The tales of two organic cation transporters, OCT-1 and OCT-2, in Caenorhabditis elegans

Solute carrier transporters, previously thought to perform roles in the transport of ions and various nutrients are now assigned broader functions. These transporters have recently been shown to permit entry of therapeutic drugs into cells. There is growing interest to understand the broad spectrum of drugs and chemical compounds that are recognized by these transporters such that specific ligands can be used as therapeutics to target definite physiological pathways. To facilitate this investigation, simpler and cost effective model systems are needed, one of such is the live whole model animal Caenorhabditis elegans (C. elegans) that offers a multitude of advantages. In general, studies with C. elegans are feasible due to the simplicity of the readouts that include lifespan, brood size, germ cell death, and visualization by epifluorescent microscopy, which can be set up in any laboratory. In C. elegans, two solute carrier transporters, the organic cation transporters OCT-1 and its paralogue OCT-2 have been partially characterized. OCT-1 mutants display a significantly reduced lifespan and brood size, as well as exhibiting an increased susceptibility towards oxidative stress and a subset of DNA damaging agents. These multiple phenotypes are directly linked to OCT-1 depletion causing upregulation of OCT-2, as RNAi-mediated downregulation of OCT-2 rescues the oct-1 mutant phenotypes. Thus, in C. elegans OCT-1 exerts control onto OCT-2, and this latter transporter plays a predominant role in the uptake of various ligands. We first showed that OCT-2 can efficiently mediate uptake of the widely used anticancer drug doxorubicin into the animals, but prevented uptake upon its downregulation. Additional ligands of OCT-2 including cisplatin and camptothecin were revealed by ligand-docking prediction studies. These analyses generated docking scores indicating that OCT-2 can make robust contact with a number of therapeutics and anticancer drugs, as well as chemical compounds that possess the ability to target specific physiological pathways. Several of the compounds displaying high docking scores with OCT-2 were validated and indeed found to be substrates that OCT-2 transported into the animals. This review provides an insight how the transporters OCT-1 and OCT-2 of a simple model organism C. elegans can be exploited to report on the cytotoxicities and genotoxicities of therapeutic agents, as well as trace amounts of undocumented toxic compounds with neoplastic potentials that are present in the environment

Cellular Responses to Anthracyclines Identify Ku70, a DNA Repair Factor that Changes Compartment and Remains Stable in Leukemic Cells

Anthracyclines such as doxorubicin and daunorubicin are anticancer drugs that act by damaging the DNA and used for treating a variety of cancers including adult acute myeloid leukemia.  To date, nearly 50 % of acute myeloid leukemia patients show resistance to anthracyclines although the cause is not known.  We first investigate if there is a relationship between the expression level of 23 DNA repair genes in three leukemic cell lines (KG-1, HL-60 and Mono-Mac1) and cellular responses to anthracyclines.  We observed that the DNA repair genes were all downregulated in these cell lines following exposure to doxorubicin.  Further analysis revealed that the general downregulation of the genes was linked to a substantial decrease in the recovery of total RNA raising the possibility that assessment of total RNA, and not specific gene or set of genes, can be used as a simple indicator of cellular responses to anthracyclines.  Furthermore, examination of total protein extracts derived from these cell lines revealed for the first time that Ku70 is a key protein that remained stable, while the majority of proteins were loss, upon anthracycline treatment.  Importantly, Ku70 redistributes from the cytoplasm to the nucleoli in a time-dependent manner in response to anthracycline exposure.  We propose that Ku70 redistribution might play a vital role in predicting cellular response to anthracycline and promoting cell death

Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy

The Peptidyl Prolyl Isomerase Rrd1 Regulates the Elongation of RNA Polymerase II during Transcriptional Stresses

Rapamycin is an anticancer agent and immunosuppressant that acts by inhibiting the TOR signaling pathway. In yeast, rapamycin mediates a profound transcriptional response for which the RRD1 gene is required. To further investigate this connection, we performed genome-wide location analysis of RNA polymerase II (RNAPII) and Rrd1 in response to rapamycin and found that Rrd1 colocalizes with RNAPII on actively transcribed genes and that both are recruited to rapamycin responsive genes. Strikingly, when Rrd1 is lacking, RNAPII remains inappropriately associated to ribosomal genes and fails to be recruited to rapamycin responsive genes. This occurs independently of TATA box binding protein recruitment but involves the modulation of the phosphorylation status of RNAPII CTD by Rrd1. Further, we demonstrate that Rrd1 is also involved in various other transcriptional stress responses besides rapamycin. We propose that Rrd1 is a novel transcription elongation factor that fine-tunes the transcriptional stress response of RNAPII

Caenorhabditis elegans organic cation transporter-2 is a novel drug uptake transporter that mediates induced mutagenesis by environmental genotoxic compounds

Uptake transporters are being studied for roles in the entry of therapeutic drugs into cells and thus can be exploited to improve the treatment of various diseases. The live whole model organism, Caenorhabditis elegans, offers an array of advantages to investigate the roles of these transporters. This organism possesses two organic cation transporters (OCTs), OCT1 and OCT2 that are involved in the uptake of clinically relevant genotoxic anticancer drugs such as doxorubicin and cisplatin into the animal. C. elegans lacking OCT1 displays a shortened lifespan, a decreased brood size, an increased susceptibility to oxidative stress, and certain DNA damaging agents. Remarkably, these phenotypes can be rescued by downregulating the OCT1 paralog, OCT2, leading to the suggestion that OCT1 exerts control on OCT2. Indeed, the loss of OCT1 led to the upregulation of OCT2. OCT2 is an uptake transporter involved in the influx of doxorubicin, as well as a number of other therapeutic agents and chemical compounds, some of which have been identified through ligand-protein docking analyses. The genotoxic compounds entering into C. elegans lead to DNA damage-induced apoptosis of germ cells, a process that can be attenuated by blocking OCT2 function. Thus, by combining the roles of the OCT1 and OCT2 transporters with defects in various DNA repair mechanisms, it is possible to engineer a set of supersensitive C. elegans strains that can serve as the most powerful living sensors to date. These tester C. elegans strains can be used to report on the cytotoxicities and genotoxicities of a battery of old and new drugs developed by pharmaceuticals, undocumented toxicants generated by various industries, and compounds that can cause cancers and are present in trace amounts in the environment around the world. These efforts are attainable as C. elegans can live in the soil and water, and a multitude of tools are available to monitor several readouts from the animals

Intracellular regulation of neurospora endo-exonuclease in response to DNA damage and heat shock

It was shown, previously that N. crassa contains an endo-exonuclease which exist in two forms, an active form and an inactive form activated by trypsin treatment in vitro. Both of these forms were found in the cytosol and in mitochondria, but only the active form is present in the vacuoles. In the present work, it has been shown that both forms are also present in the nuclei bound to the chromatin.Active endo-exonuclease was previously implicated in DNA repair and in this study additional evidence was obtained in further support of such a role: (i) the mutagen sensitive uvs-3 mutant of N. crassa was found to contain only 10% of the level of active enzyme in the two DNA-containing organelles, in comparison to the levels found in the wild-type, (ii) in response to low doses of the DNA-damaging agent 4-nitroquinoline 1-oxide (4-NQO), the inactive enzyme decrease and the active form increase in the DNA-containing organelles indicating that the inactive enzyme may have been converted proteolytically to the active form. This response did not occur in the uvs-3 mutant.Under a different stress, namely heat shock, endo-exonuclease was regulated differently in the DNA-containing organelles. Active enzyme was released from the nuclei, mitochondria, and vacuoles into the cytosol where it was nearly completely inhibited by a specific inhibitor induced by heat shock. This heat shock-induced inhibitor shared some properties in common with a constitutive endo-exonuclease inhibitor recently isolated from this laboratory. However, there were also some distinct differences between these two inhibitors. At least one difference may be explained by phosphorylation of the heat shock inhibitor

Apurinic/apyrimidinic endonuclease 1 performs multiple roles in controlling the outcome of cancer cells toward radiation and chemotherapeutic agents

Many endogenous and exogenous sources produce reactive oxygen species such as superoxide radical anions and hydrogen peroxide that are converted to the highly reactive form, hydroxyl radical. It is this latter species that can damage several macromolecules in the cells, in particular, the DNA to produce a variety of DNA lesions. These DNA lesions include oxidatively damaged purine and pyrimidine bases, as well as single-strand and double-strand breaks. These unrepaired DNA lesions lead to base substitutions, deletions, insertions, and rearrangements of the chromosome, ultimately altering the stability of the genome. Maintaining the integrity of the genome is essential to prevent various diseases such as several types of cancers. There are several DNA repair pathways including base-excision repair (BER), nucleotide-excision repair, mismatch repair, homologous recombination, and nonhomologous end joining that operate in the human cells to prevent genomic instability. Each of these DNA repair pathways consists of multiple enzymes that execute specific function (s). This review focuses on a key enzyme apurinic/apyrimidinic endonuclease 1 (APE1) that belongs to the BER pathway that plays a pivotal role in the removal of modified DNA bases. We provide an overview of the multifaceted roles performed by APE1, which also serves as a redox factor and referred to as redox effector factor 1 (Ref-1) or APE1/Ref-1. In addition, we discuss more recent findings whereby (i) peroxiredoxin 1 controls the redox activity of APE1 and (ii) CUT-like homeobox 1 protein, a transcription factor that binds to DNA and stimulates the DNA repair activities of APE1 to confer resistance to radio- and chemotherapy

Embryonic extracts derived from the nematode Caenorhabditis elegans remove uracil from DNA by the sequential action of uracil-DNA glycosylase and AP (apurinic/apyrimidinic) endonuclease.

DNA bases continuously undergo modifications in response to endogenous reactions such as oxidation, alkylation or deamination. The modified bases are primarily removed by DNA glycosylases, which cleave the N-glycosylic bond linking the base to the sugar, to generate an apurinic/apyrimidinic (AP) site, and this latter lesion is highly mutagenic. Previously, no study has demonstrated the processing of these lesions in the nematode Caenorhabditis elegans. Herein, we report the existence of uracil-DNA glycosylase and AP endonuclease activities in extracts derived from embryos of C. elegans. These enzyme activities were monitored using a defined 5'-end (32)P-labelled 42-bp synthetic oligonucleotide substrate bearing a single uracil residue opposite guanine at position 21. The embryonic extract rapidly cleaved the substrate in a time-dependent manner to produce a 20-mer product. The extract did not excise adenine or thymine opposite guanine, although uracil opposite either adenine or thymine was processed. Addition of the highly specific inhibitor of uracil-DNA glycosylase produced by Bacillus subtilis to the extract prevented the formation of the 20-mer product, indicating that removal of uracil is catalysed by uracil-DNA glycosylase. The data suggest that the 20-mer product was generated by a sequential reaction, i.e., removal of the uracil base followed by 5'-cleavage of the AP site. Further analysis revealed that product formation was dependent upon the presence of Mg(2+), suggesting that cleavage of the AP site, following uracil excision, is carried out by a Mg(2+)-dependent AP endonuclease. It would appear that these activities correspond to the first two steps of a putative base-excision-repair pathway in C. elegans