Carcinogenesis Advance Access originally published online on December 12, 2005
Carcinogenesis 2006 27(8):1526-1537; doi:10.1093/carcin/bgi311
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Investigating human cancer etiology by DNA lesion footprinting and mutagenicity analysis
Division of Biology, Beckman Research Institute of the City of Hope National Medical Center 1450 East Duarte Road, Duarte, CA 91010, USA
*To whom correspondence should be addressed. Tel: +1 626 256 4673 ext. 65918; Fax: +1 626 358 7703; Email: ania{at}coh.org
 |
Abstract |
|---|
Top
Abstract
IntroductionMany genotoxic carcinogens are known to leave unique signatures on cancer-related genes. The signature of carcinogens is manifested by the induction of characteristic mutations at distinctive nucleotide positions along oncogenes and/or tumor suppressor genes. Often, the nucleotide positions, wherein mutations occur, co-localize with the sites of initial DNA damage induced by the respective carcinogens. Thus, DNA damage-targeted mutation can be a predictor of carcinogenicity of genotoxins. Today, genomic sequencing technologies for investigating human cancer etiology are based on DNA-lesion footprinting in conjunction with mutagenicity analysis of genotoxic carcinogens. In this review article, we discuss the ligation-mediated PCR and terminal transferase-dependent-PCR, two versatile DNA-lesion footprinting techniques. We highlight the in vitro shuttle vector-based mutation systems for investigating site-specific mutagenicity of carcinogens and the in vivo transgenic rodent mutation systems for exploring DNA damaging and mutagenic properties of carcinogens. We present examples of application of each of these methodologies to human cancer etiology, and provide prospective views on investigations using these technologies for carcinogenicity testing.
Abbreviations: B[a]PDE, (+) anti-benzo[a]pyrene diol epoxide; B[g]CDE, benzo[g]chrysene-11,12-diol-13,14-epoxide; HPRT, hypoxanthine–guanine phosphoribosyltransferase; LM-PCR, ligation-mediated PCR; PAH, polycyclic aromatic hydrocarbon; TD-PCR, terminal transferase-dependent-PCR; UV, ultraviolet
|
Introduction |
|---|
The introduction of genomic sequencing techniques in the mid to late 1980s opened a new window of opportunities for analyzing DNA methylation, DNA–protein interactions, and genetic polymorphisms at the level of nucleotide resolution (1–6). This procedure has significantly evolved during the past two decades so that it is now applicable to a variety of other investigations including studying of chromatin structure, RNA structure, e.g. RNA splicing, ribozyme cleavage, and RNA–protein interactions, as well as of DNA-lesion footprinting (also referred to as the ‘DNA-damage mapping’) (7,8). The original genomic sequencing procedure has been improved considerably by the addition of the following steps (i) enrichment of the target sequence, e.g. by pre-treatment with restriction enzymes (9), (ii) linear amplification using gene-specific primer extension (5), and (iii) exponential amplification by PCR (3,4). Additionally, the development of alternatives to radioactive-labeled primers, e.g. infrared fluorochrome-labeled primers together with the advent of simultaneous gel electrophoresis and scanning technology has enabled rapid, sensitive, and specific analysis of any sequence of interest (7). The ever-increasing use of robotic systems has also allowed automated, high-throughput, and cost-effective replacements of many laborious and time-consuming genomic sequencing protocols (10,11). Today, genomic sequencing is used to study genotoxic carcinogens that are known to leave unique ‘signatures’ on cancer-related genes (12).
The signature of carcinogens is manifested by the induction of specific types of mutations, e.g. base substitutions or frameshifts, at distinctive locations along oncogenes and/or tumor suppressor genes. These characteristic mutations are usually preceded by the formation of carcinogen-induced DNA damage at the mutated sites (12). This phenomenon is best illustrated by the exemplary cases of solar ultraviolet light (UV) and tobacco smoke-derived polycyclic aromatic hydrocarbons (PAHs). The respective carcinogens induce C
T or CCTT transitions at dipyrimidine sites and GT transversions at CpG dinucleotides in both the RAS oncogenes and TP53 tumor suppressor gene. The same nucleotide positions are virtually the hotspots of DNA damage formation by each agent, respectively (12,13). Different genotoxic carcinogens may leave comparable signatures on cancer-related genes through identical mechanisms targeting similar nucleotide sequences, thereby yielding analogous patterns of mutation. The latter can cause certain degrees of overlap in the signatures of various carcinogens. Detecting and assessing such overlapping signatures remain a challenge in the molecular epidemiology of cancer. Currently, genomic sequencing technologies for investigating human cancer etiology are based on DNA-lesion footprinting in conjunction with mutagenicity analysis of genotoxic carcinogens.
In the past two decades, our laboratory together with others' has pioneered developing of genomic sequencing technologies used for studying human cancer etiology. The existing literature, however, lacks a comprehensive review on this subject. In this review article, we discuss the evolution of genomic sequencing technologies, highlight the principle methodological aspects, describe the most versatile, advanced and updated techniques, present examples of applications of various methodologies to human cancer etiology, and provide prospective views on investigations using genomic sequencing for carcinogenicity testing.
|
Investigating human cancer etiology |
|---|
Earlier works of Benzer and Freese revealed that chemical carcinogens induce specific types of mutations, e.g. transitions, transversions or deletions, in bacteria (14–16). This phenomenon was subsequently confirmed in cultured mammalian cells in vitro and experimental animals in vivo (17–19). The induced mutations, and the nucleotide positions in which they occurred, proved to be unique for each carcinogen. Often, these nucleotide positions co-localize with the sites of initial DNA damage caused by the respective carcinogen (18,19). Next, compilation of epidemiologic data identified distinctive patterns of mutation in oncogenes and/or tumor suppressor genes in specific types of human cancer (13,20). The incidence of many of these cancers was shown to be associated with occupational, dietary, medicinal, or recreational exposure to carcinogens. With a few exceptions, however, a causative link between carcinogen exposure and human cancer development is yet to be established (21). Convincing evidence does exist on a causal relationship between exposure to sunlight and skin cancer (22–24), exposure to food-borne aflatoxin and liver cancer (25–27), and exposure to tobacco smoke or smoky coal and lung cancer (28–32).
By virtue of design, most cancer epidemiology studies are observational and can ascertain malignancies associated with, but not caused by, variables such as exposure to carcinogens. To deduce whether an observed association is likely to be causal the Bradford Hill criteria (33) can be used (20). These criteria, first outlined by Sir Austin Bradford Hill (33), include strength and specificity of the association, consistency and coherence across studies, temporality of the putative cause and effect, biological gradient and plausibility, and experimental evidence and analogy. The fulfillment of these criteria adds weight to the probability that the observed association is causal. However, fulfilling all these criteria is challenging because multiple confounders might impact upon a single outcome association. Thus, adjusting for relevant confounders is an essential, yet, intricate pre-requisite for causality inference.
Generally, prospective observations are more definitive than cross-sectional ones for inferring causality (34). For instance, nested-case–control studies can establish the relationship between carcinogen exposure and cancer development by monitoring healthy individuals with known exposure to specific carcinogen(s) over a period of time (35). This time period is usually long because most human cancers develop many years after the initial exposure to carcinogens. Unfortunately, assessing human exposure for prolonged periods of time is costly and demanding considering that humans are constantly and to some extent unavoidably exposed to a plethora of carcinogens (12).
An indirect approach for investigating cancer etiology is to correlate the pattern of mutations specific for each type of human cancer with the experimentally established signature of carcinogens (13,20). Obviously, experimental exposure of humans to carcinogens is unethical and out of the question. Thus, the signature of carcinogens can only be determined in model systems. Both in vitro and in vivo model systems exist that can be used for establishing carcinogen signatures. However, caution should be taken in interpreting the findings because these model systems lack complete comparability to humans. For example, in vitro cell culture systems cannot replicate the exact physiological conditions as those existing in vivo. The systems often use a single-cell type environment that may differ from the in vivo environment in which multiple cell types interact with each other. Or, in vivo experimental animal systems may not behave similarly to humans in response to carcinogens, e.g. due to inter-species differences in xenobiotics activating and/or detoxifying capacities, and DNA repair machinery. Nonetheless, if used properly, both systems can provide invaluable information, which may help unravel many aspects of human carcinogenesis (13,20).
An in vitro model system for DNA footprinting of carcinogens is cultured primary human cells treated with a carcinogen of interest. For comparative purposes, representative cells of individuals with a history of exposure to the same carcinogen can be concurrently analyzed. For instance, cultured primary human bronchial epithelial cells treated with benzo[a]pyrene diol epoxide (B[a]PDE), a tobacco smoke-derived PAH, together with representative cells of smokers can be used for DNA footprinting of carcinogenic PAH compounds (30). Generally, DNA footprinting of carcinogens is performed on endogenous cancer-related target genes. On the other hand, mutagenicity analysis of carcinogens is performed on exogenous/endogenous reporter genes or endogenous target genes depending on the model system. To date, the in vitro shuttle vector-based mutation systems (36,37) and the in vivo transgenic rodent mutation systems (38,39) are the most widely used model systems for mutagenicity analysis of carcinogens. The increasingly popular transgenic rodent mutation systems are also suitable for simultaneous DNA footprinting and mutagenicity analysis of carcinogens. The systems provide unique opportunities for studying DNA damaging and mutagenic properties of carcinogens in a single setting (38,39).
|
DNA-lesion footprinting of carcinogens |
|---|
Two genomic sequencing assays, namely ligation-mediated (LM)-PCR and terminal transferase-dependent (TD)-PCR, can be used for DNA-lesion footprinting of carcinogens (40). Methodologically, both of these assays are based on the concept that DNA polymerase cannot synthesize DNA past certain types of lesions, i.e. bulky lesions and single strand breaks. Although many aspects of LM-PCR and TD-PCR are similar, there also exist fundamental differences between these two assays (40). Here, we describe the updated methodology for each of these two assays, and present examples of their applications to cancer etiology investigations.
|
LM-PCR |
|---|
LM-PCR is initiated by chemical or enzymatic conversion of lesion-containing DNA to single-stranded DNA breaks with 5'-phosphate termini. These fragments are used as templates and are subjected to a gene-specific primer (primer 1) extension to provide molecules with one blunt end. An asymmetric synthetic double-stranded linker is then ligated to the blunt ended molecules to generate a common sequence at all 5'-ends. The resultant molecules undergo exponential PCR amplification using a nested gene-specific primer (primer 2) together with the longer oligonucleotide of the linker (linker primer). The PCR-amplified products are subjected to a second round of primer extension with a labeled gene-specific primer (primer 3). The reaction products are resolved by gel electrophoresis and the results are quantified using a label-specific scanner (40). The outline of the LM-PCR procedure is illustrated in Figure 1.
|
A unique and challenging aspect of LM-PCR is the ligation of the DNA templates to the linker. Because the linker is unphosphorylated, it is imperative for the template DNAs to have 5'-phosphate termini. This can be achieved by special chemical or enzymatic digestion of DNA prior to the ligation. For example, the ß–
-elimination step of Maxam and Gilbert chemical reactions generates single-stranded DNA breaks with 5'-phosphate termini at each repetition of specific base(s) (41) (Figure 2). Also, hot piperidine treatment of certain UV-induced dimeric DNA lesions, i.e. pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] and their Dewar valence photoisomers (Figure 3), yields single-stranded DNA breaks with 5'-phosphate groups at the lesion-formation sites (42). Another prominent class of UV-induced DNA dimers, cis-syn cyclobutane pyrimidine-dimers (CPDs), however, can only be converted to ligatable 5'-ends by successive enzymatic digestion with T4 endonuclease V (T4 endo V) and Escherichia coli photolyase reactivation (43). The T4 endo V cleaves the glycosidic bond of the 5'-pyrimidine in a CPD and breaks the sugar–phosphate backbone between the two dimerized pyrimidines. The resulting dissociated 3'-pyrimidine retains an overhang dimer, which makes it unligatable until the E.coli photolyase reactivation step detaches the dimer, yielding a single-stranded DNA with a normal base on the 5'-sugar–phosphate terminus (43).
|
|
Other utilized enzymatic digestions include treatment with DNA glycosylases such as formamidopyrimidine DNA glycosylase, which not only generates abasic (AP) sites by releasing damaged bases but also produces single-stranded DNA breaks with 5'-phosphate termini by cleaving the sugar–phosphate backbone (44). Another versatile enzyme is the E.coli UvrABC endonuclease. This enzyme complex makes a dual incision at distinctive positions 5' and 3' of a variety of lesions induced by DNA crosslinking agents such as cisplatinum, mitomycin C, and psoralens, as well as by bulky DNA adduct forming agents such as PAH, and UV or UV mimetic agents, e.g. 4-nitroquinoline (45). As an example, the UvrABC complex incises seven bases 5' and four/five bases 3' to B[a]PDE-guanine lesions (30).
A significant improvement in the original LM-PCR protocol is the inclusion of biotinylated primers in the first extension step in conjunction with the use of magnetic streptavidin-coated beads permitting an efficient purification of the extended products (7). This purification step prior to PCR amplification rids the extension products of unbound non-specific DNA templates that could otherwise engage in the PCR. Also, the introduction of a new generation of primer labels and compatible detectors, e.g. fluorescence-based systems, has offered an expedient replacement of the earlier painstaking visualization procedures, i.e. sequencing gel transfer, electroblotting, and hybridization with radiolabeled gene-specific probes. The new methodology has also the advantage of allowing even spacing of bands during gel electrophoresis, which leads to a longer and clearer read-out (7) (Figure 2). A detailed protocol for LM-PCR is available in ref. (46).
|
TD-PCR |
|---|
The principles of TD-PCR are similar to LM-PCR with the only differences being the primer extension and ligation steps (7,47). TD-PCR can be readily applied to all 5'-phosphorylated or unphosphorylated DNA templates that contain polymerase blocking lesions, i.e. bulky lesions and single-stranded breaks (Figure. 4). As in LM-PCR, lesion-bearing DNA templates in TD-PCR initially undergo primer extension, but for multiple cycles, with a gene-specific biotinylated primer (‘repeated’ primer extension). The biotinylated extension products are captured using streptavidin-coupled magnetic beads, thus removing the unbound original DNA templates and the non-specific DNA fragments. The purified single-stranded extension products are subjected to 3'-ribotailing using terminal deoxynucleotidyl transferase (TdT) (7,47). The TdT treatment adds long deoxyribonucleotide tails to the 3'-termini of single-stranded DNA molecules (48). However, this addition can be limited to short tails if riboguanosine triphosphate (rGTP) is used as a substrate. Under standardized TD-PCR conditions, an average of three rGTPs is tailed to all extension products. The resulting homopolymeric ribotailed molecules undergo cohesive-end ligation using a phosphorylated double-stranded linker with a three-deoxycytidine overhang at the 3'-end of the longer oligonucleotide (47,48). The ligation products are PCR-amplified using a nested gene-specific primer along with a linker primer that is complementary to the lower strand of the linker. The amplified products are subjected to labeling and gel electrophoresis for visualization and analysis as described for LM-PCR (7,47). The outline of the TD-PCR procedure is illustrated in Figure 1. A detailed and updated protocol for TD-PCR is available in ref. 8.
|
TD-PCR has a higher sensitivity than LM-PCR because it utilizes ‘repeated’ primer extension to synthesize multiple copies of the original lesion-containing DNA templates. These multiplied templates can then participate in the ligation step. In LM-PCR only the original DNA templates take part in the ligation step, and solely those 5'-phosphorylated DNA templates in which primer extension has gone to completion are ligatable. Conversely, in TD-PCR, both prematurely terminated- and fully extended products made from (un) phosphorylated DNA templates are ligatable. Conventional LM-PCR and TD-PCR have a detection limit of approximately 1 lesion per 20 000 and per 50 000–100 000 unmodified nucleotides, respectively. On the other hand, LM-PCR has a greater specificity than TD-PCR because it takes advantage of the initial (chemical/enzymatic) cleavage step to permit the identification of unique and homogenous types of DNA damage prior to the ligation. In TD-PCR, however, a combination of lesions, which pause or arrest DNA polymerase progression, is involved in the ligation. If possible, a pre-digestion with enzymes or chemicals specific for certain types of damage can enrich the lesion content, and add extra layers of specificity and sensitivity to the conventional TD-PCR (7,47).
|
Mutagenicity analysis of carcinogens |
|---|
The in vitro shuttle vector-based mutation systems (36,37) for investigating site-specific mutagenicity of carcinogens and the in vivo transgenic rodent mutation systems (39) for studying DNA damaging and mutagenic properties of carcinogens are relevant model systems for determining carcinogen signatures. Conventionally, shuttle vector-based mutation systems are used as the initial approach to verify the etiologic relevance of suspected carcinogens identified by cancer epidemiology investigations. Once verified, the etiologic involvement of the carcinogen can be further explored using the in vivo transgenic rodent mutation systems. Here, we describe the general features of each of these two model systems, and provide representative methodologies and examples of their application to cancer etiology.
|
Target and reporter genes for mutagenicity analysis of carcinogens |
|---|
Both endogenous cancer-related target genes and exogenous/endogenous reporter genes can be used for mutagenicity analysis of carcinogens (36,37). Obviously, the former genes are of more relevance for cancer etiology investigations. However, the latter genes can also be of value especially when analyzing target genes is not feasible. Of the reporter genes, endogenous ones are preferable for surrogating cancer-related target genes. Albeit, assaying endogenous genes can be either technically challenging (49) or limited to specific organs or developmental stages (50).
|
Cancer-related target genes: the RAS oncogenes and TP53 tumor suppressor gene |
|---|
The RAS oncogene sequences have commonly served as the target sequences for mutagenicity analysis of carcinogens (36,37). Activation of RAS oncogenes occurs frequently in various human cancers as a result of specific mutations at only a few codons along these oncogenes (51,52). The RAS oncogene family consists of three members, N-RAS, H-RAS and K-RAS that are located on chromosomes 1, 11 and 12, respectively. The three genes all encode a 21 kDa protein (p21), which possesses a guanosine triphosphatase activity. The p21 has four domains including, non-essential, essential, nucleotide binding, and effector domains. The activated RAS oncogenes bear mutations at codons 12, 13, 59 or 61 that cause an amino acid change in the last three domains of the p21, which causes the encoded p21 protein to be constitutively active. The activated RAS oncogenes are thought to be involved in aberrant cell proliferation, altered cell cycle checkpoints and cellular differentiation (51,52). In human cancers, the most commonly activated form of RAS oncogenes is the K-RAS oncogene with mutations at codon 12 (51,52). Over 90% of pancreatic cancers, 50% of colon cancers, and 30% of smoking-associated lung cancers have an activated K-RAS oncogene with mutations at codon 12 (51,52).
Another routinely used target gene for mutagenicity analysis of carcinogens is the TP53 tumor suppressor gene (36,37). Inactivation of the TP53 gene is the most frequent event in human cancers due to mutations occurring in a large target region encompassing exons 5–9 (53,54). The multifunctional TP53 gene plays a key role in cell survival and acts as a safeguard against genetic instability caused by endogenous and/or exogenous stressors (53,54). The TP53 gene encodes a 53 kDa nuclear protein (p53; 393 amino acids), which is a transcriptional activator. There are five domains in the p53 protein with characteristic structures and functionalities (53,54). The sequence-specific DNA binding domain of the TP53 gene (amino acids: 97–300) is the major target in most human cancers. Generally, missense mutations mapping to this domain inactivate p53 function by abolishing the DNA binding and transcriptional activation properties of the protein. The overall mutation spectra, the codon position, sequence context, or DNA strand in which inactivating TP53 mutations occur are specific for several types of human cancer (13). For example, non-random TP53 mutations occurring in codons 157, 158, 245, 248, 249 and 273, predominantly at CpG dinucleotides, with a majority being GT transversions and >90% located on the non-transcribed strand, are characteristic for smoking-associated lung cancer (55) (Figure 5). Or, specific TP53 mutations in several dipyrimidine-containing codons mostly in a 5'-CCG or 5'-TCG sequence context and primarily being CT or CCTT transitions are unique for UV-associated non-melanoma skin cancer (24). Also, TP53 mutations almost exclusively at codon 249 and mainly being GT transversions are specific for aflatoxin B1-associated hepatocellular carcinoma (13,20) (Figure 6).
|
|
For the most part, mutagenicity analysis of carcinogens in the RAS and TP53 genes is restricted to tumorous tissues or even to specific cells within a tumor due to the clonal nature of tumorigenesis. Such analysis in unselected cell types in a normal tissue would not be as informative nor would it be significantly indicative of environmental exposures except in a few select cells (56).
|
Reporter genes: the hypoxanthine–guanine phosphoribosyltransferase gene |
|---|
The endogenous hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene is a commonly used reporter gene for mutagenicity analysis of carcinogens (57). The human X-chromosomal HPRT gene was first identified in an enzyme defect associated with severe neurological, psychiatric, arthritic and metabolic disorders represented by excessive purine metabolism and extreme urate production, i.e. the Lesch–Nyhan syndrome (58). HPRT is a single copy gene
44 kb in length including nine exons and eight introns. The coding region of the HPRT gene is 654 bp long. The HPRT gene product is an enzyme required for nucleic acid biosynthesis through phosphoribosylation of hypoxanthine and guanine. The HPRT enzyme also phosphoribosylates purine analogs such as 6-thioguanine and 8-azaguanine, a pre-requisite step for their cytotoxicity. The mutated enzyme is unable to phosphoribosylate these agents, thereby, causing resistance to their cytotoxicity. This is the basis for the HPRT selection system, in which only cells with a mutated HPRT can survive and grow in media containing the purine analogs. The use of HPRT as an expressed reporter gene for mutagenicity analysis of carcinogens has the advantages of being highly sensitive, i.e. relatively low frequency of spontaneous mutations, and reflecting transcriptional determinants of mutagenicity in target genes. However, it also has the disadvantages of being technically challenging, e.g. involving cell cloning, limited to specific cell types, i.e. T lymphocytes and fibroblasts, and under-representing some characteristics of target genes, e.g. low frequency of methylated CpGs. As a homozygous and not heterozygous gene, the HPRT gene also fails to permit the recovery of large chromosomal rearrangements and multi-genic deletions (this is a general limitation of transgenes, as well). Other reporter genes of exogenous origin are discussed in ‘Transgenic Rodent Mutation Systems’. |
Shuttle vector-based mutation detection systems |
|---|
Shuttle vector-based mutation detection systems utilize synthesized oligonucleotides containing defined sequences of reporter genes from plasmids or phages, e.g. supF and lacZ or of cancer-related target genes, e.g. human RAS and TP53 (36,37). A carcinogen-induced lesion of interest is placed at a specific location along the sequence and the resulting oligonucleotide is incorporated into a single- or double-stranded vector. The vector is then replicated in a compatible cellular system, i.e. bacteria or mammalian cells. The progenies are evaluated for the occurrence and types of induced mutation that represent the mutagenic potency and specificity, respectively, of the tested carcinogen.
Shuttle vector-based systems utilize single-stranded or double-stranded vectors. The single-stranded based systems have the advantage of mimicking in vivo circumstances under which translesion synthesis-derived mutations occur at a single/double-stranded DNA junction in the presence of DNA polymerases. The double-stranded based systems are advantageous for examining DNA strand-bias of mutagenesis caused by leading or lagging strand DNA replication. Both systems have been successfully used in various site-specific mutagenicity experiments to elucidate the mutagenic potency and specificity of numerous genotoxic carcinogens (59).
|
Transgenic rodent mutation detection systems |
|---|
Transgenic rodent mutation detection systems offer a chromosomally integrated exogenous gene (mostly of prokaryotic origin) as the target for both DNA footprinting and mutagenicity analysis of carcinogens. A DNA construct bearing the transgene is microinjected into the pronucleus of a zygote from mice or rats. The transgene is randomly integrated as multiple tandem copies at a single location into the genome, and transmitted to the offspring of the foster animal derived from the injected embryo. The ubiquitous and high copy number transgene in all organs of the animal can be rescued from the genomic DNA and used as a reporter gene, e.g. in bacterial expression assays (38).
A carcinogen of interest can be administered to these transgenic animals, and subsequently DNA footprinting and mutagenicity analysis can be performed on the transgene from various organs. The mutagenic potency of the administered carcinogen can be inferred from the relative increase in the mutation rate of the transgene (mutant frequency) in the genomic DNA of the treated animal. The mutagenic specificity of the carcinogen can be deduced from the molecular nature of induced mutations through DNA sequencing of phenotypically expressed mutant transgenes. Theoretically, DNA damage-targeting mutations in the transgene of target organ(s) in a carcinogen-treated animal is reflective of carcinogen exposure (38,39,60). Although selection plays a critical role in tumorigenesis, it does not significantly affect the mutation spectra recovered from a transgene. Throughout the tumorigenesis process, selection is a determinant that shapes and molds the final mutation spectrum that one observes in the tumor—along with mutation induction consequent to exposure to carcinogens. Whereas both selection and mutation induction caused by carcinogen exposure affect tumorigenesis, only the latter is of significance in shaping the mutation spectrum found in the transgene.
Transgenes are used as surrogates for endogenous cancer-related genes, although they may not necessarily possess all characteristics of the endogenous genes. For example, transgenes exist in multiple copies per haploid genome, are heavily methylated, and are not expressed. Being non-transcribed and genetically neutral (38), the transgenes are not subjected to selective pressures existing in vivo (38) as are the endogenous genes (61). The transgenes are not biased for DNA strand-specific mutagenicity because they lack transcription-coupled DNA repair (38). This characteristic allows mutations in the transgene to accumulate and persist over time, thereby making them more easily detectable (62). However, it may also preclude the transgene from mirroring the exact events occurring in endogenous active genes (38). For the most part, transgenes have shown similar responses to carcinogens relative to endogenous cancer-related genes (63–65), although there have also been some dissimilarities (66,67).
Of significance for transgenic mutation systems is determining optimal treatment conditions and sampling times, and relevant target and surrogate organs for analyses (38). Generally, multiple treatments are more preferable to a single treatment with carcinogens in terms of effectiveness. Depending on the outcome of interest, i.e. DNA damage or mutagenesis, sampling should be done immediately after treatment (to avoid removal of DNA damage by DNA repair systems) or at specific time points post-treatment (to allow the induced DNA damage to be translated into mutations after DNA replication, i.e. manifestation time). An expert consultation on transgenic mutation systems (60) has recently recommended a ‘28-daily’ treatment as optimal for all in vivo DNA damage-targeted mutagenicity experiments. Based on the assumption that the mutagenic response to carcinogens varies in different organs (depending on cellular proliferation rate), it was agreed that at a specific time such a response in all comparable organs reaches a maximum and stays at a plateau, thereafter. For highly proliferative organs, the 28-day consecutive treatment period was considered to be sufficient to reach such specific time. For highly differentiated and slowly dividing organs, a manifestation time of ‘28 days’ following the treatment period was also considered to be optimal. Moreover, there was a consensus that all analyses should be performed on target organs wherein tumors arise, although surrogate organs might also be useful once thoroughly validated. Detailed and updated guidelines for transgenic mutation systems are available in refs 39,60.
Today, compelling evidence attests the ability of transgenic rodent mutation systems to predict carcinogenicity (68). A recent evaluation of a database of 155 agents has verified that transgenic mouse mutation systems can convincingly predict carcinogenicity, i.e. 80% of proven carcinogens tested positive in at least one system, and 78% of known non-carcinogens yielded negative results throughout (60). To extrapolate such findings in rodents to humans, however, should be done cautiously because these animals may differ from humans in response to carcinogens due to, e.g. varying metabolic activation and detoxification, and DNA repair capacities (69). Currently, a wide range of transgenic rodent mutation systems exists including Big Blue® mice and rats, MutaMice, gpt delta mice, etc. As a widely used representative, the transgenic Big Blue mutation system is discussed below.
|
Big blue rodents mutation system |
|---|
Since the introduction of Big Blue rodent transgenic animals, the mouse and rat mutation systems have increasingly been used for genotoxicity of an array of carcinogens (70,71). The Big Blue mutation system offers an easily recoverable coliphage lambda shuttle vector (
LIZ), which contains two mutational reporter genes namely, the lacI and cII. This vector is present in 40 copies and integrated into the genome of the rodent at a single locus in a head-to-tail arrangement. For mutation detection, the vector is recovered from the genomic DNA and packaged into viable bacteriophages using an enzyme-based packaging extract (Stratagene; La Jolla, CA). Subsequently, the phages infect an appropriate host E.coli and then, the bacterial culture is grown on a special agar lawn. Mutations in the lacI transgene lead to a functional inactivation of the lacI protein, which is phenotypically expressed as a recovery of ß-galactosidase activity exhibited by blue plaque formation on the agar lawn. Mutations in the cII transgene lead to a lack of functional cII protein that is essential for activation of the cI repressor and integrase, both of which being necessary for lysogenization. Thus, the E.coli indicators that bear phages with a mutated cII undergo lysis and form visible plaques on the agar lawn (71,72). The relative increase in the frequency of lacI or cII mutant plaques in the genomic DNA of a carcinogen-treated animal represents the mutagenic potency of the carcinogen. The lacI and cII mutant plaques can also be isolated and subjected to DNA sequencing, thereby providing information on the molecular nature of induced mutations, i.e. mutagenic specificity of the carcinogen. Because both the lacI and cII transgene are relatively short in length i.e. 1080 and 294 bp, respectively, multiple genomic sequencing can be done from small amounts of DNA isolated from various organs of the transgenic animal (70,71). The lacI and cII transgenic rodent systems have been successfully used to study various classes of chemical and/or physical carcinogens (73–83). |
Examples of application of DNA-lesion footprinting and mutation analysis for investigating human cancer etiology |
|---|
We highlight two examples of application of genomic sequencing for exploring human cancer etiology. In both cases, mutation analysis of the suspected carcinogens verified their etiologic relevance as implicated by cancer epidemiology investigations. DNA-lesion footprinting of the carcinogens provided supportive evidence only in one case, however. In the other case, it offered suggestive clues on interactions between carcinogen exposure and other determinants, being ultimately responsible for cancer development.
|
Tobacco smoke-associated lung cancer |
|---|
Distribution of tobacco smoke-induced DNA lesions was mapped along exons 5–8 of the TP53 gene in B[a]PDE-treated normal human bronchial epithelial cells, HeLa cells, and naked DNA from normal human fibroblasts using the UvrABC-coupled LM-PCR (30). In all cases, there were three major lesion-formation sites including codons 157, 248 and 273 all containing methylated CpG dinucleotides (30) [all CpGs in the DNA binding domain of the human TP53 gene are completely and tissue-independently methylated (84)]. The preferentially formed lesions were on the non-transcribed strand of the TP53 gene (30). B[a]PDE-induced DNA lesions were removed 2–4 times less efficiently from the non-transcribed strand relative to transcribed strand of this gene (85). The selective formation of B[a]PDE-induced DNA lesions at methylated CpGs was reiterated by showing that unmethylated pAT153P53
, a plasmid containing the genomic sequence of human TP53 encompassing exons 2–11, treated with B[a]PDE had a different lesion-formation profile relative to B[a]PDE-treated genomic DNA. A pre-treatment of the plasmid with methylase SssI, which methylates all CpG dinucleotides, however, produced an identical profile of lesion formation to that of B[a]PDE-treated genomic DNA (86). Using a similar methodology, it was confirmed that cytosine methylation substantially enhances guanine alkylation at all CpG sites in the TP53 gene by a variety of carcinogens, including B[a]PDE, benzo[g]chrysene diol epoxide (B[g]CDE), N-acetoxy-2-acetylaminofluorene (N-AAAF), and aflatoxin B1 8,9-epoxide (87). Subsequent mapping of other PAH-derived DNA lesions in exons 5–8 of the TP53 gene in normal human bronchial epithelial cells treated with five PAH-diol epoxides, including chrysene diol epoxide, 5-methylchrysene diol epoxide, 6-methylchrysene diol epoxide, benzo[c]phenanthrene diol epoxide, and B[g]CDE yielded mostly similar results to those observed for B[a]PDE (31). Except for codon 249, all other TP53 mutational hotspots in lung cancer were the preferential binding sites for all or some of these PAH-diol epoxides. It was proposed that the overall spectrum of TP53 mutations in lung cancer is determined by exposure to multiple PAH, possibly having additive or multiplicative effects (31). This is in accordance with the fact that tobacco smoke contains a mixture of PAH, some of which may act synergistically or multiplicatively in relation to each other. It was also acknowledged that selection may shape the spectrum of mutations because not all sites of PAH-induced lesions are necessarily major mutational hotspots (31). These findings are compatible with the cascade hypothesis of mutations invoked by Loeb et al. (88). According to this hypothesis, the specific exposure-associated mutation spectrum recovered from a cancer-related gene in a tumor is of relevance for cancer etiology only if two things apply: (i) the mutations occur early on in the carcinogenesis process and (ii) the gene in which the specific mutations occur is essential and required for tumorigenesis and retained throughout the tumor selection process. Otherwise, the Loeb hypothesis, which may be correct, would predict chaos and a random collection of mutations in the tumor (88).
Distribution of tobacco smoke-associated DNA lesions was also mapped in the N-, H- and K-RAS oncogenes in B[a]PDE-treated normal human bronchial epithelial cells and the naked DNA from these cells, as well as normal human fibroblasts (32). In all cases, the induced DNA lesions were preferentially formed at guanine residues in codons 12 and 14 on the non-transcribed strand of the K-RAS oncogene; however, the removal of lesions from codon 12 was twice slower than that from codon 14. To further explore the specificity of smoke-derived DNA lesions targeting K-RAS mutations, DNA footprinting was performed on normal human bronchial epithelial cells treated with B[g]CDE, N-AAAF, and aflatoxin B1 8,9-epoxide. B[g]CDE and N-AAAF yielded essentially similar lesion-formation profiles to that of B[a]PDE, whereas the food-borne mycotoxin aflatoxin B1 8,9-epoxide showed slightly different results (32). Together, these findings are in good agreement with cancer epidemiology data indicating that approximately one-third of smoking-associated lung tumors harbors an activated K-RAS oncogene with mutations at codon 12 (89).
|
Aflatoxin-associated hepatocellular carcinoma |
|---|
Distribution of DNA lesions induced by aflatoxin B1 was mapped along exons 7–8 of the TP53 gene in human HepG2 cells using both LM-PCR and TD-PCR (90). In both cases, significant DNA-lesion formation was seen at codon 249 in exon 7. However, similar or even more pronounced formation of DNA lesions was observed at several other codons both in exon 7 and 8. In addition, removal of the lesions was more efficient at codon 249 than at most other lesion-formation sites. Together, these data could not per se explain the predominance of TP53 mutations at codon 249 in aflatoxin B1-associated hepatocellular carcinoma (13,20). However, cancer epidemiology investigations have documented that exposure to aflatoxin B1 and infection with hepatitis B virus (HBV) can synergistically impact upon TP53 mutations at codon 249 in hepatocellular carcinoma (91). In fact, in the geographical areas with equal prevalence of HBV infection, aflatoxin B1 exposure has been shown to be positively associated with TP53 mutations at codon 249 in hepatocellular carcinoma (92). Based on these observations, it was proposed that codon 249 mutations in the TP53 gene in hepatocellular carcinoma is a consequence of aflatoxin B1 exposure selected by a concomitant HBV infection (90). However, the exact biological mechanisms for selection of this particular TP53 mutation still remain unknown.
|
Prospective view |
|---|
Genomic sequencing technologies for investigating human cancer etiology are advancing rapidly. The highly sensitive and specific DNA footprinting methodologies are now becoming increasingly automated (10,11). This can expand the application of DNA-lesion footprinting to large-scale molecular cancer epidemiology studies. Accordingly, if sufficient sensitivity for lesion detection is obtained, high-throughput and cost-effective analyses can be performed on cancer-related genes in various target and surrogate organs of populations variably exposed to carcinogens. This approach can help identify valid and non-invasively obtainable tissues for routine biomonitoring of the general population. Furthermore, progress is being made in identifying new genes that are uniquely targeted in specific types of human cancer. These newly identified genes can provide additional target sequences for in vivo DNA-damage mapping and in vitro shuttle vector-based systems. Also, genetically altered rodent models are progressively becoming available. The utility of these models to help create transgenic mutation systems with multiple cancer-related genes over-expressed or inactivated is indispensable (93). Admittedly, however, creating these systems is challenging due to inherent complications in manipulating multiple genes (especially if they all need to be homozygous). Prospectively, generating such transgenic rodent mutation systems will provide valuable information on the mechanism of carcinogenesis and will facilitate validation of mutagenicity analysis in transgenes for predicting carcinogenicity.
Currently, cancer epidemiology investigations are identifying candidate carcinogens of etiologic relevance for human cancers other than lung, liver, and skin cancers. Of these, several common internal cancers such as breast, colon, and prostate cancers are linked to specific environmental factors (13,20). Mutation spectrometry of the induced tumors in these cancers also shows unique signatures of mutation (13,20). For example, 50% of all TP53 mutations in colon cancer are CT transitions at methylated CpG dinucleotides (94,95). Although these mutations are generally assumed to be due to deamination of 5-methylcytosine (84,96), the evidence that this phenomenon does in fact occur in vivo during initiation or progression of colon cancer awaits further confirmation. It is likely that DNA-lesion footprinting and mutagenicity analysis of candidate colon carcinogens may uncover a potential etiologic agent if such an agent would be exactly specific for modification of methylated CpG sequences and would cause C:GT:A transition mutations at the same sequences. In fact, Ambs et al. (97) have demonstrated a significant correlation between activity levels of inducible Ca2+-independent nitric oxide synthase (NOS2), which generates mutagenic nitric oxide (NO) with deaminating metabolites, and occurrence of C:GT:A transition mutations in the TP53 gene in human colon cancer. Furthermore, they have documented a significant dose–response relationship between NOS2 activity and C:GT:A transition mutations at CpG dinucleotides in carcinomas. Because TP53 mutations arise mostly at the transitional stage from adenoma to carcinoma in situ, the authors hypothesized that the observed association between NOS2 and the frequency of C:GT:A transition mutations at the CpG dinucleotides in carcinomas is a consequence of NO-induced mutagenesis (97).
|
Acknowledgments |
|---|
We would like to acknowledge Dr. Arthur D. Riggs for his original contributions to genomic sequencing technology development and for discussions. Work of the authors is supported by NIH grants ES06070 and CA84469.
|
References |
|---|
- Church G.M. and Gilbert W. (1984) Genomic sequencing. Proc. Natl Acad. Sci. USA 81:1991–1995.
[Abstract/Free Full Text] - Becker P.B., Ruppert S., Schutz G. (1987) Genomic footprinting reveals cell type-specific DNA binding of ubiquitous factors. Cell 51:435–443.[CrossRef][Web of Science][Medline]
- Mueller P.R. and Wold B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780–786.
[Abstract/Free Full Text] - Pfeifer G.P., Steigerwald S.D., Mueller P.R., Wold B., Riggs A.D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246:810–813.
[Abstract/Free Full Text] - Saluz H. and Jost J.P. (1989) A simple high-resolution procedure to study DNA methylation and in vivo DNA–protein interactions on a single-copy gene level in higher eukaryotes. Proc. Natl Acad. Sci. USA 86:2602–2606.
[Abstract/Free Full Text] - Yandell D.W. and Dryja T.P. (1989) Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing. Am. J. Hum. Genet. 45:547–555.[Web of Science][Medline]
- Chen H.H., Kontaraki J., Bonifer C., Riggs A.D. (2001) Terminal transferase-dependent PCR (TDPCR) for in vivo UV photofootprinting of vertebrate cells. Sci. STKE 2001:PL1.[Medline]
- Chen H.H., Castanotto D., LeBon J., Rossi J.J., Riggs A.D. (2004) In vivo detection of ribozyme cleavage products and RNA structure by use of terminal transferase-dependent PCR. Methods Mol. Biol. 252:109–124.[Medline]
- Becker M.M., Wang Z., Grossmann G., Becherer K.A. (1989) Genomic footprinting in mammalian cells with ultraviolet light. Proc. Natl Acad. Sci. USA 86:5315–5319.
[Abstract/Free Full Text] - Dai S.M., Chen H.H., Chang C., Riggs A.D., Flanagan S.D. (2000) Ligation-mediated PCR for quantitative in vivo footprinting. Nat. Biotechnol. 18:1108–1111.[CrossRef][Web of Science][Medline]
- Ingram R., Tagoh H., Riggs A.D., Bonifer C. (2005) Rapid, solid-phase based automated analysis of chromatin structure and transcription factor occupancy in living eukaryotic cells. Nucleic Acids Res. 33:e1.
[Abstract/Free Full Text] - Besaratinia A. and Pfeifer G.P. (2005) DNA damage and mutagenesis induced by polycyclic aromatic hydrocarbons. In Luch A. (Ed.). The Carcinogenic Effects of Polycyclic Aromatic Hydrocarbons (Imperial College Press, London, UK) pp. 171–210.
- Olivier M., Hussain S.P., Caron de Fromentel C., Hainaut P., Harris C.C. (2004) TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer. IARC Sci. Publ. 247–270.
- Freese E.B. (1961) Transitions and transversions induced by depurinating agents. Proc. Natl Acad. Sci. USA 47:540–545.
[Free Full Text] - Freese E., Bautz E., Freese E.B. (1961) The chemical and mutagenic specificity of hydroxylamine. Proc. Natl Acad. Sci. USA 47:845–855.
[Free Full Text] - Nomura M. and Benzer S. (1961) The nature of the ‘deletion’ mutants in the rII region of phage T4. J. Mol. Biol. 3:684–692.[Web of Science][Medline]
- Chu E.H. and Malling H.V. (1968) Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro. Proc. Natl Acad. Sci. USA 61:1306–1312.
[Free Full Text] - Thilly W.G. (1990) Mutational spectrometry in animal toxicity testing. Annu. Rev. Pharmacol. Toxicol. 30:369–385.[CrossRef][Web of Science][Medline]
- Skandalis A. and Glickman B.W. (1990) Endogenous gene systems for the study of mutational specificity in mammalian cells. Cancer Cells 2:79–83.[Web of Science][Medline]
- Hussain S.P. and Harris C.C. (1998) Molecular epidemiology of human cancer: contribution of mutation spectra studies of tumor suppressor genes. Cancer Res. 58:4023–4037.
[Free Full Text] - Thilly W.G. (2003) Have environmental mutagens caused oncomutations in people? Nat. Genet. 34:255–259.[CrossRef][Web of Science][Medline]
- Daya-Grosjean L. and Sarasin A. (2005) The role of UV induced lesions in skin carcinogenesis: an overview of oncogene and tumor suppressor gene modifications in xeroderma pigmentosum skin tumors. Mutat. Res. 571:43–56.[Web of Science][Medline]
- Melnikova V.O. and Ananthaswamy H.N. (2005) Cellular and molecular events leading to the development of skin cancer. Mutat. Res. 571:91–106.[Web of Science][Medline]
- Pfeifer G.P., You Y.H., Besaratinia A. (2005) Mutations induced by ultraviolet light. Mutat. Res. 571:19–31.[Web of Science][Medline]
- Wogan G.N. (1999) Aflatoxin as a human carcinogen. Hepatology 30:573–575.[CrossRef][Web of Science]
- Yu M.C. and Yuan J.M. (2004) Environmental factors and risk for hepatocellular carcinoma. Gastroenterology 127:S72–S78.[CrossRef][Web of Science]
- Groopman J.D. and Kensler T.W. (2005) Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol. Appl. Pharmacol. 206:131–137.[CrossRef][Web of Science][Medline]
- Mumford J.L., He X.Z., Chapman R.S., et al. (1987) Lung cancer and indoor air pollution in Xuan Wei, China. Science 235:217–220.
[Abstract/Free Full Text] - DeMarini D.M., Landi S., Tian D., et al. (2001) Lung tumor KRAS and TP53 mutations in nonsmokers reflect exposure to PAH-rich coal combustion emissions. Cancer Res. 61:6679–6681.
[Abstract/Free Full Text] - Denissenko M.F., Pao A., Tang M., Pfeifer G.P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 274:430–432.
[Abstract/Free Full Text] - Smith L.E., Denissenko M.F., Bennett W.P., Li H., Amin S., Tang M., Pfeifer G.P. (2000) Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons. J. Natl Cancer Inst. 92:803–811.
[Abstract/Free Full Text] - Feng Z., Hu W., Chen J.X., Pao A., Li H., Rom W., Hung M.C., Tang M.S. (2002) Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer. J. Natl Cancer Inst. 94:1527–1536.
[Abstract/Free Full Text] - Hill A.B. (1965) The environment and disease: association or causation? Proc. R. Soc. Med. 58:295–300.[Web of Science][Medline]
- Rundle A.G., Vineis P., Ahsan H. (2005) Design options for molecular epidemiology research within cohort studies. Cancer Epidemiol. Biomarkers Prev. 14:1899–1907.
[Abstract/Free Full Text] - Tang D., Phillips D.H., Stampfer M., et al. (2001) Association between carcinogen–DNA adducts in white blood cells and lung cancer risk in the physicians health study. Cancer Res. 61:6708–6712.
[Abstract/Free Full Text] - Singer B. and Essigmann J.M. (1991) Site-specific mutagenesis: retrospective and prospective. Carcinogenesis 12:949–955.
[Free Full Text] - Loechler E.L. (1996) The role of adduct site-specific mutagenesis in understanding how carcinogen–DNA adducts cause mutations: perspective, prospects and problems. Carcinogenesis 17:895–902.
[Abstract/Free Full Text] - Heddle J.A., Dean S., Nohmi T., et al. (2000) In vivo transgenic mutation assays. Environ. Mol. Mutagen. 35:253–259.[CrossRef][Web of Science][Medline]
- Lambert I.B., Singer T.M., Boucher S.E., Douglas G.R. (2005) Detailed review of transgenic rodent mutation assays. Mutat. Res. 590:1–280.[CrossRef][Web of Science][Medline]
- Pfeifer G.P., Chen H.H., Komura J., Riggs A.D. (1999) Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction. Methods Enzymol. 304:548–571.[Web of Science][Medline]
- Maxam A.M. and Gilbert W. (1977) A new method for sequencing DNA. Proc. Natl Acad. Sci. USA 74:560–564.
[Abstract/Free Full Text] - Pfeifer G.P., Drouin R., Riggs A.D., Holmquist G.P. (1991) In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction. Proc. Natl Acad. Sci. USA 88:1374–1378.
[Abstract/Free Full Text] - Pfeifer G.P., Drouin R., Riggs A.D., Holmquist G.P. (1992) Binding of transcription factors creates hot spots for UV photoproducts in vivo. Mol. Cell. Biol. 12:1798–1804.
[Abstract/Free Full Text] - O'Connor T.R. and Laval J. (1989) Physical association of the 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites. Proc. Natl Acad. Sci. USA 86:5222–5226.
[Abstract/Free Full Text] - Selby C.P. and Sancar A. (1990) Structure and function of the (A)BC excinuclease of Escherichia coli. Mutat. Res. 236:203–211.[Medline]
- Pfeifer G.P. (2005) Measuring the formation and repair of DNA damage by ligation-mediated PCR. In Henderson D.S. (Ed.). DNA Repair Protocols Mammalian Systems. (Humana Press, Totowa, NJ) pp. 201–212.
- Komura J. and Riggs A.D. (1998) Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts. Nucleic Acids Res. 26:1807–1811.
[Abstract/Free Full Text] - Schmidt W.M. and Mueller M.W. (1996) Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences. Nucleic Acids Res. 24:1789–1791.
[Abstract/Free Full Text] - McKinzie P.B., Delongchamp R.R., Heflich R.H., Parsons B.L. (2001) Prospects for applying genotypic selection of somatic oncomutation to chemical risk assessment. Mutat. Res. 489:47–78.[CrossRef][Web of Science][Medline]
- Winton D.J., Blount M.A., Ponder B.A. (1988) A clonal marker induced by mutation in mouse intestinal epithelium. Nature 333:463–466.[CrossRef][Medline]
- Sills R.C., Boorman G.A., Neal J.E., Hong H.L., Devereux T.R. (1999) Mutations in ras genes in experimental tumours of rodents. IARC Sci. Publ. 55–86.
- Malumbres M. and Barbacid M. (2003) RAS oncogenes: the first 30 years. Nat. Rev. Cancer 3:459–465.[CrossRef][Web of Science][Medline]
- Robles A.I., Linke S.P., Harris C.C. (2002) The p53 network in lung carcinogenesis. Oncogene 21:6898–6907.[CrossRef][Web of Science][Medline]
- Hofseth L.J., Hussain S.P., Harris C.C. (2004) p53: 25 years after its discovery. Trends Pharmacol. Sci. 25:177–181.[CrossRef][Medline]
- Hainaut P. and Pfeifer G.P. (2001) Patterns of p53 GT transversions in lung cancers reflect the primary mutagenic signature of DNA-damage by tobacco smoke. Carcinogenesis 22:367–374.
[Abstract/Free Full Text] - Hussain S.P., Amstad P., Raja K., et al. (2001) Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung. Cancer Res. 61:6350–6355.
[Abstract/Free Full Text] - Albertini R.J. (2001) HPRT mutations in humans: biomarkers for mechanistic studies. Mutat. Res. 489:1–16.[CrossRef][Web of Science][Medline]
- Seegmiller J.E., Rosenbloom F.M., Kelley W.N. (1967) Enzyme defect associated with a sex-linked human neurological disorder and excessive purine synthesis. Science 155:1682–1684.
[Abstract/Free Full Text] - Khalili H., Zhang F.J., Harvey R.G., Dipple A. (2000) Mutagenicity of benzo[a]pyrene-deoxyadenosine adducts in a sequence context derived from the p53 gene. Mutat. Res. 465:39–44.[Web of Science][Medline]
- Thybaud V., Dean S., Nohmi T., et al. (2003) In vivo transgenic mutation assays. Mutat. Res. 540:141–151.[Web of Science][Medline]
- Mellon I., Spivak G., Hanawalt P.C. (1987) Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene. Cell 51:241–249.[CrossRef][Web of Science][Medline]
- Tao K.S., Urlando C., Heddle J.A. (1993) Comparison of somatic mutation in a transgenic versus host locus. Proc. Natl Acad. Sci. USA 90:10681–10685.
[Abstract/Free Full Text] - Shane B.S., de Boer J., Watson D.E., Haseman J.K., Glickman B.W., Tindall K.R. (2000) LacI mutation spectra following benzo[a]pyrene treatment of Big Blue mice. Carcinogenesis 21:715–725.
[Abstract/Free Full Text] - Yoon J.H., Smith L.E., Feng Z., Tang M., Lee C.S., Pfeifer G.P. (2001) Methylated CpG dinucleotides are the preferential targets for G-to-T transversion mutations induced by benzo[a]pyrene diol epoxide in mammalian cells: similarities with the p53 mutation spectrum in smoking-associated lung cancers. Cancer Res. 61:7110–7117.
[Abstract/Free Full Text] - You Y.H. and Pfeifer G.P. (2001) Similarities in sunlight-induced mutational spectra of CpG-methylated transgenes and the p53 gene in skin cancer point to an important role of 5-methylcytosine residues in solar UV mutagenesis. J. Mol. Biol. 305:389–399.[CrossRef][Web of Science][Medline]
- Skopek T.R., Kort K.L., Marino D.R., Mittal L.V., Umbenhauer D.R., Laws G.M., Adams S.P. (1996) Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice. Environ. Mol. Mutagen. 28:376–384.[CrossRef][Web of Science][Medline]
- Mittelstaedt R.A., Manjanatha M.G., Shelton S.D., Lyn-Cook L.E., Chen J.B., Aidoo A., Casciano D.A., Heflich R.H. (1998) Comparison of the types of mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI and hprt genes of Big Blue rats. Environ. Mol. Mutagen. 31:149–156.[CrossRef][Web of Science][Medline]
- Dean S.W., Brooks T.M., Burlinson B., Mirsalis J., Myhr B., Recio L., Thybaud V. (1999) Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-of-contact mutagens. Mutagenesis 14:141–151.
[Abstract/Free Full Text] - Lijinsky W. (1993) Species differences in carcinogenesis. In Vivo 7:65–72.[Medline]
- Kohler S.W., Provost G.S., Kretz P.L., Dycaico M.J., Sorge J.A., Short J.M. (1990) Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice. Nucleic Acids Res. 18:3007–3013.
[Abstract/Free Full Text] - Jakubczak J.L., Merlino G., French J.E., Muller W.J., Paul B., Adhya S., Garges S. (1996) Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target. Proc. Natl Acad. Sci. USA 93:9073–9078.
[Abstract/Free Full Text] - Swiger R.R., Cosentino L., Shima N., Bielas J.H., Cruz-Munoz W., Heddle J.A. (1999) The cII locus in the MutaMouse system. Environ. Mol. Mutagen. 34:201–207.[CrossRef][Web of Science][Medline]
- Zhang X.B., Felton J.S., Tucker J.D., Urlando C., Heddle J.A. (1996) Intestinal mutagenicity of two carcinogenic food mutagens in transgenic mice: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and amino(alpha)carboline. Carcinogenesis 17:2259–2265.
[Abstract/Free Full Text] - Dycaico M.J., Stuart G.R., Tobal G.M., de Boer J.G., Glickman B.W., Provost G.S. (1996) Species-specific differences in hepatic mutant frequency and mutational spectrum among lambda/lacI transgenic rats and mice following exposure to aflatoxin B1. Carcinogenesis 17:2347–2356.
[Abstract/Free Full Text] - Besaratinia A. and Pfeifer G.P. (2003) Enhancement of the mutagenicity of benzo(a)pyrene diol epoxide by a nonmutagenic dose of ultraviolet A radiation. Cancer Res. 63:8708–8716.
[Abstract/Free Full Text] - Bol S.A., Horlbeck J., Markovic J., de Boer J.G., Turesky R.J., Constable A. (2000) Mutational analysis of the liver, colon and kidney of Big Blue rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline. Carcinogenesis 21:1–6.
[Abstract/Free Full Text] - Besaratinia A. and Pfeifer G.P. (2004) Biological consequences of 8-methoxypsoralen-photoinduced lesions: sequence-specificity of mutations and preponderance of T to C and T to A mutations. J. Invest. Dermatol. 123:1140–1146.[CrossRef][Web of Science][Medline]
- Chen T., Mittelstaedt R.A., Beland F.A., Heflich R.H., Moore M.M., Parsons B.L. (2005) 4-Aminobiphenyl induces liver DNA adducts in both neonatal and adult mice but induces liver mutations only in neonatal mice. Int. J. Cancer 117:182–187.[CrossRef][Web of Science][Medline]
- Besaratinia A., Synold T.W., Xi B., Pfeifer G.P. (2004) G-to-T transversions and small tandem base deletions are the hallmark of mutations induced by ultraviolet A radiation in mammalian cells. Biochemistry 43:8169–8177.[CrossRef][Medline]
- Shane B.S., Smith-Dunn D.L., de Boer J.G., Glickman B.W., Cunningham M.L. (2000) Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice. Mutat. Res. 452:197–210.[Web of Science][Medline]
- Besaratinia A. and Pfeifer G.P. (2004) Genotoxicity of acrylamide and glycidamide. J. Natl Cancer Inst. 96:1023–1029.
[Abstract/Free Full Text] - Mirsalis J.C., Shimon J.A., Johnson A., Fairchild D., Kanazawa N., Nguyen T., de Boer J., Glickman B., Winegar R.A. (2005) Evaluation of mutant frequencies of chemically induced tumors and normal tissues in lambda/cII transgenic mice. Environ. Mol. Mutagen. 45:17–35.[CrossRef][Web of Science][Medline]
- Besaratinia A. and Pfeifer G.P. (2005) Investigating DNA adduct-targeted mutagenicity of tamoxifen: preferential formation of tamoxifen–DNA adducts in the human p53 gene in SV40 immortalized hepatocytes but not endometrial carcinoma cells. Biochemistry 44:8418–8427.[CrossRef][Medline]
- Tornaletti S. and Pfeifer G.P. (1995) Complete and tissue-independent methylation of CpG sites in the p53 gene: implications for mutations in human cancers. Oncogene 10:1493–1499.[Web of Science][Medline]
- Denissenko M.F., Pao A., Pfeifer G.P., Tang M. (1998) Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers. Oncogene 16:1241–1247.[CrossRef][Web of Science][Medline]
- Denissenko M.F., Chen J.X., Tang M.S., Pfeifer G.P. (1997) Cytosine methylation determines hot spots of DNA damage in the human P53 gene. Proc. Natl Acad. Sci. USA 94:3893–3898.
[Abstract/Free Full Text] - Chen J.X., Zheng Y., West M., Tang M.S. (1998) Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots. Cancer Res. 58:2070–2075.
[Abstract/Free Full Text] - Loeb L.A., Loeb K.R., Anderson J.P. (2003) Multiple mutations and cancer. Proc. Natl Acad. Sci. USA 100:776–781.
[Abstract/Free Full Text] - Wistuba I.I., Gazdar A.F., Minna J.D. (2001) Molecular genetics of small cell lung carcinoma. Semin. Oncol. 28:3–13.[Web of Science][Medline]
- Denissenko M.F., Koudriakova T.B., Smith L., O'Connor T.R., Riggs A.D., Pfeifer G.P. (1998) The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts. Oncogene 17:3007–3014.[CrossRef][Web of Science][Medline]
- Ross R.K., Yuan J.M., Yu M.C., Wogan G.N., Qian G.S., Tu J.T., Groopman J.D., Gao Y.T., Henderson B.E. (1992) Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 339:943–946.[CrossRef][Web of Science][Medline]
- Unsal H., Yakicier C., Marcais C., Kew M., Volkmann M., Zentgraf H., Isselbacher K.J., Ozturk M. (1994) Genetic heterogeneity of hepatocellular carcinoma. Proc. Natl Acad. Sci. USA 91:822–826.
[Abstract/Free Full Text] - Hursting S.D., Nunez N.P., Patel A.C., Perkins S.N., Lubet R.A., Barrett J.C. (2005) The utility of genetically altered mouse models for nutrition and cancer chemoprevention research. Mutat. Res. 576:80–92.[Web of Science][Medline]
- Greenblatt M.S., Bennett W.P., Hollstein M., Harris C.C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54:4855–4878.
[Free Full Text] - Hollstein M., Sidransky D., Vogelstein B., Harris C.C. (1991) p53 mutations in human cancers. Science 253:49–53.
[Abstract/Free Full Text] - Rideout W.M. III, Coetzee G.A., Olumi A.F., Jones P.A. (1990) 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 249:1288–1290.
[Abstract/Free Full Text] - Ambs S., Bennett W.P., Merriam W.G., Ogunfusika M.O., Oser S.M., Harrington A.M., Shields P.G., Felley-Bosco E., Hussain S.P., Harris C.C. (1999) Relationship between p53 mutations and inducible nitric oxide synthase expression in human colorectal cancer. J. Natl Cancer Inst. 91:86–88.
[Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J.-H. Yoon, L. Prakash, and S. Prakash Highly error-free role of DNA polymerase {eta} in the replicative bypass of UV-induced pyrimidine dimers in mouse and human cells PNAS, October 27, 2009; 106(43): 18219 - 18224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Besaratinia, S.-i. Kim, and G. P. Pfeifer Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells FASEB J, July 1, 2008; 22(7): 2379 - 2392. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-i. Kim, G. P. Pfeifer, and A. Besaratinia Lack of Mutagenicity of Acrolein-Induced DNA Adducts in Mouse and Human Cells Cancer Res., December 15, 2007; 67(24): 11640 - 11647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Arlt, M. Stiborova, J. vom Brocke, M. L. Simoes, G. M. Lord, J. L. Nortier, M. Hollstein, D. H. Phillips, and H. H. Schmeiser Aristolochic acid mutagenesis: molecular clues to the aetiology of Balkan endemic nephropathy-associated urothelial cancer Carcinogenesis, November 1, 2007; 28(11): 2253 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Besaratinia and G. P. Pfeifer A review of mechanisms of acrylamide carcinogenicity Carcinogenesis, March 1, 2007; 28(3): 519 - 528. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||