Amifostine (WR2721) restores transcriptional activity of specific p53 mutant proteins in a yeast functional assay.

Many p53 mutants found in human cancer have an altered ability to bind DNA and transactivate gene expression. Re-expression of functional p53 in cells in which the endogenous TP53 gene is inactivated has been demonstrated to restore a non-tumorigenic phenotype. Pharmacological modulation of p53 mutant conformation may therefore represent a mechanism to reactivate p53 function and consequently improve response to radio- and chemotherapy. We have recently reported that the radio- and chemoprotector Amifostine (WR2721, Ethyol) activates wild-type p53 in cultured mammalian cells. In the present study, we have used a yeast functional assay to investigate the effect of WR2721 on the transcriptional activity of p53. WR2721 restored this activity in a temperature-sensitive mutant V272M (valine to methionine at codon 272) expressed at the non-permissive temperature and it also partially restored the transcriptional activity of several other conformationally flexible p53 mutants. The results indicate that the yeast functional assay may be used to identify compounds that modulate p53 activity, with potential therapeutic implications.

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Partial characterization of SUVi, a new mammalian gene induced by UV-C and expressed during the S phase of the cell cycle.

By using a lacZ-based gene-trap approach, we identified a mammalian gene induced by UV-C in a Chinese hamster ovary cell clone (Menichini P et al. [1997]: Nucleic Acids Res 25:4803-4807). The activity of the encoded protein fused to a bacterial beta-galactosidase was followed through the hydrolysis of different beta-galactosidase substrates. In this study we describe how the expression of this gene is modulated during the cell cycle and in response to UV-irradiation. We show that the beta-galactosidase activity was virtually undetectable in quiescent cells (G[0]), started to increase when cells progressed in G(1), and reached a maximum in mid-S phase, indicating a possible role of the endogenous protein during DNA synthesis. Following UV-irradiation, besides a delay of the progression through the S phase, a twofold increase of the reporter protein activity in all phases of the cell cycle was observed. The partial sequence analysis showed that this gene, here named SUVi (for S phase UV-inducible), contains a domain that is highly conserved among different helicases. Together, these data suggest that the SUVi gene could be involved in DNA synthesis, a process that takes place both in the S phase and in the processing of UV-induced damage.

Analysis of stepwise genetic changes in an AIDS-related Burkitt's lymphoma.

In this study, immunoglobulin variable (Ig V) region genes, c-myc re-arrangement and sequence and p53 status were analyzed in clones derived from a Burkitt's lymphoma cell line (LAM) in which it was previously demonstrated that Epstein-Barr virus (EBV) infection occurred late during lymphomagenesis. Such evidence was based on the finding that 2 groups of cellular clones, characterized by the same c-myc re-arrangement but different EBV-fused termini, were obtained from the LAM cell line. The Ig V gene sequences were identical for the 2 groups of clones with different EBV-fused termini. The Ig variable heavy (V(H)) gene sequence displayed a substantial accumulation of point mutations (but no intra-clonal diversification), whereas the productive Ig V lambda (V(lambda)) gene sequence was virtually unmutated. Studies on the Ig V kappa (V(kappa)) locus suggested a receptor revision event (with a switch from kappa to lambda chain production) prior to EBV infection. Likewise, it was determined that the mutations observed in both p53 alleles and in the re-arranged c-myc gene occurred before EBV infection. Based on these findings, we present a model for the various steps of lymphomagenesis. It is proposed that stimulation by an antigen or a superantigen initially favored the clonal expansion and accumulation of other cytogenetic changes, including those involved in receptor editing. These events occurred prior to or during the germinal center (GC) phase of B-cell maturation. Thereafter, possibly upon exit of the cells from the GC, EBV infection occurred, further promoting lymphomagenesis.

Gadd45 mutations are uncommon in human tumour cell lines.

GADD45 is an evolutionarily conserved gene that encodes a small acidic, nuclear protein and is an example of a p53 responsive gene. Gadd45 protein has been shown to interact with PCNA and also p21waf1. It has been implicated in growth arrest, DNA repair, chromatin structure and signal transduction. The confusing biochemical data has been clarified by the demonstration that Gadd45 null mice have a phenotype strikingly similar to that of p53 null mice, being tumour prone and showing marked genomic instability. We have tested the hypothesis that mutations in the GADD45 coding region might substitute for p53 abnormalities in tumour cell lines where p53 is wild type. After generating cDNA from mRNA in a panel of 24 cell lines we sequenced the GADD45 cDNA and have demonstrated that no mutations can be observed, even in the p53 wild type cell lines. Such data suggest that Gadd45 mutations are uncommon in human cancer. From this we postulate that, despite the phenotype of the GADD45 null mouse, GADD45 is unlikely to be the key mechanistic determinant of the tumour suppressor activity of the p53 pathway.

Defective nuclear localization of p53 protein in a Chinese hamster cell line is associated with the formation of stable cytoplasmic protein multimers in cells with gene amplification.

Many p53 functions require p53 transport into the nucleus. Mutant p53 also generally accumulates in the nucleus of transformed or neoplastic cells. However, examples of cytoplasmic accumulation of wild-type or mutant p53 have also been reported. Various explanations have been provided for defective nuclear localization. Here we propose a novel example of cytoplasmic p53 localization which occurs in cells showing gene amplification and appears to be due to the formation of stable p53 multimers. We studied a methotrexate-resistant Chinese hamster cell line (MTX M) carrying amplified dihydrofolate reductase genes and derived from a cell line with p53 nuclear accumulation. MTX M showed cytoplasmic p53 localization and, on immunoblots, several extra bands in the high molecular weight region, besides the expected 53 kDa band. p53 localization and the appearance of high molecular weight bands appeared to be correlated with the degree of DNA amplification. However, amplification of dihydrofolate reductase itself was not involved. Changing the p53 phosphorylation status quantitatively influenced the formation of high molecular weight bands. Cell fusion experiments demonstrated that p53 cytoplasmic localization in MTX M is a dominant phenotype. This result suggests that the defect causing lack of nuclear localization in this cell line does not reside in the nucleus. In the cytoplasm of MTX M and of wild-type/MTX M heterodikaryons p53 gives rise to protein complexes that are unable to re-enter the nucleus. The formation of such protein complexes is dependent on the amplification of an unknown gene product.

The yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.

The assumption of molecular epidemiology that carcinogens leave fingerprints has suggested that analysis of the frequency, type, and site of mutations in genes frequently altered in carcinogenesis may provide clues to the identification of the factors contributing to carcinogenesis. In this mini-review, we revise the development, and validation of the yeast-based p53 functional assay as a new tool for molecular epidemiology. We show that this assay has some very interesting virtues but also has some drawbacks. The yeast functional assay can be used to determine highly specific mutation fingerprints in the human p53 cDNA sequence. Discrimination is possible when comparing mutation spectra induced by sufficiently different mutagens. However, we also reported that the same carcinogen may induce distinguishable mutation spectra due to known influencing factors.

p53 mutations experimentally induced by 8-methoxypsoralen plus UVA (PUVA) differ from those found in human skin cancers in PUVA-treated patients.

8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis. PUVA therapy has been associated with an increased risk of developing skin squamous cell carcinoma (SCC). In order to determine the PUVA-induced p53 mutation spectrum, a yeast expression vector harbouring a human wild-type p53 cDNA was incubated with 8-MOP, and UVA irradiated in vitro. PUVA-damaged and undamaged DNA was transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. An 8-MOP concentration-dependent decrease in survival and increase in mutant frequency were observed. At a fixed 8-MOP concentration, survival decreased and mutant frequency increased as UVA irradiation increased. Eleven mutant clones contained 11 mutations: 10 were single base pair substitutions, the remaining one being a complex mutation. All eight T:A-targeted mutations were at 5'-TpA sites, hallmark mutations of PUVA mutagenesis. Through a rigorous statistical test, the PUVA-induced p53 mutation spectrum appears to differ significantly (P < 0.0002) from that observed in SCC in PUVA-treated patients. The present work demonstrates that a specific PUVA-induced mutational fingerprint could be obtained and recognized on human p53 cDNA. This result may suggest that PUVA therapy can be a risk factor for the development of SCC in psoriasis patients through a mechanism not involving the induction of p53 mutations.

Derivative chromosome 17 in a case of Burkitt lymphoma with 8;14 translocation.

A complex chromosome rearrangement present in a B-cell line established from a patient with Burkitt lymphoma was studied by using fluorescence in situ hybridization (FISH) and immunocytochemistry techniques. The rearranged chromosome (der17) was apparently composed of 17q, of a partially deleted 17p, and of other material of chromosome 17p origin that was interspersed with regions without any clear banding pattern. der(17) contained a functional ch17 centromere and two additional centromeres of unknown origin that were inactive by all evidence. By FISH analysis with a TP53 probe, a signal could be demonstrated on the normal ch17, but not on the rearranged chromosome, a finding which indicates that 17p deletion caused a concurrent loss of one of the two TP53 alleles. The marker chromosome was previously observed in some of the malignant cells obtained from the patient's peripheral blood. These observations therefore indicate that cells with this specific rearrangement were generated in vivo and subsequently selected. This rearrangement is likely to have conferred a selective growth advantage to a subclone present in the original malignant cell population.

N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic.

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation.

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.

p53 mutations and DNA ploidy in colorectal adenocarcinomas.

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases. Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p < 0.04, Fisher's exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation. The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.

Heterogeneous p53 mutations in a Burkitt lymphoma from an AIDS patient with monoclonal c-myc and VDJ rearrangements.

This study investigates the timing of p53 mutations detected in the malignant cells of a Burkitt's lymphoma cell line (BRG-P) with respect to other maturation or transforming events. The BRG-P cell line, derived from an AIDS patient, was of special value since it displayed subclones that had undergone an isotype switch from IgM to IgA1 (BRG-M and BRG-A cells). BRG-M and BRG-A cells were characterized by the same monoclonal c-myc and VDJ rearrangements and by the expression of Ig receptors with specificity for a 45 kDa protein of human breast cells. Analysis of p53 mutations in the different BRG subclones showed that 1) BRG-M cells displayed 2 different p53 mutations in trans; since the original BL cells also showed the same mutations, this finding indicated that both occurred in vivo; 2) one of the p53 alleles of BRG-A cells was lost, while the other showed a mutation different from those seen in BRG-M cells; and 3) all 3 mutations observed in BRG-M or BRG-A cells resulted in the functional inactivation of the transcriptional activation function of p53. Together, our data demonstrate that p53 mutations were relatively late events during lymphomagenesis. Moreover, in view of the role of p53 in cell apoptosis, it is conceivable that BRG cells were subjected to a strong selective pressure that favored p53 inactivation. Such inactivation was possibly required to counterbalance other potentially apoptotic events, including the presence of a deregulated c-myc oncogene and signals delivered by the host environment in situ.

Analysis of K-ras, p53, bcl-2 and Rb expression in non-small cell lung cancer cell lines.

Six non-small cell lung cancer (NSCLC) cell lines (A-549, Ca-Lu-6, SK-Lu-1, Ca-Lu-1, SK-Mes-1 and LX-1) were studied to assess the presence of multiple concomitant alterations of different oncogenes (K-ras, bcl-2) and tumor suppressor genes (p53, Rb) in NSCLC. K-ras (exon 1) and p53 (exons 5-8) gene mutations were determined via a PCR-based-DGGE (Denaturing Gradient Gel Electro-phoresis) and by sequencing approach. Different mutations were found in the Ist exon of K-ras gene in 5 of 6 cell lines examined. Five of six cell lines contained K-ras mutations at codon 12 (A-549, SK-Lu-1, LX-1) or codon 13 (SK-Mes-1, Ca-Lu-1). In addition, 5 of 6 cell lines showed p53 mutations of exon 8 (SK-Mes-1, Ca-Lu-1 cod. 280; LX-1 cod. 273) or exon 6 (Ca-Lu-6 cod. 196; SK-Lu-1 cod. 193). In 4 of these cell lines, p53 protein nuclear expression was also confirmed with DO-7 mAb immunocytochemistry. Expression of cytoplasmic bcl-2 protein, by anti-bcl-2 mAb flow cytometric analysis, was found in A-549, Ca-Lu-1, SK-Lu-1, SK-Mes-1 cell lines. In contrast, RT-PCR analysis of Rb gene could not identify any change in the cell lines examined. In conclusion, most NSCLC cell lines tested displayed concomitant multiple oncogene/tumor suppressor gene alterations.

Lack of mutations in K-ras codons 12 and 13 in human atherosclerotic lesions.

In the framework of a project investigating the possible involvement of cancer biomarkers in human atherogenesis, we evaluated the occurrence of K-ras mutations in the DNA extracted from smooth muscle cells of abdominal aorta atherosclerotic lesions. The molecular analysis of the DNA from 32 surgical specimens, using PCR-based denaturing gradient gel electrophoresis (DGGE), did not reveal any variant in K-ras codons 12 and 13, which are the most frequently involved codons among the ras genes mutated in various types of human tumors. Analysis of the DNA extracted from four cell lines carrying known K-ras mutational alleles showed typically positive DGGE patterns. Thus, on the whole, the conclusions of this study and of previous studies using the same biological material are consistent with the occurrence of DNA adducts in human atherosclerotic lesions but in the absence of p53 involvement or of K-ras mutations in codons 12 and 13. The search for candidate genes which may possibly be involved in the atherogenetic process warrants further studies.

p53 is frequently mutated in Barrett's metaplasia of the intestinal type.

Barrett's Esophagus (BE) is a complication of gastroesophageal reflux in which the normal squamous epithelium of the lower esophagus is replaced by metaplastic tissue. The clinical significance of this condition is the associated predisposition to adenocarcinomas (ADCs). Three types of BE have been characterized: the gastric fundic (F) type, the gastric cardial (C) type, and the intestinal (I) type. The latter is the most closely associated with the development of ADCs; the causes of this bias remain unknown. To determine whether p53 and/or K-ras gene alterations (a) are present in preneoplastic lesions and (b) are associated with a specific histotype, we performed PCR-based denaturing gradient gel electrophoresis (DGGE) analysis of exon 1 (codons 12-13) of K-ras gene and of exons 5-8 of the p53 gene in biopsies obtained from 30 patients with BE of the I type (9 patients), combined I type (I + C +/- F; 10 patients) and non-I type (C, F, or C + F; 11 patients). None of the cases under study revealed K-ras mutations, whereas biopsies from 12 patients showed at least one p53 DGGE variant. Four patients showed the exact same variants in leukocytes also (polymorphisms), whereas eight cases revealed specific DGGE variants only in biopsies. The molecular characterization of these variants revealed that four of them showed a single base pair substitution, and four showed multiple mutations. Of 17 somatic mutations, all but 1 were base pair substitutions located mainly in exons 7 and 8. The majority of these mutations were GC targeted (13 of 16; 81%), 54% (7 of 13) of which were transitions occurring at CpG sites. All somatic mutations were found in BE with at least one I component. The association with the histotype was statistically significant (P < 0.03; pure I type versus non-I type; P < 0.04, combined I type versus non-I type; Fisher's exact test). Loss of heterozygosity in the vicinity of the p53 locus was evaluated by PCR using a highly polymorphic variable number of tandem repeats marker on 25 out of 30 cases. Ninety-two % of the cases analyzed were informative, and none of them showed LOH. In conclusion, we showed that p53 mutations are frequently observed in specimens from BE patients of the I-type, whereas no involvement of K-ras (exon 1) mutational activation was observed. In light of the key roles that the p53 protein plays in controlling cell cycle and cell diploidy, this result may suggest why this type of metaplasia is the most closely associated to the development of ADCs.

Study on aneuploidy and p53 mutations in astrocytomas.

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Genetic alterations in astrocytic tumors: hints to a molecular epidemiological interpretation.

General incidence of brain tumors has significantly increased over the past two decades. Although the aetiology of this increase has not been determined, increased life span and improved diagnosis methods can partially be responsible for it. Starting from the epidemiological data on risk factors, we reviewed the molecular events known to be involved in the genesis and progression of the most common brain neoplasm in adult, namely astrocytoma. Alterations in different genes, encoding key regulatory elements in the cell cycle control, were reviewed. In light of the molecular epidemiological notion that carcinogens leave fingerprints, point mutations at the p53 locus from a panel of astrocytic tumor patients from all over the world were analysed. The results of this analysis suggest that the majority of astrocytomas may have a spontaneous origin. In particular, the kind of mutational events observed suggests a major role for mutagenic events occurring at CpG sites. In order to yield valid conclusions on the potential role of environmental mutagenic factors, well designed molecular epidemiological studies on populations clearly showing a higher relative risk of developing brain tumors are needed.

Mutational fingerprint induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea in mammalian cells.

Using the pZ189 shuttle vector approach, we determined two chloroethyl-cyclohexyl-nitrosourea (CCNU)-induced mutation spectra (3 and 6 mM) in African green monkey kidney cells (CV1). One hundred and twenty-one independent clones (101 CCNU induced, 45 at 3 mM and 56 at 6 mM; 20 spontaneous) showing functional inactivation of the supF gene were analyzed. One hundred and five plasmids (91 CCNU induced, 41 at 3 mM and 50 at 6 mM; 14 spontaneous), showing no large deletion/rearrangements, were sequenced. Ninety mutants (81 CCNU induced and 9 spontaneous) showed at least one mutation in the supF region. The analysis of the 122 CCNU-induced mutations (56 and 66 at 3 and 6 mM, respectively) revealed that: (a) the majority of the mutations were GC-targeted base pair substitutions; (b) AT-targeted mutations were significantly more frequent in the CCNU-induced (6 mM) than in the spontaneous mutational spectrum (P < 0.0006, Fisher's exact test); (c) mutational spectra obtained at 3 and 6 mM CCNU were significantly different (P < 0.008); (d) induced mutations were nonrandomly located in both spectra and generated either a common hot spot (position 123, 5'-GGG-3') or hot spots exclusive for each CCNU concentration (3 mM: position 159, 5'-AGG-3'; 6 mM: position 109, 5'-GGG-3'); (e) the occurrence of GC-->AT transitions was significantly different as a function of CCNU concentration (P < 0.02, Fisher's exact test), the mutated G being almost exclusively preceded by a purine (5'Pu G) at 6 mM and by either Pu or Py at 3 mM; and (f) by applying Calladine's rules, we found that sequences encompassing the three CCNU hot spots shared identical helix parameters for no more than 2 bp steps 5' (or 3 bp steps 3') to the mutated G. Our results are consistent with the hypothesis that O6-alkylguanine is responsible, either directly or indirectly, for the majority of GC-targeted mutations, while O4-alkylthymine and/or N3-alkyladenine are probably responsible for AT-targeted mutations. The results suggest also that, in CV1 cells, the efficiency of the repair mechanism(s) involved in the removal of O6-alkylguanine is influenced by the DNA sequence context. All of these factors determine the CCNU mutational fingerprint. CCNU has been implicated in the induction of therapy-related leukemias.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer biomarkers in human atherosclerotic lesions: no evidence of p53 involvement.

In order to assess similarities between the atherogenic and the carcinogenic processes, we investigated whether the p53 tumor suppressor gene, the most commonly altered gene in human cancer, may be also involved in human atherosclerotic lesions. The medium layers of abdominal aorta fragments taken at surgery from 32 patients were subjected to immunohistochemical analysis, using either monoclonal (Pab 1801) or polyclonal (CM-1) antibodies, and to molecular analysis by the PCR-based denaturing gradient gel electrophoresis approach. The results obtained indicated that p53 mutations are not involved in the pathogenesis of atherosclerotic lesions, and that no accumulation of the wild-type protein occurs in smooth muscle cells of these lesions. A polymorphism characterized by an AT to GC transition at codon 213 (CGA --> CGG) causing no aminoacid substitution (Arg --> Arg) was detected in the 10.5% of the examined patients. Our negative findings do not support the hypothesis that the atherosclerotic plaques may be pathogenetically akin to benign tumors yet they are not in contrast with this theory, since in most cases p53 is involved in advanced stages of the carcinogenesis process.