Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

Butz, James A.; Wickstrom, Eric; Edwards, Jeremy S.

doi:10.1186/1472-6750-3-11

Cited by 41 publications

(12 citation statements)

References 23 publications

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“…Both DLD-1 and Capan-1 cells significantly differ with respect to haplotype distribution from DNA repair proficient cells ( table 4 ). Capan-1 cells showed a significant decrease in heterozygotic loci, 2.1% compare to 3.3% for DNA repair proficient, which was anticipated due to the known trend for loss of heterozygosity in these cells as reported in the literature due to gene conversion in the absence of BRCA2 [42] , [43] . In contrast, there was an increase (5.5%) in hetereozygotic loci in DLD-1 cells, which can potentially be attributed to increased mutation due to the MMR defects responsible for the MSI in DLD-1 cells.…”

Section: Resultssupporting

confidence: 58%

Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

et al. 2014

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Microsatellites (MST), tandem repeats of 1–6 nucleotide motifs, are mutational hot-spots with a bias for insertions and deletions (INDELs) rather than single nucleotide polymorphisms (SNPs). The majority of MST instability studies are limited to a small number of loci, the Bethesda markers, which are only informative for a subset of colorectal cancers. In this paper we evaluate non-haplotype alleles present within next-gen sequencing data to evaluate somatic MST variation (SMV) within DNA repair proficient and DNA repair defective cell lines. We confirm that alleles present within next-gen data that do not contribute to the haplotype can be reliably quantified and utilized to evaluate the SMV without requiring comparisons of matched samples. We observed that SMV patterns found in DNA repair proficient cell lines without DNA repair defects, MCF10A, HEK293 and PD20 RV:D2, had consistent patterns among samples. Further, we were able to confirm that changes in SMV patterns in cell lines lacking functional BRCA2, FANCD2 and mismatch repair were consistent with the different pathways perturbed. Using this new exome sequencing analysis approach we show that DNA instability can be identified in a sample and that patterns of instability vary depending on the impaired DNA repair mechanism, and that genes harboring minor alleles are strongly associated with cancer pathways. The MST Minor Allele Caller used for this study is available at https://github.com/zalmanv/MST_minor_allele_caller.

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Section: Resultssupporting

confidence: 58%

Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

et al. 2014

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“…We further validated that the sorted cells were indeed PANC‐1 cells by performing targeted genomic sequencing. PANC‐1 cells have a loss of heterozygosity in TP53 and a missense mutation at codon 273 (p.R273H), and a heterozygous mutation in KRAS codon 12 (p.G12D) . Replicate populations of 50 recovered PANC‐1 cells from FACS sorting were amplified by multiple displacement amplification (MDA) and sequenced for a panel of 20 oncogenes representative of mutations commonly observed across multiple cancer types.…”

Section: Resultsmentioning

confidence: 99%

“…PANC-1 cells have a loss of heterozygosity in TP53 and a missense mutation at codon 273 (p.R273H), and a heterozygous mutation in KRAS codon 12 (p.G12D). [19] Replicate populations of 50 recovered PANC-1 cells from FACS sorting were amplified by multiple displacement amplification (MDA) and sequenced for a panel of 20 oncogenes representative of mutations commonly observed across multiple cancer types. Bulk genomic DNA (gDNA) from the blood of the healthy donor and the PANC-1 cell line were also sequenced.…”

Section: Selective Delivery To Circulating Tumor Cellsmentioning

confidence: 99%

A Size‐Selective Intracellular Delivery Platform

Saung

Sharei

Adalsteinsson

et al. 2016

Small

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Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 µm wide constriction is effective for delivery of cargo to primary human T-cells that have an average diameter of 6.7 µm. In contrast, a 6 or 7 µm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 µm) and PANC-1 (12.3 µm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T-cells. The application of this approach is demonstrated by selectively delivering dextran-conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.

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“…In response to various forms of cellular stress, the tumor suppressor p53 transcriptionally activates various proapoptotic proteins that can give rise to mitochondrial membrane permeabilization [32]. Although moderately differing in sensitivities, both BxPC-3 and PANC-1, two different p53-mutated pancreatic cancer cell lines, were responsive to JCTH-4 [33,34]. Thus, signalling of p53 and its transcriptional targets, all of which are cellular events upstream of mitochondrial permeabilization, play an insignificant role in JCTH-4-induced apoptosis, supporting the notion of a direct mitochondrial target by JCTH-4.…”

Section: Discussionmentioning

confidence: 99%

Induction of Apoptosis and Autophagy in Human Pancreatic Cancer Cells by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin

Ma¹,

Tremblay²,

Mahngar³

et al. 2011

Am. J. Biomed. Sci.

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Pancreatic cancer is amongst the deadliest cancers in the world. It is associated with poor prognosis, is notorious for developing chemoresistance, and very few approved chemotherapeutics are available to treat this disease. The natural compound pancratistatin (PST) has shown to effectively induce cytotoxicity selectively in numerous cancer cell types. However, it is present in only minute quantities in its natural source and many complications have burdened its chemical synthesis. We have overcome these bottlenecks by synthesizing a C-1 acetoxymethyl analogue of 7-deoxypancratistatin, JC-TH-acetate-4 (JCTH-4), which we have shown to have similar selective anti-cancer activity to that of PST. In this report, we show JCTH-4 to be a potent chemotherapeutic against pancreatic cancer cells (BxPC-3, PANC-1). It induced apoptosis selectively in BxPC-3 and PANC-1 cells by targeting the mitochondria; it dissipated mitochondrial membrane potential, caused release of apoptogenic factors, and in isolated mitochondria, increased the generation of reactive oxygen species. Furthermore, JCTH-4 selectively induced autophagy in pancreatic cancer cells while normal human fetal fibroblasts were markedly less sensitive to JCTH-4 insult. Altogether, this study outlines JCTH-4 as a potentially safe and effective chemotherapeutic agent in treating notoriously chemoresistant pancreatic cancer.

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Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

Cited by 41 publications

References 23 publications

Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

Exome-Wide Somatic Microsatellite Variation Is Altered in Cells with DNA Repair Deficiencies

A Size‐Selective Intracellular Delivery Platform

Induction of Apoptosis and Autophagy in Human Pancreatic Cancer Cells by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin

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