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Engineering ethics is a required aspect of accredited ABET programs, but there is widespread variation in how ethics is taught, to what ends, and how those ends are assessed. This variation makes it challenging to identify practices for teaching ethics to engineers aligned with extant practices in the field. In this study, we revise a recent coding framework by reviewing exemplary engineering ethics programs recognized by the National Academy of Engineering in 2016, or what we refer to as "exemplars." We pursue two primary objectives: (1) To apply and revise a prior coding framework to codify ethics learning objectives, instructional strategies, and assessment strategies in engineering education; and (2) To use the revised coding framework to identify trends in learning objectives, instructional strategies, and assessment strategies of NAE exemplars. We employ systemic review procedures to update the coding framework using 24 of 25 exemplars as a data source. The updated framework includes four primary categories associated with learning objectives, instructional strategies, assessment data collection strategies, and assessment design characteristics. Results indicate that ethical sensitivity or awareness was present in every exemplar as a learning objective, often alongside ethical reasoning-based learning objectives and the formation of professional skills. Exemplars employed numerous instructional strategies in tandem, as we coded eight out of 18 instructional strategies among at least half of the exemplars. Assignments/homework and summative reflections were the most oft-used sources of assessment data. Due to our challenges in coding assessment approaches, we offer practical suggestions for assessing engineering ethics instruction which are based on many of our coding discussions. We hope that this coding framework, the results classifying exemplary features of the NAE programs, and our practical suggestions can guide future instructors as they design, classify, assess, and report their approaches to engineering ethics education.
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Ética Profesional , Aprendizaje , IngenieríaRESUMEN
Although learning is often viewed as a unique feature of organisms with complex nervous systems, single-celled organisms also demonstrate basic forms of learning. The giant ciliate Stentor coeruleus responds to mechanical stimuli by contracting into a compact shape, presumably as a defense mechanism. When a Stentor cell is repeatedly stimulated at a constant level of force, it will learn to ignore that stimulus but will still respond to stronger stimuli. Prior studies of habituation in Stentor reported a graded response, suggesting that cells transition through a continuous range of response probabilities. By analyzing single cells using an automated apparatus to deliver calibrated stimuli, we find that habituation occurs via a single step-like switch in contraction probability within each cell, with the graded response in a population arising from the random distribution of switching times in individual cells. This step-like response allows Stentor behavior to be represented by a simple two-state model whose parameters can be estimated from experimental measurements. We find that transition rates depend on stimulus force and also on the time between stimuli. The ability to measure the behavior of the same cell to the same stimulus allowed us to quantify the functional heterogeneity among single cells. Together, our results suggest that the behavior of Stentor is governed by a two-state stochastic machine whose transition rates are sensitive to the time series properties of the input stimuli.
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Cilióforos , Habituación Psicofisiológica , Análisis de la Célula Individual , Cilióforos/fisiología , Factores de TiempoRESUMEN
The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor's transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.
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Cilióforos , Secuencia de Bases , Cilióforos/genética , Humanos , Interferencia de ARN , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.1,18 A Stentor cell is roughly 2 mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.
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Cilióforos , Proteoma , Polaridad Celular/genética , Cilióforos/genética , Morfogénesis/genética , Proteoma/metabolismo , Proteómica , Saccharomyces cerevisiaeRESUMEN
Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes-many not associated with the oral apparatus (OA)-that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.
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Rastreo Celular/normas , Regeneración/genética , AnimalesRESUMEN
The giant unicellular ciliate Stentor coeruleus can be cut into pieces and each piece will regenerate into a healthy, full-sized individual. The molecular mechanism for how Stentor regenerates is a complete mystery, however, the process of regeneration shows striking similarities to the process of cell division. On a morphological level, the process of creating a second mouth in division or a new oral apparatus in regeneration have the same steps and occur in the same order. On the transcriptional level, genes encoding elements of the cell division and cell cycle regulatory machinery, including Aurora kinases, are differentially expressed during regeneration. This suggests that there may be some common regulatory mechanisms involved in both regeneration and cell division. If the cell cycle machinery really does play a role in regeneration, then inhibition of proteins that regulate the timing of cell division may also affect the timing of regeneration in Stentor. Here we show that two well-characterized Aurora kinase A+B inhibitors that affect the timing of regeneration. ZM447439 slows down regeneration by at least one hour. PF03814735 completely suppresses regeneration until the drug is removed. Here we provide the first direct experimental evidence that Stentor may harness the cell division machinery to regulate the sequential process of regeneration.
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PURPOSE: Cervical cancer remains the second most common cancer and cancer-related death among women in Ethiopia. This is the first study, to our knowledge, describing the demographic, and clinicopathologic characteristics of cervical cancer cases in a mainly rural, Southwestern Ethiopian population with a low literacy rate to provide data on the cervical cancer burden and help guide future prevention and intervention efforts. METHODS: A descriptive analysis of 154 cervical cancer cases at the Jimma University Teaching Hospital in Southwestern Ethiopia from January 2008 -December 2010 was performed. Demographic and clinical characteristics were obtained from patient questionnaires and cervical punch biopsies were histologically examined. RESULTS: Of the 154 participants with a histopathologic diagnosis of cervical cancer, 95.36% had not heard of cervical cancer and 89.6% were locally advanced at the time of diagnosis. Moreover, 86.4% of participants were illiterate, and 62% lived in a rural area. CONCLUSION: A majority of the 154 women with cervical cancer studied at the Jimma University Teaching Hospital in Southwestern Ethiopia were illiterate, had not heard of cervical cancer and had advanced disease at the time of diagnosis. Given the low rates of literacy and knowledge regarding cervical cancer in this population which has been shown to correlate with a decreased odds of undergoing screening, future interventions to address the cervical cancer burden here must include an effective educational component.
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Neoplasias del Cuello Uterino/epidemiología , Adulto , Etiopía/epidemiología , Femenino , Conocimientos, Actitudes y Práctica en Salud , Humanos , Alfabetización , Tamizaje Masivo , Persona de Mediana Edad , Población Rural , Encuestas y Cuestionarios , Neoplasias del Cuello Uterino/diagnósticoRESUMEN
Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.
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Cilióforos/crecimiento & desarrollo , Técnicas Histológicas/métodos , Regeneración/fisiología , Animales , Cilióforos/patogenicidadRESUMEN
Measurements of the thermal transport properties of the skin can reveal changes in physical and chemical states of relevance to dermatological health, skin structure and activity, thermoregulation and other aspects of human physiology. Existing methods for in vivo evaluations demand complex systems for laser heating and infrared thermography, or they require rigid, invasive probes; neither can apply to arbitrary regions of the body, offers modes for rapid spatial mapping, or enables continuous monitoring outside of laboratory settings. Here we describe human clinical studies using mechanically soft arrays of thermal actuators and sensors that laminate onto the skin to provide rapid, quantitative in vivo determination of both the thermal conductivity and thermal diffusivity, in a completely non-invasive manner. Comprehensive analysis of measurements on six different body locations of each of twenty-five human subjects reveal systematic variations and directional anisotropies in the characteristics, with correlations to the thicknesses of the epidermis (EP) and stratum corneum (SC) determined by optical coherence tomography, and to the water content assessed by electrical impedance based measurements. Multivariate statistical analysis establishes four distinct locations across the body that exhibit different physical properties: heel, cheek, palm, and wrist/volar forearm/dorsal forearm. The data also demonstrate that thermal transport correlates negatively with SC and EP thickness and positively with water content, with a strength of correlation that varies from region to region, e.g., stronger in the palmar than in the follicular regions.
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Fenómenos Fisiológicos de la Piel , Conductividad Térmica , Sensación Térmica , Algoritmos , Humanos , Modelos Teóricos , Flujo Sanguíneo Regional , TemperaturaRESUMEN
Retroviral gene transfer is a highly efficient and effective method of stably introducing genetic material into the genome of specific cell types. The process involves the transfection of retroviral expression vectors into a packaging cell line, the isolation of viral particles, and the infection of target cell lines. Compared to traditional gene transfer methods such as liposome-mediated transfection, retroviral gene transfer allows for stable gene expression in cell populations without the need for lengthy selection and cloning procedures. This is particularly helpful when studying gene products that have negative effect on cell growth and viability. Here, we describe the retroviral transfer of cystatin cDNAs using HEK293-derived Phoenix packaging cells and human HeLa cervical carcinoma cells as target cells.
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Cistatinas/genética , Expresión Génica , Técnicas de Transferencia de Gen , Retroviridae/metabolismo , Células HEK293 , Células HeLa , Humanos , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción Genética , Transfección , Ensamble de VirusRESUMEN
Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28-29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2'O-methylation at their 3' ends. We propose models for the role of these "27macRNAs" in macronuclear development.
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Macronúcleo/genética , Oxytricha/genética , ARN Protozoario/genética , ARN Nuclear Pequeño/genética , Regulación de la Expresión Génica , Genoma de Protozoos , Macronúcleo/metabolismo , Oxytricha/fisiología , ARN Protozoario/química , ARN Nuclear Pequeño/química , Análisis de Secuencia de ARN , Telómero/genéticaRESUMEN
Senescent cells exhibit altered expression of numerous genes. Identifying the significance of the changes in gene expression may help advance our understanding of the senescence biology. Here, we report on the consistent and strong upregulation of CST1 expression during cellular senescence, independent of the initial trigger. CST1 expression at both the messenger RNA and protein levels was barely detected in control cells, which included early passage proliferating, quiescent, or immortal human fibroblasts and various human tumor cell lines. Immunoblotting and immunofluorescence cytochemical studies further suggest that CST1 accumulates intracellularly, within vesicular structures. We discuss these results in light of the known function of CST1 as a potent inhibitor of lysosomal cysteine proteases.
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Envejecimiento/genética , Senescencia Celular/genética , Expresión Génica , ARN/genética , Cistatinas Salivales/genética , Envejecimiento/patología , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunohistoquímica , Cistatinas Salivales/biosíntesisRESUMEN
Rsf-1 (HBXAP) has been reported as an amplified gene in human cancer, including the highly aggressive ovarian serous carcinoma. Rsf-1 protein interacts with SNF2H to form an ISWI chromatin remodeling complex, RSF. In this study, we investigated the functional role of Rsf-1 by observing phenotypes after expressing it in nontransformed cells. Acute expression of Rsf-1 resulted in DNA damage as evidenced by DNA strand breaks, nuclear γH2AX foci, and activation of the ATM-CHK2-p53-p21 pathway, leading to growth arrest and apoptosis. Deletion mutation and gene knockdown assays revealed that formation of a functional RSF complex with SNF2H was required for Rsf-1 to trigger DNA damage response (DDR). Gene knock-out of TP53 alleles, TP53 mutation, or treatment with an ATM inhibitor abolished up-regulation of p53 and p21 and prevented Rsf-1-induced growth arrest. Chronic induction of Rsf-1 expression resulted in chromosomal aberration and clonal selection for cells with c-myc amplification and CDKN2A/B deletion. Co-culture assays indicated Rsf-1-induced DDR as a selecting barrier that favored outgrowth of cell clones with a TP53 mutation. The above findings suggest that increased Rsf-1 expression and thus excessive RSF activity, which occurs in tumors harboring Rsf-1 amplification, can induce chromosomal instability likely through DDR.
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Daño del ADN , Inestabilidad Genómica , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Quinasa de Punto de Control 2 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Aberraciones Cromosómicas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Trophoblast cells and many cancer cells that harbor foreign antigens may evade immunity by epigenetic silencing of key immune response genes, including MHC class I and II and CD40. Chromatin active agents, such as histone deacetylase inhibitors (HDACi), induce immune response gene expression but often the expression levels are low and the cells lack a robust antigen presentation response. We show here that pre-treatment of trophoblast cells and certain cancer cells with agents that activate stress pathways (Ras oncogene, PMA or H2O2) and induce senescence can substantially enhance the induction of immune response genes (MHC class II, CD40, MICA, MICB) by HDACi and restore a vigorous IFN-gamma response in trophoblast cells and tumor cells. These results could potentially impact the development of novel anti-cancer therapeutic strategies.
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Senescencia Celular/inmunología , Coriocarcinoma/inmunología , Trofoblastos/inmunología , Neoplasias Uterinas/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Antígenos CD40/genética , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Coriocarcinoma/tratamiento farmacológico , Coriocarcinoma/genética , Coriocarcinoma/patología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/inmunología , Femenino , Silenciador del Gen/efectos de los fármacos , Silenciador del Gen/inmunología , Genes MHC Clase I/genética , Genes MHC Clase I/inmunología , Genes MHC Clase II/genética , Genes MHC Clase II/inmunología , Células HeLa , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Estrés Oxidativo/inmunología , Embarazo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/inmunología , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo , Trofoblastos/patología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/inmunología , Neoplasias Uterinas/tratamiento farmacológico , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologíaRESUMEN
The evolutionarily conserved TREX (Transcription/Export) complex physically couples transcription, messenger ribonucleoprotein particle biogenesis, RNA processing, and RNA export for a subset of genes. HPR1 encodes an essential component of the S. cerevisiae TREX complex. HPR1 loss compromises transcriptional elongation, nuclear RNA export, and genome stability. Yet, HPR1 is not required for yeast viability. Thoc1 is the recently discovered human functional orthologue of HPR1. Thoc1 is expressed at higher levels in breast cancer than in normal epithelia, and expression levels correlate with tumor size and metastatic potential. Depletion of Thoc1 protein (pThoc1) in human cancer cell lines compromises cell proliferation. It is currently unclear whether Thoc1 is essential for all mammalian cells or whether cancer cells may differ from normal cells in their dependence on Thoc1. To address this issue, we have compared the requirements for Thoc1 in the proliferation and survival of isogenic normal and oncogene-transformed cells. Neoplastic cells rapidly lose viability via apoptotic cell death on depletion of pThoc1. Induction of apoptotic cell death is coincident with increased DNA damage as indicated by the appearance of phosphorylated histone H2AX. In contrast, the viability of normal cells is largely unaffected by pThoc1 loss. Normal cells lacking Thoc1 cannot be transformed by forced expression of E1A and Ha-ras, suggesting that Thoc1 may be important for neoplastic transformation. In sum, our data are consistent with the hypothesis that cancer cells require higher levels of pThoc1 for survival than normal cells. If true, pThoc1 may provide a novel molecular target for cancer therapy.
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Proteínas de Ciclo Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/fisiología , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Transformación Celular Neoplásica , Daño del ADN , Proteínas de Unión al ADN , Evolución Molecular , Fibroblastos/metabolismo , Genoma , Células HeLa , Humanos , Modelos Biológicos , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas de Unión al ARNRESUMEN
Activated Ras signaling can induce a permanent growth arrest in osteosarcoma cells. Here, we report that a senescence-like growth inhibition is also achieved in human carcinoma cells upon the transduction of H-Ras(V12). Ras-induced tumor senescence can be recapitulated by the transduction of activated, but not wild-type, MEK. The ability for H-Ras(V12) to suppress tumor cell growth is drastically compromised in cells that harbor endogenous activating ras mutations. Notably, growth inhibition of tumor cells containing ras mutations can be achieved through the introduction of activated MEK. Tumor senescence induced by Ras signaling can occur in the absence of p16 or Rb and is not interrupted by the inactivation of Rb, p107, or p130 via short hairpin RNA or the transduction with HPV16 E7. In contrast, inactivation of p21 via short hairpin RNA disrupts Ras-induced tumor senescence. In summary, this study uncovers a senescence-like program activated by Ras signaling to inhibit cancer cell growth. This program appears to be intact in cancer cells that do not harbor ras mutations. Moreover, cancer cells that carry ras mutations remain susceptible to tumor senescence induced by activated MEK. These novel findings can potentially lead to the development of innovative cancer intervention.
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Senescencia Celular , Neoplasias , Transducción de Señal , Proteínas ras/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Mutación , Neoplasias/metabolismo , Neoplasias/patología , Proteínas ras/genéticaRESUMEN
In contrast to rodent cells, normal human fibroblasts are generally resistant to neoplastic transformation in vitro. Here, we report the derivation and characterization of a spontaneously transformed cell line from normal human IMR90 fibroblasts transduced with E1A and Ras oncogenes. Unlike the parental, non-tumorigenic E1A/Ras-expressing IMR90 cells, these spontaneously transformed cells displayed aberrant growth potential in vitro and were capable of tumorigenesis in vivo. In contrast to the parental E1A/Ras-expressing cells, both the spontaneously transformed cells and cells derived from resultant tumors displayed specific t(7q;8q) and t(5q;17) structural chromosomal changes. Chromosome 8q contains c-Myc, which is capable of activating the telomerase catalytic subunit hTERT. Notably, upregulation of c-Myc, hTERT and telomerase activity were detected only in the tumorigenic cells. Transduction of Myc siRNA into the tumorigenic cells led to a concomitant downregulation of hTERT. Furthermore, transduction of Myc or hTERT into the non-tumorigenic E1A/Ras-expressing IMR90 cells was able to confer tumorigenesis on these cells. These studies suggest that the t(7;8) translocation may result in Myc overexpression and its subsequent activation of hTERT, which may contribute to the tumorigenicity of the IMR90 cells. Furthermore, this report describes additional successful neoplastic transformation of human IMR90 fibroblasts by defined genetic elements. The spontaneously transformed cells we have derived provide a valuable model system for the study of neoplastic transformation.
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Transformación Celular Neoplásica , Fibroblastos , Transducción Genética , Proteínas E1A de Adenovirus/fisiología , Técnicas de Cultivo de Célula , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica , Genes myc , Genes ras , Predisposición Genética a la Enfermedad , Humanos , Telomerasa/biosíntesis , Translocación Genética , Células Tumorales CultivadasRESUMEN
Upregulation of the p16 tumor suppressor is a hallmark of senescence in human fibroblasts. In this study, we investigated potential protein modification of p16 in senescent human fibroblasts using 2D SDS-PAGE analysis. Three distinct p16 variants with isoelectric points of 5.2, 5.4, and 5.6, were consistently detected in normal human IMR90 fibroblasts that had undergone senescence due to forced expression of oncogenic H-ras or culture passage. Moreover, in contrast to short-term serum starvation, which induces quiescence, IMR90 fibroblasts cultured in low serum for a prolonged period exhibited senescent phenotypes and expression of the three p16 variants. All three p16 variants are unlikely phosphoproteins since they failed to react with antibodies against phospho-serine, and were resistant to the treatment with phosphatases. Functionally, co-immunoprecipitation assays using antibodies against cdk4 and/or cdk6 revealed that only the two most acidic p16 variants associated with cdk4/6. Moreover, senescence induced by the forced expression of p16 in early passage IMR90 fibroblasts or osteosarcoma U2OS cells was accompanied by expression of the two most acidic p16 variants, which also associated with cdk4/6. In summary, we report that prolonged serum starvation-induced senescence may provide an additional model for studying biochemical changes in senescence, including p16 regulation. Furthermore, induction of endogenous p16 in senescent human fibroblasts correlates with the expression of three distinct p16 variants independent of protein phosphorylation. Lastly, expression of the two cdk-bound variants is sufficient to induce senescence in human cells.
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Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Western Blotting , Medio de Cultivo Libre de Suero , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Inmunoprecipitación , Fosforilación , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.
Asunto(s)
Senescencia Celular/genética , Genes cdc/fisiología , Genes ras/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas ras/genética , Animales , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Forma de la Célula/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Senescencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Genes cdc/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción Genética , Proteínas ras/metabolismoRESUMEN
Senescence irreversibly arrests the proliferation of cells that have sustained significant cellular stress. Replicative senescence, due to the shortening and dysfunction of telomeres, appears to provide a barrier to the immortalization of cells and development of cancer. In normal human fibroblasts, senescence induced by oncogenic H-ras displays a nearly identical cellular phenotype to that of replicative senescence, suggesting the activation of a common senescence mechanism. In this study, we investigated the gene expression profile of oncogenic H-ras-induced senescent human diploid fibroblasts. We found altered gene expression of various cell cycle regulators in both oncogenic H-ras-induced senescent cells and replicative senescent cells. Similar to replicative senescent cells, H-ras-induced senescent cells exhibited specific downregulation of genes involved in G2/M checkpoint control and contained tetraploid cells that were arrested in a G1 state. This observation suggests that the inactivation of G2/M checkpoints may be involved in senescence and may play a role in the generation of senescent G1 tetraploid cells. Lastly, we have identified two genes, topoisomerase IIalpha and HDAC9, whose expression was specifically altered under several conditions associated with senescence, suggesting that these two molecules may be novel biomarkers for senescent human fibroblasts.