ei24, a p53 response gene involved in growth suppression and apoptosis

DNA damage and/or hyperproliferative signals activate the wild-type p53 tumor suppressor protein, which induces a G(1) cell cycle arrest or apoptosis. Although the mechanism of p53-mediated cell cycle arrest is fairly well defined, the p53-dependent pathway regulating apoptosis is poorly understood. Here we report the functional characterization of murine ei24 (also known as PIG8), a gene directly regulated by p53, whose overexpression negatively controls cell growth and induces apoptotic cell death. Ectopic ei24 expression markedly inhibits cell colony formation, induces the morphological features of apoptosis, and reduces the number of beta-galactosidase-marked cells, which is efficiently blocked by coexpression of Bcl-X(L). The ei24/PIG8 gene is localized on human chromosome 11q23, a region frequently altered in human cancers. These results suggest that ei24 may play an important role in negative cell growth control by functioning as an apoptotic effector of p53 tumor suppressor activities.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.     

The tumor suppressor RASSF1A in human carcinogenesis: an update

Loss of heterozygosity of the small arm of chromosome 3 is one of the most common alterations in human cancer. Most notably, a segment in 3p21.3 is frequently lost in lung cancer and several other carcinomas. We and others have identified a novel Ras effector at this segment, which was termed Ras Association Domain family 1 (RASSF1A) gene. RASSF1 consists of two main variants (RASSF1A and RASSF1C), which are transcribed from distinct CpG island promoters. Aberrant methylation of the RASSF1A promoter region is one of the most frequent epigenetic inactivation events detected in human cancer and leads to silencing of RASSF1A. Hypermethylation of RASSF1A was commonly observed in primary tumors including lung, breast, pancreas, kidney, liver, cervix, nasopharyngeal, prostate, thyroid and other cancers. Moreover, RASSF1A methylation was frequently detected in body fluids including blood, urine, nipple aspirates, sputum and bronchial alveolar lavages. Inactivation of RASSF1A was associated with an advanced tumor stage (e.g. bladder, brain, prostate, gastric tumors) and poor prognosis (e.g. lung, sarcoma and breast cancer). Detection of aberrant RASSF1A methylation may serve as a diagnostic and prognostic marker. The functional analyses of RASSF1A reveal an involvement in apoptotic signaling, microtubule stabilization and mitotic progression. The tumor suppressor RASSF1A may act as a negative Ras effector inhibiting cell growth and inducing cell death. Thus, RASSF1A may represent an epigenetically inactivated bona fide tumor suppressor in human carcinogenesis.     

Molecular basis of spontaneous regression of neuroblastoma: role of neurotrophic signals and genetic abnormalities

Neuroblastoma is one of the most common pediatric solid tumors originating from the sympathoadrenal lineage of the neural crest. The tumors found in infants very frequently regress spontaneously by differentiating and/or undergoing programmed cell death, while those in children over one year of age are very aggressive and eventually kill the patients. The recent advances in neuroblastoma research have revealed that the neurotrophin signals, especially those through nerve growth factor and its receptor, TrkA, play an important role in regulating the regression of neuroblastoma. The genetic abnormalities such as allelic loss of the distal region of chromosome 1p, gain of chromosome 17q21-q25, and MYCN amplification, all of which are associated with the tumor aggressiveness, inhibit the regression of neuroblastoma. In the regressing tumor cells, caspases are induced and surviving, which is mapped to 17q25 and is overexpressed in the advanced stage tumors, is down-regulated. Thus, the molecular mechanism of spontaneous regression of neuroblastoma is now being unveiled. However, many more questions including the developmental machinery to trigger regression still remain to be clarified.     

Refined physical mapping and genomic structure of the EXTL1 gene

Recently, the EXTL1 gene, a member of the EXT tumor suppressor gene family, has been mapped to 1p36, a chromosome region which is frequently implicated in a wide variety of malignancies, including breast carcinoma, colorectal cancer and neuroblastoma. In this study, we show that the EXTL1 gene is located between the genetic markers D1S511 and D1S234 within 200 kb of the LAP18 gene on chromosome 1p36. 1, a region which has been proposed to harbor a tumor suppressor gene implicated in MYCN-amplified neuroblastomas. In addition, we determined the genomic structure of the EXTL1 gene, revealing that the EXTL1 coding sequence spans 11 exons within a 50-kb region.     

The putative tumor suppressor gene GLTSCR2 induces PTEN-modulated cell death

Glioma tumor suppressor candidate region gene 2 (GLTSCR2/PICT-1) is localized within the well-known 1.4-Mb tumor suppressive region of chromosome 19q, which is frequently altered in various human tumors, including diffuse gliomas. Aside from its localization on the chromosome, several lines of evidence, such as PTEN phosphorylation, support that GLTSCR2 partakes in the suppression of tumor growth and development. However, much remains unknown about the molecular mechanisms of the tumor suppressive activity of GLTSCR2. The purpose of this study was to investigate the molecular mechanisms of GLTSCR2 in cell death pathways in association with its binding partner PTEN. In this work, we show that GLTSCR2 is a nucleus-localized protein with a discrete globular expression pattern. In addition to phosphorylating PTEN, GLTSCR2 induces caspase-independent PTEN-modulated apoptotic cell death when overexpressed. However, the cytotoxic activity of GLTSCR2 is independent of its ability to phosphorylate PTEN, suggesting that the GLTSCR2-induced cell death pathway is divergent from PTEN-induced death pathways. Our results suggest that the induction of PTEN-modulated apoptosis is one of the putative mechanisms of tumor suppressive activity by GLTSCR2.     

KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD

Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bbeta and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bbeta and KIF1A. We also show that DENN/MADD binds preferentially to GTP-Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bbeta and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP-Rab3 was more effectively transported than GDP-Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.     

Loss of heterozygosity and K-ras gene mutations in gastric cancer

In order to identify relevant genetic lesions in gastric carcinoma, we searched for tumor suppressor gene inactivation and K-ras gene mutations by analyzing tumor and control DNAs from 34 patients. These were from an epidemiologically defined area of Italy characterized by one of the world's highest incidences of stomach cancer. Allele losses were investigated by the Southern blotting procedure at 16 polymorphic loci on 11 different chromosomes. Our data demonstrate that chromosomal regions 5q, 11p, 17p and 18q are frequently deleted, and that 7q and 13q chromosome arms are also involved, although at a lower frequency. Loss of heterozygosity (LOH) at region 11p was not found during other surveys carried out on patients of different geographic origins. No specific combination of allelic losses could be recognized in the samples analyzed, the only exception being that tumors with 17p allelic loss also showed LOH on the 18q region. When matching frequent LOH events and the stage of progression of the tumors, we observed a trend of association between advanced stages and allelic losses on 17p and 18q chromosome arms. The analysis of K-ras, carried out by the polymerase chain reaction and denaturing gradient gel electrophoresis, demonstrated transforming mutations in only 3 out of 32 cases. Colorectal tumorigenesis proceeds by the accumulation of genetic alterations, including K-ras mutations and inactivation of tumor suppressor genes on the 5q, 17p and 18q regions. Our data indicate that, although gastric and colorectal neoplasias share common genetic alterations, they probably progress through different pathways.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.     

贲门癌中染色体8p21-p23杂合性丢失的研究

目的 研究贲门癌中染色体8p21-p23杂合性丢失的情况。方法 采用激光捕获显微切割技术获得均质的肿瘤细胞及正常的胃粘膜细胞,多重置换扩增技术扩增捕获细胞的基因组DNA。PCR结合硝酸银染色方法分析19例贲门癌染色体8p21-p23的杂合性丢失。结果 在贲门癌中染色体8p21-p23的缺失频率非常高（63.2%）,我们确定了一个最小丢失区域. 结论 进一步明确此最小丢失区域内的抑癌基因将有助于贲门癌发生机制的阐明。     

Role of p73 in malignancy: tumor suppressor or oncogene?

The recently identified p53 family member, p73, shows substantial structural and functional homology with p53. However, despite the established role of p53 as a proto-type tumor suppressor, a similar function of p73 in malignancy is questionable. Overexpression of p73 can activate typical p53-responsive genes, and activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human tumors, led to the hypothesis that p73 has tumor suppressor activity just like p53. However, unlike p53-/- mice, p73 knockout mice do not develop tumors. Extensive studies on primary tumor tissues have revealed overexpression of wild-type p73 in the absence of p73 mutations instead, suggesting that p73 may augment, rather than inhibit tumor development. In contrast to p53, differential splicing of the TP73 gene locus gives rise to a complex pattern of interacting p73 isoforms with antagonistic functions. In fact, induction of apoptosis by increased levels of p73 can be blocked by both p53 mutants and the N-terminally truncated p73 isoforms, which were recently shown to possess oncogenic potential. In the light of these new findings the contradictory role of p73 in malignancy will be discussed.     

Genomic Organization and Mutation Analysis ofp73in Oligodendrogliomas with Chromosome 1 p-Arm Deletions

p73, a protein having substantial structural and functional similarity to p53, has recently been identified and demonstrated to be a potential tumor suppressor. Its location on human chromosome 1p36.33 implicates p73 as a candidate for neuroblastoma. Like neuroblastoma, oligodendrogliomas also show a high frequency of deletions in chromosome 1p36.3. To determine whetherp73is a potential tumor suppressor gene involved in the development of oligodendrogliomas, we performed mutation analysis ofp73in oligodendrogliomas with chromosome 1 p-arm deletions. We first determined the genomic organization and the intron–exon boundary sequences of thep73gene by long PCR, vectorette PCR, and Southern hybridization. This gene spans about 65 kb with a large first intron. Primer pairs for the amplification of each of the 13 p73 encoding exons were designed in corresponding introns. The amplicons were then analyzed using the denaturing high-performance liquid chromatography system for mutations in thep73gene. Twenty oligodendroglioma samples with 1p36.3 deletions were screened, but no mutations were detected except for several polymorphisms. It is thus clear thatp73is not a candidate gene for oligodendroglioma despite its location in the frequently deleted 1p36.3 region.