Another upregulated gene located in this region was CEP72 , which regulates the localization of key centrosome proteins involved in spindle formation [41]. This gene has been found frequently amplified in non-small-cell lung cancers [42]. Most of the remaining genes that were shown to be upregulated in other studies had a fold change higher than 1.
In contrast with Chr 5, which shows clearly amplified 5p and unaltered 5q, the overall profile of Chr 3 shows that 3p is generally deleted and 3q amplified in some cell lines. They look like the inverse of one another. Furthermore, the phenomenon of amplification in 3q appears to be quite different from that in 5p. For instance, the level of gain or amplification was lower than in 5p and it did not include the entire 3q arm in all the cell lines; instead, only certain sparse regions were found altered recurrently.
The full arm CaLo and HeLa or several regions CaSki and SiHa of 3q were found to be amplified Figure 6 , similarly to the findings in previous reports [5] , [7] , [9] , [19]. This could explain the lower proportion However, even in those cell lines where most of 3q was gained, the proportion of deregulated genes did not rise CaLo or increased modestly HeLa.
In addition, in 3q, the proportion of downregulated genes was higher than the proportion of upregulated genes, particularly in 3q26, where 8 of 11 deregulated genes were downregulated, even some of them were recurrently gained.
Furthermore, similarly to 5p, deregulated genes seemed to be grouped in clusters in 3q26— These findings indicate that an increase in the copy number does not necessarily mean that genes located in those regions will be upregulated. It suggests that, in those entirely amplified regions, epigenetic mechanisms could be involved in gene repression. On the other hand, the increased frequency of downregulated genes with the number of amplified SNPs in the subset of genes located in MRRs, which seems to be not entirely amplified having less than SNPs; Figure 5C , supports that partial gene amplification may be a mechanism of gene silencing.
This idea has been proposed theoretically [44]. However, it has not been demonstrated that these genes were upregulated [5] , [7] , [9] , [19] , particularly in the same samples where the CN alterations were found.
However, conclusions with these negative results from the microarrays may be too risky without the validation with different methodologies, like qPCR and qRT-PCR. Interestingly, the gene encoding for tumor necrosis factor ligand superfamily member 10 TNFSF10 , a protein that induces apoptosis in transformed and tumor cells, was found to be downregulated in the 4 cell lines, even though the gene was recurrently gained MRR ; Figure 6B.
However, the protein encoded by ECT2 epithelial cell transforming sequence two oncogene is a transforming protein that is a nuclear guanine nucleotide exchange factor GEF and regulates RhoB-mediated cell death after DNA damage in cervical cell lines [47]. The expression of this gene is elevated with the onset of DNA synthesis and remains elevated during the G2 and M phases [48].
Increase in gene dosage by DNA amplification is a common mechanism to achieve overexpression of genes in tumors [49]. ECT2 showed the highest fold change of the upregulated genes found at 3q; therefore, it is a good candidate for the amplified driven oncogene in 3q26 for CC.
In 1q the correlation between CN and gene expression was also very poor and similar to the figures seen in 3q. This low correlation could be related to several factors. First, most genes located in the CNAs, identified formerly in the cell lines with the K microarray, were not altered in the number of copies.
Therefore, it is clear that not all amplified genes are upregulated; instead, some of them may be repressed, possibly by epigenetic mechanisms.
Third, deregulated genes were found in clusters, suggesting that, besides the segment amplification, the location in the same chromatin region may influence gene expression. Fourth, the steady rise of downregulated genes with the increase of amplified SNPs in regions CN altered discontinuously suggests that partial gene amplification could be a mechanism of silencing gene expression. All participants signed informed written consent forms prior to their inclusion in the study.
CaLo cell line was a kind gift of Dr. DNA obtained from blood samples collected from 38 healthy women was used as controls for K microarray analysis. Ten samples of normal cervical epithelium were used as controls for the analysis of gene expression. These samples were obtained from cervical specimens of patients undergoing hysterectomy due to myomatosis at the Gynecology Service in the Hospital General de Mexico. Patients were previously diagnosed with a normal cervix by colposcopy and cytology.
Immediately after receiving a cervix fragment from the operating room, the exocervical epithelium was dissected with the aid of a stereoscopic microscope to avoid stromal cells. The quality of RNA was confirmed by the presence of intact ribosomal RNA 28 s and 18 s bands by using agarose-gel electrophoresis. Cell intensity files. This algorithm performs a multiple-chip analysis that facilitates the estimation of probe effects and allele signals simultaneously and, if necessary, borrows the information of other SNPs to better predict the properties of the clusters formed by the genotypes.
To maximize the accuracy of calling, the analysis was performed in a single run including the 38 controls. The software compares the cell lines to a reference set of normal samples. For this analysis, the protocol of unpaired samples was followed. Briefly, the parameters were set as follows: quantile normalization was performed at the perfect match probe level, followed by summarization of the signal intensity for each allele of each SNP.
Genomic smoothing was set to 0. CEL intensity files from the four cell lines and 38 controls were imported into the copy number analysis module of SVS. After the normalization was performed, the log 2 ratio was calculated using the normalized probes intensities with controls as reference.
The calculated log 2 ratio of chromosomes 1, 3 and 5 from the cell lines CaSki, HeLa, SiHa and CaLo were plotted and smoothed with the median using a window radius value of Figure S2 shows that the log 2 ratio profiles of chromosomes 1, 3 and 5 obtained with both softwares are almost identical.
This array contains 33, probe sets that correspond to approximately 20, genes of the human gene reference database according to UCSC Genome Browser Assembly Mar. Then in vitro transcription amplification was performed overnight using the GeneChip amplification kit Affymetrix. A hybridization cocktail was prepared that included the labeled target DNA and control probes for hybridization.
Finally, the chips were scanned using a GeneChip Scanner Array hybridization, scanning, and image analysis were done according to the manufacturer's protocols Affymetrix GeneChip Expression Assay manual. To assess the quality of the experiments, the following parameters were used: the expression of the exogenous polyA controls, the presence of the oligo B2 used to make grid alignments, and the values of the area under the curve AUC above 0.
Only those microarrays with optimal quality controls were then analyzed. Microarrays were normalized using the RMA algorithm robust multichip average in the Affymetrix expression console.
The values of the normalized intensity were referred to as units of intensity UI. A set of 23 genes was used to validate gene expression in the 4 cell lines and 10 healthy cervical epithelium controls with qPCRs.
The rest of genes are located in other chromosomes and were selected to validate gene expression because most of them ranked throughout the first places of de-regulated genes in cell lines Table S4. Measurement of gene expression was based on a relative standard curve constructed from a fold serially diluted pool of the 4 cell-line cDNAs ranging from to 0.
The expression of target genes was normalized in each cell line and control sample to the median intensity of the internal references by using a method previously described in detail [55]. The fold-change expression was calculated by dividing the normalized intensity of each cell line by the average normalized intensity of the control samples.
The statistical difference between each cell line and the set of controls was measured with a Mann—Whitney non-parametric test. The level of correlation between the microarray results and qRT-PCR data was measured with the Pearson's correlation coefficient. Double-color FISH experiments were performed to determinate the copy numbers of 3 regions, i. For 5p, 2 cocktails of probes were used Abbott Laboratories. One included a target probe located at 5p Slide preparation, DNA hybridization, and post-hybridization washes were carried out using standard methods described in the manual.
At least 20 cells were analyzed using direct microscopic visualization and digital-imaging analysis to verify number of signals and probe location. The copy number changes were measured by calculating the ratio between the average number of signals of target and control probes. We used 6. Quantification was performed using both the relative standard curve method and the comparative C T method.
The copy number was calculated by dividing the normalized values of PARP1 of each cell line between the median values of the control samples and then multiplied by 2. Genes were classified using functional annotation clustering with consideration of the gene ontology biological processes.
Classification stringency was set at the maximum level. The CNAs of cell lines were aligned according to the position in the genome, and minimal recurrent regions MRRs common to all 4 cell lines were identified Figure S1. To identify the arms, cytobands or MRRs with the higher alterations, an enrichment analysis, based on a chi squared test, was performed.
The frequencies of the CN-altered SNPs, CN-altered genes or deregulated genes in each of those regions were compared with the frequencies found in the whole genome of cell lines.
A chi square or Fisher exact test, as appropriate, were used to evaluate the statistical significance of the differences. In addition, a Parametric Gene Set Enrichment Analysis PAGE procedure was performed to evaluate the enrichment of deregulated genes in chromosomal arms and cytobands [59]. This procedure is based on the calculation of the Z score, which takes into account the number of deregulated genes and the fold change FC.
Microsoft Excel was used to calculate p-values from Z scores. Gene sets with less than 10 genes were discarded. For 1q, 3q and 5p the genes were ordered according to its position in the chromosome and the fold change graphed.
The identification of clusters of 2 or more contiguous deregulated genes were identified by simple inspection and to test whether this distribution was statistically significant from a random distribution, a chi square test was used. Men could go extinct? Y chromosome disappearing slowly. Skip to content Main Navigation Search.
Dictionary Articles Tutorials Biology Forum. The Homo Species The evolution of the species of the genus "Homo" led to the emergence of modern humans.
Insects There are more species of insects than any other species combined. Bryophytes Bryophytes nonvascular plants are a plant group characterized by lacking vascular tissues. Muscle Muscle cells are specialized to generate force and movement. Developmental gene amplification: insights into DNA replication and gene expression.
Savelyeva L, Schwab M. Amplification of oncogenes revisited: from expression profiling to clinical application. Cancer Lett ; — Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells.
J Biol Chem ; — Gene amplification and drug resistance in cultured murine cells. Science ; —5. Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N- phosphonacetyl -L-aspartate-resistant hamster cells. Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells.
Nat Biotechnol ; —8. Effects of methotrexate on transfected DNA stability in mammalian cells. Mol Cell Biol ; —4. Inducible overproduction of the mouse c-myc protein in mammalian cells. Metaphase chromosome anomaly: association with drug resistance and cell-specific products. Science ; —7. Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes.
Schimke RT. Gene amplification in cultured animal cells. Cell ; — Gene amplification as double minutes or homogeneously staining regions in solid tumors: origin and structure. Genome Res ; — Smith CA, Vinograd J. J Mol Biol ; — Cancer Res ; — Cytogenetic features of human neuroblastomas and cell lines. Novel amplification and transcriptional activity of chorion genes in Drosophila melanogaster follicle cells. McClintock B. Chromosome organization and genic expression. Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons.
The breakage-fusion-bridge BFB cycle as a mechanism for generating genetic heterogeneity in osteosarcoma. Chromosoma ; — Futcher AB. Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae.
J Theor Biol ; — Site-specific recombination promotes plasmid amplification in yeast. The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification. EMBO J ; 7: — Watanabe T, Horiuchi T.
A novel gene amplification system in yeast based on double rolling-circle replication. EMBO J ; —8. Gene amplification system based on double rolling-circle replication as a model for oncogene-type amplification.
Nucleic Acids Res ; e On the mechanism of gene amplification induced under stress in Escherichia coli. PLoS Genet ; 2: e A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Nat Genet ; — Klein G, Klein E. Conditioned tumorigenicity of activated oncogenes. Pall ML. Gene-amplification model of carcinogenesis. Chromosome aberrations in solid tumors. Schwab M, Amler LC. Amplification of cellular oncogenes: a predictor of clinical outcome in human cancer.
Genes Chromosomes Cancer ; 1: — Detection of gene amplification by genomic hybridization to cDNA microarrays. Comparative genomic hybridization CGH -arrays pave the way for identification of novel cancer-related genes. Cancer Sci ; — Computer image analysis of comparative genomic hybridization.
Cytometry ; 10— High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing.
Nat Genet ; —9. Massively parallel sequencing approaches for characterization of structural variation. Methods Mol Biol ; — The landscape of somatic copy-number alteration across human cancers. Nature ; — Transforming mutations of RAC guanosine triphosphatases in human cancers.
Kauraniemi P, Kallioniemi A. Activation of multiple cancer-associated genes at the ERBB2 amplicon in breast cancer. Endocr Relat Cancer ; 39— In: Gunduz M, Gunduz E, editors. Breast cancer — carcinogenesis, cell growth and signalling pathways. Croatia: InTech, — Acta Oncol ; 35— Comparison of HER2 gene amplification assessed by fluorescence in situ hybridization and HER2 protein expression assessed by immunohistochemistry in gastric cancer.
Oncol Rep ; 65— Br J Cancer ; — Breast Cancer Res Treat ; — Grb7-based molecular therapeutics in cancer. Expert Rev Mol Med ; 5: 1— FEBS Lett ; — EMBO J ; — Cancer Treat Rev ; 64— Oncogene ; — MLN64 contains a domain with homology to the steroidogenic acute regulatory protein StAR that stimulates steroidogenesis. Tsujishita Y, Hurley JH. Structure and lipid transport mechanism of a StAR-related domain.
Nat Struct Biol ; 7: — Using Trusted Resources. Coronavirus Information for Patients. Clinical Trials during Coronavirus. Adolescents and Young Adults with Cancer.
Emotional Support for Young People with Cancer. Cancers by Body Location. Late Effects of Childhood Cancer Treatment. Pediatric Supportive Care. Rare Cancers of Childhood Treatment. Childhood Cancer Genomics.
Study Findings. Metastatic Cancer Research. Intramural Research. Extramural Research. Cancer Research Workforce. Partners in Cancer Research. What Are Cancer Research Studies. Research Studies. Get Involved. Cancer Biology Research. Cancer Genomics Research. Research on Causes of Cancer. Cancer Prevention Research. Cancer Treatment Research. Cancer Health Disparities. Childhood Cancers Research. Global Cancer Research. Cancer Research Infrastructure. Clinical Trials. Frederick National Laboratory for Cancer Research.
Bioinformatics, Big Data, and Cancer. Annual Report to the Nation.
0コメント