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During 1979-2022, Cameroon recorded 32 laboratory-confirmed mpox cases among 137 suspected mpox cases identified by the national surveillance network. The highest positivity rate occurred in 2022, indicating potential mpox re-emergence in Cameroon. Both clade I (n = 12) and clade II (n = 18) monkeypox virus (MPXV) were reported, a unique feature of mpox in Cameroon. The overall case-fatality ratio of 2.2% was associated with clade II. We found mpox occurred only in the forested southern part of the country, and MPXV phylogeographic structure revealed a clear geographic separation among concurrent circulating clades. Clade I originated from eastern regions close to neighboring mpox-endemic countries in Central Africa; clade II was prevalent in western regions close to West Africa. Our findings suggest that MPXV re-emerged after a 30-year lapse and might arise from different viral reservoirs unique to ecosystems in eastern and western rainforests of Cameroon.
Assuntos
Monkeypox virus , Mpox , Humanos , Camarões/epidemiologia , Monkeypox virus/genética , Ecossistema , Mpox/epidemiologia , África Ocidental/epidemiologiaRESUMO
BACKGROUND: This study focuses on the efficacy and 2-year outcomes of ultra-low-dose radiotherapy (RT) in treating primary and secondary ocular adnexal lymphoma (OAL). METHODS: A retrospective analysis was conducted on patients with OAL between 2017 and 2022, treated with 4 Gy of RT. The primary and secondary outcomes assessed were response rate, progression-free survival, and lymphoma-related death. RESULTS: Twenty-one patients with primary and secondary OAL of diverse, presentations, subtypes, and stages were included. The orbital tumors had an average size of 17 × 16 × 16 mm. Of the 14 primary OAL cases, 3 (14%) had T1N0M0 disease, 8 (38%) T2N0M0, and 3 (14%) T3N0M0 (AJCC 8th edition staging); of the 7 secondary OALs, 4 (19%) were stage IE, 2 (10%) stage IIE, and 1 (5%) stage IIIE (Ann Arbor staging). Ultra-low-dose RT yielded a 95% complete response rate and 100% progression-free survival rates, both locally and systemically at 2 years. Mild dry eyes were reported in 14% of patients as a late treatment toxicity. CONCLUSIONS: Ultra-low-dose RT emerges as an effective and well-tolerated treatment approach for OAL. Our findings support the use of 4 Gy, showcasing high complete response rates (95%) and durable disease control without significant local relapses over an average follow up of 27 months. Our results align with earlier investigations, validating the curative potential of ultra-low-dose RT and reinforcing the concept of achieving favorable outcomes with minimal intervention. This approach may potentially alleviate the burden of long-term ocular side effects associated with higher radiation doses, enhancing the overall quality of life for OAL patients.
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BACKGROUND: Day care units are an essential part of psychiatric treatment in Germany. In rheumatology they are also regularly used. Axial spondylarthritis (axSpA) is an inflammatory rheumatic disease that causes pain, diminished quality of life, limitations in activities of daily living and ability to work, especially if insufficiently treated. The multimodal rheumatologic complex treatment with at least 14 days of inpatient care is an established tool to control exacerbated disease activity. The feasibility and effect of an equivalent treatment in a day care setting has not yet been evaluated. METHODS: The effect of a therapy in a day care unit comparable to the inpatient multimodal rheumatologic complex treatment was investigated using clinically established patient reported outcomes (NAS pain, FFbH, BASDAI, BASFI). RESULTS: Selected subgroups of axSpA patients can routinely and effectively be treated in day care units. Intensified multimodal as well as nonintensified treatment forms lead to reduced disease activity. Additionally, compared to nonintensified treatment, the intensified multimodal treatment approach leads to significantly reduced pain, and disease-related and functional limitations in daily life. CONCLUSION: If available, treatment in a day care unit can complement the established inpatient treatment modalities in selected axSpA patients. In cases with high disease activity and suffering, intensified multimodal treatment should be preferred due to better outcomes.
Assuntos
Artrite Reumatoide , Espondiloartrite Axial , Espondilartrite , Espondilite Anquilosante , Humanos , Espondilartrite/terapia , Qualidade de Vida , Hospital Dia , Atividades Cotidianas , DorRESUMO
In systemic lupus erythematosus, ophthalmic manifestations are noted in up to one-third of patients. We describe a patient with an unusual initial presentation of this disorder.
Assuntos
Dacriocistite/diagnóstico , Pálpebras/patologia , Lúpus Eritematoso Sistêmico/diagnóstico , Adulto , Pálpebras/diagnóstico por imagem , Feminino , Humanos , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Current treatment strategies for head and neck cancer are associated with significant morbidity and up to 50% of patients relapse, highlighting the need for more specific and effective therapeutics. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Smac mimetics (SMs) are promising anticancer agents, but their effect on head and neck squamous cell carcinoma (HNSCC) remains unknown. METHODS: We examined the response of a panel of nine HNSCC cell lines to TRAIL and SMs and investigated the mechanism of cell type-specific response by functional analysis. RESULTS: Head and neck cancer cell lines revealed a converse response pattern with three cell lines being highly sensitive to Smac-164 (SM) but resistant to TRAIL, whereas the other six were sensitive to TRAIL but resistant to SM. Distinct protein expression and activation patterns were found to be associated with susceptibility of HNSCC cell lines to TRAIL and SM. Tumour necrosis factor-related apoptosis-inducing ligand sensitivity was associated with high caspase-8 and Bid protein levels, and TRAIL-sensitive cell lines were killed via the type II extrinsic apoptotic pathway. Smac mimetic-sensitive cells expressed low levels of caspase-8 and Bid but had high TNF-α expression. Smac mimetic-induced cell death was associated with caspase-10 activation, suggesting that in the absence of caspase-8, caspase-10 mediates response to SM. Cotreatment with TNF-α sensitised the resistant cells to SM, demonstrating a decisive role for TNF-α-driven feedback loop in SM sensitivity. CONCLUSIONS: Tumour necrosis factor-related apoptosis-inducing ligand and SMs effectively kill HNSCC cell lines and therefore represent potential targeted therapeutics for head and neck cancer. Distinct molecular mechanisms determine the sensitivity to each agent, with levels of TNF-α, caspase-8, Bid and caspase-10 providing important predictive biomarkers of response to these agents.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Caspase 10/metabolismo , Caspase 8/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Triazóis/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Biomimética , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11-13, containing a tumour targeting, integrin αvß3/αvß5 ligand portion and a pro-apoptotic SMAC mimetic portion (cyclo-RGD/SMAC mimetic conjugates) are reported. The binding strength of the two separate units is generally maintained by these dual action conjugates. In particular, the connection between the separate units (anchor points on each unit; nature, length and stability of the linker) influences the activity of each portion against its molecular targets (integrins αvß3/αvß5 for cyclo-RGD, IAP proteins for SMAC mimetics). Each conjugate portion tolerates different substitutions while preserving the binding affinity for each target.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Mitocondriais/metabolismo , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Receptores de Vitronectina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Biotinilação/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Sistema Livre de Células , Dimerização , Doxorrubicina/farmacologia , Humanos , Concentração Inibidora 50 , Ligantes , Peptídeos Cíclicos/química , Ligação Proteica/efeitos dos fármacos , Vitronectina/metabolismoRESUMO
CONTEXT: Exposure to secondhand smoke (SHS) has been reduced in the USA by banning smoking in public places. These restrictions have not had the same effect on children's exposure to SHS as adults suggesting that children are exposed to SHS in locations not covered by bans, such as private homes and cars. OBJECTIVES: Assess exposure to SHS in the backseat of a stationary vehicle where a child would sit, quantify exposures to fine particulates (PM2.5), polycyclic aromatic hydrocarbons (PAH), carbon monoxide (CO) and nicotine. Estimate the impact on a child's mean daily exposure to PM2.5. METHODS: SHS exposures in stationary vehicles with two different window configurations were monitored. A volunteer smoked three cigarettes in a one-hour period for twenty-two experiments. PM2.5, CO, nicotine and PAH where measured in the backseat of the vehicle. 16 PAH compounds were measured for in gas and particle phases as well as real-time particle phase concentrations. RESULTS: The mean PAH concentration, 1325.1 ng/m(3), was larger than concentrations measured in bars and restaurants were smoking is banned in many countries. We estimate that a child spending only ten minutes in the car with a smoker at the mean PM2.5 concentration measured in the first window configuration--1697 mg/m(3)--will cause a 30% increase to the daily mean PM2.5 personal average of a child. CONCLUSIONS: Estimates made using the measured data and previously reported PM2.5 daily mean concentrations for children in California showing that even short exposure periods are capable of creating large exposure to smoke.
Assuntos
Poluição do Ar em Ambientes Fechados/análise , Exposição Ambiental/análise , Veículos Automotores , Material Particulado/análise , Hidrocarbonetos Policíclicos Aromáticos/análise , Fumar , Poluição por Fumaça de Tabaco/análise , California , Monóxido de Carbono/análise , Criança , Humanos , Nicotina/análise , RestaurantesRESUMO
As the U.S. population becomes more racially diverse and different groups move in to previously White-dominated spaces, new techniques of exclusion and marginalization are being employed in an effort to regulate the opportunities and progress available to racialized minority groups. In this article, the author argues that mass media's preoccupation with the Williams sisters' "on-court" play and "off-court" activities constitutes a form of surveillance that is used by Whites to identify, observe, and ultimately, limit the range of available representations of Venus and Serena Williams. The author also suggests that this kind of public scrutiny produces racialized images and narratives constitutive of "race talk," a key manifestation of the new racism(s) characteristic of the politics of this sociohistorical moment.
Assuntos
Atletas , Negro ou Afro-Americano , Vigilância da População , Opinião Pública , Relações Raciais , Mulheres , Negro ou Afro-Americano/educação , Negro ou Afro-Americano/etnologia , Negro ou Afro-Americano/história , Negro ou Afro-Americano/legislação & jurisprudência , Negro ou Afro-Americano/psicologia , Atletas/educação , Atletas/história , Atletas/legislação & jurisprudência , Atletas/psicologia , História do Século XX , História do Século XXI , Humanos , Opinião Pública/história , Relações Raciais/história , Relações Raciais/legislação & jurisprudência , Relações Raciais/psicologia , Mudança Social/história , Estados Unidos/etnologia , Mulheres/educação , Mulheres/história , Mulheres/psicologiaRESUMO
BACKGROUND: XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis. METHODS: Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases. RESULTS: We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0]alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL. CONCLUSION: Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents.
Assuntos
Ácidos Borônicos/farmacologia , Morte Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Melanoma/tratamento farmacológico , Proteínas Mitocondriais/farmacologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Reguladoras de Apoptose , Bortezomib , Caspase 8/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Interações Medicamentosas , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Proteínas Mitocondriais/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagemRESUMO
Fanconi anemia (FA) is an autosomal recessive disease characterized by pancitopenia, congenital malformations, predisposition to cancers and chromosomal instability. We report the clinical and molecular features of a patient initially identified as a potential FA case only because of chemotherapy toxicity during the treatment of a T-lineage acute lymphoblastic leukemia (ALL). Cells from this patient showed a moderate chromosomal instability, increasing sensitivity to DNA crosslinking agents but normal response to ionizing radiation. The analysis of FA proteins demonstrated a marked reduction of FANCD2 (>95%), but normal levels of FANCA or FANCG. Interestingly, this defect was associated with a homozygous missense mutation of FANCD2, resulting in a novel amino-acid substitution (Leu153Ser) at residue Leu153, which is highly conserved through evolution. The FANCD2(L153S) protein, whose reduced expression was not due to impaired transcription, was detected also in its monoubiquitinated form in the nucleus, suggesting that the mutation does not affect post-translation modifications or subcellular localization but rather the stability of FANCD2. Therefore, the hypomorphic Leu153Ser mutation represents the first example of a FANCD2 defect that might promote clonal progression of tumors, such as T-ALL, and severe chemotherapy toxicity in patients without any clinical manifestations typical of FA.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/genética , Mutação , Substituição de Aminoácidos , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Antígenos CD13 , Criança , Instabilidade Cromossômica , Progressão da Doença , Anemia de Fanconi/genética , Humanos , Infecções/etiologia , Infecções/genética , Leucemia-Linfoma de Células T do Adulto/fisiopatologia , Masculino , Pancitopenia/induzido quimicamente , Pancitopenia/genética , Indução de Remissão , Lectina 3 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. Here we show that the ATM-dependent activation of Chk2 by gamma- radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2.
Assuntos
Dano ao DNA , Ativação Enzimática , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Ativação Enzimática/efeitos da radiação , Fibroblastos/metabolismo , Raios gama , Deleção de Genes , Humanos , Immunoblotting , Microscopia de Fluorescência , Mitose , Mutação , Fosforilação , Fosfotransferases/metabolismo , Testes de Precipitina , Radiação Ionizante , Fatores de Tempo , Transfecção , Fosfatases cdc25/metabolismoAssuntos
Antineoplásicos Fitogênicos/farmacologia , Paclitaxel/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Transformada , Códon , Resistencia a Medicamentos Antineoplásicos/genética , Heterozigoto , Humanos , Camundongos , Mutação , Tolerância a Radiação/genética , Células Tumorais CultivadasRESUMO
The proteasome inhibitor PSI is potently cytotoxic in vitro against human chronic myeloid leukemia (CML) and acute myeloid leukemias (AML). Here, we have tested proteasome inhibitor I (PSI) in a panel of 11 human multiple myeloma (MM) cell lines and found that it has antiproliferative activity, with an IC50 between 4.5 and 557 nM at 48 h. PSI potentiated the toxicity of a number of chemotherapeutic agents in myeloid leukemia but not in MM cell lines, while in combination with therapeutic proteasome inhibitor PS-341 (Bortezomib) it had a synergistic effect. PSI suppressed the growth of AML cell lines more effectively than PS-341. CFU-GM colony assays revealed that CD34+ bone marrow progenitors from CML and AML patients were more sensitive to PSI than those from normal subjects (IC50: 5, 15 and 50 nM for AML, CML and normal, respectively). Moreover, the growth of normal primitive progenitors (LTC-IC) was unaffected by 15 nM PSI (P=0.576). PSI-induced cell death required RNA transcription and protein synthesis, but not DNA replication, was accompanied by the upregulation of Bcl-2 and modest reduction of Bax and Bcl-XL proteins, and involved the activation of caspases 2, 3, 7 and 8. These findings lend additional support to preclinical investigations with PSI.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Leucemia Mieloide/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/farmacologia , Antineoplásicos/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Leucemia Mieloide/patologia , Mieloma Múltiplo/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Human melanoma cells freshly isolated from 20 patients with primary and 73 patients with metastatic melanomas were analyzed by indirect immunofluorescence staining with monoclonal antibodies (MoAb) to class I (HLA-A, -B, and -C) and class II (HLA-DR and -DQ) antigens and to melanoma associated antigen (MAA). The latter included the GD3-MAA and the high molecular weight MAA. HLA class I antigens were present in 91 and 93% of primary and metastatic tumors, respectively. GD3-MAA was detected in 100% of primary and 80% of metastatic tumors. Whereas the high molecular weight MAA was expressed in 75% of tumors. Sixty % of primary and 50% of metastatic melanomas were stained by anti-HLA-DR MoAb, whereas 38 and 21% of cases, respectively, were stained by anti-HLA-DQ MoAb. Marked phenotypic heterogeneity was evident among primary and metastatic tumors, including different metastases from the same patient. Moreover, in vitro culture of melanoma cells isolated from metastases was associated with an increase from 50 to 75% of tumors stained by anti-HLA-DR MoAb but not of tumors positive for HLA class I antigens and MAA. In vitro incubation with partially purified or recombinant human gamma-interferon enhanced the expression of HLA-DR antigens on all short-term cultured melanoma cells tested but induced and/or augmented the expression of HLA-DQ antigens only in 5 of the 8 cases examined. The average increase in antigenic expression was higher for HLA-DQ than for HLA-DR antigens. Flow cytometric measurement of DNA content of melanoma cells treated with gamma-interferon revealed that the increase of HLA-DR and -DQ expression induced by gamma-interferon was independent from the cell cycle of the tumor cells.
Assuntos
Antígenos de Neoplasias/análise , Antígenos HLA/análise , Antígenos de Histocompatibilidade Classe II/análise , Melanoma/imunologia , Anticorpos Monoclonais , Divisão Celular , Antígenos HLA-DQ , Antígenos HLA-DR , Humanos , Interferon gama/uso terapêutico , Melanoma/terapia , Metástase Neoplásica , Fenótipo , Proteínas Recombinantes/uso terapêuticoRESUMO
We examined the regulation of apoptosis, radiosensitivity, and spindle checkpoint in response to DNA-damaging agents in ataxia telangiectasia (AT)-derived lymphoblastoid cell lines (AT-LCLs), which lack AT mutated (ATM) protein expression. In addition to the previous findings that AT-LCLs are defective in regulation of cell cycle at the G1, S, and G2-M checkpoints in response to X-ray irradiation (X-IR) and are highly sensitive to X-IR (J. Biol. Chem., 271: 20486-20493, 1996), we showed for the first time that AT-LCLs were defective in X-IR-associated spindle checkpoint control. The cells were also resistant to early apoptosis as much as LCLs derived from patients with Li-Fraumeni syndrome (LFS-LCLs). Terminal deoxynucleotidyl transferase-mediated nick end labeling assay of LCLs, however, demonstrated a significant increase in apoptotic cells among AT-LCLs cultured over a longer period after X-IR. These findings were in contrast to those of LFS-LCL, which showed very little increase in terminal deoxynucleotidyl transferase-mediated nick end labeling-positive population, even in cells with hyperploidy. Thus, although early apoptosis and cell cycle controls in response to DNA damage are disrupted in both ATM and p53 mutations, cells from AT patients are much more susceptible to late-onset apoptosis than those of LFS. These differences may depend on the level of accumulation of DNA damage and/or threshold that triggers late-onset cell death in ATM or p53 mutations. Our findings allow a better understanding of the role of ATM in p53-dependent and independent signal transduction pathways in response to DNA damaging agents.
Assuntos
Apoptose , Ataxia Telangiectasia/genética , Proteínas Serina-Treonina Quinases , Tolerância a Radiação , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Proteínas de Ciclo Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Dano ao DNA , Proteínas de Ligação a DNA , Humanos , Proteínas/análise , Transdução de Sinais , Proteína Supressora de Tumor p53/fisiologia , Proteínas Supressoras de TumorRESUMO
Ataxia telangiectasia (AT) carrier-derived lymphoblastoid cell lines (AT-LCLs/hetero) with suboptimal ATM protein expression were examined for the regulation of radiosensitivity, apoptosis, and mitotic spindle checkpoint in response to DNA-damaging agents. Although AT-LCLs/hetero showed intermediate radiation sensitivity, as determined by clonogenic assay, they were resistant to early-onset apoptosis, as much as AT patient-derived LCLs (AT-LCLs/homo). Furthermore, two of three AT-LCLs/hetero showed defective mitotic spindle checkpoint control in response to X-ray irradiation, which is a recently characterized biological feature in AT-LCLs/homo. Our findings indicate that carriers of ATM mutation have biological abnormalities due to haploinsufficiency of ATM protein or dominant-negative effect of mutant ATM protein. Thus, although it is still controversial whether ATM mutation carriers are at higher risk for cancer during adulthood, our findings based on in vitro biological indicators support the notion that at least some of such carriers are at a higher risk for cancer development than those without ATM mutation. Our findings may help to reevaluate epidemiological studies on cancer susceptibility in AT carriers.
Assuntos
Apoptose/genética , Ataxia Telangiectasia/genética , Heterozigoto , Mitose/genética , Proteínas Serina-Treonina Quinases , Proteínas/genética , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/efeitos da radiação , Proteínas de Ligação a DNA , Humanos , Peróxido de Hidrogênio/farmacologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Mitose/fisiologia , Oxidantes/farmacologia , Tolerância a Radiação , Proteínas Supressoras de TumorRESUMO
Fourteen cases of primary cutaneous B-cell lymphomas were investigated at the immunohistochemical and molecular level to further characterize this newly defined entity. Neoplastic cells from all cases, phenotyped with a panel of monoclonal antibodies, were positive for HLA-DR, for the B-cell markers CD19, CD22, but not CD23 (except one case), and negative for the T-cell marker CD2. Monoclonal immunoglobulin light chains were demonstrated in six cases. The reactivity with the Ki-67 monoclonal antibody indicated that the neoplastic cells are proliferating. In five biopsies the presence of dendritic cells infiltrating the neoplastic areas was revealed using the monoclonal antibody Kim4b. By Southern blot analysis, clonal rearrangement of the immunoglobulin heavy chain gene (involving one or both alleles) was shown in 12 of 14 cases and of the light chain genes in 13 cases. The bcl-2 oncogene, normally involved in nodal follicular lymphomas, was in germ-line configuration. The c-myc and the beta and gamma chain genes of the T-cell receptor were also in the germ-line configuration. None of the cases presented Epstein-Barr virus sequences. These data indicate that primary cutaneous lymphomas of B-cell origin share morphological and phenotypic similarities with the nodal B-cell lymphomas of follicular histotype, are proliferating, and express in 45% of cases clear monoclonal immunoglobulin light chain; the molecular analysis confirms the B-cell derivation and the monoclonal nature of this neoplasia; it also shows that neither bcl-2 nor c-myc oncogenes are involved and that no inappropriate rearrangements of the T-cell receptor genes are found in this lymphoma.
Assuntos
Rearranjo Gênico do Linfócito T , Rearranjo Gênico , Genes de Imunoglobulinas , Linfoma/genética , Proto-Oncogenes , Neoplasias Cutâneas/genética , Adulto , Antígenos de Diferenciação de Linfócitos B/análise , Linfócitos B/imunologia , Southern Blotting , DNA de Neoplasias/análise , Feminino , Humanos , Cadeias Leves de Imunoglobulina/genética , Linfoma/imunologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Neoplasias Cutâneas/imunologiaRESUMO
N-(4-Hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of particular clinical interest in cancer chemoprevention. We have examined the in vitro effects of HPR on lymphoid and myeloid malignant cell lines and found that at concentrations between 10(-5) and 3 x 10(-7) M it induces a dose-dependent growth inhibition (the peak plasma concentration in patients treated with HPR is 1 to 2 x 10(-6) M). The antiproliferative effect of HPR was, in all cell lines except K422, more potent than that induced by an equimolar dose of all-trans retinoic acid (RA). Also, this effect was irreversible on HL60 and DoHH2 cells that had been exposed to HPR (3 x 10(-6) M) for 24 h, but reversible on Raji and DHL4 exposed to the retinoid for 48 and 72 h, respectively. Time-course growth analysis showed that HPR at 3 x 10(-6) M or below induces a rapid fall of thymidine uptake and viability (> 90%), whereas between 10(-6) and 3 x 10(-7) M exhibits cytostatic effects. Interestingly, the RA-resistant HL-60R and NB306 cells, characterized by a point mutation in the retinoic acid receptor (RAR) and by the loss of the pml/RAR protein, respectively, were, like the parental RA-inducible HL-60 and NB4 cell lines, fully responsive to HPR, thereby suggesting that HPR and RA could act through different receptors or pathways. DNA flow-cytofluorimetric analysis revealed that HPR does not block cells in a specific phase of the cell cycle but triggers programmed cell death or apoptosis. This phenomenon was evidenced both by the visualization, on gel electrophoresis, of fragmented DNA, and by the "in cell" enzymatic labeling of DNA breaks with fluorescent dUTP. With the latter method, apoptotic cells become detectable by 6 h following exposure to 3 x 10(-6) M HPR. Ultrastructural examination of HPR-treated samples showed cells with chromatin compaction and cytoplasm condensation, characteristic of apoptotic cells. In conclusion, our study demonstrates that HPR suppresses malignant cell growth and induces apoptosis at pharmacologically relevant doses. The differential responsiveness by a number of cell lines, especially HL-60R and NB306, to HPR and RA indicates that these compounds may act through different receptors. The clinical use of HPR, particularly in retinoic acid-unresponsive acute promyelocytic leukemia patients, is suggested.
Assuntos
Apoptose/efeitos dos fármacos , Fenretinida/farmacologia , Tretinoína/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA/análise , DNA/metabolismo , Resistência a Medicamentos , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/patologia , Células Tumorais CultivadasRESUMO
The cell line AR230 was established from the peripheral blood mononuclear cells of a patient with chronic myeloid leukemia and t(9;22) translocation bearing a variant type of BCR/ABL rearrangement. AR230 expresses a BCR/ABL fusion protein with a molecular mass of 230 kilodaltons (kDa) due to the insertion of 180 amino acids encoded by 3' exons of BCR (b4 to c3). An immune complex kinase assay showed that the 230-kDa BCR/ABL protein ahd autophosphorylation activity. Immunoprecipitation analysis revealed a stable complex of GRB2 and 230-kDa BCR/ABL proteins, indicating that the Ras activation pathway is involved in the process of transformation. AR230 expressed another short transcript consisting of a BCRc2/ABL junction, which is associated with a stop signal shortly after the junction. To our knowledge, this is the first cell line expressing a 230-kDa fusion product of BCR/ABL. AR230 will be useful for studying the biological function of divergent BCR/ABL proteins.
Assuntos
Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células Tumorais Cultivadas , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Superfície/análise , Sequência de Bases , Southern Blotting , Cromossomos Humanos Par 22 , Cromossomos Humanos Par 9 , Éxons , Feminino , Rearranjo Gênico , Genes abl , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfotransferases/metabolismo , Testes de Precipitina , Translocação GenéticaRESUMO
P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.