P53 Hotspot Mutants Research Articles

P53 hot-spot mutants increase tumor vascularization via ROS-mediated activation of the HIF1/VEGF-A pathway

The function of p53 tumor suppressor is often altered in various human tumors predominantly through missense-mutations resulting in accumulation of mutant proteins. We revealed that expression of p53 proteins with amino-acid substitutions at codons 175 (R175H), 248 (R248W), and 273 (R273H), representing the hot-spots of mutations in various human tumors, increased the number of vessels in HCT116 human colon carcinoma xenografts and, as a result, accelerated their growth. Stimulation of tumor angiogenesis was connected with about 2-fold increase in intracellular level of reactive oxygen species (ROS). Antioxidant N-acetyl- l-aspartate (NAC) decreased vessels number in tumors formed by cells with inactivated p53 and inhibited their growth. Effect of ROS on angiogenesis in tumors expressing hot-spot p53 mutants was correlated with their ability to increase a content of HIF1 transcriptional factor responsible for up-regulation of VEGF-A mRNAs.

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots

Tamoxifen elevates the risk of endometrial tumours in women and α-(N2-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with α-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N2-tamoxifen and dG-N2-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both α-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for α-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only α-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC→TA transversions. Application of the LwPy53 algorithm to the α-acetoxytamoxifen spectrum predicted strong GC→TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N2-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.

The R246S hot-spot p53 mutant exerts dominant-negative effects in embryonic stem cells in vitro and in vivo

p53 is the most frequently mutated tumour-suppressor gene in human cancers. Mutant p53 is thought to contribute to carcinogenesis by the acquisition of gain-of-function properties or through the exertion of dominant-negative (DN) effects over the remaining wild-type protein. However, the context in which the DN effects are observed is not well understood. We have therefore generated 'knock-in' mouse embryonic stem (ES) cells to investigate the effects of expressing a commonly found hot-spot p53 mutant, R246S -- the mouse equivalent of human R249S, which is associated with hepatocellular carcinomas. We demonstrate here that R246S mutant p53 exhibits DN effects with respect to target gene expression, cell survival and cell cycle arrest both in cells that are in the undifferentiated state and upon differentiation. The knock-in cells contain higher levels of p53 that localizes to the nucleus even in the absence of genotoxic stress and yet remains non-functional, reminiscent of mutant p53 found in human tumours. In a model based on carbon-tetrachloride-induced liver injury, these cells were consistently highly tumorigenic in vivo, similar to p53(-/-) cells and in contrast to both p53(+/+) and p53(+/-) ES cells. These data therefore indicate that the DN effects of mutant p53 are evident in the stem-cell context, in which its expression is relatively high compared with terminally differentiated cells.

Degradation of HPV20E6 by p53: ΔNp63α and mutant p53R248W protect the wild type p53 mediated caspase‐degradation

The E6 and E7 proteins of human papillomaviruses (HPV) play a crucial role in the pathogenesis of malignant tumors. E6 protein of high-risk mucosal papillomaviruses targets a number of proteins for proteosomal degradation through complex formation with ubiquitin ligase E6AP. These proteins include, amongst others, p53, paxillin and PDZ-domain proteins e.g. Dlg and MAGUK. The mechanism by which the E6 protein of cutaneous HPV types interacts with cellular proteins to induce either benign or malignant cutaneous lesions, has not been elucidated, although extensive ultraviolet exposure and mutated p53 (hot-spot mutations) are known to be associated with non-melanoma skin cancer. We demonstrate two mechanisms in which HPV20E6 may be involved in the infected cell. One pathway is the wtp53 mediated degradation of HPV20E6 through caspase-3. Mutated p53R248W and Delta Np63 alpha, as well as other unknown proteins involved in proteosome-dependent degradation, convey a protective effect on HPV20E6 under these conditions. This unveils a remarkable opposite regulation to the well-known mechanism of E6-E6AP mediated degradation of p53 for mucosal HPV types. In a second interaction, ectopically expressed HPV20E6 induces cleavage of procaspase-3 to active caspase-3. We demonstrate, in addition, in vivo binding of HPV20E6 to the intermediate filament vimentin.

Polymorphism of XRCC1 and the frequency of mutation in codon 249 of the p53 gene in hepatocellular carcinoma among guangxi population, China

In hepatocellular carcinoma (HCC), hotspot mutation in codon 249 of the p53 gene has been associated with exposure to aflatoxin B1 (AFB1). While the polymorphism of DNA repair gene X-ray repair cross-complementary group 1 (XRCC1) Arg399Gln may be related with AFB1-DNA adducts and gene mutations. Five hundred one HCCs were included in this study to investigate the role of the XRCC1 codon 399 polymorphism on hotspot mutation in codon 249 of the p53 gene. The genotypes of XRCC1 codon 399 and p53 codon 249 were examined by PCR-RFLP. The HCC patients with XRCC1 genotypes with 399 Gln (namely: XRCC1-AG/GG) exhibited a significantly higher frequency of the p53 hotspot mutations in codon 249 than those with the wild-type homozygote of XRCC1 [namely: XRCC1-AA, adjusted odds ratio (OR) = 6.77, 95% confidence interval (CI) = 4.34-10.57]. Compared with those individuals who did express XRCC1-AA as reference (OR = 1), moreover, individuals featuring XRCC1-AG/GG and AFB1-DNA adducts did experience a significantly greater frequency of the hotspot mutation in codon 249 of the p53 gene (adjusted OR = 28.37, 95% CI = 13.19-61.02, P < 0.01). This study suggests that the XRCC1 Arg399Gln polymorphism and AFB1-DNA adducts are associated with the increased frequency of the p53 mutations in codon 249.

Limitations in Molecular Detection of Lymph Node Micrometastasis From Colorectal Cancer

Colorectal cancer patients with lymph node metastasis have a shorter survival and may require adjuvant therapy after surgery of the primary tumor. It is supposed that a more reliable diagnosis can be achieved using tumor-specific DNA mutations for the detection of metastasizing cells. To design a practical approach for a molecular diagnosis of micrometastasis, we applied direct DNA sequencing to screen 48 early stage colorectal carcinomas for the most frequent mutations of the KRAS, P53, and APC tumor genes. KRAS mutations were detected as frequently as described earlier. In contrast, the frequency of P53 and APC hot spot mutations was unexpectedly low, compared with previous studies using other screening methods or including advanced tumor stages. Not more than 31% of early stage tumors showed a mutation in at least 1 of the selected hot spot codons. Applying mutant-enriched polymerase chain reaction (PCR), the mutation of the primary tumor was detected in lymph node DNA from 2 of the KRAS-positive patients. In 1 patient, the result was not verified by subtractive iterative PCR, a principally different molecular method with high sensitivity and specificity. Our data suggest that screening for suitable markers for a molecular detection of occult lymph node metastasis cannot be restricted to small-sized hot spot regions of a few tumor genes and possibly must include tumor-specific epigenetic changes. Furthermore, restriction enzyme-based methods such as mutant-enriched PCR are not suitable to detect any mutation with equal efficiency and they should be carefully controlled to avoid false-positive detection of marker mutations in lymph node DNA.

Detection and Quantitation of Acrolein-Derived 1,N2-Propanodeoxyguanosine Adducts in Human Lung by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N2-propano-dGuo adducts: (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (alpha-OH-Acr-dGuo) and (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (gamma-OH-Acr-dGuo). alpha-OH-Acr-dGuo is more mutagenic and mainly induces G --> T transversions. A recent study demonstrated that acrolein-DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in the human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from the human lung in the presence of [13C10,15N5]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion and by carrying out reactions with NaOH and NaBH4, which produced N2-(3-hydroxypropyl)-dGuo or 1,N2-(1,3-propano)-dGuo from gamma-OH-Acr-dGuo and alpha-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed, and Acr-dGuo adducts were detected in all samples. Both alpha-OH- and gamma-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16-209 adducts/109 nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in the human lung.

Cancer-derived p53 mutants suppress p53-target gene expression--potential mechanism for gain of function of mutant p53.

Tumour-derived p53 mutants are thought to have acquired ‘gain-of-function’ properties that contribute to oncogenicity. We have tested the hypothesis that p53 mutants suppress p53-target gene expression, leading to enhanced cellular growth. Silencing of mutant p53 expression in several human cell lines was found to lead to the upregulation of wild-type p53-target genes such as p21, gadd45, PERP and PTEN. The expression of these genes was also suppressed in H1299-based isogenic cell lines expressing various hot-spot p53 mutants, and silencing of mutant p53, but not TAp73, abrogated the suppression. Consistently, these hot-spot p53 mutants were able to suppress a variety of p53-target gene promoters. Analysis using the proto-type p21 promoter construct indicated that the p53-binding sites are dispensable for mutant p53-mediated suppression. However, treatment with the histone deacetylase inhibitor trichostatin-A resulted in relief of mutant p53-mediated suppression, suggesting that mutant p53 may induce hypo-acetylation of target gene promoters leading to the suppressive effects. Finally, we show that stable down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis. Together, the results demonstrate another mechanism through which p53 mutants could promote cellular growth.

Evaluation of the Combined Effect of p53 Codon 72 Polymorphism and Hotspot Mutations in Response to Anticancer Drugs

Mutations in p53 are common events during carcinogenesis and have been suggested to affect sensitivity to chemotherapy. Recently, the common polymorphism at codon 72, resulting in either an arginine (72R) or proline (72P) residue, was shown to differentially affect the response to anticancer drugs. Here, we have generated isogenic lung cancer cell lines to evaluate the effect of six p53 hotspot mutations (R175H, G245S, R248W, R249S, R273H, and R282W) in conjunction with the codon 72 polymorphism, for their response to a variety of anticancer drugs, either alone or in combination. The data indicate that 72R mutations do not confer general resistance to cisplatin, etoposide, gemcitabine, vinblastine, and taxol. For doxorubicin, cells expressing 249-72R were more resistant than the 249-72P cells. Combined treatment with cisplatin + etoposide resulted in an additive effect in cells expressing most 72R and 72P mutations, except for the 175-72R cells which were refractory to combined treatment. However, combined treatment with cisplatin + gemcitabine resulted in the absence of an additive effect in cells expressing the 273-72R and 282-72R mutants, unlike their 72P counterparts. Nonetheless, all p53 mutants (72R or 72P) equally inhibited p73-mediated transcriptional activity in lung cancer cells, suggesting that the selective resistance conferred by some 72R mutants to certain drugs is probably due to other p73-independent effects of these mutants. Together, the data show that the status of codon 72 polymorphism and p53 mutations can be used as a means for prediction of treatment response, although variables for each cancer type requires detailed evaluation.

Influence of p53 Status on the HSV-Tk/GCV-Induced Bystander Effect in a Panel of Human Ovarian Carcinoma Cell Lines

The p53 protein is mutated in 50% of all human tumors and plays a key role in apoptosis, cell cycle, and the expression of several genes. We investigated if p53 mutations influence the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV)-induced bystander effect (BE). Additionally, we studied some of the underlying mechanisms. GCV sensitivity and BE were studied in a human ovarian carcinoma cell line transfected with an empty vector (A2780/cmv) or a vector containing a p53 hotspot mutation at codon 175 (A2780/m175) or 248 (A2780/m248). In addition, expression levels of two nucleoside analogue transporters, multidrug resistance-related protein 4 and 5 (MRP4/MRP5), were determined. Finally, differences in gap junctional intercellular communication (GJIC) were studied by determining connexin 43 (Cx43) expression and by modulating GJIC with 18-alpha-glycyrrhetinic acid. Our results showed that compared to A2780/cmv, GCV sensitivity was significantly decreased in A2780/m175 and A2780/m248. Additionally, a significant BE (relative increase in cell kill) was found in A2780/cmv and A2780/m248. In contrast, an increased survival was observed in A2780/m175. No MRP4 or MRP5 expression was detected. However, all A2780 cell lines expressed Cx43. Modulating the GJIC significantly decreased the BE in A2780/cmv and A2780/ m248. In conclusion, HSV-tk/GCV-induced BE is influenced by p53 mutations. Differences in GJIC could be one of the underlying mechanisms.

Gain of Function of a p53 Hot Spot Mutation in a Mouse Model of Li-Fraumeni Syndrome

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 ( p53 +/515A ) corresponding to the p53R175H hot spot mutation in human cancers. Although p53 +/515A mice display a similar tumor spectrum and survival curve as p53 +/− mice, tumors from p53 +/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53 515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53 −/− cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53 515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53 515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.

In silico p53 mutation hotspots in lung cancer.

For cancer one of the primary aims of molecular epidemiology is to identify the endogenous or exogenous cause of mutations within a gene. Regarding exogenous mutagens, many mutation data have become available via in vitro and in vivo mutation assays and become publicly available through mutation databases such as the Mammalian Gene Mutation Database (http://lisntweb.swan.ac.uk/cmgt/index.htm). One particular mutation assay incorporates the bacterial supF tRNA gene which allows selection of mutations at virtually all nucleotides. We have developed an algorithm called LwPy53 that utilizes mutation data from supF that can be used to predict chemically induced hot-spots along the p53 gene. The prediction is based on a number of parameters: the mutability of supF dinucleotides after treatment with a mutagen of interest; DNA curvature along the p53 gene; the selectability of a mutation along the gene; the likelihood of a site being within a nucleosome. We applied LwPy53 to exons 5, 7 and 8 of p53 using benzo[a]pyrene diol epoxide (BPDE)-induced mutation data for supF to obtain a predicted BPDE G-->T transversion spectrum after hypothetical treatment with BPDE. The resulting predicted mutation distribution reveals strong mutation hot-spots at codons 157, 248 and 273 that correlate with known BPDE adduct hot-spots within p53. The predicted BPDE spectrum strongly resembles the G-->T mutation spectrum compiled from known lung cancer mutation data from smokers and further supports evidence that BPDE contributes to the overall smoking-related mutation distribution in lung cancer. The algorithm shows how BPDE target sequence specificity and DNA curvature both shape the overall mutation distribution.

P53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1.

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent "hot-spot" p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.

P53 mutations as an identification marker for the clonal origin of bladder tumors and its recurrences

The clonality of synchronous and metachronous bladder tumors has been studied for years with controversial results. Some recent studies support the 'polyclonal origin' hypothesis, i.e. that independently transformed different tumor cell clones exist in the same bladder cancer patient and arise from the field cancerogenisation affecting the entire bladder urothelium by environmental mutagens. Others could demonstrate a monoclonal origin of primary bladder tumors and its recurrences due to a single genetically transformed cell clone spread through the urinary system. With increasing understanding of the clonal origin of bladder tumors and recurrences, clonality markers might contribute to an early and accurate prediction of tumor recurrence and progression. We used p53 mutations as an identification marker permitting the prediction of clonality in bladder tumors and its recurrences. Primary tumors (n=33) and recurrences (n=63) were screened by direct genomic sequencing the p53 mutation hot spot region, exons 5-8. P53 mutations occurred in 12% in our cohort, predominantly in higher malignant (>or=G2), invasive (>or=T1) tumor samples. We were able to demonstrate intratumoral heterogeneity regarding the p53 status and that recurrences may occur from genetically unrelated primary tumor sites. Some of our results argue for a polyclonal origin of synchronous and metachronous bladder tumors possibly due to the field effect in bladder carcinogenesis. Evidence for a monoclonal origin was found in two cases: one case with a high malignant primary tumor and 3 metachronous recurrences, all of them harbouring the same exon 8 mutation found in the primary tumor; one case with identical mutations of exon 8 in the primary and one recurrent tumor. For further implications concerning clonality of recurrent bladder tumors, p53 status should be combined with a broader range of markers such as CGH and LOH pattern.

Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers.

It is estimated that cigarette smoking kills over 1 000 000 people each year by causing lung cancer as well as many other neoplasmas. p53 mutations are frequent in tobacco-related cancers and the mutation load is often higher in cancers from smokers than from nonsmokers. In lung cancers, the p53 mutational patterns are different between smokers and nonsmokers with an excess of G to T transversions in smoking-associated cancers. The prevalence of G to T transversions is 30% in smokers' lung cancer but only 12% in lung cancers of nonsmokers. A similar trend exists, albeit less marked, in laryngeal cancers and in head and neck cancers. This type of mutation is infrequent in most other tumors aside from hepatocellular carcinoma. At several p53 mutational hotspots common to all cancers, such as codons 248 and 273, a large fraction of the mutations are G to T events in lung cancers but are almost exclusively G to A transitions in non-tobacco-related cancers. Two important classes of tobacco smoke carcinogens are the polycyclic aromatic hydrocarbons (PAH) and the nicotine-derived nitrosamines. Recent studies have indicated that there is a strong coincidence of G to T transversion hotspots in lung cancers and sites of preferential formation of PAH adducts along the p53 gene. Endogenously methylated CpG dinucleotides are the preferred sites for G to T transversions, accounting for more than 50% of such mutations in lung tumors. The same dinucleotide, when present within CpG-methylated mutational reporter genes, is the target of G to T transversion hotspots in cells exposed to the model PAH compound benzo[a]pyrene-7,8-diol-9,10-epoxide. As summarized here, a number of other tobacco smoke carcinogens also can cause G to T transversion mutations. The available data suggest that p53 mutations in lung cancers can be attributed to direct DNA damage from cigarette smoke carcinogens rather than to selection of pre-existing endogenous mutations.

A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc mutant mice alters transactivation functions.

A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122-->L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc(-/-)) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122-->L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122-->L allele in p53(-/-) mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122-->L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122-->L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122-->L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122-->L mutant allele in the Xpc(-/-) background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.

Novel gain of function activity of p53 mutants: activation of the dUTPase gene expression leading to resistance to 5-fluorouracil.

Mutated forms of p53 are often expressed in a variety of human tumors. In addition to loss of function of the p53 tumor suppressor, mutant p53s contribute to malignant process by acquisition of novel functions that enhance transformed properties of cells and resistance to anticancer therapy in vitro, and increase tumorigenecity, invasiveness and metastatic ability in vivo. Searching for genes that change expression in response to p53 gain of function mutants may give a clue to the mechanisms underlying their oncogenic effects. Recently by subtraction hybridization cloning we found that the dUTPase gene is transcriptionally upregulated in p53-null mouse fibroblasts expressing the exogenous human tumor-derived His175 p53 mutant. Here we show that conditional expression of His175 and Trp248 hot-spot p53 mutants in p53-negative mouse 10(1) fibroblasts and human SK-OV3 and H1299 tumor cells results in increase in dUTPase gene transcription, an important marker predicting the efficacy of cancer therapy with fluoropyrimidine drugs. Using tetracycline-regulated retroviral vectors for conditional expression of p53 mutants, we found that transcription of the dUTPase gene is increased within 24 h after tetracycline withdrawal, and the cells acquire higher resistance to 5-FU. Additional inactivation of the N-terminal transcription activation domain of mutant p53 (substitutions in amino-acid residues 22 and 23) results in abrogation of both induction of dUTPase transcripts and 5-FU resistance.

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (−/−) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.

Stabilization of the MDM2 Oncoprotein by Mutant p53

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.

Cyclobutane pyrimidine dimers form preferentially at the major p53 mutational hotspot in UVB-induced mouse skin tumors.

The most prevalent DNA lesion induced by UV irradiation is the cyclobutane pyrimidine dimer (CPD) which forms at positions of neighboring pyrimidines. In mouse skin tumors induced by irradiation with UVB (280-320 nm) lamps or solar UV simulators, a major mutational hotspot occurs at codon 270 (Arg-->Cys) involving a sequence change from 5'-TCGT to 5'-TTGT. We have shown previously that CPD formation by UVB or sunlight is enhanced up to 10-fold at 5'-CCG and 5'-TCG sequences due to the presence of 5-methylcytosine bases. Sequence analysis showed that the CpG at codon 270 is methylated in mouse epidermis at a level of approximately 85%. Irradiation of mouse skin or mouse cells in culture produced the strongest CPD signal within exon 8 at the 5'-TCG sequence which is part of codon 270. Time course experiments showed that CPDs at this particular sequence persist longer than at several neighboring positions. The data suggest that formation of CPDs is responsible for induction of the major p53 mutational hotspot in UV-induced mouse skin tumors.