Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

• MeSH
• buněčná smrt genetika   MeSH
• lidé MeSH
• myši MeSH
• nádorová transformace buněk * genetika   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• poškození DNA * genetika   MeSH
• proliferace buněk genetika   MeSH
• proteinfosfatasa 2C * genetika   metabolismus   MeSH
• proteinfosfatasy genetika   metabolismus   MeSH
• stárnutí buněk * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH
• PPM1D protein, human MeSH Prohlížeč
• proteinfosfatasa 2C * MeSH
• proteinfosfatasy MeSH

Accumulation of senescent cells in tissues with advancing age participates in the pathogenesis of several human age-associated diseases. Specific senescent secretome, the resistance of senescent cells to apoptotic stimuli, and lack of immune system response contribute to the accumulation of senescent cells and their adverse effects in tissues. Inhibition of antiapoptotic machinery, augmented in senescent cells, by BCL-2 protein family inhibitors represents a promising approach to eliminate senescent cells from tissues. This study aimed to explore synergistic and selective senolytic effects of anti-apoptotic BCL-2 family targeting compounds, particularly BH3 mimetics. Using human non-transformed cells RPE-1, BJ, and MRC-5 brought to ionizing radiation-, oncogene-, drug-induced and replicative senescence, we found synergy in combining MCL-1 selective inhibitors with other BH3 mimetics. In an attempt to uncover the mechanism of such synergy, we revealed that the surviving subpopulation of cells resistant to individually applied ABT-737/ABT-263, MIK665, ABT-199, and S63845 BCL-2 family inhibitors showed elevated MCL-1 compared to untreated control cells indicating the presence of a subset of cells expressing high MCL-1 levels and, therefore, resistant to BCL-2 inhibitors within the original population of senescent cells. Overall, we found that combining BCL-2 inhibitors can be beneficial for eliminating senescent cells, thereby enabling use of lower, potentially less toxic, doses of drugs compared to monotherapy, thereby overcoming the resistance of the subpopulation of senescent cells to monotherapy.

• Klíčová slova
• BCL-2, MCL-1, cellular senescence, homoharringtonine, senolytics,
• MeSH
• apoptóza MeSH
• lidé MeSH
• protein MCL-1 metabolismus   MeSH
• protoonkogenní proteiny c-bcl-2 * antagonisté a inhibitory   MeSH
• stárnutí buněk * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein MCL-1 MeSH
• protoonkogenní proteiny c-bcl-2 * MeSH

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

• MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• HeLa buňky MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• nádory genetika   MeSH
• nestabilita genomu MeSH
• poškození DNA MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• replikace DNA * MeSH
• transport RNA MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetyltransferasy MeSH
• intracelulární signální peptidy a proteiny MeSH
• komplex proteinů jaderného póru MeSH
• MCM3AP protein, human MeSH Prohlížeč
• protoonkogenní proteiny MeSH
• TPR protein, human MeSH Prohlížeč

Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/- mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.

• Klíčová slova
• cancer, cell-cycle checkpoint, protein phosphatase, tumor suppressor p53,
• MeSH
• apoptóza * MeSH
• buněčný cyklus MeSH
• ionizující záření MeSH
• lymfom metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• nádory vyvolané zářením metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• proliferace buněk MeSH
• proteinfosfatasa 2C fyziologie   MeSH
• thymocyty cytologie   metabolismus   MeSH
• thymus * cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH
• Ppm1d protein, mouse MeSH Prohlížeč
• proteinfosfatasa 2C MeSH
• Trp53 protein, mouse MeSH Prohlížeč

Replication stress (RS) is a major driver of genomic instability and tumorigenesis. Here, we investigated whether RS induced by the nucleotide analog fludarabine and specific kinase inhibitors [e.g. targeting checkpoint kinase 1 (Chk1) or ataxia telangiectasia and Rad3-related (ATR)] led to apoptosis or senescence in four cancer cell lines differing in TP53 mutation status and expression of lamin A/C (LA/C). RS resulted in uneven chromatin condensation in all cell types, as evidenced by the presence of metaphasic chromosomes with unrepaired DNA damage, as well as detection of less condensed chromatin in the same nucleus, frequent ultrafine anaphase bridges, and micronuclei. We observed that responses to these chromatin changes may be distinct in individual cell types, suggesting that expression of lamin A/C and lamin B1 (LB1) may play an important role in the transition of damaged cells to senescence. MCF7 mammary carcinoma cells harboring wild-type p53 (WT-p53) and LA/C responded to RS by transition to senescence with a significant reduction of lamin B receptor and LB1 proteins. In contrast, a lymphoid cancer cell line WSU-NHL (WT-p53) lacking LA/C and expressing low levels of LB1 died after several hours, while lines MEC-1 and SU-DHL-4, both with mutated p53, and SU-DHL-4 with mutations in LA/C, died at different rates by apoptosis. Our results show that, in addition to being influenced by p53 mutation status, the response to RS (apoptosis or senescence) may also be influenced by lamin A/C and LB1 status.

• Klíčová slova
• ATR inhibitor, Chk1 inhibitor, apoptosis, lamin B receptor, replication stress, senescence,
• MeSH
• apoptóza fyziologie   MeSH
• lamin typ A metabolismus   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   MeSH
• replikace DNA fyziologie   MeSH
• stárnutí buněk fyziologie   MeSH
• vidarabin analogy a deriváty   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fludarabine MeSH Prohlížeč
• lamin typ A MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč
• vidarabin MeSH

Mutagenesis is a hallmark and enabling characteristic of cancer cells. The E3 ubiquitin ligase RAD18 and its downstream effectors, the 'Y-family' Trans-Lesion Synthesis (TLS) DNA polymerases, confer DNA damage tolerance at the expense of DNA replication fidelity. Thus, RAD18 and TLS polymerases are attractive candidate mediators of mutagenesis and carcinogenesis. The skin cancer-propensity disorder xeroderma pigmentosum-variant (XPV) is caused by defects in the Y-family DNA polymerase Pol eta (Polη). However it is unknown whether TLS dysfunction contributes more generally to other human cancers. Recent analyses of cancer genomes suggest that TLS polymerases generate many of the mutational signatures present in diverse cancers. Moreover biochemical studies suggest that the TLS pathway is often reprogrammed in cancer cells and that TLS facilitates tolerance of oncogene-induced DNA damage. Here we review recent evidence supporting widespread participation of RAD18 and the Y-family DNA polymerases in the different phases of multi-step carcinogenesis.

• Klíčová slova
• DNA damage, RAD18, cancer, genome maintenance, mutagenesis, trans-lesion synthesis (TLS),
• MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• genom lidský MeSH
• karcinogeneze genetika   metabolismus   patologie   MeSH
• lidé MeSH
• multigenová rodina MeSH
• mutageneze MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nádory genetika   metabolismus   patologie   MeSH
• poškození DNA MeSH
• regulace genové exprese u nádorů * MeSH
• signální transdukce MeSH
• ubikvitinligasy genetika   metabolismus   MeSH
• xeroderma pigmentosum genetika   metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• nádorové proteiny MeSH
• RAD18 protein, human MeSH Prohlížeč
• Rad30 protein MeSH Prohlížeč
• ubikvitinligasy MeSH

Aging involves tissue accumulation of senescent cells (SC) whose elimination through senolytic approaches may evoke organismal rejuvenation. SC also contribute to aging-associated pathologies including cancer, hence it is imperative to better identify and target SC. Here, we aimed to identify new cell-surface proteins differentially expressed on human SC. Besides previously reported proteins enriched on SC, we identified 78 proteins enriched and 73 proteins underrepresented in replicatively senescent BJ fibroblasts, including L1CAM, whose expression is normally restricted to the neural system and kidneys. L1CAM was: 1) induced in premature forms of cellular senescence triggered chemically and by gamma-radiation, but not in Ras-induced senescence; 2) induced upon inhibition of cyclin-dependent kinases by p16INK4a; 3) induced by TGFbeta and suppressed by RAS/MAPK(Erk) signaling (the latter explaining the lack of L1CAM induction in RAS-induced senescence); and 4) induced upon downregulation of growth-associated gene ANT2, growth in low-glucose medium or inhibition of the mevalonate pathway. These data indicate that L1CAM is controlled by a number of cell growth- and metabolism-related pathways during SC development. Functionally, SC with enhanced surface L1CAM showed increased adhesion to extracellular matrix and migrated faster. Our results provide mechanistic insights into senescence of human cells, with implications for future senolytic strategies.

• Klíčová slova
• MAPK pathway, SILAC, aging, mass spectrometry, proteomics,
• MeSH
• buněčná adheze fyziologie   MeSH
• buněčný cyklus MeSH
• down regulace MeSH
• fibroblasty MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• molekula buněčné adheze nervové L1 genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• pohyb buněk fyziologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• regulace genové exprese účinky léků   účinky záření   MeSH
• RNA interference MeSH
• signální transdukce MeSH
• stárnutí buněk MeSH
• transformující růstový faktor beta metabolismus   farmakologie   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• molekula buněčné adheze nervové L1 MeSH
• transformující růstový faktor beta MeSH

BACKGROUND: Senescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now. METHODS: In the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs. RESULTS: This experimental setting has three advantages that are presented and discussed: i) it covers a "gap" in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics. CONCLUSIONS: Consequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.

• Klíčová slova
• CDC6, Cancer, DNA damage response, In situ hybridization, Micro-RNAs, Oncogene-induced senescence, R loops, Replication stress, SenTraGorTM, rDNA,
• MeSH
• epitelové buňky metabolismus   MeSH
• genom MeSH
• jaderné proteiny metabolismus   MeSH
• karcinogeneze MeSH
• kultivované buňky MeSH
• lidé MeSH
• mikro RNA metabolismus   MeSH
• nádory glandulární a epitelové genetika   patologie   ultrastruktura   MeSH
• onkogeny * MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• stárnutí buněk genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDC6 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• mikro RNA MeSH
• proteiny buněčného cyklu MeSH
• proteiny MeSH

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

• Klíčová slova
• replication stress, aphidicolin, camptothecin, cancer, cisplatin, etoposide, hydroxyurea,
• MeSH
• buňky účinky léků   metabolismus   MeSH
• fyziologický stres * účinky léků   MeSH
• lidé MeSH
• protinádorové látky chemie   farmakologie   MeSH
• replikace DNA * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• protinádorové látky MeSH

Oncogene-evoked replication stress (RS) fuels genomic instability in diverse cancer types. Here we report that BRCA1, traditionally regarded a tumour suppressor, plays an unexpected tumour-promoting role in glioblastoma (GBM), safeguarding a protective response to supraphysiological RS levels. Higher BRCA1 positivity is associated with shorter survival of glioma patients and the abrogation of BRCA1 function in GBM enhances RS, DNA damage (DD) accumulation and impairs tumour growth. Mechanistically, we identify a novel role of BRCA1 as a transcriptional co-activator of RRM2 (catalytic subunit of ribonucleotide reductase), whereby BRCA1-mediated RRM2 expression protects GBM cells from endogenous RS, DD and apoptosis. Notably, we show that treatment with a RRM2 inhibitor triapine reproduces the BRCA1-depletion GBM-repressive phenotypes and sensitizes GBM cells to PARP inhibition. We propose that GBM cells are addicted to the RS-protective role of the BRCA1-RRM2 axis, targeting of which may represent a novel paradigm for therapeutic intervention in GBM.

• MeSH
• analýza přežití MeSH
• glioblastom genetika   metabolismus   patologie   MeSH
• karcinogeneze genetika   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• nádory mozku genetika   metabolismus   patologie   MeSH
• protein BRCA1 genetika   metabolismus   MeSH
• regulace genové exprese u nádorů * MeSH
• replikace DNA genetika   MeSH
• retrospektivní studie MeSH
• ribonukleosiddifosfátreduktasa genetika   metabolismus   MeSH
• RNA interference MeSH
• transplantace heterologní MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BRCA1 protein, human MeSH Prohlížeč
• protein BRCA1 MeSH
• ribonucleotide reductase M2 MeSH Prohlížeč
• ribonukleosiddifosfátreduktasa MeSH