UV induces p21WAF1/CIP1 protein in keratinocytes without p53.
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Cyclin-Dependent Kinase Inhibitor p21↗
Biomedical subjects
D E Brash
Publications and source records attributed to D E Brash.
Induction of cyclin-dependent kinase inhibitors and G(1) prolongation by the chemopreventive agent N-acetylcysteine.
Cyclin-dependent kinase (cdk) inhibitors, such as p16(INK4a) and p21(WAF1/CIP1), often inhibit G(1) cyclin kinases and result in G(1) arrest. It has been suggested that p21(WAF1/CIP1) may also play a role in other chemopreventive activities such as DNA repair, slowdown of DNA replication and induction of cellular differentiation. In this report we demonstrate that the antioxidant N-acetylcysteine (NAC), a well-known chemopreventive agent, induces p16(INK4a) and p21(WAF1/CIP1) gene expression and prolongs cell-cycle transition through G(1) phase. A portion of the G(1) arrest by NAC is governed by p16(INK4a); it is independent of p53. NAC's usual mechanism of increasing intracellular glutathione level is not required for the G(1) arrest. An antioxidant whose action is limited to scavenging radicals, Trolox, does not induce G(1) arrest. Taken together, these results suggest a potential novel molecular basis for chemoprevention by NAC.
Acetylcysteine↗
Antioxidant action via p53-mediated apoptosis.
The biological effects of antioxidants are often considered in terms of their effects on oxygen or lipid radicals. However, antioxidants can also exert their effects through altering the cellular redox potential. Herein, we report that sulfur-containing antioxidants such as N-acetylcysteine and dimercaptopropanol induced apoptosis in several transformed cell lines and transformed primary cultures but not in normal cells. In contrast, chain-breaking antioxidants such as vitamin E lacked this activity. An increased glutathione level was not required for apoptosis; however, all apoptosis-inducing antioxidants elevated the total cellular thiol levels. Antioxidant-induced apoptosis required the p53 tumor suppressor gene. N-Acetylcysteine elevated p53 expression posttranscriptionally by increasing the rate of p53 mRNA translation rather than by altering the protein stability. The p53 induction occurred in normal cells. These observations indicate a redox sensor for p53 induction in vivo, with additional transformation-specific information being required for apoptosis. Manipulating p53-dependent apoptosis with nontoxic antioxidants may have a direct clinical application.
Acetylcysteine↗
Skin precancer.
Squamous cell carcinoma of the skin and melanoma are the rare progeny of precancerous lesions that usually remain stable or regress. For SCC the sequence appears to include TP53 mutant clones in normal skin; dysplasia; carcinoma in situ; and SCC. When such lesions are contiguous, their TP53 mutations are consistent with a single clonal lineage. The set of TP53 mutations in tumours is more restricted than in precancers, suggesting additional selection. Melanoma lies at the end of a continuum including mole, dysplastic naevus, radial growth melanoma and vertical growth. The genetics of melanoma is less clear. Basal cell carcinomas seem to arise without a precancer and contain mutations in TP53 and PTCH. Childhood sunlight exposure directs the location and frequency of precancers. For melanoma, its effects on intermittently exposed body sites are superimposed on the effect at sites chronically exposed. SCC precancers and tumours, BCC tumours and melanoma cell lines contain UV induced mutations. Sun exposed skin of normal individuals contains thousands of small clones of TP53 mutated cells. Predisposition to sunlight induced precancer is a multigenic trait involving factors such as hair and skin color, DNA repair proficiency and mole type and number. These each contribute a relative risk on the order of two to four. Familial predisposition to dysplastic naevi carries a larger risk. The cell of origin for melanoma is uncontroversial, and the proposed hair follicle origin of BCC is consistent with the presence of stem cells in the bulge region. The origin of SCCs and the arrangement of interfollicular stem cell compartments are less clear. Clonal expansion of the initial mutated cell may also be driven by sunlight. When a mutation confers apoptosis resistance, as TP53 mutations do, subsequent UV exposure will be more likely to kill normal cells than mutants. The latter can expand into a clone, only one cell of which need be mutated again. Immunosuppressant drugs may have the same effect as UV, facilitating the clonal expansion of precancers. In the absence of exogenous influences, mutant clones and precancers tend to regress. There is little evidence that regression of precancers is immunological, though regression of melanoma appears to be. The chemotherapeutic agent 5-FU causes regression of dysplasias by removing initiated cells, perhaps by enhancing apoptosis. In contrast, retinoic acid temporarily suppresses clonal expansion. Most sunscreens are mutagenic, with as yet unknown consequences. Mice develop dysplasias and SCCs after UV irradiation. Initiation and clonal expansion of dysplasias is UV driven, but conversion to SCC and subsequent growth involve spontaneous events. With chemical carcinogens mice develop papillomas that usually regress and thus are precancers. Tumour promotion yields abundant low risk papillomas that contain Hras1 mutations but rarely progress to SCC. High risk papillomas are infrequent but do convert to SCC, particularly if re-treated with mutagens. Conversion to SCC is associated with TP53 mutations. The mechanisms of multiple mutation and clonal expansion observed in human and mouse systems, respectively, are beginning to converge into a coherent understanding of precancerous events in skin.
Animals↗
Sunlight and the onset of skin cancer.
The photons of sunlight precipitate a series of genetic events in skin leading to cancer. These events involve somatic mutations as well as inherited alleles. Competition between cell populations ensues, as a single mutated cell expands into a clone. Thus cancer involves both a single-cell problem and a many-cell problem; in skin cancer, sunlight appears to drive both.
Apoptosis↗
Frequent clones of p53-mutated keratinocytes in normal human skin.
The multiple genetic hit model of cancer predicts that normal individuals should have stable populations of cancer-prone, but noncancerous, mutant cells awaiting further genetic hits. We report that whole-mount preparations of human skin contain clonal patches of p53-mutated keratinocytes, arising from the dermal-epidermal junction and from hair follicles. These clones, 60-3000 cells in size, are present at frequencies exceeding 40 cells per cm2 and together involve as much as 4% of the epidermis. In sun-exposed skin, clones are both more frequent and larger than in sun-shielded skin. We conclude that, in addition to being a tumorigenic mutagen, sunlight acts as a tumor promoter by favoring the clonal expansion of p53-mutated cells. These combined actions of sunlight result in normal individuals carrying a substantial burden of keratinocytes predisposed to cancer.
Adult↗
Relationship between sunlight exposure and a key genetic alteration in basal cell carcinoma.
BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common cancer in humans. Epidemiologic studies implicate sunlight exposure as one risk factor, but the limited association between BCCs and UVB radiation (i.e., UV radiation of a wavelength of 280-320 nm) suggests that additional factors must be involved. At the molecular level, not much is known about the role of specific environmental agents in the pathogenesis of BCCs. Point mutations of the types produced by UVB radiation are seen in the p53 gene (also known as TP53; chromosome 17p) of 40%-56% of BCCs. Loss of heterozygosity (LOH) on chromosome 9q22, however, is the most frequent genetic alteration in these tumors, and its causative agent is unknown. PURPOSE: We investigated whether the genetic alteration in chromosome 9 is common to all clinical subtypes of BCCs and whether inactivation of this putative tumor suppressor is related to sunlight exposure. The presence of UVB radiation-related point mutations in the p53 gene was used as an internal control for sunlight exposure to the precursor cells. METHODS: Tumor and blood samples were obtained from skin cancer patients by a surgeon who used Mohs' micrographic surgical technique. Clinical information on each tumor included location, size, histologic, subtype and whether it was primary or recurrent and sporadic or hereditary. Sixty BCCs from 58 patients were evaluated for LOH with 12 polymorphic markers that span chromosome 9. A subset of 18 tumors was evaluated for point mutations in exons 2-11 of the p53 gene, and a subset of 26 tumors was evaluated for LOH by use of a polymorphism in exon 4 of the p53 gene. Associations between tumor characteristics and molecular alterations were tested by a two-tailed chi-squared analysis or a two-tailed Fisher's exact test, depending on sample size. RESULTS: In a clinically diverse series of 47 informative tumors, 32 (68%) showed LOH for chromosome 9q markers, irrespective of histologic characteristics or clinical behavior. Forty-four (94%) of the 47 tumors were from sun-exposed areas of the body, defined as the head and neck in both sexes, shoulders or chest in males, and legs in females. No association was found between chromosome 9q LOH and sunlight exposure, as assessed by either the location of tumors on the body or the presence of UVB radiation-related p53 mutations. Of note, there was a striking difference between the frequency of LOH on chromosome 17p (two [12.5%] of 16 informative tumors) and on chromosome 9q (32 [68%] of 47 informative tumors; P < .001). CONCLUSIONS: Inactivation of a gene on chromosome 9q22 may be a necessary event for basal cell carcinogenesis. The pathogenesis of mutations in this gene may involve factors other than sunlight in a large proportion of tumors. IMPLICATIONS: The limited association between sunlight exposure and BCC incidence may reflect an etiologic contribution of additional environmental agents.
Adult↗
Photobiology. Wrinkles waiting for GoDoT.
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Aging↗
The role of the human homologue of Drosophila patched in sporadic basal cell carcinomas.
Basal cell carcinoma (BCC) is the most common cancer in humans. The majority of sporadic BCCs have allele loss on chromosome 9q22 implying that inactivation of a tumour suppressor in this region is an important step in BCC formation. The gene for nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominant disorder characterized by multiple BCCs, maps to the same region and is presumed to be the tumour suppressor inactivated at this site. NBCCS has been identified recently and encodes a protein with strong homology to the Drosophila segment polarity gene, patched. Analysis of Drosophila mutants indicates that patched interacts with the hedgehog signalling pathway, repressing the expression of various hedgehog target genes including wingless, decapentaplegic and patched itself. Using single strand conformational polymorphism (SSCP) to screen human patched in 37 sporadic BCCs, we detected mutations in one-third of the tumours. Direct sequencing of two BCCs without SSCP variants revealed mutations in those tumours as well suggesting that inactivation of patched is probably a necessary step in BCC development. Northern blots and RNA in situ hybridization showed that patched is expressed at high levels in tumour cells but not normal skin suggesting that mutational inactivation of the gene leads to overexpression of mutant transcript owing to failure of a negative feedback mechanism.
Animals↗
Tumor suppressor gene mutations and photocarcinogenesis.
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Apoptosis↗
Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.
Sunlight is a carcinogen to which everyone is exposed. Epidemiology indicates that most carcinogenic sunlight exposure takes place several decades before the tumor arises. Some of the early events have been identified by searching for genes having ultraviolet (UV)-specific mutations. Over 90% of squamous cell carcinomas and more than 50% of basal cell carcinomas from New England patients contain UV-like mutations in the p53 tumor suppressor gene. From the mutation pattern, it can be concluded that the carcinogenic DNA lesions were pyrimidine-cytosine photoproducts caused by the UVB portion of sunlight. Particular codons of the p53 gene are most susceptible, apparently because of slower DNA repair at specific sites. Sunlight is sufficiently mutagenic often to mutate both p53 alleles. These mutations are also found in the precancer for squamous cell carcinoma, actinic keratosis, implying an early role. The function of p53 in normal skin is indicated by the observation that inactivating p53 in mouse skin reduces the appearance of sunburn cells, apoptotic keratinocytes generated by UV overexposure. Skin thus appears to possess a p53-dependent "cellular proofreading" response to DNA damage in which precancerous cells self-destruct. If this response is reduced in a single cell by a prior p53 mutation, sunburn can thereafter select for clonal expansion of the p53-mutated cell into an actinic keratosis. Sunlight appears to act twice: as tumor initiator and as tumor promoter.
Animals↗
Intragenic domains of strand-specific repair in Escherichia coli.
Heterogeneity of DNA repair has been observed at different levels of genomic organization, including chromatin domains, expressed genes and DNA strands. If heterogeneity also existed intragenically, it could reveal fine details of the excision repair mechanism in vivo. Here we measure the frequency of UV-induced cyclobutane pyrimidine dimers at individual nucleotides within defined portions of two Escherichia coli genes, lacl and lacZ, at various times after irradiation. Two domains of differential repair rates were apparent, with repair being slow at nucleotides adjacent to the transcription start sites. In lacZ, the domain of faster repair began 32 bases downstream of the transcription start site and required the mfd gene. Since mfd codes for a transcription-repair coupling factor, this transcription-coupled repair system evidently becomes operative downstream of the initiation complex region in vivo. Unexpectedly, however, (1) an mfd mutation reduced repair in the downstream domain even when transcription was at a very low level and (2) induction of lacZ transcription with isopropyl-beta-D-thiogalactoside overcame this reduction. Evidently, the Mfd transcription-repair coupling factor is required for basal levels of strand-specific repair in this gene, but induced levels of repair are related to transcription through another mechanism.
Bacterial Proteins↗
Local recurrence versus new primary: clinical analysis of 82 breast relapses and potential applications for genetic fingerprinting.
PURPOSE: The purpose of this study was to perform a detailed clinical pathological analysis of breast relapses in patients treated with conservative surgery and radiation therapy in an effort to classify those relapses as true local recurrences or second primary tumors, and to assess the prognostic and therapeutic implications of such a classification system. METHODS AND MATERIALS: Of 990 patients treated with conservative surgery and radiation therapy at our facilities prior to December 1987, 82 patients have experienced a relapse in the conservatively treated breast as the primary site of failure. Patients were classified as having new primary tumors if they fulfilled any one of the following criteria: a) breast relapse occurring at a site distinctly removed from the original tumor; b) histology of the breast relapse compared with the original tumor consistent with a new primary; or c) DNA flow cytometry converting from an aneuploid primary to a diploid relapse. RESULTS: As of 2/92, with a median follow-up of 5.4 years from the time of breast relapse, the overall 5-year survival rate following breast relapse was 55%. Forty-seven patients were classified as true recurrences and 33 patients were classified as new primaries. Patients classified as true recurrences had a shorter median time to breast relapse than patients classified as new primaries (3.16 years vs. 5.42 years, p < .05) and an inferior post breast recurrence survival rate compared to patients classified as new primaries (36% vs. 89%, p < .05). Residual disease outside of the recurrent tumor bed was also noted to be more frequent in patients classified as true recurrences compared to patients classified as new primaries (48% vs. 16%, p < .05). CONCLUSION: Based on the clinical and pathological criteria outlined, it appears that a significant portion of patients experiencing a relapse in the conservatively treated breast may have new primary tumors as opposed to true local relapses. Distinction between a true recurrence and a new primary tumor may have significant prognostic implications. Uncertainties associated with the clinical and pathological criteria are presented and further investigations with genetic fingerprinting techniques to establish the clonality of breast relapses are presented and discussed.
Adult↗
Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.
To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lacI gene, the cyclobutane pyrimidine dimer frequency was 2- to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UV-induced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mfd strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min. Wild-type E. coli's bias for producing mutations at photoproducts on the nontranscribed strand, reported to require the mfd gene, therefore appears to be due to an excision repair system specific for the transcribed strand of chromosomal DNA.
Base Sequence↗
Status of the p53 tumor suppressor gene in human squamous carcinoma cell lines.
Dominant-negative and/or loss-of-function mutations of the p53 tumor suppressor gene are frequently found in squamous cell carcinomas of the skin and of the head-and-neck region. In order to identify the precise mechanisms of inactivation of p53 in tumors of this class, we examined the status of p53 RNA, protein and DNA in a panel of eight human squamous carcinoma cell lines (head-and-neck, 3; esophagus, 1; lung, 1; uterine cervix, 2; vulva, 1). Three lines (A253, CaLu-1, SqCC/Y1) failed to express any p53 mRNA. A253 cells contained a single p53 allele without mutations in exons 2-9, suggesting that the lack of transcription was the result of mutations in the regulatory region of the gene. Both p53 alleles were deleted in CaLu-1 cells, whereas the single allele present in SqCC/Y1 cells was rearranged and carried two missense mutations in exon 5. Two cell lines (A431, FaDu) expressed only 50% of the normal level of p53 mRNA, either because only one allele was present (A431), or because only one of the two alleles was transcribed (FaDu). The two cervical carcinoma lines (CaSki, C4-1) expressed normal levels of p53 mRNA, but no wild type protein, presumably as a result of accelerated degradation by the human papillomavirus 16 or -18 E6 oncoprotein present in these cells as previously described (Scheffner et al., Proc. Natl. Acad. Sci. USA 88:5523-5527; 1991). Three of the lines expressed only mutant p53 protein (A431, FaDu, CE-48) resulting from missense mutations in codons 248 and 273.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.
Sunlight is a carcinogen to which everyone is exposed. Its UV component is the major epidemiologic risk factor for squamous cell carcinoma of the skin. Of the multiple steps in tumor progression, those that are sunlight-related would be revealed if they contained mutations specific to UV. In a series of New England and Swedish patients, we find that 14/24 (58%) of invasive squamous cell carcinomas of the skin contain mutations in the p53 tumor suppressor gene, each altering the amino acid sequence. Involvement of UV light in these p53 mutations is indicated by the presence in three of the tumors of a CC----TT double-base change, which is only known to be induced by UV. UV is also implicated by a UV-like occurrence of mutations exclusively at dipyrimidine sites, including a high frequency of C----T substitutions. p53 mutations in internal malignancies do not show these UV-specific mutations. The dipyrimidine specificity also implicates dipyrimidine photoproducts containing cytosine as oncogenic photoproducts. We believe these results identify a carcinogen-related step in a gene involved in the subsequent human cancer.
Aged↗