Genetic deletion of granzyme B does not confer resistance to the development of spontaneous diabetes in non-obese diabetic mice.

Granzyme B (GzmB) and perforin are proteins, secreted mainly by natural killer cells and cytotoxic T lymphocytes that are largely responsible for the induction of apoptosis in target cells. Because type 1 diabetes results from the selective destruction of ß cells and perforin deficiency effectively reduces diabetes in non-obese diabetic (NOD) mice, it can be deduced that ß cell apoptosis involves the GzmB/perforin pathway. However, the relevance of GzmB remains totally unknown in non-obese diabetic (NOD) mice. In this study we have focused on GzmB and examined the consequence of GzmB deficiency in NOD mice. We found that NOD.GzmB(-/-) mice developed diabetes spontaneously with kinetics similar to those of wild-type NOD (wt-NOD) mice. Adoptive transfer study with regulatory T cell (Treg )-depleted splenocytes (SPCs) into NOD-SCID mice or in-vivo Treg depletion by anti-CD25 antibody at 4 weeks of age comparably induced the rapid progression of diabetes in the NOD.GzmB(-/-) mice and wt-NOD mice. Expression of GzmA and Fas was enhanced in the islets from pre-diabetic NOD.GzmB(-/-) mice. In contrast to spontaneous diabetes, GzmB deficiency suppressed the development of cyclophosphamide-promoted diabetes in male NOD mice. Cyclophosphamide treatment led to a significantly lower percentage of apoptotic CD4(+) , CD8(+) and CD4(+) CD25(+) T cells in SPCs from NOD.GzmB(-/-) mice than those from wt-NOD mice. In conclusion, GzmB, in contrast to perforin, is not essentially involved in the effector mechanisms for ß cell destruction in NOD mice.

MtDNA control region sequence polymorphisms and phylogenetic analysis of Malay population living in or around Kuala Lumpur in Malaysia.

Control region polymorphisms in the mitochondrial DNA of 124 unrelated individuals from the Malay population living in or around Kuala Lumpur in Malaysia were investigated and phylogenetic haplogroup lineages were determined. The intergenic COII/tRNALys 9-bp deletion, 3010 and 5178 mutations, and several coding region polymorphisms were examined to discriminate some phylogenetic haplogroups. Sequence comparison of the control regions led to the identification of 117 mitochondrial haplotypes, in which 103 types were observed in only one individual and the other nine types were shared by more than two individuals. Gene diversity was estimated to be 0.997. Phylogenetic haplogroup determination revealed that the gene pool of the modern Malay population in Malaysia consisted mainly of southeast Asian, east Asian, unidentified and unique, and aboriginal southeast-specific haplogroups. These results suggest a multi-original nature for the modern Malay population. The present database may help not only in personal identification but also in determining geographic origin in forensic casework in Malaysian, Southeast Asian and East Asian populations.

Solid stress facilitates spheroid formation: potential involvement of hyaluronan.

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.

Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF.

Mesenchymal stem cells (MSC) that can differentiate to various connective tissue cells may be useful for autologous cell transplantation to defects of bone, cartilage, and tendon, if MSC can be expanded in vitro. However, a short life span of MSC and a reduction in their differentiation potential in culture have limited their clinical application. The purpose of this study is to identify a growth factor(s) involved in self-renewal of MSC and the maintenance of their multilineage differentiation potential. Fibroblast growth factor-2 (FGF-2) markedly increased the growth rate and the life span of rabbit, canine, and human bone marrow MSC in monolayer cultures. This effect of FGF-2 was more prominent in low-density cultures than in high-density cultures. In addition, all MSC expanded in vitro with FGF-2, but not without FGF-2, differentiated to chondrocytes in pellet cultures. The FGF+ MSC also retained the osteogenic and adipogenic potential throughout many mitotic divisions. These findings suggest that FGFs play a crucial role in self-renewal of MSC.

Vascular endothelial growth factor (VEGF) modulation by targeting hypoxia-inducible factor-1alpha--> hypoxia response element--> VEGF cascade differentially regulates vascular response and growth rate in tumors.

Although tumors can activate vascular endothelial growth factor (VEGF) promoter in host stromal cells, the relative contribution to VEGF production of host versus tumor cells and the resulting vascular response have not been quantitated to date. To this end, we implanted VEGF-/- and wild-type (WT) embryonic stem (ES) cells in transparent dorsal skin windows in severe combined immunodeficient mice. VEGF-/- ES cell-derived tumors produced approximately 50% of VEGF compared with the WT tumors, suggesting significant contribution of host stromal cells. To discern the hypoxia-induced hypoxia inducible factor (HIF)-1alpha --> hypoxia response element (HRE) --> VEGF signaling cascade, we also examined tumors derived from HIF-1alpha-/- and HRE-/- ES cells. As expected, the VEGF protein level in HIF-1alpha-/- ES tumors was intermediate between VEGF-/- and WT ES cell tumors. Surprisingly, HRE-/- ES tumors produced the same level of VEGF as the VEGF-/- ES tumors, suggesting a critical role of HRE in tumor cell VEGF production. Angiogenesis in these tumors was proportional to their VEGF levels (VEGF-/- approximate to HRE-/- < HIF-1alpha-/- < WT). In contrast, vascular permeability, leukocyte-endothelial adhesion, and tumor growth were reduced in VEGF-/- and HRE-/- tumors but were comparable in HIF-1a-/- and WT tumors. This discrepancy suggests that different intracellular signaling pathways may be involved in each of these functions of VEGF. More importantly, these data suggest that host cells are active players in tumor angiogenesis and growth and need to be taken into account in the design of any therapeutic strategy.

Endogenous AP-1 levels necessary for oncogenic activity are higher than those sufficient to support normal growth.

We investigated the role of endogenous AP-1 in human tumor cell lines by introducing SupJunD-1, a dominant-negative mutant of AP-1, using vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors. Single inoculation of six human tumor cell lines, originating from osteosarcomas, non-small cell lung carcinomas or cervical carcinomas, with recombinant SupJunD-1 virus at a high multiplicity of infection readily inhibited colony formation in soft agar. We detected no significant changes in expression levels of AP-1 components c-Jun or Fra-1, adhesion molecules CD44 or E-cadherin, or cell cycle regulator p53, which are encoded by genes previously reported to be under the control of AP-1 in some mouse or human cell lines. By varying the dosage of VSV-G-pseudotyped retrovirus, we were able to change the proviral copy number of supjunD-1 from 1 to approximately 10 and monitor suppression of endogenous AP-1 function as assessed by growth characteristics of the tumor cell lines, we found a SupJunD-1 dosage which significantly suppressed anchorage-independent growth without affecting the cellular growth in monolayer cultures at all. We conclude that endogenous AP-1 levels necessary for oncogenic activity are much higher than those sufficient to support normal growth.

Isolation of the regulatory regions and genomic organization of the porcine alpha1,3-galactosyltransferase gene.

BACKGROUND: Alpha1,3-galactosyltransferase (alpha1,3GT) is an enzyme that produces carbohydrate chains termed alphaGal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost alpha1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated. METHODS: We used 5'-or 3'-RACE, GenomeWalking, reverse transcriptase polymerase chain reaction (RT-PCR) and dual Luciferase reporter assay for identification of the full-length cDNA, which includes the transcription initiation site and the promoter region of porcine alpha1,3GT gene. RESULTS: The region around exon 1 is guanine and cytosine (GC)-rich (about 70%), comprising a CpG island spanning more than 1.5 kbp. The 5'-flanking region of exon 1 contains multiple transcription factor consensus motifs, including GC-box, SP1, AP2, and GATA-box sites, in the absence of TATA or CAAT-box sequences. The entire gene consists of three 5' noncoding and six coding region exons spanning more than 52 kbp. Detailed analysis of alpha1,3GT transcripts revealed two major alternative splicing patterns in the 5'-untranslated region (5'-UTR) and evidence for minor splicing activity that occurs in a tissue-specific manner. Interspecies comparison of 5'-UTR shows minimal homology between porcine and murine sequences except for exon 2, which suggests that the regulatory regions differ among species. CONCLUSIONS: These observations have important implications for experiments involving genetic manipulation of the alpha1,3GT gene in transgenic animals in terms of promoter utilization, and particularly in genetically engineering cells for the animal cloning technology by nuclear transfer.

Anti-Vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions.

Recent studies in experimental animals have shown that combining antiangiogenic therapy with radiation can enhance tumor response. Whether this enhancement is mainly attributable to angiogenesis inhibition, endothelial cell radiosensitivity, tumor cell apoptosis, or a decrease in the number of hypoxic cells (improved oxygenation) is not known. We designed this study to discern the role of tumor oxygenation. We chose an anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody (mAb) which has a known target, human VEGF. We also measured interstitial fluid pressure (IFP) to test the hypothesis that the decreased vascular permeability induced by the anti-VEGF mAb can lower IFP. The effect of anti-VEGF mAb on vascular density, partial oxygen tension (pO2), and apoptosis was also measured. Athymic NCr/Sed nu/nu mice bearing 6-mm xenograft of the human glioblastoma multiforme (U87), or colon adenocarcinoma (LS174T) were treated with anti-VEGF mAb injected i.p. on alternate days for a total of six injections at a dosage of 100 microg/injection/mouse. For combined anti-VEGF and radiation, single radiation doses were given under normal blood flow (20 and 30 Gy) or clamped hypoxic conditions (30 and 40 Gy) 24 h after the sixth injection of mAb. The inhibition of the growth of U87 and LS174T tumors by the anti-VEGF mAb was associated with a significant reduction in tumor vascular density and a relatively small increase in the number of apoptotic cells. Compared with size-matched controls, IFP decreased by 74% in LS174T, and 73% in U87 in mice treated with anti-VEGF mAb. After antibody treatment PO2 increased significantly in U87, but did not change in LS174T tumors. Combined treatment induced in U87 tumors a tumor-growth delay (TGD) which was greater than additive; in LS174T except for the 40-Gy hypoxic group, the effect was only additive. In both U87 and LS174T the TGD induced by the antibody was independent of oxygen levels in the tumor at the time of radiation. The fact that the increase in TGD occurred under both normoxic and hypoxic conditions suggests that anti-VEGF mAb treatment can compensate for the resistance to radiation induced by hypoxia.

Analysis of cartilage maturation using micromass cultures of primary chondrocytes.

A micromass culture (MM-C) system of primary immature chondrocytes for functional analysis of soluble factors involved in the maturation step of cartilage was previously developed. Ectopically expressed BMP-2 was shown to induce the expression of the Ihh and Noggin genes. Here it is demonstrated that, upon longer culture, secreted bone morphogenetic protein-2 (BMP-2) further promotes the maturation step as judged by the induction of type X collagen and BMP-6 expression, which are known to be detectable in the later phase of cartilage maturation. Induction of all of these genes by secreted BMP-2 was not inhibited by ectopic expression of parathyroid hormone-related peptide (PTHrP) induced by retrovirus vector infection, although the same virus vector showed strong inhibitory effects on the expression of type X collagen gene or alkaline phosphatase activity in mature chondrocytes. These results suggest that the maturation-promoting activity exhibited by BMP-2 is dominant over the suppressive effect of PTHrP in immature chondrocytes. When the BMP-6 gene was introduced into the same virus vector as that used for BMP-2, it induced the same sets of genes (Ihh, Noggin, type X collagen and endogenous BMP-6) as BMP-2 did. These results also suggest that BMP-6 would autonomously maintain and/or promote a later stage of chondrocytic maturation.

BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression.

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.

Corticotropin-releasing hormone stimulates angiogenesis and epithelial tumor growth in the skin.

The hypothalamic neuropeptide corticotropin-releasing hormone is the major hypothalamic regulator of the endocrine pituitary-adrenal axis. Corticotropin-releasing hormone is also expressed in many peripheral sites, where its functions are unclear. It is also secreted by diverse neoplasms, where it may be associated with malignant behavior. To provide information regarding the function of corticotropin-releasing hormone in peripheral sites and in tumors, we asked whether corticotropin-releasing hormone has angiogenic properties. In vitro, we found that human corticotropin-releasing hormone specifically stimulates endothelial chemotaxis via a corticotropin-releasing hormone receptor-dependent mechanism. In vivo, subcutaneous inoculation of nude mice with human epithelial tumor cells engineered to secrete corticotropin-releasing hormone was associated with significantly enhanced angiogenesis (2.3-fold over control) and tumor growth (5-fold over control). Peripheral corticotropin-releasing hormone may thus enhance local angiogenesis, which may provide clues to its function outside of the nervous system.

Structural basis for amide hydrolysis catalyzed by the 43C9 antibody.

Among catalytic antibodies, the well-characterized antibody 43C9 is unique in its ability to catalyze the difficult, but desirable, reaction of selective amide hydrolysis. The crystallographic structures that we present here for the single-chain variable fragment of the 43C9 antibody, both with and without the bound product p -nitrophenol, strongly support and extend the structural and mechanistic information previously provided by a three-dimensional computational model, together with extensive biochemical, kinetics, and mutagenesis results. The structures reveal an unexpected extended beta-sheet conformation of the third complementarity determining region of the heavy chain, which may be coupled to the novel indole ring orientation of the adjacent Trp H103. This unusual conformation creates an antigen-binding site that is significantly deeper than predicted in the computational model, with a hydrophobic pocket that encloses the p -nitrophenol product. Despite these differences, the previously proposed roles for Arg L96 in transition-state stabilization and for His L91 as the nucleophile that forms a covalent acyl-antibody intermediate are fully supported by the crystallographic structures. His L91 is now centered at the bottom of the antigen-binding site with the imidazole ring poised for nucleophilic attack. His L91, Arg L96, and the bound p -nitrophenol are linked into a hydrogen-bonding network by two well-ordered water molecules. These water molecules may mimic the positions of the phosphonamidate oxygen atoms of the antigen, which in turn mimic the transition state of the reaction. This network also contains His H35, suggesting that this residue may also stabilize the transition-states. A possible proton-transfer pathway from His L91 through two tyrosine residues may assist nucleophilic attack. Although transition-state stabilization is commonly observed in esterolytic antibodies, nucleophilic attack appears to be unique to 43C9 and accounts for the unusually high catalytic activity of this antibody.

Developmental patterning in chondrocytic cultures by morphogenic gradients: BMP induces expression of indian hedgehog and noggin.

BACKGROUND: The maturation of chondrocytes is essential for endochondral bone formation. The Indian Hedgehog (Ihh) gene is expressed in prehypertropic chondrocytes and has been proposed to regulate chondrocyte maturation. While such secretary factors as PTHrP and BMP are thought to be involved in Ihh expression, the mechanism of the restricted expression of Ihh is not clear. RESULTS: Using primary chondrocytes, we have developed here a modified micromass culture (MM-C) system that allows the formation of concentration gradients of secreted factors, expressed either endogenously or retrovirally, from each of plural micromass cultures on a single plate. Using this system, we determined that chondrocytes create the inhibitory micro-environment, partly dependent on PTHrP secretion, for the Ihh expression. We also showed that retrovirally induced BMP-2 induces the expression of both Ihh and Noggin (encoding the BMP-inactivating protein), and we further present evidence that a negative-feedback loop involving Noggin might account for the precise localization of BMP signalling for Ihh induction. CONCLUSION: These results suggest that the expression of the Ihh gene in cartilage is regulated by several mechanisms that include the secretion of inhibitory factors (including PTHrP) and the negative-feed back loop formed by BMPs and Noggin.

Sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cells.

High local polyamine concentrations may suppress cell growth of primary prostatic carcinomas. When cell growth assays were conducted in defined serum-free medium, spermine inhibited the growth of poorly metastatic rat prostate carcinoma cells, causing cell cycle arrest in the G1 phase. In contrast, highly metastatic prostate carcinoma cells were resistant to the growth inhibitory activity of spermine. Ornithine decarboxylase antizyme levels, measured by Western blotting, were elevated 1-2 h after spermine treatment of spermine-sensitive cells but not spermine-resistant cells. Spermine uptake was similar in both the sensitive and resistant cell lines. These results suggest that failure to induce antizyme correlates with spermine resistance in prostate carcinoma.

How platelet aggregation affects B16BL6 melanoma cell trafficking.

In blood-borne metastasis, intravasated metastatic tumor cells are thought to localize at the target site via a series of processes involving platelet aggregation, adhesion to endothelium, and invasion through the basal membrane. In the present study, we examined how platelet aggregation contributes to the trafficking of metastatic tumor cells in vivo by use of an inhibitor of platelet aggregation. Highly invasive B16BL6 melanoma cells were labeled with [2-18F]2-fluoro-2-deoxy-D-glucose and injected into mice to determine cell trafficking non-invasively by positron emission tomography. Both platelet aggregation inhibitor cyclo(RSarDPhg), which could not inhibit metastasis, and metastatic inhibitor cyclo(GRGDSPA) suppressed the accumulation of B16BL6 cells in the lung by about 12%, suggesting that platelet aggregation partly affects cell trafficking but not to a great extent, and that platelet aggregation is not the essential step for B16BL6 cell arrest in targets.

TIP49, homologous to the bacterial DNA helicase RuvB, acts as an autoantigen in human.

TATA-binding protein (TBP), a central component for transcriptional regulation, forms complexes with various transcription regulators. We have isolated a novel human cDNA for a 49-kD TBP-interacting protein (TIP49). The human TIP49 was highly homologous to bacterial RuvB proteins that function as a DNA helicase to promote branch migration of the Holliday junction. Immunofluorescence analysis using anti-TIP49 antibody showed a typical dot-shaped nuclear staining pattern, suggesting that TIP49 is included in a macromolecular structure in the nucleus and may participate in nuclear events such as transcription and recombination. Moreover, glycerol gradient analysis demonstrated that TIP49 is present in a macromolecular complex in nuclear extracts. Interestingly, we detected a high level of autoantibodies against TIP49 in sera of patients with autoimmune diseases such as polymyositis/dermatomyositis and autoimmune hepatitis. This indicates that the autoantibody against this protein is a new marker for particular connective tissue diseases. These findings provide further evidence that the macromolecular structures described above are targeted by an autoimmune mechanism. The anti-TIP49 antibodies can be useful probes for clinical diagnosis and for investigation of intranuclear structure.