Enrichment technique to allow early detection and monitor emergence of KRAS mutation in response to treatment.

Sensitivity of cell-free circulating tumour DNA (ctDNA) assays is often hampered by the limited quantity of intact mutant nucleotide fragments. To overcome the issue of substrate limitation in clinical applications, we developed an enrichment method utilizing pyrrole-imidazole (PI) polyamides and their ability to bind the minor groove of B-DNA. We present here a proof-of-concept experiment to enrich specific mutant KRAS alleles with biotinylated PI polyamides. We investigated the clinical feasibility of incorporating PI polyamides to detect KRAS mutations in ctDNA from 40 colorectal cancer (CRC) patients, of whom 17 carried mutations in KRAS. After enriching ctDNA with those polyamides, we used digital PCR to detect several common KRAS codon 12 mutations. Enrichment by biotinylated PI polyamides improved the sensitivity of ctDNA analysis (88.9% vs. 11.1%, P < 0.01) in 9 non-metastatic mutation-positive patients. We observed no differences in performance for the 8 metastatic subjects (100% vs. 75%, P = 0.47). In the remaining 23/40 patients with wild type KRAS codon 12, no mutant alleles were detected with or without polyamide-facilitated enrichment. Enriching B-form of ctDNA with PI polyamides significantly improved the assay sensitivity in detecting KRAS mutations in non-metastatic CRC patient samples.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Evaluation of benzimidazole resistance status in Ascaridia galli.

Susceptability of Ascaridia galli to benzimidazole (BZ) was investigated using faecal egg count reduction test (FECRT), in ovo larval development test (LDT) and genetic markers (mutations at codons 167, 198 and 200 of ß-tubulin gene). Six flocks (F1-F6) of a commercial laying hen farm with different number of exposure to BZ were recruited. The FECR was calculated by analyzing individual faeces (F1, F2, F4 and F5) before and 10 days after treatment. The LDT was performed on parasite eggs from pooled samples from F1 to F6 and LC50 and LC99 were calculated. DNA was extracted from 120 worms and sequenced for ß-tubulin gene. In all flocks, the FECRs were above 95% (lower CI above 90%). No significant difference was observed (p > 0·05) among obtained LC50 (F1/F4 and F2/F5 vs F3/F6) in the LDT. However, LC50 and LC99 were higher than suggested values for declaration of resistance in other nematode species. No variation was observed in codon positions involved in BZ resistance. Overall, our results indicated lack of evidence of resistance to BZ in A. galli. More research is needed to confirm these results and to further optimize the existing tools for detection and monitoring of anthelmintic resistance in A. galli.

AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249.

Aflatoxin B1 (AFB1) contamination in the food chain is a major cause of hepatocellular carcinoma (HCC). More than 60% of AFB1 related HCC carry p53 codon 249 mutations but the causal mechanism remains unclear. We found that 1) AFB1 induces two types of DNA adducts in human hepatocytes, AFB1-8,9-epoxide-deoxyguanosine (AFB1-E-dG) induced by AFB1-E and cyclic α-methyl-γ-hydroxy-1,N2-propano-dG (meth-OH-PdG) induced by lipid peroxidation generated acetaldehyde (Acet) and crotonaldehyde (Cro); 2) the level of meth-OH-PdG is >30 fold higher than the level of AFB1-E-dG; 3) AFB1, Acet, and Cro, but not AFB1-E, preferentially induce DNA damage at codon 249; 4) methylation at -CpG- sites enhances meth-OH-PdG formation at codon 249; and 5) repair of meth-OH-PdG at codon 249 is poor. AFB1, Acet, and Cro can also inhibit DNA repair and enhance hepatocyte mutational sensitivity. We propose that AFB1-induced lipid peroxidation generated aldehydes contribute greatly to hepatocarcinogenesis and that sequence specificity of meth-OH-PdG formation and repair shape the codon 249 mutational hotspot.

Quaternionic representation of the genetic code.

A heuristic diagram of the evolution of the standard genetic code is presented. It incorporates, in a way that resembles the energy levels of an atom, the physical notion of broken symmetry and it is consistent with original ideas by Crick on the origin and evolution of the code as well as with the chronological order of appearance of the amino acids along the evolution as inferred from work that mixtures known experimental results with theoretical speculations. Suggested by the diagram we propose a Hamilton quaternions based mathematical representation of the code as it stands now-a-days. The central object in the description is a codon function that assigns to each amino acid an integer quaternion in such a way that the observed code degeneration is preserved. We emphasize the advantages of a quaternionic representation of amino acids taking as an example the folding of proteins. With this aim we propose an algorithm to go from the quaternions sequence to the protein three dimensional structure which can be compared with the corresponding experimental one stored at the Protein Data Bank. In our criterion the mathematical representation of the genetic code in terms of quaternions merits to be taken into account because it describes not only most of the known properties of the genetic code but also opens new perspectives that are mainly derived from the close relationship between quaternions and rotations.

[Resistance to medicinal tumor therapy. Current status]. / Resistenzen gegenüber medikamentöser Tumortherapie. Aktueller Status.

In the past multiple mechanisms could be identified that are involved in anticancer drug resistance; however, diagnostic assays for prediction of therapy response to classical cytostatic drugs did not enter routine clinical diagnostics. Only when new targeted drugs, e.g. tyrosine kinase inhibitors or therapeutic antibodies, were introduced in oncology were diagnostics for prediction of therapy response routinely preformed. First and foremost this was the result of the development of highly standardized techniques, i.e. exact mutation analysis in functional relevant codons of genes encoding signal proteins of cancer-related signal transduction pathways targeted by the new drugs. Due to increasing costs of health systems, in the future predictive diagnostics will probably become more and more important. Therefore, it will be necessary to develop improved diagnostic assays for prediction of individual therapy response.

Impact of the specific mutation in KRAS codon 12 mutated tumors on treatment efficacy in patients with metastatic colorectal cancer receiving cetuximab-based first-line therapy: a pooled analysis of three trials.

PURPOSE: This study investigated the impact of specific mutations in codon 12 of the Kirsten-ras (KRAS) gene on treatment efficacy in patients with metastatic colorectal cancer (mCRC). PATIENTS: Overall, 119 patients bearing a KRAS mutation in codon 12 were evaluated. All patients received cetuximab-based first-line chemotherapy within the Central European Cooperative Oncology Group (CECOG), AIO KRK-0104 or AIO KRK-0306 trials. RESULTS: Patients with KRAS codon 12 mutant mCRC showed a broad range of outcome when treated with cetuximab-based first-line regimens. Patients with tumors bearing a KRAS p.G12D mutation showed a strong trend to a more favorable outcome compared to other mutations (overall survival 23.3 vs. 14-18 months; hazard ratio 0.66, range 0.43-1.03). An interaction model illustrated that KRAS p.G12C was associated with unfavorable outcome when treated with oxaliplatin plus cetuximab. CONCLUSION: The present analysis suggests that KRAS codon 12 mutation may not represent a homogeneous entity in mCRC when treated with cetuximab-based first-line therapy.

Ganglioside GM1 induces phosphorylation of mutant huntingtin and restores normal motor behavior in Huntington disease mice.

Huntington disease (HD) is a progressive neurodegenerative monogenic disorder caused by expansion of a polyglutamine stretch in the huntingtin (Htt) protein. Mutant huntingtin triggers neural dysfunction and death, mainly in the corpus striatum and cerebral cortex, resulting in pathognomonic motor symptoms, as well as cognitive and psychiatric decline. Currently, there is no effective treatment for HD. We report that intraventricular infusion of ganglioside GM1 induces phosphorylation of mutant huntingtin at specific serine amino acid residues that attenuate huntingtin toxicity, and restores normal motor function in already symptomatic HD mice. Thus, our studies have identified a potential therapy for HD that targets a posttranslational modification of mutant huntingtin with critical effects on disease pathogenesis.

[Mapping DNA damage to understand somatic mutagenesis]. / Comprendre la mutagenèse somatique grâce à la cartographie des dommages à l'ADN.

Somatic mutation theory explains how DNA damage can lead to the malignant transformation of cells. It therefore elucidates the connection between genotoxic agents and cancers. Mutational spectra, which tend to be characteristic of a cancer type, are available for certain genes like p53 which is frequently mutated in tumors. A mutational spectrum could therefore be the signature of the genotoxic agent(s) at the origin of the malignant transformation. Ligation-mediated PCR (LMPCR) is a genomic sequencing method that can be used for the mapping of DNA damage at nucleotide resolution. Such a mapping can then be compared to a mutational spectrum to test the hypothesis that implies one agent can cause mutations into one cancer type. LMPCR has been used this way to map DNA damage generated by different UV wavelengths. The frequently damaged sites following UVB irradiation correlate with the mutational spectrum of p53 in skin cancer. Similarly, BPDE, the activated form of the benzo[a]pyrene present in tobacco smoke, generates frequent adducts at sites corresponding to mutation hotspots of p53 in lung cancers. Still, the correlation between BPDE damage sites and p53 mutations is not perfect and this suggests a role of other genotoxic substances that are also present in tobacco smoke, such as the nitrosamine NNK. Finally, and beyond this objective of better understanding somatic mutagenesis, LMPCR is commonly used whenever DNA damage frequency and/or repair is to be investigated.

Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells.

BACKGROUND: Metformin, one of most commonly used antidiabetes drugs, is reported to exert its therapeutic effects by activating AMP-activated protein kinase (AMPK); however, the mechanism by which metformin activates AMPK is poorly defined. The objective of the present study was to determine how metformin activates AMPK in endothelial cells. METHODS AND RESULTS: Exposure of human umbilical vein endothelial cells or bovine aortic endothelial cells to metformin significantly increased AMPK activity and the phosphorylation of both AMPK at Thr172 and LKB1 at Ser428, an AMPK kinase, which was paralleled by increased activation of protein kinase C (PKC)-zeta, as evidenced by increased activity, phosphorylation (Thr410/403), and nuclear translocation of PKC-zeta. Consistently, either pharmacological or genetic inhibition of PKC-zeta ablated metformin-enhanced phosphorylation of both AMPK-Thr172 and LKB1-Ser428, suggesting that PKC-zeta might act as an upstream kinase for LKB1. Furthermore, adenoviral overexpression of LKB1 kinase-dead mutants abolished but LKB1 wild-type overexpression enhanced the effects of metformin on AMPK in bovine aortic endothelial cells. In addition, metformin increased the phosphorylation and nuclear export of LKB1 into the cytosols as well as the association of AMPK with LKB1 in bovine aortic endothelial cells. Similarly, overexpression of LKB1 wild-type but not LKB1 S428A mutants (serine replaced by alanine) restored the effects of metformin on AMPK in LKB1-deficient HeLa-S3 cells, suggesting that Ser428 phosphorylation of LKB1 is required for metformin-enhanced AMPK activation. Moreover, LKB1 S428A, like kinase-dead LKB1 D194A, abolished metformin-enhanced LKB1 translocation as well as the association of LKB1 with AMPK in HeLa-S3 cells. Finally, inhibition of PKC-zeta abolished metformin-enhanced coimmunoprecipitation of LKB1 with both AMPKalpha1 and AMPKalpha2. CONCLUSIONS: We conclude that PKC-zeta phosphorylates LKB1 at Ser428, resulting in LKB1 nuclear export and hence AMPK activation.

The wobble hypothesis revisited: uridine-5-oxyacetic acid is critical for reading of G-ending codons.

According to Crick's wobble hypothesis, tRNAs with uridine at the wobble position (position 34) recognize A- and G-, but not U- or C-ending codons. However, U in the wobble position is almost always modified, and Salmonella enterica tRNAs containing the modified nucleoside uridine-5-oxyacetic acid (cmo(5)U34) at this position are predicted to recognize U- (but not C-) ending codons, in addition to A- and G-ending codons. We have constructed a set of S. enterica mutants with only the cmo(5)U-containing tRNA left to read all four codons in the proline, alanine, valine, and threonine family codon boxes. From the phenotypes of these mutants, we deduce that the proline, alanine, and valine tRNAs containing cmo(5)U read all four codons including the C-ending codons, while the corresponding threonine tRNA does not. A cmoB mutation, leading to cmo(5)U deficiency in tRNA, was introduced. Monitoring A-site selection rates in vivo revealed that the presence of cmo(5)U34 stimulated the reading of CCU and CCC (Pro), GCU (Ala), and GUC (Val) codons. Unexpectedly, cmo(5)U is critical for efficient decoding of G-ending Pro, Ala, and Val codons. Apparently, whereas G34 pairs with U in mRNA, the reverse pairing (U34-G) requires a modification of U34.

Aminoglycosides and other factors promoting stop codon readthrough in human cells.

Enhanced stop codon readthrough is a potential treatment strategy for diseases caused by nonsense mutations. Here, we compare readthrough levels induced by three types of factors: aminoglycoside antibiotics, suppressor tRNAs, and factors decreasing translation termination efficiency. We show that the highest levels of readthrough were obtained by prolonged treatment with aminoglycosides and suppressor tRNAs, whereas prolonged depletion of release factors induced only a moderate increase in readthrough. We discuss the benefits and inconvenients of the three types of factors for their use in the therapy of diseases caused by premature stop codons.

Absence of beta-tubulin gene mutation in gastric carcinoma.

BACKGROUND: Effective chemotherapy for advanced gastric cancer is yet to be established. Taxanes, novel anticancer drugs which bind to beta-tubulin and prevent disruption of microtubules, are newly approved and promising agents for advanced and recurrent gastric cancer. To predict the chemoresistance to a taxan in gastric cancer, we examined the genetic mutations of the beta-tubulin gene. METHODS: Fifty pairs of gastric tumor and normal mucosa tissues were obtained from operations and the genomic DNA was extracted from each specimen. The four exons of the beta-tubulin gene were amplified for DNA mutations by single-strand conformation polymorphism (SSCP) methods and sequencing analysis. RESULTS: Nine (18%) of 50 patients with gastric cancer had two kinds of silent variations of the beta-tubulin gene in exon 4. Three kinds of intronic variations were detected in exons 1, 2, and 3. However, no genetic alterations that would change the beta-tubulin protein structure were detected in any of the 50 gastric tumors. CONCLUSION: Our findings indicate that mutations of the beta-tubulin gene, which might be a contraindication for chemotherapy based on taxans, were very rare events in gastric cancer.

Codon 64 of K-ras gene mutation pattern in hepatocellular carcinomas induced by bleomycin and 1-nitropyrene in A/J mice.

Bleomycin is a radiomimetic antitumor agent with unique genotoxic properties. 1-nitropyrene is an environmental mutagen and carcinogen that undergoes both oxidative and reductive metabolism. In the present study, hepatocellular carcinomas were induced in male A/J mice by the intraperitoneal injection of bleomycin (120 mg/kg) followed by the intraperitoneal administration of 1-nitropyrene (total dose: 1,575 mg/kg). In order to understand the mechanism by which these two compounds induce hepatocellular carcinomas, the incidence and spectrum of mutations in the K-ras proto-oncogene in these hepatocellular carcinomas were analyzed. The hepatocellular carcinomas were induced by the administration of bleomycin and 1-nitropyrene were evaluated for point mutations in exon 1 and exon 2 of the K-ras gene by the polymerase chain reaction and a sequencing analysis. No mutation was found in the hotspots regions of the K-ras gene codon 12, 13, or 61. However, the codon 64 of the K-ras gene mutation was identified in 10 of 10 (100%) hepatocellular carcinomas. All mutations showed the same pattern, which was TAC-CAC transition. Codon 64 of the K-ras gene mutation may thus play an important role in the induction of hepatocellular carcinomas by bleomycin in the existence of 1-nitropyrene. As far as we know, this is the first report of a codon 64 mutation in the K-ras gene in a chemically induced tumor.

Tumor initiating activity of Helicobacter pylori water extract on mouse skin carcinogenesis.

Helicobacter pylori (H. pylori) infection has been associated with gastric carcinogenesis, but responsible and detail mechanisms are insufficient by the absence of adequate data. To obtain direct evidence regarding the carcinogenicity of H. pylori, we investigated the initiating and promoting activity of H. pylori water extract (HPE) in two-stage mouse skin carcinogenesis model. HPE treatment, as an initiation, significantly enhanced tumor formation compared with control group. Moreover, HPE treatment increased production of 8-hydroxydeoxyguanosine in epidermal cells and HPE-initiated/TPA-promoted papillomas demonstrated a point mutation of the Ha-ras gene. These results suggest an initiating activity of HPE on two-stage mouse skin carcinogenesis.

Point mutations induced by foscarnet (PFA) in the human cytomegalovirus DNA polymerase.

BACKGROUND: In vitro selection of viruses with decreased drug susceptibility is a useful tool for mapping drug resistance-associated alterations, evaluating cross-resistance profiles, and elucidating molecular mechanisms of antiviral activity. OBJECTIVES: To provide data on mechanisms of selective drug action and features of drug resistance that may be clinically important. STUDY DESIGN: Foscarnet (PFA) and ganciclovir (GCV) were used to induce mutants of the human cytomegalovirus (HCMV) Towne strain. RESULTS: Three new mutations, selected in the presence of PFA, were identified with single base substitutions resulting in T419M, Q578H, and L773V in conserved regions of the HCMV DNA polymerase. None of these mutations have been reported previously. These mutations conferred resistance to PFA but did not change the susceptibility to GCV. A mutant was selected in the presence of GCV. This GCV-selected mutant had no mutation in the UL54 but had an amino acid alteration at codon M460V of UL97, which conferred resistance to GCV. All the mutants had the same growth phenotype as the parental laboratory strain Towne. CONCLUSIONS: We have determined three novel alterations in HCMV DNA polymerase inducing reduced susceptibility to PFA. None of these alterations changed the growth phenotype of the parental virus.

Differences in base excision repair capacity may modulate the effect of dietary antioxidant intake on prostate cancer risk: an example of polymorphisms in the XRCC1 gene.

We propose a hypothesis that differences in base excision repair capacity modulate the effect of dietary antioxidant intake on prostate cancer risk. As a preliminary test of this hypothesis, we conducted a pilot case-control study to evaluate prostate cancer risk in men with polymorphisms in the XRCC1 gene, a key player in base excision repair, across different strata of antioxidant intake. Seventy-seven prostate cancer patients and 183 community controls, for whom we have detailed dietary information, were frequency matched on age and race. We found a somewhat lower prostate cancer risk for men with one or two copies of the variant alleles at the XRCC1 codons 194 and 399 than for those who were homozygous for the common allele [codon 194: odds ratio (OR) = 0.8; 95% confidence interval (CI), 0.4-1.8 and codon 399: OR = 0.8; 95% CI, 0.5-1.3]. The variant at codon 280 was associated with a slightly increased prostate cancer risk (OR = 1.5; 95% CI, 0.7-3.6). Only the codon 399 polymorphism occurred frequently enough to investigate its joint effect with antioxidant intake. Prostate cancer risk was highest among men who were homozygous for the common allele at codon 399 and had low dietary intake of vitamin E (OR = 2.4; 95% CI, 1.0-5.6) or lycopene (OR = 2.0; 95% CI, 0.8-4.9), whereas low intake of these antioxidants in men without this genotype hardly increased prostate cancer risk. The polymorphism did not modulate risk associated with low intake of vitamin C, A, or beta-carotene. The data give some support for our hypothesis but should be regarded as preliminary, because it is limited by small sample size. We discuss what kind of data and what kind of studies are needed for future evaluation of this hypothesis.

Prostaglandin H synthase 2 variant (Val511Ala) in African Americans may reduce the risk for colorectal neoplasia.

Prostaglandin H synthase 2 (also known as cyclooxygenase-2) is thought to play a role in the prevention of colon cancer by aspirin, an inhibitor of the enzyme. We used DNA heteroduplex analysis to screen the prostaglandin H synthase 2 gene, to search for naturally occurring enzyme variants that may simulate the effects of aspirin. We found among African-Americans a single-nucleotide polymorphism that changes valine to alanine at residue 511 (V511A; GTT>GCT; g.5939T>C; allele frequency 0.045). The polymorphism was also seen among Asian-Indians (allele frequency, 0.03) but not among Chinese, Filipinos, Hispanics, Japanese, Koreans, Samoans, and Caucasians. The amino acid change is predicted to open a 53 cubic angstrom cavity near the active site of the enzyme, but no change in V(max), K(m), or thermal stability was observed for the variant enzyme in COS-1 cell transfection assays. Case-control analysis of African-Americans from two different study populations showed a 0.56 odds ratio for colorectal adenomas among polymorphism carriers (95% confidence interval, 0.25-1.27; 161 cases and 219 controls). A similar analysis of African-Americans nested in the Multiethnic Cohort Study showed a 0.67 odds ratio for colorectal cancer (95% confidence interval, 0.28-1.56; 138 cases and 258 controls). Consistency of the results across all three of the studies is potentially compatible with a protective effect of the polymorphism, mimicking aspirin.

Differences in KRAS mutation spectrum in lung cancer cases between African Americans and Caucasians after occupational or environmental exposure to known carcinogens.

Elevated mortality rates of lung cancer in the Mississippi River corridor in Louisiana have been clearly documented for the past half-century and rank among the highest in the nation. A population-based case-control study of lung cancer termed Lower Mississippi River Interagency Cancer Study was conducted in southern Louisiana. Lung tumor specimens were collected, isolated by laser capture microdissection, subjected to PCR to amplify KRAS, and sequenced to confirm mutation status and specificity. Of the 116 lung tumors analyzed to date, 32 (27.6%) contained mutations in either codon 12 or 13 of KRAS. This frequency is comparable to that reported in the literature; however, the mutation spectrum was strikingly different. Of the 32 mutations observed, 21 (65.6%) resulted in the inappropriate insertion of cysteine, 6 (18.8%) resulted in the insertion of serine, 3 (9.4%) resulted in the insertion of valine, and 1 (3.1%) each resulted in the insertion of aspartate and alanine. These data indicate that an abnormally high proportion of cysteine (P = 0.010) and serine (P = 0.002) mutations was observed in our sample group versus lung cancers reported in the literature. KRAS mutations were more common in African Americans with an odds ratio of 2.4 (P = 0.048), as were serine mutations, although the latter did not reach statistical significance (odds ratio, 2.6; P = 0.373). No association was found between the observed mutation spectrum and known lung cancer risk factors.

Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer.

BACKGROUND: Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. METHODS: We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. RESULTS: Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. CONCLUSION: These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.