RESUMO
BACKGROUND: Adipose-derived stem cell (ASC) expansion under atmospheric oxygen levels (21%) was previously shown to cause increased reactive oxygen species (ROS) accumulation and genetic instability compared to cells cultured under physiological oxygen levels (2-8%). However, since culture under physiological oxygen levels is costly and complicated, a simpler method to reduce ROS accumulation is desirable. The current study aimed to determine whether lower culture temperature can reduce ROS production in ASCs without impairing their culture expansion. METHODS: Proliferation, differentiation, ROS accumulation, and gene expression were compared between ASC cultures at 35 °C and 37 °C. ASCs isolated either from rat fat depots or from human lipoaspirates were examined in the study. RESULTS: Rat visceral ASCs (vASCs) cultured at 35 °C demonstrated reduced ROS production and apoptosis and enhanced expansion and adipogenic differentiation compared to vASCs cultured at 37 °C. Similarly, the culture of human ASCs (hASCs) at 35 °C led to reduced ROS accumulation and apoptosis, with no effect on the proliferation rate, compared to hASCs cultured at 37 °C. Comparison of gene expression profiles of 35 °C versus 37 °C vASCs uncovered the development of a pro-inflammatory phenotype in 37 °C vASCs in correlation with culture temperature and ROS overproduction. This correlation was reaffirmed in both hASCs and subcutaneous rat ASCs. CONCLUSIONS: This is the first evidence of the effect of culture temperature on ASC growth and differentiation properties. Reduced temperatures may result in superior ASC cultures with enhanced expansion capacities in vitro and effectiveness in vivo.
Assuntos
Tecido Adiposo/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Humanos , Inflamação , Estresse Oxidativo , Ratos , Ratos Endogâmicos LewRESUMO
The prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo. This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies.
Assuntos
Antígenos CD/biossíntese , Moléculas de Adesão Celular/biossíntese , Melanoma/patologia , Fatores de Transcrição SOXB1/metabolismo , Neoplasias Cutâneas/patologia , Animais , Antígenos CD/genética , Western Blotting , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Análise Serial de TecidosRESUMO
Recent structural determinations and metagenomic studies shed light on the evolution of photosystem I (PSI) from the homodimeric reaction centre of primitive bacteria to plant PSI at the top of the evolutionary development. The evolutionary scenario of over 3.5 billion years reveals an increase in the complexity of PSI. This phenomenon of ever-increasing complexity is common to all evolutionary processes that in their advanced stages are highly dependent on fine-tuning of regulatory processes. On the other hand, the recently discovered virus-encoded PSI complexes contain a minimal number of subunits. This may reflect the unique selection scenarios associated with viral replication. It may be beneficial for future engineering of productive processes to utilize 'primitive' complexes that disregard the cellular regulatory processes and to avoid those regulatory constraints when our goal is to divert the process from its original route. In this article, we discuss the evolutionary forces that act on viral reaction centres and the role of the virus-carried photosynthetic genes in the evolution of photosynthesis.