[Expression changes of RNA m6A regulators in mouse cerebellum affected by hypobaric hypoxia stimulation].

Objective: To investigate the role of RNA m6A methylation in mediating cerebellar dysplasia through analyzing the phenotypes of the mouse cerebella and the expression of several key m6A regulators upon hypobaric hypoxia treatment. Methods: Five-day old C57/BL6 mice were exposed to hypobaric hypoxia for 9 days. The status of mouse cerebellar development was analyzed by comparing the body weights, brain weights and histological features. Immunostaining of cell-type-specific markers was performed to analyze the cerebellar morphology. Real-time PCR, Western blot and immunohistochemical staining were performed to detect the expression of key m6A regulators in the mouse cerebella. Results: Compared with the control, the body weights, brain weights and cerebellar volumes of hypobaric hypoxic mice were significantly reduced (P<0.01). The expression of specific markers in different cells, including NeuN (mature neuron), Calbindin-D28K (Purkinje cell) and GFAP (astrocyte), was decreased in hypobaric hypoxic mouse cerebella (P<0.01), accompanied with disorganized cellular structure. The expression of methyltransferase METTL3 was significantly down-regulated in the cerebella of hypobaric hypoxic mice (P<0.05). Conclusions: Hypobaric hypoxia stimulation causes mouse cerebellar dysplasia, with structural abnormalities in mature granular neurons, Purkinje cells and astrocytes. Expression of METTL3 is decreased in hypobaric hypoxic mice cerebellum compared with that of normobaric normoxic mice, suggesting that its mediated RNA m6A methylation may play an important role in hypobaric hypoxia-induced mouse cerebellar dysplasia.

[Effects of RNA M6A demethylase ALKBH5 gene deficiency on morphology and function of cerebellum in aged mice].

Objective: To investigate the effects of RNA m6A demethylase ALKBH5 gene deficiency on cerebellar morphology and function in the aged mice, and to explore the role of ALKBH5 in cerebellar degeneration. Methods: Western blot was performed to detect the protein level of ALKBH5 in the cerebellum of wild-type mice of various ages. The expression of NeuN, Calbindin-D28K, MAP2, GFAP and other proteins in the cerebella of middle-aged (12-month-old) and aged (18-month-old) wild-type mice and ALKBH5-/- mice was examined using immunohistochemistry. The balance beam test and gait analysis were performed to test the balance ability and motor coordination of the mice. Results: With aging of the mice, the expression of ALKBH5 in the cerebellum increased gradually in an age-dependent manner. In the aged mice, but not middle-aged mice, the body weight, whole brain weight and cerebellum weight of ALKBH5-/- mice decreased by 15%, 10% and 21%, respectively (P<0.05). The expression of ALKBH5 in the Purkinje cells was much higher than that in other types of neural cells. Correspondingly, ALKBH5-deficiency caused 40% reduction in the number of Purkinje cells, as well as the length and density of neuronal dendrites in the aged mice (P<0.01). In addition, the time for the aged ALKBH5-/- mice to pass the balance beam was 70% longer than that of the wild type mice of the same age, with unstable gaits (P<0.01). Conclusions: Gene deficiency of RNA m6A demethylase ALKBH5 causes cerebellar atrophy, Purkinje neuron loss and damage in the aged mice. These changes eventually affect mice's motor coordination and balance ability. These results suggest that imbalanced RNA m6A methylation may lead to neurodegenerative lesions in the cerebellum of mice.

GenePiper, a Graphical User Interface Tool for Microbiome Sequence Data Mining.

Amplicon sequencing of the 16S rRNA gene is commonly performed for the assessment and comparison of microbiomes. Here, we introduce GenePiper, an open-source R Shiny application that provides an easy-to-use interface, a wide range of analytical methods, and optimized graphical outputs for offline microbiome data analyses.

[Expression of heterogeneous nuclear ribonucleo-protein A2B1 in mouse cerebellar development and human medulloblastoma].

Objective: To investigate the expression and potential role of heterogeneous nuclear ribonucleo-protein A2B1 (HNRNPA2B1) in mouse cerebellar development and the significance of HNRNPA2B1 in human medulloblastoma. Methods: The data of HNRNPA2B1 RNA expression in mouse and human cerebella were obtained from databases. Western blot and immunohistochemical staining were performed to detect the protein level of HNRNPA2B1 in mouse cerebella at different ages. The expression level of HNRNPA2B1 in control human cerebellum and medulloblastoma was detected by immunohistochemical staining. m6A-IP-qPCR method was applied to confirm whether HNRNPA2B1 RNA in Daoy cells was modified with m6A.Western blot was used to detect the effect of MG132 treatment on the HNRNPA2B1 protein level in Daoy cells. Results: The level of HNRNPA2B1 protein in postnatal mouse cerebella was higher than that in adult mouse cerebella, with weak HNRNPA2B1 staining in external granular cells while strong staining in mature Purkinje cells and molecular layer. Compared with control normal human cerebella, the RNA expression level of HNRNPA2B1 increased in medulloblastoma, while immunohistochemical staining showed that the mean intensity of HNRNPA2B1 decreased in medulloblastoma. HNRNPA2B1 RNA in medulloblastoma and Daoy cells was modified by m6A. The HNRNPA2B1 protein level in Daoy cells increased upon MG132 treatment. Conclusions: HNRNPA2B1 is dynamically expressed during mouse cerebellar development. Compared with normal human cerebella, HNRNPA2B1 is significantly up-regulated at transcriptional level but obviously down-regulated at translational level in medulloblastoma. These results indicate that HNRNPA2B1 may be involved in cerebellar development process and medulloblastoma tumorigenesis. The m6A methylation in HNRNPA2B1 transcript and protein ubiquitin-proteasome pathway may account for the down-regulation of HNRNPA2B1 at protein level.

[Expression of protein phosphatase 2AC in the brain of mice and Alzheimer's patients].

Objective: To investigate the biological functions of protein phosphatase 2AC(PP2AC) in the brain, and to detect its spatio-temporal expression and its involvement in neurological disorders in the brains of mice and Alzheimer's patients. Methods: Western blot was used to evaluate the expression level of PP2AC in different organs. Immunohistochemical staining was performed to detect the in situ expression levels of PP2AC in the brains of mice and patients, and the pathological changes were confirmed in the brains of patients with Alzheimer's disease. Results: Among all the tested organs in adult mouse, the expression of PP2AC protein was the highest in the brain. From embryonic day 18.5 to postnatal 2-year-old mice, PP2AC exhibited spatio-temporal specific expression in the brains. Furthermore, an age-dependent increased expression in the cerebral cortex at both protein and RNA levels was observed. Compared to control group, PP2AC protein expression was lower in the frontal cortex of Alzheimer's patients. Conclusions: The spatio-temporal specific expression profiles of PP2AC in mouse brain implicate its biological significance. Its diminished expression in the frontal cortex of Alzheimer's disease patients implies that PP2AC plays a potential role in the pathogenesis of Alzheimer's disease.

[Correlation between Mic60 haploid insufficiency and cardiac aging in mouse].

Objective: To investigate the role of Mic60 in cardiac aging. Methods: Wild-type and Mic60(+ /-) male mice at age of 4-6 months (young group, n=6) and 18-20 months (aged group, n=9) were used. H&E and Masson staining of frozen and paraffin sections were subjected to morphologic evaluation of the cardiac tissue samples. SA-ß-Gal staining was utilized to detect the activity of senescence-associated ß-galactosidase. Western blot was performed to detect the expression of Mic60 and p21 in cardiac tissues. Results: Expression of Mic60 in mouse cardiac tissue increased in an age-dependent manner. Haploid insufficiency of Mic60 resulted in an increased left ventricular wall thickness [(1.32±0.09) mm vs.(1.12±0.09) mm, P<0.05], cardiomyocyte hypertrophy[(474.9±27.6) µm(2) vs.(358.8±48.7) µm(2), P<0.05] and interstitial fibrosis [ (38.24±7.58) ×10(3)µm(2) vs.(25.81±4.12)×10(3)µm(2,) P<0.05], increased activity of SA-ß-Gal (2.26±0.24 vs.0.25±0.05, P<0.01) and higher expression of p21 (P<0.01) in aged mouse cardiac tissue, but not in young mice. Conclusion: Haploid insufficiency of Mic60 leads to cardiac hypertrophy, interstitial fibrosis, increased activity of SA-ß-Gal and higher expression of p21 in aged cardiac tissue in mice, suggesting that Mic60 may prevent cardiac aging.

Long noncoding RNA MIR31HG exhibits oncogenic property in pancreatic ductal adenocarcinoma and is negatively regulated by miR-193b.

Long noncoding RNAs (lncRNAs) play important regulatory roles in a variety of diseases, including many tumors. However, the functional roles of these transcripts and mechanisms responsible for their deregulation in pancreatic ductal adenocarcinoma (PDAC) are not thoroughly understood. In this study, we discovered that lncRNA MIR31HG is markedly upregulated in PDAC. Knockdown of MIR31HG significantly suppressed PDAC cell growth, induced apoptosis and G1/S arrest, and inhibited invasion, whereas enhanced expression of MIR31HG had the opposite effects. Online database analysis tools showed that miR-193b could target MIR31HG and we found an inverse correlation between MIR31HG and miR-193b in PDAC specimens. Inhibition of miR-193b expression significantly upregulated the MIR31HG level, while overexpression of miR-193b suppressed MIR31HG's expression and function, suggesting that MIR31HG is negatively regulated by miR-193b. Moreover, using luciferase reporter and RIP assays, we provide evidence that miR-193b directly targeted MIR31HG by binding to two microRNA binding sites in the MIR31HG sequence. On the other hand, MIR31HG may act as an endogenous 'sponge' by competing for miR-193b binding to regulate the miRNA targets. Collectively, these results demonstrate that MIR31HG functions as an oncogenic lncRNA that promotes tumor progression, and miR-193b targets not only protein-coding genes but also the lncRNA, MIR31HG.

DNA damage and oxidative injury are associated with hypomyelination in the corpus callosum of newborn Nbn(CNS-del) mice.

Nijmegen breakage syndrome (NBS), caused by mutation of the Nbn gene, is a recessive genetic disorder characterized by immunodeficiency, elevated sensitivity to ionizing radiation, chromosomal instability, microcephaly, and high predisposition to malignancies. To explore the underlying molecular mechanisms of NBS microcephaly, Frappart et al. previously inactivated Nbn gene in the central nervous system (CNS) of mice by the nestin-Cre targeting gene system and generated Nbn(CNS-del) mice. Here we first report that Nbn gene inactivation induces the defective proliferation and enhanced apoptosis of the oligodendrocyte precursor cells (OPCs), contributing to the severe hypomyelination of the nerve fibers of the corpus callosum. Under conditions of DNA damage and oxidative stress, the distinct regulatory roles of ATM-Chk2 signaling and AKT/mTOR signaling are responsible for the defective proliferation and enhanced apoptosis of the Nbn-deficient OPCs. In addition, specific HDAC isoforms may play distinctive roles in regulating the myelination of the Nbn-deficient OPCs. However, brain-derived neurotrophic factor and nerve growth factor stimulation attenuates the oxidative stress and thereby increases the proliferation of the Nbn-deficient OPCs, which is accompanied by upregulation of the AKT/mTOR/P70S6K signaling pathway. Taken together, these findings demonstrate that DNA damage and oxidative stress resulting from Nbn gene inactivation are associated with hypomyelination of the nerve fibers of corpus callosum.

Serine 312 phosphorylation is dispensable for wild-type p53 functions in vivo.

Cellular stimulation results in phosphorylation of the tumor suppressor p53 on multiple residues, though the functional relevance is not always clear. It is noteworthy that the serine (S) 315 residue is unique, as it has been suggested to be phosphorylated not only by genotoxic signals, but also during cell-cycle progression and by endoplasmic-reticulum stress. However, in vitro data have been conflicting as phosphorylation at this site was shown to both positively and negatively regulate p53 functions. We have thus generated knock-in mice expressing an unphosphorylable S312 (equivalent to human S315), by substitution with an alanine (A) residue, to clarify the conflicting observations and to evaluate its functional relevance in vivo. Born at Mendelian ratios, the p53(S312A/S312A) mice show no anomalies during development and adulthood. p53 activation, stability, localization and ability to induce apoptosis, cell-cycle arrest and prevent centrosome amplification are not compromised in p53(S312A/S312A) cells. p53(S312A/S312A) mice are unable to rescue mdm2(-/-) lethality, and tumorigenesis--both spontaneous and irradiation/oncogene-induced--is not accentuated. Taken together, the results show that the S312 phosphorylation site is not in itself necessary for efficient p53 function, and advocates the possibility that it is neither relevant in the mouse context nor important for p53 functions in vivo.

Poly(ADP-ribose) polymerase-1 plays a role in suppressing mammary tumourigenesis in mice.

The DNA strand break-binding molecule, poly(ADP-ribose) polymerase-1 (PARP-1), plays a role in DNA repair, chromosomal stability, transcription and cell death. Accumulating evidence suggests that dysfunction of PARP-1 contributes to tumorigenesis. Here, we report that PARP-1 deficiency causes mammary carcinoma formation in female mice, and that the introduction of Trp53 mutations accelerates the onset and shortens the latency of mammary tumorigenesis. We show that PARP-1 deficiency results in chromosomal aneuploidy and centrosome amplification, which are substantiated by the inactivation of Trp53 in primary mammary epithelial (PME) cells. In addition, PARP-1 deficiency compromises p53 activation and impairs BRCA1 recruitment to the sites of DNA damage in PME cells. PARP-1 complementation partly rescues the defective DNA damage response mediated by p53 and BRCA1. The present study thus identifies a role of PARP-1 in suppressing mammary tumorigenesis in vivo and suggests that dysfunction of PARP-1 may be a risk factor for breast cancer in humans.

UV-induced DNA damage and mutations in Hupki (human p53 knock-in) mice recapitulate p53 hotspot alterations in sun-exposed human skin.

The major etiological agent contributing to human nonmelanoma skin cancer is sunlight. The p53 tumor suppressor gene is usually mutated in these tumors, and the mutations are "UV signature" single or tandem transitions at dipyrimidine sequences in the DNA-binding domain (DBD). Cells that harbor these characteristic mutations are already present in sun-exposed skin areas of healthy individuals, and small epidermal patches that are immunoreactive to anti-p53 antibody accrue as exposure increases. To explore carcinogen-specific human p53 mutation patterns experimentally, we generated a knock-in (Hupki) mouse in which the murine DBD of the p53 gene has been replaced by the homologous human p53 DBD segment; thus, the precise base sequence context frequently targeted by mutagens or endogenous mutagenic processes in human carcinogenesis is present in this strain (J. L. Luo et al., Oncogene, 20: 320-328, 2001). Here we show that when epidermal cells of Hupki mice (p53(ki/ki)) are irradiated in vivo with a single acute dose of UVB light, they accumulate UV photoproducts at the same locations of the p53 gene as human cells. Chronic exposure of Hupki mice (4.5 kJ/m(2) 5x/week for 4 weeks) results in the appearance of cell patches that stain intensely with the anti-p53 antiserum CM1. DNA preparations from 2 cm(2) sections of chronically irradiated Hupki epidermis harbor C to T and CC to TT mutations at two mutation hotspots identified in human skin cancer, one at codons 278-279, and one at codons 247-248; the latter is the most frequent UVB-associated mutation site in humans but not in p53 wild-type mice. Thus, Hupki keratinocytes with these p53 mutations encode an aberrant DBD identical in amino acid sequence to the mutant p53 molecules in human UV-induced tumors. The Hupki mouse model offers a new experimental tool in molecular epidemiology and biomedical research.

Fibroblast growth factor-8b-stimulated myogenic cell proliferation is suppressed by the promyelocytic leukemia gene.

Muscle cell growth is regulated by growth-promoting and -inhibiting factors. In this study, the physiological effects of fibroblast growth factor (FGF)-8b and the promyelocytic leukemia (PML) gene on G8 myogenic cells were examined. FGF-8b was found to strongly stimulate myogenic cell proliferation. Signal transduction assays using AP-1/SEAP and E-box/SEAP reporters revealed that the transcriptional factors junB/c-fos and c-myc were involved in FGF-8b-stimulated G8 cell growth. Besides examining factors that positively stimulate myogenic cell growth, we also examined genes that negatively affect cell growth. PML is a growth suppressor gene and we studied its expression in G8 cells under different growth conditions. Immunohistochemical staining revealed that in the presence of low serum, PML was expressed in approximately 23.2% of all cultured G8 cells. However, under normal culture conditions (10% serum), PML expression dropped to about 2.6%. We found that the PML gene acted antagonistically to FGF-8b, as the overexpression of PML in G8 cells significantly inhibited FGF-8b-stimulated cell proliferation. It also inhibited AP-1 and E-box transactivation. However, we believe that PML functions as a stress-response gene in G8 cells rather than as a gene normally involved in regulating muscle development.

Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells.

DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ.

DNA strand break-sensing molecule poly(ADP-Ribose) polymerase cooperates with p53 in telomere function, chromosome stability, and tumor suppression.

Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP(-/-) cells showed telomere shortening and p53(-/-) cells showed normal telomere length, inactivation of PARP in p53(-/-) cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.

Knock-in mice with a chimeric human/murine p53 gene develop normally and show wild-type p53 responses to DNA damaging agents: a new biomedical research tool.

The high prevalence and great diversity of p53 tumor suppressor gene mutations in human tumors call for development of therapeutic molecules that rescue function of aberrant p53 protein. P53 mutations also offer new approaches to the study of the origins of mutations in human cancer. An experimental mouse model with a genetically modified but normal functioning p53 gene harboring the human rather than the murine core domain, would be of considerable benefit to research on both cancer therapeutics and etiology; however, it is uncertain whether such mice would permit biological functions of p53 to be retained. Using a Cre/lox P gene-targeting approach, we have constructed a human p53 knock-in (hupki) mouse strain in which exons 4-9 of the endogenous mouse p53 allele were replaced with the homologous, normal human p53 gene sequence. The chimeric p53 allele (p53(KI)) is properly spliced, transcribed in various tissues at levels equivalent to wild-type mice, and yields cDNA with the anticipated sequence, that is, with a core domain matching that of humans. The hupki p53 protein binds to p53 consensus sequences in gel mobility shift assays and accumulates in the nucleus of hupki fibroblasts in response to UV irradiation, as is characteristic of wild-type p53. Induction of various p53-regulated genes in spleen of gamma-irradiated homozygous hupki mice (p53(KI/KI)), and the kinetics of p53-dependent apoptosis in thymocytes are similar to results with wild-type (p53(+/+)) mice, further indicating normal p53 pathway function in the hupki strain. The mice are phenotypically normal and do not develop spontaneous tumors at an early age, in contrast to knock-out (p53(-/-)) strains with a defective p53 gene. The chimeric (p53(KI)) allele thus appears to provide a biological equivalent to the endogenous murine (p53(+)) gene. This strain is a unique tool for examining in vivo spontaneous and induced mutations in human p53 gene sequences for comparison with published human tumor p53 mutation spectra. In addition, the hupki strain paves the way for mouse models in pre-clinical testing of pharmaceuticals designed to modulate DNA-binding activity of human p53.

Poly(ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis.

Poly(ADP-ribosyl)ation is an immediate cellular response to DNA damage generated either exogenously or endogenously. This post-translational modification is catalyzed by poly(ADP-ribose) polymerase (PARP, PARP-1, EC 2.4.2.30). It is proposed that this protein plays a multifunctional role in many cellular processes, including DNA repair, recombination, cell proliferation and death, as well as genomic stability. Chemical inhibitors of the enzyme, dominant negative or null mutations of PARP-1 cause a high degree of genomic instability in cells. Inhibition of PARP activity by chemical inhibitors renders mice or rats susceptible to carcinogenic agents in various tumor models, indicating a role for PARP-1 in suppressing tumorigenesis. Despite the above observations, PARP-1 knockout mice are generally not prone to the development of tumors. An enhanced tumor development was observed, however, when the PARP-1 null mutation was introduced into severely compromised immune-deficient mice (a mutation in DNA-dependent protein kinase) or mice lacking other DNA repair or chromosomal guardian molecules, such as p53 or Ku80. These studies indicate that PARP-1 functions as a cofactor to suppress tumorigenesis via its role in stabilization of the genome, and/or by interacting with other DNA strand break-sensing molecules. Studies using PARP-1 mutants and chemical inhibitors have started to shed light on the role of this protein and of the specific protein post-translational modification in the control of genomic stability and hence its involvement in cancer.

Mechanism of antimitogenic action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal growth factor-stimulated cell growth.

Because the efficacy of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] in treatment of colon cancer might critically depend on its ability to specifically counteract epidermal growth factor (EGF)-stimulated tumor cell growth, we utilized human colon adenocarcinoma-derived cells in primary culture as well as the Caco-2 cell line to elucidate possible sites of interaction of 1alpha,25-(OH)2D3 with signaling from EGF receptor activation. In both types of colon cancer cells investigated, 10(-8) M 1alpha,25-(OH)2D3 reduced basal cell proliferation by about 50%, and prevented any rise in proliferation when colon cancer cells were treated with 25 ng/ml EGF: this can be explained by a marked inhibitory effect of 1alpha,25-(OH)2D3 on EGFR mRNA and protein expression. The steroid hormone also seemingly promotes EGF-induced internalization of apical and basolateral membrane EGFR. In addition, 1alpha,25-(OH)2D3 significantly reduced basal and EGF-stimulated expression of cyclin D1 at the mRNA and protein level in primary cultures as well as in the Caco-2 cell line. The ability of 1alpha,25-(OH)2D3 to interfere with a key event in cell cycle control and thereby to block mitogenic signaling from EGF could be seen as advantageous for the potential use of vitamin D compounds in colon cancer therapy.

Functions of poly(ADP-ribose) polymerase in controlling telomere length and chromosomal stability.

In most eukaryotes, poly(ADP-ribose) polymerase (PARP) recognizes DNA strand interruptions generated in vivo. DNA binding by PARP triggers primarily its own modification by the sequential addition of ADP-ribose units to form polymers; this modification, in turn, causes the release of PARP from DNA ends. Studies on the effects of the disruption of the gene encoding PARP (Adprt1, formerly Adprp) in mice have demonstrated roles for PARP in recovery from DNA damage and in suppressing recombination processes involving DNA ends. Telomeres are the natural termini of chromosomes and are, therefore, potential targets of PARP. Here, by the use of two different techniques, we show that mice lacking PARP display telomere shortening compared with wild-type mice. Telomere shortening is seen in different genetic backgrounds and in different tissues, both from embryos and adult mice. In vitro telomerase activity, however, is not altered in Adprt1-/- mouse fibroblasts. Furthermore, cytogenetic analysis of mouse embryonic fibroblasts reveals that lack of PARP is associated with severe chromosomal instability, characterized by increased frequencies of chromosome fusions and aneuploidy. The absence of PARP does not affect the presence of single-strand overhangs, naturally present at the ends of telomeres. This study therefore reveals an unanticipated role for PARP in telomere length regulation and provides insights into its functions in maintaining genomic integrity.