Reduced ATR or Chk1 Expression Leads to Chromosome Instability and Chemosensitization of Mismatch Repair–deficient Colorectal Cancer Cells

Abstract

Genomic instability in colorectal cancer is categorized into two distinct classes: chromosome instability (CIN) and microsatellite instability (MSI). MSI is the result of mutations in the mismatch repair (MMR) machinery, whereas CIN is often thought to be associated with a disruption in the APC gene. Clinical data has recently shown the presence of heterozygous mutations in ATR and Chk1 in human cancers that exhibit MSI, suggesting that those mutations may contribute to tumorigenesis. To determine whether reduced activity in the DNA damage checkpoint pathway would cooperate with MMR deficiency to induce CIN, we used siRNA strategies to partially decrease the expression of ATR or Chk1 in MMR-deficient colorectal cancer cells. The resultant cancer cells display a typical CIN phenotype, as characterized by an increase in the number of chromosomal abnormalities. Importantly, restoration of MMR proficiency completely inhibited induction of the CIN phenotype, indicating that the combination of partial checkpoint blockage and MMR deficiency is necessary to trigger CIN. Moreover, disruption of ATR and Chk1 in MMR-deficient cells enhanced the sensitivity to treatment with the commonly used colorectal chemotherapeutic compound, 5-fluorouracil. These results provide a basis for the development of a combination therapy for those cancer patients. INTRODUCTIONEukaryotic cells are continuously exposed to endogenous and exogenous insults capable of damaging DNA. To maintain the integrity of their genomes, cells have evolved a series of sophisticated and complex signaling networks that allow cells to respond to genotoxic injury. Such responses include recognition of DNA lesions, activation of cell cycle checkpoints and DNA repair mechanisms, and, in the event of irreparable damage, initiation of apoptosis. Defects in many of these surveillance mechanisms result in the inability of cells to properly process genomic stresses, often leading to genomic instability and subsequently tumorigenesis. Genomic instability can be divided into two clinically distinct classes that have been extensively studied in colorectal cancers: chromosome instability (CIN) and microsatellite instability (MSI) (Kinzler and Vogelstein, 1996;Harfe and Jinks-Robertson, 2000;Rajagopalan et al., 2003;Rustgi, 2007). Tumors with CIN comprise ϳ85% of all colorectal cancers and are characterized by gross karyotypic changes exhibiting abnormalities in chromosome structure and number (Rajagopalan et al., 2003). The pathway leading to the development of CIN is not currently clear but it has been correlated with truncations in the adenomatous polyposis coli (APC) protein (Kinzler and Vogelstein, 1996;Rajagopalan et al., 2003). Tumors with MSI account for the remaining 15% of colorectal cancers. In contrast to CIN tumors, MSI tumors have a relatively stable karyotype and harbor anomalies at the nucleotide level, resulting in frameshift and missense mutations that disrupt the normal function of proto-oncogenes and tumor suppressors. This type of instability has been dir...

RETRACTED ARTICLE: P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance

“…In this case, activation of p38MAPK appears to be dependent on both ATM and ATR proteins, as demonstrated for genotoxic stress in the absence of p53 (Reinhardt et al, 2007), indicating a redundancy in their function. Interestingly, lack of ATM or ATR has been associated to sensitivity to 5-FU (Fedier et al, 2003;Wilsker and Bunz, 2007;Ewald et al, 2008a;Jardim et al, 2009). In this regard, at least two points should be considered.…”

“…Resistance to 5-FU may occur by a number of mechanisms, including alterations in the enzymes implicated in the metabolism or in the mechanism of action of the drug, especially in the target enzyme (for review, see Peters et al, 2002), also, alterations in molecules such as p53 (Lowe et al, 1993;Bunz et al, 1999;Longley et al, 2002;Tominaga et al, 2010) and in proteins critical in the response to DNA damage, such as ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3 related (ATR) (for review, see Wyatt and Wilson III, 2009). For example, ATR has been shown to be an essential mediator in the antitumoral properties of 5-FU (Wilsker and Bunz 2007) through the control exerted on the mismatch repair system (Liu et al, 2008) or on CHK1, a regulatory component of the DNA damage checkpoint (Jardim et al, 2009). CHK1 has been recently proposed to have a major role in the autophagy triggered by antimetabolites, including 5-FU (Zeng and Kinsella, 2010).…”

Analysis of Mutations That Dissociate G2 and Essential S Phase Functions of Human Ataxia Telangiectasia-mutated and Rad3-related (ATR) Protein Kinase

“…Gene dosage is known to be important for ATR function. ATR is a haploinsufficient tumor suppressor in certain genetic backgrounds (48,49), and reducing ATR protein levels can sensitize tumor cell lines to chemotherapies (50). Additionally, a hypomorphic allele of ATR found in Seckel syndrome patients results in diminished ATR protein levels due to aberrant splicing (51,52).…”

Persimmon Leaf Extract Inhibits the ATM Activity during DNA Damage Response Induced by Doxorubicin in A549 Lung Adenocarcinoma Cells

“…DOX mainly activates ATM through the induction of double-strand break in cellular DNA, 1,25,26) whereas HU or 5FU preferentially activates ATR that is sensitive to single-strand break. [27][28][29][30] Therefore we further examined whether PLE could inhibit the phosphorylation of ATM. As expected, ATM phosphorylation was dose-dependently inhibited in the cells pre-incubated with PLE (Fig.…”