Lianghao Ding

Hypoxia-inducible mir-210 regulates normoxic gene expression involved in tumor initiation.

Previous studies have suggested that the HIF transcription factors can both activate and inhibit gene expression. Here we show that HIF1 regulates the expression of mir-210 in a variety of tumor types through a hypoxia-responsive element. Expression analysis in primary head and neck tumor samples indicates that mir-210 may serve as an in vivo marker for tumor hypoxia. By Argonaute protein immunoprecipitation, we identified 50 potential mir-210 targets and validated randomly selected ones. The majority of these 50 genes are not classical hypoxia-inducible genes, suggesting mir-210 represses genes expressed under normoxia that are no longer necessary to adapt and survive in a hypoxic environment. When human head and neck or pancreatic tumor cells ectopically expressing mir-210 were implanted into immunodeficient mice, mir-210 repressed initiation of tumor growth. Taken together, these data implicate an important role for mir-210 in regulating the hypoxic response of tumor cells and tumor growth.

Gene Expression Profiles of Normal Human Fibroblasts after Exposure to Ionizing Radiation: A Comparative Study of Low and High Doses

Abstract Ding, L-H., Shingyoji, M., Chen, F., Hwang, J-J., Burma, S., Lee, C., Cheng, J-F. and Chen, D. J. Gene Expression Profiles of Normal Human Fibroblasts after Exposure to Ionizing Radiation: A Comparative Study of Low and High Doses. Radiat. Res. 164, 17–26 (2005). Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.

Effective Gene Therapy for Pancreatic Cancer by Cytokines Mediated by Restricted Replication-Competent Adenovirus

Pancreatic cancer has a poor prognosis even when surgical treatment can be accomplished. Studies have demonstrated that pancreatic cancer is associated with various genetic abnormalities in oncogenes and tumor suppressor genes including p53. New therapeutic approaches for pancreatic cancer can be developed by targeting these genetic alterations. Adenovirus (Adv) lacking the 55-kDa E1B protein (E1B55K) replicates preferentially in p53-deficient cancer cells. We constructed E1B55K-deleted Adv (AxE1AdB), and studied its replication and cytopathic effect on pancreatic cancer cells. AxE1AdB replicated in and caused cell death of the p53-deficient pancreatic cancer cell lines tested (e.g., PANC-1, MIAPaCa-2, SU.86.86, BxPC-3, and PK-1). To enhance its therapeutic effect, we examined the combination of coinfecting this restricted replication-competent adenovirus (RRCA) with other Adv. Coinfection of E1-deficient Adv expressing the reporter lacZ gene (AxCAlacZ) together with AxE1AdB resulted in the replication of both viruses and a marked increase in reporter gene expression. PANC-1 cells coinfected with AxE1AdB and the Adv for human IL-2 (AxCAhIL2), produced 110 times more IL-2 than those infected with AxCAhIL2 alone. Similarly, coinfection of AxE1AdB and Adv for human IL-12 augmented the IL-12 production by 370-fold. Injecting AxE1AdB into the PANC-1 tumor of severe combined immunodeficient mice (SCID mice) resulted in marked reduction of the volume of the tumor. Moreover, injecting AxE1AdB with AxCAhIL2 into the PANC-1 tumor resulted in complete regression of the established tumors. These data suggest that RRCA, which augments the antitumor effect of a viral transgene (i.e., cytokines), may be a powerful tool for treating p53-deficient pancreatic cancer.

Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology

Despite the tremendous growth of microarray usage in scientific studies, there is a lack of standards for background correction methodologies, especially in single-color microarray platforms. Traditional background subtraction methods often generate negative signals and thus cause large amounts of data loss. Hence, some researchers prefer to avoid background corrections, which typically result in the underestimation of differential expression. Here, by utilizing nonspecific negative control features integrated into Illumina whole genome expression arrays, we have developed a method of model-based background correction for BeadArrays (MBCB). We compared the MBCB with a method adapted from the Affymetrix robust multi-array analysis algorithm and with no background subtraction, using a mouse acute myeloid leukemia (AML) dataset. We demonstrated that differential expression ratios obtained by using the MBCB had the best correlation with quantitative RT–PCR. MBCB also achieved better sensitivity in detecting differentially expressed genes with biological significance. For example, we demonstrated that the differential regulation of Tnfr2, Ikk and NF-kappaB, the death receptor pathway, in the AML samples, could only be detected by using data after MBCB implementation. We conclude that MBCB is a robust background correction method that will lead to more precise determination of gene expression and better biological interpretation of Illumina BeadArray data.

Pancreatic microcirculation in acute pancreatitis

We present a review of the microvascular morphology of the pancreas and microstructure of the pancreatic lobule, and report our experimental results of the investigation of pancreatic microcirculation following acute pancreatitis. Impairment of pancreatic microcirculation in the early phase of acute pancreatitis may play a key role in the progression of this disease. Possible contributory mechanisms include increased vascular permeability, reduced blood flow, leukocyte-endothelial cell interaction and intravascular thrombus formation. Using an in-vivo microscope system and off-line computer analysis, we achieved direct visualization and quantification of changes in microvascular permeability and leukocyte behavior in pancreas with acute pancreatitis. Bradykinin and oxygen radicals have been demonstrated to be involved in the increase of vascular permeability in the early stage of caerulein pancreatitis. Leukocyte adherence to the vessels in the pancreatic microcirculation is a secondary event following permeability changes in acute pancreatitis. Leukocyte infiltration during exacerbation of acute pancreatitis is mediated by leukocyte-endothelial cell interaction via leukocyte integrin CD11b/18.

Genome wide expression analysis of the CCR4-NOT complex indicates that it consists of three modules with the NOT module controlling SAGA-responsive genes.

Of the nine known members of the CCR4-NOT complex, CCR4/CAF1 are most important in mRNA deadenylation whereas the NOT1-5 proteins are most critical for transcriptional repression. Whole genome microarray analysis using deletions in seven of the CCR4-NOT genes was used to determine the overall mRNA expression patterns that are affected by members of the yeast CCR4-NOT complex. Under glucose conditions, ccr4 and caf1 displayed a high degree of similarity in the manner that they affected gene expression. In contrast, the not deletions were similar in the way they affected genes, but showed no correlation with that of ccr4/caf1. A number of groups of functionally related proteins were specifically controlled by the CCR4/CAF1 or NOT modules. Importantly, the NOT proteins preferentially affected SAGA-controlled gene expression. Also, both the CCR4/CAF1 and NOT group of proteins shared much greater similarities in their effects on gene expression during the stress of glucose deprivation. BTT1, a member of the nascent polypeptide association complex that binds the ribosome, was shown to be a tenth member of the CCR4-NOT complex, interacting through CAF130. Microarray analysis indicated that BTT1 and CAF130 correlate very highly in their control of gene expression and preferentially repress genes involved in ribosome biogenesis. These results indicate that distinct portions of the CCR4-NOT complex control a number of different cellular processes.

Gene expression changes in normal human skin fibroblasts induced by HZE-particle radiation

Abstract Ding, L-H., Shingyoji, M., Chen, F., Chatterjee, A., Kasai, K-E. and Chen, D. J. Gene Expression Changes in Normal Human Skin Fibroblasts Induced by HZE-Particle Radiation. Radiat. Res. 164, 523–526 (2005). Studies have shown that radiation exposure affects global gene expression in mammalian cells. However, little is known about the effects of HZE particles on gene expression. To study these effects, human skin fibroblasts were irradiated with HZE particles of different energies and LETs. The data obtained from these experiments indicate that changes in gene expression are dependent on the energy of the radiation source. Particles with the highest energy, i.e. iron, induced the biggest expression changes in terms of numbers of genes and magnitudes of changes. Many genes were found to undergo significant expression changes after HZE-particle irradiation, including CDKN1A/p21, MDM2, TNFRSF6/fas, PCNA and RAD52. Unlike X rays, HZE particles expose cells to two types of radiation: primary ions and δ rays. We hypothesized that the biological effects of δ rays, which are secondary electron emissions, should resemble the effects of X rays. To explore this idea, gene expression changes between cells that had been irradiated with HZE particles and X rays were compared. The results support our hypothesis since the number of genes that commonly changed after exposure to both radiations increased as a function of particle energy.

In vivo evaluation of the early events associated with liver metastasis of circulating cancer cells

The mechanism of metastasis formation remains still largely unknown. Many studies underline the importance and complexity of the initial arrest of the circulating tumour cells in the target organ, a key stage in metastasis occurrence. In our study, we evaluated by visual means the metastasis formation using an in vivo microscopy system in a murine model. Moreover, we investigated the involvement of P-selectin in these processes using immunohistochemistry and P-selectin knockout mice. The present study offers direct evidence of distinct pathways for tumour metastasis formation by a lymphoma cell – EL-4 and a solid tumour cell – C26. Off-line analysis of the images and histological data confirmed that mechanical entrapment of the solid tumour cell, which had a bigger diameter than that of the liver sinusoids, promoted metastasis without any detectable involvement of adhesion molecules. On the other hand, we observed that lymphoma cells, in spite of their smaller diameter as compared to the sinusoids, promoted liver metastasis as well, but with the essential participation in their arrest of P-selectin, indicating an adhesion molecule-mediated pathway.

Distinct transcriptome profiles identified in normal human bronchial epithelial cells after exposure to γ-rays and different elemental particles of high Z and energy

BackgroundIonizing radiation composed of accelerated ions of high atomic number (Z) and energy (HZE) deposits energy and creates damage in cells in a discrete manner as compared to the random deposition of energy and damage seen with low energy radiations such as γ- or x-rays. Such radiations can be highly effective at cell killing, transformation, and oncogenesis, all of which are concerns for the manned space program and for the burgeoning field of HZE particle radiotherapy for cancer. Furthermore, there are differences in the extent to which cells or tissues respond to such exposures that may be unrelated to absorbed dose. Therefore, we asked whether the energy deposition patterns produced by different radiation types would cause different molecular responses. We performed transcriptome profiling using human bronchial epithelial cells (HBECs) after exposure to γ-rays and to two different HZE particles (28Si and 56Fe) with different energy transfer properties to characterize the molecular response to HZE particles and γ-rays as a function of dose, energy deposition pattern, and time post-irradiation.ResultsClonogenic assay indicated that the relative biological effectiveness (RBE) for 56Fe was 3.91 and for 28Si was 1.38 at 34% cell survival. Unsupervised clustering analysis of gene expression segregated samples according to the radiation species followed by the time after irradiation, whereas dose was not a significant parameter for segregation of radiation response. While a subset of genes associated with p53-signaling, such as CDKN1A, TRIM22 and BTG2 showed very similar responses to all radiation qualities, distinct expression changes were associated with the different radiation species. Gene enrichment analysis categorized the differentially expressed genes into functional groups related to cell death and cell cycle regulation for all radiation types, while gene pathway analysis revealed that the pro-inflammatory Acute Phase Response Signaling was specifically induced after HZE particle irradiation. A 73 gene signature capable of predicting with 96% accuracy the radiation species to which cells were exposed, was developed.ConclusionsThese data suggest that the molecular response to the radiation species used here is a function of the energy deposition characteristics of the radiation species. This novel molecular response to HZE particles may have implications for radiotherapy including particle selection for therapy and risk for second cancers, risk for cancers from diagnostic radiation exposures, as well as NASA’s efforts to develop more accurate lung cancer risk estimates for astronaut safety. Lastly, irrespective of the source of radiation, the gene expression changes observed set the stage for functional studies of initiation or progression of radiation-induced lung carcinogenesis.

Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis.