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1.
Nano Lett ; 21(20): 8785-8793, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34614348

RESUMO

Monitoring dynamic processes in complex cellular environments requires the integration of uniformly distributed detectors within such three-dimensional (3D) networks, to an extent that the sensor could provide real-time information on nearby perturbations in a non-invasive manner. In this context, the development of 3D-printed structures that can function as both sensors and cell culture platforms emerges as a promising strategy, not only for mimicking a specific cell niche but also toward identifying its characteristic physicochemical conditions, such as concentration gradients. We present herein a 3D cancer model that incorporates a hydrogel-based scaffold containing gold nanorods. In addition to sustaining cell growth, the printed nanocomposite inks display the ability to uncover drug diffusion profiles by surface-enhanced Raman scattering, with high spatiotemporal resolution. We additionally demonstrate that the acquired information could pave the way to designing novel strategies for drug discovery in cancer therapy, through correlation of drug diffusion with cell death.


Assuntos
Nanocompostos , Nanotubos , Ouro , Hidrogéis , Análise Espectral Raman
2.
Cancers (Basel) ; 13(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34503116

RESUMO

Prostate cancer (PCa) is one of the most prevalent cancers in men. Androgen receptor signaling plays a major role in this disease, and androgen deprivation therapy is a common therapeutic strategy in recurrent disease. Sphingolipid metabolism plays a central role in cell death, survival, and therapy resistance in cancer. Ceramide kinase (CERK) catalyzes the phosphorylation of ceramide to ceramide 1-phosphate, which regulates various cellular functions including cell growth and migration. Here we show that activated androgen receptor (AR) is a repressor of CERK expression. We undertook a bioinformatics strategy using PCa transcriptomics datasets to ascertain the metabolic alterations associated with AR activity. CERK was among the most prominent negatively correlated genes in our analysis. Interestingly, we demonstrated through various experimental approaches that activated AR reduces the mRNA expression of CERK: (i) expression of CERK is predominant in cell lines with low or negative AR activity; (ii) AR agonist and antagonist repress and induce CERK mRNA expression, respectively; (iii) orchiectomy in wildtype mice or mice with PCa (harboring prostate-specific Pten deletion) results in elevated Cerk mRNA levels in prostate tissue. Mechanistically, we found that AR represses CERK through interaction with its regulatory elements and that the transcriptional repressor EZH2 contributes to this process. In summary, we identify a repressive mode of AR that influences the expression of CERK in PCa.

3.
Front Cell Dev Biol ; 9: 624089, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33869174

RESUMO

LUZP1 is a centrosomal and actin cytoskeleton-localizing protein that regulates both ciliogenesis and actin filament bundling. As the cytoskeleton and cilia are implicated in metastasis and tumor suppression, we examined roles for LUZP1 in the context of cancer. Here we show that LUZP1 exhibits frequent genomic aberrations in cancer, with a predominance of gene deletions. Furthermore, we demonstrate that CRISPR/Cas9-mediated loss of Luzp1 in mouse fibroblasts promotes cell migration and invasion features, reduces cell viability, and increases cell apoptosis, centriole numbers, and nuclear size while altering the actin cytoskeleton. Loss of Luzp1 also induced changes to ACTR3 (Actin Related Protein 3, also known as ARP3) and phospho-cofilin ratios, suggesting regulatory roles in actin polymerization, beyond its role in filament bundling. Our results point to an unprecedented role for LUZP1 in the regulation of cancer features through the control of actin cytoskeleton.

4.
Cancers (Basel) ; 12(9)2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32932846

RESUMO

Prostate cancer is the most frequent malignancy in European men and the second worldwide. One of the major oncogenic events in this disease includes amplification of the transcription factor cMYC. Amplification of this oncogene in chromosome 8q24 occurs concomitantly with the copy number increase in a subset of neighboring genes and regulatory elements, but their contribution to disease pathogenesis is poorly understood. Here we show that TRIB1 is among the most robustly upregulated coding genes within the 8q24 amplicon in prostate cancer. Moreover, we demonstrate that TRIB1 amplification and overexpression are frequent in this tumor type. Importantly, we find that, parallel to its amplification, TRIB1 transcription is controlled by cMYC. Mouse modeling and functional analysis revealed that aberrant TRIB1 expression is causal to prostate cancer pathogenesis. In sum, we provide unprecedented evidence for the regulation and function of TRIB1 in prostate cancer.

5.
FASEB J ; 34(5): 6907-6919, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32267586

RESUMO

DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin-binding protein with a specific function in fork progression under replication stress.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Replicação do DNA/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular , Linhagem Celular , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Técnicas do Sistema de Duplo-Híbrido
6.
Cell Rep ; 30(7): 2416-2429.e7, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32075739

RESUMO

It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Ligação Proteica/genética , Humanos
7.
Cell Mol Life Sci ; 77(4): 735-749, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31297568

RESUMO

During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.


Assuntos
Replicação do DNA/efeitos dos fármacos , Hidroxiureia/farmacologia , Linhagem Celular , DNA de Cadeia Simples/genética , Humanos , Fase S/efeitos dos fármacos
8.
FASEB J ; 32(7): 3502-3517, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29452566

RESUMO

A considerable proportion of tumors exhibit aneuploid karyotypes, likely resulting from the progressive loss of chromosomes after whole-genome duplication. Here, by using isogenic diploid and near-tetraploid (4N) single-cell-derived clones from the same parental cell lines, we aimed at exploring how polyploidization affects cellular functions and how tetraploidy generates chromosome instability. Gene expression profiling in 4N clones revealed a significant enrichment of transcripts involved in cell cycle and DNA replication. Increased levels of replication stress in 4N cells resulted in DNA damage, impaired proliferation caused by a cell cycle delay during S phase, and higher sensitivity to S phase checkpoint inhibitors. In fact, increased levels of replication stress were also observed in nontransformed, proliferative posttetraploid RPE1 cells. Additionally, replication stress promoted higher levels of intercellular genomic heterogeneity and ongoing genomic instability, which could be explained by high rates of mitotic defects, and was alleviated by the supplementation of exogenous nucleosides. Finally, our data found that 4N cancer cells displayed increased migratory and invasive capacity, both in vitro and in primary colorectal tumors, indicating that tetraploidy can promote aggressive cancer cell behavior.-Wangsa, D., Quintanilla, I., Torabi, K., Vila-Casadesús, M., Ercilla, A., Klus, G., Yuce, Z., Galofré, C., Cuatrecasas, M., Lozano, J. J., Agell, N., Cimini, D., Castells, A., Ried, T., Camps, J. Near-tetraploid cancer cells show chromosome instability triggered by replication stress and exhibit enhanced invasiveness.


Assuntos
Movimento Celular , Instabilidade Cromossômica , Dano ao DNA , Neoplasias/genética , Tetraploidia , Linhagem Celular Tumoral , Replicação do DNA , Humanos , Fase S
9.
Nucleic Acids Res ; 44(10): 4745-62, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26939887

RESUMO

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.


Assuntos
Proteínas Cdh1/metabolismo , Replicação do DNA , Origem de Replicação , Fase S/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Dano ao DNA , Ativação Enzimática , Instabilidade Genômica , Humanos , Hidroxiureia/toxicidade , Estresse Fisiológico/genética , Proteína Supressora de Tumor p53/fisiologia
10.
Oncotarget ; 6(29): 28238-56, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26318587

RESUMO

Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised.


Assuntos
Centrossomo/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dano ao DNA , Células Epiteliais/metabolismo , Telômero/genética , Western Blotting , Células Cultivadas , Centríolos/genética , Centríolos/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Hibridização in Situ Fluorescente , Glândulas Mamárias Humanas/citologia , Microscopia de Fluorescência , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , Tetraploidia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
11.
Cell Cycle ; 11(19): 3627-37, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22935704

RESUMO

Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38ß isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.


Assuntos
Replicação do DNA , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Mitose , Proteínas Quinases/metabolismo , Animais , Afidicolina/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Ciclina B1/antagonistas & inibidores , Ciclina B1/metabolismo , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Hidroxiureia/farmacologia , Cinética , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo
12.
Clin Lab ; 58(1-2): 185-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22372364

RESUMO

BACKGROUND: We explored whether the Asp42Gly polymorphism (rs12075) in the DARC gene represents a confounding factor in the interpretation of monocyte chemoattractant protein-1 (MCP-1) concentration in circulating blood. METHODS: MCP-1 concentration in serum and plasma were measured in 278 healthy Caucasian participants who are representative of our geographic area. The rs12075 genotype distribution was also assessed in this population. RESULTS: Plasma MCP-1 concentration did not vary among the rs12075 polymorphism derived genotypes [in pg/mL, AA: 171.9 (100.2 - 287.2), AG: 178.9 (105.1 - 326.4) and GG: 173.7 (94.4 - 405.7)]. However, there were significant increases in serum MCP-1 related to the presence of the A allele [in pg/mL, AA: 334.6 (180.4 - 756.4), AG: 299.1 (166.1 - 634.9) and GG: 249.1 (149.3 - 578.1)]. CONCLUSIONS: These findings limit the value of circulating MCP-1 as a biomarker and apparently indicate a pathophysiological role for silent chemokine receptors.


Assuntos
Quimiocina CCL2/sangue , Sistema do Grupo Sanguíneo Duffy , Sistema do Grupo Sanguíneo Duffy/genética , Feminino , Genótipo , Humanos , Masculino , Plasma , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Soro
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