Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Apoptotic Tumor Cell-Derived Extracellular Vesicles as Important Regulators of the Onco-Regenerative Niche

Cells undergoing apoptosis produce heterogeneous populations of membrane delimited extracellular vesicles (Apo-EVs) which vary not only in size—from tens of nanometers to several microns—but also in molecular composition and cargo. Apo-EVs carry a variety of potentially biologically active components, including small molecules, proteins, and nucleic acids. Larger forms of Apo-EVs, commonly termed “apoptotic bodies,” can carry organelles, such as mitochondria and nuclear fragments. Molecules displayed on the surface of extracellular vesicles (EVs) can contribute substantially to their size, as well as their functions. Thus far, relatively little is known of the functional significance of Apo-EVs apart from their roles in fragmentation of dying cells and indicated immunomodulatory activities. Here, we discuss EV production by dying tumor cells and consider the possible roles of Apo-EVs in a cell death-driven sector of the tumor microenvironment known as the onco-regenerative niche (ORN). We propose that tumor-derived Apo-EVs are significant vehicles of the ORN, functioning as critical intercellular communicators that activate oncogenic tissue repair and regeneration pathways. We highlight important outstanding questions and suggest that Apo-EVs may harbor novel therapeutic targets

Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1–2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin

An Innovative Cell Microincubator for Drug Discovery Based on 3D Silicon Structures

We recently employed three-dimensional (3D) silicon microstructures (SMSs) consisting in arrays of 3 μm-thick silicon walls separated by 50 μm-deep, 5 μm-wide gaps, as microincubators for monitoring the biomechanical properties of tumor cells. They were here applied to investigate the in vitro behavior of HT1080 human fibrosarcoma cells driven to apoptosis by the chemotherapeutic drug Bleomycin. Our results, obtained by fluorescence microscopy, demonstrated that HT1080 cells exhibited a great ability to colonize the narrow gaps. Remarkably, HT1080 cells grown on 3D-SMS, when treated with the DNA damaging agent Bleomycin under conditions leading to apoptosis, tended to shrink, reducing their volume and mimicking the normal behavior of apoptotic cells, and were prone to leave the gaps. Finally, we performed label-free detection of cells adherent to the vertical silicon wall, inside the gap of 3D-SMS, by exploiting optical low coherence reflectometry using infrared, low power radiation. This kind of approach may become a new tool for increasing automation in the drug discovery area. Our results open new perspectives in view of future applications of the 3D-SMS as the core element of a lab-on-a-chip suitable for screening the effect of new molecules potentially able to kill tumor cells

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures

Fluorescence properties of the Na+/H+ exchanger inhibitor HMA (5-(N,N-hexamethylene) amiloride) are modulated by intracellular Ph

HMA (5-(N,N-hexamethylene)amiloride), which belongs to a family of novel amiloride derivatives, is one of the most effective inhibitors of Na+/H+ exchangers, while uneffective against Na+ channels and Na+/Ca2+ exchangers. In this study, we provided evidence that HMA can act as a fluorescent probe. In fact, human retinal ARPE19 cells incubated with HMA show an intense bluish fluorescence in the cytoplasm when observed at microscope under conventional UV-excitation conditions. Interestingly, a prolonged observation under continuous exposure to excitation lightdoes not induce great changes in cells incubated with HMA for times up to about 5 min, while an unexpected rapid increase in fluorescence signal is observed in cells incubated for longer times. The latter phenomenon is particularly evident in the perinuclear region and in discrete spots in the cytoplasm. Since HMA modulates intracellular acidity, the dependence of its fluorescence properties on medium pH and response upon irradiation have been investigated in solution, at pH 5.0 and pH 7.2. The changes in both spectral shape and amplitude emission indicate a marked pH influence on HMA fluorescence properties, making HMA exploitable as a self biomarker of pH alterations in cell studies, in the absence of perturbations induced by the administration of other exogenous dyes

Two-color fluorescence detection of Poly (ADP-Ribose) Polymerase-1 (PARP-1) cleavage and DNA strand breaks in etoposide-induced apoptotic cells

During apoptosis, the nuclear enzyme Poly(ADPRibose) Polymerase-1 (PARP-1) catalyzes the rapid and transient synthesis of poly(ADP-ribose) from NAD+ and becomes inactive when cleaved by caspases. The regulation of these two opposite roles of PARP-1 is still unknown. We have recently investigated PARP-1 activation/degradation in Hep-2 cells driven to apoptosis by actinomycin D. In the present work, we have extended our analysis to the effect of the DNA damaging agent etoposide, and paid attention to the relationship between PARP-1 cleavage and DNA fragmentation. An original fluorescent procedure was developed to simultaneously identify in situ the p89 proteolytic fragment of PARP-1 (by immunolabeling) and DNA degradation (by the TUNEL assay). The presence of p89 was observed both in cells with advanced signs of apoptosis (where the PARP-1 fragment is extruded from the nucleus into the cytoplasm) and in TUNEL-negative cells, with only incipient signs of chromatin condensation; this evidence indicates that PARP-1 degradation in etoposide-treated apoptotic cells may precede DNA cleavage

Interaction of p21CDKN1A with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

The cell-cycle inhibitor p21CDKN1A has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA