RESUMO
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.
Assuntos
Senescência Celular/genética , Neoplasias Epiteliais e Glandulares/genética , Oncogenes , Carcinogênese , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Genoma , Humanos , MicroRNAs/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Epiteliais e Glandulares/ultraestrutura , Proteínas Nucleares/metabolismo , Proteínas/metabolismoRESUMO
Human malignancies overcome replicative senescence either by activating the reverse-transcriptase telomerase or by utilizing a homologous recombination-based mechanism, referred to as alternative lengthening of telomeres (ALT). In budding yeast, ALT exhibits features of break-induced replication (BIR), a repair pathway for one-ended DNA double-strand breaks (DSBs) that requires the non-essential subunit Pol32 of DNA polymerase delta and leads to conservative DNA replication. Here, we examined whether ALT in human cancers also exhibits features of BIR A telomeric fluorescence in situ hybridization protocol involving three consecutive staining steps revealed the presence of conservatively replicated telomeric DNA in telomerase-negative cancer cells. Furthermore, depletion of PolD3 or PolD4, two subunits of human DNA polymerase delta that are essential for BIR, reduced the frequency of conservatively replicated telomeric DNA ends and led to shorter telomeres and chromosome end-to-end fusions. Taken together, these results suggest that BIR is associated with conservative DNA replication in human cells and mediates ALT in cancer.
Assuntos
Reparo do DNA , Replicação do DNA , Neoplasias/genética , Homeostase do Telômero , Quebras de DNA de Cadeia Dupla , DNA Polimerase III/deficiência , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Reparo do DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Recombinação Homóloga/genética , Humanos , Hibridização in Situ Fluorescente , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Leveduras/genética , Leveduras/fisiologiaRESUMO
The vertebrate RECQL4 (RECQ4) gene is thought to be the ortholog of budding yeast SLD2. However, RecQL4 contains within its C-terminus a RecQ-like helicase domain, which is absent in Sld2. We established human pre-B lymphocyte Nalm-6 cells, in which the endogenous RECQL4 gene was homozygously targeted such that the entire C-terminus would not be expressed. The RECQL4(ΔC/ΔC) cells behaved like the parental cells during unperturbed DNA replication or after treatment with agents that induce stalling of DNA replication forks, such as hydroxyurea (HU). However, after exposure to ionizing radiation (IR), the RECQL4(ΔC/ΔC) cells exhibited hypersensitivity, inability to complete S phase and prematurely terminated or paused DNA replication forks. Deletion of BLM, a gene that also encodes a RecQ helicase, had the opposite phenotype; an almost wild-type response to IR, but hypersensitivity to HU. Targeting both RECQL4 and BLM resulted in viable cells, which exhibited mostly additive phenotypes compared with those exhibited by the RECQL4(ΔC/ΔC) and the BLM(-/-) cells. We propose that RecQL4 facilitates DNA replication in cells that have been exposed to IR.
Assuntos
Dano ao DNA , Replicação do DNA , RecQ Helicases/química , RecQ Helicases/genética , RecQ Helicases/metabolismo , Linhagem Celular , DNA/efeitos da radiação , Instabilidade Genômica , Humanos , Hidroxiureia/farmacologia , Células Precursoras de Linfócitos B/citologia , Estrutura Terciária de Proteína , Radiação IonizanteRESUMO
Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.
Assuntos
Células-Tronco Embrionárias/metabolismo , Inativação Gênica , Heterocromatina/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células-Tronco Embrionárias/fisiologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/genética , Histonas/metabolismo , Metilação , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Fatores de Transcrição/genéticaRESUMO
Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.
Assuntos
Instabilidade Cromossômica , Aberrações Cromossômicas , Cromossomos Humanos/metabolismo , Homeostase do Telômero/genética , Telômero/genética , Telômero/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Humanos , Cariotipagem , Translocação Genética/genéticaRESUMO
Polyploidy contributes to extensive intratumor genomic heterogeneity that characterizes advanced malignancies and is thought to limit the efficiency of current cancer therapies. It has been shown that telomere deprotection in p53-deficient mouse embryonic fibroblasts leads to high rates of polyploidization. We now show that tumor genome evolution through whole-genome duplication occurs in â¼15% of the karyotyped human neoplasms and correlates with disease progression. In a panel of human cancer and transformed cell lines representing the two known types of genomic instability (chromosomal and microsatellite), as well as the two known pathways of telomere maintenance in cancer (telomerase activity and alternative lengthening of telomeres), telomere dysfunction-driven polyploidization occurred independently of the mutational status of p53. Depending on the preexisting context of telomere maintenance, telomerase activity and its major components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA component (hTERC), exert both reverse transcriptase-related (canonical) and noncanonical functions to affect tumor genome evolution through suppression or induction of polyploidization. These new findings provide a more complete mechanistic understanding of cancer progression that may, in the future, lead to novel therapeutic interventions.
Assuntos
Transformação Celular Neoplásica/genética , Poliploidia , RNA/genética , Telomerase/genética , Proteína Supressora de Tumor p53 , Animais , Proliferação de Células , Duplicação Cromossômica/genética , Genoma Humano , Instabilidade Genômica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Mutação , RNA/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment. Therefore, in vitro models are necessary for the investigation of the phenotypic changes provoked by cytotoxic agents and more importantly for preclinical testing of new anticancer drugs. METHODS: We analyzed chromosomal, numerical, and structural changes after development of MDR, alterations in p53 and PTEN, single nucleotide polymorphisms (SNPs) in the mdr1 gene and corresponding protein expression of P-glycoprotein (P-gp) in three human MDR cancer cell lines: non-small cell lung carcinoma NCI-H460/R, colorectal carcinoma DLD1-TxR, and glioma U87-TxR. In addition, we explored how these molecular and phenotypic alterations influence the anticancer effect of new drugs. RESULTS: Cytogenetic analysis showed polyploidy reduction after development of MDR in U87-TxR. Losses of 6q in all resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. Overexpression of P-gp was observed in all MDR cancer cell lines. We evaluated the anticancer activities and MDR reversal potential of Akt inhibitor GSK690693, Ras inhibitor Tipifarnib, and two P-gp inhibitors (jatrophane diterpenoids). Their effects vary due to the cell-type differences, existence of MDR phenotype, presence of mdr1 SNP, and tumor suppressors' alterations. Tipifarnib and jatrophane diterpenoids significantly sensitized MDR cancer cells to paclitaxel. CONCLUSION: In conclusion, investigated MDR cancer cells obtained new molecular and cytogenetic characteristics that may serve as potential clinical prognostic markers. In addition, these MDR cancer cell lines present a valuable model for preclinical evaluation of new anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Glioma/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Análise Citogenética , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Neoplasias Pulmonares/genética , Oxidiazóis/farmacologia , PTEN Fosfo-Hidrolase/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Proteína Supressora de Tumor p53/genéticaRESUMO
Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero/genética , Telômero/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Expressão Gênica , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Recombinação Genética/genética , Telomerase/deficiência , Telômero/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Soft tissue sarcomas are a diverse set of fatal human tumors where few agents have demonstrable clinical efficacy, with the standard therapeutic combination of doxorubicin and ifosfamide showing only a 25% to 30% response rate in large multi-institutional trials. Although liposarcomas are the most common histologic form of adult soft tissue sarcomas, research in this area is severely hampered by the lack of experimentally tractable in vitro model systems. To this end, here we describe a novel in vitro model for human pleomorphic liposarcoma. The cell line (LS2) is derived from a pleomorphic liposarcoma that uses the alternative lengthening of telomeres (ALT) mechanism of telomere maintenance, which may be important in modulating the response of this tumor type to DNA-damaging agents. We present detailed baseline molecular and genomic data, including genome-wide copy number and transcriptome profiles, for this model compared with its parental tumor and a panel of liposarcomas covering multiple histologies. The model has retained essentially all of the detectable alterations in copy number that are seen in the parental tumor, and shows molecular karyotypic and expression profiles consistent with pleomorphic liposarcomas. We also show the utility of this model, together with two additional human liposarcoma cell lines, to investigate the relationship between topoisomerase 2A expression and the sensitivity of ALT-positive liposarcomas to doxorubicin. This model, together with its associated baseline data, provides a powerful new tool to develop treatments for this clinically poorly tractable tumor and to investigate the contribution that ALT makes to modulating sensitivity to doxorubicin.
Assuntos
Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Lipossarcoma/genética , Neoplasias de Tecidos Moles/genética , Telômero/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Aberrações Cromossômicas/induzido quimicamente , Análise Citogenética , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Instabilidade Genômica/efeitos dos fármacos , Células HeLa , Humanos , Lipossarcoma/tratamento farmacológico , Lipossarcoma/patologia , Modelos Teóricos , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/patologia , Telômero/metabolismoRESUMO
Malignant melanomas are characterized by increased karyotypic complexity, extended aneuploidy and heteroploidy. We report a melanoma metastasis to the peritoneal cavity with an exceptionally stable, abnormal pseudodiploid karyotype as verified by G-Banding, subtelomeric, centromeric and quantitative Fluorescence in Situ Hybridization (FISH). Interestingly this tumor had no detectable telomerase activity as indicated by the Telomere Repeat Amplification Protocol. Telomeric Flow-FISH and quantitative telomeric FISH on mitotic preparations showed that malignant cells had relatively short telomeres. Microsatellite instability was ruled out by the allelic pattern of two major mononucleotide repeats. Our data suggest that a combination of melanoma specific genomic imbalances were sufficient and enough for this fatal tumor progression, that was not accompanied by genomic instability, telomerase activity, or the engagement of the alternative recombinatorial telomere lengthening pathway.
RESUMO
In the alternative pathway of telomere lengthening (ALT), neoplastic cell growth is prolonged by telomere recombination. We show that ALT is unexpectedly characterized by high rates of ongoing pericentromeric chromosomal instability. Combined with telomeric recombination, ALT pericentromeric instability generates neoacrocentric chromosomes. In the present studies, we describe a subgroup of ALT neoacrocentric minute chromosomes, composed of DNA entities two to five times smaller in size than human chromosome 21. The frequencies of ALT minute chromosomes were increased by gamma-irradiation and suppressed by telomerase. Continuous growth after telomerase inhibition/depletion was followed by increased rates of telomeric sister chromatid recombination and the emergence of minute chromosomes. We show that ALT minute chromosomes were derived from true centromeric fissions and/or chromosomal breakage/fusion/bridge cycles. They exhibit a two-chromatid structure, carry genomic DNA, centromeric and telomeric repeats, and display regular mitotic functionality. These observations are important in understanding the global genomic instability that characterizes most human advanced malignancies.