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The opossum (Monodelphis domestica), with its sequenced genome, ease of laboratory care and experimental manipulation, and unique biology, is the most used laboratory marsupial. Using the mammalian methylation array, we generated DNA methylation data from n = 100 opossum samples from the ear, liver, and tail. We contrasted postnatal development and later aging effects in the opossum methylome with those in mouse (Mus musculus, C57BL/6 J strain) and other marsupial species such as Tasmanian devil, kangaroos, and wallabies. While the opossum methylome is similar to that of mouse during postnatal development, it is distinct from that shared by other mammals when it comes to the age-related gain of methylation at target sites of polycomb repressive complex 2. Our immunohistochemical staining results provide additional support for the hypothesis that PRC2 activity increases with later aging in mouse tissues but remains constant in opossum tissues. We present several epigenetic clocks for opossums that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (opossum and human). Two dual-species human-opossum pan-tissue clocks accurately measure chronological age and relative age, respectively. The human-opossum epigenetic clocks are expected to provide a significant boost to the attractiveness of opossum as a biological model. Additional epigenetic clocks for Tasmanian devil, red kangaroos and other species of the genus Macropus may aid species conservation efforts.

Naked mole rats (NMRs) live an exceptionally long life, appear not to exhibit age-related decline in physiological capacity and are resistant to age-related diseases. However, it has been unknown whether NMRs also evade aging according to a primary hallmark of aging: epigenetic changes. To address this question, we profiled n = 385 samples from 11 tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). We observed strong epigenetic aging effects and developed seven highly accurate epigenetic clocks for several tissues (pan-tissue, blood, kidney, liver, skin clocks) and two dual-species (human–NMR) clocks. The skin clock correctly estimated induced pluripotent stem cells derived from NMR fibroblasts to be of prenatal age. The NMR epigenetic clocks revealed that breeding NMR queens age more slowly than nonbreeders, a feature that is also observed in some eusocial insects. Our results show that despite a phenotype of negligible senescence, the NMR ages epigenetically.

DNA-methylation profiles have been used successfully to develop highly accurate biomarkers of age, epigenetic clocks, for many species. Using a custom methylation array, we generated DNA methylation data from n = 238 porcine tissues including blood, bladder, frontal cortex, kidney, liver, and lung, from domestic pigs (Sus scrofa domesticus) and minipigs (Wisconsin Miniature Swine™). Samples used in this study originated from Large White X Landrace crossbred pigs, Large White X Minnesota minipig crossbred pigs, and Wisconsin Miniature Swine™. We present 4 epigenetic clocks for pigs that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (pig and human). Two dual-species human-pig pan-tissue clocks accurately measure chronological age and relative age, respectively. We also characterized CpGs that differ between minipigs and domestic pigs. Strikingly, several genes implicated by our epigenetic studies of minipig status overlap with genes (ADCY3, TFAP2B, SKOR1, and GPR61) implicated by genetic studies of body mass index in humans. In addition, CpGs with different levels of methylation between the two pig breeds were identified proximal to genes involved in blood LDL levels and cholesterol synthesis, of particular interest given the minipig’s increased susceptibility to cardiovascular disease compared to domestic pigs. Thus, breed-specific differences of domestic and minipigs may potentially help to identify biological mechanisms underlying weight gain and aging-associated diseases. Our porcine clocks are expected to be useful for elucidating the role of epigenetics in aging and obesity, and the testing of anti-aging interventions. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00439-6.

Human DNA methylation profiles have been used successfully to develop highly accurate biomarkers of aging (“epigenetic clocks”). Although these human epigenetic clocks are not immediately applicable to all species of the animal kingdom, the principles underpinning them appear to be conserved even in animals that are evolutionarily far removed from humans. This is exemplified by recent development of epigenetic clocks for mice and other mammalian species. Here, we describe epigenetic clocks for the domestic cat (Felis catus), based on methylation profiles of CpGs with flanking DNA sequences that are highly conserved between multiple mammalian species. Methylation levels of these CpGs are measured using a custom-designed Infinium array (HorvathMammalMethylChip40). From these, we present 3 epigenetic clocks for cats; of which, one applies only to blood samples from cats, while the remaining two dual-species human-cat clocks apply both to cats and humans. We demonstrate that these domestic cat clocks also lead to high age correlations in cheetahs, tigers, and lions. It is expected that these epigenetic clocks for cats possess the potential to be further developed for monitoring feline health as well as being used for identifying and validating anti-aging interventions. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00445-8.

DNA methylation profiles have been used to develop biomarkers of aging known as epigenetic clocks, which predict chronological age with remarkable accuracy and show promise for inferring health status as an indicator of biological age. Epigenetic clocks were first built to monitor human aging, but their underlying principles appear to be evolutionarily conserved, as they have now been successfully developed for many mammalian species. Here, we describe reliable and highly accurate epigenetic clocks shown to apply to 93 domestic dog breeds. The methylation profiles were generated using the mammalian methylation array, which utilizes DNA sequences that are conserved across all mammalian species. Canine epigenetic clocks were constructed to estimate age and also average time to death. We also present two highly accurate human–dog dual species epigenetic clocks (R = 0.97), which may facilitate the ready translation from canine to human use (or vice versa) of antiaging treatments being developed for longevity and preventive medicine. Finally, epigenome-wide association studies here reveal individual methylation sites that may underlie the inverse relationship between breed weight and lifespan. Overall, we describe robust biomarkers to measure aging and, potentially, health status in canines.

It has been noted for quite some time that DNA methylation levels decline with age. The significance of this change remained unknown until it became possible to measure methylation status of specific sites on the DNA. It was observed that while the methylation of some sites does indeed decrease with age, that of others increase or remain unchanged. The application of machine learning methods to these quantitative changes in multiple sites, allowed the generation of a highly accurate estimator of age, called the epigenetic clock. The application of this clock on large human epidemiological data sets revealed that discordance between the predicted (epigenetic age) and chronological age is associated with many age-related pathologies, particularly when the former is greater than the latter. The epigenetic clock clearly captures to some degree, biological features that accompany the ageing process. Despite the ever-increasing scope of pathologies that are found to be associated with accelerated epigenetic ageing, the basic principles that underlie the ticking of the clock remain elusive. Here, we describe the known molecular and cellular attributes of the clock and consider their properties, and proffer opinions as to how they may be connected and what might be the underlying mechanism. Emerging from these considerations is the inescapable view that epigenetic ageing begins from very early moments after the embryonic stem cell stage and continues un-interrupted through the entire life-course. This appears to be a consequence of processes that are necessary for the development of the organism from conception and to maintain it thereafter through homeostasis. Hence, while the speed of ageing can, and is affected by external factors, the essence of the ageing process itself is an integral part of, and the consequence of the development of life.Impact statementThe field of epigenetic ageing is relatively new, and the speed of its expansion presents a challenge in keeping abreast with new discoveries and their implications. Several reviews have already addressed the great number of pathologies, health conditions, life-style, and external stressors that are associated with changes to the rate of epigenetic ageing. While these associations highlight and affirm the ability of epigenetic clock to capture biologically meaningful changes associated with age, they do not inform us about the underlying mechanisms. In this very early period since the development of the clock, there have been rather limited experimental research that are aimed at uncovering the mechanism. Hence, the perspective that we proffer is derived from available but nevertheless limited lines of evidence that together provide a seemingly coherent narrative that can be tested. This, we believe would be helpful towards uncovering the workings of the epigenetic clock.

Evidence suggests that exposure to the UV-A radiation can liberate nitric oxide from skin cells eliciting vasodilation in-vivo. However, the duration of nitric oxide release in skin cells after UV exposure is not well studied, with emphasis on UV-B mediated iNOS upregulation. The current study demonstrated persistence of nitric oxide release in a dark reaction after moderate UV-A exposure, peaking around 48h post exposure; this effect was shown in keratinocytes, fibroblasts and endothelial cells of both neonatal and aged donors and confirmed the hypothesis that UV-A exposure appeared to upregulate cNOS alongside iNOS. Release of nitric oxide in the skin cells induced by a moderate exposure to UV-A in sunlight may be especially beneficial for some demographic groups such as the elderly, hypertensive patients or those with impaired nitric oxide function, not only during exposure but many hours and days after that.

Cytosine methylation patterns have not yet been thoroughly studied in horses. Here, we profile n = 333 samples from 42 horse tissue types at loci that are highly conserved between mammalian species using a custom array (HorvathMammalMethylChip40). Using the blood and liver tissues from horses, we develop five epigenetic aging clocks: a multi-tissue clock, a blood clock, a liver clock and two dual-species clocks that apply to both horses and humans. In addition, using blood methylation data from three additional equid species (plains zebra, Grevy’s zebras and Somali asses), we develop another clock that applies across all equid species. Castration does not significantly impact the epigenetic aging rate of blood or liver samples from horses. Methylation and RNA data from the same tissues define the relationship between methylation and RNA expression across horse tissues. We expect that the multi-tissue atlas will become a valuable resource., Methylation levels of specific sites in the genome is correlated with aging. Here the authors develop a human-horse clock which could assist in translating anti-aging interventions from humans to horses and vice versa.

The opossum (Monodelphis domestica), with its sequenced genome, ease of laboratory care and experimental manipulation, and unique biology, is the most used laboratory marsupial. Using the mammalian methylation array, we generated DNA methylation data from n=100 opossum tissues including blood, liver, and tail. We contrast age-related changes in the opossum methylome to those of C57BL/6J mice. We present several epigenetic clocks for opossums that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (opossum and human). Two dual-species human-opossum pan-tissue clocks accurately measure chronological age and relative age, respectively. These human-opossum epigenetic clocks are expected to provide a significant boost to the attractiveness of opossum as a biological model.

Human DNA methylation data have previously been used to develop highly accurate biomarkers of aging (“epigenetic clocks”). Subsequent studies demonstrate that similar epigenetic clocks can also be developed for mice and many other mammals. Here, we describe epigenetic clocks for common marmosets (Callithrix jacchus) based on novel DNA methylation data generated from highly conserved mammalian CpGs that were profiled using a custom Infinium array (HorvathMammalMethylChip40). From these, we developed and present here two epigenetic clocks for marmosets that are applicable to whole blood samples. We find that the human-marmoset clock for relative age exhibits moderately high age correlations in two other non-human primate species: vervet monkeys and rhesus macaques. In a separate cohort of marmosets, we tested whether intervention with rapamycin, a drug shown to extend lifespan in mice, would alter the epigenetic age of marmosets, as measured by the marmoset epigenetic clocks. These clocks did not detect significant effects of rapamycin on the epigenetic age of marmoset blood. The common marmoset stands out from other mammals in that it is not possible to build accurate estimators of sex based on DNA methylation data: the accuracy of a random forest predictor of sex (66%) was substantially lower than that observed for other mammals (which is close to 100%). Overall, the epigenetic clocks developed here for the common marmoset are expected to be useful for age estimation of wild-born animals and for anti-aging studies in this species. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00438-7.

Age‐associated DNA‐methylation profiles have been used successfully to develop highly accurate biomarkers of age ("epigenetic clocks") in humans, mice, dogs, and other species. Here we present epigenetic clocks for African and Asian elephants. These clocks were developed using novel DNA methylation profiles of 140 elephant blood samples of known age, at loci that are highly conserved between mammalian species, using a custom Infinium array (HorvathMammalMethylChip40). We present epigenetic clocks for Asian elephants (Elephas maximus), African elephants (Loxodonta africana), and both elephant species combined. Two additional human‐elephant clocks were constructed by combining human and elephant samples. Epigenome‐wide association studies identified elephant age‐related CpGs and their proximal genes. The products of these genes play important roles in cellular differentiation, organismal development, metabolism, and circadian rhythms. Intracellular events observed to change with age included the methylation of bivalent chromatin domains, and targets of polycomb repressive complexes. These readily available epigenetic clocks can be used for elephant conservation efforts where accurate estimates of age are needed to predict demographic trends., DNA methylation aging clocks for African and Asian elephants. Some of these clocks apply to humans as well. Parts of the graphic were created by Hollis Burbank‐Hammerlund, Sandra Oviedo, and BioRender.com.

Maximum lifespan of a species is the oldest that individuals can survive, reflecting the genetic limit of longevity in an ideal environment. Here we report methylation-based models that accurately predict maximum lifespan (r=0.89), gestational time (r=0.96), and age at sexual maturity (r=0.87), using cytosine methylation patterns collected from over 12,000 samples derived from 192 mammalian species. Our epigenetic maximum lifespan predictor corroborated the extended lifespan in growth hormone receptor knockout mice and rapamycin treated mice. Across dog breeds, epigenetic maximum lifespan correlates positively with breed lifespan but negatively with breed size. Lifespan-related cytosines are located in transcriptional regulatory regions, such as bivalent chromatin promoters and polycomb-repressed regions, which were hypomethylated in long-lived species. The epigenetic estimators of maximum lifespan and other life history traits will be useful for characterizing understudied species and for identifying interventions that extend lifespan.

DNA methylation profiles have been used to develop biomarkers of aging known as epigenetic clocks, which predict chronological age with remarkable accuracy and show promise for inferring health status as an indicator of biological age. Epigenetic clocks were first built to monitor human aging but the principles underpinning them appear to be evolutionarily conserved, as they have now been successfully developed for over 120 mammalian species. Here we describe reliable and highly accurate epigenetic clocks shown to apply to 51 domestic dog breeds. The methylation profiles were generated using a custom array with DNA sequences that are conserved across all mammalian species (HorvathMammalMethylChip40). Canine epigenetic clocks were constructed to estimate age and also predict mortality risk by estimating average time-to-death. We also present two highly accurate human-dog dual species epigenetic clocks (R=0.97), which may facilitate the ready translation from canine to human use (or vice versa) of anti-aging treatments being developed for longevity and preventive medicine. Finally, epigenome-wide association studies herein reveal individual methylation sites that may underlie the inverse relationship between breed weight and lifespan. Overall, we describe robust biomarkers to measure aging and potentially health status in canines.

SummaryEpigenetics has hitherto been studied and understood largely at the level of individual organisms. Here, we report a multi-faceted investigation of DNA methylation across 11,117 samples from 176 different species. We performed an unbiased clustering of individual cytosines into 55 modules and identified 31 modules related to primary traits including age, species lifespan, sex, adult species weight, tissue type and phylogenetic order. Analysis of the correlation between DNA methylation and species allowed us to construct phyloepigenetic trees for different tissues that parallel the phylogenetic tree. In addition, while some stable cytosines reflect phylogenetic signatures, others relate to age and lifespan, and in many cases responding to anti-aging interventions in mice such as caloric restriction and ablation of growth hormone receptors. Insights uncovered by this investigation have important implications for our understanding of the role of epigenetics in mammalian evolution, aging and lifespan.

Naked mole-rats (NMRs) live an exceptionally long life, appear not to exhibit age-related decline in physiological capacity, and are seemingly resistant to age-related diseases. However, it has been unknown whether NMRs also evade aging according to a primary hallmark of aging: epigenetic changes. To address this question, we generated DNA methylation profiles from 329 tissues from animals of known age, at loci that are highly conserved between mammalian species, using a custom Infinium array (HorvathMammalMethylChip40). We observed strong aging effects on NMR DNA methylation, from which we developed seven highly accurate age estimators (epigenetic clocks) for several tissues (pan-tissue, blood, kidney clock, liver clock, skin clock) and two dual species (human-NMR) clocks. By identifying age-related cytosine methylation that are shared between NMR and humans, but not with the mouse, we identified genes and cellular pathways that impinge on developmental and metabolic processes that are potentially involved in NMR and human longevity. The NMR epigenetic clocks revealed that breeding NMR queens age more slowly than non-breeders, a feature that is also observed in some eusocial insects. CpGs associated with queen status were located near developmental genes and those that are regulated by the LHX3 transcription factor that controls pituitary development. In summary, our study demonstrates that despite a phenotype of reduced senescence, the NMR ages epigenetically through developmental and metabolic processes, and that NMR queens age more slowly than non-breeders.

Cattle are an attractive animal model of fertility in women due to their high degree of similarity relative to follicle selection, embryo cleavage, blastocyst formation, and gestation length. To facilitate future studies of the epigenetic underpinnings of aging effects in the female reproductive axis, several DNA methylation‐based biomarkers of aging (epigenetic clocks) for bovine oocytes are presented. One such clock was germane to only oocytes, while a dual‐tissue clock was highly predictive of age in both oocytes and blood. Dual species clocks that apply to both humans and cattle were also developed and evaluated. These epigenetic clocks can be used to accurately estimate the biological age of oocytes. Both epigenetic clock studies and epigenome‐wide association studies revealed that blood and oocytes differ substantially with respect to aging and the underlying epigenetic signatures that potentially influence the aging process. The rate of epigenetic aging was found to be slower in oocytes compared to blood; however, oocytes appeared to begin at an older epigenetic age. The epigenetic clocks for oocytes are expected to address questions in the field of reproductive aging, including the central question: how to slow aging of oocytes., Cattle are an attractive animal model of fertility in women due to their high degree of similarity relative to follicle selection, embryo cleavage, blastocyst formation, and gestation length. To facilitate future studies of the epigenetic underpinnings of aging effects in the female reproductive axis, several DNA methylation‐based biomarkers of aging (epigenetic clocks) for bovine oocytes are presented. One such clock was germane to only oocytes, while a dual‐tissue clock was highly predictive of age in both oocytes and blood.

DNA methylation data have been successfully used to develop highly accurate estimators of age (“epigenetic clocks”) in many mammalian species. With a view of extending such epigenetic clocks to all primate species, we analyzed DNA methylation profiles of 2400 tissues derived from 37 primate species including 11 haplorhine species (baboons, marmosets, vervets, rhesus macaque, chimpanzees, gorillas, orangutan, humans) and 26 strepsirrhine species (suborders Lemuriformes and Lorisiformes). From these we present here, pan-primate epigenetic clocks which are highly accurate for all primates including humans (age correlation R=0.98). We also carried out in-depth analysis of baboon DNA methylation profiles and generated five epigenetic clocks for baboons (Olive-yellow baboon hybrid), one of which, the pan-tissue epigenetic clock, was trained on seven tissue types (fetal cerebral cortex, adult cerebral cortex, cerebellum, adipose, heart, liver, and skeletal muscle) with ages ranging from late fetal life to 22.8 years of age. To facilitate translation of findings in baboons to humans, we further constructed two dual-species, human-baboon clocks. We also identified and present here, epigenetic predictors of sex that apply to all primate species. Low overlap can be observed between age- and sex-related CpGs. Overall, this study advances our understanding of conserved age- and sex-related epigenetic changes in primates, and provides biomarkers to study the aging of all primate species with the facility to readily translate any findings between primate species.

Human DNA methylation data have previously been used to develop highly accurate biomarkers of aging (“epigenetic clocks”). Subsequent studies demonstrate that similar epigenetic clocks can also be developed for mice and many other mammals. Here, we describe epigenetic clocks for common marmosets (Callithrix jacchus) based on novel DNA methylation data generated from highly conserved mammalian CpGs that were profiled using a custom Infinium array (HorvathMammalMethylChip40). From these, we developed and present here, two epigenetic clocks for marmosets that are applicable to whole blood samples. We find that the human-marmoset clock for relative age exhibits moderately high age correlations in two other non-human primate species: vervet monkeys and rhesus macaques. In a separate cohort of marmosets, we tested whether intervention with rapamycin, a drug shown to extend lifespan in mice, would alter the epigenetic age of marmosets, as measured by the marmoset epigenetic clocks. These clocks did not detect significant effects of rapamycin on the epigenetic age of marmoset blood. The common marmoset stands out from other mammals in that it is not possible to build accurate estimators of sex based on DNA methylation data: the accuracy of a random forest predictor of sex (66%) was substantially lower than that observed for other mammals (which is close to 100%). Overall, the epigenetic clocks developed here for the common marmoset are expected to be useful for age estimation of wild-born animals and for anti-aging studies in this species.

DNA-methylation profiles have been used successfully to develop highly accurate biomarkers of age, epigenetic clocks, for many species. Using a custom methylation array, we generated DNA methylation data from n=238 porcine tissues including blood, bladder, frontal cortex, kidney, liver and lung, from domestic pigs (Sus scrofa domesticus) and minipigs (Wisconsin Miniature Swine™). We present 4 epigenetic clocks for pigs that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (pig and human). Two dual-species human-pig pan-tissue clocks accurately measure chronological age and relative age, respectively. We also characterized CpGs that differ between minipigs and domestic pigs. Strikingly, several genes implicated by our epigenetic studies of minipig status overlap with genes (ADCY3, TFAP2B, SKOR1, and GPR61) implicated by genetic studies of body mass index in humans. In addition, CpGs with different levels of methylation between the two pig breeds were identified proximal to genes involved in blood LDL levels and cholesterol synthesis, of particular interest given the minipig’s increased susceptibility to cardiovascular disease compared to domestic pigs. Thus, inbred differences of domestic and minipigs may potentially help to identify biological mechanisms underlying weight gain and aging-associated diseases. Our porcine clocks are expected to be useful for elucidating the role of epigenetics in aging and obesity, and the testing of anti-aging interventions.

Methylation levels at specific CpG positions in the genome have been used to develop accurate estimators of chronological age in humans, mice, and other species. Although epigenetic clocks are generally species-specific, the principles underpinning them appear to be conserved at least across the mammalian class. This is exemplified by the successful development of epigenetic clocks for mice and several other mammalian species. Here, we describe epigenetic clocks for the rhesus macaque (Macaca mulatta), the most widely used nonhuman primate in biological research. Using a custom methylation array (HorvathMammalMethylChip40), we profiled n=281 tissue samples (blood, skin, adipose, kidney, liver, lung, muscle, and cerebral cortex). From these data, we generated five epigenetic clocks for macaques. These clocks differ with regards to applicability to different tissue types (pan-tissue, blood, skin), species (macaque only or both humans and macaques), and measure of age (chronological age versus relative age). Additionally, the age-based human-macaque clock exhibits a high age correlation (R=0.89) with the vervet monkey (Chlorocebus sabaeus), another Old World species. Four CpGs within the KLF14 promoter were consistently altered with age in four tissues (adipose, blood, cerebral cortex, skin). It is expected that the macaque clocks will reveal an epigenetic aging rate associated with a host of health conditions and thus lend themselves for identifying and validating anti-aging interventions.

Age-associated DNA-methylation profiles have been used successfully to develop highly accurate biomarkers of age (“epigenetic clocks”) in humans, mice, dogs, and other species. Here we present epigenetic clocks for African and Asian elephants. These clocks were developed using novel DNA methylation profiles of 140 elephant blood samples of known age, at loci that are highly conserved between mammalian species, using a custom Infinium array (HorvathMammalMethylChip40). We present epigenetic clocks for Asian elephants (Elephas maximus), African elephants (Loxodonta africana), and both elephant species combined. Two additional human-elephant clocks were constructed by combing human and elephant samples. Epigenome-wide association studies identified elephant age-related CpGs and their proximal genes. The products of these genes play important roles in cellular differentiation, organismal development, metabolism, and circadian rhythms. Intracellular events observed to change with age included the methylation of bivalent chromatin domains, targets of polycomb repressive complexes, and TFAP2C binding sites. These readily available epigenetic clocks can be used for elephant conservation efforts where accurate estimates of age are needed to predict demographic trends.

Cattle are an attractive animal model of fertility in women due to their high degree of similarity relative to follicle selection, embryo cleavage, blastocyst formation, and gestation length. To facilitate future studies of the epigenetic underpinnings of aging effects in the female reproductive axis, several DNA methylation-based biomarkers of aging (epigenetic clocks) for bovine oocytes are presented. One such clock was germane to only oocytes, while a dual-tissue clock was highly predictive of age in both oocytes and blood. Dual species clocks that apply to both humans and cattle were also developed and evaluated. These epigenetic clocks can be used to accurately estimate the chronological age of the oocyte donor. Both epigenetic clock studies and epigenome wide association studies revealed that blood and oocytes differ substantially with respect aging and the underlying epigenetic signatures that potentially influence the aging process. The rate of epigenetic aging was found to be slower in oocytes compared to blood, however, oocytes appeared to begin at an older epigenetic age. The epigenetic clocks for oocytes are expected to address questions in the field of reproductive aging, including the central question: how to slow aging of oocytes.

Human DNA methylation profiles have been used successfully to develop highly accurate biomarkers of aging (“epigenetic clocks”). Although these human epigenetic clocks are not immediately applicable to all species of the animal kingdom, the principles underpinning them appear to be conserved even in animals that are evolutionarily far removed from humans. This is exemplified by recent development of epigenetic clocks for mice and other mammalian species. Here, we describe epigenetic clocks for the domestic cat (Felis catus), based on methylation profiles of CpGs with flanking DNA sequences that are highly conserved between multiple mammalian species. Methylation levels of these CpGs are measured using a custom-designed Infinium array (HorvathMammalMethylChip40). From these, we present 3 epigenetic clocks for cats; of which, one applies only to blood samples from cats, while the remaining two dual-species human-cat clocks apply both to cats and humans. As the rate of human epigenetic ageing is associated with a host of health conditions and pathologies, it is expected that these epigenetic clocks for cats would do likewise and possess the potential to be further developed for monitoring feline health as well as being used for identifying and validating anti-aging interventions.

Young blood plasma is known to confer beneficial effects on various organs in mice. However, it was not known whether young plasma rejuvenates cells and tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly-accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=593 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=850 human tissue samples to the training data. We employed these six clocks to investigate the rejuvenation effects of a plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. Cellular senescence, which is not associated with epigenetic aging, was also considerably reduced in vital organs. Overall, this study demonstrates that a plasma-derived treatment markedly reverses aging according to epigenetic clocks and benchmark biomarkers of aging.

Over the past decades, there have been huge advances in understanding cellular responses to ionising radiation (IR) and DNA damage. These studies, however, were mostly executed with cell lines and mice using single or multiple acute doses of radiation. Hence, relatively little is known about how continuous exposure to low dose ionising radiation affects normal cells and organisms, even though our cells are constantly exposed to low levels of radiation. We addressed this issue by examining the consequences of exposing human primary cells to continuous ionising γ-radiation delivered at 6–20 mGy/h. Although these dose rates are estimated to inflict fewer than a single DNA double-strand break (DSB) per hour per cell, they still caused dose-dependent reductions in cell proliferation and increased cellular senescence. We concomitantly observed histone protein levels to reduce by up to 40%, which in contrast to previous observations, was not mainly due to protein degradation but instead correlated with reduced histone gene expression. Histone reductions were accompanied by enlarged nuclear size paralleled by an increase in global transcription, including that of pro-inflammatory genes. Thus, chronic irradiation, even at low dose-rates, can induce cell senescence and alter gene expression via a hitherto uncharacterised epigenetic route. These features of chronic radiation represent a new aspect of radiation biology.

The paramount role of senescent cells in ageing has prompted suggestions that re-expression of telomerase may prevent ageing; a proposition that is predicated on the assumption that senescent cells are the sole cause of ageing. Recently, several DNA methylation-based age estimators (epigenetic clocks) have been developed and they revealed that increased epigenetic age is associated with a host of age-related conditions, and is predictive of lifespan. Employing these clocks to measure epigenetic age in vitro, we interrogated the relationship between epigenetic ageing and telomerase activity. Although hTERT did not induce any perceptible change to the rate of epigenetic ageing, hTERT-expressing cells, which bypassed senescence, continued to age epigenetically. Employment of hTERT mutants revealed that neither telomere synthesis nor immortalisation is necessary for the continued increase in epigenetic age by these cells. Instead, the extension of their lifespan is sufficient to support continued epigenetic ageing of the cell. These characteristics, observed in cells from numerous donors and cell types, reveal epigenetic ageing to be distinct from replicative senescence. Hence, while re-activation of hTERT may stave off physical manifestation of ageing through avoidance of replicative senescence, it would have little impact on epigenetic ageing which continues in spite of telomerase activity.

DNA methylation (DNAm)-based biomarkers of aging have been developed for many tissues and organs. However, these biomarkers have sub-optimal accuracy in fibroblasts and other cell types used in ex vivo studies. To address this challenge, we developed a novel and highly robust DNAm age estimator (based on 391 CpGs) for human fibroblasts, keratinocytes, buccal cells, endothelial cells, lymphoblastoid cells, skin, blood, and saliva samples. High age correlations can also be observed in sorted neurons, glia, brain, liver, and even bone samples. Gestational age correlates with DNAm age in cord blood. When used on fibroblasts from Hutchinson Gilford Progeria Syndrome patients, this age estimator (referred to as the skin & blood clock) uncovered an epigenetic age acceleration with a magnitude that is below the sensitivity levels of other DNAm-based biomarkers. Furthermore, this highly sensitive age estimator accurately tracked the dynamic aging of cells cultured ex vivo and revealed that their proliferation is accompanied by a steady increase in epigenetic age. The skin & blood clock predicts lifespan and it relates to many age-related conditions. Overall, this biomarker is expected to become useful for forensic applications (e.g. blood or buccal swabs) and for a quantitative ex vivo human cell aging assay.

Aging is often perceived as a degenerative process caused by random accrual of cellular damage over time. In spite of this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, it is difficult to ignore the likelihood that a defined and shared mechanism instead, underlies the aging process. To address this, we generated over 10,000 methylation arrays, each profiling up to 37,000 cytosines in highly-conserved stretches of DNA, from over 59 tissue-types derived from 128 mammalian species. From these, we identified and characterized specific cytosines, whose methylation levels change with age across mammalian species. Genes associated with these cytosines are greatly enriched in mammalian developmental processes and implicated in age-associated diseases. From the methylation profiles of these age-related cytosines, we successfully constructed three highly accurate universal mammalian clocks for eutherians, and one universal clock for marsupials. The universal clocks for eutherians are similarly accurate for estimating ages (r>0.96) of any mammalian species and tissue with a single mathematical formula. Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species - a notion that was long-debated without the benefit of this new and compelling evidence.

// Sylwia Kabacik 1 and Ken Raj 1 1 Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Chilton, UK Correspondence to: Sylwia Kabacik, email: sylwia.kabacik@phe.gov.uk Keywords: permeability, VE-cadherin, ADAM10, cell junctions, ionising radiation Received: February 14, 2017 Accepted: May 03, 2017 Published: May 30, 2017 ABSTRACT The association between ionising radiation (IR) exposure and risk of cardiovascular diseases (CVD) is well documented, but the underlying mechanism is still poorly understood. As atherosclerotic plaques are the most common cause of CVD, we investigated the effects of IR on one of the critical parameters for atherosclerotic plaque formation – endothelium permeability to macromolecules. We used endothelial cells from human coronary artery as a model of the endothelial layer. Our results show that exposure of this endothelial layer to IR increased its permeability to macromolecules of various sizes in a dose-dependent manner. Immunofluorescence analysis revealed disruption of cell junctions caused by decreased amounts of two junction proteins, one of which is vascular endothelial cadherin (VE-cadherin). The reduction in the level of this protein was not due to diminished transcription but to protein processing instead. We observed a radiation dose-dependent increase in the cleavage of VE-cadherin by ADAM10. This was not mediated through the canonical VEGF route but was instead accompanied by intra-cellular calcium release. Importantly, inhibition of ADAM10 activity rescued IR-induced permeability. Our observations demonstrate that exposure to IR activates ADAM10 to cleave VE-cadherin leading to augmented endothelium permeability; a feature that can lead to the development of atherosclerotic plaques and increase the risk of cardiovascular disease.

Telomere length (TL) is associated with several aging-related diseases. Here, we present a DNA methylation estimator of TL (DNAmTL) based on 140 CpGs. Leukocyte DNAmTL is applicable across the entire age spectrum and is more strongly associated with age than measured leukocyte TL (LTL) (r ~-0.75 for DNAmTL versus r ~ -0.35 for LTL). Leukocyte DNAmTL outperforms LTL in predicting: i) time-to-death (p=2.5E-20), ii) time-to-coronary heart disease (p=6.6E-5), iii) time-to-congestive heart failure (p=3.5E-6), and iv) association with smoking history (p=1.21E-17). These associations are further validated in large scale methylation data (n=10k samples) from the Framingham Heart Study, Women's Health Initiative, Jackson Heart Study, InChianti, Lothian Birth Cohorts, Twins UK, and Bogalusa Heart Study. Leukocyte DNAmTL is also associated with measures of physical fitness/functioning (p=0.029), age-at-menopause (p=0.039), dietary variables (omega 3, fish, vegetable intake), educational attainment (p=3.3E-8) and income (p=3.1E-5). Experiments in cultured somatic cells show that DNAmTL dynamics reflect in part cell replication rather than TL per se. DNAmTL is not only an epigenetic biomarker of replicative history of cells, but a useful marker of age-related pathologies that are associated with it.

A confounding aspect of biological ageing is the nature and role of senescent cells. It is unclear whether the three major types of cellular senescence, namely replicative senescence, oncogene-induced senescence and DNA damage-induced senescence are descriptions of the same phenomenon instigated by different sources, or if each of these is distinct, and how they are associated with ageing. Recently, we devised an epigenetic clock with unprecedented accuracy and precision based on very specific DNA methylation changes that occur in function of age. Using primary cells, telomerase-expressing cells and oncogene-expressing cells of the same genetic background, we show that induction of replicative senescence (RS) and oncogene-induced senescence (OIS) are accompanied by ageing of the cell. However, senescence induced by DNA damage is not, even though RS and OIS activate the cellular DNA damage response pathway, highlighting the independence of senescence from cellular ageing. Consistent with this, we observed that telomerase-immortalised cells aged in culture without having been treated with any senescence inducers or DNA-damaging agents, re-affirming the independence of the process of ageing from telomeres and senescence. Collectively, our results reveal that cellular ageing is distinct from cellular senescence and independent of DNA damage response and telomere length.

Increasing evidence regarding positive effects of exposure to sunlight has led to suggestions that current advice may be overly weighted in favour of avoidance. UV-A has been reported to lower blood pressure, possibly through nitric oxide (NO) production in skin. Here, we set out to investigate effects of UV-A and solar-simulated radiation on the potential source of dermal NO, the effective doses and wavelengths, the responsiveness of different human skin cells, the magnitude of inter-individual differences and the potential influence of age. We utilised isogenic keratinocytes, microvascular endothelial cells, melanocytes and fibroblasts isolated from 36 human skins ranging from neonates to 86 years old. We show that keratinocytes and microvascular endothelial cells show greatest NO release following biologically relevant doses of UV-A. This was consistent across multiple neonatal donors and the effect is maintained in adult keratinocytes. Our observations are consistent with a bi-phasic mechanism by which UV-A can trigger vasodilatory effects. Analyses of NO-production spectra adds further evidence that nitrites in skin cells are the source of UV-mediated NO release. These potentially positive effects of ultraviolet radiation lend support for objective assessment of environmental influence on human health and the idea of “healthy sun exposure”.

DNA methylation age is an accurate biomarker of chronological age and predicts lifespan, but its underlying molecular mechanisms are unknown. In this genome-wide association study of 9907 individuals, we find gene variants mapping to five loci associated with intrinsic epigenetic age acceleration (IEAA) and gene variants in three loci associated with extrinsic epigenetic age acceleration (EEAA). Mendelian randomization analysis suggests causal influences of menarche and menopause on IEAA and lipoproteins on IEAA and EEAA. Variants associated with longer leukocyte telomere length (LTL) in the telomerase reverse transcriptase gene (TERT) paradoxically confer higher IEAA (P, Epigenetic clocks based on DNA methylation levels are estimators of chronological age. Here, the authors perform a GWAS of epigenetic aging rates in blood and find SNP variants in the TERT locus associated with increased intrinsic epigenetic age are also associated with longer telomeres.

DNA methylation age is an accurate biomarker of chronological age and predicts lifespan, but its underlying molecular mechanisms are unknown. In this genome-wide association study of 9,907 individuals, we found gene variants mapping to five loci associated with intrinsic epigenetic age acceleration (IEAA) and gene variants in 3 loci associated extrinsic epigenetic age acceleration (EEAA). Mendelian randomization analysis suggested causal influences of menarche and menopause on IEAA and lipid levels on IEAA and EEAA. Variants associated with longer leukocyte telomere length (LTL) in the telomerase reverse transcriptase gene (TERT) locus at 5p15.33 confer higher IEAA (P-11). Causal modelling indicatesTERT-specific and independent effects on LTL and IEAA. Experimental hTERT expression in primary human fibroblasts engenders a linear increase in DNA methylation age with cell population doubling number. Together, these findings indicate a critical role for hTERT in regulating the DNA methylation clock, in addition to its established role of compensating for cell replication-dependent telomere shortening.

PURPOSE: Ionizing radiation induces cardiovascular disease, the endothelium being the main target. The exact mechanism of the damage is unclear but the involvement of multiple signaling pathways is probable. Reversible lysine acetylation is a posttranslational protein modification that regulates activity across a broad range of signaling pathways. The goal of this study was to determine if a low radiation dose results in acetylome alteration in endothelial cells. MATERIALS AND METHODS: Human coronary artery endothelial cell line was irradiated with Cs-137 gamma-rays (0.5 Gy) and proteomics analysis was performed using enriched acetylated peptides and all peptides. Data were validated using immunoblotting, deacetylase activity assay, and RhoA activity assay. RESULTS: Nearly a hundred proteins were found to have an altered acetylation status 24 h after irradiation, primarily due to an overall decrease in acetylation. The expression of specific deacetylases was significantly increased, coinciding with an enhancement in global deacetylase activity. Proteins changed in their acetylation status belonged to several pathways including protein synthesis, cytoskeleton-related processes, protein folding and calcium signaling. The predicted changes in the RhoA/actin cytoskeleton pathway were validated by immunoassay. CONCLUSIONS: This study shows that protein acetylation is an important mediator of radiation responses in human cardiac coronary endothelial cells. Increased knowledge of the endothelial response to radiation is crucial for the development of normal tissue sparing modalities during radiation therapy.

Age is undoubtedly a major risk factor for heart disease. However, the reason for this is not entirely clear. In the course of our investigation into the mechanism of radiation-induced cardiovascular disease, we made several unexpected findings that inform us on this question. We observed that human coronary endothelial cells, while being able to initiate repair of radiation-induced DNA damage, often fail to complete the repair and become senescent. Such radiation-induced cellular aging occurs through a mutation-independent route. Endothelial cells that aged naturally through replication or as a result of radiation exhibited indistinguishable characteristics. The promoter regions of the CD44 gene in aging endothelial cells become demethylated, and the proteins are highly expressed on the cell surface, making the cells adhesive for monocytes. Adhesion is a cardinal feature that recruits monocytes to the endothelium, allowing them to infiltrate the vessel wall and initiate atherosclerosis. The epigenetic activation of CD44 expression is particularly significant as it causes persistent elevated CD44 protein expression, making senescent endothelial cells chronically adhesive. In addition to understanding why cardiovascular disease increases with age, these observations provide insights into the puzzling association between radiation and cardiovascular disease and highlight the need to consider premature aging as an additional risk of radiation to human health.

Adeno-associated virus (AAV) infection triggers a DNA damage response in the cell. This response is not induced by viral proteins but by virtue of the structure of AAV ssDNA being recognized by the cell as damaged DNA. The consequence of this is the killing of cells lacking p53 activity. We have observed that cells that lack p21 or pRb activity are also sensitive to AAV-induced cell death. We report that cells respond to AAV infection by activating two DNA damage signaling cascades. The first activates the p84N5 protein, which in turn activates caspase-6, leading to cell death. The second cascade activates the p53-21-pRb pathway, which inhibits activation of the p84N5 protein and thus prevents cell death. The result of the antagonistic interaction between these two pathways is that cells that do not exhibit functional p53-p21-pRb signaling undergo apoptosis as a consequence of AAV infection. Cells with a functional p53-21-pRb pathway are refractory to AAV-induced cell death. These results show that p53, although a proapoptotic protein, together with pRb and p21 proteins, is a member of an antiapoptotic cellular mechanism. As such, these experiments reveal features that may be exploited to specifically kill cells that lack the p53-p21-pRb pathway, such as cancer cells. The use of AAV to expose these subtle characteristics of intracellular signaling further highlights the advantages of using viruses as precision tools with which to address questions of cell biology. [Cancer Res 2007;67(16):7631–7]

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Adeno-associated virus Rep78 protein has antiproliferative effects on cells. It inhibits cell cycle progression, and, in particular, Rep78 induces a complete arrest within S phase, a response rarely seen after cell DNA damage. We examined how Rep78 achieves such an efficient S phase block. Rep78 inhibits Cdc25A activity by a novel means in which binding between the two proteins stabilizes Cdc25A, thus increasing its abundance, while at the same time preventing access to its substrates cyclin-dependent kinase (Cdk) 2 and Cdk1. This effect alone does not induce a complete S phase block. In addition, Rep78, as well as Rep68, produces nicks in the cellular chromatin, inducing a DNA damage response mediated by ataxia telangiectasia mutated (ATM) leading to G 1 and G 2 blocks. Mutational analysis shows that the zinc finger domain and nuclease activity of Rep78 are both required for the S phase block. The results suggest that a true S phase block cannot be achieved through a single pathway, and that adeno-associated virus Rep78 protein arrests cells within S phase by interfering with two pathways that would normally lead to an S phase slow-down.

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1∧E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1∧E4 arrests HeLa cervical epithelial cells in G 2 . 16E1∧E4 also caused a G 2 arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1∧E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1∧E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1∧E4 to be important for arrest. This region, which we have termed the “arrest domain,” contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1∧E4-mediated G 2 arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G 2 arrest was also mediated by the E1∧E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1∧E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G 2 arrest. We conclude that, like other papillomavirus proteins, 16E1∧E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1∧E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1∧E4.L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1∧E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1∧E4 (in vivo and in vitro) and could be immunoprecipitated as an E1∧E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1∧E4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1∧E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae , which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1∧E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1∧E4-associated protein indicates a possible role for E1∧E4 in virus synthesis.

The abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1 ∧ E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1 ∧ E4 into amyloid-like fibrils, which bind to thioflavin T. The C-terminal region is highly amyloidogenic, and its deletion abolishes amyloid staining and prevents E1 ∧ E4 accumulation. Amyloid-imaging probes can detect 16E1 ∧ E4 in biopsy material, as well as 18E1 ∧ E4 and 33E1 ∧ E4 in monolayer cells, indicating structural conservation. Our results suggest a role for fibril formation in facilitating the accumulation of E1 ∧ E4 during HPV infection.

There have been innumerate demonstrations of p53's activity as a tumour suppressor protein with the ability to stimulate cell signalling that can lead to cell cycle arrest and cell death in the event of DNA damage. Despite the solid body of evidence to support these properties of p53, reports have emerged that suggest a role for p53 in protecting cells from cell death. Our recent report highlighted a mechanism by which p53 activity can promote cell survival in the event of DNA damage. Here we present the various mechanisms that are activated by p53 signalling that can confer protection to cells with damaged DNA and emphasise the practical and clinical implications of a more balanced and context-dependent understanding of p53's pro-apoptotic and pro-survival activities.

5-Azacytidine, a DNA methyl transferase inhibitor, is effective in patients with myelodysplastic syndromes (MDS). Whether responses to 5-Azacytidine are achieved by demethylation of key genes or by cytotoxicity is unclear. Of 34 patients with MDS or acute myeloid leukaemia (AML) treated with 5-Azacytidine, 7 achieved complete remissions (CR) (21%) and 6 achieved haematological improvement. All six had less than 5% bone marrow (BM) blasts at the time of haematological improvements (HI) (2 had pre-existing refractory anaemia (RA), 4 had refractory anaemia with excess blasts (RAEB)). A further patient with RAEB had blast reduction to less than 5% without HI. Five of the seven (71%) complete responders had chromosome 7 abnormalities. BM CR predicted longer overall survival (OS) (median 23 versus 9 months, P=0.015). Bisulphite genomic sequencing (BGS) of the CDKN2B (p15(INK4b)) promoter showed low level, heterogeneous pretreatment methylation (mean 12.2%) in 14/17 (82%) patients analysed. Lower baseline methylation occurred in responders (9.8% versus 16.2% in non-responders P=0.07). No response was seen in patients with24% methylation, in whom p15(INK4b) mRNA was not expressed. 5-Azacytidine reduced CDKN2B methylation by mean 6.8% in 8/17 (47%) patients, but this did not correlate with response. At 75 mg/m(2), cell death (reduced BM cellularity (P=0.001) and increased apoptosis (P=0.02)) rather than demethylation of CDKN2B correlates with response. Patients with24% methylation may benefit from alternative dosing or combination strategies.

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1 ∧ E4 protein is lost during malignant progression, but in premalignant lesions, E1 ∧ E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 ∧ E4 in cell culture causes G 2 cell cycle arrest. Here we show that unlike many other G 2 arrest mechanisms, 16E1 ∧ E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1 ∧ E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1 ∧ E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G 2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 ∧ E4-expressing cells. We hypothesize that E1 ∧ E4-induced G 2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1 ∧ E4 expression may contribute to malignant progression.