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D Hanahan

Publications and source records attributed to D Hanahan.

At least 37 records · Page 2Linked to original sources
PubMed1998

MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes.

Homozygous mice with a null mutation in the MMP-9/gelatinase B gene exhibit an abnormal pattern of skeletal growth plate vascularization and ossification. Although hypertrophic chondrocytes develop normally, apoptosis, vascularization, and ossification are delayed, resulting in progressive lengthening of the growth plate to about eight times normal. After 3 weeks postnatal, aberrant apoptosis, vascularization, and ossification compensate to remodel the enlarged growth plate and ultimately produce an axial skeleton of normal appearance. Transplantation of wild-type bone marrow cells rescues vascularization and ossification in gelatinase B-null growth plates, indicating that these processes are mediated by gelatinase B-expressing cells of bone marrow origin, designated chondroclasts. Growth plates from gelatinase B-null mice in culture show a delayed release of an angiogenic activator, establishing a role for this proteinase in controlling angiogenesis.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Fibroblast growth factor receptor 4, implicated in progression of islet cell carcinogenesis by its expression profile, does not contribute functionally.

Fibroblast growth factor receptor 4 (FGFR4) gene expression is activated in late-stage beta-cell tumors that develop in transgenic mice harboring SV40 large T antigen (Tag) gene that is under the transcriptional control of the insulin promoter (RIP-Tag). The FGFR4 gene was active in cell lines derived from tumors but not in cells derived from hyperplastic islets. We used both gain-of-function and loss-of-function FGFR4 transgenic mice to determine whether FGFR4 modulates islet cell tumorigenesis and, if so, to identify the nature of the effect. Both types of FGFR4 transgenic mice were viable and fertile and developed islet tumors when crossed with RIP-Tag mice. Remarkably, there was no significant perturbation in the tumorigenesis pathway resulting from either chronic up-regulation or absence of FGFR4 gene expression. Analyses included the incidence and size of tumors, rate of cell proliferation, cell density, and life span. We conclude that FGFR4 gene activation is a marker of but is not causal for beta-cell transformation.

Adenoma, Islet Cell↗
PubMed1998

Angiogenesis and apoptosis are cellular parameters of neoplastic progression in transgenic mouse models of tumorigenesis.

The epidemiology and histopathology of human cancers and studies of animal models of tumorigenesis have led to a widely-accepted notion that multiple genetic and epigenetic changes have to accrue for the successful development of a malignant phenotype. Tumor growth and expansion requires an ability not only to proliferate, but also to down-modulate cell death (apoptosis) and activate angiogenesis to produce a tumor neovasculature. This review will describe the interplay between apoptosis and proliferation, as well as the characteristics of the angiogenic phenotype in two transgenic mouse models of multi-step tumorigenesis, namely, pancreatic islet cell carcinomas and squamous cell carcinomas of the skin.

Animals↗
PubMed1997

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67-/- animals, which are born with developmental abnormalities and die shortly after birth, GAD65-/- mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5' phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65-/- mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65-/- mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65-/- mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65-/- animals as a model of epilepsy involving GABA-ergic pathways.

Animals↗
PubMed1997

FISH probes for mouse chromosome identification.

P1 clones near the telomeres and centromeres of each mouse chromosome except Y have been selected from a mouse genomic library and mapped using fluorescence in situ hybridization (FISH). Each clone was selected to contain a genetically mapped polymorphic DNA sequence as close as possible to the centromere or telomere of a chromosome. The genetic distance from the various P1 clones to the most distal genetically mapped polymorphic sequence ranged from 0 for about half of the clones to 6.7 cM for the probe at the telomere of chromosome 14. The average distance to the most distal or proximal chromosome marker was 1.5 cM. The use of FISH with these probes for mouse chromosome identification during comparative genomic hybridization is illustrated.

Animals↗
PubMed1997

Up-regulation of vascular endothelial growth factor expression in a rat glioma is conferred by two distinct hypoxia-driven mechanisms.

Up-regulation of vascular endothelial growth factor (VEGF) expression is a major event leading to neovascularization in malignant gliomas. Hypoxia is believed to be the crucial environmental stimulus for this up-regulation. To critically assess this hypothesis, we asked whether the mechanisms defined previously for hypoxia-induced VEGF expression in vitro are similarly involved and sufficient for up-regulation of VEGF gene expression in vivo, using a lacZ reporter gene under the control of VEGF regulatory sequences in an experimental glioma model. Inclusion of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5' regulatory sequences used in the hybrid gene produced weak beta-galactosidase staining in a special tumor cell subtype, the so-called perinecrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 binding site abolished reporter gene expression in the PNP cells, indicating that transcriptional activation of VEGF expression in gliomas is mediated by HIF 1. Inclusion of 3' untranslated sequences from the VEGF gene in the reporter constructs resulted in an increased beta-galactosidase staining in the PNP cells, suggesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma cells growing as solid tumors. Combination of the 5' flanking region including the HIF 1 site along with 3' untranslated sequences produced increased levels of beta-galactosidase expression in PNP cells. EF 5 immunostaining for regions of low oxygen partial pressure covered the same PNP cells that were stained for beta-galactosidase. Collectively, the data provide experimental evidence that VEGF gene expression is activated in a distinct tumor cell subpopulation, the perinecrotic palisading cells of gliomas, by two distinct hypoxia-driven regulatory mechanisms.

Animals↗
PubMed1997

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.

Adoptive Transfer↗
PubMed1997

Cross-species comparison of angiogenesis during the premalignant stages of squamous carcinogenesis in the human cervix and K14-HPV16 transgenic mice.

Infection of the human cervix with certain papillomavirus subtypes is associated with the development of neoplastic squamous lesions that can progress to overt cervical malignancies. Recently, multistage squamous carcinogenesis has been achieved in K14-HPV16 transgenic mice, wherein expression of the human papillomavirus (HPV) type 16 early genes is targeted to basal squamous epithelial cells by regulatory elements of the human keratin-14 (K14) promoter. Immunostaining of the endothelial marker vWf revealed a parallel upregulation of angiogenesis during the early neoplastic stages in both human cervix and the epidermis of K14-HPV16 transgenic mice. Moreover, high-grade premalignant lesions and cancers in humans and transgenic mice were characterized by an additional increment in the number of new capillaries and close apposition of the microvasculature to the overlying neoplastic epithelium. Expression of the potent angiogenic factor VEGF was progressively up-regulated during carcinogenesis in both species, correlating with the increased density and altered distribution of the microvasculature. Thus, angiogenesis occurs during the premalignant stages of squamous carcinogenesis in both human cervical disease and a relevant transgenic model and may be controlled by similar molecular mechanisms in both species. These results validate the use of the transgenic model to elucidate the role of angiogenesis during HPV-associated neoplastic progression.

Animals↗
PubMed1997

Mouse models for the study of telomerase.

The ends of chromosomes, or telomeres, consist of short repeated sequences that are synthesized by a ribonucleoprotein-DNA polymerase called telomerase. The RNA component of telomerase is essential for enzyme activity. The maintenance of telomere length by telomerase has been proposed to be essential for cellular viability and to play an important role in cellular senescence and immortalization. We are interested in using the mouse as a model system for the study of telomerase. We studied telomerase activity and expression of the mouse telomerase RNA component (mTR) in two different transgenic mouse models of multistage tumorigenesis: models of islet cell carcinoma and squamous cell carcinoma. In both tumour models, telomerase activity was detected only in late-stage tumours, whereas the telomerase RNA was present at higher than normal levels in pre-neoplastic stages and increased further in late-stage tumours. However, the RNA levels did not parallel the amounts of telomerase activity detected, suggesting that regulation of telomerase activity does not correlate with the regulation of its RNA component. These results establish a direct correlation between progression to late-stage tumours and induction of telomerase activity, and suggest that the initial upregulation of telomerase RNA is an early event. To address the role of telomerase during normal mouse development and tumour formation, we have constructed a knockout mouse for the mouse telomerase RNA, mTR-/-. These mice and the cell lines derived from them are telomerase deficient.

Animals↗
PubMed1997

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.

Adenoviridae↗
PubMed1997

Pancreatic gene expression in rare cells of thymic medulla: evidence for functional contribution to T cell tolerance.

We report the initial characterization of rare cells within the thymus that express 'peripheral' self-antigens and are capable of inducing partial tolerance to a model protein. Mice from two transgenic families that express SV40 T antigen (Tag) in pancreatic islet beta cells under control of a rat insulin promoter (RIP) develop T cell tolerance toward this neo-self antigen. These mice express low levels of Tag mRNA in the thymus. Transplantation of thymus from tolerant RIP-Tag mice into athymic hosts is sufficient to confer tolerance by CD4+ Th cells and elicits variable tolerance by CD8+ cytotoxic T cells. Thymic medulla is shown to contain rare cells that express the endogenous insulin and somatostatin genes, and in the transgenic animals, Tag. These cells are referred to as 'peripheral antigen-expressing' (PAE) cells. Thymic cell fractionation reveals the PAE cells expressing insulin and Tag to be present in a fraction enriched for non-lymphoid, MHC class II+ cells. Notably, absence of thymic expression of the RIP-Tag gene in another transgenic family correlates with failure to establish self-tolerance and susceptibility to autoimmunity. Thus, expression of tissue-restricted genes such as insulin in PAE cells of thymic medulla may serve to limit development of potentially autoimmune T cells.

Animals↗
PubMed1997

DNA copy number changes associated with characteristic LOH in islet cell carcinomas of transgenic mice.

Comparative genomic hybridization (CGH) provides a method of surveying the entire tumor genome for regional variations in DNA sequence copy number. Such variations, if found recurrently, may indicate the locations of genes that contribute to tumor development through upregulation of oncogenes (copy number increase), inactivation of tumor-suppressor genes (copy number decrease), or changes in the level of expression through gene dosage effects. Thus, CGH is a powerful tool for screening for new cancer genes. Although CGH is widely applied to human genome analysis, application to the mouse is only beginning. The present study is designed to compare results obtained by CGH with those obtained by other techniques used for analysis of the murine genome. We report CGH analysis of several control cell lines with cytogenetically established regional copy number changes, as well as analysis of 16 primary insulinomas, four tumor-derived cell lines, and three hyperplasia-derived cell lines from transgenic mice expressing the SV40 large T antigen under control of the rat insulin promoter. Loss of heterozygosity (LOH) on chromosomes 9 and 16 had previously been found to be frequent in primary insulinomas, and specimens were selected for the present study based on the LOH status of these chromosomes. We found complete concordance of the CGH results with the cytogenetically described copy number changes in the control cell lines and with the LOH on chromosomes 9 and 16 in the tumors. Thus, CGH can provide accurate data in murine systems, and it reveals that the LOH in these islet cell tumors most frequently results from deletion of one of the alleles.

Adenoma, Islet Cell↗
PubMed1996

Upregulation of fibroblast growth factors and their receptors during multi-stage epidermal carcinogenesis in K14-HPV16 transgenic mice.

Upregulation of acidic and basic fibroblast growth factors (FGF-1 and -2), and their cognate receptors FGFR-1 and -2, has been demonstrated in a variety of epithelial malignancies. However, the patterns of FGF/FGFR expression at specific stages of epithelial carcinogenesis have not been extensively characterized. In this report, the levels of FGF-1, FGF-2, FGF-7 mRNA and their receptors FGFR-1 and FGFR-2, were investigated during epidermal carcinogenesis in transgenic mice expressing the early region of the 'high risk' papillomavirus type 16 (HPV16) under control of the human keratin-14 enhancer/promoter (K14-HPV16 transgenic mice). FGF-1 was first upregulated in dysplasias, while FGF-2 was constitutively expressed in non-transgenic, neoplastic, and malignant keratinocytes throughout carcinogenesis. Expression of FGF-7 was undetectable in non-transgenic epidermis, and remained at threshold levels at all stages of progression. In well differentiated squamous cancers, FGFR-1 was upregulated and co-localized with angiogenic capillaries in the dermis underlying dysplastic lesions and within papillary fronds of invasive cancers. In contrast, FGFR-1 was upregulated specifically within the malignant squamous cells of moderate-poorly differentiated squamous cancers. The expression of FGFR-2 was essentially constitutive in both non-transgenic and neoplastic epidermis. Collectively the data suggest that the FGF/FGFR signaling pathways may potentially contribute to several facets of multi-stage epithelial carcinogenesis, including auto- or paracrine growth stimulation, upregulation of angiogenesis, and stromal remodeling.

Animals↗
PubMed1996

The rise and fall of apoptosis during multistage tumorigenesis: down-modulation contributes to tumor progression from angiogenic progenitors.

In a mouse model of multistage tumorigenesis of islet beta-cells, apoptosis was activated concomitant with T-antigen oncogene-induced cell proliferation, further increased in the angiogenic stage, and markedly reduced in solid tumors. Crosses to p53-null mice confirmed this stage-specific variation as a p53-independent apoptotic process. Several apoptosis regulators were expressed, of which bcl-xL was up-regulated in tumors. When overexpressed throughout the pathway, bcl-xL protected most oncogene-expressing cells from apoptosis, enhancing progression from angiogenic progenitor to tumor without affecting earlier transitions. Further, two classes of solid tumor are described, distinguished by size and apoptotic incidence, implicating apoptosis regulation in expansive tumor growth. Thus, down-modulation of apoptosis selectively contributes to late steps in a tumorigenesis pathway.

Animals↗