Luis M. Criado

Increased phosphoinositide 3-kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo

Alterations in the cell division:cell death ratio induce multiple autoimmune and transformation processes. Phosphoinositide 3‐kinase (PI3K) controls cell division and cell death in vitro, but its effect on the function of the cellular immune system and on tumor formation in mammals is poorly characterized. Here we show that transgenic mice expressing in T lymphocytes an active form of PI3K derived from a thymic lymphoma, p65PI3K, developed an infiltrating lymphoproliferative disorder and autoimmune renal disease with an increased number of T lymphocytes exhibiting a memory phe‐notype and reduced apoptosis. This pathology was strikingly similar to that described in mice exhibiting heterozygous loss of the tumor suppressor PTEN, a lipid and protein phosphatase. We show that overexpression of PTEN selectively blocks p65PI3K‐induced 3T3 fibroblast transformation. Moreover, the early development of T cell lymphomas in p65PI3K Tg p53_/_ mice indicated that PI3K contributes to tumor development. These observations demonstrate that constitutive activation of PI3K extends T cell survival in vivo, affects T cell homeostasis, and contributes to tumor generation, supporting a model in which selective increases in one type of PTEN substrate, the PI3K‐derived lipid products, induce tumorigenesis. PI3K thus emerges as a potential target in autoimmune disease and cancer therapy.—R.‐Borlado, L., Redondo, C., Alvarez, B., Jimenez, C., Criado, L. M., Flores, J., Marcos, M. A. R., Martinez‐A., C., Balomenos, D., Carrera, A. C. Increased phosphoinositide 3‐kinase activity induces a lymphoproliferative disorder and contributes to tumor generation in vivo. FASEB J. 14, 895–903 (2000)

Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease

Interaction of chemokines with their specific receptors results in tight control of leukocyte migration and positioning. CCR8 is a chemokine receptor expressed mainly in CD4+ single-positive thymocytes and Th2 cells. We generated CCR8-deficient mice (CCR8−/−) to study the in vivo role of this receptor, and describe in this study the CCR8−/− mouse response in OVA-induced allergic airway disease using several models, including an adoptive transfer model and receptor-blocking experiments. All CCR8−/− mice developed a pathological response similar to that of wild-type animals with respect to bronchoalveolar lavage cell composition, peripheral blood and bone marrow eosinophilia, lung infiltrates, and Th2 cytokine levels in lung and serum. The results contrast with a recent report using one of the OVA-induced asthma models studied here. Similar immune responses were also observed in CCR8−/− and wild-type animals in a different model of ragweed allergen-induced peritoneal eosinophilic inflammation, with an equivalent number of eosinophils and analogous increased levels of Th2 cytokines in peritoneum and peripheral blood. Our results show that allergic diseases course without critical CCR8 participation, and suggest that further work is needed to unravel the in vivo role of CCR8 in Th2-mediated pathologies.

Blocked negative selection of developing T cells in mice expressing the baculovirus p35 caspase inhibitor

Clonal deletion in the thymus by apoptosis is involved in purging the immune system of self‐reactive T lymphocytes (negative selection). Cysteine proteases (caspases) belonging to the CPP32 family are activated during this process. We have produced transgenic mice expressing baculovirus p35, a broad‐range caspase inhibitor. Thymocytes from p35 transgenic mice were resistant in vitro to several apoptosis‐inducing agents; this resistance correlated with the inhibition of CPP32‐like activity. Negative selection in vivo of thymocytes triggered by two exogenous antigens, staphylococcal enterotoxin B superantigen and an antigenic peptide in the F5 T‐cell receptor transgenic model, was specifically inhibited in p35 transgenic mice. Our results provide direct evidence for caspase involvement in negative selection during thymocyte development.

Dido gene expression alterations are implicated in the induction of hematological myeloid neoplasms

The myelodysplastic/myeloproliferative diseases (MDS/MPDs) are a heterogeneous group of myeloid neoplasms that share characteristics with chronic myeloproliferative diseases and myelodysplastic syndromes. The broad spectrum of clinical manifestations makes MDS/MPDs extremely difficult to diagnose and treat, with a median survival time of 1-5 years. No single gene defect has been firmly associated with MDS/MPDs, and no animal models have been developed for these diseases. The association of deletions on chromosome 20q with myeloid malignancies suggests the presence of unidentified tumor suppressor genes in this region. Here we show that the recently identified death inducer-obliterator (Dido) gene gives rise to at least 3 polypeptides (Dido1, Dido2, and Dido3) through alternative splicing, and we map the human gene to the long arm of chromosome 20. We found that targeting of murine Dido caused a transplantable disease whose symptoms and signs suggested MDS/MPDs. Furthermore, 100% of human MDS/MPD patients analyzed showed Dido expression abnormalities, which we also found in other myeloid but not lymphoid neoplasms or in healthy donors. Our findings suggest that Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPD diseases and testing new approaches to their diagnosis and treatment.

Genetic inactivation of Par4 results in hyperactivation of NF-κB and impairment of JNK and p38

The Par4 gene was first identified in prostate cells undergoing apoptosis after androgen withdrawal. PAR4 was subsequently shown to interact with, and inhibit, atypical protein kinase C isoforms, functioning as a negative regulator of the NF‐κB pathway. This may explain its pro‐apoptotic function in overexpression experiments. To determine the physiological role of PAR4, we have derived primary embryonic fibroblasts (EFs) from Par4−/− mice. We show here that loss of PAR4 leads to a reduction in the ability of tumour necrosis factor‐α (TNF‐α) to induce apoptosis by increased activation of NF‐κB. Consistent with recent reports demonstrating the antagonistic actions of NF‐κB and c‐Jun amino‐terminal kinase (JNK) signalling, we have found that Par4−/− cells show a reduced activation of the sustained phase of JNK and p38 stimulation by TNF‐α and interleukin 1. Higher levels of an anti‐apoptotic JNK‐inhibitor protein, X‐chromosome‐linked inhibitor of apoptosis, in Par4−/− EFs might explain the inhibition of JNK activation in these cells.

Ectopic Meis1 expression in the mouse limb bud alters P-D patterning in a Pbx1-independent manner

During limb development, expression of the TALE homeobox transcription factor Meis1 is activated by retinoic acid in the proximal-most limb bud regions, which give rise to the upper forelimb and hindlimb. Early subdivision of the limb bud into proximal Meis-positive and distal Meis-negative domains is necessary for correct proximo-distal (P-D) limb development in the chick, since ectopic Meis1 overexpression abolishes distal limb structures, produces a proximal shift of limb identities along the P-D axis, and proximalizes distal limb cell affinity properties. To determine whether Meis activity is also required for P-D limb specification in mammals, we generated transgenic mice ectopically expressing Meis1 in the distal limb mesenchyme under the control of the Msx2 promoter. Msx2:Meis1 transgenic mice display altered P-D patterning and shifted P-D Hox gene expression domains, similar to those previously described for the chicken. Meis proteins function in cooperation with PBX factors, another TALE homeodomain subfamily. Meis-Pbx interaction is required for nuclear localization of both proteins in cell culture, and is important for their DNA-binding and transactivation efficiency. During limb development, Pbx1 nuclear expression correlates with the Meis expression domain, and Pbx1 has been proposed as the main Meis partner in this context; however, we found that Pbx1 deficiency did not modify the limb phenotype of Msx2:Meis1 mice. Our results indicate a conserved role of Meis activity in P-D specification of the tetrapod limb and suggest that Pbx function in this context is either not required or is provided by partners other than Pbx1.

Inhibitor of apoptosis protein from Orgyia pseudotsugata nuclear polyhedrosis virus provides a costimulatory signal required for optimal proliferation of developing thymocytes.

The inhibitors of apoptosis proteins (IAPs) constitute a family of endogenous inhibitors that control apoptosis in the cell by inhibiting caspase processing and activity. IAPs are also implicated in cell division, cell cycle regulation, and cancer. To address the role of IAPs in thymus development and homeostasis, we generated transgenic mice expressing IAP generated from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP). Developing thymocytes expressing OpIAP show increased nuclear levels of NF-κB and reduced cytoplasmic levels of its inhibitor, IκBα. In mature thymocytes, OpIAP induces optimal activation and proliferation after TCR triggering in the absence of a costimulatory signal. OpIAP expression in immature thymocytes blocks TCR-induced apoptosis. Taken together, our data illustrate the pleiotropism of OpIAP in vivo.

Normal cellular senescence and cancer susceptibility in mice genetically deficient in Ras-induced senescence-1 (Ris1).

Oncogenic Ras triggers a permanent cell-cycle arrest known as oncogene-induced senescence (OIS) that constitutes a relevant tumor suppressor mechanism. Ris1 (Ras-induced senescence-1) is a novel gene that was identified in a screen as specifically upregulated during Ras-induced senescence, and that is located at a chromosomal region, 3p21.3, frequently lost in human cancer. Moreover, Ris1 is highly conserved in vertebrates, does not present paralogs, and its sequence does not reveal similarities with other proteins or domains. To analyse the physiological function of Ris1 and test its putative role as a tumor suppressor gene, we have generated mutant mice deficient for this gene. Ris1-null mice are viable, fertile, develop normally and do not display any obvious abnormalities. Of relevance, Ris1-deficient mice had a normal lifespan and did not exhibit predisposition to spontaneous tumors or to tumors induced by chemical carcinogens. Finally, Ris1-deficient embryonic fibroblasts were indistinguishable from wild-type cells regarding their proliferation properties, immortalization, senescence and oncogenic transformation. These findings do not support a role of Ris1 in tumor suppression or in OIS.

Exacerbated inflammatory responses in transgenic mice expressing an inhibitor of apoptosis protein (OpIAP)

AbstractMembers of the inhibitor of apoptosis protein family are involved not only in suppressing apoptosis, but also in signal transduction, cell division, and are associated with some types of cancers. Here we show that transgenic expression of the inhibitor of apoptosis protein OpIAP in murine T lymphocytes leads to a significant increase in T-cell receptor-induced cell activation, proliferation and cytokine production. Transgenic T lymphocytes expressing OpIAP have a lower proliferation threshold in response to T-cell receptor stimulation. Unstimulated OpIAP transgenic T lymphocytes show elevated nuclear levels of NF-κB transcription factor that increase after in vivo antigen peptide treatment. OpIAP transgenic animals present an exacerbated inflammatory response in an experimental contact hypersensitivity model, suggesting increased T-cell activation in vivo. These data indicate a new role for the inhibitor of apoptosis proteins in T-lymphocyte activation and proliferation.