Role of human CYP1A1 and NAT2 in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mutagenicity and DNA adducts.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in multiple organs and numerous species. Bioactivation of PhIP is initiated by PhIP N(2)-hydroxylation catalysed by cytochrome P450s. Following N-hydroxylation, O-acetylation catalysed by N-acetyltransferase 2 (NAT2) is considered a further possible activation pathway. Genetic polymorphisms in NAT2 may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were used to test the effect of CYP1A1 and NAT2 polymorphism on PhIP genotoxicity. Cells transfected with NAT2*4 had significantly higher levels of N-hydroxy-PhIP O-acetyltransferase (p = 0.0150) activity than cells transfected with NAT2*5B. Following PhIP treatment, CHO cell lines transfected with CYP1A1, CYP1A1/NAT2*4 and CYP1A1/NAT2*5B each showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis not observed in untransfected CHO cells. dG-C8-PhIP was the primary DNA adduct formed and levels were dose dependent in transfected CHO cells in the order: CYP1A1 < CYP1A1 and NAT2*5B < CYP1A1 and NAT2*4, although levels did not differ significantly (p > 0.05) following one-way analysis of variance. These results strongly support activation of PhIP by CYP1A1 with little effect of human NAT2 genetic polymorphism on mutagenesis and DNA damage.

Differentially Expressed mRNA Targets of Differentially Expressed miRNAs Predict Changes in the TP53 Axis and Carcinogenesis-Related Pathways in Human Keratinocytes Chronically Exposed to Arsenic.

Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite software, and mapped to their pathways using MetaCore software. MDM2, HMGB1 and TP53 mRNA, and protein levels were assayed by RT-qPCR and Western blot. Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis.

Adenosine deaminase (ADA) deficiency due to deletion of the ADA gene promoter and first exon by homologous recombination between two Alu elements.

In 15-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The goal of this study was to determine the precise molecular defect in a patient with ADA-deficient SCID whom we previously have shown to have a total absence of ADA mRNA and a structural alteration of the ADA gene. By detailed Southern analysis, we now have determined that the structural alteration is a deletion of approximately 3.3 kb, which included exon 1 and the promoter region of the ADA gene. DNA sequence analysis demonstrates that the deletion created a novel, complete Alu repeat by homologous recombination between two existing Alu repeats that flanked the deletion. The 26-bp recombination joint in the Alu sequence includes the 10-bp "B" sequence homologous to the RNA polymerase III promoter. This is the first example of homologous recombination involving the B sequence in Alu repeats. Similar recombination events have been identified involving Alu repeats in which the recombination joint was located between the A and B sequences of the polymerase III split promoter. The nonrandom location of these events suggests that these segments may be hot spots for recombination.

Characterization of the human XPA promoter.

We cloned the human xeroderma pigmentosum group A gene (XPA) and characterized the XPA promoter (pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.

A gel electrophoresis system for resolving over 500 nucleotides with a single sample loading.

A procedure for the resolution of over 500 nucleotides in a single loading on a sequencing gel is described. The method uses exponential wedge spacers to achieve compression of the band spacing in the lower portion of the gel. The method is simple and reliable and reduces the cost of electrophoresis and autoradiography supplies by at least a factor of two.

Enhanced XPA mRNA levels in cisplatin-resistant human ovarian cancer are not associated with XPA mutations or gene amplification.

Enhanced expression of the nucleotide excision repair gene XPA is associated with resistance to cisplatin treatment in human ovarian cancer. Understanding the cause of enhanced XPA expression will provide new molecular targets for therapy directed at overcoming chemoresistance. Enhanced gene expression in cancer cells is often caused by mutations or gene amplification. Molecular analyses of the XPA genes in human ovarian cancers indicate that gene mutation and amplification are not the cause of enhanced XPA mRNA levels in ovarian cancers overexpressing XPA. Altered nucleotide excision repair (NER) gene regulation in chemoresistant tumors is discussed.

The Cockayne syndrome group B DNA repair protein as an anti-cancer target.

Cells from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair (TCR), which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS (of which there are two complementation groups, CSA and CSB) do not demonstrate an elevated incidence of cancer. Recently, we demonstrated that disruption of the CSB gene reduces the spontaneous tumor rate in cancer predisposed Ink4a/ARF-/- mice as well as causing their embryo fibroblasts to proliferate more slowly and be more sensitive to UV-induced apoptosis. In the present study we characterized phosphorothioate backbone antisense oligodeoxynucleotides (AOs) that reduced the levels of CSB mRNA in A2780/CP70 ovarian carcinoma cells. The AOs caused the cells to proliferate more slowly and made them more sensitive to either cisplatin or oxaliplatin. The AOs also enhanced the cytotoxicity of hydrogen peroxide and gamma-radiation, both of which can induce oxidative DNA lesions, which are subject to TCR. The AOs did not potentiate the cytotoxicity of topotecan, which induces DNA strand breaks. Chemically modified () AOs (MBOs) targeting CSB were able to potentiate the anti-tumor effect of cisplatin against A2780/CP70 tumor xenografts formed in nude mice. The MBOs enabled a non-toxic (3 mg/kg) dose of cisplatin to have the same degree of anti-tumor efficacy as a more toxic (5 mg/kg) cisplatin dose. Collectively, these results suggest that the CSB gene product may be viewed as an anti-cancer target.

Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis.

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.

Splice site mutations in a xeroderma pigmentosum group A patient with delayed onset of neurological disease.

XP12BE is a commonly studied XP-A cell line that exhibits slightly increased resistance to UV compared with the majority of XP-A cell lines. The elevated UV survival is common to a subset of XP-A cell lines and correlates with delayed onset of the neurological disease in patients. We identified the XPA mutations in XP12BE by single strand conformation polymorphism (SSCP) analyses and nucleotide sequencing. XP12BE is a compound heterozygote and both mutations affect mRNA splicing. One mutation is a G to C transversion within the splice donor site of intron 4 that is common to several cell lines from XP-A patients with delayed onset of neurological disease. The other mutation is a G to T transversion at the same position as a G to C transversion in the splice acceptor site of intron 3 that is common in Japanese XP-A patients. We also demonstrated the persistence of the XP12BE mutations in cell line 2-O-A2 which has been shown to express XPA protein. These results suggest that the intron 4 splice donor mutation likely produces some, at least partially functional, XPA protein that accounts for the increased UV survival of XP-A cell lines derived from patients with delayed onset of neurological disease.

Isolation and fractionation of total nucleic acids from tissues and cells.

A simple and efficient method was devised to permit the isolation of total nucleic acids (poly(A+) and poly(A-) RNAs and DNA) from cells and tissues (e.g. testis) enriched in DNA. Chaotropic reagents (guanidine thiocyanate and LiBr) were utilized to inactivate nucleases rapidly, minimize the viscosity of the homogenization solution and to precipitate RNA selectively (LiBr). Both total and poly(A+)-enriched mRNAs were recovered in a biologically active form as demonstrated by their ability to programme an in vitro translation reaction. High molecular weight DNA (greater than 22 kilobases) was recovered from the LiBr-soluble supernatants by selective ethanol precipitation, subsequently purified and was in a form suitable for further biochemical analysis.

The DNA damage-recognition problem in human and other eukaryotic cells: the XPA damage binding protein.

The capacity of human and other eukaryotic cells to recognize a disparate variety of damaged sites in DNA, and selectively excise and repair them, resides in a deceptively small simple protein, a 38-42 kDa zinc-finger binding protein, XPA (xeroderma pigmentosum group A), that has no inherent catalytic properties. One key to its damage-recognition ability resides in a DNA-binding domain which combines a zinc finger and a single-strand binding region which may infiltrate small single-stranded regions caused by helix-destabilizing lesions. Another is the augmentation of its binding capacity by interactions with other single-stranded binding proteins and helicases which co-operate in the binding and are unloaded at the binding site to facilitate further unwinding of the DNA and subsequent catalysis. The properties of these reactions suggest there must be considerable conformational changes in XPA and associated proteins to provide a flexible fit to a wide variety of damaged structures in the DNA.

Preferential DNA damage in the p53 gene by benzo[a]pyrene metabolites in cytochrome P4501A1-expressing xeroderma pigmentosum group A cells.

Gene-specific DNA damage levels were determined by quantitative polymerase chain reaction (QPCR) after treating cytochrome P450 (CYP) 1A1-expressing xeroderma pigmentosum fibroblasts with [3H]benzo[a]pyrene-trans-7,8-dihydrodiol ([3H]BPD) or [3H]benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ([3H]BPDE). DNA damage in the p53 gene (which is transcriptionally active) and the beta-globin gene (which is transcriptionally inactive) was measured in cells treated with [3H](+/-)-anti-BPDE, [3H](+/-)-BPD, and [3H](-)-BPD. DNA adduct formation in the genome overall was determined by measuring the incorporation of 3H into DNA. DNA damage in a p53 gene fragment (exons 8-9, 445 bp) was readily detected by QPCR. DNA damage was either not detected or much reduced in a similarly sized target in the beta-globin gene (exons 1-2, 551 bp). At equivalent levels of genomic DNA adducts, BPD treatment induced more damage in the p53 gene than BPDE treatment did. The lesion frequencies in the p53 and beta-globin genes in purified DNA treated with BPDE in vitro were the same, indicating that there was no sequence-specific basis for preferential lesion formation in the p53 gene in treated cells. DNA damage in both the p53 and beta-globin genes showed a dose response to [3H](-)-BPD. The frequency of BPD-induced lesions in the p53 gene was sixfold to sevenfold greater than in the beta-globin gene and 200- to 300-fold greater than in bulk DNA. The BPD-induced lesion frequency in the beta-globin gene was 30- to 50-fold greater than in bulk DNA. The data indicate that the distribution of BPDE-induced DNA lesions is dramatically nonrandom and suggest that the nonrandomness is governed by DNA sequence composition, chromatin structure, and dose rate.

Evidence of sequences resembling avian retrovirus long terminal repeats flanking the trout protamine gene.

Additional TATA boxes are present in the flanking regions of trout protamine genes. Their activity as promoters was assayed using an in vitro transcription system. These additional TATA boxes, together with polyadenylation signals that include the consensus AATAAA and CACTG sequences very close to the promoters, suggest that these sequences may be closely related to retroviral long terminal repeat (LTR) sequences. Other features of retroviral LTRs that are also present are short inverted repeats. The LTR-like sequences flanking the trout protamine gene show significant homology to the avian sarcoma virus LTR over a 40-bp region. The trout protamine gene falls into the relatively rare intronless class of eukaryotic genes. This suggests that the gene could have been derived from a processed gene introduced into the genome by reverse transcription of a mature mRNA. The protamine-mRNA-coding region is flanked by AACA... TGTT sequences, which might represent vestigial traces of past recombination events and whose presence supports the notion that the protamine gene sequence was of foreign origin. Recent attempts in this laboratory to transfer the protamine gene into mouse cells have resulted in a high frequency of deletions similar to those observed with constructs in which a retrovirus was used as a vector to transfect foreign DNA with promoters. The distribution of protamine genes in the animal kingdom is very sporadic, which suggests that protamine genes appeared relatively late in evolution. The nonuniform occurrence of the gene among lower vertebrates may have been the result of its horizontal transmission only to certain species, possibly by infection with retroviruses that acquired it from a different species.

Phosphorylation and activation of brain tryptophan hydroxylase: identification of serine-58 as a substrate site for protein kinase A.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is activated by protein kinase A and calcium/calmodulin-dependent protein kinase. One important aspect of the regulation of any enzyme by a phosphorylation-dephosphorylation cascade, and one that is lacking for tryptophan hydroxylase, lies in the identification of its site of phosphorylation by protein kinases. Recombinant forms of brain tryptophan hydroxylase were expressed as glutathione S-transferase fusion proteins and exposed to protein kinase A. This protein kinase phosphorylates and activates full-length tryptophan hydroxylase. The inactive regulatory domain of the enzyme (corresponding to amino acids 1-98) was also phosphorylated by protein kinase A. The catalytic core of the hydroxylase (amino acids 99-444), which expresses high levels of enzyme activity, was neither phosphorylated nor activated by protein kinase A. Conversion of serine-58 to arginine resulted in the expression of a full-length tryptophan hydroxylase mutant that, although remaining catalytically active, was neither phosphorylated nor activated by protein kinase A. These results indicate that the activation of tryptophan hydroxylase by protein kinase A is mediated by the phosphorylation of serine-58 within the regulatory domain of the enzyme.

Stable transformation of xeroderma pigmentosum group A cells with an XPA minigene restores normal DNA repair and mutagenesis of UV-treated plasmids.

The ability of an XPA minigene construct to complement the DNA repair defect in xeroderma pigmentosum group A (XP-A) cells was demonstrated. XP-A cells (XP12BE-SV) were stably transformed with an XPA minigene linked to a neomycin resistance (neor) expression cassette. The G418-resistant clone XAN1 was isolated and its DNA repair phenotype compared with XP12BE-SV cells transformed with a cosmid containing a human chromosome 8 gene and a neo(r) cassette and selected for G418 resistance (2-0-A2), DNA repair-normal human fibroblasts and untransfected XP12BE-SV cells. Colony forming ability after UV-irradiated reactivation of a UV-irradiated chloramphenicol acetyltransferase (CAT) expression vector and UV-induced mutagenesis in a supF tRNA shuttle vector (pSP189) were all restored to normal levels in XAN1 cells. In addition, mutation spectra in the supF gene of pSP189 after replication in all four cell lines were compiled at low (100 J/m2) and high (1000 J/m2) UV doses. The majority of mutations were point mutations and these were predominately G:C-->A:T transitions regardless of dose for all cell lines. Dose-dependent differences were observed in the positions of mutation hot spots in pSP189 mutation spectra after replication in all four cell lines. Mutation spectra for XAN1 and GM0637 cells had only minor differences. An increase in the proportion of transversions was observed only in plasmids irradiated with a low UV dose and replicated in XAN1 cells. 2-0-A2 cells were reported to have partial restoration of DNA repair that was later suggested to be caused by a reversion. 2-0-A2 cells were nearly identical to XP12BE-SV cells in all aspects investigated, indicating that transformation to neor had no effect on DNA repair in these cells.

General method for isolation of DNA sequences that interact with specific nuclear proteins in chromosomes: binding of the high mobility group protein HMG-T to a subset of the protamine gene family.

A general method is described for the isolation of the DNA with which specific nuclear proteins interact in chromosomes. This method is based on the covalent photo-cross-linking of nuclear proteins to the DNA sequences, to which they normally bind, by means of irradiation with UV light and the selective retrieval of specific subsets of protein-DNA adducts by using specific antibodies. The application of this procedure to isolate the DNA sequences with which the trout high mobility group protein (HMG-T) interacts has shown that in trout liver this protein associates specifically with DNA sequences in proximity to a subset of the family of protamine genes but not with the histone or vitellogenin genes. From these observations, it appears that the HMG-T protein may be associated with inactive gene sequences.