Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca 2؉ from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

A candidate tumor suppressor gene, p33ING1, was previously identified by using the genetic suppressor element methodology. p33ING1 cooperates with p53 and plays a significant role in p53-mediated cellular processes. Recently, we have identified p33ING2, which shows a sequence homology similar to p33ING1 and modulates p53 function. In the present study, we identified and characterized another 'ING family' gene. The estimated molecular weight of the encoded protein is 46.8 kDa, thus, we named it p47ING3. The p47ING3 gene is located at chromosome 7q31.3 and consists of 12 exons that encode 418 amino acids. A computational domain search revealed a C-terminal PHD-finger motif. Such motifs are common in proteins involved in chromatin remodeling. p47ING3 is highly expressed in some normal human tissues or organs, including the spleen, testis, skeletal muscle, and heart. p47ING3 expression levels varied among cancer cell lines. p47ING3 overexpression resulted in a decreased population of cells in S phase, a diminished colony-forming efficiency, and induced apoptosis in RKO cells, but not in RKO-E6 cells with inactivated p53. p47ING3 activates p53-transactivated promoters, including promoters of p21/waf1 and bax. Thus, we have isolated a novel ING family gene, p47ING3, which modulates p53-mediated transcription, cell cycle control, and apoptosis.

Irinotecan-induced severe neutropenia is associated with homozygosity for the UGT1A1*28 or UGT1A1*6 alleles. In this study, we determined the maximum-tolerated dose (MTD) of irinotecan in patients with UGT1A1 polymorphisms. Patients who had received chemotherapy other than irinotecan for metastatic gastrointestinal cancer were enrolled. Patients were divided into three groups according to UGT1A1 genotypes: wild-type (*1 ⁄ *1); heterozygous (*28 ⁄ *1, *6 ⁄ *1); or homozygous (*28 ⁄ *28, *6 ⁄ *6, *28 ⁄ *6). Irinotecan was given every 2 weeks for two cycles. The wild-type group received a fixed dose of irinotecan (150 mg ⁄ m 2 ) to serve as a reference. The MTD was guided from 75 to 150 mg ⁄ m 2 by the continual reassessment method in the heterozygous and homozygous groups. Dose-limiting toxicity (DLT) and pharmacokinetics were evaluated during cycle 1. Of 82 patients enrolled, DLT was assessable in 79 patients (wild-type, 40; heterozygous, 20; and homozygous, 19). Dose-limiting toxicity occurred in one patient in the wild-type group, none in the heterozygous group, and six patients (grade 4 neutropenia) in the homozygous group. In the homozygous group, the MTD was 150 mg ⁄ m 2 and the probability of DLT was 37.4%. The second cycle was delayed because of neutropenia in 56.3% of the patients given the MTD. The AUC 0-24 h of SN-38 was significantly greater (P < 0.001) and more widely distributed in the homozygous group. Patients homozygous for the UGT1A1*28 or UGT1A1*6 allele can receive irinotecan in a starting dose of 150 mg ⁄ m 2 , but many required dose reductions or delayed treatment in subsequent cycles.

Background Japan Clinical Oncology Group (JCOG) 0212 (http://clinicaltrials.gov NCT00190541) was a non‐inferiority phase III trial of patients with clinical stage II–III rectal cancer without lateral pelvic lymph node enlargement. The trial compared mesorectal excision (ME) with ME and lateral lymph node dissection (LLND), with a primary endpoint of recurrence‐free survival (RFS). The planned primary analysis at 5 years failed to confirm the non‐inferiority of ME alone compared with ME and LLND. The present study aimed to compare ME alone and ME with LLND using long‐term follow‐up data from JCOG0212. Methods Patients with clinical stage II–III rectal cancer below the peritoneal reflection and no lateral pelvic lymph node enlargement were included in this study. After surgeons confirmed R0 resection by ME, patients were randomized to receive ME alone or ME with LLND. The primary endpoint was RFS. Results A total of 701 patients from 33 institutions were assigned to ME with LLND (351) or ME alone (350) between June 2003 and August 2010. The 7‐year RFS rate was 71.1 per cent for ME with LLND and 70·7 per cent for ME alone (hazard ratio (HR) 1·09, 95 per cent c.i. 0·84 to 1·42; non‐inferiority P = 0·064). Subgroup analysis showed improved RFS among patients with clinical stage III disease who underwent ME with LLND compared with ME alone (HR 1·49, 1·02 to 2·17). Conclusion Long‐term follow‐up data did not support the non‐inferiority of ME alone compared with ME and LLND. ME with LLND is recommended for patients with clinical stage III disease, whereas LLND could be omitted in those with clinical stage II tumours.

ING2 (inhibitor of growth family, member 2) is a member of the plant homeodomain (PHD)-containing ING family of putative tumor suppressors. As part of mSin3A-HDAC corepressor complexes, ING2 binds to tri-methylated lysine 4 of histone H3 (H3K4me3) to regulate chromatin modification and gene expression. ING2 also functionally interacts with the tumor suppressor protein p53 to regulate cellular senescence, apoptosis and DNA damage response in vitro, and is thus expected to modulate carcinogenesis and aging. Here we investigate the developmental and physiological functions of Ing2 through targeted germline disruption. Consistent with its abundant expression in mouse and human testes, male mice deficient for Ing2 showed abnormal spermatogenesis and were infertile. Numbers of mature sperm and sperm motility were significantly reduced in Ing2 −/− mice (∼2% of wild type, P<0.0001 and ∼10% of wild type, P<0.0001, respectively). Their testes showed degeneration of seminiferous tubules, meiotic arrest before pachytene stage with incomplete meiotic recombination, induction of p53, and enhanced apoptosis. This phenotype was only partially abrogated by concomitant loss of p53 in the germline. The arrested spermatocytes in Ing2 −/− testes were characterized by lack of specific HDAC1 accumulation and deregulated chromatin acetylation. The role of Ing2 in germ cell maturation may extend to human ING2 as well. Using publicly available gene expression datasets, low expression of ING2 was found in teratozoospermic sperm (>3-fold reduction) and in testes from patients with defective spermatogenesis (>7-fold reduction in Sertoli-cell only Syndrome). This study establishes ING2 as a novel regulator of spermatogenesis functioning through both p53- and chromatin-mediated mechanisms, suggests that an HDAC1/ING2/H3K4me3-regulated, stage-specific coordination of chromatin modifications is essential to normal spermatogenesis, and provides an animal model to study idiopathic and iatrogenic infertility in men. In addition, a bona fide tumor suppressive role of Ing2 is demonstrated by increased incidence of soft-tissue sarcomas in Ing2− /− mice.

Inhibitor of growth 2 (ING2) is associated with chromatin remodeling and regulation of gene expression by binding to a methylated histone H3K4 residue and recruiting HDAC complexes to the region. The aim of our study is to investigate the regulation of ING2 expression and the clinical significance of upregulated ING2 in colon cancer. Here, we show that the ING2 mRNA level in colon cancer tissue increased to more than twice than that in normal mucosa in the 45% of colorectal cancer cases that we examined. A putative NF-jB binding site was found in the ING2 promoter region. We confirmed that NF-jB could bind to the ING2 promoter by EMSA and luciferase assays. Subsequent microarray analyses revealed that ING2 upregulates expression of matrix metalloproteinase 13 (MMP13), which enhances cancer invasion and metastasis. Inhibitor of growth 2 (ING2), which is a member of ING gene family, was identified by searching EST databases for any similarity to the ING1 gene. A previous report has shown that the ING2 mRNA is expressed in testis at the highest level among normal tissues, and also emphasizes that ING2 mRNA expression is remarkably increased in colon cancer tissue compared to nonmalignant tissue.2 ING2 as well as ING1b are considered to be candidate tumor suppressor genes, which cooperate with p53 and share many biological functions, such as apoptosis, cell cycle regulation and senescence.3-7 Our recent report has shown that ING2 expression is suppressed by nutlin-3a-induced p53, resulting in the induction of senescence in normal human fibroblasts and a cancer cell line.8 ING family proteins functionally form complexes with several factors possessing intrinsic histone acetyltransferase (HAT) and histone deacetylase complex (HDAC) activities, thus, linking the function of ING genes to chromatin remodeling and transcriptional regulation through histone modifications.9-15 Among ING proteins, ING2 especially has a potential ability to bind to the trimethylated histone H3 at lysine 4 (H3K4me3). H3K4me3 correlates with transcriptional activation, while methylations occurred at many other histone residues are associated with transcriptional suppression. [16][17][18] Previous reports suggest that ING2 only induces gene expression and does not repress expression of any known genes in normal fibroblasts. 19ING2 also interacts with phosphatidylinositol 5-phosphate (PtdIns5P). 20,21 PtdIns5P, which may function in a nuclear signaling pathway, has been implicated as a critical regulator of nuclear signaling events during cell-cycle progression.22,23 Cellular stress, such as UV irradiation or oxidative stress, causes the accumulation of nuclear PtdIns5P, resulting in the activation of ING2. 24,25 Oxidative and nitrosative stress are linked with inflammation-associated cancer including colon carcinoma. 26 We report here evidence that pathophysiological expression of ING2 may enhance invasion and metastasis of colon cancer by increasing the expression of MMP13. Material and methods Clinical samples and RNA extractionFive colorectal...

We found aberrant DNA methylation of the WNT10B promoter region in 46% of primary hepatocellular carcinoma (HCC) and 15% of colon cancer samples. Three of 10 HCC and one of two colon cancer cell lines demonstrated low or no expression, and 5-aza-2'deoxycytidine reactivated WNT10B expression with the induction of demethylation, indicating that WNT10B is silenced by DNA methylation in some cancers, whereas WNT10B expression is up-regulated in seven of the 10 HCC cell lines and a colon cancer cell line. These results indicate that WNT10B can be deregulated by either overexpression or silencing in cancer. We found that WNT10B up-regulated beta-catenin/Tcf activity. However, WNT10B-overexpressing cells demonstrated a reduced growth rate and anchorage-independent growth that is independent of the beta-catenin/Tcf activation, because mutant beta-catenin-transduced cells did not suppress growth, and dominant-negative hTcf-4 failed to alleviate the growth suppression by WNT10B. Although WNT10B expression alone inhibits cell growth, it acts synergistically with the fibroblast growth factor (FGF) to stimulate cell growth. WNT10B is bifunctional, one function of which is involved in beta-catenin/Tcf activation, and the other function is related to the down-regulation of cell growth through a different mechanism. We suggest that FGF switches WNT10B from a negative to a positive cell growth regulator.