The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (Denissenko et al, 1996, 1998). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors. To test whether a selectivity of AFB1 adduct formation is related to this peculiar mutational spectrum, we have mapped AFB1-DNA adducts at nucleotide resolution using ligation-mediated PCR and terminal transferase-dependent PCR. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat liver microsomes. Significant adduct formation was seen at the third base of codon 249. However, this was not the major site of AFB1 adducts and strong adduction was also observed at codons 226, 243, 244, 245 and 248 in exon 7 of the p53 gene and at several codons in exon 8. The damage at codon 249 does not consist of a unique abasic site or ring-opened aflatoxin B1 adduct but rather is consistent with the principal N7-guanine adduct of AFB1. Time course experiments indicate that, under the conditions used, AFB1 adducts are not removed in a strand-selective manner and adduct removal from the third base of codon 249 proceeds at a relatively fast rate (50% in 7 h). The incomplete correspondence between sites of persistent AFB1 damage and the specific codon 249 mutation suggests that AFB1 may not be involved in mutation of this site or that additional mechanisms such as parallel infection with hepatitis B virus may be required for selection of codon 249 mutants in HCC.     

Strand asymmetry of CpG transitions as indicator of G1 phase-dependent origin of multiple tumorigenic p53 mutations in stem cells

In dividing cells, expression of mutations is DNA strand symmetric. Of all mutations originating de novo in nondividing cells, only those in the transcribed (noncoding) strand are immediately expressed in mRNA and protein. In contrast, any new mutation in the nontranscribed (coding) strand remains unexpressed until the cells enter S phase and begin proliferation. This previously unrecognized difference enables us to examine the cell cycle-dependent origin of multiple tumorigenic mutations in stem cells. The human p53 gene, which acts as a gatekeeper in the control of G1 to S phase transition, was chosen for the analysis. Of all multiple mutations contained in p53 databases, we have tested in detail CpG transitions. Three features of CpG sites dictate this choice: C --> T transitions at methylated mCpG are the direct product of mC deamination and are replication-independent; it is easy to identify the strand bearing a primary mC --> T event because C --> T on the transcribed strand appears as G --> A on the nontranscribed strand; and CpG transitions are the most frequent (as both singular and multiple occurrences) tumor-related p53 mutations. The origin of double nonsilent CpG transitions in nondividing cells predicts a significant excess of the heterostrand (C --> T, G --> A) doublets over the homostrand (C --> T, C --> T and G --> A, G --> A) doublets. For p53, we found such an excess. Based on this result, along with the results of three other tests reported here, we conclude that the majority of multiple p53 mutations from human tumors occurred in quiescent stem cells.     

Mitral inflow and pulmonary venous Doppler measurements do not predict pulmonary capillary wedge pressure in heart transplant recipients

We examined the spectrum of p53 mutations found in 40 UV-induced skin tumors of xeroderma pigmentosum group A gene (XPA)-deficient mice. p53 mutations were detected in 48% of the tumors. Nearly all of the mutations were induced at dipyrimidine sites. Ninety-three % of the mutations were G.C-->A.T transitions at dipyrimidine sites, including tandem transitions (CC-->TT), which are the hallmark of the UVB-induced mutation. Seventy-two % of the mutations at dipyrimidine sites could be ascribed to damage on the transcribed strand. In addition, no evident mutational hot spots were detected. This is in contrast to the UVB-induced skin tumors of normal mice, in which 92% of p53 mutations occurred as a result of DNA damage on the nontranscribed strand, and clear hot spots were observed. Thus, XPA-deficient mice showed significant mutation features that might be characteristic of the absence of nucleotide excision repair and may provide a good animal model for the analysis of the high incidence of skin cancer in xeroderma pigmentosum group A patients.     

Growth and cuticular synthesis in Ascaris suum larvae during development from third to fourth stage in vitro

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10(8) nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (> 1 adduct/10(9) nucleotide). Current heavy smokers (> 20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 (28%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid chromatography (HPLC). Six of 33 (18%) samples were positive for BPDE-DNA adducts by both postlabeling and HPLC/SFS. No correlation was observed between the SFS and 32P-postlabeling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.     

Secular trends in the epidemiology of nosocomial fungal infections at a teaching hospital in Taiwan, 1981 to 1993

PURPOSE: p53 is a tumour suppressor gene encoding a nuclear phosphoprotein that plays an important role in the control of normal cell proliferation. We have tried to establish the value of expression of the p53 protein in malignant lymphomas and its correlation with the presence of structural gene abnormalities. MATERIAL AND METHODS: 230 cases of lymphomas (11 Hodgkin's disease and 219 non-Hodgkin's) were studied by immunohistochemistry using an anti-p53 monoclonal antibody (DO-7, DAKO). Sections were heated by pressure cooker in 0.01 M sodium citrate buffer pH 6 as a method for antigen unmasking. The quantification of levels of nuclear p53 protein were measured with an Image Analyzer (CAS-200, Becton-Dickinson S.A.). We have also searched for mutations in a series of 94 lymphomas using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis in exons 5 to 9 of the p53 gene and the sample showing abnormal pattern were sequenced. RESULTS: p53 immunoreactivity has been detected in 82.6% of cases. The percentage of positive cells varies in a wide range which was divided into 0% (17.39%), less than 5% (57.39%), between 5-10% (13.91%) and more than 10% (11.30%). In all lymphoma subtypes, we have found the majority of cases show levels less than 10% and few more than 10%. We found abnormal bands in 9 of 94 lymphomas with different diagnosis (1 ALCL, 1 T-LNH, 2 B-LNH, 2 centroblastic, 1 lymphoblastic and 2 MALT). Two cases resulted to be a silent base change which not alter the function of the p53 protein and represent a common polymorphism. Seven cases showed single base pair missense mutations in all of them. Mutations in codons 179, 248 and 273 correspond to some of the typical hotspots described in p53 gene. CONCLUSIONS: p53 expression, is a frequent finding in malignant lymphomas, is variable and relates to histological subtype. The results suggest that positive immunocytochemistry cannot be used to determine which tumours have mutations of p53 because the existence of cases with discrepancies between overexpression of the protein and presence of mutations.     

8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene

We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand.     

Expression of CD30 mRNA, CD30L mRNA and a variant form of CD30 mRNA in restimulated peripheral blood mononuclear cells (PBMC) of patients with helminthic infections resembling a Th2 disease

The reactivity of guanines in an oligonucleotide containing mutational hot spots within the p53 gene (codons 248 and 249), 5'-CCG1G2AG3G4CCCA-3', toward dimethyl sulfate (DMS) and aflatoxin B1-8,9-epoxide (AFB1-8,9-epoxide) was investigated by a modified Maxam-Gilbert technique. 5-Methylcytosine in the CpG site of codon 248 did not appear to modulate the reactivity of target guanines G1, G2, G3, and G4 toward either genotoxin when compared to the sequence containing a nonmethylated CpG site. A similar experiment was conducted in which a 0.5-kb fragment of the human HPRT gene containing exon 1 and several CpG sites was treated with UV-activated aflatoxin B1. Results showed that guanine adduct formation was independent of the methylation status of the CpG site. These findings are discussed in relation to other studies that have shown that cytosine methylation has an inhibiting effect, an enhancing effect, or no effect on adduct formation with nearby guanine nucleotides.     

The C-terminal domain of p53 catalyzes DNA-renaturation and strand exchange toward annealing between intact ssDNAs and toward eliminating damaged ssDNA from duplex formation through preferential recognition of damaged DNA by a duocarmycin

The C-terminal domain of p53 may bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules, suggesting a possible direct role for p53 in DNA repair (Proc. Natl. Acad. Sci. USA, 92, 9455-9459, 1995). We found that DU-86, a duocarmycin derivative which alkylates DNA, bound ssDNA and enhanced the DNA binding activity of the p53 C-terminus. DU-86 weakened p53-mediated catalysis of complementary ssDNA renaturation. p53 C-terminus catalyzed DNA strand transfer toward annealing between intact ssDNAs and toward eliminating DU-86-damaged ssDNA from duplex formation. These results suggest that p53, via the C-terminal domain, may play a direct role in DNA repair by preferential recognization and elimination of damaged DNA.     

Relative involvement of chromosome #21 in radiation induced exchange aberrations in lymphocytes of Down syndrome patients

The reaction of chemical carcinogens with DNA appears to be one of the earliest events in the initiation phase of cancer. These DNA reactions can be base- and position-specific, are affected by sequence context, and are repaired at different rates depending on whether or not they are on the transcribed or nontranscribed strand of DNA and which nucleotide sequence is modified. Thus, measurement of total genomic DNA reaction of carcinogens is only a crude first step in dissecting out which are the critical lesions for cancer initiation. On the other hand, we know that DNA adducts, which have been primarily characterised in experimental studies, appear to have similar structures in human DNA arising from occupational or environmental exposures. A number of different methods have been developed to detect and measure DNA adducts in man. These include physico-chemical methods such as mass spectrometry, 32P-postlabelling, fluorescence and accelerator mass spectrometry (AMS) and biological methods such as immunoassay. All these methods have their strengths and weaknesses. Human studies, using 32P-postlabelling, demonstrate that this method can be used to examine the effect of potential chemoprotective agents on DNA adduct level. AMS has been used to measure DNA adducts in human tissue after patients have ingested trace quantities of the food mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a heterocyclic amine formed during the cooking of meat and the naturally occurring mycotoxin, aflatoxin B1. These studies can assist in assessing the risks associated with low-level exposure to food genotoxins.