Winter Temperature and UV Are Tightly Linked to Genetic Changes in the p53 Tumor Suppressor Pathway in Eastern Asia

The tumor suppressor p53 is a master sensor of stress. Two human-specific polymorphisms, p53 codon 72 and MDM2 SNP309, influence the activities of p53. There is a tight association between cold winter temperature and p53 Arg72 and between low UV intensity

U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia

Upregulated gene 19 (U19)/ELL-associated factor 2 (Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepato cellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 defficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

Background: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. Methods: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. Results: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. Conclusions: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes.

Comparative Expression of Zebrafish Lats1 and Lats2 and Their Implication in Gastrulation Movements

Large tumor suppressor (Lats) is a Ser/Thr kinase, and it presents an important function in tumor suppression. lats was originally identified in Drosophila and recently in mammals. In mammals, it contains two homologues, lats1 and lats2. In the present study, lats1 and lats2 were characterized from zebrafish (Danio rerio), which is the first report of lats in a nonmammalian vertebrate. The primary structure, genomic organization, and phylogenesis of lats from different species were studied, and the results suggest that lats1 is the direct descendant of invertebrate lats, whereas lats2 is formed by genome duplication. In zebrafish, both lats genes are maternally expressed, while they show distinctly different expression profiles during gastrulation. lats1 is almost ubiquitously expressed through development, and lats2 is more prominently expressed in the non-neural ectoderm region of zebrafish gastrula. Most intriguingly, as revealed by cell tracing and gene expression analysis, morpholino-mediated knockdown of either lats1 or lats2 led to obvious defects of cell migration in gastrulation, indicating the functional significance of lats in gastrulation movements. Developmental Dynamics 238:28502859, 2009. (C) 2009 Wiley-Liss, Inc.

U19/Eaf2 Binds to and Stabilizes von Hippel-Lindau Protein

Studies have firmly established a key regulatory role for the tumor suppressor pVHL in the regulation of the vascular system and normal spermatogenesis. Here, we report that knockout of the newly identified tumor suppressor U19/Eaf2 also caused vascular system abnormalities and aspermatogenesis, suggesting a potential link between U19/Eaf2 and pVHL. Coimmunoprecipitation and in vitro binding assays showed an association between U19/Eaf2 and pVHL, whereas deletion mutagenesis revealed the requirement of the NH2 terminus of U19/Eaf2 and both the alpha and beta domains of pVHL for this binding. U19/Eaf2 stabilizes pVHL, as shown by protein stability and pulse-chase studies. Testes and mouse embryonic fibroblasts (MEF) derived from U19/Eaf2 knockout mice expressed reduced levels of pVHL, indicating that full in vivo expression of pVHL indeed requires U19/Eaf2. As expected, U19/Eaf2 knockout MEF cells exhibited an increased level and activity of hypoxia-inducible factor 1 alpha (HIF1 alpha), a protein typically regulated via a pVHL-mediated degradation pathway. Furthermore, angiogenesis in a Matrigel plug assay was significantly increased in U19/Eaf2 knockout mice. The above observations argue that U19/Eaf2 can modulate HIF1 alpha and angiogenesis, possibly via direct binding and stabilization of pVHL. [Cancer Res 2009;69(6):2599-606]

FB1, an E2A fusion partner in childhood leukemia, interacts with U19/EAF2 and inhibits its transcriptional activity

Background: U19/EAF2 is a potential tumor suppressor exhibiting frequent down-regulation and allelic loss in advanced human prostate cancer specimens. U 19/EAF2 has also been identified as ELL-associated factor 2 (EAF2) based on its binding to ELL, a fusion partner of MLL in acute myeloid leukemia. U19/EAF2 is a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Methods: Yeast-two-hybrid-screening was used to identify U19/EAF2-binding partners. Co-immunoprecipitation and mammalian 1-hybrid assay were used to characterize a U19/EAF2-binding partner. Results: FB1, an E2A fusion partner in childhood leukemia, was identified as a binding-partner of U19/EAF2. FB1 also binds to EAF1, the only homologue of U19/EAF2. FB1 also interacts and co-localizes with ELL in the nucleus. Interestingly, FB1 inhibited the transcriptional activity of U19/EAF2 but not EAF1. Conclusions: FB1 is an important binding partner and a functional regulator of U19/EAF2, EAF1, and/or ELL. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activatep21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.

A Proteomic Study on Cell Cycle Progression of Endothelium Exposed to Tumor Conditioned Medium and the Possible Role of Cyclin D-1/E

This study was designed to comprehensively analyze the differential expression of proteins from human umbilical vein endothelial cells (HUVECs) exposed to tumor conditioned medium (TCM) and to identify the key regulator in the cell cycle progression. The HUVECs were exposed to TCM from breast carcinoma cell line MDA-MB-231, then their cell cycle distribution was measured by flow cytometer (FCM). The role of protein in cell cycle progression was detected via two-dimensional polyacrylamide gel electrophoresis (2-DE) and western blotting. Following the stimulation of TCM, HUVECs showed a more cells in the S phase than did the negative control group (ECGF-free medium with 20% FBS), but the HUVECs' level was similar to the positive control group (medium with 25 mug/ml ECGF and 20% FBS). Increased expression of cyclin D-1/E and some changes in other related proteins occurred after incubation with TCM. From our results, we can conclude that breast carcinoma cell line MDA-MB-231 may secrete soluble pro-angiogenic factors that induce the HUVEC angiogenic switch, during which the expression of cell cycle regulator cyclin D-1/E increases and related proteins play an important role in this process.

Bioinformatic identification of genes encoding C1q-domain-containing proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

A novel tumor necrosis factor ligand superfamily member (CsTL) from Ciona savignyi: Molecular identification and expression analysis

A novel invertebrate TNF ligand was identified and characterized in Ciona savignyi. The CsTL cDNA consisted of 995 nucleotides and encoded 281 amino acids. A conserved TNF family signature and several motifs of TNF ligand superfamily were identified in deduced amino acid sequence of CsTL. Phylogenetic analysis grouped CsTL, CiTNF (predicted TNF ligand superfamily homolog in Ciona intestinalis) and urchin TL1A with their own cluster apart from mammalian TNF alpha, LTA, TNFSF15 and fish TNFa proteins. Expression studies demonstrated that CsTL mRNA is present in all tested tissues from unchallenged ascidians and its expression was significantly upregulated in hemocytes following LIPS injection. The recombinant CsTL protein expressed using a baculovirus expression system showed potential cytotoxic activity in L929 cells. Present results indicated that TNF ligand superfamity molecules are present in marine invertebrates. (C) 2008 Elsevier Ltd. All rights reserved.