Detection of mutations in the DNA polymerase ? gene of human sporadic colorectal cancers and colon cancer cell lines

Flohr, Thomas; Dai, Jia-Chun; Büttner, Jens; Popanda, Odilia; Hagmüller, E.; Thielmann, Heinz Walter

doi:10.1002/(sici)1097-0215(19990315)80:6<919::aid-ijc19>3.0.co;2-u

Cited by 72 publications

(81 citation statements)

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“…Polymerase proofreading mutation in cancer R Yoshida et al POLD1 has previously been explored in a limited number of human colorectal carcinoma tissue specimens, and no sequence alterations causing amino-acid substitutions were found in the genomic sequences encoding the proofreading domain. 32 Similarly, we identified no POLD1 alterations in a relatively large panel of colorectal cancer patients.…”

Section: Resultsmentioning

confidence: 66%

“…32 However, apart from the previously reported R506H mutation in DLD-1 cells, no sequence alterations resulting in amino-acid substitutions were found in the genomic regions encoding the proofreading domain. In this present study, using a relatively large panel of colorectal carcinomas, we first elucidated that POLD1 proofreading domain mutations were indeed infrequent in colorectal cancer patients.…”

Section: Discussionmentioning

confidence: 84%

“…Other types of sequence alterations such as insertions/deletions were not found. Among these sequence alterations, POLD1 R506H in DLD-1 cells has been previously reported 32,44 and therefore is not novel. On the other hand, POLD1 V312M in LoVo cells has not thus far been reported in the literature.…”

Section: Resultsmentioning

confidence: 92%

“…However, the genomic sequences that encode proofreading domains of the both replicative DNA polymerases, pol delta and epsilon, have not previously been explored simultaneously in human diseases, particularly in human neoplasms, although the POLD1 gene encoding the p125 subunit of pol delta has been sequenced in several human colorectal tumours. 32 In this present study, we first sequenced both the genomic sequences encoding the pol delta and epsilon proofreading domains in a large panel of human colorectal carcinomas, including six established human colorectal cancer cell lines, and found novel mutations in both the genes. In particular, the mutation in the POLE gene that encodes p261 catalytic subunit of pol epsilon is the first found in human cells.…”

Section: Introductionmentioning

confidence: 98%

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Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

et al. 2010

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Genomic sequences encoding the 3¢ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms. Keywords: polymerase delta; polymerase epsilon; proofreading domain; colorectal cancer INTRODUCTION Mutation rates on the genome are invariably regulated and typically suppressed to an extremely low level, such as 10 À10 per base replicated, in the organisms. 1 The high fidelity of DNA replication is achieved by several molecular mechanisms. The frequency of erroneous nucleotide incorporation is indeed extremely low compared with that expected from base-pairing energetics, and the vast reduction of the misincorporation frequency involves three steps of molecular events: (a) correct incorporation of complementary nucleotides by DNA polymerases, (b) removal of the newly added nucleotides, particularly incorrectly paired nucleotides, by the 3¢ exonuclease activities associated with the DNA polymerases (proofreading) and (c) postreplicative scanning of mispaired bases by DNA mismatch repair (MMR). 2,3 Various Escherichia coli (E. coli) mutator strains have been used to study these molecular mechanisms by which organisms maintain their mutation rates at very low levels. The dnaQ + (mutD + ) gene of E. coli encodes the epsilon subunit of the DNA polymerase III holoenzyme that is involved in 3¢ exonuclease proofreading activity, 4 and, therefore, the mutation frequencies in the dnaQ mutators are sometimes 1000-10 000 times higher than the wild-type levels. 4,5 In E. coli, MMR is chiefly directed by the proteins encoded by the three genes: mutS + , mutL + and mutH + . 6 The mutS or mutL mutators exhibit an approximately 100 times increase in the mutation frequency. 7 Thus, polymerase proofreading and MMR are the two major

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Section: Resultsmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 98%

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Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

et al. 2010

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“…20 RPA protein overexpression in tumor cells does not seem to result from mutation of the encoding gene since Popanda et al 24 failed to detect amino-acid changes in the RPA and other DNA replication genes analyzed in patients with sporadic colon cancer. 25 The authors have suggested that this scarcity of mutations is due to stringent selection, eliminating functionally impaired replication proteins. 24 The mechanism of RPA1 and RPA2 protein overexpression in colon carcinomas merits further investigation.…”

Section: Discussionmentioning

confidence: 99%

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

et al. 2007

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Replication protein A (RPA), a component of the origin recognition complex, is required for stabilization of single-stranded DNA at early and later stages of DNA replication being thus critical for eukaryotic DNA replication. Experimental studies in colon cancer cell lines have shown that RPA protein may be the target of cytotoxins designed to inhibit cellular proliferation. This is the first study to investigate the expression of RPA1 and RPA2 subunits of RPA protein and assess their prognostic value in colon cancer patients. We analyzed immunohistochemically the expression of RPA1 and RPA2 proteins in a series of 130 colon cancer resection specimens in relation to conventional clinicopathological parameters and patients' survival. Statistical significant positive associations emerged between: (a) RPA1 and RPA2 protein expressions (P ¼ 0.0001), (b) RPA1 and RPA2 labelling indices (LIs) and advanced stage of the disease (P ¼ 0.001 and 0.003, respectively), (c) RPA1 and RPA2 LIs and the presence of lymph node metastasis (P ¼ 0.002 and 0.004, respectively), (d) RPA1 LI and the number of infiltrated lymph nodes (P ¼ 0.021), (e) RPA2 LI and histological grade of carcinomas (P ¼ 0.05). Moreover, a statistical significant higher RPA1 LI was observed in the metastatic sites compared to the original ones (P ¼ 0.012). RPA1 and RPA2 protein expression associated with adverse patients' outcome in both univariate (log rank test: Po0.00001 and 0.00001, respectively) and multivariate (Cox model: P ¼ 0.092 and 0.0001, respectively) statistical analysis. Statistical significant differences according to the expression of RPA1 and RPA2 proteins were also noticed in the survival of stage II (Po0.00001 and 0.0016, respectively) and stage III (P ¼ 0.0029 and 0.0079, respectively) patients. In conclusion, RPA1 and RPA2 proteins appear to be useful prognostic indicators in colon cancer patients and attractive therapeutic targets for regulation by tumor suppressors or other proteins involved in the control of cell proliferation. Modern Pathology (2007) 20, 159-166.

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Mutation analysis of replicative genes encoding the large subunits of DNA polymerase ? and replication factors A and C in human sporadic colorectal cancers

et al. 2000

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We examined cDNAs of the catalytic subunit of DNA polymerase α (185 kDa), the 70 kDa subunit of replication protein A (single‐stranded DNA‐binding protein) and the 140 kDa subunit of replication factor C for mutations. Surgical specimens from 12 patients with sporadic colon cancer and normal mucosae from the same patients were investigated. In addition, we analyzed 3 human colon cancer cell lines that exhibited defects in mismatch repair (DLD‐1, HCT116, SW48) and 3 colon cancer cell lines without such a defect (HT29, SW480 and SW620). For detection of mutations, we used reverse transcription of mRNA, amplification of cDNAs by PCR, analysis of single‐strand conformation polymorphism and DNA sequencing. Eleven colon cancers and 6 colon cancer cell lines were analyzed for DNA polymerase α. Only 2 silent point mutations were detected, in 1 colon carcinoma and in cell line HCT116. Two sequence alterations of the 70 kDa subunit of replication factor A were identified in 15 specimens (9 colon carcinomas and 6 cell lines). Colon carcinomas from 2 patients (CC5MA and CC25HN) exhibited an ACA→GCA transition in codon 351, which caused a Thr→Ala exchange. In carcinomas CC5MA and CC8MA, a TCC→TCT (Ser→Ser) transition in codon 352 was observed. The deviations in codons 351 and 352 occurred in both cancer tissues and normal mucosae, suggesting a genetic polymorphism. No mutation was found in the 140 kDa subunit of replication factor C from 16 specimens (10 tumors and 6 cell lines). Point mutations were identified in the p53 tumor‐suppressor gene in 4 of the 6 colon cancer cell lines and 3 of the 8 carcinoma specimens. We did not find tumor‐associated DNA sequence alterations that resulted in amino acid changes in the DNA replication genes analyzed. We infer that the scarcity of mutations found is due to stringent selection, eliminating functionally impaired replication proteins. Int. J. Cancer 86:318–324, 2000. © 2000 Wiley‐Liss, Inc.

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Detection of mutations in the DNA polymerase ? gene of human sporadic colorectal cancers and colon cancer cell lines

Cited by 72 publications

References 24 publications

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

Mutation analysis of replicative genes encoding the large subunits of DNA polymerase ? and replication factors A and C in human sporadic colorectal cancers

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