ICF, An Immunodeficiency Syndrome: DNA Methyltransferase 3B Involvement, Chromosome Anomalies, and Gene Dysregulation

The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) is the only disease known to result from a mutated DNA methyltransferase gene, namely, DNMT3B. Characteristic of this recessive disease are decreases in serum immunoglobulins despite the presence of B cells and, in the juxtacentromeric heterochromatin of chromosomes 1 and 16, chromatin decondensation, distinctive rearrangements, and satellite DNA hypomethylation. Although DNMT3B is involved in specific associations with histone deacetylases, HP1, other DNMTs, chromatin remodelling proteins, condensin, and other nuclear proteins, it is probably the partial loss of catalytic activity that is responsible for the disease. In microarray experiments and real-time RT-PCR assays, we observed significant differences in RNA levels from ICF vs. control lymphoblasts for pro- and anti-apoptotic genes (BCL2L10, CASP1, and PTPN13); nitrous oxide, carbon monoxide, NF-κB, and TNFa signalling pathway genes (PRKCH, GUCY1A3, GUCY1B3, MAPK13; HMOX1, and MAP4K4); and transcription control genes (NR2F2 and SMARCA2). This gene dysregulation could contribute to the immunodeficiency and other symptoms of ICF and might result from the limited losses of DNA methylation although ICF-related promoter hypomethylation was not observed for six of the above examined genes. We propose that hypomethylation of satellite 2at1qh and 16qh might provoke this dysregulation gene expression by trans effects from altered sequestration of transcription factors, changes in nuclear architecture, or expression of noncoding RNAs

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Arginase II expressed in cancer-associated fibroblasts indicates tissue hypoxia and predicts poor outcome in patients with pancreatic cancer.

An adequate level of arginine in the tissue microenvironment is essential for T cell activity and survival. Arginine levels are regulated by the arginine-catabolizing enzyme, arginase (ARG). It has been reported that arginase II (ARG2), one of two ARGs, is aberrantly expressed in prostate cancer cells, which convert arginine into ornithine, resulting in a lack of arginine that weakens tumor-infiltrating lymphocytes and renders them dysfunctional. However, immune suppression mediated by ARG2-expressing cancer cells in lung cancer has not been observed. Here we studied the expression of ARG2 in pancreatic ductal carcinoma (PDC) tissue clinicopathologically by examining over 200 cases of PDC. In contrast to prostate cancer, ARG2 expression was rarely demonstrated in PDC cells by immunohistochemistry, and instead ARG2 was characteristically expressed in α-smooth muscle actin-positive cancer-associated fibroblasts (CAFs), especially those located within and around necrotic areas in PDC. The presence of ARG2-expressing CAFs was closely correlated with shorter overall survival (OS; P  = 0.003) and disease-free survival (DFS; P  = 0.0006). Multivariate Cox regression analysis showed that the presence of ARG2-expressing CAFs in PDC tissue was an independent predictor of poorer OS (hazard ratio [HR]  = 1.582, P  = 0.007) and DFS (HR  = 1.715, P  = 0.001) in PDC patients. In addition to the characteristic distribution of ARG2-expressing CAFs, such CAFs co-expressed carbonic anhydrase IX, SLC2A1, or HIF-1α, markers of hypoxia, in PDC tissue. Furthermore, in vitro experiments revealed that cultured fibroblasts extracted from PDC tissue expressed the ARG2 transcript after exposure to hypoxia, which had arginase activity. These results indicate that cancer cell-mediated immune suppression through ARG2 expression is not a general event and that the presence of ARG2-expressing CAFs is an indicator of poor prognosis, as well as hypoxia, in PDC tissue

Correlation of expression of ARG2 in stromal cells with clinicopathological characteristics.

<p>W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated.</p><p>tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma.</p>*<p>Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society.</p>**<p>Comparisons of qualitative variables are performed using the χ² test and otherwise Fisher’s exact test.</p>***<p>Total number of patients are 211 and otherwise 214.</p

ARG2 was expressed mostly in CAFs under hypoxic conditions.

<p>Histology of PDC tissue in low- (upper columns) and high-power view (lower columns). HE staining and immunohistochemistry for ARG2, CAIX, and SLC2A1 in serial tissue sections. Necrotic areas are surrounded by star marks in the upper HE photo and the rectangle (light blue) corresponds to the area of the lower column. Double immunostaining (the right-most columns) reveals that most of the granular ARG2 staining (brown) is present in spindle-shaped cells stained for CAIX (purple). Inset is a very high-power view.</p

Univariate and multivariate analyses of prognostic factors associated with disease-free survival in patients with ductal carcinoma of the pancreas.

<p>W/D, well differentiated tubular adenocarcinoma and papillary carcinoma; M/D, moderately differentiated.</p><p>tubular adenocarcinoma; P/D, poorly differentaited adenocarcinoma.</p>*<p>Classified according to the classification of pancreatic carcinoma of Japan Pancreas Society.</p

Kaplan-Meier survival curves.

<p>(A, C) Kaplan-Meier survival curve showing a comparison of overall survival between the presence (grades 1 and 2) and absence (grade 0) of ARG2 expression in stromal cells (<i>P</i>  = 0.003) in A and that among expression grades (grades 0 to 2) of ARG2 in stromal cells (grade 0 vs. grade 1, log-rank test, <i>P</i>  = 2.08; grade 0 vs. grade 2, <i>P</i><0.0001; grade 1 vs. grade 2, <i>P</i>  = 0.0009) in C. (B, D) Kaplan-Meier survival curve showing a comparison of disease-free survival between the presence (grades 1 and 2) and absence (grade 0) of ARG2 expression in stromal cells (<i>P</i>  = 0.0006) in B and that among expression grades (grades 0 to 2) of ARG2 in stromal cells (grade 0 vs. grade 1, <i>P</i>  = 0.069; grade 0 vs. grade 2, <i>P</i><0.0001; grade 1 vs. grade 2, <i>P</i>  = 0.013) in D. Black circle and white circle represent censoring and failure, respectively.</p

Pancreatic cancer cells and ARG2-expressing CAFs.

<p>(A) ARG2-expressing CAFs do not support proliferation of pancreatic cancer cells. CAFs extracted from PDC tissues and MiaPaCa-2 cells were co-cultured in medium with or without 2 mM DFMO under normoxic or hypoxic conditions for 48 hrs and the numbers of living cells were calculated the basis of data obtained by flow cytometry. The absolute number of MiaPaCa-2 cells cultured under hypoxic conditions decreased significantly in comparison with normoxic conditions, although this effect was not significantly affected by the presence of DFMO in the culture medium. Data represent one of three independent experiments. Significance value (Student’s <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><0.01 (**). (B) Oxidative stress-induced apoptosis was induced in MiaPaCa-2 cells by exposure to various concentrations (0–500 µM) of H₂O₂ for 7 hrs. The dead cells and living cells were detected by flow cytometry after staining with Annexin V and PI. (C) ARG2-expressing CAFs did not protect pancreatic cancer cells from oxidative-induced apoptosis. After 48 hrs of co-culture of CAFs extracted from PDC tissues and MiaPaCa-2 cells in medium with or without 2 mM DFMO under normoxic or hypoxic conditions, all the cells were cultured for another 4 hrs under oxidative stress (50 µM H₂O₂) using the same conditions as before. The percentages of living cells were measured by flow cytometry (left column). In order to evaluate the effect of oxidative stress, the percentages of living cells after exposure to oxidative stress were divided by the percentages of living cells cultured under the same conditions before oxidative stress (right column). The ratio of living cells before and after oxidative stress decreased significantly in both MiaPaCa-2 cells and CAFs cultured under hypoxic conditions. Blocking the synthesis of polyamines with DFMO increased significantly the degree of oxidative stress-induced apoptosis in the CAFs. Data represent one of three independent experiments. Significance value (Student’s <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><0.01 (**).</p

ARG2 was expressed mostly in CAFs within and around necrotic areas in PDC tissue.

<p>(A) Triple immunofluorescence shows that most of the dot-like staining of ARG2 (red) is present in α-SMA-positive fibroblasts (green). Cancer cells are positive for cytokeratins (blue). Nuclei are stained by DAPI (white). (B) Histology and immunohistochemistry of PDC tissue in low- (upper-photo of each pair of photos) and high-power view (lower-photo of each pair of photos). HE staining and immunohistochemistry for several antigens in serial tissue sections. Necrotic areas are surrounded by star marks in the low-power HE photo and the rectangle (light blue) corresponds to the area of the high-power view.</p

ARG2-expressing CAFs potentially affect the immune reaction.

<p>(A) T cell proliferation assay. CFSE-labeled CD3⁺ T cells were stimulated with anti-CD3/CD28 antibody-conjugated beads in fresh medium supplemented with the indicating L-arginine (control) or in conditioned medium after culture of CAFs under normoxic conditions (normoxia) or hypoxic conditions (hypoxia) in the medium with the indicating L-arginine (including 2.0 µM arginine of serum contents) for four days, and their proliferation profiles were analyzed by flow cytometry. Non-proliferated cells showed the highest fluorescence intensity and the intensity of the labeled T cells was halved with every cell division. The numbers represent the ratio of numbers of cells having undergone multiple divisions relative to the original number of cells. Data represent one of six independent experiments. (B) Percentages of cells having undergone cell divisions. Data represent one of six independent experiments. (C, D) Comparison of absolute number of tumor-infiltrating CD3⁺ T cells (C) with their proliferating index (the proportion of Ki-67-positive proliferating cells among the CD3⁺ T cells) (D) between the area around ARG2-expressing CAFs (white) and the area within the tumor except for necrotic tissue (speckled). We counted tumor-infiltrating CD3⁺ T cells and their Ki-67 positivity in each twenty different high-power fields per area categories using two PDC cases. Each data column represents the mean± SE for triplicate determinations. Significance value (Student’s <i>t</i> test) of <i>P</i><0.05 (*) and <i>P</i><0.001 (**).</p