Anna A. Kondratova

The circadian clock and pathology of the ageing brain

Ageing leads to a functional deterioration of many brain systems, including the circadian clock — an internal time-keeping system that generates ∼24-hour rhythms in physiology and behaviour. Numerous clinical studies have established a direct correlation between abnormal circadian clock functions and the severity of neurodegenerative and sleep disorders. Latest data from experiments in model organisms, gene expression studies and clinical trials imply that dysfunctions of the circadian clock contribute to ageing and age-associated pathologies, thereby suggesting a functional link between the circadian clock and age-associated decline of brain functions. Potential molecular mechanisms underlying this link include the circadian control of physiological processes such as brain metabolism, reactive oxygen species homeostasis, hormone secretion, autophagy and stem cell proliferation.

Poliovirus Protein 3A Inhibits Tumor Necrosis Factor (TNF)-Induced Apoptosis by Eliminating the TNF Receptor from the Cell Surface

ABSTRACT Viral infections often trigger host defensive reactions by activating intrinsic (intracellular) and extrinsic (receptor-mediated) apoptotic pathways. Poliovirus is known to encode an antiapoptotic function(s) suppressing the intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on cell sensitivity to tumor necrosis factor (TNF)-induced (i.e., receptor-mediated) apoptosis was studied. This sensitivity is dramatically enhanced by the viral proteinase 2A, due, most likely, to inhibition of cellular translation. On the other hand, cells expressing poliovirus noncapsid proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes the proapoptotic activity of 2A and results in a specific suppression of TNF signaling, including the lack of activation of NF-κB, due to elimination of the TNF receptor from the cell surface. In agreement with this, poliovirus infection results in a dramatic decrease in TNF receptor abundance on the surfaces of infected cells as early as 4 h postinfection. Poliovirus proteins that confer resistance to TNF interfere with endoplasmic reticulum-Golgi protein trafficking, and their effect on TNF signaling can be imitated by brefeldin A, suggesting that the mechanism of poliovirus-mediated resistance to TNF is a result of aberrant TNF receptor trafficking.

Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis.

The mammalian circadian system has been implicated in the regulation of various biological processes including those involved in genotoxic stress responses and tumor suppression. Here we report that mice with the functional deficiency in circadian transcription factor CLOCK (Clock/Clock mutant mice) do not display predisposition to tumor formation both during their normal lifespan or when challenged by γ-radiation. This phenotype is consistent with high apoptotic and low proliferation rate in lymphoid tissues of Clock mutant mice and is supported by the gene expression profiling of a number of apoptosis and cell cycle-related genes, as well as by growth inhibition of cells with CLOCK downregulation. At the same time, Clock mutant mice respond to low-dose irradiation by accelerating their aging program, and develop phenotypes that are reminiscent of those in Bmal1-deficient mice. Taken together, our results demonstrate the dichotomy in biological consequences of the disruption of the circadian clock with respect to ageing and cancer. They also highlight the existence of a complex interconnection between ageing, carcinogenesis and individual components of the circadian clock machinery.

Dual role of the CLOCK/BMAL1 circadian complex in transcriptional regulation

The basic helix–loop–helix (bHLH) –PAS domain containing transcription factors CLOCK and BMAL1 are two major components of the circadian molecular oscillator. It is known that the CLOCK/BMAL1 complex positively regulates the activity of E‐box containing promoters. Here we demonstrate that the CLOCK/BMAL1 complex can also suppress the activity of some promoters upon its interaction with CRYPTOCHROME (CRY). Such a dual function of the circadian transcriptional complex provides a mechanistic explanation for the unpredicted pattern of circadian gene expression in the tissues of Bmal1 null mice. We speculate that the switch from transcriptional activation to transcriptional repression may provide a highly efficient mechanism for circadian control of gene expression. We also show that CLOCK/BMAL1 can interfere with promoter regulation by other, non‐circadian, transcription factors including N‐MYC and ETS, leading to attenuation or abrogation of transcription of CLOCK/BMAL1‐controlled stress‐induced genes. We propose that, based upon these results, both circadian repression and activation of the transcription of different target genes are required for circadian responses to various external stimuli, including genotoxic stress induced by anticancer treatment.

Post-translational regulation of circadian transcriptional CLOCK(NPAS2)/BMAL1 complex by CRYPTOCHROMES.

Mammalian CLOCK(NPAS2), BMAL1 and CRYPTOCHROMEs are core components of the circadian oscillatory mechanism. The active CLOCK/BMAL1 or NPAS2/BMAL1 complexes regulate expression of numerous genes including two Cryptochromes. The products of these genes, CRY1 and CRY2, in turn repress CLOCK/BMAL1 transcriptional activity by an unknown mechanism. We have examined the effect of CRYPTOCHROMEs on posttranslational modifications and intracellular distribution of endogenous and ectopically expressed CLOCK(NPAS2) and BMAL1 proteins. We found that ectopic coexpression with CRY led to stabilization and nuclear accumulation of unphosphorylated forms of the proteins, which directly correlated with the inhibition of their transcriptional activity. This effect was CRY-specific, as other known repressors of CLOCK/BMAL1 and NPAS2/BMAL1 transcriptional activity were not able to induce similar effects. CRYs had no effect on CLOCK(NPAS2)/BMAL1 complex formation or its ability to bind DNA. Altogether, these results demonstrate that CRYs regulate the functional activity of circadian transcriptional complex at the posttranslational level. Importantly, the posttranslational modifications and intracellular distribution of CLOCK and BMAL1 proteins were critically impaired in the tissues of mice with targeted disruption of both Cry genes, thus confirming the suggested role of CRY in clock function in vivo. Based on these findings we propose a modified model of the circadian transcriptional control, which implies CRY-mediated periodic rotation of transcriptionally active and inactive forms of CLOCK/BMAL1 on the promoter. This model provides mechanistic explanation for previously reported dual functional activity of CLOCK/BMAL1 and highlights the involvement of the circadian system in modulating the organism’s response to various types of genotoxic stress, including chemotherapy and radiation.

Circadian clock protein BMAL1 regulates cellular senescence in vivo

Deficiency of the circadian clock transcriptional factor BMAL1 results in the development of premature aging in mice. In agreement with the accelerated aging phenotype, we observed an increase in the number of senescent cells in different tissues (lungs, liver and spleen) of Bmal1-/- mice, which suggests the important role of BMAL1 in the control of senescence in vivo. However, no difference in the rate of proliferation and senescence between primary fibroblasts isolated from wild-type and Bmal1-/- mice has been detected, suggesting that BMAL1 does not play a significant role in replicative senescence in vitro. BMAL1 deficient fibroblasts had an increased sensitivity to hydrogen peroxide treatment, and reduced sensitivity to DNA damaging anticancer drugs etoposide and daunorubicin. Increased sensitivity of Bmal1-/- cells to oxidative stress was p53 independent and correlated with the disrupted regulation of reactive oxygen species (ROS) homeostasis in BMAL1 deficient cells: indeed, circadian oscillations of ROS level can be induced in wild-type but not in Bmal1-/- cells. We propose that BMAL1 is important for the regulation of oxidative stress and DNA damage responses, while deregulation of these processes upon BMAL1 deficiency leads to development of stress induced senescence in vivo.

Poliovirus Protein 3A Binds and Inactivates LIS1, Causing Block of Membrane Protein Trafficking and Deregulation of Cell Division

Poliovirus infection results in resistance of infected cells to apoptotic stimuli. Viral proteins involved in such functions usually target key cellular regulators. Here we demonstrate that viral protein 3A binds and inactivates LIS1, a component of the dynein/dynactin motor complex, encoded by the gene mutated in patients with type I lissencephaly („smooth brain‰), thus causing rapid disappearence of TNF and interferon receptors from the plasma membrane. Like 3A, truncated derivatives of LIS, acting in a dominant negative manner, deregulate endoplasmatic reticilum -to-Golgi vesicular transport and eliminate unstable receptors from the cell surface. Protein 3A locks Golgi-targeted YFP in endoplasmatic reticilum, while expression of LIS1 mutants results in a dispersed cytoplasmic localization of the reporter protein. LIS1 dysfunction caused by ectopic expressing 3A or LIS1 mutants, as well as by overexpression of wild type LIS1, leads to cell trap at a postmitotic stage associated with inability to undergo cytokinesis. Thus, using vural protein as a research tool, we revealed the role of cellular protein LIS1 in endoplasmatic reticilum -to-Golgi transport, maintenance of Golgi integrity and cell cycle progression. Supplemental Material 1 Supplemental Material 2 Supplemental Material 3

Cutaneous Tumors Cease CXCL9/Mig Production as a Result of IFN-γ–Mediated Immunoediting

During growth in the host, tumor cells are subjected to the stresses of innate and adaptive immunity (immunoediting), which provoke epigenetic changes in the tumor and increase tumor resistance to these immune responses. Our recent studies in methylcholanthrene-induced fibrosarcomas have indicated the appearance and rapid growth of tumor variants deficient in producing the T cell chemoattractant chemokine CXCL9/Mig, an important component of antitumor immunity. In the current report, we demonstrate that highly tumorigenic Mig-deficient tumor variants arise in both cutaneous fibrosarcoma and melanoma as a result of immune stress imposed by IFN-γ and T cells. The consequence of the loss of tumor-derived Mig expression is the increased resistance of Mig-deficient tumors to T cell-mediated immunity, which promotes the accelerated growth of these tumor variants. Remarkably, the ability of Mig-deficient tumor cells to express another CXCR3 ligand, CXCL10/IFN-γ–inducible protein, does not compensate for the absent antitumor functions of Mig, suggesting a nonredundant role for this chemokine in the suppression of tumor growth. To our knowledge, these studies report for the first time that IFN-γ–mediated stress leads to the loss of specific chemokine expression by tumor cells, which in turn promotes tumor growth and evasion of the immune response.

A common copy-number breakpoint of ERBB2 amplification in breast cancer colocalizes with a complex block of segmental duplications.

IntroductionSegmental duplications (low-copy repeats) are the recently duplicated genomic segments in the human genome that display nearly identical (> 90%) sequences and account for about 5% of euchromatic regions. In germline, duplicated segments mediate nonallelic homologous recombination and thus cause both non-disease-causing copy-number variants and genomic disorders. To what extent duplicated segments play a role in somatic DNA rearrangements in cancer remains elusive. Duplicated segments often cluster and form genomic blocks enriched with both direct and inverted repeats (complex genomic regions). Such complex regions could be fragile and play a mechanistic role in the amplification of the ERBB2 gene in breast tumors, because repeated sequences are known to initiate gene amplification in model systems.MethodsWe conducted polymerase chain reaction (PCR)-based assays for primary breast tumors and analyzed publically available array-comparative genomic hybridization data to map a common copy-number breakpoint in ERBB2-amplified primary breast tumors. We further used molecular, bioinformatics, and population-genetics approaches to define duplication contents, structural variants, and haplotypes within the common breakpoint.ResultsWe found a large (> 300-kb) block of duplicated segments that was colocalized with a common-copy number breakpoint for ERBB2 amplification. The breakpoint that potentially initiated ERBB2 amplification localized in a region 1.5 megabases (Mb) on the telomeric side of ERBB2. The region is very complex, with extensive duplications of KRTAP genes, structural variants, and, as a result, a paucity of single-nucleotide polymorphism (SNP) markers. Duplicated segments are varied in size and degree of sequence homology, indicating that duplications have occurred recurrently during genome evolution.ConclusionsAmplification of the ERBB2 gene in breast tumors is potentially initiated by a complex region that has unusual genomic features and thus requires rigorous, labor-intensive investigation. The haplotypes we provide could be useful to identify the potential association between the complex region and ERBB2 amplification.

Replication fork integrity and intra-S phase checkpoint suppress gene amplification

Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs.