Angelo Theodoratos

A mutation in a chromosome condensin II subunit, kleisin β, specifically disrupts T cell development

Condensins are ubiquitously expressed multiprotein complexes that are important for chromosome condensation and epigenetic regulation of gene transcription, but whose specific roles in vertebrates are poorly understood. We describe a mouse strain, nessy, isolated during an ethylnitrosourea screen for recessive immunological mutations. The nessy mouse has a defect in T lymphocyte development that decreases circulating T cell numbers, increases their expression of the activation/memory marker CD44, and dramatically decreases the numbers of CD4+CD8+ thymocytes and their immediate DN4 precursors. A missense mutation in an unusual alternatively spliced first exon of the kleisin β gene, a member of the condensin II complex, was shown to be responsible and act in a T cell-autonomous manner. Despite the ubiquitous expression and role of condensins, kleisin βnes/nes mice were viable, fertile, and showed no defects even in the parallel pathway of B cell lymphocyte differentiation. These data define a unique lineage-specific requirement for kleisin β in mammalian T cell differentiation.

The impact of glutathione transferase kappa deficiency on adiponectin multimerisation in vivo.

Glutathione transferase Kappa (GSTK1-1) also termed disulfide bond-forming oxidoreductase A-like protein (DsbA-L) has been implicated in the post-translational multimerization of adiponectin and has been negatively correlated with obesity in mice and humans. We investigated adiponectin in Gstk1−/− mice and surprisingly found no difference in the levels of total serum adiponectin or the level of high molecular weight (HMW) multimers when compared with normal controls. Non-reducing SDS-polyacrylamide gel electrophoresis and western blotting also showed a similar distribution of low, middle and HMW multimers in normal and Gstk1−/− mice. Variation in adiponectin has been correlated with glucose tolerance and with the levels of phosphorylated AMP-kinase but we found similar glucose tolerance and similar levels of phospho 5-AMP-activated protein kinase in normal and Gstk1−/− mice. Consequently, our findings suggest that GSTK1-1 is not absolutely required for adiponectin multimerization in vivo and alternate pathways may be activated in GSTK1-1 deficiency.

Mouse strains with point mutations in TAP1 and TAP2

We report two new mouse strains: Jasmine (C57BL/6J/Apb‐Tap2jas/Apb), with a point mutation in the transporter associated with antigen processing (TAP)2 ; and Rose, (C57BL/6J/Apb‐Tap1rose/Apb), with a point mutation in TAP1. These strains were detected as the result of ethyl nitroso urea (ENU) screens for recessive point mutations affecting the immune system. As expected in cases of defective TAP expression, the mice have very low major histocompatibility complex (MHC)‐I cell‐surface expression, and few CD8+ T cells. The Rose strain has an A to T substitution in exon 10 of TAP1, resulting in an asparagine to valine substitution at position 643. Jasmine has an A to C transversion in exon 5 of TAP2, resulting in a threonine to proline substitution at position 293 of the protein. The mutation does not affect mRNA levels, but results in a very severe reduction in TAP2 protein. TAP1 protein levels are also decreased in Jasmine mice, demonstrating a new role for mouse TAP2 in stabilizing TAP1 protein expression. Jasmine is the first strain available with defective TAP2. The two mouse strains provide additional animal models for the human condition Bare Lymphocyte syndrome type 1, and identify new residues important for TAP function.

Phenylalanine-induced leucopenia in genetic and dichloroacetic acid generated deficiency of glutathione transferase Zeta

Glutathione transferase Zeta (GSTZ1-1) is identical to maleylacetoacetate isomerase and catalyses a significant step in the catabolism of phenylalanine and tyrosine. Exposure of GSTZ1-1 deficient mice to high dietary phenylalanine causes a rapid loss of circulating white blood cells (WBCs). The loss was significant (P<0.05) after 2 days and total WBCs were reduced by 60% after 6 days. The rapid loss of WBCs was attributed to the accumulation of the catabolic intermediates maleylacetoacetate or maleylacetone (MA) in the circulation. Serum from GSTZ1-1 deficient mice treated with phenylalanine was cytotoxic to splenocytes from normal BALB/c mice and direct incubation of normal splenocytes with MA caused a rapid loss of viability. Dichloroacetic acid (DCA) has been used therapeutically to treat lactic acidosis and is potentially of use in cancer chemotherapy. Since DCA can inactivate GSTZ1-1 there is a possibility that long-term treatment of patients with DCA could cause GSTZ1-1 deficiency and susceptibility to oxidative stress and phenylalanine/tyrosine-induced WBC loss. However, although we found that DCA at 200mg/(kg day) causes a severe loss of hepatic GSTZ1-1 activity in BALB/c mice, it did not induce WBC cytotoxicity when combined with high dietary phenylalanine.

Glutathione transferase kappa deficiency causes glomerular nephropathy without overt oxidative stress

Glutathione transferase kappa (GSTK1-1) is a highly conserved, mitochondrial enzyme potentially involved in redox reactions. GSTK1-1-deficient mice were generated to further study the enzymes biological role. Reduced and total glutathione levels in liver and kidney were unchanged by GSTK1-1 deficiency and NADPH quinone oxidoreductase 1 expression was not elevated indicating that there is no general underlying oxidative stress in Gstk1−/− mice. Electron microscopy of liver and kidney showed no changes in mitochondrial morphology with GSTK1-1 deficiency. The death of a number of Gstk1−/− males with urinary tract problems prompted close examination of the kidneys. Electron microscopy revealed glomerular basement membrane changes at 3 months, accompanied by detectable microalbuminuria in male mice (albumin:creatinine ratio of 2.66±0.83 vs 1.13±0.20 mg/mmol for Gstk1−/− and wild-type (WT), respectively, P=0.001). This was followed by significant foot process effacement (40–55% vs 10% for Gstk1−/− and WT, respectively) at 6 months of age in all Gstk1−/− mice examined. Kidney tubules were ultrastructurally normal. Compared with human disease, the Gstk1−/− kidneys show changes seen in glomerulopathies causing nephrotic syndrome. Gstk1−/− mice may offer insights into the early development of glomerular nephropathies.

Identification of chromatin accessibility domains in human breast cancer stem cells

ABSTRACT Epithelial-to-mesenchymal transition (EMT) is physiological in embryogenesis and wound healing but also associated with the formation of cancer stem cells (CSCs). Many EMT signaling pathways are implicated in CSC formation, but the precise underlying mechanisms of CSC formation remain elusive. We have previously demonstrated that PKC is critical for EMT induction and CSC formation in inducible breast EMT/CSC models. Here, we used formaldehyde-assisted isolation of regulatory elements-sequencing (FAIRE-seq) to investigate DNA accessibility changes after PKC activation and determine how they influence EMT and CSC formation. During EMT, DNA accessibility principally increased in regions distant from transcription start sites, low in CpG content, and enriched with chromatin enhancer marks. ChIP-sequencing revealed that a subset of these regions changed from poised to active enhancers upon stimulation, with some even more acteylated in CSCs. While regions with increased accessibility were enriched for FOX, AP-1, TEAD, and TFAP2 motifs, those containing FOX and AP-1 motif were associated with increased expression of CSC-associated genes, while those with TFAP2 were associated with genes with increased expression in non-CSCs. Silencing of 2 members of the FOX family, FOXN2 and FOXQ1, repressed CSCs and the mesenchymal phenotype and inhibited the CSC gene signature. These novel, PKC-induced DNA accessibility regions help explain how the epigenomic plasticity of cells undergoing EMT leads to CSC gene activation.

Dichloroacetic acid up-regulates hepatic glutathione synthesis via the induction of glutamate-cysteine ligase

Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. Summary: Memory T cells have a rapid transcriptional program upon re-stimulation. Chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit this transcriptional memory in T cells.

Splice variants of the condensin II gene Ncaph2 include alternative reading frame translations of exon 1

Condensins I and II are five‐protein complexes that are important for the condensation of chromatin. They are essential for mitosis and important for regulating gene expression during interphase. Here, we investigated the transcription and translation of the mouse Ncaph2 gene, which encodes a subunit of condensin II. We identified three splice variants within the first exon, a NAGNAG splice variant at the beginning of exon 16 and alternative 3′‐UTRs. In total, Ncaph2 is potentially capable of generating 12 unique mRNA transcripts and six unique proteins. We confirm that Ncaph2 can generate three different N‐termini, all encoded by exon 1, one of which is translated from an alternative reading frame. This alternative reading frame splice variant appears to be a novel outcome of splicing. If this is applicable to other genes, it would account for a previously unappreciated level of eukaryotic protein diversity.

12. Prevention of cisplatin-induced nephrotoxicity by dichloroacetate

Background: There is a continuing need to improve the efficacy of anticancer agents and to reduce the toxicity associated with them. Recently, reversal of the glycolytic phenotype of cancer cells with novel agents such as DCA (dichloroacetate) has been recognised as an important new target for cancer therapy. DCA is currently being assessed as a novel chemotherapeutic in clinical trials. It is anticipated that DCA will be used in combination with other well-established chemotherapeutic drugs. It is therefore critically important to determine the effects of DCA on therapeutic efficacy, and off-target effects of existing frontline anti-cancer drugs. We have recently discovered that DCA induces glutamate cysteine ligase, the rate-limiting step in glutathione (GSH) synthesis, a major cellular antioxidant. We hypothesised that the ability of DCA to stimulate GSH-synthesising capacity could reduce cisplatin-induced nephrotoxicity, which is thought to be mediated in part by oxidative stress. Aims: To determine whether DCA can attenuate cisplatin-induced nephrotoxicity, and to determine if DCA influences the anti-cancer properties of cisplatin. Methods: 120 Balb/c mice were injected subcutaneously with the syngeneic 4T1 breast cancer cell line and then co-treated with DCA and cisplatin weekly for 1 month. Controls included treatment with cisplatin alone, DCA alone, or normal saline injections. Serum blood urea nitrogen (BUN) and creatinine were measured and kidney damage was assessed histologically. Tumour size was monitored throughout. Results and conclusions: DCA prevented increases in serum creatinine, BUN, and renal proximal tubule apoptosis evident in cisplatin-only treated mice. The tumour size in mice in the co-treated group decreased at the same rate as the cisplatin-only treated mice. We have thus concluded that DCA largely prevents the development of cisplatin-induced nephrotoxicity, and does not attenuate its anti-cancer properties.