Professor Mieczysław Chorąży and His Works.

This essay is in memoriam of Professor Mieczysław Chorąży (1925 - 2021). Eminent Man, outstanding scientist, soldier of the Warsaw Uprising, moral authority for generations of fellow researchers and an exceptionally warm person. His character and life works are recalled here against the background of the times he lived in.

Wonderful World of Nucleic Acids.

Wonderful World of Nucleic Acids.

The Role of Different TET Proteins in Cytosine Demethylation Revealed by Mathematical Modeling.

In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2'-deoxycytidine (5-mdC) in DNA to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult.

Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity.

The regulation of translation by RNA-induced silencing complexes (RISCs) composed of Argonaute proteins and micro-RNAs is well established; however, the mechanisms underlying specific cellular responses to miRNAs and how specific complexes arise are not completely clear. To explore these questions, we performed experiments with and firefly luciferase reporter genes transfected in a psiCHECK-2 plasmid into human HCT116 or Me45 cells, where only the gene contained sequences targeted by microRNAs (miRNAs) in the 3'UTR. The effects of targeting were miRNA-specific; miRNA-21-5p caused strong inhibition of translation, whereas miRNA-24-3p or Let-7 family caused no change or an increase in reporter luciferase synthesis.

Involvement of miRNAs in cellular responses to radiation.

Purpose: Exposure of living cells to ionizing radiation has different consequences, depending on the dose and cell type. Changes in gene expression at the level of transcription and translation, including those regulated by microRNAs (miRNAs), play a role in the intrinsic radiosensitivity of different cells and define their fate, survival or death. The aim of our work was to examine how ionizing radiation may influence the expression of genes regulated by different miRNAs and miRNA biogenesis.

Micro RNAs in Regulation of Cellular Redox Homeostasis.

In living cells Reactive Oxygen Species (ROS) participate in intra- and inter-cellular signaling and all cells contain specific systems that guard redox homeostasis. These systems contain both enzymes which may produce ROS such as NADPH-dependent and other oxidases or nitric oxide synthases, and ROS-neutralizing enzymes such as catalase, peroxiredoxins, thioredoxins, thioredoxin reductases, glutathione reductases, and many others. Most of the genes coding for these enzymes contain sequences targeted by micro RNAs (miRNAs), which are components of RNA-induced silencing complexes and play important roles in inhibiting translation of their targeted messenger RNAs (mRNAs).

Circuits Regulating Superoxide and Nitric Oxide Production and Neutralization in Different Cell Types: Expression of Participating Genes and Changes Induced by Ionizing Radiation.

Superoxide radicals, together with nitric oxide (NO), determine the oxidative status of cells, which use different pathways to control their levels in response to stressing conditions. Using gene expression data available in the Cancer Cell Line Encyclopedia and microarray results, we compared the expression of genes engaged in pathways controlling reactive oxygen species and NO production, neutralization, and changes in response to the exposure of cells to ionizing radiation (IR) in human cancer cell lines originating from different tissues. The expression of NADPH oxidases and NO synthases that participate in superoxide radical and NO production was low in all cell types.

A mathematical model as a tool to identify microRNAs with highest impact on transcriptome changes.

Background: Rapid changes in the expression of many messenger RNA (mRNA) species follow exposure of cells to ionizing radiation. One of the hypothetical mechanisms of this response may include microRNA (miRNA) regulation, since the amounts of miRNAs in cells also vary upon irradiation. To address this possibility, we designed experiments using cancer-derived cell lines transfected with luciferase reporter gene containing sequences targeted by different miRNA species in its 3'- untranslated region.

Cell type-specific differences in redox regulation and proliferation after low UVA doses.

Ultraviolet A (UVA) radiation is harmful for living organisms but in low doses may stimulate cell proliferation. Our aim was to examine the relationships between exposure to different low UVA doses, cellular proliferation, and changes in cellular reactive oxygen species levels. In human colon cancer (HCT116) and melanoma (Me45) cells exposed to UVA doses comparable to environmental, the highest doses (30-50 kJ/m2) reduced clonogenic potential but some lower doses (1 and 10 kJ/m2) induced proliferation.

Correction: Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

[This corrects the article DOI: 10.1371/journal.pone.

Modulation by neighboring cells of the responses and fate of melanoma cells irradiated with UVA.

UVA radiation, which accounts for about 95% of the solar spectrum, contributes to and may be the etiological factor of skin cancers of which malignant melanoma is the most aggressive. UVA causes oxidative stress in various types of cells in the skin, keratinocyte, melanocytes, and fibroblasts, which is responsible for its cytotoxic effect. Here we used a transwell system to explore how the responses of melanoma cells to a low dose of UVA (20kJ/m, ~10% of the minimal erythema dose) are influenced by neighboring co-cultured melanoma cells or fibroblasts.

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo).

The Impact of DIDS-Induced Inhibition of Voltage-Dependent Anion Channels (VDAC) on Cellular Response of Lymphoblastoid Cells to Ionizing Radiation.

Background: The voltage-dependent anion channels (VDAC) play an essential role in the cross talk between mitochondria and the rest of the cell. Their implication in cell life and cell death has been studied extensively in recent years. In this work we studied the impact of mitochondrial membrane (VDACs) on cell survival and response to X-ionizing radiation (IR) of human lymphoblastoid K562 cells.

Vitamin C enhances substantially formation of 5-hydroxymethyluracil in cellular DNA.

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells.

The classification, structure and functioning of Ago proteins in Eukaryotes.

Ago proteins are members of the highly specialized and conserved Argonaute family, primarily responsible for regulation of gene expression. As a part of RNA-induced silencing complexes (RISCs) Ago proteins are responsible for binding a short RNA and cleavage/inhibition of translation of target mRNAs. Phosphorylation may work as the switch between those two functions, but the role of magnesium ion concentration is also taken into consideration.

Development of a population of cancer cells: Observation and modeling by a Mixed Spatial Evolutionary Games approach.

Living cells, like whole living organisms during evolution, communicate with their neighbors, interact with the environment, divide, change their phenotypes, and eventually die. The development of specific ways of communication (through signaling molecules and receptors) allows some cellular subpopulations to survive better, to coordinate their physiological status, and during embryonal development to create tissues and organs or in some conditions to become tumors. Populations of cells cultured in vitro interact similarly, also competing for space and nutrients and stimulating each other to better survive or to die.

The expression pattern of PFKFB3 enzyme distinguishes between induced-pluripotent stem cells and cancer stem cells.

Induced pluripotent stem cells (iPS) have become crucial in medicine and biology. Several studies indicate their phenotypic similarities with cancer stem cells (CSCs) and a propensity to form tumors. Thus it is desirable to identify a trait which differentiates iPS populations and CSCs.

Microarray experiments and factors which affect their reliability.

Oligonucleotide microarrays belong to the basic tools of molecular biology and allow for simultaneous assessment of the expression level of thousands of genes. Analysis of microarray data is however very complex, requiring sophisticated methods to control for various factors that are inherent to the procedures used. In this article we describe the individual steps of a microarray experiment, highlighting important elements and factors that may affect the processes involved and that influence the interpretation of the results.

The different radiation response and radiation-induced bystander effects in colorectal carcinoma cells differing in p53 status.

Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system.

Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway.

Sources of high variance between probe signals in Affymetrix short oligonucleotide microarrays.

High density oligonucleotide microarrays present a big challenge for statistical data processing methods which aim to separate changes induced by experimental factors from those caused by artifacts and measurement inaccuracies. Despite huge advances in the field of microarray probe design methods, the signal variation between probes that target a single transcript is substantially larger than their between-replicate array variability, suggesting a large influence of various probe-specific effects that introduce bias to the data. In this work we present the influence of probe-related design variations on the expression intensities of individual probes, focusing on five potential sources of high probe signal variance: the GC composition of the probe, the distance between individual probe target sites, G-quadruplex formation in the probe sequence, the occurrence of sequence motifs complementary to the oligo(dT) primer, and the specificity of unrecognized alternative splicing probeset assignment.

Induction of bystander effects by UVA, UVB, and UVC radiation in human fibroblasts and the implication of reactive oxygen species.

Radiation-induced bystander effects are various types of responses displayed by nonirradiated cells induced by signals transmitted from neighboring irradiated cells. This phenomenon has been well studied after ionizing radiation, but data on bystander effects after UV radiation are limited and so far have been reported mainly after UVA and UVB radiation. The studies described here were aimed at comparing the responses of human dermal fibroblasts exposed directly to UV (A, B, or C wavelength range) and searching for bystander effects induced in unexposed cells using a transwell co-incubation system.

MicroRNAs and reactive oxygen species: are they in the same regulatory circuit?

Interactions of living organisms with their environment mainly involve modulation of gene expression by stimulation or silencing. One of the epigenetic mechanisms that regulate translation and the levels of transcripts is RNA interference, in which microRNAs (miRNAs) address RNA-induced silencing complexes (RISCs) to degrade specific mRNAs or to silence their translation. In this mechanism, double stranded RNA structure is crucial for miRNA biogenesis and the action of RISCs.

Changes of reactive oxygen and nitrogen species and mitochondrial functioning in human K562 and HL60 cells exposed to ionizing radiation.

Free radicals generated by mitochondria are candidates for mediating long-lasting effects of radiation on cells, including genetic instability. To better understand the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in these long-term effects we assayed ROS and RNS levels, the mitochondrial membrane potential and mass, and the frequency of DNA strand breaks, apoptosis and necrosis in human leukemic cells (K562 and HL60) after 12 Gy of X irradiation. An increase in intracellular ROS level was observed immediately post-irradiation, and about 24 h later a second increase of ROS was accompanied by increase in nitrogen oxide, mitochondrial potential and mitochondrial mass in both cell types.

The influence of XPD, APE1, XRCC1, and NBS1 polymorphic variants on DNA repair in cells exposed to X-rays.

Polymorphism of genes coding for proteins which participate in DNA repair may predispose to or protect against development of cancer. Here we studied how common polymorphisms of the genes XPD (Asp312Asn and Lys751Gln), APE1 (Asp148Glu), XRCC1 (Arg399Gln), and NBS1 (Gln185Glu) influence DNA repair and other responses after X-irradiation of lymphocytes from colon carcinoma patients. Genotypes with polymorphic Asp148Glu APE1 and Asp312Asn XPD showed a significantly higher level of DNA incisions immediately after irradiation (p=0.