[Intrinsic factors, genes, and skin aging]. / Endogene Faktoren, Gene und Hautalterung.

Skin aging is determined by a combination of endogenous and environmental influences, including epigenetic, posttranslational, microbial, and lifestyle factors. In particular genetic changes, programmed or not, play a pivotal role and understanding of these complex mechanisms may contribute to the prevention of age-related diseases and extension of healthy lifespan. In this article, new knowledge about genes and biological processes that can significantly affect skin homeostasis in old age and can lead to the typical morphological and physiological characteristics of aging skin are summarized.

A new verdict for an old convict.

Certain human cancers and carcinogen-induced rodent tumors commonly contain Kras2 mutations. This activated form of ras has always been described as a dominant oncogene. A new study indicates that wildtype Kras2 has properties of a tumor suppressor gene and may have the capacity to reduce the transforming potential of oncogenically activated ras.

Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3.

Allelic loss at the short arm of chromosome 3 is one of the most common and earliest events in the pathogenesis of lung cancer, and is observed in more than 90% of small-cell lung cancers (SCLCs) and in 50-80% of non-small-cell lung cancers (NSCLCs). Frequent and early loss of heterozygosity and the presence of homozygous deletions suggested a critical role of the region 3p21.3 in tumorigenesis and a region of common homozygous deletion in 3p21.3 was narrowed to 120 kb (ref. 5). Several putative tumour-suppressor genes located at 3p21 have been characterized, but none of these genes appear to be altered in lung cancer. Here we describe the cloning and characterization of a human RAS effector homologue (RASSF1) located in the 120-kb region of minimal homozygous deletion. We identified three transcripts, A, B and C, derived from alternative splicing and promoter usage. The major transcripts A and C were expressed in all normal tissues. Transcript A was missing in all SCLC cell lines analysed and in several other cancer cell lines. Loss of expression was correlated with methylation of the CpG-island promoter sequence of RASSF1A. The promoter was highly methylated in 24 of 60 (40%) primary lung tumours, and 4 of 41 tumours analysed carried missense mutations. Re-expression of transcript A in lung carcinoma cells reduced colony formation, suppressed anchorage-independent growth and inhibited tumour formation in nude mice. These characteristics indicate a potential role for RASSF1A as a lung tumour suppressor gene.

Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer.

Ultraviolet light has been linked with the development of human skin cancers. Such cancers often exhibit mutations in the p53 tumor suppressor gene. Ligation-mediated polymerase chain reaction was used to analyze at nucleotide resolution the repair of cyclobutane pyrimidine dimers along the p53 gene in ultraviolet-irradiated human fibroblasts. Repair rates at individual nucleotides were highly variable and sequence-dependent. Slow repair was seen at seven of eight positions frequently mutated in skin cancer, suggesting that repair efficiency may strongly contribute to the mutation spectrum in a cancer-associated gene.

Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53.

Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

Genomic sequencing and methylation analysis by ligation mediated PCR.

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.

Frequent epigenetic inactivation of cystatin M in breast carcinoma.

Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fisher's exact test). Treatment of breast cancer cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.

Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function.

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 (Igf2) gene and the closely linked H19 gene are regulated by an intervening 2 kb imprinting control region (ICR), which displays parentspecific differential DNA methylation [1] [2]. Four 21 bp repeats are embedded within the ICR and are conserved in the putative ICR of human and rat Igf2 and H19, suggesting that the repeats have a function [3] [4]. Here, we report that prominent DNA footprints were found in vivo on the unmethylated maternal ICR at all four 21 bp repeats, demonstrating the presence of protein binding. The methylated paternal ICR displayed no footprints. Significantly, the maternal-specific footprints were localized to putative binding sites for CTCF, a highly conserved zinc-finger DNA-binding protein with multiple roles in gene regulation including that of chromatin insulator function [5] [6]. These results strongly suggest that the maternal ICR functions as an insulator element in regulating mutually exclusive expression of Igf2 and H19 in cis.

X-chromosome inactivation and cell memory.

Mammalian X-chromosome inactivation is an excellent example of the faithful maintenance of a determined chromosomal state. As such, it may provide insight into the mechanisms for cell memory, defined as the faithful maintenance of a determined state in clonally derived progeny cells. We review here the aspects of X-chromosome inactivation that are relevant to cell memory and discuss the various molecular mechanisms that have been proposed to explain its occurrence, with emphasis on DNA methylation and a recently proposed mechanism that depends on the timing of replication.

Mutagenesis at methylated CpG sequences.

5-Methylcytosine in DNA is genetically unstable. Methylated CpG (mCpG) sequences frequently undergo mutation resulting in a general depletion of this dinucleotide sequence in mammalian genomes. In human genetic disease- and cancer-relevant genes, mCpG sequences are mutational hotspots. It is an almost universally accepted dogma that these mutations are caused by random deamination of 5-methylcytosines. However, it is plausible that mCpG transitions are not caused simply by spontaneous deamination of 5-methylcytosine in double-stranded DNA but by other processes including, for example, mCpG-specific base modification by endogenous or exogenous mutagens or, alternatively, by secondary factors operating at mCpG sequences and promoting deamination. We also discuss that mCpG sequences are favored targets for specific exogenous mutagens and carcinogens. When adjacent to another pyrimidine, 5-methylcytosine preferentially undergoes sunlight-induced pyrimidine dimer formation. Certain polycyclic aromatic hydrocarbons form guanine adducts and induce G to T transversion mutations with high selectivity at mCpG sequences.

In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response.

Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.

Lack of gene- and strand-specific DNA repair in RNA polymerase III-transcribed human tRNA genes.

UV light induces DNA lesions which are removed by nucleotide excision repair. Genes transcribed by RNA polymerase II are repaired faster than the flanking chromatin, and the transcribed strand is repaired faster than the coding strand. Transcription-coupled repair is not seen in RNA polymerase I-transcribed human rRNA genes. Since repair of genes transcribed by RNA polymerase III has not been analyzed before, we investigated DNA repair of tRNA genes after irradiation of human fibroblasts with UVC. We studied the repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution by ligation-mediated PCR. A single-copy gene encoding selenocysteine tRNA, a tRNA valine gene, and their flanking sequences were analyzed. Protein-DNA footprinting showed that both genes were occupied by regulatory factors in vivo, and Northern blotting and nuclear run-on analysis of the tRNA indicated that these genes were actively transcribed. We found that both genes were repaired slower than RNA polymerase II-transcribed genes. No major difference between repair of the transcribed and the coding DNA strands was detected. Transcribed sequences of the tRNA genes were not repaired faster than flanking sequences. Indeed, several sequence positions in the 5' flanking region of the tRNA(Val) gene were repaired more efficiently than the gene itself. These results indicate that unlike RNA polymerase II, RNA polymerase III has no stimulatory effect on DNA repair. Since tRNA genes are covered by the regulatory factor TFIIIC and RNA polymerase III, these proteins may actually inhibit the DNA's accessibility to repair enzymes.

In vivo structure of the human cdc2 promoter: release of a p130-E2F-4 complex from sequences immediately upstream of the transcription initiation site coincides with induction of cdc2 expression.

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of cells to reenter the cell cycle results in induction of cdc2 expression, beginning at the G1-to-S transition and reaching maximum levels during late S and G2 phases. To investigate cdc2 transcriptional regulation throughout cell cycle progression, we monitored protein-DNA interactions by in vivo footprinting along 800 bp of the human cdc2 promoter in quiescent fibroblasts and at different time points following serum stimulation. We found 11 in vivo protein-binding sites, but no protein binding was observed at a high-affinity E2F site that had previously been implicated in cdc2 regulation. Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Sp1 sites, and one ets-2 site) bind transcription factors constitutively throughout the cell cycle. However, at two elements located at positions -60 and -20 relative to the transcription start site, the binding pattern changes significantly as the cells are entering S phase. A G0- and G1-specific protein complex disappears at the -20 element at the beginning of S phase. This sequence deviates at one base position from known E2F consensus binding sites. We found that the major E2F activity in human fibroblasts contains E2F-4 and p130. The -20 element of the cdc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G0 cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutant cdc2 promoter constructs indicate that the -20 element is involved in suppressing cdc2 activity in quiescent cells. We suggest that the presence of the p130-E2F-4 complex in G0/G1 blocks access of components of the basal transcription machinery or prevents transaction by the constitutively bound upstream activator proteins.

Characterization of novel parent-specific epigenetic modifications upstream of the imprinted mouse H19 gene.

Genomic imprinting results in parent-specific monoallelic expression of a small number of genes in mammals. The identity of imprints is unknown, but much evidence points to a role for DNA methylation. The maternal alleles of the imprinted H19 gene are active and hypomethylated; the paternal alleles are inactive and hypermethylated. Roles for other epigenetic modifications are suggested by allele-specific differences in nuclease hypersensitivity at particular sites. To further analyze the possible epigenetic mechanisms determining monoallelic expression of H19, we have conducted in vivo dimethylsulfate and DNase I footprinting of regions upstream of the coding sequence in parthenogenetic and androgenetic embryonic stem cells. These cells carry only maternally and paternally derived alleles, respectively. We observed the presence of maternal-allele-specific dimethylsulfate and DNase I footprints at the promoter indicative of protein-DNA interactions at a CCAAT box and at binding sites for transcription factors Sp1 and AP-2. Also, at the boundary of a region further upstream for which existent differential methylation has been suggested to constitute an imprint, we observed a number of strand-specific dimethylsulfate reactivity differences specific to the maternal allele, along with an unusual chromatin structure in that both strands of maternally derived DNA were strongly hypersensitive to DNase I cutting over a distance of 100 nucleotides. We therefore reveal the existence of novel parent-specific epigenetic modifications, which in addition to DNA methylation, could constitute imprints or maintain monoallelic expression of H19.

Binding of transcription factors creates hot spots for UV photoproducts in vivo.

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

Genome-wide DNA methylation profiling reveals cancer-associated changes within early colonic neoplasia.

Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study, we have combined laser-capture microdissection with reduced representation bisulfite sequencing to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2. Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. Although aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Altogether, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

Cell cycle-independent removal of UV-induced pyrimidine dimers from the promoter and the transcription initiation domain of the human CDC2 gene.

To assess whether removal of UV-induced cyclobutane pyrimidine dimers (CPDs) occurs with equal efficiency at different stages of the cell cycle in a cell cycle-regulated gene, we have analyzed repair of CPDs, following a single dose of UV, in normal human fibroblasts that were synchronized in either G(0) or S phase. Based on a single nucleotide resolution analysis, we established a detailed map of DNA repair rates along the promoter region and the transcription initiation area of the human CDC2 gene. The promoter of this gene is covered by an array of sequence-specific transcription factors located between nt -280 and -9 relative to the major transcription start site. In both quiescent and S phase-synchronized fibroblasts the majority of these sequences were poorly repaired even after 24 h, probably as a result of the constitutive binding of transcription factors throughout the cell cycle. A domain of fast repair was found at sequences surrounding the transcription initiation site and continuing downstream for approximately 80 nt. CPD removal from this domain was preferential in both quiescent and proliferating fibroblasts, despite lower levels of global genome repair and a lack of CDC2 transcription in quiescent cells. We suggest that sequences involved in transcription initiation may be book-marked for efficient repair throughout the cell cycle, even when the gene is temporarily not expressed.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing.

We describe a fluorescence-based directed termination PCR (fluorescent DT-PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT-PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT-PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT-PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT-PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in combustion products of organic matter, including cigarette smoke. Metabolically activated diol epoxides of these compounds, including benzo[a]pyrene diol epoxide (B[a]PDE), have been suggested as causative agents in the development of lung cancer. We previously mapped the distribution of B[a]PDE adducts within the p53 tumor suppressor gene (also known as TP53), which is mutated in 60% of human lung cancers, and found that B[a]PDE adducts preferentially form at lung cancer mutational hotspots (codons 154, 157, 158, 245, 248, and 273). Other PAHs may be important in lung cancer as well. METHODS: Here we have mapped the distribution of adducts induced by diol epoxides of additional PAHs: chrysene (CDE), 5-methylchrysene (5-MCDE), 6-methylchrysene (6-MCDE), benzo[c]phenanthrene (B[c]PDE), and benzo[g]chrysene (B[g]CDE) within exons 5, 7, and 8 of the p53 gene in human bronchial epithelial cells. RESULTS: CDE exposure produced only low levels of adducts. Exposure of cells to the other activated PAHs resulted in DNA damage patterns similar to those previously observed with B[a]PDE but with some distinct differences. 5-MCDE, 6-MCDE, B[g]CDE, and B[c]PDE efficiently induced adducts at guanines within codons 154, 156, 157, 158, and 159 of exon 5, codons 237, 245 and 248 of exon 7, and codon 273 of exon 8, but the relative levels of adducts at each site varied for each compound. B[g]CDE, B[c]PDE, and 5-MCDE induced damage at codon 158 more selectively than 6-MCDE or B[a]PDE. The sites most strongly involved in PAH adduct formation were also the sites of highest mutation frequency (codons 157, 158, 245, 248, and 273). CONCLUSION: The data suggest that PAHs contribute to the mutational spectrum in human lung cancer.

Hypermethylation of the cpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers.

The human Ras association domain family 1A gene (RASSF1A), recently cloned from the lung tumor suppressor locus 3p21.3, was shown to be hypermethylated in primary lung tumors, and reexpression of RASSF1A suppressed the growth of lung cancer cells (R. Dammann et al., Nat. Genet., 25: 315-319, 2000). In this study, we analyzed the expression and possible alterations of RASSF1A in breast cancer. In five breast cancer cell lines (MCF7, MDAMB157, MDAMB231, T47D, and ZR75-1), the CpG island and promoter of RASSF1A was completely methylated, and transcription was silenced. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the expression of RASSF1A. In 28 of 45 (62%) primary mammary carcinomas, the promoter of RASSF1A was highly methylated at its CpG sites. Coincident with methylation, the expression level of RASSF1A was lower in tumors compared with matching normal tissues. No somatic mutations were found in the samples that were unmethylated. The data suggest that hypermethylation of the CpG island promoter of RASSF1A may play an important role in breast cancer pathogenesis.