Frequent mutation related with overexpression of DNA polymerase beta in primary tumors and precancerous lesions of human stomach
Introduction
DNA repair system is critical to the integrity of the genome and may be altered in early stages of carcinogenesis. Maintenance of intact genome requires different DNA synthesis operated by various DNA polymerases [1]. Polymerase beta (pol β) bears primary responsibility for a single nucleotide gap and mainly plays a critical role in base excision repair (BER) [2]. Pol β can be distinguished from other DNA polymerases by the lack of associated proofreading activity and a poor ability to discriminate nucleotides, and it belongs to specialized DNA polymerases [3], [4], [5], [6], [7], [8], [9], which perform translesion DNA synthesis of specific types of damage with high genetic fidelity. But they exhibit reduced fidelity when copying on undamaged DNA or on DNA with lesions that are (apparently) not cognate substrates, which induces genomic instability [10], [11], [12], [13], [14], [15]. In light of these characteristics, it is essential that pol β be regulated at a lower level and independent from cell cycles [16], so that it can function normally in BER and its mutagenic potential is limited. Abnormal pol β could hinder BER and/or increase error-prone DNA synthesis and leads to accumulation of oncogene and tumor suppressor genes mutations, and consequently induces the cancer risk [17], [18].
Thus far, alteration of pol β gene has been reported in tumors [19], [20], [21]. Recent investigations revealed that frequent mutations within the pol β coding sequence were present in tumors analyzed, but not in normal tissues from the same patients and normal individuals [22]. Furthermore, loss of heterozygosity in the pol β locus on the short arm of chromosome 8 is a frequent event in a variety of human tumors [23], [24]. In addition, it is also reported that the percentage of alternative splicing of pre-mRNA variant of pol β increased in some tumors [25]. And the alteration of pol β expression appeared to induce major genetic changes associated with a malignant phenotype [18], [26]. However, these studies on pol β concentrated mainly on different malignancy, but rarely denoted early stages of carcinoma.
Gastric cancer (GC) is the leading cause of cancer death in China [27]. Epidemiological investigations suggest a pathogenetic model of GC involves intermediate stages including chronic atrophic gastritis (CAG), intestinal metaplasias (IM) and dysplasias (Dys) [28], [29], during which gastric mucosa is constantly exposed to a wide variety of genotoxic agents of either endogenous and exogenous sources, with the later such as salted food and Helicobacter pylori prevails [30], [31]. It is quite possible that the event of DNA damage and repair may occur in the early and intermediate stages of the GC. In this study, we analyzed alterations of pol β in 13 GC cell lines and 104 tissues from Chinese patients with GC and precancerous lesions.
Section snippets
Cell lines and tissue samples
Eight GC cell lines AGS, N-87, SUN1, SUN5, SNU16, RF1, RF48 and MKN45 were obtained from American Type Culture Collection (ATCC). Cell lines BGC823, MGC803, SGC7901, PAMC82, and GES were established in China [32], [33]. One hundred and four human gastric specimens including 21 superficial gastritis (SG), 19 CAG, 14 IM, 10 Dys, and 40 GC and their matched normal gastric tissues were collected by gastroscopy or surgery (Table 1). None of the patients had received any chemotherapy or radiotherapy
Detection of pol β mutations in tumor cell lines, primary tumors and precancerous lesions of human stomach
In order to examine whether a dynamic alteration of pol β occurred in the tissues from different stages of gastric precancerous lesions to GC, total coding region of pol β cDNA from 13 cell lines and 104 gastric tissues was amplified by RT-PCR and screened by DHPLC followed by sequencing. The resultant sequences were compared with normal counter part reported in GeneBank. As the data shown in Table 3, nine types of point mutation in both mRNA and genomic DNA of pol β were detected in 23 of 104
Discussion
The pathogenesis of GC is thought to involve a multistep and multifactorial process, in which DNA repair systems are critical to the malignant transformation of the cell and are the first defensive line against cancer [40]. The DNA repair enzyme pol β plays a rate-determining role in BER, in addition to its involvement in the multiple steps before and after DNA synthesis. Malfunction of pol β includes hypofunction, which leads to deficiency or ineffective BER, and hyperfunction, which leads to
Acknowledgements
We are thankful to Dr Qimin Zhan (National Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Science) for critical reading and insight comments on the manuscript. And we thank Dr Zhi-Feng Pang and Dr Qi-Feng Zhang for providing tissue specimens. This study was supported by grants from the National Key Basic Research Program Project (Grants 199803-1200), National Science Foundation in China NSFC (Grants: 39725016) and National Bio-Tech 863 Program (2002-AA2Z 2001;
References (49)
- et al.
Low fidelity DNA synthesis by a y family DNA polymerase due to misalignment in the active site
J. Biol. Chem.
(2002) Damage repair DNA polymerases Y
Curr. Opin. Struct. Biol.
(2003)- et al.
The Y-family of DNA polymerases
Mol. Cell
(2001) - et al.
Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases
J. Biol. Chem.
(1997) - et al.
Increased activity and fidelity of DNA polymerase beta on single-nucleotide gapped DNA
J. Biol. Chem.
(1997) - et al.
The fidelity of DNA polymerase beta during distributive and processive DNA synthesis
J. Biol. Chem.
(1999) - et al.
The base substitution fidelity of DNA polymerase beta-dependent single-nucleotide base excision repair
J. Biol. Chem.
(2003) - et al.
Mutation analysis of p53 in ovarian tumors by DHPLC
J. Biochem. Biophys. Methods
(2001) - et al.
Involvement of DNA polymerase beta in DNA replication and mutagenic consequences
J. Mol. Biol.
(2002) - et al.
Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain
J. Biol. Chem.
(1990)