DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells.

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.

Altered Ig hypermutation pattern and frequency in complementary mouse models of DNA polymerase ζ activity.

To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.

Replication of damaged genomes.

Cellular DNA is continuously assaulted by chemical and physical agents that arise from both endogenous metabolic processes as well as exogenous insults. Commonly encountered environmental agents include polyaromatic hydrocarbons, polycyclic aromatic amines, the ultraviolet component of sunlight, and ionizing radiation, among many others. Although the kinds of damages and the mechanisms involved in their interaction with DNA vary widely, genotoxic agents alter the structure of DNA in ways that may result in permanent alterations in the DNA sequence or in cell death. To avoid these consequences, cells have evolved countermeasures to reduce the biological consequences of DNA damage. These mechanisms are highly conserved and are present in all eukaryotic cells. In general, cellular responses include the detection of damage, signal transduction to halt cell cycle progression, and the recruitment of repair mechanisms that are tailored to the specific kind of damage. If replication-blocking damage remains when cells enter S-phase, then tolerance mechanisms in the form of complex recombination mechanisms or translesion DNA synthesis using accessory DNA polymerases exist. These mechanisms complete the replication of damaged genomes and suppress cytotoxicity, but at the potential cost of mutagenesis and genomic instability. This review focuses on error-prone mechanisms, including a discussion of the Y-family of DNA polymerases, current concepts of DNA polymerase switching mechanisms, and their relevance to cancer and cancer prevention.

Sequential assembly of translesion DNA polymerases at UV-induced DNA damage sites.

In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Polη and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Polζ. Here we describe the detection of endogenous human Polη, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Polη are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Polη. In contrast, neither Rev1 nor Polη recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Polη and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.

Translesion synthesis polymerases in the prevention and promotion of carcinogenesis.

A critical step in the transformation of cells to the malignant state of cancer is the induction of mutations in the DNA of cells damaged by genotoxic agents. Translesion DNA synthesis (TLS) is the process by which cells copy DNA containing unrepaired damage that blocks progression of the replication fork. The DNA polymerases that catalyze TLS in mammals have been the topic of intense investigation over the last decade. DNA polymerase η (Pol η) is best understood and is active in error-free bypass of UV-induced DNA damage. The other TLS polymerases (Pol ι, Pol κ, REV1, and Pol ζ) have been studied extensively in vitro, but their in vivo role is only now being investigated using knockout mouse models of carcinogenesis. This paper will focus on the studies of mice and humans with altered expression of TLS polymerases and the effects on cancer induced by environmental agents.

Micro- and nanotechnology approaches for capturing circulating tumor cells.

Circulating tumor cells (CTC) are cells that have detached from primary tumors and circulate in the bloodstream where they are carried to other organs, leading to seeding of new tumors and metastases. CTC have been known to exist in the bloodstream for more than a century. With recent progress in the area of micro- and nanotechnology, it has been possible to adopt new approaches in CTC research. Microscale and nanoscale studies can throw some light on the time course of CTC appearance in blood and CTC overexpression profiles for cancer-related markers and galvanize the development of drugs to block metastases. CTC counts could serve as endpoint biomarkers and as prognostic markers for patients with a metastatic disease. This paper reviews some of the recent researches on using micro- and nanotechnology to capture and profile CTC.

Effect of rapid human N-acetyltransferase 2 haplotype on DNA damage and mutagenesis induced by 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx).

Heterocyclic amines such as 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) are dietary carcinogens generated when meats are cooked well-done. Bioactivation includes N-hydroxylation catalyzed by cytochrome P4501A2 (CYP1A2) followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were treated with IQ or MeIQx to examine the effect of NAT2 genetic polymorphism on IQ- or MeIQx-induced DNA adducts and mutagenesis. MeIQx and IQ both induced decreases in cell survival and significantly (p<0.001) greater number of endogenous hypoxanthine phosphoribosyl transferase (hprt) mutants in the CYP1A2/NAT2*4 than the CYP1A2/NAT2*5B cell line. IQ- and MeIQx-induced hprt mutant cDNAs were sequenced and over 85% of the mutations were single-base substitutions with the remainder exon deletions likely caused by splice-site mutations. For the single-base substitutions, over 85% were at G:C base pairs. Deoxyguanosine (dG)-C8-IQ and dG-C8-MeIQx adducts were significantly (p<0.001) greater in the CYP1A2/NAT2*4 than the CYP1A2/NAT2*5B cell line. DNA adduct levels correlated very highly with hprt mutants for both IQ and MeIQx. These results suggest substantially increased risk for IQ- and MeIQx-induced DNA damage and mutagenesis in rapid NAT2 acetylators.

REV1 is implicated in the development of carcinogen-induced lung cancer.

The somatic mutation hypothesis of cancer predicts that reducing the frequency of mutations induced by carcinogens will reduce the incidence of cancer. To examine this, we developed an antimutator strategy based on the manipulation of the level of a protein required for mutagenic bypass of DNA damage induced by the ubiquitous carcinogen benzo[a]pyrene. The expression of this protein, REV1, was reduced in mouse cells using a vector encoding a gene-specific targeting ribozyme. In the latter cells, mutagenesis induced by the activated form of benzo[a]pyrene was reduced by >90%. To examine if REV1 transcripts could be lowered in vivo, the plasmid was complexed with polyethyleneimine, a nonviral cationic polymer, and delivered to the lung via aerosol. The endogenous REV1 transcript in the bronchial epithelium as determined by quantitative real-time PCR in laser capture microdissected cells was reduced by 60%. There was a significant decrease in the multiplicity of carcinogen-induced lung tumors from 6.4 to 3.7 tumors per mouse. Additionally, REV1 inhibition completely abolished tumor formation in 27% of the carcinogen-exposed mice. These data support the central role of the translesion synthesis pathway in the development of lung cancer. Further, the selective modulation of members of this pathway presents novel potential targets for cancer prevention. The somatic mutation hypothesis of cancer predicts that the frequency of cancers will also be reduced.

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage.

Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or poleta only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In the absence of damage these proteins are not physically associated within the nucleoplasm.

RAD18 signals DNA polymerase IOTA to stalled replication forks in cells entering S-phase with DNA damage.

Endogenously generated reactive oxygen species and genotoxic carcinogens can covalently modify bases in cellular DNA. If not recognized and removed prior to S-phase of the cell cycle, such modifications can block DNA replication fork progression. If blocked forks are not are not resolved, they result in double strand breaks and cell death. Recent data indicate that the process of translesion DNA synthesis (TLS) is a highly conserved mechanism for bypassing lesions in template DNA. Although not fully understood, in yeast a ubiquitin ligase (RAD18) signals error-prone Y family polymerases to the blocked fork to bypass the damage with potentially mutagenic consequences. Homologs of the yeast proteins are found in higher eukaryotic cells, including human. We are examining the hypothesis that RAD18 acts as a proximal signal to Y-family polymerases to bypass damage, in a manner analogous to yeast but with additional layers of complexity. Here we report that RAD18 accumulates in nuclear foci after UV irradiation only in cells entering S-phase with DNA damage. These foci co-localize with proliferating cell nuclear antigen (PCNA). In addition, a newly described DNA polymerase, pol iota, also forms nuclear foci in a damage- and S-phase dependent manner. These data support our overall hypothesis that RAD18 accumulates at blocked forks and initiates the signal to recruit TLS polymerases.

Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts.

Comparative mutagenesis of 1,N(6)-ethenoadenine (epsilonA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past epsilonA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, epsilonA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G-->T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of epsilonA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the epsilonA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, epsilonA-->T mutations increased 3-fold in products derived from the mismatched epsilonA: G construct compared with those derived from the epsilonA: T construct. More efficient repair of epsilonA in the epsilonA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol eta have shown that this polymerase is highly error-prone when bypassing epsilonA. To examine if pol eta is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The epsilonA: T construct induced epsilonA-->G and epsilonA-->C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, epsilonA-->T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol eta-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.

2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced DNA adducts and genotoxicity in chinese hamster ovary (CHO) cells expressing human CYP1A2 and rapid or slow acetylator N-acetyltransferase 2.

Heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) are present in diet and cigarette smoke. Bioactivation in humans includes N-hydroxylation catalyzed by cytochrome P4501A2 possibly followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A2 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A2 catalytic activity levels did not differ significantly (P > 0.05) among the CYP1A2-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of N-acetyltransferase (P = 0.0001) and N-hydroxy-PhIP O-acetyltransferase (P = 0.0170) catalytic activity than cells transfected with NAT2*5B. PhIP caused dose-dependent decreases in cell survival and significant (P < 0.001) increases in mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in all the CYP1A2-transfected cell lines. Transfection with NAT2*4 or NAT2*5B did not further increase hprt mutagenesis. PhIP-induced hprt mutant cDNAs were sequenced, and 80% of the mutations were single base substitutions at G:C base pairs. dG-C8-PhIP DNA adduct levels were dose-dependent in the order: untransfected < transfected with CYP1A2 < transfected with CYP1A2 and NAT2*5B < transfected with CYP1A2 and NAT2*4. Following incubation with 1.2 microM PhIP, DNA adduct levels were significantly (P < 0.05) higher in CHO cells transfected with CYP1A2/NAT2*4 versus CYP1A2/NAT2*5B. These results strongly support an activation role for CYP1A2 in PhIP-induced mutagenesis and DNA damage and suggest a modest effect of human NAT2 and its genetic polymorphism on PhIP DNA adduct levels.

Army field surgical experience.

In the recent Falklands campaign four Army Field Surgical Teams were deployed in the two phases of the war. They functioned as Advanced Surgical Centres and operated on 233 casualties. There were 3 deaths. The patterns of wounding and the methods of casualty management are discussed and compared with other recent campaigns.

Participation of mouse DNA polymerase iota in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer.

DNA polymerase iota (pol iota) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol iota performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol eta, pol iota, or both. A deficiency in mouse DNA polymerase eta (pol eta) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol iota, indicating that pol iota participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol eta- and pol iota-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol iota status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol eta, pol iota, or both. Although pol iota deficiency alone had no effect, UV-induced skin tumors in pol eta-deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol iota. Collectively, these data reveal functions for pol iota in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

Cytotoxic and mutagenic effects of tobacco-borne free fatty acids.

Tobacco smoke contains substances capable of binding iron in an aqueous medium and transferring the metal into both organic solvents and intact mammalian red cells. This iron-binding activity is due to free fatty acids which are abundant in tobacco smoke and form 2:1 (free fatty acid:iron) chelates with ferrous iron. These earlier observations suggested that smoke-borne free fatty acids and the associated delocalization of iron within the lung might contribute to both the chronic pulmonary inflammation and the carcinogenesis associated with smoking. We now report that micromolar concentrations of iron or free fatty acid are not toxic to cultured human lung fibroblasts. However, when combined, the same low concentrations of iron and free fatty acid exert synergistic toxicity. Furthermore, the combination of free fatty acid and iron is highly mutagenic, inducing almost as many selectable mutations in the gene for hypoxanthine/guanine phosphoribosyl transferase as does benzo[a]pyrenediolepoxide, a class I carcinogen generated from benzo[a]pyrene present in cigarette smoke. The combination of free fatty acid and iron also promotes transformation of NIH 3T3 cells into an anchorage-independent phenotype. We conclude that free fatty acids in tobacco smoke may be important contributors to both the pulmonary damage and the carcinogenesis associated with smoking.

Translesion DNA replication proteins as molecular targets for cancer prevention.

Mutations in DNA are generally considered to have an etiologic role in the development of cancer. If so, it follows that reducing the frequency of such mutations will reduce the incidence of cancer induced by mutagens. Recent advances in elucidating the molecular mechanisms of carcinogen-induced mutagenesis indicate that replication of DNA templates that contain replication-blocking adducts is accomplished with error-prone DNA polymerases. These polymerases have relaxed base-pairing requirements, and can insert bases across from adducted templates, but with potentially mutagenic consequences. In principle, these proteins present new and attractive molecular targets to reduce mutagenesis. If this can be done in vivo without increasing cytotoxic responses to carcinogens, then novel chemopreventive strategies can be designed to reduce the risk of cancer in exposed populations prior to the appearance of disease symptoms.

REV1 accumulates in DNA damage-induced nuclear foci in human cells and is implicated in mutagenesis by benzo[a]pyrenediolepoxide.

The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene.

In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.