RESUMO
CD40, a member of the TNF receptor family, is expressed on all mature B cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40 signaling in B cells induced by a fusion protein consisting of the transmembrane part of the Epstein-Barr viral latent membrane protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Because LMP1/CD40-expressing B cells showed an upregulation of CD19, we investigated CD19's function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced lifespan of mature B cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B cells are dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival and proliferation signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K-dependent Erk phosphorylation in human diffuse large B-cell lymphoma cell lines. Our data provide evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40 signaling.
Assuntos
Antígenos CD19/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Linfoma/imunologia , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Humanos , Ativação Linfocitária/imunologia , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Transgênicos , FosforilaçãoRESUMO
Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development.
Assuntos
Sistema Cardiovascular/embriologia , Proteínas Repressoras/fisiologia , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Padronização Corporal , Diferenciação Celular , Proliferação de Células , Primers do DNA , Camundongos , Camundongos Knockout , Músculo Liso/citologia , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Repressoras/genéticaRESUMO
Formalin fixation and paraffin embedding is the standard technique for preserving biological material for both storage and histological analysis. Although recent progress has been made in the molecular analysis of formalin-fixed, paraffin-embedded (FFPE) tissues, proteomic applications are a special challenge due to the cross-linking property of formalin. Here we present the results of a new formalin-free tissue fixative, PAXgene, and demonstrate successful extraction of nondegraded and immunoreactive protein for subsequent standard protein assays, such as Western blot analysis and reverse-phase protein arrays. High amounts of protein can be obtained from PAXgene-fixed, paraffin-embedded (PFPE) mouse liver and human spleen, breast, duodenum, and stomach tissues, similar to frozen material. By Western blot analysis, we found that the detection of membrane, cytoplasmic, nuclear, and phosphorylated protein from PAXgene-fixed human tissue samples was comparable to cryopreserved samples. Furthermore, the distribution of protein in PAXgene-fixed human tissue specimens is adequate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry for in situ proteomic analysis. Taken together, we demonstrate here that PAXgene has great potential to serve as a novel multimodal fixative for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples.
Assuntos
Proteoma/análise , Proteômica/métodos , Análise Serial de Tecidos/métodos , Fixação de Tecidos/métodos , Animais , Biomarcadores/análise , Western Blotting , Eletroforese em Gel Bidimensional , Fixadores , Formaldeído , Humanos , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição Box Pareados/análise , Fatores de Transcrição Box Pareados/genética , Inclusão em Parafina , Proteoma/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Baço/metabolismoRESUMO
Peripheral T cell lymphomas (PTCLs) are highly aggressive malignancies with poor prognosis. Their molecular pathogenesis is not well understood and small animal models for the disease are lacking. Recently, the chromosomal translocation t(5;9)(q33;q22) generating the interleukin-2 (IL-2)-inducible T cell kinase (ITK)-spleen tyrosine kinase (SYK) fusion tyrosine kinase was identified as a recurrent event in PTCL. We show that ITK-SYK associates constitutively with lipid rafts in T cells and triggers antigen-independent phosphorylation of T cell receptor (TCR)-proximal proteins. These events lead to activation of downstream pathways and acute cellular outcomes that correspond to regular TCR ligation, including up-regulation of CD69 or production of IL-2 in vitro or deletion of thymocytes and activation of peripheral T cells in vivo. Ultimately, conditional expression of patient-derived ITK-SYK in mice induces highly malignant PTCLs with 100% penetrance that resemble the human disease. Our work demonstrates that constitutively enforced antigen receptor signaling can, in principle, act as a powerful oncogenic driver. Moreover, we establish a robust clinically relevant and genetically tractable model of human PTCL.
Assuntos
Linfoma de Células T Periférico/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 9/genética , Modelos Animais de Doenças , Células-Tronco Embrionárias/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-2/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/genética , Linfoma de Células T Periférico/patologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , Camundongos , Proteínas Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Baço/enzimologia , Quinase Syk , Translocação GenéticaRESUMO
Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell-like (ABC) subtype of DLBCL is characterized by constitutive NF-kappaB activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11-BCL10-MALT1 (CBM) complexes couple upstream events to IkappaB kinase (IKK)/NF-kappaB activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-kappaB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell-like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-kappaB activity, and decreases the expression of NF-kappaB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.
Assuntos
Inibidores de Caspase , Citotoxicidade Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.
Assuntos
Caspases/metabolismo , Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Ubiquitina/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspases/genética , Linhagem Celular , Proteínas de Ligação a DNA , Ativação Enzimática , Humanos , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.
Assuntos
Caspases/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular , Guanilato Ciclase/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Interleucina-2/metabolismo , Células Jurkat , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Linfócitos T/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , UbiquitinaçãoRESUMO
NF-kappaB (Rel) transcription factors control physiological and pathological immune cell function. The scaffold proteins Bcl-10 and MALT1 couple antigen-receptor signals to the canonical NF-kappaB pathway and are pivotal in lymphomagenesis. Here we found that Bcl-10 and MALT1 differentially regulated B cell receptor-induced activation of RelA and c-Rel. Bcl-10 was essential for recruitment of the kinase IKK into lipid rafts for the activation of RelA and c-Rel, for blocking apoptosis and for inducing division after B cell receptor ligation. In contrast, MALT1 participated in survival signaling but was not involved in IKK recruitment or activation and was dispensable for RelA induction and proliferation. MALT1 selectively activated c-Rel to control a distinct subprogram. Our results provide mechanistic insights into B cell receptor-induced survival and proliferation signals and demonstrate the selective control of c-Rel in the canonical NF-kappaB pathway.
Assuntos
Caspases/metabolismo , Ativação Linfocitária/imunologia , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/fisiologia , Proteína 10 de Linfoma CCL de Células B , Caspases/imunologia , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Citometria de Fluxo , Imunofluorescência , Immunoblotting , Imunoprecipitação , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/metabolismoRESUMO
Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Microdomínios da Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome de Alstrom , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspases/metabolismo , Ativação Enzimática/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Microdomínios da Membrana/genética , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Complexos Multiproteicos/genética , Proteínas de Neoplasias/metabolismo , Proteína Quinase C/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor fas/metabolismoRESUMO
Bcl10 plays an essential role in the adaptive immune response, because Bcl10-deficient lymphocytes show impaired Ag receptor-induced NF-kappaB activation and cytokine production. Bcl10 is a phosphoprotein, but the physiological relevance of this posttranslational modification remains poorly defined. In this study, we report that Bcl10 is rapidly phosphorylated upon activation of human T cells by PMA/ionomycin- or anti-CD3 treatment, and identify Ser(138) as a key residue necessary for Bcl10 phosphorylation. We also show that a phosphorylation-deficient Ser(138)/Ala mutant specifically inhibits TCR-induced actin polymerization yet does not affect NF-kappaB activation. Moreover, silencing of Bcl10, but not of caspase recruitment domain-containing MAGUK protein-1 (Carma1) induces a clear defect in TCR-induced F-actin formation, cell spreading, and conjugate formation. Remarkably, Bcl10 silencing also impairs FcgammaR-induced actin polymerization and phagocytosis in human monocytes. These results point to a key role of Bcl10 in F-actin-dependent immune responses of T cells and monocytes/macrophages.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de IgG/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína 10 de Linfoma CCL de Células B , Células Cultivadas , Humanos , Ativação Linfocitária , NF-kappa B/metabolismo , Fagocitose/genética , Fosforilação , Proteínas Proto-Oncogênicas c-vav/metabolismo , Serina/genética , Serina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
During T cell development in the thymus, high-affinity/avidity TCR engagement induces negative selection by apoptosis, while lower affinity/avidity TCR interactions lead to positive selection and survival of thymocytes. Yet, the mechanisms that discriminate between positive and negative selection are not fully understood. One major regulator of survival and apoptosis in lymphoid cells is the transcription factor NF-kappaB. Several reports have indicated key roles for NF-kappaB in positive and negative selection. In peripheral T cells, TCR ligation activates NF-kappaB through a selective pathway that involves protein kinase Ctheta, Bcl10, and Malt1. While protein kinase Ctheta is dispensable for thymic TCR signaling, the molecular roles of Bcl10 and Malt1 in thymocytes have not been investigated. In the present study, we show that both Bcl10 and Malt1 are essential for TCR signaling in thymocytes as a genetic disruption of either molecule blocks TCR-induced NF-kappaB activation in these cells. To investigate the function of this pathway in thymic selection, we introduced the Bcl10 or Malt1 mutations into three well-established TCR transgenic mouse models. Surprisingly, using several in vivo or in vitro assays, we were unable to demonstrate a role for TCR-induced NF-kappaB activation in either positive or negative selection. Thus, while TCR signaling to NF-kappaB controls the activation of mature T cells, we suggest that this pathway is not involved in the positive or negative selection of thymocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caspases/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Proteína 10 de Linfoma CCL de Células B , Caspases/deficiência , Caspases/genética , Células Cultivadas , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos T CD4-Positivos/imunologia , Caspases/metabolismo , Guanilato Quinases/metabolismo , Quinase I-kappa B/fisiologia , Ativação Linfocitária , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Complexos Multiproteicos/metabolismo , Fosforilação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mutations of the Sonic hedgehog (SHH) receptor, Patched1 (PTCH1), have been identified in a variety of tumors. PTCH1 is usually considered to be a tumor suppressor gene. However, one normal allele is retained in many tumors. We investigated the mechanism of tumorigenesis in murine heterozygous Ptch1 knock-out mice. Here we show that Ptch1 transcripts, which are consistently overexpressed in tumors in these mice, are derived predominantly from the mutated allele. These transcripts give rise to a mutant protein incapable of pathway inhibition. In contrast, the expression of wild-type transcripts in the tumor is reduced. The transcriptional activity of a Ptch1 promoter is sensitive to methylation. Based on these results, we propose a model, in which tumorigenesis begins with the transcriptional silencing of one PTCH1/Ptch1 allele. This alone has no functional consequences. Upon mutational inactivation of the other allele, the resulting loss of PTCH1/Ptch1 function activates PTCH1/Ptch1 transcription from the non-silenced, i.e. the mutant, allele. These events can occur in an opposite order. This model is consistent with the expression of PTCH1/Ptch1-derived transcripts and proteins found in tumors, with the sensitivity of the murine Ptch1 promoter to methylation, and with the recently reported effect of demethylating agents on Ptch1 expression. These latter agents could be effective in treatment of, at least, some tumors associated with loss of PTCH1 function.