Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones. We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays. Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration. These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression.

Key Points: Tolerance of oxidative DNA lesions ensures the genomic and functional integrity of hematopoietic stem and precursor cells. Endogenous DNA damage–induced replication stress is associated with mitochondrial dysfunction.

Several mechanisms have been proposed to explain the vascular toxicity of arsenic. Some of them are described in this work, such as stress-induced premature senescence (SIPS), dedifferentiation, and medial vascular calcification, and they all affect vascular smooth muscle cells (VSMC). Rat aortic VSMC were treated with 1–100µM of either sodium arsenate (As^V), sodium arsenite (As^III), monomethylarsonic acid, or dimethylarsinic acid. None of the treatments induced VSMC calcification in the presence of 1mM inorganic phosphate (Pi), but 1µM As^III did increase calcification when induced with 2.5mM Pi. A lactate dehydrogenase assay revealed that this increase was explained by a rise in cytotoxicity due to simultaneous incubation with 1µM As^III and 2.5mM Pi. This calcification increase was also observed in the aortas of a vascular calcification model: 5/6 nephrectomized rats fed with a high Pi diet and treated with vitamin D₃. Several known mechanisms that might explain arsenic toxicity in our experimental model were discarded: apoptosis, oxidative stress, and inflammasome activation. Nevertheless, both senescence-associated β-galactosidase activity and p21 expression were increased by As^III, which reveals the induction of SIPS. As^III also caused dedifferentiation of VSMC, as shown by the reduced expression of the VSMC markers SM22α and calponin. Senescence and gene expression were also observed in the aortas of healthy rats treated with 50 ppm As^V in drinking water for 1 month. In conclusion, both premature senescence in aortic VSMC with phenotypic dedifferentiation and the increase of Pi-induced calcification are novel mechanisms of arsenic vasculotoxicity.

Deoxyribonucleic acid (DNA) represents the main reservoir of genetic information in the cells, which is why it is protected in the nucleus. Entry into the nucleus is, in general, difficult, as the nuclear membrane is a selective barrier to molecules longer than 40 kDa. However, in some cases, the size of certain nanoparticles (NPs) allows their internalization into the nucleus, thus causing a direct effect on the DNA structure. NPs can also induce indirect effects on DNA through reactive oxygen species (ROS) generation. In this context, nanomaterials are emerging as a disruptive tool for the development of novel therapies in a broad range of biomedical fields; although their effect on cell viability is commonly studied, further interactions with DNA or indirect alterations triggered by the internalization of these materials are not always clarified, since the small size of these materials makes them perfectly suitable for interaction with subcellular structures, such as the nucleus. In this context, and using as a reference the predicted interactions presented in a computational model, we describe and discuss the observed direct and indirect effects of the implicated nanomaterials on DNA.

Nanoparticles (NPs) have unique physicochemical properties that are useful for a broad range of biomedical and industrial applications; nevertheless, increasing concern exists about their biosafety. This review aims to focus on the implications of nanoparticles in cellular metabolism and their outcomes. In particular, some NPs have the ability to modify glucose and lipid metabolism, and this feature is especially interesting to treat diabetes and obesity and to target cancer cells. However, the lack of specificity to reach target cells and the toxicological evaluation of nontargeted cells can potentially induce detrimental side effects, closely related to inflammation and oxidative stress. Therefore, identifying the metabolic alterations caused by NPs, independent of their application, is highly needed. To our knowledge, this increase would lead to the improvement and safer use with a reduced toxicity, increasing the number of available NPs for diagnosis and treatment of human diseases.

Nucleic acids, which constitute the genetic material of all organisms, are continuously exposed to endogenous and exogenous damaging agents, representing a significant challenge to genome stability and genome integrity over the life of a cell or organism. Unrepaired DNA lesions, such as single- and double-stranded DNA breaks (SSBs and DSBs), and single-stranded gaps can block progression of the DNA replication fork, causing replicative stress and/or cell cycle arrest. However, translesion synthesis (TLS) DNA polymerases, such as Rev1, have the ability to bypass some DNA lesions, which can circumvent the process leading to replication fork arrest and minimize replicative stress. Here, we show that Rev1-deficiency in mouse embryo fibroblasts or mouse liver tissue is associated with replicative stress and mitochondrial dysfunction. In addition, Rev1-deficiency is associated with high poly(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD⁺, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via the PARP-NAD⁺-SIRT1-PGC1α axis.