John D. Kelly

Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers

A number of histone demethylases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human disease like cancer have not been well understood. Here, we demonstrate important roles of lysine‐specific demethylase 1 (LSD1) in human carcinogenesis. Expression levels of LSD1 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001). cDNA microarray analysis also revealed its transactivation in lung and colorectal carcinomas. LSD1‐specific small interfering RNAs significantly knocked down its expression and resulted in suppression of proliferation of various bladder and lung cancer cell lines. Concordantly, introduction of exogenous LSD1 expression promoted cell cycle progression of human embryonic kidney fibroblast cells. Expression profile analysis showed that LSD1 could affect the expression of genes involved in various chromatin‐modifying pathways such as chromatin remodeling at centromere, centromeric heterochromatin formation and chromatin assembly, indicating its essential roles in carcinogenesis through chromatin modification.

Dysregulation of PRMT1 and PRMT6, Type I arginine methyltransferases, is involved in various types of human cancers

Protein arginine methylation is a novel post‐translational modification regulating a diversity of cellular processes, including histone functions, but the roles of protein arginine methyltransferases (PRMTs) in human cancer are not well investigated. To address this issue, we first examined expression levels of genes belonging to the PRMT family and found significantly higher expression of PRMT1 and PRMT6, both of which are Type I PRMTs, in cancer cells of various tissues than in non‐neoplastic cells. Abrogation of the expression of these genes with specific siRNAs significantly suppressed growth of bladder and lung cancer cells. Expression profile analysis using the cells transfected with the siRNAs indicated that PRMT1 and PRMT6 interplay in multiple pathways, supporting regulatory roles in the cell cycle, RNA processing and also DNA replication that are fundamentally important for cancer cell proliferation. Furthermore, we demonstrated that serum asymmetric dimethylarginine (ADMA) levels of a number of cancer cases are significantly higher than those of nontumor control cases. In summary, our results suggest that dysregulation of PRMT1 and PRMT6 can be involved in human carcinogenesis and that these Type I arginine methyltransferases are good therapeutic targets for various types of cancer.

Towards a phonology of conversation: turn-taking in Tyneside English

Remarkably little is known in detail about the phonetics and phonology of naturally occurring conversational talk. Virtually nothing of interest is known of the interactional implications of particular kinds of phonetic events in everyday talk: in particular about the ways in which participants in talk deploy general phonetic resources to accomplish specific interactional tasks. This is in part a consequence of the tendency of recent research on the phonological aspect of discourse to limit itself to ‘intonation’ as an area of primary interest. This work has moved away from the type of phonological analysis, such as that of Halliday (1967), that states intonational systems in terms of grammatically defined units or sentence types. Workers such as Brazil (1975, 1978, 1981), Brown, Currie and Kenworthy (1981), and Coulthard and Brazil (1981) have pursued Bolingers suggestion that the relationship between intonation and grammar is ‘casual not causal’ and have sought to relate ‘intonation’ to discourse categories rather than to grammatical ones. These, and similar attempts to deal with aspects of discourse phonology, have suggested some organizational features which traditional linguistic accounts have not dealt with. On the whole, however, these recent attempts have been less than satisfactory for one or more of the following reasons.

Overexpression of the JmjC histone demethylase KDM5B in human carcinogenesis: involvement in the proliferation of cancer cells through the E2F/RB pathway

BackgroundAlthough an increasing number of histone demethylases have been identified and biochemically characterized, their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. We investigated the role of KDM5B, a JmjC histone demethylase, in human carcinogenesis. Quantitative RT-PCR and microarray analyses were used to examine the expression profiles of histone demethylases in clinical tissue samples. We also examined the functional effects of KDM5B on the growth of cancer cell lines treated with small interfering RNAs (siRNAs). Downstream genes and signal cascades induced by KDM5B expression were identified from Affymetrix Gene Chip experiments, and validated by real-time PCR and reporter assays. Cell cycle-dependent characteristics of KDM5B were identified by immunofluorescence and FACS.ResultsQuantitative RT-PCR analysis confirmed that expression levels of KDM5B are significantly higher in human bladder cancer tissues than in their corresponding non-neoplastic bladder tissues (P < 0.0001). The expression profile analysis of clinical tissues also revealed up-regulation of KDM5B in various kinds of malignancies. Transfection of KDM5B-specific siRNA into various bladder and lung cancer cell lines significantly suppressed the proliferation of cancer cells and increased the number of cells in sub-G1 phase. Microarray expression analysis indicated that E2F1 and E2F2 are downstream genes in the KDM5B pathway.ConclusionsInhibition of KDM5B may affect apoptosis and reduce growth of cancer cells. Further studies will explore the pan-cancer therapeutic potential of KDM5B inhibition.

Evaluation of the Therapeutic Potential of the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Gefitinib in Preclinical Models of Bladder Cancer

The epidermal growth factor receptor (EGFR) is associated with aggressive phenotypes and is an independent predictor of stage progression and mortality in bladder cancer. Gefitinib (‘Iressa,’ ZD1839) is an orally active EGFR-tyrosine kinase inhibitor. The objective of this study was to evaluate the in vitro and in vivo effects of gefitinib in the EGFR-expressing human bladder cancer cell lines 253J B-V, RT-112, and T24. EGFR expression was 3- and 2-fold higher in 253J B-V and RT-112, respectively, compared with T24 cells. Ten μm gefitinib inhibited EGFR, p42/44 extracellular signal-regulated kinase (ERK), and Akt/protein kinase B phosphorylation in all three of the cell lines. Inhibition of ERK by gefitinib was significantly greater in 253J B-V compared with RT-112 and T24 cells (9:2:1 in 253J B-V:RT-112:T24), whereas inhibition of Akt phosphorylation was less in 253J B-V compared with RT-112 and T24 cells (1:9:30 in 253J B-V:RT-112:T24). When cultured in serum-free medium supplemented with epidermal growth factor, 10 μm gefitinib inhibited DNA synthesis in T24 and RT-112 cells, whereas 1 μm gefitinib was sufficient to inhibit DNA synthesis in 253J B-V cells. Similarly, in the presence of serum, 10 μm gefitinib induced a significant reduction in S-phase and viable cell number in T24 and RT-112 cells, whereas 1–10 μm gefitinib caused a dose-dependent effect on these phenotypes in 253J B-V cells. Gefitinib significantly enhanced the ability of ionizing radiation to reduce colony forming ability in 253J B-V and RT-112 cells. In nude mice, a daily oral dose of 150 mg/kg gefitinib induced regression of tumors produced by 253J B-V cells growing at s.c. sites and suppression of tumors produced by these cells at orthotopic sites but had no effect on tumors produced by RT-112 cells growing at s.c. sites. The data indicates that gefitinib has potential therapeutic value, alone or in combination with ionizing radiation, in a subset of EGFR-expressing bladder cancers. However, there is a differential response to gefitinib in these EGFR-expressing bladder cancer cell lines. Although gefitinib can inhibit phosphorylation of EGFR, ERK, and Akt, and inhibit growth of bladder cancer cells in vitro, it does not necessarily inhibit growth of bladder cancer cells in vivo. It is likely that optimized therapy approaches will require an accurate “molecular” diagnosis allowing effective, selective, tailored therapeutic strategies to be designed.

UHRF1 is a novel molecular marker for diagnosis and the prognosis of bladder cancer

Background:Bladder cancer is the second most common cancer of the urinary system. Early diagnosis of this tumour and estimation of risk of future progression after initial transuretherial resection have a significant impact on prognosis. Although there are several molecular markers for the diagnosis and prognosis for this tumour, their accuracy is not ideal. Previous reports have shown that UHRF1 (ubiquitin-like with PHD and ring-finger domains 1) is essential for cellular proliferation. In this study, we examined whether UHRF1 can be a novel molecular marker of bladder cancer.Methods:We performed real-time TaqMan quantitative reverse transcription–PCR and immunohistochemistry to examine expression levels of UHRF1 in bladder and kidney cancers.Results:Significant overexpression of UHRF1 was observed in bladder cancer. The overexpression was correlated with the stage and grade of the cancer. Although UHRF1 expression in muscle-invasive cancer was greater than in non-invasive (pTa) or superficially invasive (pT1) cancers, UHRF1 could still be detected by immunohistochemistry in these early-stage cancers. Overexpression of UHRF1 in bladder cancer was associated with increased risk of progression after transurethral resection. High expression of UHRF1 in kidney cancer was also observed. But the increased levels of UHRF1 in kidney cancer were less significant compared with those in bladder cancer.Conclusion:Our result indicates that an immunohistochemistry-based UHRF1 detection in urine sediment or surgical specimens can be a sensitive and cancer-specific diagnostic and/or prognosis method, and may greatly improve the current diagnosis based on cytology.

Comprehensive profiling and localisation of the matrix metalloproteinases in urothelial carcinoma.

The matrix metalloproteinases (MMPs) are endopeptidases which break down the extracellular matrix and regulate cytokine and growth factor activity. Several MMPs have been implicated in the promotion of invasion and metastasis in a broad range of tumours including urothelial carcinoma. In this study, RNA from 132 normal bladder and urothelial carcinoma specimens was profiled for each of the 24 human MMPs, the four endogenous tissue inhibitors of MMPs (TIMPs) and several key growth factors and their receptors using quantitative real time RT–PCR. Laser capture microdissection (LCM) of RNA from 22 tumour and 11 normal frozen sections was performed allowing accurate RNA extraction from either stromal or epithelial compartments. This study confirms the over expression in bladder tumour tissue of well-documented MMPs and highlights a range of MMPs which have not previously been implicated in the development of urothelial cancer. In summary, MMP-2, MT1-MMP and the previously unreported MMP-28 were very highly expressed in tumour samples while MMPs 1, 7, 9, 11, 15, 19 and 23 were highly expressed. There was a significant positive correlation between transcript expression and tumour grade for MMPs 1, 2, 8, 10, 11, 12, 13, 14, 15 and 28 (P<0.001). At the same confidence interval, TIMP-1 and TIMP-3 also correlated with increasing tumour grade. LCM revealed that most highly expressed MMPs are located primarily within the stromal compartment except MMP-13 which localised to the epithelial compartment. This work forms the basis for further functional studies, which will help to confirm the MMPs as potential diagnostic and therapeutic targets in early bladder cancer.

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

Toward Detection and Localization of Instruments in Minimally Invasive Surgery

Methods for detecting and localizing surgical instruments in laparoscopic images are an important element of advanced robotic and computer-assisted interventions. Robotic joint encoders and sensors integrated or mounted on the instrument can provide information about the tools position, but this often has inaccuracy when transferred to the surgeons point of view. Vision sensors are currently a promising approach for determining the position of instruments in the coordinate frame of the surgical camera. In this study, we propose a vision algorithm for localizing the instruments pose in 3-D leaving only rotation in the axis of the tools shaft as an ambiguity. We propose a probabilistic supervised classification method to detect pixels in laparoscopic images that belong to surgical tools. We then use the classifier output to initialize an energy minimization algorithm for estimating the pose of a prior 3-D model of the instrument within a level set framework. We show that the proposed method is robust against noise using simulated data and we perform quantitative validation of the algorithm compared to ground truth obtained using an optical tracker. Finally, we demonstrate the practical application of the technique on in vivo data from minimally invasive surgery with traditional laparoscopic and robotic instruments.

The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene.

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogensis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real‐time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di‐methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down‐regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G1 arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.