Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system

Hypoxia enhances the expression of autocrine motility factor and the motility of human pancreatic cancer cells

The incidence of distant metastases is higher in the tumours with low oxygen pressure than in those with high oxygen pressure. It is well known that hypoxia induces the transcription of various genes involved in angiogenesis and anaerobic metabolism necessary for the growth of tumour cells in vivo, suggesting that hypoxia may also induce the transcription of metastasis-associated genes. We sought to identify the metastasis-associated genes differentially expressed in tumour cells under hypoxic conditions with the use of a DNA microarray system. We found that hypoxia enhanced the expression of autocrine motility factor mRNA in various cancer cells and also enhanced the random motility of pancreatic cancer cells. Autocrine motility factor inhibitors abrogated the increase of motility under hypoxic conditions. In order to explore the roles of hypoxia-inducible factor-1α, we established hypoxia-inducible factor-1α-transfectants and dominant negative hypoxia-inducible factor-1α-transfectants. Transfection with hypoxia-inducible factor-1α and dominant-negative hypoxia-inducible factor-1α enhanced and suppressed the expression of autocrine motility factor/phosphohexase isomerase/neuroleukin mRNA and the random motility, respectively. These results suggest that hypoxia may promote the metastatic potential of cancer cells through the enhanced autocrine motility factor/phosphohexase isomerase/neuroleukin mRNA expression and that the disruption of the hypoxia-inducible factor-1 pathway may be an effective treatment for metastasis

Control of Alzheimer's Amyloid Beta Toxicity by the High Molecular Weight Immunophilin FKBP52 and Copper Homeostasis in Drosophila

FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Aβ toxicity. Towards this goal, we generated Aβ transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Aβ and increased lifespan in Aβ flies, whereas loss of function of FKBP52 exacerbated these Aβ phenotypes. Interestingly, the Aβ pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (−/−) cells have increased intracellular copper and higher levels of Aβ. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Aβ peptides

Dexamethasone potentiates the antiangiogenic activity of docetaxel in castration-resistant prostate cancer

We sought to characterise whether dexamethasone (DEX) may enhance tumour response to docetaxel in in vitro and in vivo models of metastatic prostate cancer (CaP). In vitro experiments conducted on PC3 and human bone marrow endothelial cells (hBMECs) determined that administration of DEX (10 nM) reduced constitutive nuclear factor-κB (NF-κB) activity, decreasing interleukin (IL)-8, CXCL1 and VEGF gene expression in PC3 cells. Dexamethasone also attenuated docetaxel-induced NF-κB and activator protein-1 transcription and reduced docetaxel-promoted expression/secretion of IL-8 and CXCL1 in PC3 and hBMECs. Although DEX failed to enhance docetaxel cytotoxicity on PC3 cells, DEX potentiated the antiangiogenic activity of docetaxel in vitro, further reducing vessel area and vessel length in developing endothelial tubes (P<0.05). Docetaxel had a potent antiangiogenic activity in the dorsal skin flap-implanted PC3 tumours in vivo. Small blood vessel formation was further suppressed in tumours co-treated with docetaxel and DEX, substantiated by an increased average vessel diameter and segment length and a decreased number of branch points in the residual tumour vasculature (P<0.001). Our data show that DEX potentiates the antiangiogenic activity of docetaxel, suggesting a putative mechanism for the palliative and survival benefits of these agents in metastatic CaP

HIF-1α determines the metastatic potential of gastric cancer cells

Gastric adenocarcinoma is characterised by rapid emergence of systemic metastases, resulting in poor prognosis due to vanished curative treatment options. Better understanding of the molecular basis of gastric cancer spread is needed to design innovative treatments. The transcription factor HIF-1α (hypoxia-inducible factor 1α) is frequently overexpressed in human gastric cancer, and inhibition of HIF-1α has proven antitumour efficacy in rodent models, whereas the relevance of HIF-1α for the metastatic phenotype of gastric adenocarcinoma remains elusive. Therefore, we have conducted a comprehensive analysis of the role of HIF-1α for pivotal metastasis-associated processes of human gastric cancer. Immunhistochemistry for HIF-1α showed specific staining at the invading tumour edge in 90% of human gastric cancer samples, whereas normal gastric tissue was negative and only a minority of early gastric cancers (T1 tumours) showed specific staining. Hypoxia-inducible factor 1α-deficient cells showed a significant reduction of migratory, invasive and adhesive properties in vitro. Furthermore, the HIF-1α-inhibitor 2-methoxy-estradiol significantly reduced metastatic properties of gastric cancer cells. The accentuated expression at the invading edge together with the in vitro requirement of HIF-1α for migration, invasion and adherence argues for a pivotal role of HIF-1α in local invasion and, ultimately, systemic tumour spread. These results warrant the exploration of HIF-1α-inhibiting substances in clinical treatment studies of advanced gastric cancer

Clinicopathologic significance of HIF-1α, p53, and VEGF expression and preoperative serum VEGF level in gastric cancer

<p>Abstract</p> <p>Background</p> <p>Hypoxia influences tumor growth by inducing angiogenesis and genetic alterations. Hypoxia-inducible factor 1α (HIF-1α), p53, and vascular endothelial growth factor (VEGF) are all important factors in the mechanisms inherent to tumor progression. In this work, we have investigated the clinicopathologic significance of HIF-1α, p53, and VEGF expression and preoperative serum VEGF (sVEGF) level in gastric cancer.</p> <p>We immunohistochemically assessed the HIF-1α, p53, and VEGF expression patterns in 114 specimens of gastric cancer. Additionally, we determined the levels of preoperative serum VEGF (sVEGF).</p> <p>Results</p> <p>The positive rates of p53 and HIF-1α (diffuse, deep, intravascular pattern) were 38.6% and 15.8%, respectively. The VEGF overexpression rate was 57.9%. p53 and HIF-1α were correlated positively with the depth of invasion (<it>P </it>= 0.015, <it>P </it>= 0.001, respectively). Preoperative sVEGF and p53 levels were correlated significantly with lymph node involvement (<it>P </it>= 0.010, <it>P </it>= 0.040, respectively). VEGF overexpression was more frequently observed in the old age group (≥ 60 years old) and the intestinal type (<it>P </it>= 0.013, <it>P </it>= 0.014, respectively). However, correlations between preoperative sVEGF level and tissue HIF-1α, VEGF, and p53 were not observed. The median follow-up duration after operation was 24.5 months. HIF-1α was observed to be a poor prognostic factor of disease recurrence or progression (<it>P </it>= 0.002).</p> <p>Conclusion</p> <p>p53, HIF-1α and preoperative sVEGF might be markers of depth of invasion or lymph node involvement. HIF-1α expression was a poor prognostic factor of disease recurrence or progression in patients with gastric cancers.</p

Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells

<p>Abstract</p> <p>Background</p> <p>Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions.</p> <p>Methods</p> <p>Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1α) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1α), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1α. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax.</p> <p>Results</p> <p>Overexpression of pcDNA3-DN-Hif-1α led to a significant reduction in hypoxia -induced apoptosis (17 ± 2%, <it>P </it>< 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1α transfected cells. Moreover, selective ablation of HIF-1α protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1α exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1α led to a two-fold increase in Hif-1α levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1α also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1α constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression.</p> <p>Conclusion</p> <p>These data demonstrate that HIF-1α is an important component of the apoptotic signaling machinery in the two cell types.</p

Dietary glycemic load and gastric cancer risk in Italy

We investigated gastric cancer risk in relation to dietary glycemic index (GI) and glycemic load (GL), which represent indirect measures of carbohydrate absorption and consequently of dietary insulin demand, in a case-control study conducted in northern Italy between 1997 and 2007, including 230 patients with the incident, histologically confirmed gastric cancer and 547 frequency matched controls, admitted to the same hospitals as cases with acute non-neoplastic conditions. We used conditional logistic regression models, including terms for major recognised gastric cancer risk factors and non-carbohydrate energy intake. The odds ratios (ORs) in the highest vs lowest quintile were 1.9 (95% CI: 1.0–3.3) for GI and 2.5 (95% CI: 1.3–4.9) for GL. Compared with participants reporting low GL and high fruits/vegetables intake, the OR rose across strata of high GL and low fruits/vegetables, to reach 5.0 (95% CI: 2.2–11.5) for those reporting low fruits/vegetables intake and high GL. Our study may help to explain the direct relation observed in several studies between starchy foods and gastric cancer risk

International Conference on Advances in Radiation Oncology (ICARO): Outcomes of an IAEA Meeting

The IAEA held the International Conference on Advances in Radiation Oncology (ICARO) in Vienna on 27-29 April 2009. The Conference dealt with the issues and requirements posed by the transition from conventional radiotherapy to advanced modern technologies, including staffing, training, treatment planning and delivery, quality assurance (QA) and the optimal use of available resources. The current role of advanced technologies (defined as 3-dimensional and/or image guided treatment with photons or particles) in current clinical practice and future scenarios were discussed