Thomas V. O’Halloran

Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs

In last few hours of maturation, the mouse oocyte takes up over twenty billion zinc atoms and arrests after the first meiotic division, until fertilization or pharmacological intervention stimulates cell cycle progression toward a new embryo. Using chemical and physical probes, we show that fertilization of the mature, zinc-enriched egg triggers the ejection of zinc into the extracellular milieu in a series of coordinated events termed zinc sparks. These events immediately follow the well-established series of calcium oscillations within the activated egg and are evolutionarily conserved in several mammalian species, including rodents and nonhuman primates. Functionally, the zinc sparks mediate a decrease in intracellular zinc content that is necessary for continued cell cycle progression, as increasing zinc levels within the activated egg results in the reestablishment of cell cycle arrest at metaphase. The mammalian egg thus uses a zinc-dependent switch mechanism to toggle between metaphase arrest and resumption of the meiotic cell cycle at the initiation of embryonic development.

Nano-Encapsulation of Arsenic Trioxide Enhances Efficacy against Murine Lymphoma Model while Minimizing Its Impact on Ovarian Reserve In Vitro and In Vivo

Advances in cancer therapy have increased the rate of survival of young cancer patients; however, female lymphoma patients frequently face a temporary or permanent loss of fertility when treated with traditional cytotoxic agents. The potential loss of fertility is an important concern that can influence treatment decisions for many premenopausal cancer patients. The negative effect of chemotherapeutic agents and treatment protocols to patients’ fertility–referred to as fertotoxicity–are thus an increasingly important cancer survivorship issue. We have developed a novel nanoscale formulation of arsenic trioxide, a potent drug for treatment of hematological malignancies, and demonstrate that it has significantly better activity in a murine lymphoma model than the free drug. In parallel, we have developed a novel in vitro assay of ovarian follicle function that predicts in vivo ovarian toxicity of therapeutic agents. Our results reveal that the nanotherapeutic agent is not only more active against lymphoma, but is fertoprotective, i.e., it is much less deleterious to ovarian function than the parent drug. Thus, our in vitro assay allows rapid evaluation of both established and experimental anticancer drugs on ovarian reserve and can inform the selection of efficacious and fertility-sparing treatment regimens for reproductive-age women diagnosed with cancer.

Accumulation of cadmium in insulin-producing β cells

Evidence suggests that chronic low level cadmium exposure impairs the function of insulin-producing β cells and may be associated with type-2 diabetes mellitus. Herein, we describe the cadmium content in primary human islets and define the uptake kinetics and effects of environmentally relevant cadmium concentrations in cultured β cells. The average cadmium content in islets from 10 non-diabetic human subjects was 29 ± 7 nmol/g protein (range 7 to 72 nmol/g protein). Exposure of the β-cell line MIN6 to CdCl2 concentrations between 0.1 and 1.0 µmol/L resulted in a dose- and time-dependent uptake of cadmium over 72 h. This uptake resulted in an induction of metallthionein expression, likely enhancing cellular cadmium accumulation. Furthermore, cadmium accumulation resulted in an inhibition of glucose stimulated insulin secretion in MIN6 cells and primary mouse islets. Our results indicate that this impairment in β-cell function is not due to an increase in cell death or due to an increase in oxidative stress. We conclude that mouse β cells accumulate cadmium in a dose- and time-dependent manner over a prolonged time course at environmentally relevant concentrations. This uptake leads to a functional impairment of β-cell function without significant alterations in cell viability, expression of genes important for β-cell function or increase in oxidative stress.

Patient-Derived Tumor Xenografts Are Susceptible to Formation of Human Lymphocytic Tumors

Patient-derived xenograft (PDX) tumor models have emerged as a new approach to evaluate the effects of cancer drugs on patients’ personalized tumor grafts enabling to select the best treatment for the cancer patient and providing a new tool for oncology drug developers. Here, we report that human tumors engrafted in immunodeficient mice are susceptible to formation of B-and T-cell PDX tumors. We xenografted human primary and metastatic tumor samples into immunodeficient mice and found that a fraction of PDX tumors generated from patients’ samples of breast, colon, pancreatic, bladder and renal cancer were histologically similar to lymphocytic neoplasms. Moreover, we found that the first passage of breast and pancreatic cancer PDX tumors after initial transplantation of the tumor pieces from the same human tumor graft could grow as a lymphocytic tumor in one mouse and as an adenocarcinoma in another mouse. Whereas subcutaneous PDX tumors resembling human adenocarcinoma histology were slow growing and non-metastatic, we found that subcutaneous PDX lymphocytic tumors were fast growing and formed large metastatic lesions in mouse lymph nodes, liver, lungs, and spleen. PDX lymphocytic tumors were comprised of B-cells which were Epstein-Barr virus positive and expressed CD45 and CD20. Because B-cells are typically present in malignant solid tumors, formation of B-cell tumor may evolve in a wide range of PDX tumor models. Although PDX tumor models show great promise in the development of personalized therapy for cancer patients, our results suggest that confidence in any given PDX tumor model requires careful screening of lymphocytic markers.

The zinc spark is an inorganic signature of human egg activation

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent “zinc spark.” The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.

Evolution of a Heavy Metal Homeostasis/Resistance Island Reflects Increasing Copper Stress in Enterobacteria

Copper homeostasis in bacteria is challenged by periodic elevation of copper levels in the environment, arising from both natural sources and human inputs. Several mechanisms have evolved to efflux copper from bacterial cells, including the cus (copper sensing copper efflux system), and pco (plasmid-borne copper resistance system) systems. The genes belonging to these two systems can be physically clustered in a Copper Homeostasis and Silver Resistance Island (CHASRI) on both plasmids and chromosomes in Enterobacteria. Increasing use of copper in agricultural and industrial applications raises questions about the role of human activity in the evolution of novel copper resistance mechanisms. Here we present evidence that CHASRI emerged and diversified in response to copper deposition across aerobic and anaerobic environments. An analysis of diversification rates and a molecular clock model suggest that CHASRI experienced repeated episodes of elevated diversification that could correspond to peaks in human copper production. Phylogenetic analyses suggest that CHASRI originated in a relative of Enterobacter cloacae as the ultimate product of sequential assembly of several pre-existing two-gene modules. Once assembled, CHASRI dispersed via horizontal gene transfer within Enterobacteriaceae and also to certain members of Shewanellaceae, where the original pco module was replaced by a divergent pco homolog. Analyses of copper stress mitigation suggest that CHASRI confers increased resistance aerobically, anaerobically, and during shifts between aerobic and anaerobic environments, which could explain its persistence in facultative anaerobes and emergent enteric pathogens.

Zinc, insulin, and the liver: a ménage à trois

Insulin and Zn2+ enjoy a multivalent relationship. Zn2+ binds insulin in pancreatic β cells to form crystalline aggregates in dense core vesicles (DCVs), which are released in response to physiological signals such as increased blood glucose. This transition metal is an essential cofactor in insulin-degrading enzyme and several key Zn2+ finger transcription factors that are required for β cell development and insulin gene expression. Studies are increasingly revealing that fluctuations in Zn2+ concentration can mediate signaling events, including dynamic roles that extend beyond that of a static structural or catalytic cofactor. In this issue of the JCI, Tamaki et al. propose an additional function for Zn2+ in relation to insulin: regulation of insulin clearance from the bloodstream.

3D tumor tissue analogs and their orthotopic implants for understanding tumor-targeting of microenvironment-responsive nanosized chemotherapy and radiation

UNLABELLED An appropriate representation of the tumor microenvironment in tumor models can have a pronounced impact on directing combinatorial treatment strategies and cancer nanotherapeutics. The present study develops a novel 3D co-culture spheroid model (3D TNBC) incorporating tumor cells, endothelial cells and fibroblasts as color-coded murine tumor tissue analogs (TTA) to better represent the tumor milieu of triple negative breast cancer in vitro. Implantation of TTA orthotopically in nude mice, resulted in enhanced growth and aggressive metastasis to ectopic sites. Subsequently, the utility of the model is demonstrated for preferential targeting of irradiated tumor endothelial cells via radiation-induced stromal enrichment of galectin-1 using anginex conjugated nanoparticles (nanobins) carrying arsenic trioxide and cisplatin. Demonstration of a multimodal nanotherapeutic system and inclusion of the biological response to radiation using an in vitro/in vivo tumor model incorporating characteristics of tumor microenvironment presents an advance in preclinical evaluation of existing and novel cancer nanotherapies. FROM THE CLINICAL EDITOR Existing in-vivo tumor models are established by implanting tumor cells into nude mice. Here, the authors described their approach 3D spheres containing tumor cells, enodothelial cells and fibroblasts. This would mimic tumor micro-environment more realistically. This interesting 3D model should reflect more accurately tumor response to various drugs and would enable the design of new treatment modalities.

The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development

Upon activation, mammalian eggs release billions of zinc ions in an exocytotic event termed the “zinc spark.” The zinc spark is dependent on and occurs coordinately with intracellular calcium transients, which are tightly associated with embryonic development. Thus, we hypothesized that the zinc spark represents an early extracellular physicochemical marker of the developmental potential of the zygote. To test this hypothesis, we monitored zinc exocytosis in individual mouse eggs following parthenogenetic activation or in vitro fertilization (IVF) and tracked their development. Retrospective analysis of zinc spark profiles revealed that parthenotes and zygotes that developed into blastocysts released more zinc than those that failed to develop. Prospective selection of embryos based on their zinc spark profile significantly improved developmental outcomes and more than doubled the percentage of embryos that reached the blastocyst stage. Moreover, the zinc spark profile was also associated with embryo quality as the total cell number in the resulting morulae and blastocysts positively correlated with the zinc spark amplitude (R = 0.9209). Zinc sparks can thus serve as an early biomarker of zygote quality in mouse model.

Chemical and Genetic Studies of Copper Resistance in E. coli

Copper is an essential trace element required for bacterial growth, but is toxic at high levels of free ions. Bacteria are thus presented with the complex problem of obtaining and storing sufficient quantities of copper for normal function of several enzymes while, on die other hand, being able to survive when confronted with concentrations of copper that exceed a toxic threshold. The molecular mechanisms of metal ion detoxification are well understood only for a few systems, most notably for mercury resistance.1,2 For recent reviews of a number of bacterial metal resistance systems, see Plasmid, Vol. 27(1), 1992 and reference 3. The discovery of plasmid-borne copper resistance in bacteria has provided accessible systems for genetic and phenomenological study of copper metabolism. Study of these extrachromosomal systems has recently provided the impetus and the methods for identification of chromosomally-encoded copper homeostasis systems in Pseudomonas syringae pv. tomato (P. syringae)4,5,6 and in Echerichia coli (E. coli) 7,8,9 The two best characterized copper resistance systems are the plasmid-encoded systems cop in P. syringae and pco in E. coli. These two determinants are remarkably similar genetically, but as will be shown they differ fundamentally in the mechanism of copper resistance. This paper will focus first on the genetics and regulation of copper resistance in the cop and pco systems, then on the chemical mechanisms of copper resistance.