Deregulation of both imprinted and expressed alleles of the insulin-like growth factor 2 gene during beta-cell tumorigenesis.

In a mouse model of multistage carcinogenesis elicited by the SV40 large T-antigen (Tag) oncogene in pancreatic beta cells, the gene for insulin-like growth factor IGF2 is focally up-regulated and functionally implicated in tumour development. The IGF2 gene is differentially regulated in normal tissues: the paternal allele is transiently expressed during embryogenesis, whereas the maternal allele is genomically imprinted and inactive. Crossbred mice carrying the Tag oncogene and a disruption of either the paternal or maternal allele of IGF2 reveal that both alleles are co-activated early during tumour development, and that each contributes to malignant hyperproliferation and consequent tumour volume.

Differential regulation of telomerase activity and telomerase RNA during multi-stage tumorigenesis.

Telomeres consist of short repeated sequences that are synthesized by telomerase, a ribonucleo-protein DNA polymerase. Telomerase activity is present in many tumours and not detected in many normal tissues. Telomere shortening in human and mouse tissues and primary cell cultures may be due to the absence of telomerase activity. To determine when telomerase is activated during tumour development and progression, we examined telomerase activity and expression of the recently cloned mouse telomerase RNA component (mTR) in two different transgenic mouse models of multi-stage tumorigenesis. These mouse models allow examination of many independent tumours from genetically identical individuals. These mice reproducibly develop pancreatic islet cell carcinoma and squamous cell carcinoma of the skin. In both carcinoma types, we detected telomerase activity only in late-stage tumours; in contrast, we found mTR levels were upregulated in the early preneoplastic stages, and further increased during progression. Surprisingly, mTR levels did not parallel the amount of telomerase activity detected and a subset of tumours lacked telomerase activity and yet expressed telomerase RNA. Regulation of telomerase activity may therefore be separable from expression of its RNA component. These results clearly demonstrate telomerase is activated in late stages of tumour progression, and show for the first time that the initial up regulation of telomerase RNA is an early event, concurrent with the hyperproliferation elicited by viral oncogenes.

Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas.

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.

During carcinogenesis of pancreatic islets in transgenic mice, an angiogenic switch activates the quiescent vasculature. Paradoxically, vascular endothelial growth factor (VEGF) and its receptors are expressed constitutively. Nevertheless, a synthetic inhibitor (SU5416) of VEGF signalling impairs angiogenic switching and tumour growth. Two metalloproteinases, MMP-2/gelatinase-A and MMP-9/gelatinase-B, are upregulated in angiogenic lesions. MMP-9 can render normal islets angiogenic, releasing VEGF. MMP inhibitors reduce angiogenic switching, and tumour number and growth, as does genetic ablation of MMP-9. Absence of MMP-2 does not impair induction of angiogenesis, but retards tumour growth, whereas lack of urokinase has no effect. Our results show that MMP-9 is a component of the angiogenic switch.

Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction.

Fas ligand is believed to mediate immune privilege in a variety of tissues, including the eye, testis, and a subset of tumors. We tested whether expression of Fas ligand on pancreatic islets either following adenoviral or germline gene transfer could confer immune privilege after transplantation. Islets were infected with an adenoviral vector containing the murine Fas ligand cDNA (AdFasL), and were transplanted into allogenic diabetic hosts. Paradoxically, AdFasL-infected islets underwent accelerated neutrophilic rejection. The rejection was T cell and B cell independent and required Fas protein expression by host cells, but not on islets. Similarly, transgenic mice expressing Fas ligand in pancreatic beta cells developed massive neutrophilic infiltrates and diabetes at a young age. Thus, Fas ligand expression on pancreatic islets results in neutrophilic infiltration and islet destruction. These results have important implications for the development of Fas ligand-based immunotherapies.

Self antigens expressed by solid tumors Do not efficiently stimulate naive or activated T cells: implications for immunotherapy.

Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic beta-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP x Tag2) mice (137 +/- 18 d) as opposed to control RIP-Tag2 mice (88 +/- 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP-specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP x Tag2) mice further prolonged survival (168 +/- 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.

Transgenic mice as probes into complex systems.

The transfer of genetic information into mouse embryos to stably alter the genetic constitution of mice is affording new insights into and opportunities in a wide variety of biological problems. Higher eukaryotes are composed of many interacting cells and organs. The properties of individual cell systems are often discernible only by studying natural or induced disruptions in their functions. Transgenic mice represent a new form of perturbation analysis whereby the selective expression of novel or altered genes can be used to perturb complex systems in ways that are informative about their development, their functions, and their malfunctions. The utility of this strategy is illustrated by recent research into immunological self-tolerance, oncogenes and cancer, and development.

Betacellulin: a mitogen from pancreatic beta cell tumors.

Betacellulin, a member of the epidermal growth factor family, has been identified in the conditioned medium of cell lines derived from mouse pancreatic beta cell tumors. Betacellulin is a 32-kilodalton glycoprotein that appears to be processed from a larger transmembrane precursor by proteolytic cleavage. The carboxyl-terminal domain of betacellulin has 50 percent sequence similarity with that of rat transforming growth factor alpha. Betacellulin is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells.

Effects of angiogenesis inhibitors on multistage carcinogenesis in mice.

Solid tumors depend on angiogenesis for their growth. In a transgenic mouse model of pancreatic islet cell carcinogenesis (RIP1-Tag2), an angiogenic switch occurs in premalignant lesions, and angiogenesis persists during progression to expansive solid tumors and invasive carcinomas. RIP1-Tag2 mice were treated so as to compare the effects of four angiogenesis inhibitors at three distinct stages of disease progression. AGM-1470, angiostatin, BB-94, and endostatin each produced distinct efficacy profiles in trials aimed at preventing the angiogenic switch in premalignant lesions, intervening in the rapid expansion of small tumors, or inducing the regression of large end-stage cancers. Thus, anti-angiogenic drugs may prove most efficacious when they are targeted to specific stages of cancer.

Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic alpha cells.

The regulatory region of the rat preproglucagon gene targets expression of the SV40 large T oncoprotein to two cell types in transgenic mice, the pancreatic alpha cells and a set of neurons localized in the hindbrain, both of which normally produce preproglucagon. Additional neurons in the forebrain and midbrain stain for T antigen but do not express the endogenous glucagon gene. Synthesis of T antigen in endocrine alpha cells results in the heritable development of pancreatic glucagonomas. In brains of transgenic mice from three independent lineages, expression of the hybrid gene begins at embryonic day 12 in neuroblasts of the hindbrain, where it continues throughout adult life, most notably in the medulla. Remarkably, oncoprotein expression in both proliferating neuroblasts and mature neurons has no apparent consequences, either phenotypic or tumorigenic. Expression of the hybrid glucagon gene in both neurons and islet cells supports a possible interrelationship between these cell types.

Human platelet stimulation by acetyl glyceryl ether phosphorylcholine.

Acetyl glyceryl ether phosphorylcholine (AGEPC) induced dose-dependent platelet aggregation and release of [3H]serotonin and platelet factor 4 in citrated human platelet-rich plasma. ADP scavengers or indomethacin prevented irreversible platelet aggregation responses induced by 0.2 microM AGEPC but had no effect upon platelet secretion; prostacyclin inhibited AGEPC-induced aggregation and secretion. EDTA or EGTA inhibited AGEPC-induced aggregation but had no effect on platelet secretion.

Modulation of L-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice.

Immune surveillance depends on lymphocyte access to tissue. Lymphocytes emigrate from blood when adhesion receptors such as L-selectin and the alpha 4 beta 7 integrin on these cells bind to ligands expressed on venular endothelium. Among transgenic mouse lines expressing an oncoprotein (Tag) in islet beta cells, some recognize Tag as nonself. In these mice, Tag expression elicits both beta cell hyperplasia with subsequent progression to tumors and lymphocytic infiltration. Endothelial ligands for L-selectin and alpha 4 beta 7 were upregulated in infiltrated islets in these transgenic mice. These ligands were not expressed in tumors, which were devoid of lymphocytic infiltration. In contrast, the adhesion molecules PECAM-1, ICAM-1, and VCAM-1 were expressed on endothelium in both noninfiltrated tumors and infiltrated islets. Thus, upregulation of expression of endothelial ligands for L-selectin and alpha 4 beta 7 may contribute to autoimmune infiltration. Repression of expression of these same ligands may be involved in the failure of tumor immunity.

Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.

This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.

Altered function of insulin receptor substrate-1-deficient mouse islets and cultured beta-cell lines.

Insulin receptor substrate-1 (IRS-1) is pivotal in mediating the actions of insulin and growth factors in most tissues of the body, but its role in insulin-producing beta islet cells is unclear. Freshly isolated islets from IRS-1 knockout mice and SV40-transformed IRS-1-deficient beta-cell lines exhibit marked insulin secretory defects in response to glucose and arginine. Furthermore, insulin expression is reduced by about 2-fold in the IRS-1-null islets and beta-cell lines, and this defect can be partially restored by transfecting the cells with IRS-1. These data provide evidence for an important role of IRS-1 in islet function and provide a novel functional link between the insulin signaling and insulin secretion pathways. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Peripheral-antigen-expressing cells in thymic medulla: factors in self-tolerance and autoimmunity.

The thymus expresses many genes previously thought to be specific for cell types in other organs. Thus, insulin genes are expressed in rare cells of the thymic medulla. Thymus transplantation demonstrates a functional capability of such expression for self-tolerance induction. Correlative studies suggest that impaired thymic expression confers susceptibility to autoimmune disease.

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.

Bidirectional activity of the rat insulin II 5'-flanking region in transgenic mice.

We have identified a new transcription initiation site in the 5'-flanking regulatory region of the rat insulin II gene. This site is located on the opposite strand with respect to the insulin gene promoter, upstream of the insulin gene transcriptional enhancer. The cell-specific activity of this reverse promoter element is demonstrated in two lineages of transgenic mice, in which it directs expression of simian virus 40 T antigen specifically to the beta cells of the endocrine pancreas, resulting in development of pancreatic tumors. Analysis of RNA from the tumor cells demonstrates bidirectional transcription from the insulin regulatory region of the transgene. These data raise the possibility that bidirectional activity is a quality of the regulatory region of the insulin gene in its natural genomic context.

Diabetes induced in male transgenic mice by expression of human H-ras oncoprotein in pancreatic beta cells.

Transgenic mice expressing an insulin-promoted H-ras hybrid gene in pancreatic beta cells developed beta-cell degeneration and diabetes. The disease was manifested in male mice by hyperglycemia, glycosuria, and reduced plasma insulin levels, which appeared around 5 months of age and led to premature death. Histological analyses revealed large holes within the islets of Langerhans and a reduced number of beta cells. The destruction of the islets was not associated with an obvious inflammatory activity. Ultrastructural analysis showed extensive engorgement in the endoplasmic reticulum of the residual beta cells from diabetic males. The females carrying the insulin-promoted ras gene did not manifest any of the physiological abnormalities observed in males and showed only minor histological and ultrastructural changes, even at much greater ages.

Cell-heritable stages of tumor progression in transgenic mice harboring the bovine papillomavirus type 1 genome.

Tumorigenesis of dermal fibroblasts in a line of transgenic mice carrying the BPV-1 genome was found to involve distinct proliferative stages. Cell cultures derived from normal skin, from benign proliferative fibromatoses, and from malignant fibrosarcomas each evidenced distinguishable, cell-heritable characteristics. The latent viral genome was transcriptionally inactive in normal-appearing skin and was activated in the dermal fibromatoses. Fibrosarcoma cells grew continuously in culture, formed domelike foci, and had a more rounded, anaplastic appearance. Independent cultures derived from the fibromatoses varied in their proliferative characteristics, which correlated well with the levels of viral gene expression. In contrast, progression to malignancy was not accompanied by a further increase in transgene activity, which strongly implicated cellular genetic changes in the later stages of tumorigenesis.

Cellular transformation and malignancy induced by ras require c-jun.

ras is an important oncogene in experimental animals and humans. In addition, activated ras proteins are potent inducers of the transcription factor AP-1, which is composed of heterodimeric complexes of Fos and Jun proteins. Together with the fact that deregulated expression of some AP-1 proteins can cause neoplastic transformation, this finding suggests that AP-1 may function as a critical ras effector. We have tested this hypothesis directly by analyzing the response to activated ras in cells that harbor a null mutation in the c-jun gene. The transcriptional response of AP-1-responsive genes to activated ras is severely impaired in c-jun null fibroblasts. Compared with wild-type cells, the c-jun null cells lack many characteristics of ras transformation, including loss of contact inhibition, anchorage independence, and tumorigenicity in nude mice; these properties are restored by forced expression of c-jun. Rare tumorigenic variants of ras-expressing c-jun null fibroblasts do arise. Analysis of these variants reveals a consistent restoration of AP-1 activity. The results provide genetic evidence that c-jun is a crucial effector for transformation by activated ras proteins.