RESUMEN
CSL proteins [named after the homologs CBF1 (RBP-Jκ in mice), Suppressor of Hairless and LAG-1] are conserved transcription factors found in animals and fungi. In the fission yeast Schizosaccharomyces pombe, they regulate various cellular processes, including cell cycle progression, lipid metabolism and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central ß-trefoil domain. Here, we investigated the importance of DNA binding for CSL protein functions in fission yeast. We created CSL protein mutants with disrupted DNA binding and found that the vast majority of CSL protein functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse phenotypes associated with CSL protein functions. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Factores de Transcripción , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Unión Proteica , Metabolismo de los Lípidos/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ciclo Celular/genética , Regulación Fúngica de la Expresión Génica , ADN de Hongos/metabolismo , ADN de Hongos/genéticaRESUMEN
Caddisfly larvae produce silk containing heavy and light fibroins, similar to the silk of Lepidoptera, for the construction of underwater structures. We analyzed the silk of Limnephilus lunatus belonging to the case-forming suborder Integripalpia. We analyzed the transcriptome, mapped the transcripts to a reference genome and identified over 80 proteins using proteomic methods, and checked the specificity of their expression. For comparison, we also analyzed the transcriptome and silk proteome of Limnephilus flavicornis. Our results show that fibroins and adhesives are produced together in the middle and posterior parts of the silk glands, while the anterior part produces enzymes and an unknown protein AT24. The number of silk proteins of L. lunatus far exceeds that of the web-spinning Plectrocnemia conspersa, a previously described species from the suborder Annulipalpia. Our results support the idea of increasing the structural complexity of silk in rigid case builders compared to trap web builders.
Asunto(s)
Seda , Animales , Seda/metabolismo , Seda/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Transcriptoma , Insectos/metabolismo , Insectos/genética , Fibroínas/genética , Fibroínas/metabolismo , Fibroínas/química , Proteómica/métodos , Proteoma , Perfilación de la Expresión GénicaRESUMEN
The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).
Asunto(s)
Archamoebae/genética , Evolución Biológica , Entamoeba histolytica/genética , Genoma de Protozoos , Parásitos/genética , Adaptación Biológica/genética , Anaerobiosis/genética , Animales , Archamoebae/metabolismo , Transferencia de Gen Horizontal , Tamaño del Genoma , TranscriptomaRESUMEN
The incidence of cutaneous malignant melanoma is increasing worldwide. While the treatment of initial stages of the disease is simple, the advanced disease frequently remains fatal despite novel therapeutic options . This requires identification of novel therapeutic targets in melanoma. Similarly to other types of tumours, the cancer microenvironment plays a prominent role and determines the biological properties of melanoma. Importantly, melanoma cell-produced exosomes represent an important tool of intercellular communication within this cancer ecosystem. We have focused on potential differences in the activity of exosomes produced by melanoma cells towards melanoma-associated fibroblasts and normal dermal fibroblasts. Cancer-associated fibroblasts were activated by the melanoma cell-produced exosomes significantly more than their normal counterparts, as assessed by increased transcription of genes for inflammation-supporting cytokines and chemokines, namely IL-6 or IL-8. We have observed that the response is dependent on the duration of the stimulus via exosomes and also on the quantity of exosomes. Our study demonstrates that melanoma-produced exosomes significantly stimulate the tumour-promoting proinflammatory activity of cancer-associated fibroblasts. This may represent a potential new target of oncologic therapy .
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Exosomas/metabolismo , Fibroblastos/metabolismo , Melanoma Experimental/metabolismo , Fibroblastos/patología , Humanos , Melanoma Experimental/patología , Células Tumorales CultivadasRESUMEN
Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined P. prasinana silk using 'omics' technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.
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Bombyx/genética , Evolución Molecular , Fibroínas/genética , Animales , Bombyx/clasificación , Bombyx/metabolismo , Glándulas Exocrinas/metabolismo , Fibroínas/química , Filogenia , Proteoma/genética , Proteoma/metabolismo , TranscriptomaRESUMEN
Arthrospira platensis, a blue-green alga, is a popular nutraceutical substance having potent antioxidant properties with potential anti-carcinogenic activities. The aim of our study was to assess the possible anti-angiogenic effects of A platensis in an experimental model of pancreatic cancer. The effects of an A platensis extract were investigated on human pancreatic cancer cells (PA-TU-8902) and immortalized endothelial-like cells (Ea.hy926). PA-TU-8902 pancreatic tumours xenografted to athymic mice were also examined. In vitro migration and invasiveness assays were performed on the tested cells. Multiple angiogenic factors and signalling pathways were analysed in the epithelial, endothelial and cancer cells, and tumour tissue. The A platensis extract exerted inhibitory effects on both migration and invasion of pancreatic cancer as well as endothelial-like cells. Tumours of mice treated with A platensis exhibited much lesser degrees of vascularization as measured by CD31 immunostaining (P = .004). Surprisingly, the VEGF-A mRNA and protein expressions were up-regulated in pancreatic cancer cells. A platensis inhibited ERK activation upstream of Raf and suppressed the expression of ERK-regulated proteins. Treatment of pancreatic cancer with A platensis was associated with suppressive effects on migration and invasiveness with various anti-angiogenic features, which might account for the anticancer effects of this blue-green alga.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Spirulina/química , Animales , Antioxidantes/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Neoplasias PancreáticasRESUMEN
Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding ß and ß' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.
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ARN Polimerasas Dirigidas por ADN/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/metabolismo , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , ARN Pequeño no Traducido/genéticaRESUMEN
BACKGROUND: Departures from the standard genetic code in eukaryotic nuclear genomes are known for only a handful of lineages and only a few genetic code variants seem to exist outside the ciliates, the most creative group in this regard. Most frequent code modifications entail reassignment of the UAG and UAA codons, with evidence for at least 13 independent cases of a coordinated change in the meaning of both codons. However, no change affecting each of the two codons separately has been documented, suggesting the existence of underlying evolutionary or mechanistic constraints. RESULTS: Here, we present the discovery of two new variants of the nuclear genetic code, in which UAG is translated as an amino acid while UAA is kept as a termination codon (along with UGA). The first variant occurs in an organism noticed in a (meta)transcriptome from the heteropteran Lygus hesperus and demonstrated to be a novel insect-dwelling member of Rhizaria (specifically Sainouroidea). This first documented case of a rhizarian with a non-canonical genetic code employs UAG to encode leucine and represents an unprecedented change among nuclear codon reassignments. The second code variant was found in the recently described anaerobic flagellate Iotanema spirale (Metamonada: Fornicata). Analyses of transcriptomic data revealed that I. spirale uses UAG to encode glutamine, similarly to the most common variant of a non-canonical code known from several unrelated eukaryotic groups, including hexamitin diplomonads (also a lineage of fornicates). However, in these organisms, UAA also encodes glutamine, whereas it is the primary termination codon in I. spirale. Along with phylogenetic evidence for distant relationship of I. spirale and hexamitins, this indicates two independent genetic code changes in fornicates. CONCLUSIONS: Our study documents, for the first time, that evolutionary changes of the meaning of UAG and UAA codons in nuclear genomes can be decoupled and that the interpretation of the two codons by the cytoplasmic translation apparatus is mechanistically separable. The latter conclusion has interesting implications for possibilities of genetic code engineering in eukaryotes. We also present a newly developed generally applicable phylogeny-informed method for inferring the meaning of reassigned codons.
Asunto(s)
Núcleo Celular/genética , Codón/genética , Código Genético , Animales , Cilióforos/genética , Evolución Molecular , Glutamina/genética , Insectos/parasitología , Leucina/genética , Sistemas de Lectura Abierta/genética , Filogenia , Rhizaria/genéticaRESUMEN
σ factors are considered as positive regulators of gene expression. Here we reveal the opposite, inhibitory role of these proteins. We used a combination of molecular biology methods and computational modeling to analyze the regulatory activity of the extracytoplasmic σE factor from Streptomyces coelicolor. The direct activator/repressor function of σE was then explored by experimental analysis of selected promoter regions in vivo. Additionally, the σE interactome was defined. Taken together, the results characterize σE, its regulation, regulon, and suggest its direct inhibitory function (as a repressor) in gene expression, a phenomenon that may be common also to other σ factors and organisms.
Asunto(s)
Streptomyces coelicolor , Streptomyces coelicolor/genética , Simulación por Computador , Factor sigma/genéticaRESUMEN
Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancerassociated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced coexpression of keratins8/14 in the EMG3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin8, 14, 18, 19) and epithelialmesenchymal transitionassociated markers (SLUG, SNAIL, ZEB1, E/Ncadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGFA and MFGE8 attenuated the modulatory effect of CAFs on EMG3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EMG3 cells in vitro. CAFs of different origins support the proinflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.
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Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Femenino , Humanos , Antígenos de Superficie , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Línea Celular Tumoral , Fibroblastos/metabolismo , Queratinas/genética , Queratinas/metabolismo , Células MCF-7 , Proteínas de la Leche/genética , Proteínas de la Leche/metabolismo , Pronóstico , Transcriptoma , Microambiente Tumoral/genética , Melanoma Cutáneo MalignoRESUMEN
Similar to Lepidoptera, the larvae of Trichoptera are also capable of producing silk. Plectrocnemia conspersa, a predatory species belonging to the suborder Annulipalpia, builds massive silken retreats with preycapturing nets. In this study, we describe the silk glands of P. conspersa and use the multi-omics methods to obtain a complete picture of the fiber composition. A combination of silk gland-specific transcriptome and proteomic analyses of the spun-out fibers yielded 27 significant candidates whose full-length sequences and gene structures were retrieved from the publicly available genome database. About one-third of the candidates were completely novel proteins for which there are no described homologs, including a group of five pseudofibroins, proteins with a composition similar to fibroin heavy chain. The rest were homologs of lepidopteran silk proteins, although some had a larger number of paralogs. On the other hand, P. conspersa fibers lacked some proteins that are regular components in moth silk. In summary, the multi-omics approach provides an opportunity to compare the overall composition of silk with other insect species. A sufficient number of such studies will make it possible to distinguish between the basic components of all silks and the proteins that represent the adaptation of the fibers for specific purposes or environments.
RESUMEN
Silk is a secretory product of numerous arthropods with remarkable mechanical properties. In this work, we present the complete sequences of the putative major silk proteins of E. kuehniella and compare them with those of G. mellonella, which belongs to the same moth family Pyralidae. To identify the silk genes of both species, we combined proteomic analysis of cocoon silk with a homology search in transcriptomes and genomic sequences to complement the information on both species. We analyzed structure of the candidate genes obtained, their expression specificity and their evolutionary relationships. We demonstrate that the silks of E. kuehniella and G. mellonella differ in their hydrophobicity and that the silk of E. kuehniella is highly hygroscopic. In our experiments, we show that the number of genes encoding sericins is higher in G. mellonella than in E. kuehniella. By analyzing the synteny of the chromosomal segment encoding sericin genes in both moth species, we found that the region encoding sericins is duplicated in G. mellonella. Finally, we present the complete primary structures of nine fibH genes and proteins from both families of the suborder Pyraloidea and discuss their specific and conserved features. This study provides a foundation for future research on the evolution of silk proteins and lays the groundwork for future detailed functional studies.
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Concentrative nucleoside transporters (Cnts) are unidirectional carriers that mediate the energy-costly influx of nucleosides driven by the transmembrane sodium gradient. Cnts are transmembrane proteins that share a common structural organization and are found in all phyla. Although there have been studies on Cnts from a biochemical perspective, no deep research has examined their role at the organismal level. Here, we investigated the role of the Drosophila melanogaster cnt1 gene, which is specifically expressed in the testes. We used the CRISPR/Cas9 system to generate a mutation in the cnt1 gene. The cnt1 mutants exhibited defects in the duration of copulation and spermatid maturation, which significantly impaired male fertility. The most striking effect of the cnt1 mutation in spermatid maturation was an abnormal structure of the sperm tail, in which the formation of major and minor mitochondrial derivatives was disrupted. Our results demonstrate the importance of cnt1 in male fertility and suggest that the observed defects in mating behavior and spermatogenesis are due to alterations in nucleoside transport and associated metabolic pathways.
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Many lepidopteran species produce silk, cocoons, feeding tubes, or nests for protection from predators and parasites for caterpillars and pupae. Yet, the number of lepidopteran species whose silk composition has been studied in detail is very small, because the genes encoding the major structural silk proteins tend to be large and repetitive, making their assembly and sequence analysis difficult. Here we have analyzed the silk of Yponomeuta cagnagella, which represents one of the early diverging lineages of the ditrysian Lepidoptera thus improving the coverage of the order. To obtain a comprehensive list of the Y. cagnagella silk genes, we sequenced and assembled a draft genome using Oxford Nanopore and Illumina technologies. We used a silk-gland transcriptome and a silk proteome to identify major silk components and verified the tissue specificity of expression of individual genes. A detailed annotation of the major genes and their putative products, including their complete sequences and exon-intron structures is provided. The morphology of silk glands and fibers are also shown. This study fills an important gap in our growing understanding of the structure, evolution, and function of silk genes and provides genomic resources for future studies of the chemical ecology of Yponomeuta species.
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Mariposas Nocturnas , Animales , Mariposas Nocturnas/genética , Seda/genética , Pupa , Genómica , ProteomaRESUMEN
Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.
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Chromatium/metabolismo , Euglénidos/genética , Evolución Biológica , Chromatium/genética , Euglénidos/metabolismo , Exones/genética , Genoma , Procesos Heterotróficos , Intrones/genética , Mitocondrias/genética , Filogenia , Análisis de Secuencia de ADN/métodos , Transcriptoma/genéticaRESUMEN
In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.
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Discoveries of diverse microbial eukaryotes and their inclusion in comprehensive phylogenomic analyses have crucially re-shaped the eukaryotic tree of life in the 21st century.1 At the deepest level, eukaryotic diversity comprises 9-10 "supergroups." One of these supergroups, the Metamonada, is particularly important to our understanding of the evolutionary dynamics of eukaryotic cells, including the remodeling of mitochondrial function. All metamonads thrive in low-oxygen environments and lack classical aerobic mitochondria, instead possessing mitochondrion-related organelles (MROs) with metabolisms that are adapted to low-oxygen conditions. These MROs lack an organellar genome, do not participate in the Krebs cycle and oxidative phosphorylation,2 and often synthesize ATP by substrate-level phosphorylation coupled to hydrogen production.3,4 The events that occurred during the transition from an oxygen-respiring mitochondrion to a functionally streamlined MRO early in metamonad evolution remain largely unknown. Here, we report transcriptomes of two recently described, enigmatic, anaerobic protists from the genus Anaeramoeba.5 Using phylogenomic analysis, we show that these species represent a divergent, phylum-level lineage in the tree of metamonads, emerging as a sister group of the Parabasalia and reordering the deep branching order of the metamonad tree. Metabolic reconstructions of the Anaeramoeba MROs reveal many "classical" mitochondrial features previously not seen in metamonads, including a disulfide relay import system, propionate production, and amino acid metabolism. Our findings suggest that the cenancestor of Metamonada likely had MROs with more classical mitochondrial features than previously anticipated and demonstrate how discoveries of novel lineages of high taxonomic rank continue to transform our understanding of early eukaryote evolution.
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Eucariontes , Orgánulos , Anaerobiosis , Eucariontes/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Orgánulos/genética , Orgánulos/metabolismo , Oxígeno/metabolismo , FilogeniaRESUMEN
BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) still represents one of the most aggressive cancers. Understanding of the epithelial-mesenchymal crosstalk as a crucial part of the tumor microenvironment should pave the way for therapies to improve patient survival rates. Well-established cell lines present a useful and reproducible model to study PDAC biology. However, the tumor-stromal interactions between cancer cells and cancer-associated fibroblasts (CAFs) are still poorly understood. MATERIALS AND METHODS: We studied interactions between four PDAC cell lines (Panc-1, CAPAN-2, MIAPaCa-2, and PaTu-8902) and conditioned media derived from primary cultures of normal fibroblasts/PDAC-derived CAFs (PANFs). RESULTS: When the tested PDAC cell lines were stimulated by PANF-derived conditioned media, the most aggressive behavior was acquired by the Panc-1 cell line (increased number and size of colonies, remaining expression of vimentin and keratin 8 as well as increase of epithelial-to-mesenchymal polarization markers), whereas PaTu-8902 cells were rather inhibited. Of note, administration of the conditioned media to MIAPaCa-2 cells resulted in an inverse effect on the size and number of colonies, whereas CAPAN-2 cells were rather stimulated. To explain the heterogeneous pattern of the observed PDAC crosstalk at the in vitro level, we further compared the phenotype of primary cultures of cells derived from ascitic fluid with that of the tested PDAC cell lines, analyzed tumor samples of PDAC patients, and performed gene expression profiling of PANFs. Immuno-cyto/histo-chemical analysis found specific phenotype differences within the group of examined patients and tested PDAC cell lines, whereas the genomic approach in PANFs found the key molecules (IL6, IL8, MFGE8 and periostin) that may contribute to the cancer aggressive behavior. CONCLUSION: The desmoplastic patient-specific regulation of cancer cells by CAFs (also demonstrated by the heterogeneous response of PDAC cell lines to fibroblasts) precludes simple targeting and development of an effective treatment strategy and rather requires establishment of an individualized tumor-specific treatment protocol.
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Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/patología , Fibroblastos/patología , Neoplasias Pancreáticas/patología , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Fibroblastos/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Microambiente TumoralRESUMEN
Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.
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ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Recombinantes/biosíntesis , Trypanosoma brucei brucei , Proteínas Virales/metabolismo , Dineínas/biosíntesis , Expresión Génica , Genes Protozoarios , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Aberrant regulation of the cell cycle is a typical feature of all forms of cancer. In head and neck squamous cell carcinoma (HNSCC), it is often associated with the overexpression of cyclin D1 (CCND1). However, it remains unclear how CCND1 expression changes between tumor and normal tissues and whether human papillomavirus (HPV) affects differential CCND1 expression. Here, we evaluated the expression of D-type cyclins in a cohort of 94 HNSCC patients of which 82 were subjected to whole genome expression profiling of primary tumors and paired normal mucosa. Comparative analysis of paired samples showed that CCND1 was upregulated in 18% of HNSCC tumors. Counterintuitively, CCND1 was downregulated in 23% of carcinomas, more frequently in HPV-positive samples. There was no correlation between the change in D-type cyclin expression and patient survival. Intriguingly, among the tumors with downregulated CCND1, one-third showed an increase in cyclin D2 (CCND2) expression. On the other hand, one-third of tumors with upregulated CCND1 showed a decrease in CCND2. Collectively, we have shown that CCND1 was frequently downregulated in HNSCC tumors. Furthermore, regardless of the HPV status, our data suggested that a change in CCND1 expression was alleviated by a compensatory change in CCND2 expression.