Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

C3HeB/FeJ Mice mimic many aspects of gene expression and pathobiological features of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

The paternal age effect: a multifaceted phenomenon.

Birth rates for older fathers have increased 30% since 1980. When combined with the increased risk for genetic and multifactorial disorders in children conceived by older fathers, paternal age has become an important health issue for modern society. Laboratory research in this area has been minimal, perhaps because of significant experimental barriers, not the least of which is inadequate access to fresh, disease-free human testicular tissue. Regardless, progress has been made and intriguing models supported by experimental evidence have been proposed. The putative mechanisms range from reduced DNA repair activity, leading to increased mutagenesis, to positive selection of germ cells harboring specific disease-causing mutations. There remain many important venues for research in this increasingly relevant phenomenon that impacts future generations.

Enhanced genetic integrity in mouse germ cells.

Genetically based diseases constitute a major human health burden, and de novo germline mutations represent a source of heritable genetic alterations that can cause such disorders in offspring. The availability of transgenic rodent systems with recoverable, mutation reporter genes has been used to assess the occurrence of spontaneous point mutations in germline cells. Previous studies using the lacI mutation reporter transgenic mouse system showed that the frequency of spontaneous mutations is significantly lower in advanced male germ cells than in somatic cell types from the same individuals. Here we used this same mutation reporter transgene system to show that female germ cells also display a mutation frequency that is lower than that in corresponding somatic cells and similar to that seen in male germ cells, indicating this is a common feature of germ cells in both sexes. In addition, we showed that statistically significant differences in mutation frequencies are evident between germ cells and somatic cells in both sexes as early as mid-fetal stages in the mouse. Finally, a comparison of the mutation frequency in a general population of early type A spermatogonia with that in a population enriched for Thy-1-positive spermatogonia suggests there is heterogeneity among the early spermatogonial population such that a subset of these cells are predestined to form true spermatogonial stem cells. Taken together, these results support the disposable soma theory, which posits that genetic integrity is normally maintained more stringently in the germ line than in the soma and suggests that this is achieved by minimizing the initial occurrence of mutations in early germline cells and their subsequent gametogenic progeny relative to that in somatic cells.

Human O6 -methylguanine-DNA methyltransferase containing C145A does not prevent hepatocellular carcinoma in C3HeB/FeJ transgenic mice.

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT.

EEPD1 promotes repair of oxidatively-stressed replication forks.

Unrepaired oxidatively-stressed replication forks can lead to chromosomal instability and neoplastic transformation or cell death. To meet these challenges cells have evolved a robust mechanism to repair oxidative genomic DNA damage through the base excision repair (BER) pathway, but less is known about repair of oxidative damage at replication forks. We found that depletion or genetic deletion of EEPD1 decreases clonogenic cell survival after oxidative DNA damage. We demonstrate that EEPD1 is recruited to replication forks stressed by oxidative damage induced by H2O2 and that EEPD1 promotes replication fork repair and restart and decreases chromosomal abnormalities after such damage. EEPD1 binds to abasic DNA structures and promotes resolution of genomic abasic sites after oxidative stress. We further observed that restoration of expression of EEPD1 via expression vector transfection restores cell survival and suppresses chromosomal abnormalities induced by oxidative stress in EEPD1-depleted cells. Consistent with this, we found that EEPD1 preserves replication fork integrity by preventing oxidatively-stressed unrepaired fork fusion, thereby decreasing chromosome instability and mitotic abnormalities. Our results indicate a novel role for EEPD1 in replication fork preservation and maintenance of chromosomal stability during oxidative stress.

Ionizing radiation-induced mutant frequencies increase transiently in male germ cells of older mice.

Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.

Epigenetic regulation of genetic integrity is reprogrammed during cloning.

Cloning by somatic cell nuclear transfer (SCNT) circumvents processes that normally function during gametogenesis to prepare the gamete genomes to support development of new progeny following fertilization. One such process is enhanced maintenance of genetic integrity in germ cells, such that germ cells typically carry fewer spontaneously acquired mutations than somatic cells in the same individual. Thus, embryos produced from somatic cells by SCNT could directly inherit more mutations than naturally conceived embryos. Alternatively, they could inherit epigenetic programming that predisposes more rapid accumulation of de novo mutations during development. We used a transgenic mouse system to test these possibilities by producing cloned midgestation mouse fetuses from three different donor somatic cell types carrying significantly different initial frequencies of spontaneous mutations. We found that on an individual locus basis, mutations acquired spontaneously in a population of donor somatic cells are not likely to be propagated to cloned embryos by SCNT. In addition, we found that the rate of accumulation of spontaneous mutations was similar in fetuses produced by either natural conception or cloning, indicating that cloned fetuses do not acquire mutations more rapidly than naturally conceived fetuses. These results represent the first direct demonstration that the process of cloning by SCNT does not lead to an increase in the frequency of point mutations. These results also demonstrate that epigenetic mechanisms normally contribute to the regulation of genetic integrity in a tissue-specific manner, and that these mechanisms are subject to reprogramming during cloning.

Age-related instability in spermatogenic cell nuclear and mitochondrial DNA obtained from Apex1 heterozygous mice.

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.

Short form glutathione peroxidase 4 is the essential isoform required for survival and somatic mitochondrial functions.

Glutathione peroxidase 4 (Gpx4) is an essential antioxidant enzyme having multiple functions. A long form Gpx4 protein and a short form Gpx4 protein, which are distinguishable by the presence or lack of a mitochondrial signal peptide at the N terminus, are generated from the Gpx4 gene. In this study, we generated transgenic mice using mutated GPX4 genes encoding either the long form Gpx4 (lGPX4 gene) or the short form Gpx4 (sGPX4 gene). Our results showed that transgenic mice with the sGPX4 gene had increased Gpx4 protein in all tissues and were protected against diquat-induced apoptosis in liver. Moreover, the sGPX4 gene was able to rescue the lethal phenotype of the mouse Gpx4-null mutation. In contrast, transgenic mice with the lGPX4 gene had increased Gpx4 protein only in the testes, and the lGPX4 gene failed to rescue the lethal phenotype of the mouse Gpx4-null mutation. In Gpx4-null mice rescued by the sGPX4 gene, the Gpx4 protein was present in mitochondria isolated from somatic tissues, and the submitochondrial distribution pattern of the Gpx4 protein in these mice was identical to that in wild-type mice. Interestingly, the male Gpx4-null mice rescued by the sGPX4 gene were infertile and exhibited sperm malformation. Together, our results demonstrated for the first time that the short form Gpx4 protein is present in somatic tissue mitochondria and is essential for survival and protection against apoptosis in mice, whereas the long form Gpx4 protein is important for male fertility.

BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

Fluorescent Light Incites a Conserved Immune and Inflammatory Genetic Response within Vertebrate Organs (Danio Rerio, Oryzias Latipes and Mus Musculus).

Fluorescent light (FL) has been utilized for ≈60 years and has become a common artificial light source under which animals, including humans, spend increasing amounts of time. Although the solar spectrum is quite dissimilar in both wavelengths and intensities, the genetic consequences of FL exposure have not been investigated. Herein, we present comparative RNA-Seq results that establish expression patterns within skin, brain, and liver for Danio rerio, Oryzias latipes, and the hairless mouse (Mus musculus) after exposure to FL. These animals represent diurnal and nocturnal lifestyles, and ≈450 million years of evolutionary divergence. In all three organisms, FL induced transcriptional changes of the acute phase response signaling pathway and modulated inflammation and innate immune responses. Our pathway and gene clustering analyses suggest cellular perception of oxidative stress is promoting induction of primary up-stream regulators IL1B and TNF. The skin and brain of the three animals as well as the liver of both fish models all exhibit increased inflammation and immune responses; however, the mouse liver suppressed the same pathways. Overall, the conserved nature of the genetic responses observed after FL exposure, among fishes and a mammal, suggest the presence of light responsive genetic circuitry deeply embedded in the vertebrate genome.

Base excision repair, aging and health span.

DNA damage and mutagenesis are suggested to contribute to aging through their ability to mediate cellular dysfunction. The base excision repair (BER) pathway ameliorates a large number of DNA lesions that arise spontaneously. Many of these lesions are reported to increase with age. Oxidized guanine, repaired largely via base excision repair, is particularly well studied and shown to increase with age. Spontaneous mutant frequencies also increase with age which suggests that mutagenesis may contribute to aging. It is widely accepted that genetic instability contributes to age-related occurrences of cancer and potentially other age-related pathologies. BER activity decreases with age in multiple tissues. The specific BER protein that appears to limit activity varies among tissues. DNA polymerase-beta is reduced in brain from aged mice and rats while AP endonuclease is reduced in spermatogenic cells obtained from old mice. The differences in proteins that appear to limit BER activity among tissues may represent true tissue-specific differences in activity or may be due to differences in techniques, environmental conditions or other unidentified differences among the experimental approaches. Much remains to be addressed concerning the potential role of BER in aging and age-related health span.

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis.

Humans are exposed to ionizing radiation (IR) under various circumstances, e.g. cosmic radiation, diagnostic X-rays and radiotherapy for cancer. It has been shown that IR can impair spermatogenesis and can cause mutations in germ cells. However, the mutagenic responses of germ cells exposed to IR at different stages of testicular maturation have not been examined by directly assessing the mutant frequency in defined spermatogenic cell types. This study was performed to address whether preadult exposure to IR can increase mutations in adult germ cells that could in turn have a major impact on adult reproductive function and the health of ensuing offspring. Male Lac I transgenic mice were irradiated with a single dose of 2.5 Gy of gamma-ray at different ages before adulthood, reflecting different stages of testicular maturation, and then mutant frequency (MF) was determined directly in spermatogenic cell types emanating from the irradiated precursor cells. The results showed that (1) preadult exposure to IR did not significantly increase MF in adult epididymal spermatozoa; (2) spermatogenic stages immediately following the irradiated stage(s) displayed an elevated mutant frequency; but (3) the mutant frequency was restored to unirradiated levels in later stages of spermatogenesis. These findings provide evidence that there is a mechanism(s) to prevent spermatogenic cells with elevated mutant frequencies from progressing through spermatogenesis.

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice.

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.

Spontaneous mutagenesis is enhanced in Apex heterozygous mice.

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.

A mild mutator phenotype arises in a mouse model for malignancies associated with neurofibromatosis type 1.

Defects in genes that control DNA repair, proliferation, and apoptosis can increase genomic instability, and thus promote malignant progression. Although most tumors that arise in humans with neurofibromatosis type 1 (NF1) are benign, these individuals are at increased risk for malignant peripheral nerve sheath tumors (MPNST). To characterize additional mutations required for the development of MPNST from benign plexiform neurofibromas, we generated a mouse model for these tumors by combining targeted null mutations in Nf1 and p53, in cis. CisNf1+/-; p53+/- mice spontaneously develop PNST, and these tumors exhibit loss-of-heterozygosity at both the Nf1 and p53 loci. Because p53 has well-characterized roles in the DNA damage response, DNA repair, and apoptosis, and because DNA repair genes have been proposed to act as modifiers in NF1, we used the cisNf1+/-; p53+/- mice to determine whether a mutator phenotype arises in NF1-associated malignancies. To quantitate spontaneous mutant frequencies (MF), we crossed the Big Blue mouse, which harbors a lacI transgene, to the cisNf1+/-; p53+/- mice, and isolated genomic DNA from both tumor and normal tissues in compound heterozygotes and wild-type siblings. Many of the PNST exhibited increased mutant frequencies (MF=4.70) when compared to normal peripheral nerve and brain (MF=2.09); mutations occurred throughout the entire lacI gene, and included base substitutions, insertions, and deletions. Moreover, the brains, spleens, and livers of these cisNf1+/-; p53+/- animals exhibited increased mutant frequencies when compared to tissues from wild-type littermates. We conclude that a mild mutator phenotype arises in the tumors and tissues of cisNf1+/-; p53+/- mice, and propose that genomic instability influences NF1 tumor progression and disease severity.

Pluripotent cells display enhanced resistance to mutagenesis.

Pluripotent cells have been reported to exhibit lower frequencies of point mutations and higher levels of DNA repair than differentiated cells. This predicts that pluripotent cells are less susceptible to mutagenic exposures than differentiated cells. To test this prediction, we used a lacI mutation-reporter transgene system to assess the frequency of point mutations in multiple lines of mouse pluripotent embryonic stem cells and induced pluripotent cells, as well as in multiple lines of differentiated fibroblast cells, before and after exposure to a moderate dose of the mutagen, methyl methanesulfonate. We also measured levels of key enzymes in the base excision repair (BER) pathway in each cell line before and after exposure to the mutagen. Our results confirm that pluripotent cells normally maintain lower frequencies of point mutations than differentiated cells, and show that differentiated cells exhibit a large increase in mutation frequency following a moderate mutagenic exposure, whereas pluripotent cells subjected to the same exposure show no increase in mutations. This result likely reflects the higher levels of BER proteins detectable in pluripotent cells prior to exposure and supports our thesis that maintenance of enhanced genetic integrity is a fundamental characteristic of pluripotent cells.

DNA Alkylating Agent Protects Against Spontaneous Hepatocellular Carcinoma Regardless of O6-Methylguanine-DNA Methyltransferase Status.

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O(6)-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 µmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O(6)-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma.

Aging is associated with an expansion of CD49fhi mammary stem cells that show a decline in function and increased transformation potential.

Breast cancer incidence increases during aging, yet the mechanism of age-associated mammary tumorigenesis is unclear. Mammary stem cells are believed to play an important role in breast tumorigenesis, but how their function changes with age is unknown. We compared mammary epithelial cells isolated from young and old mammary glands of different cohorts of C57BL6/J and BALB/c mice, and our findings revealed that old mammary glands were characterized by increased basal cell pool comprised of mostly CD49fhi cells, altered luminal-to-basal cell ratio, and irregular ductal morphology. More interestingly, basal stem cells in old mice were increased in frequency, but showed a functional decline of differentiation and increased neoplastic transformation potential. Gene signature enrichment analysis revealed a significant enrichment of a luminal cell gene expression signature in the basal stem cell-enriched population from old mice, suggesting some luminal cells were expressing basal markers. Immunofluorescence staining confirmed the presence of luminal cells with high CD49f expression in hyperplastic lesions implicating these cells as undergoing luminal to basal phenotypic changes during aging. Whole transcriptome analysis showed elevated immune and inflammatory responses in old basal stem cells and stromal cells, which may be the underlying cause for increased CD49fhi basal-like cells in aged glands.