Dominant-negative p53 mutant R248Q increases the motile and invasive activities of oral squamous cell carcinoma cells.

The tumor suppressor gene TP53 (gene) codes for a transcription factor which transactivates its target genes responsible for cell cycle arrest, DNA repair, apoptosis, and senescence. TP53 is well known to be the most frequent target of genetic mutations in nearly half of human cancers including oral squamous cell carcinoma (OSCC). Many p53 mutants including R248Q and R248W not only lose its tumor-suppressor activities, but also interfere with the functions of wild-type p53; this is so-called dominant-negative (DN) mutation. The DN p53 mutation is a predictor of poor outcome in patients with various cancers, and also a risk factor for metastatic recurrence in patients with OSCC. Recently it has been reported that DN p53 mutants acquire new oncogenic activities, which is named gain-of-function (GOF). This study aimed at determining whether R248Q and R248W were involved in OSCC cells' acquiring aggressive phenotypes, using SAS, HSC4 and Ca9-22 cell lines. First, two mutants p53, R248Q and R248W, were respectively transfected into SAS cells harboring recessive-type p53 (E336X). As a result, SAS cells expressing R248Q showed highly spreading, motile and invasive activities compared to parent or mock-transfected cells whereas those expressing R248W did not increase those activities. Secondly, in HSC4 cells harboring R248Q and Ca9-22 cells harboring R248W, expressions of the mutants p53 were inhibited by the transfection with siRNAs targeting p53. The inhibition of the mutants p53 decreased spreading, motile and invasive activities of HSC4 cells whereas it did not affect those activities of Ca9-22 cells. These findings suggest that R248Q p53 mutation, but not R248W p53 mutation, induces more motile and invasive potentials in human OSCC cells.

Evaluation of the Japanese Metabolic Syndrome Risk Score (JAMRISC): a newly developed questionnaire used as a screening tool for diagnosing metabolic syndrome and insulin resistance in Japan.

OBJECTIVES: To prevent the onset of lifestyle-related diseases associated with metabolic syndrome (MetS) in Japan, research into the development of a useful screening method is strongly desired. We developed a new screening questionnaire (JAMRISC) utilizing a logistic regression model and evaluated its ability to predict the development of MetS, type 2 diabetes and other lifestyle-related diseases in Japanese populace. METHODS: JAMRISC questionnaire was sent to 1,850 individuals in Rumoi, a small city in Hokkaido. We received a total of 1,054 valid responses. To maximize the target individuals accurately diagnosed with MetS, logistic regression analysis was used to generate a unique metabolic syndrome score calculation formula as taking into consideration the clinical relevance of each question item as individual coefficients. RESULTS: The results of our comparative research utilizing both JAMRISC and Finnish Diabetes Risk Score (FINDRISC) questionnaires revealed the usefulness of JAMRISC for its ability to detect risks for MetS, pre-MetS, diabetes, and pre-diabetes. Study of disease risk detection via JAMRISC questionnaire targeting the 4283 residents of Rumoi indicated a high detection rate for pre-MetS (98.8 %), MetS (94.2 %), pre-diabetes (85.1 %) and type 2 diabetes (94.9 %). In addition, JAMRISC was useful not only as a MetS risk score test, but also as a screening tool for diagnosing insulin resistance. CONCLUSIONS: JAMRISC questionnaire is a useful instrument for the detection of early risk of not only MetS and type 2 diabetes but also insulin resistance.

Antidepressant dose and treatment response in bipolar depression: Reanalysis of the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD) data.

The aim of this study is to evaluate whether an adjunct antidepressant therapy at a higher dose to a mood stabilizer would make a difference in the treatment of bipolar depression. This is a post-hoc analysis of the data from the randomized treatment for acute depression of the Systematic Treatment Enhancement Program for Bipolar Disorder (STEP-BD), in which patients with bipolar depression were randomly assigned to treatment with a mood stabilizer plus adjunctive antidepressant drugs or placebo. According to the highest dose received in the course of treatment, the subjects were divided into one of the following three groups: high-dose, low-dose and placebo groups. The primary and secondary outcomes were durable recovery (which was operationally defined as eight consecutive weeks with

Comparison of clinical management of young and elderly asthmatics by respiratory specialists and general practitioners.

BACKGROUND: Asthmatic death in the elderly is a serious problem worldwide. Differences in clinical skill between respiratory specialists (RS) and general practitioners (GP) are important in asthma control. The aim of this study was to compare asthma management between RS and GP. METHODS: A cross-sectional survey was carried out in Shimane, Japan, in February 2009 using a questionnaire about patient background, treatment, asthma control test (ACT) and adherence to treatment. We secured the cooperation of 48 clinics (39 private clinics and 9 general hospitals). Asthmatics were divided into the elderly and young groups, and also into the RS and GP groups. RESULTS: Clinical data of 779 patients were available for analysis. Elderly patients constituted 464 (RS group: 192, GP group: 272), while those of the young group were 315 (RS group: 207, GP group: 108). RS prescribed inhaled corticosteroids (ICSs) to their elderly and young patients more than GP. The total ACT score was higher in young RS group than in young GP group, but no such difference was noted in the elderly. Despite more asthma-related symptoms, the ACT showed that elderly GP asthmatics used fewer rescue inhalers than elderly RS. Self-assessment was higher in elderly GP than elderly RS asthmatics. Adherence to therapy was better in elderly patients than young patients. CONCLUSIONS: Elderly asthmatics treated by GPs underestimated the severity of their asthma and asthmatics seen by GPs were undertreated. The results stress the need to engage patients in educational activities, to adhere to guidelines, and to improve the coordination between GP and RS.

Phase I clinical and pharmacokinetic study of bi-weekly carboplatin/paclitaxel chemotherapy in elderly patients with advanced non-small cell lung cancer.

AIM: We evaluated the pharmacokinetics and quality of life of elderly patients with advanced non-small-cell lung cancer (NSCLC) treated with bi-weekly carboplatin and paclitaxel chemotherapy, and determined the maximum tolerated dose (MTD) of this treatment. PATIENTS AND METHODS: Eligible patients had histologically- or cytologically-proven inoperable NSCLC, age of 70 years or older, no prior treatment, and Eastern Cooperative Oncology Group performance status 0-2. Paclitaxel was administered in combination with carboplatin under a bi-weekly schedule. We determined the plasma concentrations of both drugs during therapy. RESULTS: The median patient age was 80 years. Using carboplatin at AUC 3, the MTD of paclitaxel was 100 mg/m(2). Both hematological and non-hematological toxicities were mostly mild and manageable. Although paclitaxel is predominantly metabolized in the liver, clearance was decreased in patients with lower estimated glomerular filtration rate. CONCLUSION: Bi-weekly treatment, as described here, is feasible for elderly patients as a conventional regimen, particularly in the outpatient setting, due to its lower toxicity.

Increased expression of the PRL-3 gene in human oral squamous cell carcinoma and dysplasia tissues.

Phosphatase of regenerating liver (PRL) belongs to a class of the protein tyrosine phosphatase family, which is known so far to consist of 3 members, PRL-1, PRL-2, and PRL-3. The aim of this study was to uncover the role of PRL genes in development of oral malignancy. We analyzed expression levels of the 3 PRL genes in 50 human oral squamous cell carcinomas (OSCCs), 11 dysplasia and 12 normal mucosa tissues by a real-time RT-PCR method. PRL-3 but not PRL-1 or PRL-2 expressions were significantly higher in OSCC and dysplasia than in normal mucosa tissues. Additionally, PRL-3 expressions were significantly higher in OSCC tissues harboring dominant-negative p53 or recessive p53 mutation than in those harboring wild-type p53. These results suggest that PRL-3 plays a role in oral cancer development and can be useful as a marker of pre-malignant and malignant lesion of oral mucosa.

Mutant p53 R248Q but not R248W enhances in vitro invasiveness of human lung cancer NCI-H1299 cells.

More than half of all human cancers are associated with mutations of the TP53 gene. In regard to the functional interaction with the remaining wild-type (WT) p53 allele, p53 mutations are classified into two types, recessive and dominant-negative (DN) mutations. The latter mutant protein has a DN activity over the remaining WT allele. We previously showed that the DN p53 mutant was useful as a predictor of poor outcome or a risk factor for metastatic recurrence in patients with some types of cancers, regardless of the presence or absence of loss of heterozygosity (LOH) of WT p53, suggesting that the DN p53 had 'gain-of-function (GOF)' activity besides the transdominance function. In this study, we investigated GOF activity of two DN p53 mutants which had a point mutation at codon 248 (R248Q and R248W), one of the hot spots, by transfecting them respectively into H1299 cells which originally expressed no p53 protein. Growth activity of the transfectants with the two mutants was not different from that of parent or Mock transfectants. Meanwhile, in vitro invasions of Matrigel and type I collagen gel by R248Q-transfectants were significantly higher than those by R248W-transfectants or the control cells. However, there were no differences in cell motile activities, expressions of extracellular matrix-degradative enzymes such as matrix metalloproteinases, urokinase-type plasminogen activator and heparanase, and their inhibitors, between R248Q- and R248W-transfectants. These findings indicate that the p53 mutants have a different quality in GOF activities even if the mutations occurred at the same codon. And detailed information of the status of p53, including transdominancy and GOF activity, is expected to be useful for diagnosis and therapeutic strategy fitting the individual patients.

A multiple primary carcinoma consisting of leukoplakia and SCC: a case report with p53 mutation analysis.

Patients with an oral squamous cell carcinoma (OSCC) often develop multiple malignant lesions. This report examined whether individual tumours developed in a patient show the same genetic alteration, such as p53 mutations. This case study describes three SCCs and three leukoplakias which developed simultaneously in a single 67-year-old Japanese man. A p53 mutation was detected in two of the three SCCs and one of the three leukoplakias. One SCC had a missense mutation at codon 285 (GAG>AAG, Glu>Lys) and the other a nonsense mutation at codon 336, and the leukoplakia had a missense mutation at codon 273 (CGT>CAT, Arg>His). This case showed that individual oral tumours may have different genetic changes even when they develop in a single patient. Therefore, this report provided strong evidence that in cases of multiple tumours it is necessary to design tailor-made therapies for each individual tumour rather than a single standardised therapy for all multiple tumours.

Imatinib mesylate inhibits the proliferation-stimulating effect of human lung cancer-associated stromal fibroblasts on lung cancer cells.

Platelet-derived growth factor (PDGF) is a significant mediator in the proliferation of cancer-associated stromal fibroblasts (CAFs). The inhibition of CAF proliferation by blocking PDGF signaling could lead to a development of novel cancer therapy. We analyzed whether inhibiting proliferation of lung CAFs by imatinib mesylate, which has inhibitory activity on PDGF-receptor tyrosine kinase, could suppress the proliferative activity of lung cancer cells which coexisted in the tumor tissue. First, we established primary cultured fibroblasts from human lung cancer tissues. RT-PCR analysis showed that PDGF-receptors (PDGFRalpha and beta) were more highly expressed in the fibroblasts, whereas PDGFs (PDGF-A, and -B) were more in lung cancer cell lines. Western blotting showed that imatinib treatment inhibited phosphorylation of PDGFRbeta, Akt, and Erk1/2 in the fibroblasts. The treatment also significantly inhibited the proliferative activity of the fibroblasts. The inhibitory effects were exerted more definitely in co-administering imatinib and PDGF-BB, a dimer of the polypeptide chains of B, than in administering imatinib alone. The conditioned media of the fibroblasts significantly increased the proliferative activity of human lung cancer cell line A549 compared to control culture medium. The proliferation-stimulating effect on A549 cells decreased significantly in the conditioned media of the primary cultured fibroblasts that had been treated with imatinib. Our results suggest that imatinib has antitumor activity which is exerted by reducing the proliferation-stimulating effect of CAFs on lung cancer cells, as well as inhibiting the proliferation of CAFs, by way of blocking PDGF signaling.

Array comparative genomic hybridization analysis revealed four genomic prognostic biomarkers for primary gastric cancers.

Unlike the case with some other solid tumors, whole genome array screening has not revealed prognostic genetic aberrations in primary gastric cancer. Comparative genomic hybridization (CGH) using bacterial artificial chromosome (BAC) arrays for 56 primary gastric cancers resulted in identification of four prognostic loci in this study: 6q21 (harboring FOXO3A; previously FKHRL1), 9q32 (UGCG), 17q21.1 approximately q21.2 (CASC3), and 17q21.32 (HOXB3 through HOXB9). If any one of these four loci was deleted, the prognosis of the patient was significantly worse (P = 0.0019). This was true even for advanced tumors (stage IIIA, IIB, or IV, n = 39) (P = 0.0113), whereas only 1 of the 17 patients with less advanced tumors (stage IA, IB, or II; n = 17) died of disease after surgery. Multivariate analysis according to the status of four BACs or pathological stage based on the Japanese Classification of Gastric Carcinoma (stages IA, IB, and II vs. stages IIIA, IIIB, and IV) demonstrated that the BAC clone status was also an independent prognostic factor (P = 0.006). These findings may help predict which patients with malignant potential need more intensive therapy, or may point to new therapeutic approaches especially for advanced tumors. The parameter here termed the integrated genomic prognostic biomarker may therefore be of clinical utility as a prognostic biomarker.

Aberrant expressions of HOX genes in colorectal and hepatocellular carcinomas.

HOX genes are known as master regulator genes which give cells positional information in embryogenesis. In this study, we compared the expression patterns of 39 HOX genes among human colorectal carcinomas from the right large intestine (cecum, ascending and transverse colon), those from the left large intestine (discending and sigmoid colon, and rectum) and hepatocellular carcinoma. The expression levels of each HOX gene were quantified by analysis based on the real-time RT-PCR. The expression patterns of HOX genes in colorectal and hepatocellular carcinoma tissues differed from those in their normal or non-cancerous tissues. Between the tumor tissues in the right-side large intestine and those in the left-side, different HOX genes were expressed in association with cancer. Further, the expression levels of HOXD8 in liver-metastatic tissues of colorectal carcinomas were as low as in non-cancerous liver tissues, and were significantly lower than those in the primary tissues. These results suggest that dysregulated expressions of HOX genes play an important role in carcinogenesis and malignant progression of colorectal and hepatocellular carcinomas.

Laminin-421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells.

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC-CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat-treatment. The chemotactic activity of LEC-CM was abolished by immunodepletion with anti-laminin-1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC-CM contained laminin-421. When melanoma C8161 cells were treated with function-blocking antibodies to integrin alpha3 or alpha6, their chemotactic responses to LEC-CM were markedly reduced. Furthermore, the knock-down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC-CM. These data suggest that laminin-421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.

PAX4 has the potential to function as a tumor suppressor in human melanoma.

We hypothesize that dysregulated expression levels of the developmental regulatory genes in the adult body result in tumor development and malignant progression. PAX genes discovered as human orthologous genes of Drosophila 'paired' encode transcription factors, which control the expression of target genes to go on along the program of development. In this study, we first quantified expression of 9 PAX genes in human nevus pigmentosus tissues, melanoma tissues and melanoma cell lines by the real-time reverse transcription-PCR method. As a result, we found that the expression levels of PAX4 and PAX9 were extremely low in melanoma tissues and cell lines compared to nevus pigmentosus tissues. We then established melanoma cells overexpressing PAX4 and examined roles of PAX4 in cell growth. PAX4-overexpression reduced in vitro cell growth of human melanoma C8161 and MeWo cells. BrdU-uptake assay and cell cycle analysis by flow cytometry indicated that the retardation of cell proliferation by PAX4-overexpression was due to decreased DNA synthesis and cell cycle arrest at the G0/G1 phase. Furthermore, treatment of C8161 and MeWo cells with 5-azacytidine, a DNA demethylating agent, induced the expression of PAX4, suggesting that DNA methylation repressed the PAX4 gene expression in human melanoma. These results suggest that PAX4 functions as a potent tumor suppressor.

Presence of dominant negative mutation of TP53 is a risk of early recurrence in oral cancer.

Genetic alteration of p53 is a significant determining factor in the carcinogenesis. The loss of function, mutant p53 can possess a dominant negative effect on wild-type p53 and may also exert gain-of-function activity. It is, however, not clear how p53 functional status due to various types of mutation results in outcome of patients with oral cancer. A total of 60 oral SCC samples were subjected to yeast functional assay that screens human p53 function in yeast, and sequencing for determination of p53 mutations. The detected mutants were further investigated for their dominant negative activity using a yeast-based transdominance assay that tests dominant negative activity of a mutant p53 over wild-type p53 by coexpressing the mutant and wild-type p53 in a yeast transcriptional reporter system. p53 mutation was found in 42 out of 60 of which 10 (24%) exhibited dominant negative activity and 32 (76%) without dominant activity (recessive mutation). The remaining 18 (30%) were considered to have wild-type p53. The patients with dominant negative mutation had significantly shorter disease-free survival than patients with no mutation (log-rank test, p<0.001) and those with a recessive mutation (p<0.016). There were slight significant differences in disease-free survival were found between the patients with tumours harbouring a recessive p53 mutation and those with tumours harbouring a wild-type p53 (p<0.038). The presence and absence of a dominant negative p53 mutation may thus provide a predictor of early recurrence in oral SCC patients.

Infiltration of Epstein-Barr virus-harboring lymphocytes occurs in a large subset of bladder cancers.

OBJECTIVE: Epstein-Barr virus (EBV) has been implicated in the genesis of a variety of human cancers. We aimed to confirm the presence and define the role of EBV in bladder cancer. METHODS: A total of 39 bladder cancer specimens were analyzed. Ten urinary bladder tissues obtained at autopsy were used as a normal control. EBV-encoded RNA (EBER) was evaluated by in situ hybridization (ISH). Frozen material available from 18 EBER-positive cases was analyzed by using reverse transcription-polymerase chain reaction for BZLF1, an early lytic gene product. The expression of CD20, CD3, ZEBRA (BZLF1 product) and transforming growth factor beta1 (TGFbeta-1) was assessed using an immunohistochemical technique. RESULTS: Infiltration of EBER-expressing lymphocytes was detected in 26 of 39 bladder cancer cases (66.7%). A small fraction of the tumor cells as well as the infiltrating lymphocytes were positive in two cases. All normal urinary bladder specimens showed negative results. The incidence of EBV-positive lymphocyte infiltration was significantly higher for advanced stage cancers than those in earlier stages (Ta-152% vs T2-4 93%, P = 0.013). The presence of BZLF1 mRNA was demonstrated in seven out of the 18 EBER-positive cases. CONCLUSIONS: Infiltration of EBV-harboring lymphocytes occurs in a large subset of bladder cancers cases. It is more frequently associated with advanced stages. EBV infection in tumor cells is very limited. Our findings suggest that EBV-positive lymphocytes might play a role in bladder cancer progression.

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics.

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.

High MMP-1 mRNA expression is a risk factor for disease-free and overall survivals in patients with invasive breast carcinoma.

BACKGROUND: Matrix metalloproteinase 1 (MMP-1) degrades extracellular matrix and thereby promotes tumor invasion and progression. In this study we examined the prognostic significance of tissue expression levels of MMP-1 mRNA in patients with invasive breast carcinoma. MATERIALS AND METHODS: We assessed the prognostic value of MMP-1 mRNA expression in tumor tissue specimens from 85 breast carcinoma patients with a median follow-up time of 38 months (range, 2-48 months). MMP-1 mRNA levels were measured by real-time quantitative reverse transcriptase polymerase chain reaction (real time RT-PCR). The results were correlated with various clinicopathological parameters and clinical outcomes. RESULTS: mRNA expression levels of MMP-1 were higher in tumor tissue specimens than in adjacent normal breast tissue specimens from 15 patients (P < 0.023). MMP-1 mRNA levels showed no significant relationship with either tumor size or axillary node status but correlated inversely with estrogen receptor levels (P < 0.0043). High MMP-1 mRNA expression as determined by real-time RT-PCR correlated significantly with a high frequency of recurrence and fatal outcome (P < 0.025 and P < 0.020). Multivariate analysis using the Cox regression model indicated that high MMP-1 mRNA expression was an independent unfavorable prognostic factor (risk ratio, 6.37; P < 0.019). CONCLUSIONS: We have demonstrated for the first time the high mRNA expression of MMP-1 in patients whose carcinomas lack estrogen receptor expression. Our results suggest that MMP-1 is an important gene implicated in the progression of human breast cancer.

Prognostic values of matrix metalloproteinase family expression in human colorectal carcinoma.

BACKGROUND: We examined expression patterns of matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP), and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in colorectal cancer tissues to assess their prognostic significance. MATERIALS AND METHODS: mRNA expressions of 17 MMPs, 4 TIMPs, and RECK were measured in 112 colorectal cancerous tissues, 20 normal mucosa tissues, and 11 metastatic liver lesions by real-time reverse-transcriptional-polymerase chain reaction. The protein level expressions were confirmed with immunohistochemistry. RESULTS: Cancers and normal mucosa displayed highly significant differences (P < 0.01) in expression of nine genes (MMP-1, -3, -7, -9, -10, -11, -12, -14, and RECK). Primary cancers and metastatic lesions showed highly significant differences (P < 0.01) in MMP-1, -10, -11, and TIMP-1. MMP-12 expression was higher in the primary tumors that were associated without hepatic metastasis than those with metastasis (P < 0.01). High expression of MMP-15 was related to longer disease-free survival (generalized Wilcoxon test, P < 0.0062; Cox hazard model, P < 0.028, hazard ratio, 0.099). CONCLUSIONS: MMP, TIMP, RECK expression patterns may provide an insight into extracellular matrix degrading (which is characteristic of colorectal cancers) and its role in metastasis.