ACTB

Gene Summary

Gene:ACTB; actin, beta
Aliases: BRWS1, PS1TP5BP1
Location:7p22
Summary:This gene encodes one of six different actin proteins. Actins are highly conserved proteins that are involved in cell motility, structure, and integrity. This actin is a major constituent of the contractile apparatus and one of the two nonmuscle cytoskeletal actins. [provided by RefSeq, Jul 2008]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:actin, cytoplasmic 1
HPRD
Source:NCBIAccessed: 27 February, 2015

Ontology:

What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 28 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • DNA Methylation
  • Reference Standards
  • Base Sequence
  • Colorectal Cancer
  • Ribosomal Proteins
  • Genes, Essential
  • Tissue Fixation
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Models, Genetic
  • Algorithms
  • Breast Cancer
  • Oligonucleotide Array Sequence Analysis
  • Chromosome 7
  • Case-Control Studies
  • Cancer DNA
  • Molecular Sequence Data
  • Reproducibility of Results
  • Lung Cancer
  • Gene Expression
  • Transcription Factors
  • Cancer RNA
  • Actins
  • RT-PCR
  • Translocation
  • RNA-Binding Proteins
  • Gene Expression Profiling
  • Bladder Cancer
  • DNA-Binding Proteins
  • Liver Cancer
  • Transition Temperature
  • Polymerase Chain Reaction
  • Messenger RNA
  • DNA
  • Hepatocellular Carcinoma
  • Cancer Gene Expression Regulation
  • RNA
  • Paraffin Embedding
  • Kidney Cancer
  • TATA-Box Binding Protein
Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

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).

Latest Publications: ACTB (cancer-related)

Brandwein JM, Kassis J, Leber B, et al.
Phase II study of targeted therapy with temozolomide in acute myeloid leukaemia and high-risk myelodysplastic syndrome patients pre-screened for low O(6) -methylguanine DNA methyltransferase expression.
Br J Haematol. 2014; 167(5):664-70 [PubMed] Related Publications
Resistance to temozolomide is largely mediated by the DNA repair enzyme O(6) -methylguanine DNA methyltransferase (MGMT). We conducted a prospective multicentre study of patients with previously untreated acute myeloid leukaemia (AML) or high-risk myelodysplastic syndrome (MDS) who were not candidates for intensive therapy. Patient selection was based on MGMT expression by Western blot. Patients with MGMT:ACTB (β-actin) ratio <0·2 were eligible to receive temozolomide 200 mg/m(2) /d ×7 d. Patients achieving a complete response (CR) could receive up to 12 monthly cycles of temozolomide ×5/28 d. Of 166 patients screened, 81 (49%) demonstrated low MGMT expression; 45 of these were treated with temozolomide. The overall response rate was 53%; 36% achieved complete clearance of blasts, with 27% achieving a CR/CR with incomplete platelet recovery (CRp). Factors associated with a trend toward a higher response rate included MDS, methylated MGMT promoter and standard cytogenetic risk group. Induction and post-remission cycles were well-tolerated and most patients were treated on an outpatient basis. Patient who achieved CR/CRp had a superior overall survival compared to partial or non-responders. In conclusion, targeted therapy based on pre-selection for low MGMT expression was associated with a higher response rate to temozolomide compared to previous reports of unselected patients.

Santillán-Benítez JG, Mendieta-Zerón H, Gómez-Oliván LM, et al.
JAK2, STAT3 and SOCS3 gene expression in women with and without breast cancer.
Gene. 2014; 547(1):70-6 [PubMed] Related Publications
INTRODUCTION: Breast cancer is a disease that arises from the accumulation of alterations in the genome of cells that make up the mammary gland. The Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway has been reported to participate in the development of breast cancer and is activated by adipocytokines such as leptin, which are elevated in obesity. In contrast, alterations in suppressor of cytokine signaling 3 (SOCS3) gene expression have been found in patients with decreased breast cancer metastasis.
OBJECTIVE: The current study sought to identify whether JAK2, STAT3 and SOCS3 gene expression is associated with body mass index (BMI) and breast cancer.
METHODS: This was a cross-sectional prospective study. JAK2, STAT3 and SOCS3 gene expression levels were determined using RT-qPCR from the biopsies of 26 patients with breast cancer and 43 patients with benign breast lesions. We compared the expression of these genes, relative to the housekeeping genes, ACTB and GAPDH, against BMI, clinical stage and immunohistochemistry.
RESULTS: STAT3 gene expression was increased in breast cancer patients (p≤0.001; AUC=0.65; AUC 95% CI: 0.5-0.8), and SOCS3 expression was decreased in obese patients with benign breast lesions (p≤0.001; AUC=0.51; AUC 95% CI: 0.36-0.65). With regard to the clinical stage, there were significant differences in STAT3 gene expression between stage II and III (p≤0.011) and stage II and IV (p≤0.033) breast cancers. Among all women, there was a positive correlation between JAK2 and STAT3 expression (R=0.493, p=0.000). In addition, breast cancers that were negative for HER2 were associated with JAK2 and SOCS3 (R=0.645, p=0.003).
CONCLUSION: High levels of STAT3 expression were associated with early stages of breast cancer development and patients in the control group with obesity showed higher expression of SOCS3 regarding overweight.

Krzystek-Korpacka M, Diakowska D, Bania J, Gamian A
Expression 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.

Partin AW, Van Neste L, Klein EA, et al.
Clinical validation of an epigenetic assay to predict negative histopathological results in repeat prostate biopsies.
J Urol. 2014; 192(4):1081-7 [PubMed] Free Access to Full Article Related Publications
PURPOSE: The DOCUMENT multicenter trial in the United States validated the performance of an epigenetic test as an independent predictor of prostate cancer risk to guide decision making for repeat biopsy. Confirming an increased negative predictive value could help avoid unnecessary repeat biopsies.
MATERIALS AND METHODS: We evaluated the archived, cancer negative prostate biopsy core tissue samples of 350 subjects from a total of 5 urological centers in the United States. All subjects underwent repeat biopsy within 24 months with a negative (controls) or positive (cases) histopathological result. Centralized blinded pathology evaluation of the 2 biopsy series was performed in all available subjects from each site. Biopsies were epigenetically profiled for GSTP1, APC and RASSF1 relative to the ACTB reference gene using quantitative methylation specific polymerase chain reaction. Predetermined analytical marker cutoffs were used to determine assay performance. Multivariate logistic regression was used to evaluate all risk factors.
RESULTS: The epigenetic assay resulted in a negative predictive value of 88% (95% CI 85-91). In multivariate models correcting for age, prostate specific antigen, digital rectal examination, first biopsy histopathological characteristics and race the test proved to be the most significant independent predictor of patient outcome (OR 2.69, 95% CI 1.60-4.51).
CONCLUSIONS: The DOCUMENT study validated that the epigenetic assay was a significant, independent predictor of prostate cancer detection in a repeat biopsy collected an average of 13 months after an initial negative result. Due to its 88% negative predictive value adding this epigenetic assay to other known risk factors may help decrease unnecessary repeat prostate biopsies.

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.

Wisnieski F, Calcagno DQ, Leal MF, et al.
Reference genes for quantitative RT-PCR data in gastric tissues and cell lines.
World J Gastroenterol. 2013; 19(41):7121-8 [PubMed] Free Access to Full Article Related Publications
AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines.
METHODS: The suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent non-neoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The ranking of the best single and combination of reference genes was determined by NormFinder, geNorm™, BestKeeper, and DataAssist™. In addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization.
RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. The level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44).
CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested.

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.

Iio A, Takagi T, Miki K, et al.
DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.
Biochim Biophys Acta. 2013; 1829(10):1102-10 [PubMed] Related Publications
In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

Wierzbicki PM, Adrych K, Kartanowicz D, et al.
Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation.
World J Gastroenterol. 2013; 19(27):4363-73 [PubMed] Free Access to Full Article Related Publications
AIM: To investigate large tumor suppressor 1 (LATS1) expression, promoter hypermethylation, and microsatellite instability in colorectal cancer (CRC).
METHODS: RNA was isolated from tumor tissue of 142 CRC patients and 40 colon mucosal biopsies of healthy controls. After reverse transcription, quantitative polymerase chain reaction (PCR) was performed, and LATS1 expression was normalized to expression of the ACTB and RPL32 housekeeping genes. To analyze hypermethylation, genomic DNA was isolated from 44 tumor CRC biopsies, and methylation-specific PCR was performed. Microsatellite instability (MSI) status was checked with PCR using BAT26, BAT25, and BAT40 markers in the genomic DNA of 84 CRC patients, followed by denaturing gel electrophoresis.
RESULTS: Decreased LATS1 expression was found in 127/142 (89.4%) CRC cases with the average ratio of the LATS1 level 10.33 ± 32.64 in CRC patients vs 32.85 ± 33.56 in healthy controls. The lowest expression was found in Dukes' B stage tumors and G1 (well-differentiated) cells. Hypermethylation of the LATS1 promoter was present in 25/44 (57%) CRC cases analyzed. LATS1 promoter hypermethylation was strongly associated with decreased gene expression; methylated cases showed 162× lower expression of LATS1 than unmethylated cases. Although high-grade MSI (mutation in all three markers) was found in 14/84 (17%) cases and low-grade MSI (mutation in 1-2 markers) was found in 30/84 (36%) cases, we found no association with LATS1 expression.
CONCLUSION: Decreased expression of LATS1 in CRC was associated with promoter hypermethylation, but not MSI status. Such reduced expression may promote progression of CRC.

Tramm T, Sørensen BS, Overgaard J, Alsner J
Optimal reference genes for normalization of qRT-PCR data from archival formalin-fixed, paraffin-embedded breast tumors controlling for tumor cell content and decay of mRNA.
Diagn Mol Pathol. 2013; 22(3):181-7 [PubMed] Related Publications
Reliable determination of gene-expression levels from qRT-PCR requires accurate normalization of target genes to reference genes in order to remove nonbiological variation. Reference genes are ideally constitutively expressed in every cell, but many genes used for normalization has been shown to vary with tissue type, cellular proliferation, cancer progression, and degradation of nucleic acids. Gene-expression analysis is increasingly performed on degraded mRNA from formalin-fixed, paraffin-embedded tissue (FFPE), giving the option of examining retrospective cohorts. The aim of this study was to select robust reference genes showing stable expression over time in FFPE, controlling for various content of tumor tissue and decay of mRNA because of variable length of storage of the tissue. Sixteen reference genes were quantified by qRT-PCR in 40 FFPE breast tumor samples, stored for 1 to 29 years. Samples included 2 benign lesions and 38 carcinomas with varying tumor content. Stability of the reference genes were determined by the geNorm algorithm. mRNA was successfully extracted from all samples, and the 16 genes quantified in the majority of samples. Results showed 14% loss of amplifiable mRNA per year, corresponding to a half-life of 4.6 years. The 4 most stable expressed genes were CALM2, RPL37A, ACTB, and RPLP0. Several of the other examined genes showed considerably instability over time (GAPDH, PSMC4, OAZ1, IPO8). In conclusion, we identified 4 genes robustly expressed over time and independent of neoplastic tissue content in the FFPE block. Other widely used reference genes were concluded to be less suited for retrospective analysis of FFPE breast samples.

Ma X, Wehland M, Aleshcheva G, et al.
Interleukin-6 expression under gravitational stress due to vibration and hypergravity in follicular thyroid cancer cells.
PLoS One. 2013; 8(7):e68140 [PubMed] Free Access to Full Article Related Publications
It is known that exposing cell lines in vitro to parabolic flights changes their gene expression and protein production patterns. Parabolic flights and spaceflight in general are accompanied by transient hypergravity and vibration, which may impact the cells and therefore, have to be considered too. To estimate the possible impact of transient hypergravity and vibration, we investigated the effects of these forces separately using dedicated ground-based facilities. We placed follicular thyroid ML-1 and CGTH W-1 cancer cells in a specific centrifuge (MuSIC Multi Sample Incubator Centrifuge; SAHC Short Arm Human Centrifuge) simulating the hypergravity phases that occur during one (P1) and 31 parabolas (P31) of parabolic flights, respectively. On the Vibraplex device, the same cell lines were treated with vibration waves corresponding to those that occur during a whole parabolic flight lasting for two hours. After the various treatments, cells were harvested and analyzed by quantitative real-time PCR, focusing on the genes involved in forming (ACTB, MYO9, TUBB, VIM, TLN1, and ITGB1) and modulating (EZR, RDX, and MSN) the cytoskeleton, as well as those encoding growth factors (EGF, CTGF, IL6, and IL8) or protein kinases (PRKAA1 and PRKCA). The analysis revealed alterations in several genes in both cell lines; however, fewer genes were affected in ML-1 than CGTH W-1 cells. Interestingly, IL6 was the only gene whose expression was changed in both cell lines by each treatment, while PKCA transcription remained unaffected in all experiments. We conclude that a PKCa-independent mechanism of IL6 gene activation is very sensitive to physical forces in thyroid cells cultured in vitro as monolayers.

Kwon JY, Weon JI, Koedrith P, et al.
Identification of molecular candidates and interaction networks via integrative toxicogenomic analysis in a human cell line following low-dose exposure to the carcinogenic metals cadmium and nickel.
Oncol Rep. 2013; 30(3):1185-94 [PubMed] Related Publications
Cadmium and nickel have been classified as carcinogenic to humans by the World Health Organization's International Agency for Research on Cancer. Given their prevalence in the environment, the fact that cadmium and nickel may cause diseases including cancer even at low doses is a cause for concern. However, the exact mechanisms underlying the toxicological effects induced by low-dose exposure to cadmium and nickel remain to be elucidated. Furthermore, it has recently been recognized that integrative analysis of DNA, mRNA and proteins is required to discover biomarkers and signaling networks relevant to human toxicant exposure. In the present study, we examined the deleterious effects of chronic low-dose exposure of either cadmium or nickel on global profiling of DNA copy number variation, mRNA and proteins. Array comparative genomic hybridization, gene expression microarray and functional proteomics were conducted, and a bioinformatics tool, which predicted signaling pathways, was applied to integrate data for each heavy metal separately and together. We found distinctive signaling networks associated with subchronic low-dose exposure to cadmium and nickel, and identified pathways common to both. ACTB, HSP90AA1, HSPA5 and HSPA8, which are key mediators of pathways related to apoptosis, proliferation and neoplastic processes, were key mediators of the same pathways in low-dose nickel and cadmium exposure in particular. CASP-associated signaling pathways involving CASP3, CASP7 and CASP9 were observed in cadmium-exposed cells. We found that HSP90AA1, one of the main modulators, interacted with HIF1A, AR and BCL2 in nickel-exposed cells. Interestingly, we found that HSP90AA1 was involved in the BCL2-associated apoptotic pathway in the nickel-only data, whereas this gene interacted with several genes functioning in CASP-associated apoptotic signaling in the cadmium-only data. Additionally, JUN and FASN were main modulators in nickel-responsive signaling pathways. Our results provide valuable biomarkers and distinctive signaling networks that responded to subchronic low-dose exposure to cadmium and nickel.

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.

Kılıç Y, Çelebiler AÇ, Sakızlı M
Selecting housekeeping genes as references for the normalization of quantitative PCR data in breast cancer.
Clin Transl Oncol. 2014; 16(2):184-90 [PubMed] Related Publications
OBJECTIVE: The common reference genes of choice in relative gene expression studies based on quantitative real time polymerase chain reaction, ACTB and B2M, were shown to be regulated differently in respect to tissue type. In this study, the stability of the selected housekeeping genes for normalizing the qPCR data were identified in the tumor and its adjacent tissues in invasive breast cancer, and the variability of their levels according to the stages and the histopathologic subtypes was analyzed.
METHODS: Four housekeeping genes: PUM1, RPL13A, B2M, and ACTB were analyzed in 99 surgically excised tissue specimens (50 tumor, 45 tumor adjacent and 4 normal breast tissues). Three of the most common softwares (GeNorm, NormFinder, and BestKeeper) were used for calculation purposes.
RESULTS: When all of the tissue samples were included in analyses, PUM1 was the most stable gene according to calculations made with both NormFinder and BestKeeper; while PUM1/RPL13A combination was the most stable by GeNorm software. The PUM1 gene was also identified as the most stable gene among the four in all sample groups (in both Estrogen Receptor positive and Estrogen Receptor negative subgroups of invasive breast carcinoma and in normal breast tissue) according to calculations made using the NormFinder software.
CONCLUSION: While suggesting PUM1 is one of the most stable single gene and the PUM1/RPL13A pair as one of the best housekeeping genes for the normalization of expression studies in invasive breast tumor studies, it will be more practical to evaluate stability once more and decide upon the reference gene accordingly within the sample group itself.

Søes S, Sørensen BS, Alsner J, et al.
Identification of accurate reference genes for RT-qPCR analysis of formalin-fixed paraffin-embedded tissue from primary non-small cell lung cancers and brain and lymph node metastases.
Lung Cancer. 2013; 81(2):180-6 [PubMed] Related Publications
Lung cancer is the most common cause of cancer-related deaths worldwide, and metastatic spread of the cancer rather than the primary tumor is the main cause of death. However, the molecular alterations of cancer cells leading to the formation of metastasis are poorly understood. This is partly a result of most solid tumor samples available for retrospective studies being archived as formalin-fixed paraffin-embedded (FFPE) specimens causing the nucleic acids to be highly degraded. Furthermore, stably expressed reference genes for normalization of gene expression data using reverse transcriptase quantitative PCR (RT-qPCR) have not been identified for combined analysis of primary lung tumors and the tissues where to the cancer metastasize. Using an optimized RT-qPCR workflow we have analyzed the expression of 23 candidate reference genes in a total of 54 FFPE specimens derived from primary Non-Small Cell Lung Cancer tumors, brain metastases, and lymph node metastases as well as normal lung, lymph node, and brain tissues. We show that every aspect of the workflow is highly reproducible, and the PUM1, TBP, and IPO8 genes were identified as the most stably expressed reference genes among the candidates, by using the GeNorm and NormFinder software programs. Furthermore, we demonstrate that commonly used reference genes such as ACTB (β-actin), GAPDH, and rRNA18S are less stably expressed in the studied samples. The presented workflow and the identified reference genes may facilitate more reliable gene expression studies in lung cancer using RNA from FFPE tissues.

Januszkiewicz-Lewandowska D, Nowicka K, Rembowska J, et al.
Env gene expression of human endogenous retrovirus-k and human endogenous retrovirus-w in childhood acute leukemia cells.
Acta Haematol. 2013; 129(4):232-7 [PubMed] Related Publications
INTRODUCTION: The etiopathogenesis of childhood leukemia is not fully understood. It is suggested that endogenous viral sequences may play a role in leukemogenesis. Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. Most HERVs are dysfunctional because of numerous mutations and deletions. Some HERVs, however, contain sequences capable of transcription. In patients with leukemia, the presence of antibodies against HERV-K has been identified, which could suggest increased expression of HERV-K in leukemic cells. To elucidate the role of endogenous retroviruses in leukemogenesis, studies were undertaken to assess env gene expression of HERV-K and HERV-W in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).
RESULTS: This study was performed in 170 children with ALL, 38 subjects with AML, and 30 healthy subjects. Expression of the env gene of HERV-K and HERV-W and the control gene ACTB was studied by real-time PCR using specific oligonucleotide primers and SYBR Green marker. Env gene expression was assessed on the basis of the absolute threshold-Ct, as well as normalized against ACTB expression and double normalized expression relative to ACTB and reference cells - normal peripheral blood lymphocytes (PBL). Env gene expression of HERV-K normalized against ACTB, as well as double normalized expression relative to ACTB and normal PBL, was significantly higher only in AML. There were no statistically significant differences in env gene expression of HERV-W normalized to ACTB in ALL and AML as compared to normal PBL.
CONCLUSION: High normalized expression of the env gene of HERV-K in AML strongly suggests a possible contribution of this gene in the pathogenesis of AML.

Souza AF, Brum IS, Neto BS, et al.
Reference gene for primary culture of prostate cancer cells.
Mol Biol Rep. 2013; 40(4):2955-62 [PubMed] Related Publications
Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

Guo C, Liu S, Wang J, et al.
ACTB in cancer.
Clin Chim Acta. 2013; 417:39-44 [PubMed] Related Publications
Beta-actin (ACTB) has traditionally been regarded as an endogenous housekeeping gene and has been widely used as a reference gene/protein in quantifying expression levels in tumors. However, ACTB is closely associated with a variety of cancers and accumulating evidence indicates that ACTB is de-regulated in liver, melanoma, renal, colorectal, gastric, pancreatic, esophageal, lung, breast, prostate, ovarian cancers, leukemia and lymphoma. ACTB is generally found to be up-regulated in the majority of tumor cells and tissues. The abnormal expression and polymerization of ACTB and the resulting changes to the cytoskeleton are revealed to be associated with the invasiveness and metastasis of cancers. The current review explores relevant mechanisms, integrates current understandings, and provides suggestions for future studies of the roles of ACTB in tumors.

Forthun RB, Sengupta T, Skjeldam HK, et al.
Cross-species functional genomic analysis identifies resistance genes of the histone deacetylase inhibitor valproic acid.
PLoS One. 2012; 7(11):e48992 [PubMed] Free Access to Full Article Related Publications
The mechanisms of successful epigenetic reprogramming in cancer are not well characterized as they involve coordinated removal of repressive marks and deposition of activating marks by a large number of histone and DNA modification enzymes. Here, we have used a cross-species functional genomic approach to identify conserved genetic interactions to improve therapeutic effect of the histone deacetylase inhibitor (HDACi) valproic acid, which increases survival in more than 20% of patients with advanced acute myeloid leukemia (AML). Using a bidirectional synthetic lethality screen revealing genes that increased or decreased VPA sensitivity in C. elegans, we identified novel conserved sensitizers and synthetic lethal interactors of VPA. One sensitizer identified as a conserved determinant of therapeutic success of HDACi was UTX (KDM6A), which demonstrates a functional relationship between protein acetylation and lysine-specific methylation. The synthetic lethal screen identified resistance programs that compensated for the HDACi-induced global hyper-acetylation, and confirmed MAPKAPK2, HSP90AA1, HSP90AB1 and ACTB as conserved hubs in a resistance program for HDACi that are drugable in human AML cell lines. Hence, these resistance hubs represent promising novel targets for refinement of combinatorial epigenetic anti-cancer therapy.

Marhefka JN, Abbud-Antaki RA
Validation of the Cancer BioChip System as a 3D siRNA screening tool for breast cancer targets.
PLoS One. 2012; 7(9):e46086 [PubMed] Free Access to Full Article Related Publications
Genomic studies have revealed that breast cancer consists of a complex biological process with patient-specific genetic variations, revealing the need for individualized cancer diagnostic testing and selection of patient-specific optimal therapies. One of the bottlenecks in translation of genomic breakthroughs to the clinic is the lack of functional genomic assays that have high clinical translatability. Anchorage-independent three-dimensional (3D) growth assays are considered to be the gold-standard for chemosensitivity testing, and leads identified with these assays have high probability of clinical success. The Cancer BioChip System (CBCS) allows for the simultaneous, quantitative, and real time evaluation of multitudes of anchorage-independent breast cancer cell growth inhibitors. We employed a Test Cancer BioChip that contains silencing RNAs (siRNAs) targeting cancer-related genes to identify 3D-specific effectors of breast cancer cell growth. We compared the effect of these siRNAs on colony growth of the hormone receptor positive (MCF7) and Human Epidermal Growth Factor Receptor 2/c- Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2/c-erb-b2) positive (SK-BR-3) cells on the Test Cancer BioChip. Our results confirmed cell-specific inhibition of MCF7 and SK-BR-3 colony formation by estrogen receptor α (ESR1) and (ERBB2) siRNA, respectively. Both cell lines were also suppressed by Phosphoinositide-3-kinase Catalytic, alpha Polypeptide (PIK3CA) siRNA. Interestingly, we have observed responses to siRNA that are unique to this 3D setting. For example, ß-actin (ACTB) siRNA suppressed colony growth in both cell types while Cathepsin L2 (CTSL2) siRNA caused opposite effects. These results further validate the importance of the CBCS as a tool for the identification of clinically relevant breast cancer targets.

Larysz D, Zebracka-Gala J, Rudnik A, et al.
Expression of genes FOLR1, BAG1 and LAPTM4B in functioning and non-functioning pituitary adenomas.
Folia Neuropathol. 2012; 50(3):277-86 [PubMed] Related Publications
INTRODUCTION: The mechanism of pathogenesis of adenomas pituitary is still unknown; differences between pituitary cells of different origin are observed. Identification of genes specific to pituitary adenomas should give better understanding of differences in their response to therapy, especially to radiotherapy. The aim of our study was to independently validate differences in the expression of FOLR1, BAG1, LAPTM4B between functioning (FA) and non-functioning (NFA) pituitary adenomas reported by microarray-based studies.
MATERIAL AND METHODS: Analysis of gene expression was performed by real-time quantitative PCR (QPCR) in 76 pituitary adenomas, 25 functioning and 51 non-functioning ones. The expression of the examined genes was normalized to the reference index, obtained by calculation of the geometric mean of reference genes expression: GUS-B, B2M, ACTB, EIF3S10, UBE2D2 and ATP6V1E.
RESULTS: Two genes showed significant differences in expression between non-functioning adenomas and functioning ones (FA) (FOLR1 32.4 x greater p = 0.022, BAG1 2.2 x lower p = 0.0002). The expression of LAPTM4B (1.1 x lower) was only insignificantly changed. The expression of FOLR1 in all tumours (functioning and non-functioning) was higher in older patients (over 50 years of age) (p = 0.018). Expression of BAG1 was significantly lower in older patients (p = 0.015). In a subgroup of pure non-functioning adenomas there was a higher expression of FOLR1 in older patients (p = 0.006). Analysis of expression profiles and invasiveness of tumours did not reveal any significant differences both in non-functioning and functioning tumours.
CONCLUSIONS: Among pituitary adenomas, the highest level of expression FOLR1 is seen in NFA which are negative by immunohistochemistry to all pituitary hormones while GH-producing adenomas are the only class of pituitary tumours where FOLR1 expression is virtually absent. For BAG1 we confirm a significantly higher expression in functioning (both PRL and GH producing) adenomas than non-functioning ones, while LAPTM4B does not exhibit any expression changes between different classes of pituitary tumours.

Ndossi DG, Frizzell C, Tremoen NH, et al.
An in vitro investigation of endocrine disrupting effects of trichothecenes deoxynivalenol (DON), T-2 and HT-2 toxins.
Toxicol Lett. 2012; 214(3):268-78 [PubMed] Related Publications
Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins. H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1-1000ng/ml), T-2 toxin (0.0005-5ng/ml) or HT-2 toxin (0.005-50ng/ml) for 48h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes. Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P<0.05) by DON (100ng/ml), T-2 toxin (0.5ng/ml) and HT-2 toxin (5ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study. Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Das Ghosh D, Bhattacharjee B, Sen S, et al.
Some novel insights on HPV16 related cervical cancer pathogenesis based on analyses of LCR methylation, viral load, E7 and E2/E4 expressions.
PLoS One. 2012; 7(9):e44678 [PubMed] Free Access to Full Article Related Publications
This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Goh WW, Lee YH, Ramdzan ZM, et al.
A network-based maximum link approach towards MS identifies potentially important roles for undetected ARRB1/2 and ACTB in liver cancer progression.
Int J Bioinform Res Appl. 2012; 8(3-4):155-70 [PubMed] Free Access to Full Article Related Publications
Hepatocellular Carcinoma (HCC) ranks among the deadliest of cancers and has a complex etiology. Proteomics analysis using iTRAQ provides a direct way to analyse perturbations in protein expression during HCC progression from early- to late-stage but suffers from consistency and coverage issues. Appropriate use of network-based analytical methods can help to overcome these issues. We built an integrated and comprehensive Protein-Protein Interaction Network (PPIN) by merging several major databases. Additionally, the network was filtered for GO coherent edges. Significantly differential genes (seeds) were selected from iTRAQ data and mapped onto this network. Undetected proteins linked to seeds (linked proteins) were identified and functionally characterised. The process of network cleaning provides a list of higher quality linked proteins, which are highly enriched for similar biological process gene ontology terms. Linked proteins are also enriched for known cancer genes and are linked to many well-established cancer processes such as apoptosis and immune response. We found that there is an increased propensity for known cancer genes to be found in highly linked proteins. Three highly-linked proteins were identified that may play an important role in driving HCC progression - the G-protein coupled receptor signalling proteins, ARRB1/2 and the structural protein beta-actin, ACTB. Interestingly, both ARRB proteins evaded detection in the iTRAQ screen. ACTB was not detected in the original dataset derived from Mascot but was found to be strongly supported when we re-ran analysis using another protein detection database (Paragon). Identification of linked proteins helps to partially overcome the coverage issue in shotgun proteomics analysis. The set of linked proteins are found to be enriched for cancer-specific processes, and more likely so if they are more highly linked. Additionally, a higher quality linked set is derived if network-cleaning is performed prior. This form of network-based analysis complements the cluster-based approach, and can provide a larger list of proteins on which to perform functional analysis, as well as for biomarker identification.

Vajda A, Marignol L, Barrett C, et al.
Gene expression analysis in prostate cancer: the importance of the endogenous control.
Prostate. 2013; 73(4):382-90 [PubMed] Related Publications
BACKGROUND: Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer.
METHODS: Prostate cancer (LNCaP, 22Rv1, PC3, and DU145) and normal (PWR1E and RWPE1) cell lines were cultured in air and hypoxia. The performance of 16 HKGs was assessed using Normfinder and coefficient of variation. In silico promoter analysis was performed to identify putative hypoxia response elements (HREs). The impact of the endogenous control on expression levels of HIF1A and GSTP1 was investigated by qRT-PCR in cell lines and tissue specimens respectively.
RESULTS: Hypoxia altered expression of several HKGs: IPO8, B2M, and PGK1. The most stably expressed HKGs were ACTB, PPIA, and UBC. Both UBC and ACTB showed constitutive expression of HIF1A in air and hypoxia, while PGK1 falsely implied a sixfold hypoxia-induced down-regulation. In prostate tumors, UBC and PGK1 both revealed down-regulation of GSTP1 relative to matched benign, whereas ACTB showed variability.
CONCLUSIONS: This study demonstrates that no universal endogenous control exists for gene expression studies, even within one disease type. It highlights the importance of validating expression of intended HKGs between different sample types and environmental exposures.

Chiba M, Kimura M, Asari S
Exosomes secreted from human colorectal cancer cell lines contain mRNAs, microRNAs and natural antisense RNAs, that can transfer into the human hepatoma HepG2 and lung cancer A549 cell lines.
Oncol Rep. 2012; 28(5):1551-8 [PubMed] Free Access to Full Article Related Publications
Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

Rienzo M, Schiano C, Casamassimi A, et al.
Identification of valid reference housekeeping genes for gene expression analysis in tumor neovascularization studies.
Clin Transl Oncol. 2013; 15(3):211-8 [PubMed] Related Publications
INTRODUCTION: Real time RT-PCR is a widely used technique to evaluate and confirm gene expression data obtained in different cell systems and experimental conditions. However, there are many conflicting reports about the same gene or sets of gene expression. A common method is to report the interest gene expression relative to an internal control, usually a housekeeping gene (HKG), which should be constant in cells independently of experimental conditions.
MATERIALS AND METHODS: In this study, the expression stability of ten HKGs was considered in parallel in two cell systems (endothelial and osteosarcoma cells): beta actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Cyclophilin A (PPIA), beta-2-microglobulin (B2M), glucuronidase beta (GUSB), eukaryotic translation elongation factor 1 alpha1 (EEF1A1), transferrin receptor (TFRC), ribosomal protein S18 (RPS18). In order to study the stability of candidate reference genes, data have been also analyzed by several algorithms (geNorm, NormFinder, BestKeeper and delta-Ct method).
RESULTS AND CONCLUSIONS: The overall analysis obtained by the comprehensive ranking showed that RPS18 and PPIA are appropriate internal reference genes for tumor neovascularization studies where it is necessary to analyze both systems at the same time.

Ma Y, Dai H, Kong X, Wang L
Impact of thawing on reference gene expression stability in renal cell carcinoma samples.
Diagn Mol Pathol. 2012; 21(3):157-63 [PubMed] Related Publications
More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M=0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M=1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P<0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P<0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods.

Com E, Clavreul A, Lagarrigue M, et al.
Quantitative proteomic Isotope-Coded Protein Label (ICPL) analysis reveals alteration of several functional processes in the glioblastoma.
J Proteomics. 2012; 75(13):3898-913 [PubMed] Related Publications
Glioblastoma (GB), the most frequent primary tumor of the central nervous system, remains one of the most lethal human cancers despite intensive researches. Current paradigm in the study of GB has been focused on inter-patient variability and on trying to isolate new classification elements or prognostic factors. Here, using ICPL, a technique for protein relative quantification by mass spectrometry, we investigated protein expression between the four regions of GB on clinically relevant biopsies from 5 patients. We identified 584 non-redundant proteins and 31 proteins were found to be up-regulated in the tumor region compared to the peri-tumoral brain tissue, among which, 24 proteins belong to an interaction network linked to 4 biological processes. The core of this network is mainly constituted of interactions between beta-actin (ACTB) with heat shock proteins (HSP90AA1, HSPA8) and 14-3-3 proteins (YWHAZ, YWHAG, YWHAB). A cluster of three isoforms of the sodium pump α-subunit (ATP1A1, ATP1A2, ATP1A3) was also identified outside this network. The differential expression observed for ACTB and 14-3-3γ was further validated by western blot and/or immunohistochemistry. Our study confirms the identity of previously proposed molecular targets, highlights several functional processes altered in GB such as energy metabolism and synaptic transmission and could thus provide added value to new therapeutic trails.

Lohr JG, Stojanov P, Lawrence MS, et al.
Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing.
Proc Natl Acad Sci U S A. 2012; 109(10):3879-84 [PubMed] Free Access to Full Article Related Publications
To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase-mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease.

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