Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
1.
Cell ; 166(1): 167-80, 2016 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27368103

RESUMO

Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Ciclo Celular , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas F-Box/metabolismo , Humanos , Mitógenos/toxicidade , Fosforilação , Proteína do Retinoblastoma/metabolismo
2.
Genes Dev ; 37(19-20): 913-928, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37932011

RESUMO

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.


Assuntos
Replicação do DNA , Neoplasias , Humanos , Replicação do DNA/genética , DNA Helicases/metabolismo , Repetições de Microssatélites , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
3.
Nature ; 619(7969): 363-370, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37407814

RESUMO

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3-5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.


Assuntos
Ciclo Celular , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Fase G2 , Fase S , Animais , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/deficiência , Quinase 4 Dependente de Ciclina/metabolismo , Mitógenos/deficiência , Mitógenos/metabolismo , Mitose , Fosforilação , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Quinase 6 Dependente de Ciclina/deficiência , Quinase 6 Dependente de Ciclina/metabolismo , Fase G1
4.
Cell ; 155(2): 369-83, 2013 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-24075009

RESUMO

Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity.


Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Mitose , Células 3T3 , Animais , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Camundongos , Proteína do Retinoblastoma/metabolismo
5.
Mol Cell ; 76(4): 562-573.e4, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31543423

RESUMO

Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.


Assuntos
Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular , Mitógenos/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismo
7.
J Biol Chem ; 299(6): 104786, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37146968

RESUMO

The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD-deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADDWT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADDKEN) fails to interact with Cdh1 and results in a G1 arrest due to its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADDWT but not FADDKEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F-independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.


Assuntos
Proteínas de Ciclo Celular , Ubiquitina-Proteína Ligases , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Expressão Gênica , Células HEK293 , Mutação , Domínios Proteicos , Transporte Proteico/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
8.
Nature ; 558(7709): 313-317, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29875408

RESUMO

Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle1-4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/CCDH1, also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/CCDH1, which suggests that APC/CCDH1 inactivation is the point of no return for cell-cycle entry 3 . Rapid inactivation of APC/CCDH1 requires early mitotic inhibitor 1 (EMI1)3,5, but the molecular mechanism that controls this cell-cycle commitment step is unknown. Here we show using human cell models that cell-cycle commitment is mediated by an EMI1-APC/CCDH1 dual-negative feedback switch, in which EMI1 is both a substrate and an inhibitor of APC/CCDH1. The inactivation switch triggers a transition between a state with low EMI1 levels and high APC/CCDH1 activity during G1 and a state with high EMI1 levels and low APC/CCDH1 activity during S and G2. Cell-based analysis, in vitro reconstitution and modelling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Our study suggests that mammalian cells commit to the cell cycle by increasing CDK2 activity and EMI1 mRNA expression to trigger a one-way APC/CCDH1 inactivation switch that is mediated by EMI1 transitioning from acting as a substrate of APC/CCDH1 to being an inhibitor of APC/CCDH1.


Assuntos
Proteínas Cdh1/antagonistas & inibidores , Proteínas Cdh1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas F-Box/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas F-Box/genética , Retroalimentação Fisiológica , Fase G1 , Células HeLa , Humanos , Fase S
9.
PLoS Biol ; 18(11): e3000920, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33137094

RESUMO

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3' splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.


Assuntos
Ribossomos/metabolismo , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Substituição de Aminoácidos , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Processamento Pós-Transcricional do RNA , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , Ribossomos/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
Mol Cell ; 38(5): 746-57, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20542006

RESUMO

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify additional signaling components, we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly represented. Included in this group are members of the Skp, Cullin, F box (SCF) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCF(Cdc4) acts together with the Cdc34 ubiquitin-conjugating enzyme at the level of the G protein; promotes degradation of the G protein alpha subunit, Gpa1, in vivo; and catalyzes Gpa1 ubiquitination in vitro. These insights to the G protein signaling network reveal the essential genome as an untapped resource for identifying new components and regulators of signal transduction pathways.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Análise por Conglomerados , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Humanos , Fenótipo , Feromônios/genética , Feromônios/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
11.
bioRxiv ; 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-39372752

RESUMO

Cyclin-dependent kinases (CDK) are key regulatory enzymes that regulate proliferation dynamics and cell fate in response to extracellular inputs. It remains largely unknown how CDK activity fluctuates and influences cell commitment in vivo during early mammalian development. Here, we generated a transgenic mouse model expressing a CDK kinase translocation reporter (KTR) that enabled quantification of CDK activity in live single cells. By examining pre- and post-implantation mouse embryos at different stages, we observed a progressive decrease in CDK activity in cells from the trophectoderm (TE) prior to implantation. This drop correlated with the establishment of an FGF4-dependent signaling gradient through the embryonic-abembryonic axis. Furthermore, we showed that CDK activity levels do not determine cell fate decisions during pre-implantation development. Finally, we uncovered the existence of conserved regulatory mechanisms in mammals by revealing lineage-specific regulation of CDK activity in TE-like human cells.

12.
Neuro Oncol ; 26(6): 1083-1096, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38134889

RESUMO

BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.


Assuntos
Antineoplásicos Alquilantes , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Temozolomida/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Camundongos , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistemas CRISPR-Cas , Camundongos Nus , Linhagem Celular Tumoral , Células Tumorais Cultivadas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Reparo do DNA/efeitos dos fármacos
13.
Proc Natl Acad Sci U S A ; 107(15): 7066-71, 2010 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-20351284

RESUMO

G-protein heterotrimers, composed of a guanine nucleotide-binding G alpha subunit and an obligate G betagamma dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by G alpha-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of "regulator of G-protein signaling" (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a G alpha switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP x AlF(4)(-)-bound conformation of the G202A mutant was found to be nearly identical to wild-type, G alpha(i1)(G202A) x GDP assumed a divergent conformation more closely resembling the GDP x AlF(4)(-)-bound state. When placed within Saccharomyces cerevisiae G alpha subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose-response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus G alpha-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein-coupled receptor (GPCR) signaling in a cellular context.


Assuntos
GTP Fosfo-Hidrolases/química , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Alanina/química , Relação Dose-Resposta a Droga , Glicina/química , Humanos , Hidrólise , Cinética , Luminescência , Modelos Moleculares , Mutação , Feromônios/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
14.
bioRxiv ; 2023 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-37662356

RESUMO

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN, knowledge that would be helpful for informing clinical development of WRN-targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system wherein the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We find that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we find no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provided the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggested a potential therapeutical rationale for dual targeting of WRN and ATR.

15.
Nat Cancer ; 4(3): 419-435, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36973439

RESUMO

Most tumor cells undergo apoptosis in circulation and at the metastatic organ sites due to host immune surveillance and a hostile microenvironment. It remains to be elucidated whether dying tumor cells have a direct effect on live tumor cells during the metastatic process and what the underlying mechanisms are. Here we report that apoptotic cancer cells enhance the metastatic outgrowth of surviving cells through Padi4-mediated nuclear expulsion. Tumor cell nuclear expulsion results in an extracellular DNA-protein complex that is enriched with receptor for advanced glycation endproducts (RAGE) ligands. The chromatin-bound RAGE ligand S100a4 activates RAGE receptors in neighboring surviving tumor cells, leading to Erk activation. In addition, we identified nuclear expulsion products in human patients with breast, bladder and lung cancer and a nuclear expulsion signature correlated with poor prognosis. Collectively, our study demonstrates how apoptotic cell death can enhance the metastatic outgrowth of neighboring live tumor cells.


Assuntos
Neoplasias Pulmonares , Proteína A4 de Ligação a Cálcio da Família S100 , Humanos , Apoptose , Neoplasias Pulmonares/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Microambiente Tumoral
16.
Cell Rep ; 42(9): 113079, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37656618

RESUMO

Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.


Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Divisão Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos
17.
J Biol Chem ; 286(17): 14852-60, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21388955

RESUMO

Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane.


Assuntos
1-Fosfatidilinositol 4-Quinase/fisiologia , Membranas Intracelulares/química , Sistema de Sinalização das MAP Quinases , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Epistasia Genética , Proteínas Fúngicas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
J Biol Chem ; 286(23): 20208-16, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21521692

RESUMO

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G(1) phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.


Assuntos
Ciclo Celular/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquitinação/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Fosforilação/fisiologia , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Nat Commun ; 13(1): 6364, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289220

RESUMO

The F-box protein beta-transducin repeat containing protein (ß-TrCP) acts as a substrate adapter for the SCF E3 ubiquitin ligase complex, plays a crucial role in cell physiology, and is often deregulated in many types of cancers. Here, we develop a fluorescent biosensor to quantitatively measure ß-TrCP activity in live, single cells in real-time. We find ß-TrCP remains constitutively active throughout the cell cycle and functions to maintain discreet steady-state levels of its substrates. We find no correlation between expression levels of ß-TrCP and ß-TrCP activity, indicating post-transcriptional regulation. A high throughput screen of small-molecules using our reporter identifies receptor-tyrosine kinase signaling as a key axis for regulating ß-TrCP activity by inhibiting binding between ß-TrCP and the core SCF complex. Our study introduces a method to monitor ß-TrCP activity in live cells and identifies a key signaling network that regulates ß-TrCP activity throughout the cell cycle.


Assuntos
Técnicas Biossensoriais , Proteínas F-Box , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Tirosina Quinases/metabolismo
20.
Sci Adv ; 7(3)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523889

RESUMO

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.


Assuntos
Dano ao DNA , Ciclo Celular/genética , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fase S
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA