Research IndicatorsGraph generated 13 March 2017 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 13 March, 2017 using data from PubMed, MeSH and CancerIndex
Specific Cancers (4)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
GEO Profiles, NCBI
Search the gene expression profiles from curated DataSets in the Gene Expression Omnibus (GEO) repository.
Latest Publications: HPRT1 (cancer-related)
Thumfart J, Weschke B, Ringe H, et al.Acute renal failure unmasking Lesch-Nyhan disease in a patient with tuberous sclerosis complex.
Eur J Paediatr Neurol. 2016; 20(4):649-51 [PubMed
] Related Publications
CASE REPORT: We report on a male patient with Tuberous Sclerosis Complex (TSC), which was prenatally diagnosed. At the age of 3 months the patient developed acute renal failure with excessive hyperuricemia. Kidney function improved after rehydration and application of rasburicase, however without full recovery. Due to the inappropriate high levels of uric acid compared to kidney function, screening of hypoxanthine-guanine phosphoribosyltransferase (HPRT) related diseases was initiated. Mutation analysis revealed a deletion of exon 2 and 3 of the HPRT gene confirming the diagnosis of Lesch-Nyhan Disease (LND). After initiation of allopurinol therapy renal function further improved. In the following months the patient developed clinically a typical neurological phenotype of LND and TSC with seizures, severe dystonia and developmental delay.
CONCLUSION: Acute renal failure is a rare complication of HPRT related diseases. Combination of two inherited diseases may lead to a delayed diagnosis due to a mixed and maybe misleading phenotype.
Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and identify potential anticancer compounds selective for the high-ploidy state. Among 354 human breast cancers, 10% have mean chromosome copy number exceeding 3, and this is most common in triple-negative and HER2-positive types. Women with high-ploidy breast cancers have higher risk of recurrence and death in two patient cohorts, demonstrating that it represents an important group for improved treatment. Because high-ploidy cancers are aneuploid, rather than triploid or tetraploid, we devised a two-step screen to identify selective compounds. The screen was designed to assure both external validity on diverse karyotypic backgrounds and specificity for high-ploidy cell types. This screen identified novel therapies specific to high-ploidy cells. First, we discovered 8-azaguanine, an antimetabolite that is activated by hypoxanthine phosphoribosyltransferase 1 (HPRT1), suggesting an elevated gene-dosage of HPRT1 in high-ploidy tumors can control sensitivity to this drug. Second, we discovered a novel compound, 2,3-diphenylbenzo[g]quinoxaline-5,10-dione (DPBQ). DPBQ activates p53 and triggers apoptosis in a polyploid-specific manner, but does not inhibit topoisomerase or bind DNA. Mechanistic analysis demonstrates that DPBQ elicits a hypoxia gene signature and its effect is replicated, in part, by enhancing oxidative stress. Structure-function analysis defines the core benzo[g]quinoxaline-5,10 dione as being necessary for the polyploid-specific effects of DPBQ. We conclude that polyploid breast cancers represent a high-risk subgroup and that DPBQ provides a functional core to develop polyploid-selective therapy. Mol Cancer Ther; 15(1); 48-59. ©2015 AACR.
Sharan RN, Vaiphei ST, Nongrum S, et al.Consensus reference gene(s) for gene expression studies in human cancers: end of the tunnel visible?
Cell Oncol (Dordr). 2015; 38(6):419-31 [PubMed
] Related Publications
BACKGROUND: Gene expression studies are increasingly used to provide valuable information on the diagnosis and prognosis of human cancers. Also, for in vitro and in vivo experimental cancer models gene expression studies are widely used. The complex algorithms of differential gene expression analyses require normalization of data against a reference or normalizer gene, or a set of such genes. For this purpose, mostly invariant housekeeping genes are used. Unfortunately, however, there are no consensus (housekeeping) genes that serve as reference or normalizer for different human cancers. In fact, scientists have employed a wide range of reference genes across different types of cancer for normalization of gene expression data. As a consequence, comparisons of these data and/or data harmonizations are difficult to perform and challenging. In addition, an inadequate choice for a reference gene may obscure genuine changes and/or result in erroneous gene expression data comparisons.
METHODS: In our effort to highlight the importance of selecting the most appropriate reference gene(s), we have screened the literature for gene expression studies published since the turn of the century on thirteen of the most prevalent human cancers worldwide.
CONCLUSIONS: Based on the analysis of the data at hand, we firstly recommend that in each study the suitability of candidate reference gene(s) should carefully be evaluated in order to yield reliable differential gene expression data. Secondly, we recommend that a combination of PPIA and either GAPDH, ACTB, HPRT and TBP, or appropriate combinations of two or three of these genes, should be employed in future studies, to ensure that results from different studies on different human cancers can be harmonized. This approach will ultimately increase the depth of our understanding of gene expression signatures across human cancers.
Manzotti G, Parenti S, Ferrari-Amorotti G, et al.Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database.
Cell Cycle. 2015; 14(16):2578-89 [PubMed
] Free Access to Full Article Related Publications
The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.
Shinmura K, Kato H, Kawanishi Y, et al.NEIL1 p.Gln282Stop variant is predominantly localized in the cytoplasm and exhibits reduced activity in suppressing mutations.
Gene. 2015; 571(1):33-42 [PubMed
] Related Publications
Human NEIL1 protein is a DNA glycosylase known to be involved in the repair of oxidized DNA lesions. A c.C844T germline variant of the NEIL1 gene has recently been identified in the Japanese population, however, the p.Q282Stop-type protein produced from this variant gene has not yet been characterized. In this study to determine whether the NEIL1 c.C844T variant might be a defective allele, we investigated the subcellular localization of the p.Q282Stop-type protein and its ability to suppress the development of mutations in mammalian cells. In contrast to the nuclear localization of wild-type (WT) NEIL1, the p.Q282Stop-type protein tagged with GFP or FLAG was localized predominantly in the cytoplasm of human H1299 cells. Mutant forms of the putative nuclear localization signal (NLS, amino acid sequences 359 to 378) of NEIL1-GFP resulted in predominant cytoplasmic localization of the mutants, suggesting that the abnormal localization of p.Q282Stop-type NEIL1 may also be caused by a loss of the putative NLS in the protein. Next, V79 mammalian cell lines inducibly expressing WT NEIL1 or p.Q282Stop-type NEIL1 were established using the piggyBac transposon vector system, and the mutation frequency was compared between the cell lines by HPRT assay. The frequency of mutations induced by glucose oxidase, an oxidative stress inducer, was higher in the p.Q282Stop-type NEIL1-transposed cells than that in the WT NEIL1-transposed cells. Finally, the Cancer Genome Atlas (TCGA) data showed an increased number of somatic mutations in primary carcinomas containing a truncating NEIL1 mutation. These results suggest that p.Q282Stop-type NEIL1 is predominantly localized in the cytoplasm, possibly due to a loss of the NLS, and possesses a reduced ability to suppress the onset of mutations, both findings suggesting that NEIL1 c.C844T is a defective allele.
Lawlor H, Meunier A, McDermott N, et al.Identification of suitable endogenous controls for gene and miRNA expression studies in irradiated prostate cancer cells.
Tumour Biol. 2015; 36(8):6019-28 [PubMed
] Related Publications
This study aimed to to evaluate the stability of commonly used endogenous control genes for messenger RNA (mRNA) (N = 16) and miRNAs (N = 3) expression studies in prostate cell lines following irradiation. The stability of endogenous control genes expression in irradiated (6 Gy) versus unirradiated controls was quantified using NormFinder and coefficient of variation analyses. HPRT1 and 18S were identified as most and least stable endogenous controls, respectively, for mRNA expression studies in irradiated prostate cells. SNORD48 and miR16 miRNA endogenous controls tested were associated with low coefficient of variations following irradiation (6 Gy). This study highlights that commonly used endogenous controls can be responsive to radiation and validation is required prior to gene/miRNAs expression studies.
Zhang J, Wang Y, Shang D, et al.Characterizing and optimizing human anticancer drug targets based on topological properties in the context of biological pathways.
J Biomed Inform. 2015; 54:132-40 [PubMed
] Related Publications
One of the challenging problems in drug discovery is to identify the novel targets for drugs. Most of the traditional methods for drug targets optimization focused on identifying the particular families of "druggable targets", but ignored their topological properties based on the biological pathways. In this study, we characterized the topological properties of human anticancer drug targets (ADTs) in the context of biological pathways. We found that the ADTs tended to present the following seven topological properties: influence the number of the pathways related to cancer, be localized at the start or end of the pathways, interact with cancer related genes, exhibit higher connectivity, vulnerability, betweenness, and closeness than other genes. We first ranked ADTs based on their topological property values respectively, then fused them into one global-rank using the joint cumulative distribution of an N-dimensional order statistic to optimize human ADTs. We applied the optimization method to 13 anticancer drugs, respectively. Results demonstrated that over 70% of known ADTs were ranked in the top 20%. Furthermore, the performance for mercaptopurine was significant: 6 known targets (ADSL, GMPR2, GMPR, HPRT1, AMPD3, AMPD2) were ranked in the top 15 and other four out of the top 15 (MAT2A, CDKN1A, AREG, JUN) have the potentialities to become new targets for cancer therapy.
Gueugnon F, Barascu A, Mavridis K, et al.Kallikrein-related peptidase 13: an independent indicator of favorable prognosis for patients with nonsmall cell lung cancer.
Tumour Biol. 2015; 36(7):4979-86 [PubMed
] Related Publications
The KLK13 gene is dysregulated in several carcinomas, and its expression levels seem to be associated with disease prognosis. The aim of our study was to investigate the prognostic potential of KLK13 mRNA expression for patients with nonsmall cell lung cancer (NSCLC). Total RNA was isolated from cancerous and normal tissues from a cohort of 128 NSCLC patients. The KLK13 mRNA transcription levels were measured using a sensitive quantitative RT-PCR method. The results were normalized by dividing the KLK13 mRNA values with the geometric mean of mRNA expression from four reference genes: beta-actin, TATA-binding protein, hypoxanthine phosphoribosyltransferase 1, and acidic ribosomal phosphoprotein P0. The malignant tissues from the majority of patients (59.3 %) contained significantly more KLK13 mRNA transcripts than did the paired nonmalignant tissues (median difference 11.1-fold, P = 0.008). KLK13 was expressed at higher levels in females than that in males (P = 0.021). No other statistically significant association with clinicopathological data was observed. Kaplan-Meier survival analyses demonstrated that patients with KLK13-positive tumors survived significantly longer than those with KLK13-negative ones (P = 0.009). KLK13 expression was also shown to be able to stratify high-risk individuals among patients with early disease stages (P = 0.030). Multivariate Cox regression analysis showed that KLK13 expression is a favorable, independent prognostic indicator of overall survival (OS) (P = 0.024). Our results suggest that KLK13 mRNA expression constitutes a novel biomarker for the prediction of overall survival in NSCLC and that its quantitative assessment in tumor tissues can aid in treatment decision making.
Ali H, Du Z, Li X, et al.Identification of suitable reference genes for gene expression studies using quantitative polymerase chain reaction in lung cancer in vitro.
Mol Med Rep. 2015; 11(5):3767-73 [PubMed
] Related Publications
The present study aimed to examine 10 housekeeping genes (HKGs), including 18s ribosomal RNA (18S), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), ribosomal protein large P0 (RPLP0), β‑actin (ACTB), peptidylprolyl isomerase A (PPIA), phosphoglycerate kinase‑1 (PGK1), β‑2‑microglobulin (B2M), ribosomal protein LI3a (RPL13A), hypoxanthine phosphoribosyl transferase‑1 (HPRT1) and TATA box binding protein (TBP) in order to identify the most stable and suitable reference genes for use in expression studies in non‑small cell lung cancer. The mRNA expression encoding the panel of the 10 HKGs was determined using reverse transcription‑quantitative PCR (RT‑qPCR) in human lung cancer cell lines. Three software programs, BestKeeper, NormFinder and geNorm, were used to ascertain the most suitable reference genes to normalize the RNA input. The present study examined three lung cancer cell lines (A549, NCI‑H446 and NCI‑H460). The analysis of the experimental data using BestKeeper software revealed that all 10 HKGs were stable, with GADPH, followed by 18S being the most stable genes and PPIA and HPRT1 being the least stable genes. The NormFinder software results demonstrated that PPIA followed by ACTB were the most stable and B2M and RPLP0 were the least stable. The geNorm software results revealed that ACTB and PGK1, followed by PPIA were the most stable genes and B2M and RPLP0 were identified as the least stable genes. Due to discrepancies in the ranking orders of the reference genes obtained by different analyzing software programs, it was not possible to determine a single universal reference gene. The suitability of selected reference genes requires unconditional validation prior to each study. Based on the three analyzing programs, ACTB, PPIA and PGK1 were the most stable reference genes in lung cancer cell lines.
Romani C, Calza S, Todeschini P, et al.Identification of optimal reference genes for gene expression normalization in a wide cohort of endometrioid endometrial carcinoma tissues.
PLoS One. 2014; 9(12):e113781 [PubMed
] Free Access to Full Article Related Publications
Accurate normalization is a primary component of a reliable gene expression analysis based on qRT-PCR technique. While the use of one or more reference genes as internal controls is commonly accepted as the most appropriate normalization strategy, many qPCR-based published studies still contain data poorly normalized and reference genes arbitrarily chosen irrespective of the particular tissue and the specific experimental design. To date, no validated reference genes have been identified for endometrial cancer tissues. In this study, 10 normalization genes (GAPDH, B2M, ACTB, POLR2A, UBC, PPIA, HPRT1, GUSB, TBP, H3F3A) belonging to different functional and abundance classes in various tissues and used in different studies, were analyzed to determine their applicability. In total, 100 endometrioid endometrial cancer samples, which were carefully balanced according to their tumor grade, and 29 normal endometrial tissues were examined using SYBR Green Real-Time RT-PCR. The expression stability of candidate reference genes was determined and compared by means of geNorm and NormFinder softwares. Both algorithms were in agreement in identifying GAPDH, H3F3A, PPIA, and HPRT1 as the most stably expressed genes, only differing in their ranking order. Analysis performed on the expression levels of all candidate genes confirm HPRT1 and PPIA as the most stably expressed in the study groups regardless of sample type, to be used alone or better in combination. As the stable expression of HPRT1 and PPIA between normal and tumor endometrial samples fulfill the basic requirement of a reference gene to be used for normalization purposes, HPRT1 expression showed significant differences between samples from low-grade and high-grade tumors. In conclusion, our results recommend the use of PPIA as a single reference gene to be considered for improved reliability of normalization in gene expression studies involving endometrial tumor samples at different tumor degrees.
Yu S, Yang Q, Yang JH, et al.Identification of suitable reference genes for investigating gene expression in human gallbladder carcinoma using reverse transcription quantitative polymerase chain reaction.
Mol Med Rep. 2015; 11(4):2967-74 [PubMed
] Related Publications
Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT‑qPCR data. The key aim of this method is to identify an applicable internal reference gene, however, there are currently no suitable reference genes for gene analysis in gallbladder carcinoma. In the present study, screening was performed using 12 common reference genes, which were selected in order to provide an experimental basis for the investigation of gene expression in gallbladder carcinoma. A total of 16 tissue samples of gallbladder carcinoma and their matched normal gallbladder tissues were used. The gene expression stability and applicability of the 12 reference gene candidates were determined using the geNorm, NormFinder and BestKeeper software programs. Following comparison of the results of the three software programs, HPRT1 was identified as the most stably expressed reference gene. In the normal gallbladder group, the relative stably expressed reference gene was PPIA and in the entire sample group, the relatively stably expressed reference gene was PPIA. The present study also demonstrated that the combination of the three reference genes was the most appropriate. The recommended combinations were PPIA + PUM1 + ACTB for the total sample group, GAPDH + PBGD + ALAS1 for the gallbladder carcinoma group and PPIA + PUM1 + TBP for the paired normal gallbladder group.
Autsavapromporn N, Plante I, Liu C, et al.Genetic changes in progeny of bystander human fibroblasts after microbeam irradiation with X-rays, protons or carbon ions: the relevance to cancer risk.
Int J Radiat Biol. 2015; 91(1):62-70 [PubMed
] Related Publications
PURPOSE: Radiation-induced bystander effects have important implications in radiotherapy. Their persistence in normal cells may contribute to risk of health hazards, including cancer. This study investigates the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of harmful effects in progeny of bystander cells.
MATERIALS AND METHODS: Confluent human skin fibroblasts were exposed to microbeam radiations with different linear energy transfer (LET) at mean absorbed doses of 0.4 Gy by which 0.036-0.4% of the cells were directly targeted by radiation. Following 20 population doublings, the cells were harvested and assayed for micronucleus formation, gene mutation and protein oxidation.
RESULTS: Our results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET. The progeny of bystander cells exposed to X-rays (LET ∼6 keV/μm) or protons (LET ∼11 keV/μm) showed persistent oxidative stress, which correlated with increased micronucleus formation and mutation at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus. Such effects were not observed after irradiation by carbon ions (LET ∼103 keV/μm). Interestingly, progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultures were exposed to X-rays.
CONCLUSIONS: These findings suggest that cellular exposure to proton and heavy charged particle with LET properties similar to those used here can reduce the risk of lesions associated with cancer. The ability of cells to communicate via gap junctions at the time of irradiation appears to impact residual damage in progeny of bystander cells.
Krzystek-Korpacka M, Diakowska D, Bania J, Gamian AExpression stability of common housekeeping genes is differently affected by bowel inflammation and cancer: implications for finding suitable normalizers for inflammatory bowel disease studies.
Inflamm Bowel Dis. 2014; 20(7):1147-56 [PubMed
] Related Publications
Instability of housekeeping genes (HKG), supposedly unregulated and hence used as normalizers, may dramatically change conclusions of quantitative PCR experiments. The effect of bowel inflammation on HKG remains unknown. Expression stability of 15 HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in 166 bowel specimens (91 normal, 35 cancerous, and 40 inflamed) was ranked by coefficients of variation (CV%) or using dedicated software: geNorm and NormFinder. The RPS23, PPIA, and RPLP0 were top-ranked, whereas IPO8, UBC and TBP were the lowest-ranked HKG across inflamed/cancerous/normal colonic tissues. The pairs RPS23/RPLP0, PGK1/MRPL19, or PPIA/RPLP0 were optimal reference by CV%, NormFinder, and geNorm, respectively. Colon inflammation affected HKG more pronouncedly than cancer with ACTB significantly down- and B2M upregulated. In inflammatory bowel disease (IBD), different genes were top-ranked in a large and small bowel, whereas TBP, UBC, and IPO8 were lowest-ranked in both. For patients with IBD at large, RPS23/PPIA, PGK1/MRPL19, and PPIA/RPLP0 were found optimal by CV%, NormFinder, and geNorm, respectively. ACTB and B2M expression was related to CRC stage and positively correlated with clinical activity of IBD. Although GAPDH was upregulated neither in CRC nor IBD, it tended to positively correlate with tumor depth and Crohn's disease activity index. Normalizing against GAPDH affected experimental conclusions in a small but not large bowel. Bowel inflammation significantly affects several classic HKG. The pair PPIA/RPLP0 is a common optimal reference for studies encompassing tissues sampled from colorectal cancer and IBD patients. Using ACTB or B2M is not recommended.
Multiple myeloma (MM) is a clonal plasma cell malignancy that is initiated by a number of mutations and the process of disease progression is characterized by further acquisition of mutations. The identification and functional characterization of these myelomagenic mutations is necessary to better understand the underlying pathogenic mechanisms in this disease. Recent advancements in next-generation sequencing have made the identification of most of these mutations a reality. However, the functional characterization of these mutations has been hampered by the lack of proper and efficient tools to dissect these mutations. Here we explored the possible utility of transcription activator-like effector nuclease (TALEN) genome engineering technology to tailoring the genome of MM cells. To test this possibility, we targeted the HPRT1 gene and found that TALENs are a very robust and efficient genome-editing tool in MM cells. Using cotransfected green fluorescent protein as an enrichment marker, single-cell subclones with desirable TALEN modifications in the HPRT1 gene were obtained in as little as 3-4 weeks of time. We believe that TALENs will greatly facilitate the functional study of somatic mutations in MM as well as other cancers.
Almeida TA, Quispe-Ricalde A, Montes de Oca F, et al.A high-throughput open-array qPCR gene panel to identify housekeeping genes suitable for myometrium and leiomyoma expression analysis.
Gynecol Oncol. 2014; 134(1):138-43 [PubMed
] Related Publications
OBJECTIVE: To evaluate 51 different housekeeping genes for their use as internal standards in myometrial and matched leiomyoma samples in proliferative and secretory phases.
METHODS: RNA from 6 myometrium and matched leiomyoma samples was obtained from pre-menopausal women who underwent hysterectomy. Reverse-transcription and real-time quantitative PCR were achieved using TaqMan high-density open-array human endogenous control panel.
RESULTS: Expression stability of 51 candidate genes was determined by GeNorm and NormFinder softwares. We identified 10 housekeeping genes, ARF1, MRPL19, FBXW2, PUM1, UBE2D2, EIF2B1, HPRT1, GUSB, ALAS1, and TRIM27, as the best set of normalization genes for comparing relative expression between leiomyoma and myometrium samples in proliferative and secretory phases.
CONCLUSIONS: Adequate reference genes for accurate normalization are essential to compare gene expression between leiomyoma and myometrial samples. Ideal housekeeping genes must have stable expression patterns regardless of the sample type and menstrual cycle phase. In this study, we propose a set of 10 candidate genes with greater expression stability than those housekeeping genes commonly used in leiomyoma and myometrium tissues. Their use will improve the sensitivity and specificity of the gene expression analysis in these tissues.
Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.
DNA polymerase η (pol η), of the Y-family, is well known for its in vitro DNA lesion bypass ability. The most well-characterized lesion bypassed by this polymerase is the cyclobutane pyrimidine dimer (CPD) caused by ultraviolet (UV) light. Historically, cellular and whole-animal models for this area of research have been conducted using UV-C (λ=100-280 nm) owing to its ability to generate large quantities of CPDs and also the more structurally distorting 6-4 photoproduct. Although UV-C is useful as a laboratory tool, exposure to these wavelengths is generally very low owing to being filtered by stratospheric ozone. We are interested in the more environmentally relevant wavelength range of UV-B (λ=280-315 nm) for its role in causing cytotoxicity and mutagenesis. We evaluated these endpoints in both a normal human fibroblast control line and a Xeroderma pigmentosum variant cell line in which the POLH gene contains a truncating point mutation, leading to a nonfunctional polymerase. We demonstrate that UV-B has similar but less striking effects compared to UV-C in both its cytotoxic and its mutagenic effects. Analysis of the mutation spectra after a single dose of UV-B shows that a majority of mutations can be attributed to mutagenic bypass of dipyrimidine sequences. However, we do note additional types of mutations with UV-B that are not previously reported after UV-C exposure. We speculate that these differences are attributed to a change in the spectra of photoproduct lesions rather than other lesions caused by oxidative stress.
Zhan C, Zhang Y, Ma J, et al.Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq.
Acta Biochim Biophys Sin (Shanghai). 2014; 46(4):330-7 [PubMed
] Related Publications
Although the accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing (RNA-Seq) to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression profiles of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes (B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, and YWHAZ), the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes (ACTB) and the 5 additional candidate reference genes (EEF1A1, FAU, RPS9, RPS11, and RPS14) were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEF1A1, FAU, RPS9, RPS11, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.
Liu S, Zhu P, Zhang L, et al.Selection of reference genes for RT-qPCR analysis in tumor tissues from male hepatocellular carcinoma patients with hepatitis B infection and cirrhosis.
Cancer Biomark. 2013; 13(5):345-9 [PubMed
] Related Publications
BACKGROUND: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the high specificity, sensitivity and accuracy of this technique. However, its reliability is strongly depends on the expression stability of reference gene used for data normalization. Therefore, identification of reliable and condition specific reference genes is critical for the success of RT-qPCR.
OBJECTIVE: Hepatitis B virus (HBV) infection, male gender and the presence of cirrhosis are widely recognized as the leading independent risk factors for the development of hepatocellular carcinoma (HCC). This study aimed to select reliable reference gene for RT-qPCR analysis in HCC patients with all of those risk factors.
METHODS: Six candidate reference genes were analyzed in 33 paired tumor and non-tumor tissues from untreated HCC patients. The genes expression stabilities were assessed by geNorm and NormFinder.
RESULTS: C-terminal binding protein 1(CTBP1) was the most stable gene among the 6 candidate genes evaluated by both geNorm and NormFinder. The expression stability values were 0.08 for CTBP1 and UBC, 0.09 for HPRT1, 0.12 for HMBS, 0.14 for GAPDH and 0.18 for 18S with geNorm analysis. The stability values suggested by NormFinder software were CTBP1: 0.044, UBC: 0.063, HMBS: 0.072, HPRT1: 0.072, GAPDH: 0.098 and 18S rRNA: 0.161.
CONCLUSION: This is the first systematic analysis which suggested CTBP1 as the highest expression-stable gene in human male HBV infection related-HCC with cirrhosis. We recommend CTBP1 as the best candidate reference gene when RT-qPCR was used to determine gene(s) expression in HCC. This may facilitate the relevant HBV related HCC studies in the future.
The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p < 0.001). The CNV assay was validated investigating HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.
Tsaur I, Renninger M, Hennenlotter J, et al.Reliable housekeeping gene combination for quantitative PCR of lymph nodes in patients with prostate cancer.
Anticancer Res. 2013; 33(12):5243-8 [PubMed
] Related Publications
BACKGROUND: To reliably compare the results of gene expression studies, the expression of the target gene should be normalized to the expression of a reference gene. For lymph node metastases of prostate cancer, no data on polymerase chain reaction (PCR) normalization have yet been reported. We aimed to determine the most reliable reference gene combination for this purpose in patients with prostate cancer.
MATERIALS AND METHODS: Ten histologically- positive and ten negative lymph nodes of patients with prostate cancer were analyzed respectively. Expression of six candidate reference genes was comparatively assessed with quantitative Real-time PCR. The most stably-expressed gene combination was determined with geNorm software version 3.4.
RESULTS: Hypoxanthine phosphoribosyltransferase-1 (HPRT1) and TATA box binding protein (TPB) were found to be the most stably expressed genes, with their combination having an expression stability value of M=0.17.
CONCLUSION: Gene combination HPRT1 and TPB has the potential to be utilized for normalization in gene profiling assessment of metastatic and non-metastatic pelvic lymph node tissue from patients with prostate cancer.
Seuter S, Pehkonen P, Heikkinen S, Carlberg CDynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes.
Biochim Biophys Acta. 2013; 1829(12):1266-75 [PubMed
] Related Publications
The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.
Walter RF, Mairinger FD, Wohlschlaeger J, et al.FFPE tissue as a feasible source for gene expression analysis--a comparison of three reference genes and one tumor marker.
Pathol Res Pract. 2013; 209(12):784-9 [PubMed
] Related Publications
BACKGROUND: Formalin-fixation, paraffin-embedding is the standard processing technique for tumor tissue in modern pathology. New techniques such as cryo-conservation allow rapid fixation and long-time storage but come along with increased costs and enlarged storage complexity. However, formalin-fixed, paraffin-embedded (FFPE) tissue is available in a large quantity, making it the ideal material for retrospective studies. The following study was designed to investigate the influence of formalin-fixation on the quality of mRNA and applicability of FFPE-derived mRNA for gene expression analysis. Three potential reference genes for pulmonary tumors with neuroendocrine differentiation were included and tested for their robust expression.
MATERIALS AND METHODS: Eighty specimens collected from 2005 to 2012 at the Institute of Pathology and Neuropathology at the University Hospital Essen were analyzed for their gene expression by using TaqMan(®) gene expression assays on demand (AoD). Three distinct potential reference genes (ACTB, GAPDH, HPRT1) were evaluated for their expression, and a proteasome subunit (PSMA1) was included in the analysis as tumor marker and functioned as an internal technical control.
CONCLUSION: For GAPDH and ACTB, a highly significant correlation and consistent expression between the investigated entities was found, making them reliable reference genes for further research. Additionally, the feasibility for a FFPE tissue-based gene expression analysis was verified by showing that the mRNA quality is sufficient. When standardized FFPE preparation is performed carefully, sufficient mRNA can be isolated for reliable and successful gene expression analysis. That provides the basis the door for large, retrospective studies that correlate molecular and clinical follow-up data.
To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.
Mutations in human DNA polymerase (Pol) ε, one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol ε mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol ε that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias toward mispairs opposite template pyrimidine bases, particularly T • dTTP mispairs. Expression of mutant Pol ε in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T • dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol ε causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ε. Since tumors from familial and somatic colorectal patients arise with Pol ε mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ε mutation requiring additional factors to promote tumor development.
In previous studies we described a decreased alpha-L-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-L-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-L-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-L-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-L-fucosidase could be regulated at transcriptional level in colorectal cancer.
Wu C, Wang X, Zhong M, et al.Evaluation of potential reference genes for qRT-PCR studies in human hepatoma cell lines treated with TNF-α.
Acta Biochim Biophys Sin (Shanghai). 2013; 45(9):780-6 [PubMed
] Related Publications
In this study, the expression of eight candidate reference genes, B2M, ACTB, GAPDH, HMBS, HPRT1, TBP, UBC, and YWHAZ, was examined to identify optimal reference genes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in two human hepatoma cell lines, BEL-7402 and SMMC-7721, treated with tumor necrosis factor-α (TNF-α) for different time periods. The expression stability of these genes was analyzed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Results showed that TBP was the most stably expressed gene in BEL-7402 and SMMC-7721 cell lines under current experimental conditions, and that the optimal set of reference genes required for accurate normalization was TBP and HMBS, based on the pairwise variation value determined with geNorm. UBC and ACTB were ranked as the least stable genes by same algorithms. Our findings provide evidence that using TBP alone or in combination with HMBS as endogenous controls could be a reliable method for normalizing qRT-PCR data in human hepatoma cell lines treated with TNF-α.
Gábelová A, Poláková V, Prochazka G, et al.Sustained induction of cytochrome P4501A1 in human hepatoma cells by co-exposure to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole underlies the synergistic effects on DNA adduct formation.
Toxicol Appl Pharmacol. 2013; 271(1):1-12 [PubMed
] Related Publications
To gain a deeper insight into the potential interactions between individual aromatic hydrocarbons in a mixture, several benzo[a]pyrene (B[a]P) and 7H-dibenzo[c,g]carbazole (DBC) binary mixtures were studied. The biological activity of the binary mixtures was investigated in the HepG2 and WB-F344 liver cell lines and the Chinese hamster V79 cell line that stably expresses the human cytochrome P4501A1 (hCYP1A1). In the V79 cells, binary mixtures, in contrast to individual carcinogens, caused a significant decrease in the levels of micronuclei, DNA adducts and gene mutations, but not in cell survival. Similarly, a lower frequency of micronuclei and levels of DNA adducts were found in rat liver WB-F344 cells treated with a binary mixture, regardless of the exposure time. The observed antagonism between B[a]P and DBC may be due to an inhibition of Cyp1a1 expression because cells exposed to B[a]P:DBC showed a decrease in Cyp1a1 mRNA levels. In human liver HepG2 cells exposed to binary mixtures for 2h, a reduction in micronuclei frequency was also found. However, after a 24h treatment, synergism between B[a]P and DBC was determined based on DNA adduct formation. Accordingly, the up-regulation of CYP1A1 expression was detected in HepG2 cells exposed to B[a]P:DBC. Our results show significant differences in the response of human and rat cells to B[a]P:DBC mixtures and stress the need to use multiple experimental systems when evaluating the potential risk of environmental pollutants. Our data also indicate that an increased expression of CYP1A1 results in a synergistic effect of B[a]P and DBC in human cells. As humans are exposed to a plethora of noxious chemicals, our results have important implications for human carcinogenesis.
Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl⁻ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses.
Mavridis K, Stravodimos K, Scorilas AQuantified KLK15 gene expression levels discriminate prostate cancer from benign tumors and constitute a novel independent predictor of disease progression.
Prostate. 2013; 73(11):1191-201 [PubMed
] Related Publications
BACKGROUND: Several transcript variants of the kallikrein-related peptidase 15 gene (KLK15) have been identified up to now. The classical KLK15 mRNA isoform encodes for a non-truncated, functional protein. Aberrant KLK15 expression is found in breast, ovarian, and prostate cancers. Our aim in this present study was the specific quantitative expression analysis of the classical KLK15 mRNA transcript in prostate tumors and the examination of its clinical significance in prostate cancer.
METHODS: We isolated total RNA from 150 prostate tissue specimens and, following cDNA synthesis, the expression of KLK15 classical mRNA transcript was measured via quantitative Real-Time PCR using the TaqMan® chemistry. HPRT1 was used as a reference gene, and the absolute quantification approach, through the incorporation of standard curves, was applied for the calculation of normalized KLK15 expression.
RESULTS: KLK15 expression levels were significantly upregulated in malignant compared to benign samples (P < 0.001). The discriminatory value of KLK15 was confirmed by ROC curve and logistic regression analysis (both P < 0.001). KLK15 was also associated with advanced pathological stage (P = 0.023). KLK15-positive prostate cancer patients presented significantly shorter progression-free survival intervals, determined by biochemical relapse (P = 0.006), compared to KLK15-negative ones. Multivariate Cox regression analysis revealed that KLK15 expression is an independent predictor of biochemical recurrence (HR = 3.36, P = 0.038).
CONCLUSIONS: The present study unravels the important role of quantified KLK15 classical mRNA expression levels as a novel biomarker helpful for the differential diagnosis and prognosis of prostate cancer patients.