Validity and reliability of the Korean version of the Speech Handicap Index in patients with oral cavity cancer.

The aims of this study were to evaluate the cross-cultural adaptation of the Speech Handicap Index (SHI) for Korean subjects and to determine its reliability and utility in patients with oral cavity cancer. The Korean version of the SHI was administered to 50 healthy subjects and 56 patients with speech problems resulting from treatment for oral cavity cancers. The content and construct validity, internal consistency, and test-retest reliability were examined. Healthy subject and patient group scores were compared, and the Mann-Whitney U-test was used to determine discriminatory ability. The Korean version of the SHI had high internal consistency (Cronbach's alpha=0.99) and test-retest reliability for the total and subscales: total (T) 0.98, speech (S) 0.99, and psychosocial (P) 0.97. Mean scores in the healthy group were 0.5 (T), 0.2 (S), and 0.2 (P), whereas those in the patient group were 34.3 (T), 16.6 (S), and 15.5 (P). The scores differed significantly between the groups (P<0.05). The Korean version of the SHI can be a useful tool to evaluate a patient's self-perception of their speech dysfunction in daily life and to better understand postoperative speech disorders in patients with oral cavity cancer.

Cigarette smoke mediates epigenetic repression of miR-217 during esophageal adenocarcinogenesis.

Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

JIP1 binding to RBP-Jk mediates cross-talk between the Notch1 and JIP1-JNK signaling pathway.

Notch1 signaling has a critical function in maintaining a balance among cell proliferation, differentiation, and apoptosis. Our earlier work showed that the Notch1 intracellular domain interferes with the scaffolding function of c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1), yet the effect of JIP1 for Notch1-recombining binding protein suppressor of hairless (RBP-Jk) signaling remains unknown. Here, we show that JIP1 suppresses Notch1 activity. JIP1 was found to physically associate with either intracellular domain of Notch1 or RBP-Jk and interfere with the interaction between them. Furthermore, we ascertained that JIP1 caused the cytoplasmic retention of RBP-Jk through an interaction between the C-terminal region of JIP1 including Src homology 3 domain and the proline-rich domain of RBP-Jk. We also found that RBP-Jk inhibits JIP1-mediated activation of the JNK1 signaling cascade and cell death. Our results suggest that direct protein-protein interactions coordinate cross-talk between the Notch1-RBP-Jk and JIP1-JNK pathways.

Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate.

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-beta, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

DJ-1 modulates UV-induced oxidative stress signaling through the suppression of MEKK1 and cell death.

DJ-1 is a multifunctional protein that performs functions in transcriptional regulation and oxidative stress, and the loss of its function is believed to result in the onset of Parkinson's disease (PD). In this study, we report that DJ-1 protects against UV-induced cell death through the suppression of the JNK1 signaling pathway. The results of both binding and kinase studies have revealed that MEKK1 is the direct target of DJ-1. The C-terminus of DJ-1 was crucial to the inhibition of the MEKK1 kinase activity. Wild-type DJ-1 sequesters MEKK1 within the cytoplasm and the L166P mutant facilitates the translocation of MEKK1 toward the nucleus without physical association. Both DJ-1 knockdown and pathogenic L166P mutant were determined to be highly susceptible to the UV-induced activation of the MEKK1-SEK1-JNK1 signaling cascade and cell death. Taken together, our findings show that missense mutation in DJ-1 sensitizes cells to stress-induced cell death through the MEKK1-SEK1-JNK1 signaling pathway, a process, which may trigger the early onset of PD.

Dynamic transcriptional regulatory complexes including BORIS, CTCF and Sp1 modulate NY-ESO-1 expression in lung cancer cells.

Previously, we reported that the paralogous zinc-finger proteins--CTCF and brother of the regulator of imprinted sites (BORIS), directly contribute to transcriptional regulation of NY-ESO-1 in lung cancer cells. To further examine mechanisms that mediate expression of this cancer-testis gene, we performed software-guided analysis of the NY-ESO-1 promoter region, which revealed several potential Sp1-binding motifs. Sequential 5-aza-2'deoxycytidine/depsipeptide FK228 treatment markedly induced BORIS expression and enhanced nuclear translocation of Sp1 in lung cancer cells. Transient transfection assays using promoter-reporter constructs, as well as gel-shift and chromatin immunoprecipitation experiments revealed that NY-ESO-1 promoter activity coincided with occupancy of the proximal Sp1-binding site in lung cancer cells. Mutations within the Sp1 recognition sequence specifically eliminated binding of Sp1 to this motif in vitro, and markedly diminished NY-ESO-1 promoter activity in vivo. siRNA-mediated inhibition of Sp1 expression decreased NY-ESO-1 promoter activity, whereas knock down of CTCF expression augmented NY-ESO-1 transcription in lung cancer cells. Co-immunoprecipitation experiments indicated that Sp1 physically interacts with BORIS but not with CTCF in vivo. Collectively, these findings suggest that BORIS recruits Sp1 to mediate de-repression of NY-ESO-1 during pulmonary carcinogenesis.

Sequential 5-Aza-2 deoxycytidine-depsipeptide FR901228 treatment induces apoptosis preferentially in cancer cells and facilitates their recognition by cytolytic T lymphocytes specific for NY-ESO-1.

Global alterations in chromatin structure profoundly influence gene expression in thoracic neoplasms, silencing tumor suppressors while facilitating the expression of various cancer testis antigens such as NY-ESO-1. Although recent studies have shown that histone deacetylase inhibitors can potentiate tumor suppressor gene induction mediated by demethylating agents in cancer cells, the ability of these agents to augment cancer testis antigen expression have not been fully defined. The authors designed the current study to determine whether the histone deacetylase inhibitor, depsipeptide FR901228 (DP), could enhance NY-ESO-1 induction mediated by the DNA demethylating agent 5-Aza-2'-deoxycytidine (DAC) in cell lines established primarily from thoracic cancers. Quantitative reverse-transcriptase polymerase chain reaction analysis revealed that, under exposure conditions potentially achievable in clinical settings, DAC dramatically induced NY-ESO-1 expression in cultured cancer lines. DP alone mediated negligible target gene induction but significantly augmented DAC-mediated induction of NY-ESO-1. After DAC or sequential DAC-DP treatment, HLA-A*0201 cancer cells were recognized by an HLA-A*0201 CTL specific for NY-ESO-1. Although sequential DAC/DP exposure did not uniformly enhance immune recognition of target cells compared with DAC alone, this treatment mediated profound induction of apoptosis in cancer cells but not normal human bronchial epithelia. The apoptotic effects of DAC, DP, or sequential DAC-DP did not correlate in an obvious manner with histology, or the magnitude of NY-ESO-1 induction in cancer cells. Although the mechanisms have not been fully defined, sequential DAC-DP treatment may be a novel strategy to augment antitumor immunity in cancer patients.

Identification and isolation of differentially expressed genes in osmotically stressed human oral keratinocytes.

Complementary DNA fragments which showed differential expression relative to unstressed controls were identified and isolated from human oral keratinocytes exposed to hyperosmotic stress. The up- or downregulation of the expression of nine of these cDNAs in response to osmotic stress was determined by Northern blotting. Sequence analysis showed that clones K-5 and K-46 contained identical sequences. Homology searches revealed that K-13 and K-33 were fragments of unknown genes. Among the upregulated cDNAs, K-16 and K-32 were 94 and 83% identical to chromosome 16 bacterial artificial chromosome (CIT987K-A-418G10) and a cDNA (ai49b01.sl) clone, respectively. Another clone, K-34, encoded a protein 73% identical to Bax epsilon. Among the downregulated genes, K-5/46 and K-45 were 99% identical to the og24d08.s1 cDNA clone and to mitochondrial genes for tRNAs and 12S and 16S ribosomal RNAs, respectively, while K-50 was 100% identical to KIAA0905 protein. The gene expression induced by osmotic stress occurred in parallel with the induction of apoptosis and a reduction in protein biosynthesis. This observation, together with the characteristics of the some of the differentially expressed genes, suggests that among the major events induced in oral keratinocytes by hyperosmotic stress are the induction of apoptosis and a decrease in protein biosynthesis, brought about by upregulation of pro-apoptotic genes and downregulation of genes involved in protein biosynthesis.

Induction of MAGE-3 expression in lung and esophageal cancer cells.

BACKGROUND: Although MAGE-3 has been detected in approximately 40% of lung and esophageal cancers, expression of this cancer testis antigen appears to be below the threshold for immune recognition in patients with these malignancies. The aim of this study was to determine if the demethylating agent, 5-Aza-2'-deoxycytidine (DAC) and if the histone deacetylase inhibitor Depsipeptide FR901228 (DP) could enhance MAGE-3 expression in lung and esophageal cancer cells. METHODS: Eleven lung and esophageal cancer lines and cultured normal human bronchial epithelial (NHBE) cells were exposed to normal media (NM), DAC, DP, or combination DAC/DP at varying concentrations and exposure durations. MAGE-3 expression was evaluated by quantitative RT-PCR (TaqMan) and immunohistochemistry techniques. Trypan blue exclusion techniques were used to examine the proliferation of cancer cells after drug exposure. RESULTS: Relative to untreated controls, MAGE-3 expression was enhanced 32-fold (range 3.9 to 110) by DAC alone (0.1 micromol/L x 72 h), 2.1-fold (0.4 to 4.2) by DP alone (25 ng/mL x 6h), and 57-fold (4.6 to 209) by sequential DAC/DP exposure. Increased MAGE-3 mRNA copy numbers coincided with enhanced protein levels in these cells. MAGE-3 expression persisted after drug exposure. Flow cytometry confirmed the presence of functional HLA class I expression in these cells. Sequential DAC/DP treatment mediated pronounced growth inhibition in cancer cells but not NHBE. CONCLUSIONS: Sequential DAC/DP treatment may be a novel strategy to simultaneously augment MAGE-3 expression and induce growth arrest in thoracic malignancies.

The dynamics of the vestibulo-ocular reflex after peripheral vestibular damage. II. Comparison with dynamics after optically induced learning.

The vestibulo-ocular reflex (VOR) stabilizes gaze adequately under a variety of conditions because it is capable of a simple form of motor learning. Learning is induced by changed visual conditions or to compensate for vestibular sensory loss. We asked whether the mechanisms that are triggered by visual signals can fully account for recovery from vestibular damage. We addressed this question by comparing the effects of optically induced motor learning (i.e., changes in gain induced by telescopic lenses) and recovery from a unilateral horizontal canal plug on the dynamics of the cat VOR. Optically induced learning modified the gain of the VOR more effectively for rotation at low frequencies (below 5 Hz) than for higher-frequency stimuli. During recovery from a plug, the gain of the VOR increased at all frequencies tested, with a similar time course for all frequencies. After recovery the gain for rotation at 5 Hz or above was relatively enhanced. After recovery reached its upper limit, optically induced learning could bring about further changes in gain. The results are interpreted with respect to partially (but not completely) shared mechanisms for optically induced learning and recovery after a unilateral canal plug.

Increased transmission by direct vestibulo-ocular reflex pathways after peripheral vestibular damage: a preliminary report.

The vestibulo-ocular reflex (VOR) allows clear vision during head movements by generating compensatory eye movements. Its response is reduced following damage to the vestibular endorgan, but recovers over time. The VOR is mediated by both direct and indirect anatomical pathways; most direct pathways include only two central synapses, both located in the brainstem. To investigate the possibility that a direct pathway is modified during the recovery of VOR gain, we measured the oculomotor response to single current pulses delivered to the vestibular labyrinth of two alert cats after plugging the contralateral horizontal canal. The response was also measured after motor learning induced by continuously worn lenses (optically induced motor learning) in two cats. The gain of the VOR was monitored concurrently. The eye movement evoked by a current pulse increased more than 100% during recovery from a plug. The electrically evoked eye movement did not change during optically induced motor learning either before the plug or after recovery. The gain of the VOR was modified in both situations. We conclude that direct VOR pathways are modified significantly during recovery after a plug, but not during optically induced learning. Our results suggest that significant modification of direct pathways may require a change in vestibular sensory input.