Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1α, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1α by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1α ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

Acquired, nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice

We previously generated a transgenic mouse model for acute promyelocytic leukemia (APL) by expressing the promyelocytic leukemia (PML)–retinoic acid receptor (RARα) cDNA in early myeloid cells. This fusion protein causes a myeloproliferative disease in 100% of animals, but only 15–20% of the animals develop acute leukemia after a long latency period (6–13 months). PML-RARα is therefore necessary, but not sufficient, for APL development. The coexpression of a reciprocal form of the fusion, RARα-PML, increased the likelihood of APL development (55–60%), but did not shorten latency. Together, these results suggested that additional genetic events are required for the development of APL. We therefore evaluated the splenic tumor cells from 18 transgenic mice with APL for evidence of secondary genetic events, by using spectral karyotyping analysis. Interstitial or terminal deletions of the distal region of one copy of chromosome 2 [del(2)] were found in 1/5 tumors expressing PML-RARα, but in 11/13 tumors expressing both PML-RARα and RARα-PML (P < 0.05). Leukemic cells that contained a deletion on chromosome 2 often contained additional chromosomal gains (especially of 15), chromosomal losses (especially of 11 or X/Y), or were tetraploid (P ≤ 0.001). These changes did not commonly occur in nontransgenic littermates, nor in aged transgenic mice that did not develop APL. These results suggest that expression of RARα-PML increases the likelihood of chromosome 2 deletions in APL cells. Deletion 2 appears to predispose APL cells to further chromosomal instability, which may lead to the acquisition of additional changes that provide an advantage to the transformed cells.

Vascular tumors in livers with targeted inactivation of the von Hippel–Lindau tumor suppressor

von Hippel–Lindau (VHL) disease is a pleomorphic familial tumor syndrome that is characterized by the development of highly vascularized tumors. Homozygous disruption of the VHL gene in mice results in embryonic lethality. To investigate VHL function in the adult we have generated a conditional VHL null allele (2-lox allele) and null allele (1-lox allele) by Cre-mediated recombination in embryonic stem cells. We show here that mice heterozygous for the 1-lox allele develop cavernous hemangiomas of the liver, a rare manifestation of the human disease. Histologically these tumors were associated with hepatocellular steatosis and focal proliferations of small vessels. To study the cellular origin of these lesions we inactivated VHL tissue-specifically in hepatocytes. Deletion of VHL in the liver resulted in severe steatosis, many blood-filled vascular cavities, and foci of increased vascularization within the hepatic parenchyma. These histopathological changes were similar to those seen in livers from mice heterozygous for the 1-lox allele. Hypoxia-inducible mRNAs encoding vascular endothelial growth factor, glucose transporter 1, and erythropoietin were up-regulated. We thus provide evidence that targeted inactivation of mouse VHL can model clinical features of the human disease and underline the importance of the VHL gene product in the regulation of hypoxia-responsive genes in vivo.

A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells

Ovarian carcinomas are thought to arise from cells of the ovarian surface epithelium by mechanisms that are poorly understood. Molecules associated with neoplasia are potentially immunogenic, but few ovarian tumor antigens have been identified. Because ovarian carcinomas can elicit humoral responses in patients, we searched for novel tumor antigens by immunoscreening a cDNA expression library with ovarian cancer patient serum. Seven clones corresponding to the homeobox gene HOXB7 were isolated. ELISAs using purified recombinant HOXB7 protein revealed significant serologic reactivity to HOXB7 in 13 of 39 ovarian cancer patients and in only one of 29 healthy women (P < 0.0001). Ovarian carcinomas were found to express HOXB7 at markedly higher levels than normal ovarian surface epithelium, suggesting that immunogenicity of HOXB7 in patients could be associated with its elevated expression in ovarian carcinomas. Overexpression of HOXB7 in immortalized normal ovarian surface epithelial cells dramatically enhanced cellular proliferation. Furthermore, HOXB7 overexpression increased intracellular accumulation and secretion of basic fibroblast growth factor, a potent angiogenic and mitogenic factor. These results reveal HOXB7 as a tumor antigen whose up-regulated expression could play a significant role in promoting growth and development of ovarian carcinomas.

Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1

The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.

T cell-specific FADD-deficient mice: FADD is required for early T cell development

FADD/Mort1, initially identified as a Fas-associated death-domain containing protein, functions as an adapter molecule in apoptosis initiated by Fas, tumor necrosis factor receptor-I, DR3, and TRAIL-receptors. However, FADD likely participates in additional signaling cascades. FADD-null mutations in mice are embryonic-lethal, and analysis of FADD−/− T cells from RAG-1−/− reconstituted chimeras has suggested a role for FADD in proliferation of mature T cells. Here, we report the generation of T cell-specific FADD-deficient mice via a conditional genomic rescue approach. We find that FADD-deficiency leads to inhibition of T cell development at the CD4−CD8− stage and a reduction in the number of mature T cells. The FADD mutation does not affect apoptosis or the proximal signaling events of the pre-T cell receptor; introduction of a T cell receptor transgene fails to rescue the mutant phenotype. These data suggest that FADD, through either a death-domain containing receptor or a novel receptor-independent mechanism, is required for the proliferative phase of early T cell development.

Glutamate antagonists limit tumor growth

Neuronal progenitors and tumor cells possess propensity to proliferate and to migrate. Glutamate regulates proliferation and migration of neurons during development, but it is not known whether it influences proliferation and migration of tumor cells. We demonstrate that glutamate antagonists inhibit proliferation of human tumor cells. Colon adenocarcinoma, astrocytoma, and breast and lung carcinoma cells were most sensitive to the antiproliferative effect of the N-methyl-d-aspartate antagonist dizocilpine, whereas breast and lung carcinoma, colon adenocarcinoma, and neuroblastoma cells responded most favorably to the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate antagonist GYKI52466. The antiproliferative effect of glutamate antagonists was Ca2+ dependent and resulted from decreased cell division and increased cell death. Morphological alterations induced by glutamate antagonists in tumor cells consisted of reduced membrane ruffling and pseudopodial protrusions. Furthermore, glutamate antagonists decreased motility and invasive growth of tumor cells. These findings suggest anticancer potential of glutamate antagonists.

CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia

This report describes a tumor-associated antigen, termed CML66, initially cloned from a chronic myelogenous leukemia (CML) cDNA expression library. CML66 encodes a 583-aa protein with a molecular mass of 66 kDa and no significant homology to other known genes. CML66 gene is localized to human chromosome 8q23, but the function of this gene is unknown. CML66 is expressed in leukemias and a variety of solid tumor cell lines. When examined by Northern blot, expression in normal tissues was restricted to testis and heart, and no expression was found in hematopoietic tissues. When examined by quantitative reverse transcription–PCR, expression in CML cells was 1.5-fold higher than in normal peripheral blood mononuclear cells. The presence of CML66-specific antibody in patient serum was confirmed by Western blot and the development of high titer IgG antibody specific for CML66 correlated with immune induced remission of CML in a patient who received infusion of normal donor lymphocytes for treatment of relapse. CML66 antibody also was found in sera from 18–38% of patients with lung cancer, melanoma, and prostate cancer. These findings suggest that CML66 may be immunogenic in a wide variety of malignancies and may be a target for antigen-specific immunotherapy.

Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.

Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.

Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.

Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.

Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53.

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these effects are unclear. One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects. In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53. p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction. We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain. As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner. In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1. In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements. These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells.

Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11.

Prostate cancer is the second leading cause of male cancer deaths in the United States. Yet, despite a large international effort, little is known about the molecular mechanisms that underlie this devastating disease. Prostate secretory epithelial cells and androgen-dependent prostate carcinomas undergo apoptosis in response to androgen deprivation and, furthermore, most prostate carcinomas become androgen independent and refractory to further therapeutic manipulations during disease progression. Definition of the genetic events that trigger apoptosis in the prostate could provide important insights into critical pathways in normal development as well as elucidate the perturbations of those key pathways in neoplastic transformation. We report the functional definition of a novel genetic locus within human chromosome 10pter-q11 that mediates both in vivo tumor suppression and in vitro apoptosis of prostatic adenocarcinoma cells. A defined fragment of human chromosome 10 was transferred via microcell fusion into a prostate adenocarcinoma cell line. Microcell hybrids containing only the region 10pter-q11 were suppressed for tumorigenicity following injection of microcell hybrids into nude mice. Furthermore, the complemented hybrids undergo programmed cell death in vitro via a mechanism that does not require nuclear localization of p53. These data functionally define a novel genetic locus, designated PAC1, for prostate adenocarcinoma 1, involved in tumor suppression of human prostate carcinoma and furthermore strongly suggest that the cell death pathway can be functionally restored in prostatic adenocarcinoma.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.