Early detection and prognosis of colon cancers

A technology for colorectal cancer, colon, in the field of aberrant methylation patterns

Inactive Publication Date: 2010-03-31
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The precise number of such epigenetic lesions in any given tumor is not precisely known, and while an increasing number of random screening methods are identifying an increasing number of candidate genes (5-12), none of these methods The method effectively covers the whole genome

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  • Early detection and prognosis of colon cancers
  • Early detection and prognosis of colon cancers
  • Early detection and prognosis of colon cancers

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Experimental program
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Embodiment 1

[0096] Materials and Method

[0097] [88] Cell culture and processing. HCT116 cells and isogenic genetic knockout derivatives are maintained as previously described (14). For drug treatment, logarithmic phase CRC cells were cultured in McCoys 5A medium (Invitrogen) for 96 hours. McCoys 5A medium contained 10% BCS and 1× penicillin / streptomycin and 5 μM 5 aza-deoxycytidine (DAC ) (Sigma; stock solution: 1 mM in PBS), changing the medium and DAC every 24 hours. The cells were treated with 300 nM Trichostatin A (Sigma; stock solution: 1.5 mM dissolved in ethanol) for 18 hours. The control cells were simulated in parallel, and an equal volume of PBS or ethanol without drugs was added.

[0098] [89] Microarray analysis. Following the manufacturer's instructions, including DNase digestion steps, use Triazol (Invitrogen) and RNeasy kit (Qiagen) to harvest total RNA from log phase cells. NanoDropND-100 was used, followed by 2100 Bioanalyzer (Agilent Technologies) for quality evaluatio...

Embodiment 2

[0103] [93] For the comprehensive identification of hypermethylation-dependent gene expression changes, our first step is to compare wild-type HCT116CRC cells with a single cell carrying two major human DNA methyltransferases in a genome-wide expression array-based method. And the isogenic chaperone cell of the combined gene deletion ( Figure 1A , 14). Importantly, in DNMT1 (- / -) DNMT3b (- / -) Double knockout (DKO) HCT116 cells-which actually completely lost all 5-methylcytosine, all previously single-checked hypermethylated genes-lacking basic expression in wild-type cells-suffered Promoters accompanying gene re-expression are demethylated (14-17). By grading genes according to the altered signal intensity on the 44K Agilent Technologies array platform, when compared with isogenic wild-type parent cells or isogenic cell lines, we observed a unique peak of increased gene expression in DKO cells. Gene wild-type parent cell or isogenic cell line 1 or 3b of DNMT has been deleted ...

Embodiment 3

[0106] [95] To illustrate the sensitivity of our new array method to identify CpG island hypermethylated genes, we first examined 11 genes known to be hypermethylated, completely silenced, and re-expressed after DAC treatment in HCT116 cells (Figure 4(S1A))(14-17). All tested genes remained in the TSA unreacted zone (Figure 4 (S1B and C)), and the direction of expression change was fully correlated in DAC treatment and DKO cells (Figure 4 (S1D)). Importantly, for the increase in DAC, 5 guide genes (45%) increased by a factor of 2 or more, and another 3 genes or a total of 73% of genes increased by a factor of 1.3 or more.

[0107] [96] Based on the observed sensitivity difference between DKO and DAC-induced gene increase and the behavior of the guide gene in the array platform, in the TSA negative region, we specified the upper layer of the gene (two-fold increase or more) and The lower layer (increase between 1.4-fold and 2-fold) to identify hypermethylated oncogenes ( Figure ...

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Abstract

We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island hypermethylation and transcriptional silencing in colorectal cancer (CRC). By screening cell lines andvalidating tumor specific hypermethylation in a panel of primary human CRC samples, we estimate that nearly 5% of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find a much larger number of genes hypermethylated in individual tumors, and much higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken. The genes we identified can be used inter alia diagnostically to detect cancer, pre-cancer, and likelihood of developing cancer.

Description

[0001] [01] The present invention was made using US government funds from the National Institute of Environmental Health Sciences grant No. ES11858 and from the National Cancer Institute Grant No. CA043318 . The U.S. government reserves certain rights in this invention under the terms of these grants. [0002] Invention Technical Field [0003] [02] The present invention relates to the field of cancer diagnosis and treatment. In particular, it relates to abnormal methylation patterns (patterns) of specific genes in colon cancer and pre-cancer. Background of the invention [0004] DNA methylation and its role in carcinogenesis [0005] [03] The information on the cells that make all living organisms is contained in their DNA. DNA is composed of a unique sequence of four bases, the four bases are: adenine (A), guanine (G), thymine (T) and cytosine (C). On the two strands forming the DNA double helix, these bases A and T and G and C are paired. These chains of base pairs store infor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C07H21/04
CPCC12Q2523/125C12Q2600/154C12Q1/6886C12Q2600/106A61P35/00
Inventor S·B·拜林K·E·舒贝尔W·V·克里金格
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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