RESUMO
Cervical cancer is the most frequent malignancy of the female genital tract and is associated with persistent infection of the uterine cervix with high-risk human papillomaviruses (HPV). The two HPV oncoproteins, E6 and E7, cooperatively immortalize cervical cells and are essential but insufficient for inducing tumorigenicity. During the progression of HPV-associated cervical dysplasia to carcinoma, the cellular telomerase reverse transcriptase (TERT) gene is activated and the TERC gene amplified. We questioned whether these increases in telomerase components might mediate the acquisition of the tumorigenic phenotype. We therefore transduced the TERT and TERC genes into E6/E7 immortalized keratinocytes that were anchorage-dependent and nontumorigenic. The resultant cells showed a profound morphological change characteristic of epithelial-mesenchymal transition as well as a corresponding increase in expression of vimentin, N-cadherin, Zinc finger E-Box binding homeobox 1, snail family transcriptional repressor 1 and matrix Metallopeptidase 2 and decrease in keratin and E-cadherin. More important, the transduced cells were now anchorage-independent and formed tumors in immunodeficient mice. Our findings indicate that overexpression of the telomerase holoenzyme in HPV-immortalized cells is sufficient to induce the complete transformed phenotype.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Telomerase , Neoplasias do Colo do Útero , Feminino , Humanos , Animais , Camundongos , Proteínas Oncogênicas Virais/genética , Telomerase/genética , Telomerase/metabolismo , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Queratinócitos/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Neoplasias do Colo do Útero/genética , Papillomaviridae/genéticaRESUMO
The high-risk alpha human papillomaviruses (HPVs) are responsible for 99% of cervical cancers. While the biological functions of the HPV E6 and E7 oncoproteins are well-characterized, the function of E5 has remained elusive. Here, we examined gene expression changes induced by E5 proteins from high-risk HPV-16 and low-risk HPV-6b in multiple pools of primary human keratinocytes. Surprisingly, microarray analysis revealed that over 700 genes were significantly regulated by HPV-6b E5, while only 25 genes were consistently and significantly regulated by HPV-16 E5 in three biological replicates. However, we observed that more than thousand genes were altered in individual sample compared with vector. The gene expression profile induced by 16E5 in primary genital keratinocytes was very different from what has been previously published using immortalized HaCaT cells. Genes altered by HPV-16 E5 were unaffected by HPV-6b E5. Our data demonstrate that E5 proteins from the high- and low-risk HPVs have different functions in the HPV-host cell. Interestingly, conversion of two amino acids in HPV-16 E5 to the low-risk HPV-6b sequence eliminated the induction of high-risk related cellular genes.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Proteínas Viroporinas , Aminoácidos , Feminino , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Viroporinas/genéticaRESUMO
The high-risk human papillomaviruses (HPV-16, -18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ-containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high-risk E6 but not low-risk E6 expression led to increases in AIB1 transcript levels by real-time RT-PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV-16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Telomerase , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Animais , Biomarcadores , Feminino , Humanos , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/complicações , Telomerase/genética , Telomerase/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53RESUMO
Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Treatment of the disease represents an unsolved clinical problem, as survival of patients with aggressive form of NB remains below 50%. Despite recent identification of numerous potential therapeutic targets, clinical trials validating them are challenging due to the rarity of the disease and its high patient-to-patient heterogeneity. Hence, there is a need for the accurate preclinical models that would allow testing novel therapeutic approaches and prioritizing the clinical studies, preferentially in personalized way. Here, we propose using conditional reprogramming (CR) technology for rapid development of primary NB cell cultures that could become a new model for such tests. This newly established method allowed for indefinite propagation of normal and tumor cells of epithelial origin in an undifferentiated state by their culture in the presence of Rho-associated kinase (ROCK) inhibitor, Y-27632, and irradiated mouse feeder cells. Using a modification of this approach, we isolated cell lines from tumors arising in the TH-MYCN murine transgenic model of NB (CR-NB). The cells were positive for neuronal markers, including Phox2B and peripherin and consisted of two distinct populations: mesenchymal and adrenergic expressing corresponding markers of their specific lineage. This heterogeneity of the CR-NB cells mimicked the different tumor cell phenotypes in TH-MYCN tumor tissues. The CR-NB cells preserved anchorage-independent growth capability and were successfully passaged, frozen and biobanked. Further studies are required to determine the utility of this method for isolation of human NB cultures, which can become a novel model for basic, translational, and clinical research, including individualized drug testing.
Assuntos
Linhagem Celular Tumoral , Neuroblastoma/patologia , Animais , Biomarcadores/metabolismo , Técnicas de Reprogramação Celular , Humanos , Camundongos Transgênicos , Neoplasias Experimentais , Neuroblastoma/metabolismo , Fenótipo , RatosRESUMO
BACKGROUND: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood. METHODS: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC. RESULTS: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients. CONCLUSION: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.
Assuntos
Claudinas/metabolismo , Ácidos Graxos/metabolismo , Metabolômica/métodos , Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Humanos , TransfecçãoRESUMO
Patient-derived xenografts (PDXs) are widely recognised as a more physiologically relevant preclinical model than standard cell lines, but are expensive and low throughput, have low engraftment rate and take a long time to develop. Our newly developed conditional reprogramming (CR) technology addresses many PDX drawbacks, but lacks many in vivo factors. Here we determined whether PDXs and CRCs of the same cancer origin maintain the biological fidelity and complement each for translational research and drug development. Four CRC lines were generated from bladder cancer PDXs. Short tandem repeat (STR) analyses revealed that CRCs and their corresponding parental PDXs shared the same STRs, suggesting common cancer origins. CRCs and their corresponding parental PDXs contained the same genetic alterations. Importantly, CRCs retained the same drug sensitivity with the corresponding downstream signalling activity as their corresponding parental PDXs. This suggests that CRCs and PDXs can complement each other, and that CRCs can be used for in vitro fast, high throughput and low cost screening while PDXs can be used for in vivo validation and study of the in vivo factors during translational research and drug development.
Assuntos
Neoplasias da Bexiga Urinária/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Modelos Animais de Doenças , Desenvolvimento de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Mutação , Pesquisa Translacional Biomédica , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/economiaRESUMO
Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research, particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y), termed conditionally reprogrammed cell (CRC) technology, enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings, we then created six CF cell CRC populations, three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted, and cells were subcultured, without irradiated feeders plus Y, into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y, dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage, but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus, CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers.
Assuntos
Brônquios/patologia , Reprogramação Celular , Fibrose Cística/patologia , Células Epiteliais/patologia , Animais , Linhagem Celular , Proliferação de Células , Forma Celular , Fibrose Cística/fisiopatologia , Fenômenos Eletrofisiológicos , Humanos , CamundongosRESUMO
A patient with a 20-year history of recurrent respiratory papillomatosis had progressive, bilateral tumor invasion of the lung parenchyma. We used conditional reprogramming to generate cell cultures from the patient's normal and tumorous lung tissue. Analysis revealed that the laryngeal tumor cells contained a wild-type 7.9-kb human papillomavirus virus type 11 (HPV-11) genome, whereas the pulmonary tumor cells contained a 10.4-kb genome. The increased size of the latter viral genome was due to duplication of the promoter and oncogene regions. Chemosensitivity testing identified vorinostat as a potential therapeutic agent. At 3 months after treatment initiation, tumor sizes had stabilized, with durable effects at 15 months.
Assuntos
Antineoplásicos/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias Pulmonares/patologia , Pulmão/citologia , Infecções por Papillomavirus/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Células Cultivadas , DNA Viral/isolamento & purificação , Expressão Gênica , Genoma Viral , Papillomavirus Humano 11/genética , Humanos , Neoplasias Laríngeas/cirurgia , Neoplasias Laríngeas/virologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/virologia , Masculino , Mutação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/cirurgia , RNA Mensageiro/metabolismo , RNA Viral/análise , Infecções Respiratórias/complicações , Infecções Respiratórias/patologia , Infecções Respiratórias/cirurgia , Células Tumorais Cultivadas , Vorinostat , Adulto JovemRESUMO
Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.
Assuntos
Transformação Celular Viral , Papillomavirus Humano 16/metabolismo , Queratinócitos/fisiologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Repressoras/metabolismo , Telomerase/metabolismo , Papillomavirus Humano 16/genética , Humanos , Queratinócitos/citologia , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Complexo Repressor Polycomb 1/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Telomerase/genética , Telômero/genética , Telômero/fisiologiaRESUMO
The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and ß1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear ß-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.
Assuntos
Células-Tronco Adultas/citologia , Amidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Piridinas/farmacologia , Células-Tronco Adultas/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Western Blotting , Reprogramação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Alimentadoras , Citometria de Fluxo , Humanos , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica , Integrina beta1/metabolismo , Cariotipagem , Fator 4 Semelhante a Kruppel , Reação em Cadeia da Polimerase em Tempo Real , Telomerase/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative "ATI-like" cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a "conditionally reprogrammed cell" phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and "ATI-like" mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air-liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases.
Assuntos
Células Epiteliais Alveolares/metabolismo , Proliferação de Células , Alvéolos Pulmonares/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , Transdiferenciação Celular , Técnicas de Cocultura , Impedância Elétrica , Células Alimentadoras , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Transporte de Íons , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells.
Assuntos
Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Raios gama , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Células 3T3 , Animais , Linhagem Celular Transformada , Meios de Cultivo Condicionados/farmacologia , Células Alimentadoras , Humanos , Queratinócitos/citologia , Masculino , CamundongosRESUMO
BACKGROUND: Acute colonic pseudo-obstruction (ACPO) is characterized by severe colonic distension without mechanical obstruction. It has an uncertain pathogenesis and poses diagnostic challenges. This study aims to explore risk factors and clinical outcomes of ACPO in polytrauma patients, and contributing information to the limited literature on this condition. METHODS: This retrospective study, conducted at a Level 1 Trauma Centre, analysed data from trauma patients with ACPO admitted between July 2009 and June 2018. A control cohort of major trauma patients was utilized. Data review encompassed patient demographics, abdominal imaging, injury characteristics, analgesic usage, interventions, complications, and mortality. Statistical analyses, including logistic regression and correlation coefficients, were employed to identify risk factors. RESULTS: There were 57 cases of ACPO, with an incidence of 1.7 / 1000 patients, rising to 4.86 in major trauma. Predominantly affecting those over 50 years of age (75%) and males (75%), with motor vehicle accidents (50.8%) and falls from height (36.8%) being the commonest mechanisms. Noteworthy associated injuries included retroperitoneal bleeds (RPB) (37%), spinal fractures (37%), and pelvic fractures (37%). Analysis revealed significant associations between ACPO and Shock Index >0.9, Injury Severity Score > 18, opioid use, RPB, and pelvic fractures. A caecal diameter of ≥12 cm had a significant association with caecal ischemia or perforation. CONCLUSION: This study underscores the significance of ACPO in polytrauma patients, demonstrating associations with risk factors and clinical outcomes. Clinicians should maintain a high index of suspicion, particularly in older patients with RPB, pelvic fractures, and opioid use. Early supportive therapy, vigilant monitoring, and timely interventions are crucial for a favourable outcome. Further research and prospective trials are warranted to validate these findings and enhance understanding of ACPO in trauma patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological, Level IV.
RESUMO
Papillomaviruses are epitheliotropic, nonenveloped, circular, double-stranded DNA viruses within the family Papillomaviridae that are associated with benign and malignant tumors in humans and animals. We report the complete genome sequence of canine papillomavirus type 10 identified from a pigmented plaque located on the head of a mixed-breed bloodhound.
Assuntos
Doenças do Cão/virologia , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/veterinária , Animais , Sequência de Bases , DNA Viral/análise , DNA Viral/genética , Cães , Genoma Viral/genética , Dados de Sequência Molecular , Infecções por Papillomavirus/virologia , Análise de Sequência de DNARESUMO
Papillomaviruses are nonenveloped, double-stranded DNA viruses that are associated with both benign and malignant tumors in animals and humans. We report the complete genome sequence of canine papillomavirus type 9 isolated from a solitary pigmented plaque on a mixed-breed bloodhound.
Assuntos
Genoma Viral , Sequência de Bases , Dados de Sequência MolecularRESUMO
The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.
Assuntos
Células Epiteliais/química , Papillomavirus Humano 16/química , Proteínas Oncogênicas Virais/análise , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Espectrometria de Massas/métodos , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Mapeamento de Peptídeos , Neoplasias do Colo do Útero/química , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.
Assuntos
Amidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Alimentadoras/fisiologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Mama/citologia , Técnicas de Cultura de Células , Reprogramação Celular/efeitos dos fármacos , Colágeno , Combinação de Medicamentos , Células Epiteliais/citologia , Células Alimentadoras/citologia , Feminino , Humanos , Laminina , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Próstata/citologia , Neoplasias da Próstata/patologia , Proteoglicanas , Transplante HeterólogoRESUMO
The nuclear receptor coactivator amplified in breast cancer 1 (AIB1/SRC-3) has a well-defined role in steroid and growth factor signaling in cancer and normal epithelial cells. Less is known about its function in stromal cells, although AIB1/SRC-3 is up-regulated in tumor stroma and may, thus, contribute to tumor angiogenesis. Herein, we show that AIB1/SRC-3 depletion from cultured endothelial cells reduces their proliferation and motility in response to growth factors and prevents the formation of intact monolayers with tight junctions and of endothelial tubes. In AIB1/SRC-3(+/-) and (-/-) mice, the angiogenic responses to subcutaneous Matrigel implants was reduced by two-thirds, and exogenously added fibroblast growth factor (FGF) 2 did not overcome this deficiency. Furthermore, AIB1/SRC-3(+/-) and (-/-) mice showed similarly delayed healing of full-thickness excisional skin wounds, indicating that both alleles were required for proper tissue repair. Analysis of this defective wound healing showed reduced recruitment of inflammatory cells and macrophages, cytokine induction, and metalloprotease activity. Skin grafts from animals with different AIB1 genotypes and subsequent wounding of the grafts revealed that the defective healing was attributable to local factors and not to defective bone marrow responses. Indeed, wounds in AIB1(+/-) mice showed reduced expression of FGF10, FGFBP3, FGFR1, FGFR2b, and FGFR3, major local drivers of angiogenesis. We conclude that AIB1/SRC-3 modulates stromal cell responses via cross-talk with the FGF signaling pathway.
Assuntos
Neovascularização Fisiológica/fisiologia , Coativador 3 de Receptor Nuclear/fisiologia , Pele/lesões , Cicatrização/fisiologia , Animais , Células Cultivadas , Colágeno , Combinação de Medicamentos , Fatores de Crescimento de Fibroblastos/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Inflamação/fisiopatologia , Laminina , Masculino , Camundongos , Camundongos Knockout , Coativador 3 de Receptor Nuclear/deficiência , Proteoglicanas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transdução de Sinais/fisiologia , Pele/irrigação sanguínea , Pele/metabolismo , Fenômenos Fisiológicos da Pele , Transplante de Pele/métodos , Células Estromais/fisiologiaRESUMO
INTRODUCTION: Morel-Lavallée lesions (MLL), also referred to as closed degloving injuries, result from traumatic shearing forces with separation of the subcutaneous fat from the underlying fascia. The aim of this study was to determine the incidence and treatment of MLLs at a level 1 trauma centre. METHODS: Single-centre retrospective cross-sectional study of consecutive patients with an imaging diagnosis of a Morel-Lavallee lesion from 1/1/2010-31/12/2019. Demographic data, mechanism of injury, volume of lesion, management and outcome data were collated. RESULTS: Sixty-six MLLs were identified in 63 patients (64% Male) with a median age of 49.5 years (19-94 years). Mechanism of injury were road traffic accidents in the majority (66%). Median injury severity score (ISS) was 17 (range 1-33). Patients on oral anti-coagulants had significantly larger lesions (181.9 cc v 445.5 cc, P = 0.044). The most common lesion location was the thigh (60.5%). Patients that underwent imaging within 72 h of injury had significantly larger lesions than those imaged more than 72 h after the inciting trauma (65 cc v 167 cc, P < 0.05). Management data were documented in 59% of lesions (39/66) in which 66.6% (n = 26) had invasive treatment. In the 31 patients where follow-up was available, 64.5% (n = 20) were persistent but decreasing in size. There was no significant difference in follow-up size for those who had invasive compared to conservative treatment (P = 0.3). CONCLUSION: The diagnosis of MLL should be considered for soft-tissue swelling in the context of shearing trauma. A variety of management options have been employed, with good overall outcomes.
Assuntos
Avulsões Cutâneas , Lesões dos Tecidos Moles , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Avulsões Cutâneas/diagnóstico por imagem , Avulsões Cutâneas/terapia , Lesões dos Tecidos Moles/diagnóstico por imagem , Lesões dos Tecidos Moles/epidemiologia , Lesões dos Tecidos Moles/terapia , Incidência , Centros de Traumatologia , Estudos Retrospectivos , Estudos Transversais , Resultado do TratamentoRESUMO
OBJECTIVE: Haemorrhagic shock is a life-threatening complication of trauma, but remains a preventable cause of death. Early recognition of retroperitoneal haemorrhage (RPH) is crucial in preventing deleterious outcomes including mortality. Injury to the 9-11th intercostal arteries (i.e. arteries of the lower thoracic region) are complicit in RPH. However, the associated injuries, implications and management of such bleeds remain poorly characterised. METHODS: We performed a retrospective review of the medical records of patients diagnosed with RPH who presented to our level-1 trauma centre (2009-2019). We described the associated injuries, management and outcomes relating to RPH of the lower thoracic region (the 9-11th intercostal arteries) from this cohort to identify potential predictors and evaluate the impact of early identification and management of non-cavitary bleeds. RESULTS: Haemorrhage of the lower intercostal arteries (LIA) into the retroperitoneal space is associated with an increased number of posterior lower rib fractures and pneumothorax/haemothorax. A higher proportion of patients in the LIA group required massive transfusion, angioembolisation or surgical ligation when compared to other causes of RPH. CONCLUSION: The present study highlights the importance of injury patterns, particularly posterior lower rib fractures, as predictors for early recognition and management of RPH in the prevention of deleterious patient outcomes. RPH secondary to bleeding of the LIA may require early and aggressive management of haemorrhage through massive transfusion, and angioembolisation or surgical ligation when compared to RPH because of other causes.