Plzf regulates limb and axial skeletal patterning.

The promyelocytic leukaemia zinc finger (Plzf) protein (encoded by the gene Zfp145) belongs to the POZ/zinc-finger family of transcription factors. Here we generate Zfp145-/- mice and show that Plzf is essential for patterning of the limb and axial skeleton. Plzf inactivation results in patterning defects affecting all skeletal structures of the limb, including homeotic transformations of anterior skeletal elements into posterior structures. We demonstrate that Plzf acts as a growth-inhibitory and pro-apoptotic factor in the limb bud. The expression of members of the abdominal b (Abdb) Hox gene complex, as well as genes encoding bone morphogenetic proteins (Bmps), is altered in the developing limb of Zfp145-/- mice. Plzf regulates the expression of these genes in the absence of aberrant polarizing activity and independently of known patterning genes. Zfp145-/- mice also exhibit anterior-directed homeotic transformation throughout the axial skeleton with associated alterations in Hox gene expression. Plzf is therefore a mediator of anterior-to-posterior (AP) patterning in both the axial and appendicular skeleton and acts as a regulator of Hox gene expression.

Pten is essential for embryonic development and tumour suppression.

The PTEN gene encodes a dual-specificity phosphatase mutated in a variety of human cancers. PTEN germline mutations are found in three related human autosomal dominant disorders, Cowden disease (CD), Lhermitte-Duclos disease (LDD) and Bannayan-Zonana syndrome (BZS), characterized by tumour susceptibility and developmental defects. To examine the role of PTEN in ontogenesis and tumour suppression, we disrupted mouse Pten by homologous recombination. Pten inactivation resulted in early embryonic lethality. Pten-/- ES cells formed aberrant embryoid bodies and displayed an altered ability to differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/- mice and chimaeric mice derived from Pten+/- ES cells showed hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell, gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhanced the ability of ES cells to generate tumours in nude and syngeneic mice, due to increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development.

Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse.

The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27(KIP1) expression have been detected in most advanced prostate cancers. But mice deficient for Cdkn1b (encoding p27(Kip1)) do not develop prostate cancer. PTEN activity leads to the induction of p27(KIP1) expression, which in turn can negatively regulate the transition through the cell cycle. Thus, the inactivation of p27(KIP1) may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten(+/-)/Cdkn1b(-/-) mice. Moreover, Pten(+/-)/Cdkn1b(-/-) mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27(Kip1) through the control of cell-cycle progression.

Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL.

Acute promyelocytic leukaemia (APL), associated with chromosomal translocations involving the retinoic acid receptor alpha gene (RARA) and the PML gene, is sensitive to retinoic acid (RA) treatment, while APL patients harbouring translocations between RARA and the PLZF gene do not respond to RA. We have generated PML-RARA and PLZF-RARA transgenic mice and show here that these fusion proteins play a critical role in leukaemogenesis and in determining responses to RA in APL, because PLZF-RARA transgenic mice develop RA-resistant leukaemia, while PML-RARA mice are responsive to RA treatment. We demonstrate that both PML-RARalpha and PLZF-RARalpha fusion proteins can act as transcriptional repressors and are able to interact with nuclear receptor transcriptional co-repressors, such as SMRT. PLZF-RARalpha, but not PML-RARalpha, can form, via its PLZF moiety, co-repressor complexes which are insensitive to RA. Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARalpha and PLZF-RARalpha as well as the unresponsiveness of PLZF-RARalpha-expressing leukaemic cells to RA. Thus, our findings unravel a crucial role for transcriptional silencing in APL pathogenesis and resistance to RA in APL.

PML is essential for multiple apoptotic pathways.

The PML gene of acute promyelocytic leukaemia (APL) encodes a cell growth and tumour suppressor, however, the mechanisms by which PML suppresses tumorigenesis are poorly understood. We show here that Pml is required for Fas- and caspase-dependent DNA-damage-induced apoptosis. We also found that Pml is essential for induction of programmed cell death by Fas, tumour necrosis factor alpha (TNF), ceramide and type I and II interferons (IFNs). As a result, Pml-/- mice and cells are protected from the lethal effects of ionizing radiation and anti-Fas antibody. Pml is required for caspase 1 and caspase 3 activation upon exposure to these stimuli. The PML-RAR alpha fusion protein of APL renders haemopoietic progenitor cells resistant to Fas-, TNF- and IFN-induced apoptosis with a lack of caspase 3 activation, thus acting as a Pml dominant-negative product. These results demonstrate that Pml is a mediator of multiple apoptotic signals, and implicate inhibition of apoptosis in the pathogenesis of APL.

Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.

The complement system plays a paradoxical role in the development and expression of autoimmunity in humans. The activation of complement in systemic lupus erythematosus (SLE) contributes to tissue injury. In contrast, inherited deficiency of classical pathway components, particularly C1q (ref. 1), is powerfully associated with the development of SLE. This leads to the hypothesis that a physiological action of the early part of the classical pathway protects against the development of SLE (ref. 2) and implies that C1q may play a key role in this respect. C1q-deficient (C1qa-/-) mice were generated by gene targeting and monitored for eight months. C1qa-/- mice had increased mortality and higher titres of autoantibodies, compared with strain-matched controls. Of the C1qa-/- mice, 25% had glomerulonephritis with immune deposits and multiple apoptotic cell bodies. Among mice without glomerulonephritis, there were significantly greater numbers of glomerular apoptotic bodies in C1q-deficient mice compared with controls. The phenotype associated with C1q deficiency was modified by background genes. These findings are compatible with the hypothesis that C1q deficiency causes autoimmunity by impairment of the clearance of apoptotic cells.

A RA-dependent, tumour-growth suppressive transcription complex is the target of the PML-RARalpha and T18 oncoproteins.

PML and Tif1a are fused to RARA and Braf, respectively, resulting in the production of PML-RARalpha and Tif1alpha-B-Raf (T18) oncoproteins. Here we show that PML, Tif1alpha and RXRalpha/RARalpha function together in a transcription complex that is dependent on retinoic acid (RA). We found that PML acts as a ligand-dependent coactivator of RXRalpha/RARalpha. PML interacts with Tif1alpha and CBP. In Pml-/- cells, the RA-dependent induction of genes such as RARB2 and the ability of Tif1alpha and CBP to act as transcriptional coactivators on RA are impaired. We show that both PML and Tif1alpha are growth suppressors required for the growth-inhibitory activity of RA. T18, similar to PML-RARalpha, disrupts the RA-dependent activity of this complex in a dominant-negative manner resulting in a growth advantage. Our data define a new pathway for the control of cell growth and tumorigenesis, and provide a new model for the pathogenesis of acute promyelocytic leukaemia (APL).

The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation.

Structural alterations of the promoter region of the BCL-6 proto-oncogene represent the most frequent genetic alteration associated with non-Hodgkin lymphoma, a malignancy often deriving from germinal-centre B cells. The BCL-6 gene encodes a zinc-finger transcriptional repressor normally expressed in both B cells and CD4+ T cells within germinal centres, but its precise function is unknown. We show that mice deficient in BCL-6 displayed normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell-dependent antibody responses. This defect included a complete lack of affinity maturation and was due to the inability of follicular B cells to proliferate and form germinal centres. In addition, BCL-6-deficient mice developed an inflammatory response in multiple organs characterized by infiltrations of eosinophils and IgE-bearing B lymphocytes typical of a Th2-mediated hyperimmune response. Thus, BCL-6 functions as a transcriptional switch that controls germinal centre formation and may also modulate specific T-cell-mediated responses. Altered expression of BCL-6 in lymphoma represents a deregulation of the pathway normally leading to B cell proliferation and germinal centre formation.

The transcriptional role of PML and the nuclear body.

The PML gene encodes a tumour suppressor protein associated with a distinct subnuclear domain, the nuclear body. Various functions have been attributed to the PML nuclear body, but its main biochemical role is still unclear. Recent findings indicate that PML is essential for the proper formation of the nuclear body and can act as a transcriptional co-factor. Here we summarize the current understanding of the biological functions of PML and the nuclear body, and discuss a role for these intra-nuclear structures in the regulation of transcription.

The function of PML in p53-dependent apoptosis.

The PML gene of acute promyelocytic leukaemia (APL) encodes a growth- and tumour-suppresor protein that is essential for several apoptotic signals. The mechanisms by which PML exerts its pro-apoptotic function are still unknown. Here we show that PML acts as a transcriptional co-activator with p53. PML physically interacts with p53 both in vitro and in vivo and co-localizes with p53 in the PML nuclear body (PML-NB). The co-activatory role of PML depends on its ability to localize in the PML-NB. p53-dependent, DNA-damage-induced apoptosis, transcriptional activation by p53, the DNA-binding ability of p53, and the induction of p53 target genes such as Bax and p21 upon gamma-irradiation are all impaired in PML-/- primary cells. These results define a new PML-dependent, p53-regulatory pathway for apoptosis and shed new light on the function of PML in tumour suppression.

Promyelocytic leukemia protein (PML) and Daxx participate in a novel nuclear pathway for apoptosis.

The promyelocytic leukemia protein (PML) gene of acute promyelocytic leukemia (APL) encodes a cell growth and tumor suppressor essential for multiple apoptotic signals. Daxx was identified as a molecule important for the cytoplasmic transduction of the Fas proapoptotic stimulus. Here, we show that upon mitogenic activation of mature splenic lymphocytes, Daxx is dramatically upregulated and accumulates in the PML nuclear body (NB) where PML and Daxx physically interact. In the absence of PML, Daxx acquires a dispersed nuclear pattern, and activation-induced cell death of splenocytes is profoundly impaired. PML inactivation results in the complete abrogation of the Daxx proapoptotic ability. In APL cells, Daxx is delocalized from the NB. Upon retinoic acid treatment, which induces disease remission in APL, Daxx relocalizes to the PML NBs. These results indicate that PML and Daxx cooperate in a novel NB-dependent pathway for apoptosis and shed new light in the role of PML in tumor suppression.

Role of promyelocytic leukemia (PML) protein in tumor suppression.

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor alpha (RARalpha) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARalpha oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML(-/-) mice with human cathepsin G (hCG)-PMLRARalpha or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARalpha TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARalpha TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARalpha are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.

FES-Cre targets phosphatidylinositol glycan class A (PIGA) inactivation to hematopoietic stem cells in the bone marrow.

A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA(-) cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA(-) blood cells. The loss of GPI-linked proteins occurred mainly in c-kit(+)CD34(+)Lin(-) progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA(-) cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.

p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).

p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).

Phosphoinositide 3-kinase-dependent membrane recruitment of p62(dok) is essential for its negative effect on mitogen-activated protein (MAP) kinase activation.

A major pathway by which growth factors, such as platelet-derived growth factor (PDGF), regulate cell proliferation is via the receptor tyrosine kinase/Ras/mitogen-activated protein kinase (MAPK) signaling cascade. The output of this pathway is subjected to tight regulation of both positive and negative regulators. One such regulator is p62(dok), the prototype of a newly identified family of adaptor proteins. We recently provided evidence, through the use of p62(dok)-deficient cells, that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation and the Ras/MAPK pathway. We show here that reintroduction of p62(dok) into p62(dok)-(/)- cells can suppress the increased cell proliferation and prolonged MAPK activity seen in these cells, and that plasma membrane recruitment of p62(dok) is essential for its function. We also show that the PDGF-triggered plasma membrane translocation of p62(dok) requires activation of phosphoinositide 3-kinase (PI3-kinase) and binding of its pleckstrin homology (PH) domain to 3'-phosphorylated phosphoinositides. Furthermore, we demonstrate that p62(dok) can exert its negative effect on the PDGFR/MAPK pathway independently of its ability to associate with RasGAP and Nck. We conclude that p62(dok) functions as a negative regulator of the PDGFR/Ras/MAPK signaling pathway through a mechanism involving PI3-kinase-dependent recruitment of p62(dok) to the plasma membrane.

Mitochondrial basis for immune deficiency. Evidence from purine nucleoside phosphorylase-deficient mice.

We generated purine nucleoside phosphorylase (PNP)-deficient mice to gain insight into the mechanism of immune deficiency disease associated with PNP deficiency in humans. Similar to the human disease, PNP deficiency in mice causes an immunodeficiency that affects T lymphocytes more severely than B lymphocytes. PNP knockout mice exhibit impaired thymocyte differentiation, reduced mitogenic and allogeneic responses, and decreased numbers of maturing thymocytes and peripheral T cells. T lymphocytes of PNP-deficient mice exhibit increased apoptosis in vivo and higher sensitivity to gamma irradiation in vitro. We propose that the immune deficiency in PNP deficiency is a result of inhibition of mitochondrial DNA repair due to the accumulation of dGTP in the mitochondria. The end result is increased sensitivity of T cells to spontaneous mitochondrial DNA damage, leading to T cell depletion by apoptosis.

Rearrangements and aberrant expression of the retinoic acid receptor alpha gene in acute promyelocytic leukemias.

Although acute promyelocytic leukemias (APLs) are consistently associated with a reciprocal chromosome 15;17 translocation, the gene(s) directly affected by the breakpoints have never been isolated. The chromosome 17 breakpoint maps to near the retinoic acid receptor alpha (RAR alpha) locus. Investigation of 20 APLs and a large series of other neoplastic patients and normal controls revealed RAR alpha gene rearrangements and aberrant transcripts only in the APL cases. These findings suggest that the RAR alpha gene is involved in the APL chromosome 17 breakpoint, is implicated in leukemogenesis, and could be used as a marker for identifying leukemic promyelocytes.

Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation.

Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) alpha expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARalpha and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARalpha degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.

Mouse models for multistep tumorigenesis.

The mouse is an ideal model system for studying the molecular mechanisms underlying the pathogenesis of human cancer. The generation of transgenic and gene-knockout mice has been instrumental in determining the role of major determinants in this process, such as oncogenes and tumor-suppressor genes. In the past few years, modeling cancer in the mouse has increased in its complexity, allowing in vivo dissection of the fundamental concepts underlying cooperative oncogenesis in various tumor types. In this review, we discuss how this transition has been facilitated, providing relevant examples. We also review how, in the post-genome era, novel methodologies will further accelerate the study of multi-step tumorigenesis in the mouse.