RESUMO
Recently, we showed that the ubiquitin ligase E6AP stabilizes ß-catenin and activates its transcriptional activity. These activities were enhanced by the human papillomavirus (HPV) E6 protein. In the present study, we explored the function of E6AP, which increases ß-catenin stabilization and transcriptional activation. Here, we report that E6AP interacts with ß-catenin and mediates its nonproteolytic ubiquitylation, as evidenced in transiently transfected cell-based and in vitro reconstitution ubiquitylation assays. Overexpression of E6AP increased ß-catenin polyubiquitylation and, consistent with that, knockdown or knock-out of E6AP expression reduced ß-catenin polyubiquitylation. The ubiquitylation of ß-catenin by E6AP was dependent on its E3 ubiquitin ligase activity, but it was proteasome-independent and did not require HPV-E6, phosphorylation of ß-catenin by glycogen synthase kinase 3ß (GSK3ß) or activity of the ß-catenin 'destruction complex'. We also show that transcriptional activation of ß-catenin by E6AP is coupled with ß-catenin protein stabilization, but not its ubiquitylation. In contrast to ß-catenin ubiquitylation, ß-catenin protein stability and its transcriptional activity were absolutely dependent on the activity of the destruction complex and phosphorylation by GSK3ß. Collectively, our data uncover a dual role for E6AP in the regulation of ß-catenin ubiquitylation, stability and transcriptional activity, with HPV-E6 enhancing only part of E6AP activities.
Assuntos
Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/virologia , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/enzimologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteínas Repressoras/genética , Transdução de Sinais , Ativação Transcricional , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , beta Catenina/genéticaRESUMO
The global agricultural sector faces a significant number of challenges for a sustainable future, and one of the tools proposed to address these challenges is the use of automation in agriculture. In particular, robotic systems for agricultural tasks are being designed, tested, and increasingly commercialised in many countries. Much touted as an environmentally beneficial technology with the ability to improve data management and reduce the use of chemical inputs while improving yields and addressing labour shortages, agricultural robotics also presents a number of potential ethical challenges - including rural unemployment, the amplification of economic and digital inequalities, and entrenching unsustainable farming practices. As such, development is not uncontroversial, and there have been calls for a responsible approach to their innovation that integrates more substantive inclusion into development processes. This study investigates current approaches to participation and inclusion amongst United Kingdom (UK) agricultural robotics developers. Through semi-structured interviews with key members of the UK agricultural robotics sector, we analyse the stakeholder engagement currently integrated into development processes. We explore who is included, how inclusion is done, and what the inclusion is done for. We reflect on how these findings align with the current literature on stakeholder inclusion in agricultural technology development, and suggest what they could mean for the development of more substantive responsible innovation in agricultural robotics.
RESUMO
Current therapies for treatment of advanced cervical cancer involve the use of cisplatin, often in combination with radiotherapy. These treatments do not lead to a high survival rate and furthermore, serious side effects are dose-limiting factors. Methyl jasmonate (MJ) was recently identified as potent and selective cytotoxic agent towards cervical cancer cells. In the present study we evaluated the effectiveness of combined treatments of MJ with cisplatin or X-irradiation on a variety of cervical cancer cells including SiHa, CaSki, HeLa and C33A. Cytotoxicity of alpha particles, emitted from (224)Ra atoms, was also evaluated as a single agent and in combination with MJ. Cooperation between MJ and cisplatin in reducing cell viability (XTT assays) and survival (clonogenicity assays) was exhibited towards several cancer cell lines at a range of combination doses. MJ effectively cooperated also with X-ray irradiation, significantly lowering the radiation doses required to inhibit cell survival (ID50) of all tested cells lines. We show for the first time, that alpha irradiation selectively reduced cell viability and survival of cervical cancer cells. Lower doses of α irradiation were required as compared to X-irradiation to inhibit cell survival. Cooperation with MJ was demonstrated in part of the cancer cell lines. In conclusion, our studies point to α irradiation and MJ, novel anticancer agents, as potent candidates for treatment of cervical cancer, in single agent regiments and in combination. MJ can be added also to conventional X-ray and cisplatin therapies to increase their cytotoxic effect while lowering the effective dose.
Assuntos
Acetatos/farmacologia , Partículas alfa , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Cisplatino/farmacologia , Ciclopentanos/farmacologia , Raios gama , Oxilipinas/farmacologia , Neoplasias do Colo do Útero/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Terapia Combinada , Feminino , Humanos , Reguladores de Crescimento de Plantas/farmacologia , Radiossensibilizantes/farmacologia , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/radioterapiaRESUMO
We recently showed that E6 protein of human papillomavirus (HPV) 16, a mucosal high-risk α-PV type, can potentiate Wnt/ß-catenin/TCF signaling. Here we investigated the transcriptional activities of E6 proteins of cutaneous HPV types from the ß and α genera. Results from reporter-gene assays showed that similar to HPV16 E6, E6 of HPV10, a cutaneous α-HPV type that is prevalent in skin warts, efficiently enhances and stimulates Wnt/ß-catenin/TCF transcription. HPV10 E6 also effectively elevated the expression levels of ß-catenin and promoted its nuclear accumulation. E6 proteins of ß-HPV types 8, 24, 38 and 49, which are prevalent in skin cancer, exhibited lower activities in all tested functions. The differences in activity correlated with E6's competence to interact with the ubiquitin ligase E6AP. This study reveals a role for E6 proteins of diverse cutaneous HPV types in potentiation of Wnt/ß-catenin signaling, irrespective of their carcinogenic potential.
Assuntos
Interações Hospedeiro-Patógeno , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/fisiologia , Neoplasias Cutâneas/virologia , Verrugas/virologia , Via de Sinalização Wnt , Humanos , Papillomaviridae/isolamento & purificação , Transcrição Gênica , Proteínas Wnt/biossíntese , beta Catenina/biossínteseRESUMO
The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/ß-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated ß-catenin level, promoted its nuclear accumulation, and activated ß-catenin/TCF transcription. A knockdown of E6AP lowered ß-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize ß-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3ß and the susceptibility of ß-catenin to GSK3ß phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/ß-catenin signaling, thereby possibly contributing to HPV carcinogenesis.
Assuntos
Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Células Cultivadas , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Queratinócitos , Plasmídeos , Complexo de Endopeptidases do Proteassoma , Estabilidade Proteica , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
In the present study, E6E7 and E6 proteins of human papillomaviruses (HPVs) associated with skin warts and cancer were compared for their transforming and carcinogenic abilities in primary human keratinocytes (PHKs). We show that E6E7 of cancer associated beta HPV types, notably 49 and 24, were able to extend the life span and enhance the clonogenic efficiency of PHKs when maintained in serum free/low calcium medium. Activities of the beta HPV E6E7 were lower than those of HPV16 E6E7. In contrast, E6 proteins from HPV types detected in skin warts or cancer, notably 10, 49 and 38, attenuated UVB induced protective responses in PHKs including cell death, proliferation arrest and accumulation of the proapoptotic proteins, p53, bax or bak. Together, this investigation revealed functional differences and commonalities between HPVs associated with skin warts and cancer, and allowed the identification of specific properties of beta HPVs supporting their involvement in skin carcinogenesis.
Assuntos
Transformação Celular Viral/fisiologia , Queratinócitos/efeitos da radiação , Queratinócitos/virologia , Papillomaviridae/classificação , Neoplasias Cutâneas/virologia , Verrugas/virologia , Animais , Sobrevivência Celular/efeitos da radiação , Regulação Viral da Expressão Gênica , Humanos , Camundongos , Células NIH 3T3 , Papillomaviridae/genética , Papillomaviridae/metabolismo , Plasmídeos , Raios Ultravioleta , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The present study further investigated the mode of action of methyl jasmonate (MJ) in different cervical cancer cell lines. We show that in addition to the short term cytotoxicity, MJ effectively reduced the survival of cervical cancer cells (clonogenicity assays). MJ induced apoptosis in all cervical cancer cells. In some cell lines, MJ caused elevation of the mitochondrial superoxide anion, notably, in HeLa and CaSki. Changes in the expression of p53 and bax were variable, yet, downregulation of survivin was common to all cervical cancer cells. MJ significantly reduced the levels of the human papillomavirus (HPV) E6 and E7 proteins without alteration of the mRNA levels. Moreover, ectopic expression of E6, E7 or both in cervical cancer cells that lack HPV (C33A), did not alter significantly their response to MJ. Our studies point to MJ as an effective anticancer agent against a variety of cervical cancer cells acting through shared and different pathways to induce cell death regardless of the presence of HPV.
Assuntos
Acetatos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclopentanos/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Regulação para Baixo , Proteínas Inibidoras de Apoptose/efeitos dos fármacos , Proteínas Oncogênicas Virais/efeitos dos fármacos , Oxilipinas/farmacologia , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Survivina , Neoplasias do Colo do Útero/metabolismoRESUMO
In this study we investigated the effect of HPV16 E6 on the Wnt/beta-catenin oncogenic signaling pathway. Luciferase reporter assays indicated that ectopically expressed E6 significantly augmented the Wnt/beta-catenin/TCF-dependent signaling response in a dose-dependent manner. This activity was independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets. Epistasis experiments suggested that the stimulatory effect is independent of GSK3beta or APC. Coexpression, half-life determination, cell fractionation and immunofluorescence analyses indicated that E6 did not alter the expression levels, stability or cellular distribution of beta-catenin. Further experiments using E6 mutants defective for E6AP binding and E6AP knockdown cells indicated the absolute requirement of the ubiquitin ligase E6AP for enhancement of the Wnt signal by E6. Thus, this study suggests a role for the E6/E6AP complex in augmentation of the Wnt signaling pathway which may contribute to HPV induced carcinogenesis.
Assuntos
Proteínas Oncogênicas Virais/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Wnt/fisiologia , Células Cultivadas , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Fatores de Transcrição TCF/fisiologia , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia , beta Catenina/fisiologiaRESUMO
In the present study the effectiveness of methyl jasmonate (MJ) against cervical cancer cell lines was investigated. We show that MJ is cytotoxic to a range of cervical cancer lines including SiHa, CaSki and HeLa that carry human papillomavirus (HPV) DNA and wild type p53, and C33A that is negative for HPV and contains mutant p53. Primary human foreskin keratinocytes were almost resistant to the drug. Cytotoxicity of MJ was dose and time dependent, and associated mainly with the induction of cell death and to a less extent with inhibition of cell growth. Cell death induced by MJ displayed features characteristic to both apoptosis and necrosis, and was associated with different changes in the levels of p53, p21, bcl-2 and bax in the various cervical cancer lines. In conclusion, MJ a novel anticancer agent, acts via multiple pathways to induce death of cervical cancer cells, thus making it a promising candidate for treatment of cervical cancer.
Assuntos
Acetatos/farmacologia , Morte Celular/efeitos dos fármacos , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Neoplasias do Colo do Útero/patologia , Trifosfato de Adenosina/metabolismo , Western Blotting , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Humanos , Proteínas de Neoplasias/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.
Assuntos
Variação Genética , Papillomavirus Humano 16/patogenicidade , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Transformada , Células Cultivadas , Regulação para Baixo , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Queratinócitos/citologia , Camundongos , Células NIH 3T3 , Proteínas Oncogênicas Virais/genética , Polimorfismo Genético , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.
Assuntos
Papillomavirus Humano 16 , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Polimorfismo Genético/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Proteína 1 Homóloga a Discs-Large , Papillomavirus Humano 16/genética , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120-132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Proteínas Oncogênicas Virais/farmacologia , Papillomaviridae/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/farmacologia , Regulação para Baixo , Humanos , RNA Mensageiro/metabolismo , Proteína X Associada a bcl-2RESUMO
Transfection of the E6 gene of human papillovirus (HPV) 16 into primary human keratinocytes (PHKs) generates proliferating cell colonies which are resistant to serum- and calcium-induced terminal differentiation. The extreme C-terminus of E6 was shown to be dispensable for this activity. To map further the amino acid sequences required for inducing resistance to serum and calcium, and to address the functional significance of E6 interactions with p53 and E6BP (ERC-55) in this function, we evaluated the activities of a series of E6 mutants. Small deletions within the central portion of the second putative zinc-finger abolished, or markedly reduced, E6 biological activity, while mutations affecting the cysteine residues in the base of the finger were less effective in this respect. When these mutants were assayed for their ability to degrade p53 in vitro and in vivo and to inhibit p53 transcriptional activation (TA), we found that there was a dissociation of these activities in some mutants. We mapped one mutant which was highly efficient in p53 degradation and inhibition of p53 TA, yet displayed severely reduced activity in the biological assay, and conversely, a subset of mutants that showed moderate activities in the colony assay while being severely impaired in p53 degradation and inhibition of p53 TA. These data argue that p53 inactivation or even elimination are not sufficient, and may not be essential, for altering the response of PHKs to serum and calcium. When these E6 mutants were evaluated for E6BP binding in vitro, there was a similar dissociation between the biological and biochemical activities of several mutants. We mapped mutants with moderate activity in the biological assay that lacked the ability to bind to E6BP and a mutant that showed high biological activity with only marginal binding to E6BP. Thus, there is no absolute correlation between the ability of E6 mutant proteins to induce alterations in keratinocyte differentiation responses to calcium and serum and to induce p53 degradation, inhibit p53 mediated transactivation, or bind E6BP. Evidently there are additional cellular targets for E6 which mediate this alteration in cellular differentiation.