Heterochromatin marks HP1γ, HP1α and H3K9me3, and DNA damage response activation in human testis development and germ cell tumours.

Heterochromatinization has been implicated in fundamental biological and pathological processes including differentiation, senescence, ageing and tumourigenesis; however, little is known about its regulation and roles in human cells and tissues in vivo. Here, we show distinct cell-type- and cancer-stage-associated patterns of key heterochromatin marks: histone H3 trimethylated at lysine 9 (H3K9me3) and heterochromatic adaptor proteins HP1α and HP1γ, compared with the γH2AX marker of endogenously activated DNA damage response (DDR) and proliferation markers in normal human foetal (n=4) and adult (n=29) testes, pre-invasive carcinoma in situ (CIS; n=26) lesions and a series of overt germ cell tumours, including seminomas (n=26), embryonal carcinomas (n=18) and teratomas (n=11). Among striking findings were high levels of HP1γ in foetal gonocytes, CIS and seminomas; enhanced multimarker heterochromatinization without DDR activation in CIS; and enhanced HP1α in teratoma structures with epithelial and neuronal differentiation. Differential expression of the three heterochromatin markers suggests their partly non-overlapping roles, and separation of heterochromatinization from DDR activation highlights distinct responses of germ cells vs. somatic tissues in early tumourigenesis. Conceptually interesting findings were that subsets of human cells in vivo proliferate despite enhanced heterochromatinization, and that cells can strongly express even multiple heterochromatin features in the absence of functional retinoblastoma protein and without DDR activation. Overall, these results provide novel insights into cell-related and tumour-related diversity of heterochromatin in human tissues in vivo, relevant for andrology and intrinsic anti-tumour defence roles attributed to activated DDR and cellular senescence.

Addiction to DUSP1 protects JAK2V617F-driven polycythemia vera progenitors against inflammatory stress and DNA damage, allowing chronic proliferation.

Inflammatory and oncogenic signaling converge in disease evolution of BCR-ABL-negative myeloproliferative neoplasms, clonal hematopoietic stem cell disorders characterized by gain-of-function mutation in JAK2 kinase (JAK2V617F), with highest prevalence in patients with polycythemia vera (PV). Despite the high risk, DNA-damaging inflammatory microenvironment, PV progenitors tend to preserve their genomic stability over decades until their progression to post-PV myelofibrosis/acute myeloid leukemia. Using induced pluripotent stem cells-derived CD34+ progenitor-enriched cultures from JAK2V617F+ PV patient and from JAK2 wild-type healthy control, CRISPR-modified HEL cells and patients' bone marrow sections from different disease stages, we demonstrate that JAK2V617F induces an intrinsic IFNγ- and NF-κB-associated inflammatory program, while suppressing inflammation-evoked DNA damage both in vitro and in vivo. We show that cells with JAK2V617F tightly regulate levels of inflammatory cytokines-induced reactive oxygen species, do not fully activate the ATM/p53/p21waf1 checkpoint and p38/JNK MAPK stress pathway signaling when exposed to inflammatory cytokines, suppress DNA single-strand break repair genes' expression yet overexpress the dual-specificity phosphatase (DUSP) 1. RNAi-mediated knock-down and pharmacological inhibition of DUSP1, involved in p38/JNK deactivation, in HEL cells reveals growth addiction to DUSP1, consistent with enhanced DNA damage response and apoptosis in DUSP1-inhibited parental JAK2V617F+ cells, but not in CRISPR-modified JAK2 wild-type cells. Our results indicate that the JAK2V617F+ PV progenitors utilize DUSP1 activity as a protection mechanism against DNA damage accumulation, promoting their proliferation and survival in the inflammatory microenvironment, identifying DUSP1 as a potential therapeutic target in PV.

IFNγ induces oxidative stress, DNA damage and tumor cell senescence via TGFß/SMAD signaling-dependent induction of Nox4 and suppression of ANT2.

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNFα) and interferon gamma (IFNγ) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic ß-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNFα and IFNγ induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFNγ and TNFα, short exposure to IFNγ alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFNγ-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGFß) secretion and consequent autocrine/paracrine activation of the TGFß/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFNγ contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFNγ/TNFα by DDR and senescence correlated with the lack of TGFß and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGFß/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFNγ/TNFα-evoked genotoxicity and cellular senescence.

Radiotherapy-induced plasticity of prostate cancer mobilizes stem-like non-adherent, Erk signaling-dependent cells.

Fractionated ionizing radiation combined with surgery or hormone therapy represents the first-choice treatment for medium to high-risk localized prostate carcinoma. One of the main reasons for the failure of radiotherapy in prostate cancer is radioresistance and further dissemination of surviving cells. In this study, exposure of four metastasis-derived human prostate cancer cell lines (DU145, PC-3, LNCaP and 22RV1) to clinically relevant daily fractions of ionizing radiation (35 doses of 2 Gy) resulted in generation of two radiation-surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2 and Nanog. While a subset of the radiation-surviving adherent cells resumed proliferation shortly after completion of the irradiation regimen, the non-adherent cells started to proliferate only on their reattachment several weeks after the radiation-induced loss of adhesion. Like the parental non-irradiated cells, radiation-surviving re-adherent DU145 cells were tumorigenic in immunocompromised mice. The radiation-induced loss of adhesion was dependent on expression of Snail, as siRNA/shRNA-mediated knockdown of Snail prevented cell detachment. On the other hand, survival of the non-adherent cells required active Erk signaling, as chemical inhibition of Erk1/2 by a MEK-selective inhibitor or Erk1/2 knockdown resulted in anoikis-mediated death in the non-adherent cell fraction. Notably, whereas combined inhibition of Erk and PI3K-Akt signaling triggered cell death in the non-adherent cell fraction and blocked proliferation of the adherent population of the prostate cancer cells, such combined treatment had only marginal if any impact on growth of control normal human diploid cells. These results contribute to better understanding of radiation-induced stress response and heterogeneity of human metastatic prostate cancer cells, document treatment-induced plasticity and phenotypically distinct cell subsets, and suggest the way to exploit their differential sensitivity to radiosensitizing drugs in overcoming radioresistance.

High expression of uncoupling protein 2 in foetal liver.

To assess the putative role of mitochondrial uncoupling protein 2 (UCP2) during perinatal development, its expression was analysed in mice and rats. Expression was detected in a large range of foetal tissues. A unique developmental pattern of UCP2 expression was found in liver, where the level of UCP2 mRNA was about 30-fold higher in foetuses than in adults (mice data), and started to decline immediately after birth. Neither UCP1 nor UCP3 mRNA was expressed in foetal liver. As in adult liver, immunohistochemical analysis suggested exclusive localisation of UCP2 in the monocyte/macrophage cells. Our results indicate a role of UCP2 in haematopoietic system development.

AP-2 enhances Sp1-dependent activation of the growth-regulated human ATP/ADP translocator.

The mammalian ATP/ADP translocator isoform-2 (ANT2) gene is growth-activated. Regulation of the gene appears to involve Sp1, as an essential activator, and a suppressor through an Sp1 core element next to the transcription start. We show here that the proximal promoter also binds AP-2 strongly and specifically to two sites, one of which overlaps the Sp1 proximal suppressor site. AP-2 binds with an affinity of 10 to15 fold higher than that of Sp1. AP-2 alone does not alter the ANT2 promoter activity in transfected SL2 cells, but enhances the Sp1-dependent activation of the promoter several fold. Enhancement by AP-2 is observed only when Sp1 is limiting for transcription activation. These data suggest that the cellular AP-2/Sp1 ratio might influence ANT2 expression in developing or differentiating cells.

The interference of cysteine thiols in the detection of glycated and non-glycated proteins by modified silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

In the previous study a staining intensification of in vitro glycated collagen type I versus a non-glycated one after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under a modified silver staining procedure was observed (Hodny, Z., Struzinsky, R. and Deyl, Z. (1992) J. Chromatogr. 578, 53-62). While investigating the specificity of this stain to glycation product(s) on protein we have observed that a great number of proteins (e.g. bovine serum albumin) was sensitive to this stain even in a non-glycated state. It is proposed from results of the analysis of amino acid composition of these proteins that their better stainability correlates with the amount of cysteine present in the protein. Modification of SH groups by iodoacetamide (or N-ethylmaleimide) had an inhibitory effect on the staining of bovine serum albumin (and some other proteins) in its 'native' state but had no visible inhibitory effect on their staining in the glycated state. However, the positive staining response of a great number of components from cellular lysates even after iodoacetamide treatment indicates the existence of further chemical groups (either of protein or nucleic acid origin) participating in this silver staining method.

Non-enzymatic glycosylation of myosin: effects of diabetes and ageing.

The influence of diabetes mellitus, streptozotocin-induced diabetes and ageing on the non-enzymatic glycosylation of myosin from cardiac and skeletal muscles was investigated. In cardiac muscle, and to a lesser extent also in skeletal muscles of the rat, non-enzymatic glycosylation of myosin increases with the age, as measured in 6-, 12- and 29-month-old animals. Skeletal muscle myosin from diabetic humans and also that from diabetic rat cardiac muscle are more glycosylated when compared with control myosin preparations. Ca(2+)-ATPase activity of myosin is lower in muscles of diabetic individuals as compared with control muscles.

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1.

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-ß interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-ß pathway and implicate this mechanism in the maintenance of genome integrity.

Cytokine expression and signaling in drug-induced cellular senescence.

Cellular senescence guards against cancer and modulates aging; however, the underlying mechanisms remain poorly understood. Here, we show that genotoxic drugs capable of inducing premature senescence in normal and cancer cells, such as 5-bromo-2'-deoxyuridine (BrdU), distamycin A (DMA), aphidicolin and hydroxyurea, persistently activate Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling and expression of interferon-stimulated genes (ISGs), such as MX1, OAS, ISG15, STAT1, PML, IRF1 and IRF7, in several human cancer cell lines. JAK1/STAT-activating ligands, interleukin 10 (IL10), IL20, IL24, interferon gamma (IFNgamma), IFNbeta and IL6, were also expressed by senescent cells, supporting autocrine/paracrine activation of JAK1/STAT. Furthermore, cytokine genes, including proinflammatory IL1, tumor necrosis factor and transforming growth factor families, were highly expressed. The strongest inducer of JAK/STAT signaling, cytokine production and senescence was BrdU combined with DMA. RNA interference-mediated knockdown of JAK1 abolished expression of ISGs, but not DNA damage signaling or senescence. Thus, although DNA damage signaling, p53 and RB activation, and the cytokine/chemokine secretory phenotype are apparently shared by all types of senescence, our data reveal so far unprecedented activation of the IFNbeta-STAT1-ISGs axis, and indicate a less prominent causative role of IL6-JAK/STAT signaling in genotoxic drug-induced senescence compared with reports on oncogene-induced or replicative senescence. These results highlight shared and unique features of drug-induced cellular senescence, and implicate induction of cancer secretory phenotype in chemotherapy.

Body height, body mass index, waist-hip ratio, fluctuating asymmetry and second to fourth digit ratio in subjects with latent toxoplasmosis.

Between 20% and 60% of the population of most countries are infected with the protozoan Toxoplasma gondii. Subjects with clinically asymptomatic life-long latent toxoplasmosis differ from those who are Toxoplasma free in several behavioural parameters. Case-control studies cannot decide whether these differences already existed before infection or whether they were induced by the presence of Toxoplasma in the brain of infected hosts. Here we searched for such morphological differences between Toxoplasma-infected and Toxoplasma-free subjects that could be induced by the parasite (body weight, body height, body mass index, waist-hip ratio), or could rather correlate with their natural resistance to parasitic infection (fluctuating asymmetry, 2D : 4D ratio). We found Toxoplasma-infected men to be taller and Toxoplasma-infected men and women to have lower 2D : 4D ratios previously reported to be associated with higher pre-natal testosterone levels. The 2D : 4D ratio negatively correlated with the level of specific anti-Toxoplasma antibodies in Toxoplasma-free subjects. These results suggest that some of the observed differences between infected and non-infected subjects may have existed before infection and could be caused by the lower natural resistance to Toxoplasma infection in subjects with higher pre-natal testosterone levels.

Staining and quantification of proteins separated by polyacrylamide gel electrophoresis.

The present review concentrates on techniques for the staining and quantification of proteins separated by polyacrylamide gel electrophoresis. Staining with organic dyes has been used for approximately thirty years; the silver staining technique was introduced in 1979. The problems of silver staining are presented separately because the mechanism of this staining is in principle different from staining with organic dyes. Less attention has been devoted to quantification of two-dimensional gels, because this autoradiography is preferred because of its high sensitivity and fewer problems with accurate quantification in contrast to silver staining.

In vitro non-enzymatic glycosylation of myofibrillar proteins.

1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered. 2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered. 3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated. 4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation. 5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

Silver staining of collagen type I after sodium dodecylsulphate polyacrylamide gel electrophoresis: effect of Maillard reaction.

Differences in the acidic silver staining, after sodium dodecylsulphate polyacrylamide gel electrophoresis, between the alpha 1 and alpha 2 collagen chains, as well as between rat-tail tendon and calf-skin collagen type I, were observed. The staining conditions at which the staining differences are both most expressed and reproducible were characterized. Age differences between staining of the alpha 1 CB6 fragment from young rats (2 and 12 months) and old rats (29 months) indicated that different susceptibilities of collagen species to the silver staining can be the result of different extents of some age-dependent post-translational modification, such as glycation. In vitro incubation of acid-soluble rat-tail tendon collagen with various sugars led to an increase in silver staining compared with samples incubated in the absence of sugar. This effect was inhibited by sodium cyanoborohydride, diethylenetriamine pentaacetic acid and aminoguanidine, i.e. compounds inhibiting the Maillard reaction at various stages. It can be concluded that the enhanced silver susceptibility of glycated collagen is related to advanced-phase Maillard reaction products attached to collagen.

Chromatographic separation of glycated nucleotides.

A high-performance liquid chromatographic method for the separation of nucleotide glycation reaction product(s) has been developed. The product(s) arising during in vitro glycation of a nucleotide (AMP, GMP or CMP) with a reducing sugar (ribose or glucose) were clearly resolved from the non-glycated constituents of the reaction mixture. The peak(s) of the glycated product(s) increased when an amino acid (lysine, arginine, beta-alanine or N epsilon-acetyllysine) was added to the reaction mixture. This increase probably corresponds to the formation of a new product with a different absorption maximum (250 versus 260 nm). Conversely, formation of this product(s) was inhibited by the presence of the metal-chelating agent diethylenetriamine pentaacetic acid and by aminoguanidine.

In vivo mapping of the human adenine nucleotide translocator-2 (ANT2) promoter provides support for regulation by a pair of proximal Sp1-activating sites and an upstream silencer element.

Regulatory factors bound to the human adenine nucleotide translocator-2 (ANT2) promoter have been mapped in HeLa cells by in vivo DNase I protection and ligation-mediated PCR amplification. Protein binding was detected at only three sites within the extended promoter region (to nt -703). One, starting at nt -61 and covering the TATA box and transcription start, most probably represents occupation by the transcription-initiation machinery. A repeated Sp1 element determined by in vitro studies to be the major activation element for the promoter was also protected in vivo on nucleotides responsible for strong binding to the zinc fingers. Occupation of two additional upstream Sp1 elements was not observed. The third site occupied in vivo was identified previously by in vitro studies as a unique silencer element. Treatment of cells with trichostatin A to induce hyperacetylation released the silencer-binding protein after 1 h, but had no effect on the Sp1-activating elements. Prolonged treatment (24 h) displaced Sp1 from the activating elements. These findings confirm and extend in vitro studies indicating that regulation of the ANT2 promoter is most probably exerted through a single pair of proximal Sp1-activating elements and an upstream silencer, and that chromatin organization plays a role in the interaction between the two.

Sp1 acts as a repressor of the human adenine nucleotide translocase-2 (ANT2) promoter.

The human adenine nucleotide translocator-2 promoter is activated by adjacent Sp1 activation elements centered at nucleotides -79 and -68 (Abox and Bbox, respectively), and is repressed by Sp1 bound to a GC element (Cbox) that lies adjacent to transcription start. Here, we address the mechanism of this unique Sp1-mediated repression using transfected Drosophila SL2 and mammalian cell lines. We show that repression is not due to steric interference with assembly of the transcription machinery, as Sp1 bound to the Cbox can, under certain conditions, activate the promoter. Furthermore, ectopic expression of Sp1 deletion mutants in SL2 cells demonstrates that both the Sp1-mediated repression and activation require the D transactivation domain of Sp1 bound to the Cbox. In addition, repression of ABbox-mediated activation is eliminated by separating the Abox and Bbox. Thus, for Cbox-bound Sp1 to repress, Sp1 must be precisely positioned at the region of the ABboxes. Together, these data suggest that the D transactivation domain mediates interactions by Sp1 complexes on separate GC elements that results in repression of the activating Sp1 species.

Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter.

The influence of chromatin on the human adenine nucleotide translocase isoform 2 (ANT2) promoter was investigated in transfected cells treated with the deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin and activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, indicating that the Sp1 elements are repressed by chromatin structure. Repressive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of basal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induced by TSA, but those with high basal expression were less effected. These data indicate that chromatin environment surrounding the integrated DNA exerts a strong influence on chromatin-dependent repression of the ANT2 promoter, and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.

Sp1 activates and inhibits transcription from separate elements in the proximal promoter of the human adenine nucleotide translocase 2 (ANT2) gene.

Expression of the adenine nucleotide translocator 2 (ANT2) gene is growth regulated. We report a feature of the ANT2 promoter that involves a novel regulatory function for the Sp1 transfactor. We show that expression from the ANT2 proximal promoter is modulated through three Sp1 elements, two of which activate and one of which partially inhibits transcription. The inhibitor site, box C, is juxtaposed to transcription start (nucleotides -7 to -2). Sp1 bound to box C decreases transcription initiation. This was demonstrated by introducing mutations in box C which (a) increased chloramphenicol acetyltransferase expression in the transient transfection assay and (b) inhibited binding of both purified Sp1 and Sp1 in crude nuclear extracts. The activating elements (A and B boxes) are located at adjacent sites in the distal region of the proximal promoter. Mutation of either box inhibits transfection by 90%, indicating that they act in a synergistic manner. Supershift experiments with crude nuclear extracts showed that only Sp1 was bound to the three GC boxes. The finding that Sp1 acts as an activator/inhibitor within the same promoter region was verified in NIH3T3, HeLa, JEG3, and COS-1, indicating that this dual effect of Sp1 is widely preserved. These data suggest a unique role for Sp1 and raise the possibility that growth activation of the ANT2 gene is regulated by the interaction of Sp1 on the A, B, and C boxes.