scholarly journals BRCA1 Gene Expression in Breast Cancer: A Correlative Study between Real-time RT-PCR and Immunohistochemistry

2005 ◽  
Vol 53 (5) ◽  
pp. 621-629 ◽  
Author(s):  
Fahd Al-Mulla ◽  
Mahera Abdulrahman ◽  
Govindarajulu Varadharaj ◽  
Nadeem Akhter ◽  
Jehoram T. Anim
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Protein Expression ◽  
Real Time ◽  
Brca1 Gene ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Brca1 Protein ◽  
Brca1 Mrna ◽  
Cancer Tissues

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard.

scholarly journals Analysis of gene expression in Lassa virus-infected HuH-7 cells

2007 ◽  
Vol 88 (5) ◽  
pp. 1568-1575 ◽  
Author(s):  
Stefanie Müller ◽  
Robert Geffers ◽  
Stephan Günther
Keyword(s):  
Gene Expression ◽  
Virus Infection ◽  
Real Time ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Lassa Virus ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Array Data ◽  
U133a Array

The pathogenesis of Lassa fever is poorly understood. As the liver is a major target organ of Lassa virus, gene expression in Lassa virus-infected HuH-7 cells, a differentiated human hepatoma cell line, was studied. Cellular mRNA levels were measured at the late phase of acute infection, when virtually all cells expressed large amounts of nucleoprotein, and virus RNA concentration had reached >108 copies (ml supernatant)−1. Two types of transcription array were used: cDNA-based macroarrays with a set of 3500 genes (Atlas Human 1.2 arrays; Clontech) and oligonucleotide-based microarrays covering 18 400 transcripts (Human Genome U133A array; Affymetrix). Data analysis was based on statistical frameworks controlling the false-discovery rate. Atlas array data were considered relevant if they could be verified by U133A array or real-time RT-PCR. According to these criteria, there was no evidence for true changes in gene expression. Considering the precision of the U133A array and the number of replicates tested, potential expression changes due to Lassa virus infection are probably smaller than twofold. To substantiate the array data, beta interferon (IFN-β) gene expression was studied longitudinally in Lassa virus-infected HuH-7 and FRhK-4 cells by using real-time RT-PCR. IFN-β mRNA levels increased only twofold upon Lassa virus infection, although there was no evidence that the virus inhibited poly(I : C)-induced IFN-β gene expression. In conclusion, Lassa virus interferes only minimally with gene expression in HuH-7 cells and poorly induces IFN-β gene transcription.

Comprehensive Analysis of the Expression and Prognostic Significance of THBSs in Breast Cancer

2021 ◽  
Author(s):  
Jun Wang ◽  
Xuebing Zhan ◽  
Qian Luo ◽  
Yunshu Kuang ◽  
Xiao Liang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Protein Expression ◽  
Cancer Patients ◽  
Prognostic Value ◽  
Breast Cancer Patients ◽  
Free Survival ◽  
Expression Levels ◽  
Cancer Tissues ◽  
Mrna Expression Levels

Abstract Background: Breast cancer is one of the most common tumors for women worldwide. Thrombospondins (THBSs) are reported to play important roles in various cellular processes and are involved in the occurrence and development of human cancers. However, the expression and prognostic value of THBSs family in breast cancer remain unclear.Methods: In this study, we examined the genes and protein expression levels of THBSs and their prognostic value by synthesizing several mainstream databases, including Oncomine, Human Protein Atlas (HPA), UALCAN, and KM Plotter. We also analyzed THBS interaction networks, genetic alterations, functional enrichment, and drug sensitivity with several publicly accessible databases, including GEPIA, GeneMANIA, STRING, cBioPortal, Metascape and NCI-60 database.Results: The results showed that the mRNA expression levels of THBS1, THBS2, THBS3, and THBS5 in breast cancer tissues were significantly higher than in normal tissues. The mRNA expression levels of THBS4 were different in different subtypes of breast cancer, and the protein expression levels of THBS1, THBS2, and THBS4 in breast cancer tissues were higher than in normal breast tissues. Survival analysis showed that breast cancer patients with high THBS1 gene expression showed worse overall survival (OS), relapse-free survival (RFS), and post-progression survival (PPS), and breast cancer patients with high THBS2 gene expression also showed worse RFS. Conversely, lower THBS3 levels predicted worse RFS, and lower THBS4 levels predicted worse OS, RFS, and distant metastasis-free survival (DMFS). Conclusions: These results suggest that THBSs may be potential biomarkers for breast cancer.

scholarly journals BRCA1 participates in the expression of noncoding XIST RNA

2019 ◽  
Vol 18 (1) ◽  
pp. 67-74
Author(s):  
E. A. Shestakova ◽  
T. A. Bogush
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Brca1 Gene ◽  
Breast Cancer Cell Lines ◽  
Rt Pcr ◽  
Brca1 Protein ◽  
Xist Rna

Introduction . Noncoding RNA of XIST gene (X inactivation-specific transcript) initiates inactivation of one of X chromosomes in cells of female organism. Further stages of this process include chromatin epigenetic modifications leading to the inhibition of the most genes on X chromosome. Recently the data were obtained that tumor suppressor BRCA1 is associated with inactive X chromosome (Xi) participating in XIST RNA localization on Xi and influencing XIST RNA expression.Objective: to reveal the role of BRCA1 in XIST RNA expression.Materials and methods . The objects of the study were mutant breast cancer cell lines (BRCA1–/–): HCC1395, HCC1937, SUM149PT, and, as controls – cell lines containing wild type of BRCA1 gene (BRCA1+/+): IMR90 и 293T. Method of reverse transcription coupled with polymerase chain reaction (RT-PCR) was used for the analysis of XIST RNA expression.Results . In the clone of doxycycline-inducible HCC1937 breast cancer cell line XIST RNA expression was observed upon BRCA1 induction. In HCC1395, HCC1937 and SUM149PT breast cancer cell lines containing mutant BRCA1 gene (BRCA1–/–) and nonfunctional BRCA1 protein the absence of XIST RNA expression was observed using RT-PCR. This observation indicates the indispensable role of functional BRCA1 protein in XIST RNA expression.Conclusion . Altogether, the data obtained in this study confirm the role of BRCA1 in the expression of noncoding inhibiting XIST RNA and suggest the involvement of BRCA1 in the inhibition of gene expression on Xi.

Gene Transcript Assay by Real-Time Rt-PCR in Epithelial Breast Cancer Cells Selected by Laser Microdissection

2004 ◽  
Vol 19 (2) ◽  
pp. 100-108 ◽  
Author(s):  
V. Becette ◽  
S. Vignaud ◽  
C. Régnier ◽  
M. Labroquère ◽  
E. Fourme ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Target Gene ◽  
Laser Microdissection ◽  
Mrna Level ◽  
Cancer Tissue ◽  
Gene Transcript ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Tissue Samples

The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERα, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor ho-mogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.

scholarly journals A Mutation in the 5′ Untranslated Region of the BRCA1 Gene in Sporadic Breast Cancer Causes Downregulation of Translation Efficiency

2007 ◽  
Vol 35 (4) ◽  
pp. 564-573 ◽  
Author(s):  
J Wang ◽  
C Lu ◽  
D Min ◽  
Z Wang ◽  
X Ma
Keyword(s):  
Breast Cancer ◽  
Untranslated Region ◽  
Novel Mutation ◽  
Brca1 Gene ◽  
Breast Tumour ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Wild Type ◽  
Brca1 Protein

We screened 117 breast tumour samples in Chinese females for mutations in the breast cancer 1 ( BRCA1) gene and identified a novel mutation in the 5′ untranslated region (5′ UTR) in two patients with grade III infiltrating ductal breast carcinoma. We examined whether this 5′ UTR mutation affected the translational efficiency of BRCA1 protein. A vector was constructed containing the mutated 5′ UTR up-stream of luciferase and we compared its translational efficiency with a wild-type 5′ UTR. The expression of BRCA1 protein in breast tumour samples was evaluated using immunohistochemistry. The mutated 5′ UTR of BRCA1 resulted in less luciferase activity compared with the wild-type 5′ UTR, while there were no significant differences in luciferase mRNA levels. BRCA1 protein was much less expressed in breast tumour tissue from patients with the 5′ UTR mutation than in samples from patients without the mutation. Our results show that a mutation in the 5′ UTR of the BRCA1 gene downregulates translational efficiency of the protein.

scholarly journals ALDH1 expression in inflammatory breast cancer tumor using Real-time RT-PCR gene expression quantifications: Moroccan prospective study

2021 ◽  
Vol 29 (2) ◽  
pp. 153-160
Author(s):  
Fouzia Mamouch ◽  
Abdelilah Laraqui ◽  
Narjiss Berrada ◽  
Hicham El Rhaffouli ◽  
Basma Elkhannousi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Prospective Study ◽  
Real Time ◽  
Inflammatory Breast Cancer ◽  
Rt Pcr ◽  
Cancer Tumor

scholarly journals IGF-independent effects of IGFBP-2 on the human breast cancer cell line Hs578T

2006 ◽  
Vol 37 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Klaus W Frommer ◽  
Katharina Reichenmiller ◽  
Burkhardt S Schutt ◽  
Andreas Hoeflich ◽  
Michael B Ranke ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Growth Factor ◽  
Cell Line ◽  
Real Time ◽  
Breast Cancer Cell ◽  
Breast Cancer Cell Line ◽  
Cancer Cell Line ◽  
Rt Pcr ◽  
Independent Effects

There is evidence that insulin-like growth factor-binding protein (IGFBP-2), a modulator of the actions of IGFs, also has IGF-independent effects in human tumor cell lines. These involve specific binding of IGFBP-2 to α5β1-integrin, followed by alterations in the phosphorylation status of downstream signaling molecules. Previously, IGFBP-2 has also been shown to be associated with cell proliferation, adhesion and migration. Here, we investigated direct effects of IGFBP-2 on apoptosis and alterations in the expression of related proteins. The breast cancer cell line Hs578T, which shows no IGFBP-2 production of its own and is independent of the IGF-I receptor, was treated with human recombinant IGFBP-2 in order to study the changes in gene expression induced by IGFBP-2. The methods employed for this purpose were oligonucleotide microarrays, real-time RT-PCR, western blotting, and immunoassays. Out of the 440 genes covered by the Oligo GEArray Human Cancer Microarray OHS-802, the expression of 77 genes was directly influenced by IGFBP-2. By the use of real-time quantitative RT-PCR, the gene expression of Nuclear Factor (NF)κB, p53, transforming growth factor β (TGF β-1), LAMB1 (Laminin, Beta 1), Bcl-2, and IIp45 was found to be significantly upregulated (by 1.2- to 3.05-fold; all P < 0.001). Accordingly, NFκB, p53, and TGF β-1 proteins, as measured by Western blotting and immunoassay, were upregulated > 1.5-fold. By using an ELISA-based and a flow cytometry-based apoptosis assay, IGFBP-2 was found to have a pro-apoptotic effect on Hs578T cells. Our results suggest that IGFBP-2-induced gene expressions are of functional significance for proliferation, cell adhesion, cell migration and apoptosis, and showed that IGFBP-2 can promote apoptosis in tumor cells independent of IGF.

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