination of rs2853669 and C228T increased the promoter activity even more strongly than C228T alone. <b><i>Conclusion:</i></b> We conclude that the presence of rs2853669 within the <i>TERT</i> promoter could be as significant as the C228T mutation in thyroid carcinoma.
20Some cancer cells lengthen their telomeres by alternative lengthening of telomeres 21 (ALT); these are referred to as ALT cancer cells and do not express telomerase. The ALT 22 mechanism involves the elongation of telomeric DNA repeats by homologous 23 recombination. In interphase nuclei of ALT cancer cells, giant telomeres can be 24 specifically observed by fluorescence in situ hybridization (FISH) to detect telomeric 25 repeats, and they are co-localized with promyelocytic-leukemia nuclear-bodies (PML-26 NBs). However, it how large telomeres specific to ALT and how they form a structural 27 relation with PML-NBs. We refer to giant telomeres specific to interphase ALT cancer 28 cells giant-telomeric nuclear-bodies (GT-NBs). To quantitatively define GT-NBs, we 29 performed telomeric FISH of both interphase nuclei and metaphase chromosomes and 30 analyzed telomere sizes by integrated FISH signals. The distributions of telomere sizes 31 in telomerase-positive cells were similar in interphase nuclei and chromosomes. However, 32 the distribution of telomere sizes in ALT cancer cells differed between interphase nuclei 33 and chromosomes. Giant telomeres that were larger than those at chromosomal ends were 34 only observed in interphase nuclei of ALT cancer cells. Accordingly, GT-NBs could be 35 quantitatively defined as larger than the maximum size of telomeres at chromosomal ends. 36 Furthermore, ALT cancer cells demonstrated fewer telomeric signals in interphase nuclei 3 37 than in chromosomes. These findings indicate that GT-NBs could be formed by the 38 aggregation of two or more telomeres at chromosomal ends. Furthermore, super-39 resolution microscopy showed that GT-NBs contain one or two PML-NBs. GT-NBs are 40 considered aggregates of telomeres and could contain multiple sites of homologous 41 recombination accompanied by PML-NBs. These findings may contribute to the 42 development of therapeutic approaches for ALT cancer. (251 words) 43 44 4 55 of Tel (+) cancer cells [3-4]. Telomeres can be detected by fluorescence in situ 56 hybridization (FISH) and quantified by integrated brightness fluorescence intensities for 57 telomere signals, reflecting the amount of telomeric DNA. The signals of giant telomeres 58 are observed by FISH in interphase nuclei of ALT cancer cells [4,12-15]. However, it is 59 not clear how large telomeres are specific to ALT cancer cells and how they form in their 60 interphase nuclei. In the present study, we refer to ALT-specific giant telomeres as "giant-61 telomeric nuclear-bodies (GT-NBs)" and attempted to define GT-NBs in a quantitative 62 manner and to clarify their morphogenesis. 63 Promyelocytic leukemia nuclear bodies (PML-NBs) are intranuclear structures 64 containing the PML protein; they were first discovered in patients with acute 65 promyelocytic leukemia [13,16-19]. Typically measuring around 0.2-1.0 nm, PML-NBs 66 assume globular structures with doughnut-shaped cross-sections. PML-NBs are involved 67 in various cellular functions, such as the regulation of the cell cycl...
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