Tools and methods for cell ablation and cell inhibition in Caenorhabditis elegans.

To understand the function of cells such as neurons within an organism, it can be instrumental to inhibit cellular function, or to remove the cell (type) from the organism, and thus to observe the consequences on organismic and/or circuit function and animal behavior. A range of approaches and tools were developed and used over the past few decades that act either constitutively or acutely and reversibly, in systemic or local fashion. These approaches make use of either drugs or genetically encoded tools. Also, there are acutely acting inhibitory tools that require an exogenous trigger like light. Here, we give an overview of such methods developed and used in the nematode Caenorhabditis elegans.

OLA-1, an Obg-like ATPase, integrates hunger with temperature information in sensory neurons in C. elegans.

Animals detect changes in both their environment and their internal state and modify their behavior accordingly. Yet, it remains largely to be clarified how information of environment and internal state is integrated and how such integrated information modifies behavior. Well-fed C. elegans migrates to past cultivation temperature on a thermal gradient, which is disrupted when animals are starved. We recently reported that the neuronal activities synchronize between a thermosensory neuron AFD and an interneuron AIY, which is directly downstream of AFD, in well-fed animals, while this synchrony is disrupted in starved animals. However, it remained to be determined whether the disruption of the synchrony is derived from modulation of the transmitter release from AFD or from the modification of reception or signal transduction in AIY. By performing forward genetics on a transition of thermotaxis behavior along starvation, we revealed that OLA-1, an Obg-like ATPase, functions in AFD to promote disruption of AFD-AIY synchrony and behavioral transition. Our results suggest that the information of hunger is delivered to the AFD thermosensory neuron and gates transmitter release from AFD to disrupt thermotaxis, thereby shedding light onto a mechanism for the integration of environmental and internal state to modulate behavior.

Variant analysis of prostate cancer in Japanese patients and a new attempt to predict related biological pathways.

There are regional and/or ethnic differences in tumorigenic pathways among several types of cancer, including prostate cancer (PCa). However, information on genome­wide gene alterations and the transcriptome is currently only available for PCa patients from Western countries. In order to profile the genetic alterations in Japanese patients with PCa, new panels were created to examine nucleotide sequence variations in 71 selected PCa­related genes (KCC71) and to detect all fusion RNA transcripts known in PCa (PCaFusion). An analysis of 21 Japanese PCa cases identified 33 different somatic variants in 24 genes in the KCC71 panel, including 2 in SPOP (F102V and F133L), 2 in BRCA2 (I1859fs and R2318ter, resulting in premature termination of the polypeptide), and 1 each in BRAF (K601E), CDH1 (E880K) and RB1 (R621S), as pathogenic alterations. Unexpectedly, the TMPRSS2­ERG fusion transcript was detected in only 1 case, although the SLC45A3­ELK4 and USP9Y­TTTY15 fusion transcripts, known as transcription­mediated chimeric RNAs, were detected in all examined cases. A new pathway analysis with The Cancer Network Galaxy (TCNG), a cancer gene regulatory network database, was also applied in an attempt to predict molecular pathways implicated in PCa in the Japanese population. Based on the 24 genes having somatic variants identified by the panel analysis as initial seed genes, a putative core network was finally established, including 5 identified genes, namely TNK2, SOX9, CDH1, FOXA1 and TP53, with high commonality from TCNG datasets. These genes are expected to be involved in tumor development, as revealed by the results of an enrichment analysis with Gene Ontology terms. This analysis must be further extended to include more cases in order to verify this method and also to elucidate the characteristics of PCa in Japanese patients.

Atypical Protein Kinase C λ/ι Expression Is Associated with Malignancy of Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Atypical protein kinase C λ/ι (aPKCλ/ι) is a cell polarity-regulator localized in the tight junction and apical membrane in epithelial cells. Previous studies suggested that aPKCλ/ι overexpression and abnormal localization were involved in tumor progression in several cancers. We investigated the relationship between aPKCλ/ι and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: The correlation between the aPKCλ/ι expression and the clinicopathological parameters in 76 OSCC cases was examined using immunohistochemical analyses. RESULTS: aPKCλ/ι overexpression was observed in 36.8% of cases. aPKCλ/ι expression was more intense in poorly differentiated OSCC and younger patients (<60 years of age). Although expression of aPKCλ/ι was not significantly associated with clinical parameters, the correlation was found between aPKCλ/ι localization and progression-free survival. CONCLUSION: This is the first study to assess the association of aPKCλ/ι expression in OSCC with clinical results. Expression and localization of aPKCλ/ι may be involved in the degree of malignancy in OSCC.

SLO potassium channels antagonize premature decision making in C. elegans.

Animals must modify their behavior with appropriate timing to respond to environmental changes. Yet, the molecular and neural mechanisms regulating the timing of behavioral transition remain largely unknown. By performing forward genetics to reveal mechanisms that underlie the plasticity of thermotaxis behavior in C. elegans, we demonstrated that SLO potassium channels and a cyclic nucleotide-gated channel, CNG-3, determine the timing of transition of temperature preference after a shift in cultivation temperature. We further revealed that SLO and CNG-3 channels act in thermosensory neurons and decelerate alteration in the responsiveness of these neurons, which occurs prior to the preference transition after a temperature shift. Our results suggest that regulation of sensory adaptation is a major determinant of latency before animals make decisions to change their behavior.

GWAS-identified CCR1 and IL10 loci contribute to M1 macrophage-predominant inflammation in Behçet's disease.

BACKGROUND: Low C-C chemokine receptor 1 (CCR1) and interleukin (IL)-10 expression is associated with risk of Behçet's disease (BD). The objective of the present study was to clarify the pathological roles of CCR1 and IL10 loci identified by previous BD genome-wide association studies (GWASs). METHODS: M1 and M2 macrophages (Mφ) were differentiated with granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor (M-CSF) from peripheral monocytes of healthy control subjects (HC) and patients with BD. Expression of CD68 and CD163 was evaluated to test for Mφ polarization. CCR1 and IL-10 messenger RNA (mRNA) and protein expression was compared according to CCR1 and IL10 single-nucleotide polymorphism (SNP) genotypes. The migratory ability of M1 and M2 Mφ toward CCR1 ligand macrophage inflammatory protein (MIP)-1α was compared. The ratio of M1 and M2 Mφ in skin lesions of BD and systemic sclerosis (SSc), which was reported to be M2 Mφ-dominant, was compared. To examine the plasticity of polarized Mφ, the differentiated cells were cultured with either the same or the other culture condition. RESULTS: Preferential expression of CD163, CCR1, and IL-10 was found in M2 Mφ compared with M1 Mφ. M2 Mφ migrated more sensitively to low concentrations of MIP-1α than M1 Mφ did. BD-derived M1 Mφ showed higher CCR1 surface expression than HC-derived M1 Mφ did. IL10 and CCR1 mRNA expression differences were observed by GWAS-identified SNP genotypes in polarized Mφ. BD skin lesions showed M1 Mφ predominance compared with SSc skin lesions. A plasticity assay revealed that M-CSF restored IL-10 synthesis and reduced IL-6 production by M1 Mφ. CONCLUSIONS: The present study reveals that GWAS-identified SNPs contribute to M1 Mφ-predominant inflammation in BD. Our data also suggest that the skewed Mφ polarization is correctable by immunological intervention.

Dysregulated heme oxygenase-1low M2-like macrophages augment lupus nephritis via Bach1 induced by type I interferons.

BACKGROUND: Innate immunity including macrophages (Mϕ) in lupus nephritis (LN) has been gaining attention, but roles of Mϕ in LN remain uncertain. METHODS: Immunohistochemical staining was performed to determine CD68, CD163, heme oxygenase (HO)-1 (a stress-inducible heme-degrading enzyme with anti-inflammatory property), pSTAT1, and CMAF-expressing Mϕ in the glomeruli of patients with LN. Effects of type I interferons on the expression levels of CD163, HO-1, BTB and CNC homology 1 (Bach1; a transcriptional HO-1 repressor), interleukin (IL)-6, and IL-10 by human M2-like Mϕ, which were differentiated in vitro from peripheral monocytes with macrophage colony-stimulating factor, were assessed by RT-PCR and immunocytostaining. Clinical manifestations, anti-double-stranded DNA (anti-dsDNA), and local HO-1 expression were compared in Bach1-deficient and wild-type MRL/lpr mice. RESULTS: The number of glomerular M2-like Mϕ correlated with the amounts of proteinuria in patients with LN. Unlike monocyte-derived M2-like Mϕ, HO-1 expression was defective in the majority of glomerular M2-like Mϕ of patients with LN. Stimulation of human M2-like Mϕ with type I interferons led to reduced HO-1 expression and increased Bach1 and IL-6 expression. Bach1-deficient MRL/lpr mice exhibited increased HO-1 expression in kidneys, prolonged survival, reduced urine proteins, and serum blood urea nitrogen levels, but serum anti-dsDNA antibody levels were comparable. Increased expression of CD163 and HO-1 was found in peritoneal Mϕ from Bach1-deficient MRL/lpr mice. CONCLUSIONS: Our data suggest that dysregulated M2-like Mϕ play a proinflammatory role in LN. Bach1 is a potential therapeutic target that could restore the anti-inflammatory property of M2 Mϕ.

Accumulated α-synuclein affects the progression of GM2 gangliosidoses.

The accumulation of α-synuclein (ASyn) has been observed in several lysosomal storage diseases (LSDs) but it remains unclear if ASyn accumulation contributes to LSD pathology. ASyn also accumulates in the neurons of Sandhoff disease (SD) patients and SD model mice (Hexb-/- ASyn+/+ mice). SD is a lysosomal storage disorder caused by the absence of a functional ß-subunit on the ß-hexosaminidase A and B enzymes, which leads to the accumulation of ganglioside in the central nervous system. Here, we explored the role of accumulated ASyn in the progression of Hexb-/- mice by creating a Hexb-/- ASyn-/- double-knockout mice. Our results show that Hexb-/- ASyn-/- mice demonstrated active microglia levels and less dopaminergic neuron loss, without altering the neuronal storage of ganglioside. The autophagy and ubiquitin proteasome pathways are defective in the neurons of Hexb-/- ASyn+/+ mice. In ultrastructural physiological studies, the mitochondria structures look degenerated and dysfunctional. As a result, expression of manganese superoxide dismutase 2 are reduced, and reactive oxygen species-mediated oxidative damage in the neurons of Hexb-/- ASyn+/+ mice. Interestingly, these dysfunctions improved in Hexb-/- ASyn-/- mice. But any clinical improvement were hardly observed in Hexb-/- ASyn-/- mice. Taken together, these findings suggest that ASyn accumulation plays an important role in the pathogenesis of neuropathy in SD and other LSDs, and is therefore a target for novel therapies.

FOSL1 immunohistochemistry clarifies the distinction between desmoplastic fibroblastoma and fibroma of tendon sheath.

AIMS: Although desmoplastic fibroblastoma (DFB) and fibroma of tendon sheath (FTS) are well-established entities, they may show overlapping clinicopathological features. In addition, cytogenetic data showing a shared 11q12 rearrangement in a small number of cases suggest a close link between these entities. A recent microarray study revealed up-regulation of FOSL1 mRNA in DFBs with 11q12 rearrangement. The aim of this study was to clarify the relationship between DFB and FTS. METHODS AND RESULTS: We tested 42 cases diagnosed originally as either DFBs or FTSs for interobserver concordance based on the existing histological criteria and correlated the diagnosis with FOSL1 immunohistochemistry. In addition, FOSL1 gene status was determined by chromogenic in-situ hybridization (CISH). Using joint histological evaluation, 41 of 42 tumours were classified unanimously by three pathologists into 25 DFBs and 16 FTSs, whereas only one case received discordant opinions. Immunohistochemically, all DFBs showed diffuse, strong FOSL1 nuclear immunoreactivity (25 of 25, 100%), while none of the FTSs showed such overexpression. None of the selected 42 DFB mimics overexpressed FOSL1. FOSL1 was not rearranged in seven DFBs tested by CISH. CONCLUSIONS: We confirm here that DFB and FTS are two distinct entities that can be distinguished using the existing histological criteria. This distinction corresponds perfectly with FOSL1 immunohistochemical expression status, and diffuse strong FOSL1 expression specific to DFBs sharpens the border between the two categories. FOSL1 overexpression in DFB may not be caused directly by FOSL1 gene rearrangement. FOSL1 may also be a diagnostic aid for differentiating DFB from other histological mimics.

M2-polarized macrophages contribute to neovasculogenesis, leading to relapse of oral cancer following radiation.

Despite the fact that radiation is one of the standard therapies in the treatment of patients with oral cancer, tumours can recur even in the early stages of the disease, negatively impacting prognosis and quality of life. We previously found that CD11b(+) bone marrow-derived cells (BMDCs) were recruited into human glioblastoma multiforme (GBM), leading to re-organization of the vasculature and tumour regrowth. However, it is not yet known how these cells contribute to tumour vascularization. In the present study, we investigated the role of infiltrating CD11b(+) myeloid cells in the vascularization and recurrence of oral squamous cell carcinoma (OSCC). In a xenograft mouse model, local irradiation caused vascular damage and hypoxia in the tumour and increased infiltration of CD11b(+) myeloid cells. These infiltrating cells showed characteristics of M2 macrophages (M2Mφs) and are associated with the promotion of vascularization. M2Mφs promoted tumour progression in recurrence after irradiation compared to non-irradiated tumours. In addition, we found that CD11b(+) myeloid cells, as well as CD206(+) M2Mφs, are increased during recurrence after radiotherapy in human OSCC specimens. Our findings may lead to the development of potential clinical biomarkers or treatment targets in irradiated OSCC patients.

A morphometric study to establish criteria for fetal and neonatal cerebellar hypoplasia: A special emphasis on trisomy 18.

Cerebellar hypoplasia (CH) is one of the congenital abnormalities of the central nervous system and is seen in several diseases and syndromes. This study was conducted in order to examine methods for evaluating CH in fetus and neonate because CH has been diagnosed without any morphometric criteria at autopsy. We sampled 140 autopsied cases including nineteen trisomy 18 (T18), four non-T18 with presumed CH, and 117 control cases without any brain malformation. Statistical significance was present in the cerebellar weight and weight ratio of cerebellum per total brain between T18 and the control. The exponential regression models (ERM) showed that cerebral weight, cerebellar weight, and weight ratio of cerebellum per total brain increased gradually relative to gestational age in both T18 and the control. However, cerebellar weight and weight ratio of cerebellum per total brain of T18 showed growth delay with clear distinction between the two groups. The non-T18 with presumed CH showed similar results. Body weight, total brain, and gestational age should be considered totally when evaluating fetal and neonatal cerebellar development. Furthermore, the ERM results may be useful to evaluate the cerebellar development of fetus and neonate at autopsy.

Molecular biology of thermosensory transduction in C. elegans.

As the environmental temperature prominently influences diverse biological aspects of the animals, thermosensation and the subsequent information processing in the nervous system has attracted much attention in biology. Thermotaxis in the nematode Caenorhabditis elegans is an ideal behavioral paradigm by which to address the molecular mechanism underlying thermosensory transduction. Molecular genetic analysis in combination with other physiological and behavioral studies revealed that sensation of ambient temperature is mediated mainly by cyclic guanosine monophosphate (cGMP) signaling in thermosensory neurons. The information of the previously perceived temperature is also stored within the thermosensory neurons, and the consequence of the comparison between the past and the present temperature is conveyed to the downstream interneurons to further regulate the motor-circuits that encode the locomotion.

Establishment of successively transplantable rabbit VX2 cancer cells that express enhanced green fluorescent protein.

Morphological detection of cancer cells in the rabbit VX2 allograft transplantation model is often difficult in a certain region such as serosal cavity where reactive mesothelial cells mimic cancer cells and both cells share common markers such as cytokeratins. Therefore, tagging VX2 cells with a specific and sensitive marker that easily distinguishes them from other cells would be advantageous. Thus, we tried to establish a successively transplantable, enhanced green fluorescent protein (EGFP)-expressing VX2 model. Cancer cells obtained from a conventional VX2-bearing rabbit were cultured in vitro and transfected with an EGFP-encoding vector, and then successively transplanted in Healthy Japanese White rabbits (HJWRs) (n = 8). Besides, conventional VX2 cells were transplanted in other HJWRs (n = 8). Clinicopathological comparison analyses were performed between the two groups. The success rate of transplantation was 100% for both groups. The sensitivity and specificity of EGFP for immunohistochemical detection of VX2 cells were 84.3 and 100%, respectively. No significant differences in cancer cell morphology, tumor size (P = 0.742), Ki-67 labeling index (P = 0.878), or survival rate (P = 0.592) were observed between the two. VX2 cells can be genetically altered, visualized by EGFP, and successively transplanted without significant alteration of morphological and biological properties compared to those of the conventional model.

SIRT1 expression is associated with a poor prognosis, whereas DBC1 is associated with favorable outcomes in gastric cancer.

Clinical trials of histone deacetylase (HDAC) inhibitors as antitumor therapy have been conducted for gastric cancer. Expression of SIRT1, a class III HDAC, is related to poor prognosis in some malignancies. We investigated the correlation between SIRT1 expression and progression and prognosis of gastric cancers comparing with molecules linked to SIRT1 in order to better predict the efficacy of HDAC inhibitors in treating this disease. We evaluated SIRT1 expression by western blot in 51 cases and SIRT1, DBC1, acetylated H4K16 (H4K16Ac), acetylated H3K9 (H3K9Ac), and p53 by immunohistochemistry (IHC) in 557 cases of gastric cancer. Western blotting showed that SIRT1 high expression related with statistics to advanced tumor progression, positive lymphatic invasion, positive venous invasion, and advanced stage but not to poor prognosis. IHC revealed that SIRT1 high expression correlated with worse clinico-pathological prognostic factors as same as in western blotting and related poor prognosis both by univariate and multivariate analyses. By the contrast, DBC1 and H4K16Ac were related to favorable prognostic factors and linked to favorable prognosis by univariate analysis but not by multivariate analysis. H3K16Ac correlated only favorable prognostic factors. Results of p53 were very similar to those of SIRT1. We found that SIRT1 high expression closely correlates with progression and prognosis in gastric cancer patients. And it was also indicated that SIRT1 acts as an oncogene by the results of DBC1, H4K16Ac, and H3K9Ac and might be a target molecule of HDAC inhibitor treatment for gastric cancer patients.

[Pathological diagnosis of renal tumors: up to date].

Renal cell carcinoma (RCC) is composed of heterogenous histological types, with different clinical features and molecular biological characteristics. The "General Rule for Clinical and Pathological Studies on RCC" was revised in 2011, according to the World Health Organization system (WHO) (2004). Although most RCC is clear cell RCC (approximately 70-80% of RCC), papillary and chromophobe RCCs are occasionally encountered (approximately 10 and 5%, respectively). Collecting duct carcinoma is a rare and highly aggressive adenocarcinoma derived from the collecting duct. Additionally, several histological types have been introduced, and "granular RCC" has been omitted as a diagnostic term. For precise diagnosis, careful gross and histological evaluations are required along with immunohistochemical and molecular biological analyses. Additionally, novel histological types have recently been emerging, i.e., 6p21 translocation-associated RCC, dialysis-associated RCC, and tubulocystic carcinoma. Furthermore, mimics of RCC are always considered as differential diagnostic candidates, i.e., metanephric adenoma, epithelioid angiomyolipoma, juxtaglomerular cell tumor, and carcinoid tumor.

HER2 protein overexpression and gene amplification in upper urinary tract urothelial carcinoma-an analysis of 171 patients.

Upper urinary tract urothelial carcinomas (UUTUC) are infrequent and show an occurrence of about 5-10% of all urothelial carcinomas. In this study, we investigated the HER2 status of 171 UUTUC patients with nephroureterectomy. The number of patients is the largest of any HER2 study. All 171 cases were analyzed for both HER2 overexpression using immunohistochemistry and HER2 gene amplification using dual-color in situ hybridization. The scoring system proposed by the ASCO/CAP and ToGA trials was used. Out of 171 patients, 140 patients had a HER2 score-0 or score-1 (81.9%), 17 a score-2 (9.9%), and 14 a score-3 (8.2%) with immunohistochemistry. HER2 gene amplification was observed in 31 out of 171 cases (18.1%). A good correlation was observed between protein overexpression and gene amplification (p<0.0001). Twenty-three UUTUC (13.5%) were determined as HER2-positive cancer according to ASCO/CAP and ToGA criteria. HER2 positivity in patients over 70 years old was higher than that of patients under 70 years old (p=0.0132). HER2 expression correlated to a high histological grade (p=0.0003) and the coexistence of a high grade carcinoma in situ (p=0.0089). No HER2-positive cancer was observed in patients with renal pelvic UUTUC (0 out of 76, p<0.0001). HER2-positive UUTUC showed a shorter recurrence time in the residual urinary bladder after nephroureterectomy with Kaplan-Meier analysis (p=0.0284) and multivariate analysis (p=0.0034). The results suggest that HER2 positivity in UUTUC is an independent predictive marker for early recurrence of urothelial carcinoma in the residual urinary bladder after surgery.

Pathology of asthma.

Asthma is a serious health and socioeconomic issue all over the world, affecting more than 300 million individuals. The disease is considered as an inflammatory disease in the airway, leading to airway hyperresponsiveness, obstruction, mucus hyper-production and airway wall remodeling. The presence of airway inflammation in asthmatic patients has been found in the nineteenth century. As the information in patients with asthma increase, paradigm change in immunology and molecular biology have resulted in an extensive evaluation of inflammatory cells and mediators involved in the pathophysiology of asthma. Moreover, it is recognized that airway remodeling into detail, characterized by thickening of the airway wall, can be profound consequences on the mechanics of airway narrowing and contribute to the chronic progression of the disease. Epithelial to mesenchymal transition plays an important role in airway remodeling. These epithelial and mesenchymal cells cause persistence of the inflammatory infiltration and induce histological changes in the airway wall, increasing thickness of the basement membrane, collagen deposition and smooth muscle hypertrophy and hyperplasia. Resulting of airway inflammation, airway remodeling leads to the airway wall thickening and induces increased airway smooth muscle mass, which generate asthmatic symptoms. Asthma is classically recognized as the typical Th2 disease, with increased IgE levels and eosinophilic inflammation in the airway. Emerging Th2 cytokines modulates the airway inflammation, which induces airway remodeling. Biological agents, which have specific molecular targets for these Th2 cytokines, are available and clinical trials for asthma are ongoing. However, the relatively simple paradigm has been doubted because of the realization that strategies designed to suppress Th2 function are not effective enough for all patients in the clinical trials. In the future, it is required to understand more details for phenotypes of asthma.

Anti-interleukin-5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin-5 levels.

Atherosclerotic diseases, such as coronary artery disease and peripheral artery disease, are systemic disorders and among the leading causes of mortality and morbidity throughout the world. However, the exact pathophysiological mechanisms underlying the development of atherosclerosis remain unknown; currently, atherosclerosis is thought to involve an inflammatory process. Systemic inflammatory reactions and accumulation of immune cells in atherosclerotic lesions in situ are considered essential. We have comprehensively analyzed autoantibodies in patients with atherosclerosis by means of a newly developed high-throughput autoantibody analysis system. A wide range of autoantibodies was found in sera from patients with atherosclerosis. After we statistically analyzed the titers of each autoantibody with conventional techniques, the results underwent text-mining analyses based on natural language processing. Combinatory analysis revealed a close association between anti-interleukin (IL)-5 antibody and atherosclerosis. Titers of anti-IL-5 antibodies and serum IL-5 concentrations were also closely associated with other risk factors, such as low-density lipoprotein cholesterol, serum creatinine, fasting plasma glucose, gender, and age, suggesting that suppressed IL-5 function mediated by autoantibodies in patients with atherosclerosis plays an important role in the disease process. To validate the clinical significance of these findings, we computed the specificity and sensitivity of titers of anti-IL-5 autoantibodies for human atherosclerosis. When antibody titers of 1.49 were assumed to predict the presence of atherosclerosis, the sensitivity was 95.0% and the specificity 91.0%, with an area under the curve of 0.940. Our results provide important clues to understanding the role of autoantibody-mediated immune reactions in human atherosclerosis and suggest novel therapeutic opportunities for management of the disease.

SIRT1 expression is associated with good prognosis for head and neck squamous cell carcinoma patients.

OBJECTIVES: SIRT1, class III histone deacetylase, has been described to be up-regulated in various malignancies. However, the opposite results have been reported in other malignancies. Therefore, we investigated SIRT1 expression to clarify its biological behavior and identify its usefulness as a biomarker for head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: SIRT1 expression was assessed by immunohistochemistry (IHC) conducted using samples from 437 consecutive HNSCC patients. Acetylated histone status and p53 expression were also examined. RESULTS AND CONCLUSION: IHC revealed 79.6% staining of SIRT1 in HNSCC, while almost all normal tissues showed positive staining. SIRT1 expression predominated in cases involving patients aged >65 years, lymph node negative, and early clinical stage cases. It was positively and statistically correlated with expression of acetylated histone H3K9 and H4K16, but not with p53. Multivariate analyses revealed that expression of SIRT1 was an independent and good indicator of prognosis.

POU domain transcription factor BRN2 is crucial for expression of ASCL1, ND1 and neuroendocrine marker molecules and cell growth in small cell lung cancer.

BRN2 is a developmental neural cell-specific POU domain transcription factor and is crucial for cell lineage determination. We investigated the importance of BRN2 in the expression of the lineage-specific transcription factors (achaete-scute homolog-like 1 (ASCL1) and NeuroD1 (ND1)) and neural/neuroendocrine marker molecules (neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYP) and chromogranin A (CHGA)) in small cell lung cancer (SCLC) using cultured lung cancer cells. All examined SCLC cell lines expressed BRN2, as well as ASCL1, ND1, NCAM1, SYP and CHGA. The expression levels of ASCL1, ND1, NCAM1, SYP and CHGA considerably decreased when BRN2 was knocked down in SCLC cells, and the addition of a BRN2 transgene into non-SCLC (NSCLC) cells induced the expression of ASCL1, ND1, NCAM1, SYP and CHGA. However, the BRN2 gene was not activated by the forced expression of ASCL1 or ND1 in NSCLC cells. The knockdown of BRN2 caused significant growth retardation with decrease of S to G2 phase population and mitotic cell rates and unaltered Ki-67-labeled or apoptotic cell rates in SCLC cells, indicating increase of G1 phase population. These findings suggest that BRN2 is a higher level regulator than ASCL1 and ND1 and BRN2 might be involved in aggressiveness of SCLC.