Activation Of DNA Damage Pathways Research Articles

Compound 225# inhibits the proliferation of human colorectal cancer cells by promoting cell cycle arrest and apoptosis induction.

Colorectal cancer (CRC) ranks as the second leading cause of cancer‑related death worldwide due to its aggressive nature. After surgical resection, >50% of patients with CRC require adjuvant therapy. As a result, eradicating cancer cells with medications is a promising method to treat patients with CRC. In the present study, a novel compound was synthesized, which was termed compound 225#. The inhibitory activity of compound 225# against CRC was determined by MTT assay, EdU fluorescence labeling and colony formation assay; the effects of compound 225# on the cell cycle progression and apoptosis of CRC cells were detected by flow cytometry and western blotting; and the changes in autophagic flux after the administration of compound 225# were detected using the double fluorescence fusion protein mCherry‑GFP‑LC3B and western blotting. The results demonstrated that compound 225# exhibited antiproliferative properties, inhibiting the proliferation and expansion of CRC cell lines in a time‑ and dose‑dependent manner. Furthermore, compound 225# triggered G2/M cell cycle arrest by influencing the expression of cell cycle regulators, such as CDK1, cyclin A1 and cyclin B1, which is also closely related to the activation of DNA damage pathways. The cleavage of PARP and increased protein expression levels of PUMA suggested that apoptosis was triggered after treatment with compound 225#. Moreover, the increase in LC3‑II expression and stimulation of autophagic flux indicated the activation of an autophagy pathway. Notably, compound 225# induced autophagy, which was associated with endoplasmic reticulum (ER) stress. In accordance with the in vitro findings, the in vivo results demonstrated that compound 225# effectively inhibited the growth of HCT116 tumors in mice without causing any changes in their body weight. Collectively, the present results demonstrated that compound 225# not only inhibited proliferation and promoted G2/M‑phase cell cycle arrest and apoptosis, but also initiated cytoprotective autophagy in CRC cells by activating ER stress pathways. Taken together, these findings provide an experimental basis for the evaluation of compound 225# as a novel potential medication for CRC treatment.

Cytotoxicities of Co-occurring alternariol, alternariol monomethyl ether and tenuazonic acid on human gastric epithelial cells.

Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) are the three major Alternaria toxin contaminants in food. In the present study, we conducted their single and combined toxicity analyses using human gastric epithelial cell line (GES-1) that was first exposed to the toxins when they entered the human body. By comparing the cytotoxicity IC50, we found that compared to several other mycotoxins with limit standards there was cytotoxicity DON>OTA>AME>AOH>ZEN>TeA. Further, we obtained combination index (CI)-isobologram equation by the Chou-Talalay method according to a toxin ratio of 1:1:2 and carried out the combined toxicity analysis of the three binary and ternary compounds, and the results showed that AOH+AME+TeA showed synergistic toxic effects. Based on the co-occurring status, we also carried out the combined toxicity analysis of AME and AOH at different ratios and found antagonistic effects at low cytotoxic concentrations as well as synergistic and additive effects at high concentrations. Also, we found that all three and their combinations caused apoptosis, activation of caspase-3 cleavage, activation of DNA damage pathways ATR-Chk1-P53 and ATM-Chk2-P53. In conclusion, we used GES-1cells to inform the risk of coaction of AOH, AME, and TeA in dietary exposure.

P610: SYNERGISTIC ACTIVITY OF FTO INHIBITOR FB23-2 WITH IBRUTINIB IN XENOGRAFT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Several studies have documented that FB23-2, as the selective inhibitor of RNA N6-methyladenosine (m6A) demethylase FTO, exerts crucial role in dampening tumorigenesis. Indeed, FTO deactivation was proved to render resistant leukemia cells sensitive to targeted agents, which suggests a potential combination therapy strategy of FB23-2. Aims: The present study was aimed to identify the synergistic utility of FB23-2 combination with ibrutinib in CLL. Methods: A disseminated leukemia xenograft murine model was established to explore the efficacy of FB23-2 in CLL. Leukemia burden of CLL mice was quantified by luciferase intensity using bioluminescence imaging. Bone marrow and spleen infiltration of leukemic cells was assessed by flow cytometry. And cell survival was determined by cell counting kit-8. Results: To investigate the combinatory effect of FB23-2 and ibrutinib, cell viability assay was performed firstly. FB23-2 displayed potent synergistic activity with ibrutinib in promoting cytotoxicity in CLL cells (Figure 1A). Consistently, FTO silencing mediated by lentivirus exhibited exquisite sensitivity to ibrutinib in CLL cells (Figure 1B). Besides, CLL primary cells and MEC-1 cells with treatment of FB23-2 and ibrutinib simultaneously triggered increased cell apoptosis compared to single agent (Figure 1C). Further underlying cooperative mechanism investigation demonstrated an aberrant activation of DNA damage pathway after serial dilution of FB23-2 treatment. Hence, enhanced expression of p-ATM, p-chk2 and p-H2AX was detected in FTO knockdown and ibrutinib co-treatment group, indicating FTO inhibition inducing ibrutinib-sensitivity via regulating DNA damage (Figure 1D). In addition, we investigated the efficacy of FB23-2 alone and corporation in CLL xenograft murine model. Compared to ibrutinib alone, combination treatment with FB23-2 significantly prolonged the lifespan of CLL mice (median survival time 20 versus 22.5 days, respectively, p=0.02, Figure 1E). FB23-2+ibrutinib group showed an obviously declined proportion of CLL cells compared to ibrutinib-treated alone both in bone marrow(20.93%±2.95% versus 37.17%±4.22%, p=0.02) and spleen (23.58%±6.03% and 54.58%±9.01%, p=0.03, Figure 1F-G). Similar results were obtained with three treated group relative to control group (all p<0.05). Moreover, FB23-2 combined with ibrutinib diminished leukemia burden and splenomegaly in CLL mice (Figure 1H-I). As shown in Figure 1J, leukemia involvement and malignant proliferation were more evident in control group, confirming the potent anti-leukemic activity of FB23-2. Image:Summary/Conclusion: Taken together, our investigation provides pre-clinical evidence for the utility of FB23-2 in CLL, especially combinatory benefit with ibrutinib. FB23-2 represents a promising strategy to novel treatment optimum in CLL.

Replication Stress Response Modifies Sarcomeric Cardiomyopathy Remodeling.

BackgroundSarcomere gene mutations lead to cardiomyocyte hypertrophy and pathological myocardial remodeling. However, there is considerable phenotypic heterogeneity at both the cellular and the organ level, suggesting modifiers regulate the effects of these mutations. We hypothesized that sarcomere dysfunction leads to cardiomyocyte genotoxic stress, and this modifies pathological ventricular remodeling.Methods and ResultsUsing a murine model deficient in the sarcomere protein, Mybpc3−/− (cardiac myosin‐binding protein 3), we discovered that there was a surge in cardiomyocyte nuclear DNA damage during the earliest stages of cardiomyopathy. This was accompanied by a selective increase in ataxia telangiectasia and rad3‐related phosphorylation and increased p53 protein accumulation. The cause of the DNA damage and DNA damage pathway activation was dysregulated cardiomyocyte DNA synthesis, leading to replication stress. We discovered that selective inhibition of ataxia telangiectasia and rad3 related or cardiomyocyte deletion of p53 reduced pathological left ventricular remodeling and cardiomyocyte hypertrophy in Mybpc3−/− animals. Mice and humans harboring other types of sarcomere gene mutations also had evidence of activation of the replication stress response, and this was associated with cardiomyocyte aneuploidy in all models studied.ConclusionsCollectively, our results show that sarcomere mutations lead to activation of the cardiomyocyte replication stress response, which modifies pathological myocardial remodeling in sarcomeric cardiomyopathy.

Nuclear ErbB-2-Induced Transcriptome Drives Triple Negative Breast Cancer Growth

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2 tyrosine kinase receptor. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Gene expression analyses have categorized TNBC into distinct molecular subtypes. Up to 78% of clinical TNBCs belong to the basal-like (BL) subtype. Here we found ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing, using a PCR-sequencing approach combined with RNA interference, revealed that BL TNBC cells express the canonical ErbB-2 (WTErbB-2), encoded by transcript 1, and the non-canonical isoform c, encoded by alternative transcript 3 (T3). The latter was not previously reported in normal or malignant cells. To characterize the isoform c we designed siRNAs targeting T3 (T3 siRNAs), which silenced up to 93% of said isoform. Transfection of T3 siRNAs into BL cells expressing only isoform c or both isoform c and WTErbB-2 was sufficient to decrease cell proliferation. Intratumoral injections of T3 siRNAs into mice bearing BL TN tumors also blocked in vivo growth. To explore whether isoform c growth-promoting effect is due to its functions as a transcriptional regulator, we performed RNA-seq in BL cells expressing only this isoform. We identified a set of genes differentially regulated in BL cells where we evicted isoform c from the nucleus, as compared to control cells. In the up-regulated group, we found enrichment of pro-apoptotic and tumor suppressor genes and in the down-regulated one, genes involved in proliferation and stemness. We used gene set enrichment analysis (GSEA) to identify the biological processes associated with these isoform c-regulated genes. We found a pronounced enrichment of gene sets related to apoptosis, activation of DNA damage pathways and cell cycle arrest in response to eviction of nuclear isoform c. GSEA also revealed negative regulation of gene sets involved in cell motility, cellular differentiation and growth pathways in BL cells lacking nuclear isoform c expression. These results suggest that NErbB-2 function modulates tumor growth and promotes a metastatic phenotype in TNBC. Furthermore, our clinical findings identified NErbB-2 as an independent predictor of shorter OS(HR 2.54; 95% CI 1.22-5.28; P = 0.013), DFS (HR 2.91; 95% CI 1.44-5.87; P = 0.003), and DMFS (HR 2.59; 95% CI 1.20-5.60; P = 0.015) in 99 TN primary tumors. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type proteins, which conserve the canonical domains and are located in their classical cellular compartments, highlighting the potential of NErbB-2 isoforms as novel therapeutic targets and clinical biomarkers in TNBC.

APOBEC3B is induced by activation of DNA damage pathway and regulates the survival and treatment response in human multiple myeloma

Apolipoprotein B mRNA editing catalytic polypeptide-like 3B (APOBEC3B), a DNA cytosine deaminase causing cytosine-to-uracil (C-to-U) deamination in single-stranded DNA, plays a prominent role in inducing mutations in multiple human cancers. In MM, high APOBEC3B activity is associated with a poor prognosis and expression of APOBEC3B is significantly up-regulated in MM patients with t(14;16) and t(14:20). However, only approximately 23 % of patients with high APOBEC3B are linked to MAF/MAFB/MAFA translocations.

Suppression of MicroRNA 424 Levels by Human Papillomaviruses Is Necessary for Differentiation-Dependent Genome Amplification.

High-risk human papillomaviruses (HPVs) link their life cycle to epithelial differentiation and require activation of DNA damage pathways for efficient replication. HPVs modulate the expression of cellular transcription factors, as well as cellular microRNAs (miRNAs) to control these activities. One miRNA that has been reported to be repressed in HPV-positive cancers of the cervix and oropharynx is miR-424. Our studies show that miR-424 levels are suppressed in cell lines that stably maintain HPV 31 or 16 episomes, as well as cervical cancer lines that contain integrated genomes such as SiHa. Introduction of expression vectors for miR-424 reduced both the levels of HPV genomes in undifferentiated cells and amplification upon differentiation. Our studies show that the levels of two putative targets of miR-424 that function in DNA damage repair, CHK1 and Wee1, are suppressed in HPV-positive cells, providing an explanation for why this microRNA is targeted in HPV-positive cells.IMPORTANCE We describe here for the first time a critical role for miR-424 in the regulation of HPV replication. HPV E6 and E7 proteins suppress the levels of miR-424, and this is important for controlling the levels of CHK1, which plays a central role in viral replication.

Abstract 5740: Aberration of (pro)renin receptor induces genomic instablity in human pancreatic ductal epithelial cells

Abstract Background: We have recently shown the potential contribution of (pro)renin receptor ((P)RR) to tumorigenesis of pancreatic ductal adenocarcinoma (PDAC) through an activation of Wnt/β-catenin signaling pathway (Sci. Rep., 2015). We also found that significant (P)RR expression was synchronized with an appearance of atypical nuclei in PanIN-2 of PDAC tissues. Furthermore, (P)RR knockdown reduced a total of DNA amount in human PDAC cell lines. Based on our findings, we have hypothesized that an aberration of (P)RR leads to a failure of genome homeostasis. Accordingly, we explored whether an aberrant expression of (P)RR induces genomic instability in human pancreatic ductal epithelial (HPDE) cells which are capable of developing to the PDAC. Methods: HPDE cells transfected by episomal vector inserted with ATP6ap2 encoding (P)RR were established. HPDE cells transfected by vector without the insertion of ATP6ap2 was noted as Mock. We also performed a comet assay to evaluate the defects of DNA repair capacity, flow-FISH to measure the length of telomere and western blotting to confirm the activation of DNA damage pathway by measuring protein levels of γ- H2AX and p53. Then, comparative analyses of human whole genome in Mock- expressing and (P)RR-expressing HPDE cells were examined. Furthermore, we implanted 14-passage HPDE cells expressed by either Mock or (P)RR into the renal subcapsules of immunodeficient mice to investigate whether (P)RR-expressing HPDE cells show a precancerous status in vivo. Results: (P)RR overexpression led to inactivation of γ-H2AX and p53 as the components of DNA damage pathway, which were associated with a disappearance of comet tail and shorten telomere length. Substantial structural variations, e.g., inter- and intra- chromosomal translocations throughout all the chromosomes were observed in HPDE cells expressed by (P)RR under the analysis by BreakDancer. Analyses with Bcftool showed that the number of all the somatic mutations was approximately 2-fold greater in HPDE cells expressed by (P)RR than those expressed by Mock (Mock: 124,579 vs. (P)RR: 239,080). (P)RR overexpression also significantly increased the number of somatic mutations in several genes which were commonly detected in PDAC tissues. The area occupied by (P)RR-expressing HPDE cell population remarkably expanded after the implantation to renal subcapsules of all the immunodeficient mice. However, there were no KRAS 12 codon mutations which are prevalent in PDAC patients. Conclusion: We have demonstrated that (P)RR overexpression induces genomic instability on global genome level in HPDE cells. These data are consistent with our hypothesis that (P)RR is essentially involved in the early carcinogenesis of PDAC. Citation Format: Yuki Shibayama, Jun Yasuda, Daisuke Yamazaki, Asadur Rahman, Shinichi Yachida, Akira Nishiyama. Aberration of (pro)renin receptor induces genomic instablity in human pancreatic ductal epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5740. doi:10.1158/1538-7445.AM2017-5740

RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest

The 90 kDa Ribosomal S6 Kinase (RSK), downstream of the ERK signaling pathway, has recently been implicated in a wide variety of cancers, ranging from lung cancer to medulloblastoma, as a driver of cancer cell proliferation and survival. However, its role in Acute Myeloid Leukemia (AML) remains unknown. Thus, the goal of this study was to characterize RSK-dependent signaling pathways in AML, with the overall hypothesis that disruption of this pathway represents a potential strategy for the treatment of AML. The RSK family consists of four gene isoforms, RSK1-4 (RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA3 (RSK2), RPS6KA4 (RSK4). Knockdown (KD) of RSK1 by shRNA in HL-60 and KG-1 cell lines resulted in reduced AML cell growth in vitro. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with 2x106 HL-60 or KG-1 RSK1KD cells and vector control transduced cells in order to investigate the effects of RSK1 KD on AML cell growth and survival in vivo. Mice injected with RSK1 KD cells exhibited prolonged survival by 17 and 21 days respectively for HL-60 and KG-1 cell induced disease (p=0.0023 and 0.0018 respectively). These data indicate that RSK1 knockdown inhibits leukemia progression, and RSK1 is required for maximal proliferation of AML cells in vivo. Pharmacological inhibition of total RSK (RSK1-4) by the small molecule inhibitor BI-D1870 reduced AML cell growth and induced cell death in both AML cell lines and patient samples after treatment for 48 hours. The IC50 for growth inhibition was 1.8 uM for MOLM-13, 1.6 uM for MV-4-11, and 1.9 uM for HL-60 cells. In methylcellulose colony assays, normal hematopoietic stem and progenitor cell proliferation was not affected by RSK inhibition up to a concentration of 15 uM, establishing an approximately 10-fold therapeutic index. To elucidate the mechanism by which RSK inhibition suppresses AML proliferation, we performed cell cycle analysis with HL-60 cells. RSK inhibition by BI-D1870 resulted in delayed S-phase progression and accumulation of cells in late S-phase with increased pH2AX, cPARP, and CDK2/Cyclin A expression, as measured by flow cytometry. These data indicate that inhibition of RSK leads to activation of DNA damage pathways and arrest in S-phase, resulting in apoptosis. Inhibition of CDK activity rescued S-phase arrest, demonstrating that activation and dysregulation of CDK are crucial mediators of RSK inhibitor-induced S-phase arrest. In summary, this is the first study to demonstrate that RSK plays an important role in maintaining AML cell survival and proliferation and to position RSK as a promising target for treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

The acetyltransferase Tip60 is a critical regulator of the differentiation-dependent amplification of human papillomaviruses.

The life cycle of human papillomaviruses (HPVs) is dependent upon differentiation of the infected host epithelial cell as well as activation of the ataxia telangiectasia mutated (ATM) DNA repair pathway that in normal cells acts to repair double-strand DNA breaks. In normal cells, following DNA damage the acetyltransferase Tip60 must acetylate ATM proteins prior to their full activation by autophosphorylation. E6 proteins have been shown to induce the degradation of Tip60, suggesting that Tip60 action may not be required for activation of the ATM pathway in HPV-positive cells. We investigated what role, if any, Tip60 plays in regulating the differentiation-dependent HPV life cycle. Our study indicates that Tip60 levels and activity are increased in cells that stably maintain complete HPV genomes as episomes, while low levels are seen in cells that express only HPV E6 and E7 proteins. Knockdown of Tip60 with short hairpin RNAs in cells that maintain HPV episomes blocked ATM induction and differentiation-dependent genome amplification, demonstrating the critical role of Tip60 in the viral life cycle. The JAK/STAT transcription factor STAT-5 has previously been shown to regulate the phosphorylation of ATM. Our studies demonstrate that STAT-5 regulates Tip60 activation and this occurs in part by targeting glycogen synthase kinase 3β (GSK3β). Inhibition of either STAT-5, Tip60, or GSK3β blocked differentiation-dependent genome amplification. Taken together, our findings identify Tip60 to be an important regulator of HPV genome amplification whose activity during the viral life cycle is controlled by STAT-5 and the kinase GSK3β. Human papillomaviruses (HPVs) are the etiological agents of cervical and other anogenital cancers. HPVs regulate their differentiation-dependent life cycle by activation of DNA damage pathways. This study demonstrates that HPVs regulate the ATM DNA damage pathway through the action of the acetyltransferase Tip60. Furthermore, the innate immune regulator STAT-5 and the kinase GSK3β mediate the activation of Tip60 in HPV-positive cells. This study identifies critical regulators of the HPV life cycle.

Effect of hypothermia on doxorubicin-induced cardiac myoblast signaling and cell death

Anthracyclines (AC) are useful chemotherapeutic agents whose principal limitation is cardiac toxicity, which may progress to heart failure, transplantation or even death. We have shown that this toxicity involves oxidative stress-induced activation of the DNA damage pathway. Hypothermia has been shown to be protective against other diseases involving oxidative stress but has not been studied in models of AC toxicity. In the current experiments, H9C2 cardiac myoblasts were treated with varying concentrations of the AC doxorubicin (DOX) during normothermia (37°C) or mild hypothermia (35°C). Total cell death was assayed using trypan blue exclusion and apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine-biotin nick end labeling (TUNEL) staining. Oxidative stress was assayed using the fluorescent indicator 2'7'-dichlorofluorescein diacetate. DNA damage pathway activation was assayed by immunostaining for H2AX and p53. Mitochondrial membrane potential was assayed by JC-1 staining. At all concentrations of DOX examined (1, 2.5 and 5 μM), hypothermia reduced oxidative stress, activation of H2AX and p53, loss of mitochondrial membrane potential and total and apoptotic cell death (P=.001-.03 for each observation). The reduction of oxidative stress-induced activation of the DNA damage pathway and consequent cell death by mild hypothermia supports a possible protective role to reduce the clinical impact of DOX-induced cardiac toxicity. Such an approach may allow expanded use of these effective chemotherapeutic agents to increase cancer cure rates.

Telomere Maintenance as Therapeutic Target in Embryonal Tumours

Embryonal tumours most commonly occur in the first few years of life and account for approximately 30% of childhood malignancies. Knowledge of these tumours' genetics has already impacted on their clinical management and further knowledge of their cellular immortalization will hopefully result in novel therapies. The ends of human chromosomes are capped and protected by telomeres; cellular replication, however, causes their loss. A critical length of telomere repeats is required to ensure proper telomere function and avoid the activation of DNA damage pathways that result in senescence and cell death. To proliferate beyond the senescence checkpoint, cells must restore their telomere length. Hence stabilization of telomere is an important step in cell immortalization and carcinogenesis. Telomere maintenance is evident in virtually all types of malignant cells, including embryonal tumours, where either a telomerase-dependent or alternative lengthening of telomeres (ALT) mechanism is employed in order to ensure their limitless replicative potential. For this reason effective strategies targeting telomere maintenance in cancer cells require a combination of telomerase and ALT inhibitors. In this review, we are giving an overview about telomere maintenance in childhood tumours and discussing its potential as a new therapeutic target.

Role of Chk1 in the differentiation program of hematopoietic stem cells.

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation

Telomere shortening limits the proliferative lifespan of human cells by activation of DNA damage pathways, including upregulation of the cell cycle inhibitor p21 (encoded by Cdkn1a, also known as Cip1 and Waf1)) (refs. 1-5). Telomere shortening in response to mutation of the gene encoding telomerase is associated with impaired organ maintenance and shortened lifespan in humans and in mice. The in vivo function of p21 in the context of telomere dysfunction is unknown. Here we show that deletion of p21 prolongs the lifespan of telomerase-deficient mice with dysfunctional telomeres. p21 deletion improved hematolymphopoiesis and the maintenance of intestinal epithelia without rescuing telomere function. Moreover, deletion of p21 rescued proliferation of intestinal progenitor cells and improved the repopulation capacity and self-renewal of hematopoietic stem cells from mice with dysfunctional telomeres. In these mice, apoptotic responses remained intact, and p21 deletion did not accelerate chromosomal instability or cancer formation. This study provides experimental evidence that telomere dysfunction induces p21-dependent checkpoints in vivo that can limit longevity at the organismal level.

Characterization of specific HHV-6 and cell cycle genes implicated in virus-mediated G1/S cell-cycle arrest of glial precursors

Human herpesvirus 6 (HHV-6), a common resident virus of the human central nervous system (CNS), has been implicated in both acute and chronic inflammatory-demyelinating diseases. HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes and astrocytes. We recently demonstrated that HHV-6 infects human glial precursor cells, the ancestors of myelin producing oligodendrocytes, in vitro. Productive infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins. Infection led to impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by BrdU uptake and demonstrated strong G1/S-phase inhibition by FACS. We now present evidence detailing both specific viral and cell cycle genes inducing cellular G1/S cell cycle arrest. In parallel with human cells, the well characterized rodent oligodendroglial progenitor cell (OPC) system was used. Restricted infection of the murine cells was demonstrated by quantitative PCR, EM, and RT-PCR, and IFA for viral late proteins but, intriguingly, reproduced the G1/S arrest seen after productive virus infection of human progenitors. Restricted infection of murine OPCs was also found to be sufficient to induce the activation of DNA damage pathways previously reported by our group in human glial progenitors (ISNV 2004) and after productive infection in human lymphocytes (Oster et al. 2005). IFA and immunoblot again demonstrated expression of phospho-ATM, CHK-2, and p53 in infected murine precursors and two viral IE proteins were sufficient to induce G1/S arrest. Virus-induced cell-cycle arrest was accompanied by GalC+ differentiation with corresponding loss of the OPC pool as had been previously seen in the human precursors. Enhanced GFP-expressing murine OPCs have been created that can be readily infected with ALEXA-632-labeled HHV-6, allowing both sorting for infected cells and their identification in evaluating repair in demyelinated mice. In light of recent observations that repair of CNS remyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in human demyelinating diseases and the disruption of normal glial differentiation.

Tumor Formation via Loss of a Molecular Motor Protein

Aneuploidy has long been suggested to be causal in tumor formation. Direct testing of this hypothesis has been difficult because of the absence of methods to specifically induce aneuploidy. The chromosome-associated kinesin motor KIF4 plays multiple roles in mitosis, and its loss leads to multiple mitotic defects including aneuploidy. Here, we have taken advantage of the direct formation of aneuploidy in the absence of KIF4 to determine whether loss of a molecular motor and generation of aneuploidy during mitosis can trigger tumorigenesis. We find that embryonic stem cells genetically depleted of KIF4 support anchorage-independent growth and form tumors in nude mice. In cells lacking KIF4, mitotic spindle checkpoints and DNA-damage response pathways are activated. Down regulation or loss of KIF4 is physiologically relevant because reduced KIF4 levels are present in 35% of human cancers from several tissues. Our results support the notion that loss of a molecular motor leads to tumor formation and that aneuploidy can act as a primary trigger of tumorigenesis.