Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Expression of E-cadherin repressors SNAIL, ZEB1 and ZEB2 by tumour and stromal cells influences tumour-budding phenotype and suggests heterogeneity of stromal cells in pancreatic cancer.

BACKGROUND There is evidence that tumour-stroma interactions have a major role in the neoplastic progression of pancreatic ductal adenocarcinoma (PDAC). Tumour budding is thought to reflect the process of epithelial-mesenchymal transition (EMT); however, the relationship between tumour buds and EMT remains unclear. Here we characterize the tumour-budding- and stromal cells in PDAC at protein and mRNA levels concerning factors involved in EMT. METHODS mRNA in situ hybridisation and immunostaining for E-cadherin, β-catenin, SNAIL1, ZEB1, ZEB2, N-cadherin and TWIST1 were assessed in the main tumour, tumour buds and tumour stroma on multipunch tissue microarrays from 120 well-characterised PDACs and associated with the clinicopathological features, including peritumoural (PTB) and intratumoural (ITB) budding. RESULTS Tumour-budding cells showed increased levels of ZEB1 (P<0.0001) and ZEB2 (P=0.0119) and reduced E-cadherin and β-catenin (P<0.0001, each) compared with the main tumour. Loss of membranous β-catenin in the main tumour (P=0.0009) and tumour buds (P=0.0053), without nuclear translocation, as well as increased SNAIL1 in tumour and stromal cells (P=0.0002, each) correlated with high PTB. ZEB1 overexpression in the main tumour-budding and stromal cells was associated with high ITB (P=0.0084; 0.0250 and 0.0029, respectively) and high PTB (P=0.0005; 0.0392 and 0.0007, respectively). ZEB2 overexpression in stromal cells correlated with higher pT stage (P=0.03), lymphatic invasion (P=0.0172) and lymph node metastasis (P=0.0152). CONCLUSIONS In the tumour microenvironment of phenotypically aggressive PDAC, tumour-budding cells express EMT hallmarks at protein and mRNA levels underlining their EMT-type character and are surrounded by stromal cells expressing high levels of the E-cadherin repressors ZEB1, ZEB2 and SNAIL1, this being strongly associated with the tumour-budding phenotype. Moreover, our findings suggest the existence of subtypes of stromal cells in PDAC with phenotypical and functional heterogeneity.

Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Functional Characterization and Comparison of Intercellular Communication in Stem Cell-Derived Cardiomyocytes

One novel treatment strategy for the diseased heart focuses on the use of pluripotent stem cell-derived cardiomyocytes (SC-CMs) to overcome the heart's innate deficiency for self-repair. However, targeted application of SC-CMs requires in-depth characterization of their true cardiogenic potential in terms of excitability and intercellular coupling at cellular level and in multicellular preparations. In this study, we elucidated the electrical characteristics of single SC-CMs and intercellular coupling quality of cell pairs, and concomitantly compared them with well-characterized murine native neonatal and immortalized HL-1 cardiomyocytes. Firstly, we investigated the electrical properties and Ca2+ signaling mechanisms specific to cardiac contraction in single SC-CMs. Despite heterogeneity of the new cardiac cell population, their electrophysiological activity and Ca2+ handling were similar to native cells. Secondly, we investigated the capability of paired SC-CMs to form an adequate subunit of a functional syncytium and analyzed gap junctions and signal transmission by dye transfer in cell pairs. We discovered significantly diminished coupling in SC-CMs compared with native cells, which could not be enhanced by a coculture approach combining SC-CMs and primary CMs. Moreover, quantitative and structural analysis of gap junctions presented significantly reduced connexin expression levels compared with native CMs. Strong dependence of intercellular coupling on gap junction density was further confirmed by computational simulations. These novel findings demonstrate that despite the cardiogenic electrophysiological profile, SC-CMs present significant limitations in intercellular communication. Inadequate coupling may severely impair functional integration and signal transmission, which needs to be carefully considered for the prospective use of SC-CMs in cardiac repair.

A novel CYP17A1 deletion causes a functional knockout of the steroid enzyme 17-hydroxylase and 17,20-lyase in a Turkish family and illustrates the precise role of the CYP17A1 gene

A novel homozygous long-range deletion of the CYP17A1 gene abolished protein expression and caused the severest form of 17-hydroxylase deficiency in one kindred of a Turkish family. The affected subjects presented with 46,XY sex reversal and 46,XX lack of pubertal development as well as severe hypertension.

siRNA mediated knockdown of tissue factor expression in pigs for xenotransplantation.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

A TEMPERATURE-SENSITIVE DEFECT IN THE EXPRESSION OF A SARCOMA VIRUS TRANSFORMING GENE

The viral proteins synthesized by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in a function required for the maintenance of the transformed state (ts110) were examined. Normal rat kidney cells (NRK) were infected with the ts110 virus and a non-virus-producing cell clone, termed 6m2, was isolated. This cell clone had a malignant phenotype at 33(DEGREES), the permissive temperature, but changed to a normal phenotype at 39(DEGREES).^ Two viral proteins were detected in 6m2 cells. A 58,000 dalton protein (P58) was detected at both 33(DEGREES) and 39(DEGREES) and contained only core protein (gag) coded sequences. An 85,000 dalton protein (P85) was detected only at 33(DEGREES) and contained sequences of viral core proteins p15, pp12, and part of p30 as well as protein sequences attributed by peptide mapping to P23 and P38, two candidate viral mouse src (v-mos) gene products. These results provide good evidence that P85 is a gag-mos polyprotein. As expected for a functional mos-gene product, P85 synthesis preceded parameters characteristic of the transformed state, including changes in cell morphology, in the cytoplasmic microtubule complex (CMTC) and in the rate of hexose uptake.^ Other studies were conducted to ascertain the defect which prohibited the synthesis of P85 at 39(DEGREES), the non-permissive temperature. When 6m2 cells were treated with actinomycin D at 39(DEGREES) and shifted to 33(DEGREES), the cells were unable to synthesize P85, but P58 continued to be made. P85 mRNA, active at 33(DEGREES), continued to be translated for two to three hours after shifting to 39(DEGREES) as judged by pulse-labeling experiments. Virus harvested at 33(DEGREES) from ts110 MuSV producer cells packaged both P85 and P58 coding RNAs while virus harvested at 39(DEGREES) was deficient in the amount of P85 coding RNA. Agarose gel electrophoresis of 6m2 cellular RNA showed that RNA harvested at 33(DEGREES) contained the 4.0 and 3.5 kb RNAs. Similar experiments on cells maintained at 39(DEGREES) have detected only the 4.0 kb RNA, suggesting that the 3.5 kb RNA codes for P85. The defect appeared to be in the long term stability of the P85 coding RNA at 39(DEGREES), since, in shift-up experiments (33(DEGREES) (--->) 39(DEGREES)), P85 was translated for only three hours at 39(DEGREES), while P58 was synthesized for at least eight hours. However, at 33(DEGREES) in the presence of actinomycin D, the ratio of P85 and P58 synthesis at hourly intervals was similar throughout a 12 hour period. ^

INCREASED EXPRESSION OF ONE OF TWO ADENOSINE DEAMINASE ALLELES IN A HUMAN CHORIOCARCINOMA CELL LINE FOLLOWING SELECTION WITH ADENINE NUCLEOSIDES

The human choriocarcinoma cell line JEG-3 is heterozygous at the adenosine deaminase (ADA) gene locus. Both allelic genes are under strong but incomplete repression causing a very low level expression of the gene locus. Because cytotoxic adenosine analogues such as 9-(beta)-D arabinofuranosyladenine (ara-A) and 9-(beta)-D xylofuranosyladenine (xyl-A) can be specifically detoxified by the action of ADA, these analogues were used to select for JEG-3 derived cells which had increased ADA expression. When JEG-3 cells were subjected to a multi-step, successively increasing dosage of either ara-A or xyl-A, resistant cells with increased ADA expression were generated. This increased ADA expression in the resistant cells was unstable, so that when the selective pressure was removed, cellular ADA expression would decrease. Subclone analysis of xyl-A resistant cells revealed that compared to parental JEG-3 cells, individual resistant cells had either elevated ADA levels or decreased adenosine kinase (ADK) levels or both. This altered ADA and ADK expression in the resistant cells were found to be independent events. Because of high endogenous tissue conversion factor (TCF) expression in the JEG-3 cells, the allelic nature of the increased ADA expression in most of the resistant cells could not be determined. However, several resistant subcloned cells were found to have lost TCF expression. These TCF('-) cells expressed only the ADA*2 allelic gene product. Cell fusion experiments demonstrated that the ADA*1 allelic gene was intact and functional in the A3-1A7 cell line. Chromosomal analysis of the A3-1A7 cells showed that they had no double-minutes or homogeneously staining chromosomal regions, although a pair of new chromosomes were found in these cells. Segregation analysis of the hybrid cells indicated that an ADA*2 allelic gene was probably located on this new chromosome. The analysis of the A3-1A7 cell line suggested that the expression of only ADA 2 in these cells was the result of possibly a cis-deregulation of the ADA gene locus or more probably an amplification of the ADA*2 allelic gene. Two effective positive selection systems for ADA('+) cells were also developed and tested. These selection systems should eventually lead to the isolation of the ADA gene.^

GLUCAGON GENE EXPRESSION: ANALYSIS OF PREPROGLUCAGON

Glucagon is a 29 amino acid polypeptide hormone produced in the (alpha) cells of the pancreatic islets. The purpose of this research was to understand better the role of glucagon in the regulation of metabolic processes. As with other polypeptide hormones, the synthesis of glucagon is thought to involve a larger precursor, which is then enzymatically cleaved to the functional form. The specific research objectives were to obtain cloned copies of the messenger RNA (mRNA) for pancreatic glucagon, to determine their primary sequences, and from this coding information to deduce the amino acid sequence of the initial glucagon precursor. From this suggested preproglucagon sequence and prior information on possible proglucagon intermediate processing products, the overall objective of this research is to propose a possible pathway for the biosynthesis of pancreatic glucagon.^ Synthetic oligodeoxynucleotide probes of 14-nucleotides (14-mer) and 17-nucleotides (a 17-mer) complementary to codons specifying a unique sequence of mature glucagon were synthesized. The ('32)P-labeled-14-mer was hybridized with size-fractionated fetal bovine pancreatic poly(A('+))RNA bound to nitrocellulose. RNA fractions of (TURN)14S were found to hybridize specifically, resulting in an (TURN)10-fold enrichment for these sequences. These poly(A('+))RNAs were translated in a cell-free system and the products analyzed by gel electrophoresis. The translation products were found to be enriched for a protein of the putative size of mammalian preproglucagon ((TURN)21 kd). These enriched RNA fractions were used to construct a complementary DNA (cDNA) library is plasmid pBR322.^ Screening of duplicate colony filters with the ('32)P-labeled-17-mer and a ('32)P-labeled-17-mer-primed cDNA probe indicated 25 possible glucagon clones from 3100 colonies screened. Restriction mapping of 6 of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1200 base pair DNA insert revealed that it contained essentially a full-length copy of glucagon cDNA.^ Analaysis of the cDNA suggested that it encoded an initial translation product of 180 amino acids with an M(,r) = 21 kd. The first initiation codon (ATG, methionine) followed by the longest open reading frame of 540 nucleotides was preceded by a 5'-untranslated region of 90 nucleotides, and was followed by a longer 3'-untranslated region of 471 nucleotides, resulting in a total of 1101 nucleotides. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Expression cloning of a candidate prostate cancer oncogene

Genetic analysis, both karyotyping and comparative genomic hybridization, of prostate cancer cell lines and specimens have revealed multiple areas of concordant increases in DNA content. An increase of DNA in specific regions of the genome in cancer is often associated with the amplification of oncogenes. Based on these observations we have hypothesized that oncogenes are involved in the initiation or progression of prostate cancer. An expression cloning approach was utilized to identify candidate oncogenes in prostate cancer. ^ A full-length, unidirectional cDNA expression library was constructed from DU145 prostate cancer cells. The cDNA library was screened using CP12, a rat prostate epithelial cell line. In soft agarose assays, CP12 (parental or vector transfected) do not form colonies. However, upon the introduction of a number of known oncogenes CP12 becomes anchorage independent in soft agarose. Based on this in-vitro phenotypic shift, a DU145 cDNA library was stably transfected into CP12, and selected for anchorage independence. Two hundred fifty nine anchorage independent clones were isolated. Some colonies contained more than one insert, bringing the candidate oncogene pool to approximately 400. Seven inserts were sequenced at random. Using the sequences obtained, GenBank was screened, and matches were found with p53, PARG1, a mitochondrial ATPase, RNF6, and three unknown genes that mapped to Unigene clusters. As the pool of cDNA inserts appeared promising, overexpressed genes were further selected. From 259 clones, 17 clones were overexpressed more than 6-fold in DU145 compared to Normal Prostate. From the 17 clones, 12 cDNA inserts were strongly expressed in DU145 and were isolated for sequencing. ^ Two of the sequences, 1G6 and 3E9, were identical. Expression of 1G6/2G9/3E9 was tested by RT-PCR. 1G6/2G9/3E9 was not expressed in normal prostate, but was expressed in all prostate cancer cell lines tested as well as six prostate cancer samples. When retransfected into CP12, 1G6/2G9/3E9 induced the formation of foci and anchorage independent colonies. Thus, functional and expression data suggest that 1G6/2G9/3E9 may be a prostate cancer oncogene. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

Functional analysis of BMP4 signaling in mouse neural development

During early mouse neural development, bone morphogenetic protein (BMP) signaling patterns the dorsal neural tube and defines distinct neural progenitor cell domains along the dorsoventral axis. Unlike the ventral signaling molecule Sonic hedgehog, which has long-range activity by establishing a concentration gradient in the ventral neural tube, these dorsally expressed BMPs appear to have a limited domain of action. This raises questions as to how BMP activity is restricted locally and how restricted BMP signaling directs dorsal neural patterning and differentiation. I hypothesize that BMPs are restricted in the dorsal neural tube for correct dorsoventral patterning. ^ Previous studies have shown that the positively charged basic amino acids located at the N-terminus of several BMPs are essential for heparin binding and diffusion. This provides a novel tool to address these questions. Here I adapted a UAS/GAL4 bigenic mouse system to control the ectopic expression of BMP4 and a mutant form of BMP4 that lacks a subset of the N-terminal basic amino acids. The target genes, UAS-Bmp4 and UAS-mBmp4 , were introduced into the Hprt locus by gene targeting in mouse embryonic stem cells. The expression of the GAL4 transactivator was driven by a roof plate specific Wnt1 promoter. ^ The bigenic mouse embryos exhibit phenotype variations, ranging from mid/hindbrain defects, hemorrhage, and eye abnormalities to vasculture formation. Embryonic death starts around E11.5 because of severe hemorrhage. The different expression levels of the activated transgene may account for the phenotype variation. Further marker analysis reveals that mutant BMP4 induces ectopic expression of the dorsal markers MSX1/2 and PAX7 in the ventral neural tube. In addition, the expression of the ventral neural marker NKX2.2 is affected by the expanded BMP4 activity, indicating that ectopic BMP signaling can antagonize ventral signaling. Comparison of the phenotypes of the Wnt1/ Bmp4 and Wnt1/mBmp4 bigenic embryos that express transgenes at the same level, respectively, shows that mutant BMP4 causes the expansion of dorsal neural fates ventrally while wild type BMP4 does not, suggesting that mutant BMP4 acts farther than wild type BMP4. Together, these data suggest that the N-terminus basic amino acid core controls BMP4 long-range activity in neural development, and that BMP signaling patterns the dorsal neural tube through a secondary signaling pathway that involves homeodomain transcription factors MSX1/2 and PAX7. ^

Gene regulation during placenta development: Murine adenosine deaminase as a model to study placenta specific expression

The formation of the placenta is one of the first and most important developmental events that occur in early mammalian embryogenesis. Even given this importance of the placenta, the academic community has largely ignored studying gene regulation during the development and maturation of the placenta. For this reason, an in-depth study of gene regulation in the trophoblast layer of the placenta using murine Adenosine Deaminase (Ada) as a model system has been undertaken. It has been determined that Ada is highly expressed in the placenta and is critical for embryo development. Dr. Kellems' laboratory has previously described a 1.8 kb fragment of the Ada 5 ′ flanking region that is capable of directing trophoblast specific expression in a transgenic model system. Preliminary studies have demonstrated several critical portions of this fragment that are necessary for the correct tissue specific expression in the placenta. My first specific aim was to elucidate the trans factor binding to one of these sequences, the FP3. Through electromobility shift assays (EMSA), the 30 bp FP3 was narrowed to a 5 bp sequence which computer databases predicted bound to Acute Myeloid Leukemia 1 (AML-1). This was confirmed by supershift analysis. The functional importance of this binding was demonstrated by a transgenic approach. A significant difference in expression of the reporter in the placenta was seen when the 5 bp sequence was mutated. This finding is a novel use for the AML-1 transcription factor which is the DNA binding portion of the heterodimer Core Binding Protein (CBP). The 5′ 240 bp region has also been demonstrated to contain functionally significant sequence. Through EMSA assays and computer predictions, the area has been narrowed to two pertinent regions that are predicted to contain GATA binding motifs. ^

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^