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Stephen G Chaney

Publications and source records attributed to Stephen G Chaney.

10 recordsLinked to original sources
PubMed2006

p53 and p21 regulate error-prone DNA repair to yield a lower mutation load.

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.

Animals↗
PubMed2005

Recognition and processing of cisplatin- and oxaliplatin-DNA adducts.

The cytotoxicity of platinum compounds is thought to be determined primarily by their DNA adducts. Cisplatin and oxaliplatin are structurally distinct, but form the same types of adducts at the same sites on DNA. However, the DNA adducts are differentially recognized by a number of cellular proteins. For example, mismatch repair proteins and some damage-recognition proteins bind to cisplatin-GG adducts with higher affinity than to oxaliplatin-GG adducts, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both DNA polymerase beta (pol beta) and DNA polymerase eta (pol eta) catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta may explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins.

Base Pair Mismatch↗
PubMed2004

The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts.

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase eta is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase eta in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase eta-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase eta expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase eta at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase eta. These results indicate that polymerase eta is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase eta, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.

Antineoplastic Agents↗
PubMed2004

NMR solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer duplex.

We have determined, at high resolution, the NMR solution structure of an oxaliplatin-GG DNA dodecamer in the AGGC sequence context by 2D NMR studies. Homonuclear assignment strategies resulted in unambiguous assignment of 203 out of 249 protons, which corresponds to assignment of approximately 81% of the protons. Assignments of H5' and H5" protons were tentative due to resonance overlap. The structure of the oxaliplatin duplex was calculated using the program CNS with a simulated annealing protocol. A total of 510 experimental restraints were employed in the structure calculation. Of 20 calculated structures, the 15 with the lowest energy were accepted as a family. The RMSD of the 15 lowest energy structures was 0.68 A, indicating good structural convergence. The theoretical NOESY spectrum obtained by back-calculation from the final average structure showed excellent agreement with the experimental data, indicating that the final structure was in good agreement with the experimental NMR data. Significant conformational differences were observed between the oxaliplatin-GG 12-mer DNA we studied and all previous solution structures of cisplatin-GG DNA duplexes. For example, the oxaliplatin-GG adduct shows much less distortion at the AG base-pair step than the cisplatin-GG adducts. In addition, the oxaliplatin-GG structure also has a narrow minor groove and an overall axis bend of about 31 degrees, both of which are very different from the recent NMR structures for the cisplatin-GG adducts. These structural differences may explain some of the biological differences between oxaliplatin- and cisplatin-GG adducts.

Antineoplastic Agents↗
PubMed2004

Protein interactions with platinum-DNA adducts: from structure to function.

Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol beta and pol eta catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.

Base Sequence↗
PubMed2003

Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases beta and eta in vitro.

DNA polymerases beta and eta are among the few eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Our laboratory has previously established that both polymerases misincorporated dTTP with high frequency across from cisplatin- and oxaliplatin-GG adducts. This decrease in polymerase fidelity on platinum-damaged DNA could lead to in vivo mutations, if this base substitution were efficiently elongated. In this study, we performed a steady-state kinetic analysis of the steps required for fixation of dTTP misinsertion during translesion synthesis past cisplatin- and oxaliplatin-GG adducts by pol beta and pol eta. The efficiency of translesion synthesis by pol eta past Pt-GG adducts was very similar to that observed for this polymerase when the template contains thymine-thymine dimers. This finding suggested that pol eta could play a role in translesion synthesis past platinum-GG adducts in vivo. On the other hand, translesion synthesis past platinum-GG adducts by pol beta was much less efficient. Translesion synthesis by pol eta is likely to be predominantly error-free, since the probability of correct insertion and extension by pol eta was 1000-2000-fold greater than the probability of incorrect insertion and extension. Our results also indicated that for pol eta the frequency of misincorporation is the same across from the 3'G and the 5'G of the platinum-GG adducts for both cisplatin and oxaliplatin adducts. On the other hand, pol beta is more likely to misinsert at the 3'G of the adducts and misinsertion occurs at higher frequency for oxaliplatin-GG than for cisplatin-GG adducts.

Base Pair Mismatch↗
PubMed2003

Translesion synthesis past platinum DNA adducts by human DNA polymerase mu.

DNA polymerase mu (pol mu) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase beta (pol beta) and terminal deoxynucleotidyl transferase (TdT). Because pol beta has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol mu to bypass these lesions. Pol mu bypassed CP and OX adducts with an efficiency of 14-35% compared to chain elongation on undamaged DNA, which is second only to pol eta in terms of bypass efficiency. The relative ability of pol mu to bypass CP and OX adducts was dependent on both template structure and sequence context. Since pol mu has been shown to be more efficient on gapped DNA templates than on primed single-stranded DNA templates, we determined the ability of pol mu to bypass Pt-DNA adducts on both primed single-stranded and gapped templates. The bypass of Pt-DNA adducts by pol mu was highly error-prone on all templates, resulting in 2, 3, and 4 nt deletions. We postulate that bypass of Pt-DNA adducts by pol mu may involve looping out the Pt-GG adduct to allow chain elongation downstream of the adduct. This reaction appears to be facilitated by the presence of a downstream "acceptor" and a gap large enough to provide undamaged template DNA for elongation past the adduct, although gapped DNA is clearly not required for bypass.

Catalysis↗
PubMed2002

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta.

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.

Base Sequence↗
PubMedDate not supplied

Oxaliplatin biotransformation and pharmacokinetics: a pilot study to determine the possible relationship to neurotoxicity.

BACKGROUND: Both oxaliplatin and ormaplatin undergo biotransformation to Pt(dach)Cl2 with studies suggesting a predictive relationship between systemic exposure to Pt(dach)Cl2 and the severity of the delayed sensory neuropathy associated with ormaplatin. Studies characterizing the pharmacokinetic parameters of oxaliplatin and Pt(dach)Cl2 in humans have not been reported. This study was conducted to characterize the pharmacokinetic parameters of oxaliplatin and Pt(dach)Cl2 and to determine the extent to which oxaliplatin undergoes biotransformation to Pt(dach)Cl2 in humans. MATERIALS AND METHODS: Ten adult patients with metastatic colon cancer received oxaliplatin with or without fluorouracil-based chemotherapy. Blood samples were obtained during cycles 1 and 2. Total platinum, oxaliplatin and Pt(dach)Cl2 in the plasma ultrafiltrate were measured using high performance liquid chromatography and atomic absorption spectrometry. All patients underwent a thorough neurological evaluation after each cycle. RESULTS: The median steady-state concentration (C(SS)) (interquartile range, 25% to 75%) for oxaliplatin 85 mg/m2 was 0.33 microg Pt/ml (0.28 to 0.38 microg Pt/ml). The area under the curve (AUC) was 0.79 microg Pt/ml/h (0.62 to 0.88 microg Pt/ml/h) and the elimination half-life was 0.32 h (0.27 to 0.46 h). The median C(SS) for Pt(dach)Cl2 was 0.008 microg Pt/ml (0.004 to 0.014 microg Pt/ml). The C(SS) ratio of oxaliplatin to Pt(dach)Cl2 was 31 (24 to 51). All patients reported acute cold-induced neuropathy following cycles 1 and 2. Only two patients reported delayed sensory neuropathy (grade 1). CONCLUSION: The parent drug oxaliplatin is the major active platinum complex detected in plasma ultrafiltrate for at least the first few hours following oxaliplatin infusion in humans. Therefore, the plasma biotransformation products of oxaliplatin are unlikely to contribute to its efficacy or toxicity. In particular, plasma Pt(dach)Cl2 is unlikely to significantly contribute to the delayed sensory neuropathy associated with oxaliplatin, since only a limited amount (<3%) of oxaliplatin undergoes biotransformation to Pt(dach)Cl2.

Adenocarcinoma↗