Biomedical subjects

Hiroyuki Miyoshi

Publications and source records attributed to Hiroyuki Miyoshi.

At least 37 records · Page 2Linked to original sources

PubMed2005

A specific region of 37 amino acid residues in the SPRY (B30.2) domain of African green monkey TRIM5alpha determines species-specific restriction of simian immunodeficiency virus SIVmac infection.

Human immunodeficiency virus type 1 (HIV-1) efficiently enters cells of Old World monkeys but encounters a block before reverse transcription. This restriction is mediated by a dominant repressive factor. Recently, a member of the tripartite motif (TRIM) family proteins, TRIM5alpha, was identified as a blocking factor in a rhesus macaque cDNA library. Among Old World monkey cell lines, the African green monkey kidney cell line CV1 is highly resistant to not only HIV-1 but also simian immunodeficiency virus SIVmac infection. We analyzed TRIM5alpha of CV1 cells and HSC-F cells, a T-cell line from a cynomolgus monkey, and found that both CV1- and HSC-F-TRIM5alphas could inhibit CD4-dependent HIV-1 infection, as well as vesicular stomatitis virus glycoprotein-mediated infection. CV1-TRIM5alpha could also inhibit SIVmac infection, whereas HSC-F-TRIM5alpha could not. In the SPRY (B30.2) domain of CV1-TRIM5alpha, there was a 20-amino-acid duplication that was not present in HSC-F-TRIM5alpha. A chimeric TRIM5alpha containing 37 amino acid residues from CV1-TRIM5alpha, which spanned the 20-amino-acid duplication, in the background of HSC-F-TRIM5alpha fully gained the ability to inhibit SIVmac infection. Conversely, the mutant CV1-TRIM5alpha lacking the 20-amino-acid duplication completely lost the ability to restrict SIVmac infection. These findings clearly indicated that a specific region of 37 amino acid residues in the SPRY domain of CV1-TRIM5alpha contained a determinant of species-specific restriction of SIVmac.

Amino Acid Sequence↗

PubMed2004

A targeted mutation of Nkd1 impairs mouse spermatogenesis.

Nkd1 is an antagonist of the canonical Wnt/beta-catenin signaling pathway. The EF-hand motif of Nkd1 is required for its inhibitory function. Early studies suggested that Nkd1 might play important roles in mouse embryonic development and tumorigenesis. We constructed Nkd1(-/-) mice whose Nkd1 protein lacked the EF-hand and was unable to inhibit Wnt/beta-catenin signaling. The homozygotes were viable and grew normally, but their fertility in males was reduced. In wild-type adult testes, Nkd1 mRNA was expressed more abundantly in the elongating spermatids than in the round spermatids. Lack of EF-hand caused reductions in the testis weight and sperm count by 30 and 60%, respectively. During testis development, Nkd1 mRNA expression started at the 25th day after birth, coincident with the onset of Wnt1 expression. Nuclear localization of beta-catenin increased in the elongating spermatids, suggesting that the mutant Nkd1 failed to inhibit the Wnt/beta-catenin pathway. These results suggest that deletion of the EF-hand from Nkd1 reduces the number of the elongating spermatids at haploid stage. In contrast, the mutant Nkd1 did not affect intestinal polyposis in Apc(Delta716) mice.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Mac-1(low) early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration.

Recent studies have shown that bone marrow (BM) cells, including the BM side population (BM-SP) cells that enrich hematopoietic stem cells (HSCs), are incorporated into skeletal muscle during regeneration, but it is not clear how and what kinds of BM cells contribute to muscle fiber regeneration. We found that a large number of SP cells migrated from BM to muscles following injury in BM-transplanted mice. These BM-derived SP cells in regenerating muscles expressed different surface markers from those of HSCs and could not reconstitute the mouse blood system. BM-derived SP/Mac-1(low) cells increased in number in regenerating muscles following injury. Importantly, our co-culture studies with activated satellite cells revealed that this fraction carried significant potential for myogenic differentiation. By contrast, mature inflammatory (Mac-1(high)) cells showed negligible myogenic activities. Further, these BM-derived SP/Mac-1(low) cells gave rise to mononucleate myocytes, indicating that their myogenesis was not caused by stochastic fusion with host myogenic cells, although they required cell-to-cell contact with myogenic cells for muscle differentiation. Taken together, our data suggest that neither HSCs nor mature inflammatory cells, but Mac-1(low) early myeloid cells in the BM-derived SP fraction, play an important role in regenerating skeletal muscles.

Animals↗

PubMed2004

Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference.

Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BRAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and beta(1) integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.

Cell Division↗

PubMed2004

A novel maxizyme vector targeting a bcr-abl fusion gene induced specific cell death in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Patients with Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) generally have a poor prognosis and would benefit from the development of new therapeutic approaches. We previously demonstrated that an allosterically controllable ribozyme, maxizyme (Mz), can induce apoptosis in chronic myelogenous leukemia (CML) cells. Ph(+) ALL cells harbor a bcrabl fusion gene (e1a2) encoding a 190-kDa fusion protein (p190) involved in disease pathogenesis. In this study, we have designed a Mz that specifically cleaves e1a2 mRNA and transduced this e1a2Mz into Ph(+) ALL cells using a third-generation lentiviral vector system. In 3 of 5 Ph(+) ALL cell lines, e1a2Mz transduction resulted in a significant decrease in viability and increased cell apoptosis. We observed a decrease in e1a2 mRNA in all Ph(+) ALL cells transduced with e1a2Mz, and the e1a2 mRNA level was higher in e1a2Mz-resistant cells than in e1a2Mz-sensitive cells. All samples of primary Ph(+) ALL cells tested showed e1a2Mz-induced growth inhibition and apoptosis. Importantly, e1a2Mz did not influence the colony formation of normal CD34(+) cord blood cells. These results indicate that e1a2Mz kills Ph(+) ALL cells specifically, suggesting that it may be used as a novel gene therapy strategy for Ph(+) ALL.

Apoptosis↗

PubMed2004

RNA interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARgamma also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARgamma expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARgamma-knockdown method will serve to clarify the PPARgamma pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

3T3-L1 Cells↗

PubMed2004

Generation and targeting of human tumor-specific Tc1 and Th1 cells transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor.

CD4+ Th cells, in particular IFN-gamma-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3zeta. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-gamma in response to CEA+ tumor cell lines but not CEA- tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-gamma and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2(-/-) mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods for the development of effective adoptive immunotherapies.

Animals↗

PubMed2004

Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells.

BACKGROUND: RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells. METHODS: We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors. RESULTS: As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five. CONCLUSION: The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi.

Base Sequence↗

PubMed2004

Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells.

RNA interference (RNAi) is a sequence-specific RNA degradation process mediated by short double-stranded RNAs termed small interfering RNAs. Here, we describe the lentivirus-based vector small interfering RNA system expressing. As a pilot study, we generated constructs expressing small hairpin RNA (shRNA) specific for luciferase gene (shLuc) or green fluorescence protein (shGFP) under the control of human H1 RNA polymerase III promoter. The effect of the shRNA was evaluated against HIV-1 infection in a single-round or multiple-round infectious system using an HIV-1 molecular clone carrying the luc or GFP gene. In the single-round infectious system, cells transduced with shLuc by lentiviral vector significantly reduced (approximately 90% reduction) viral gene expression after challenge infection at a multiplicity of infection of 10. These transduced cells continued to resist against at least four sequentially repeated challenge infections. Importantly, this efficient antiviral activity persisted over 35 days in culture. In a multiple-round infectious system using a replication-competent HIV-1 molecular clone carrying the GFP gene, we also observed that a lentiviral vector expressing shGFP could inhibit HIV-1 replication for at least 1 week. The profound effect of lentiviral shRNA was also observed in human primary monocyte-derived macrophages. Thus, shRNA introduced through the lentiviral vector can be useful for efficient and stable gene suppression in human cells including primary non-dividing cells. Moreover, quantitative analysis of viral cDNA synthesis on challenge infection showed that viral genomic RNAs packaged in incoming virus core might not be targeted by shLuc. Instead, the degradation of transcripts from integrated proviral DNAs might be a major cause of the profound reduction in HIV-1 gene expression by shRNA in our system.

Cell Division↗

PubMed2004

A lentiviral cDNA library employing lambda recombination used to clone an inhibitor of human immunodeficiency virus type 1-induced cell death.

Expression cloning technology of cDNAs is a suitable tool for identifying novel functional properties of genes. Here, we generated a lentiviral cDNA library-expressing system for human T cells based on a site-specific recombination system of phage lambda for transferring cDNA libraries with a minimum loss of its complexity. The library-transduced CD4(+) T cells were challenged with wild-type human immunodeficiency virus type 1 (HIV-1), and the cells that acquired resistance to HIV-1-induced cytopathic effect (CPE) were selected. From these cells, CD14 was isolated and proved to inhibit the entry of HIV-1 and the HIV-1-induced CPE. This cloning system allows rapid identification of genes encoding novel properties in human T cells and probably other mammalian cells.

Apoptosis↗

PubMed2004

Gene delivery to hematopoietic stem cells using lentiviral vectors.

Hematopoietic stem cells (HSCs) are clonogenic cells capable of both selfrenewal and multilineage differentiation. An efficient method for gene transfer into HSCs is required for exploring HSC biology as well as for gene therapy of hematopoietic disorders. Retroviral vectors have been the most widely used vectors for gene transfer to HSCs. However, retroviral vectors require cell division for integration, limiting their use for gene transfer into HSCs that are exclusively quiescent. Although prestimulation of HSCs with cytokines can enhance gene-transfer efficiency, exposure to cytokines also stimulates HSCs to differentiate, resulting in the reduction of long-term repopulating capacity (8-15). In contrast, lentiviral vectors based on the human immunodeficiency virus type 1 (HIV-1) can efficiently transduce human CD34+ cells without cytokine prestimulation and long-term multilineage expression of the transgene is detected in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice after transplantation (16-22). Murine HSCs can also be easily transduced with lentiviral vectors without cytokine prestimulation (23-26). In this chapter, the protocols for transduction of human CD34+ cells that contain HSCs are described. The procedures include the isolation of human CD34+ cells, preparation of lentiviral vectors, transduction of human CD34+ cells, and in vivo analysis of transduced HSCs using NOD/SCID mice.

Animals↗

PubMed2004

Lentiviral gp34/OX40L gene transfer into dendritic cells facilitates alloreactive CD4+ T-cell response in vitro.

Gene-modified dendritic cells (DCs) are promising targets for cancer immunotherapy. In this study, we demonstrated that lentiviral transduction of DCs with the gp34/OX40L gene, one of the costimulatory molecules, facilitates alloreactive CD4+ T-cell response in vitro. We achieved a 20% to 40% efficiency of gp34/OX40L gene transfer into DCs by the lentiviral vector, and lentiviral gp34/OX40L gene transfer did not alter the surface phenotype of either immature or mature DCs, suggesting that expression of gp34/OX40L did not induce the maturation of immature DCs and that gp34/OX40L-transduced DCs could fully differentiate into mature DCs. gp34/OX40L gene transfer facilitated an allogeneic CD4+ T-cell response in vitro by mature DCs but not by immature DCs. Dose escalation of the transgene induced an increasing amount of gp34/OX40L expression, leading to an increasing level of up-regulation of the allogeneic CD4+ T-cell response. The addition of anti-gp34 monoclonal antibody totally abrogated this up-regulation. These results suggest that this facilitation of allogeneic CD4+ T-cell response is specifically dependent on gp34/OX40L expressed on transduced DCs. Taken together, our findings show that gp34/ OX40L plays an important role in allogeneic CD4+ T-cell activation by DCs and that lentiviral gp34/OX40L gene transfer into DCs may be a useful strategy for cancer immunotherapy.

Antigens, Surface↗

PubMed2003

CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation.

Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2003

IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-alpha production in macrophages.

Interleukin-10 (IL-10) plays an important role in prevention of chronic inflammation in vivo. However, the molecular mechanism by which IL-10 exerts its anti-inflammatory response is poorly understood. Here, we performed a microarray analysis and identified Bcl-3 as an IL-10-inducible gene in macrophages. Lentiviral vector-mediated expression of Bcl-3 inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNF-alpha), but not IL-6, in macrophages. In Bcl-3-transduced and IL-10-pretreated macrophages, LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) p65 was not impaired. However, DNA binding by NF-kappaB p50/p65 was profoundly inhibited. Nuclear localization of Bcl-3 was associated with inhibition of LPS-induced TNF-alpha production. Overexpression of Bcl-3 suppressed activation of the TNF-alpha promoter, but not the IL-6 promoter. Bcl-3 interacted with NF-kappaB p50 and was recruited to the TNF-alpha promoter, but not the IL-6 promoter, indicating that Bcl-3 facilitates p50-mediated inhibition of TNF-alpha expression. Furthermore, Bcl-3-deficient macrophages showed defective IL-10-mediated suppression of LPS induction of TNF-alpha, but not IL-6. These findings suggest that IL-10-induced Bcl-3 is required for suppression of TNF-alpha production in macrophages.

Active Transport, Cell Nucleus↗

PubMed2003

Sporicidal kinetics of Bacillus subtilis spores by heated scallop shell powder.

Scallop shell powder heated at 1,000 degrees C for 1 h exhibited sporicidal action against Bacillus subtilis spores. The sporicidal kinetics of this action were analyzed with the use of a nonlogarithmic model. Apparent death rate constants (k) were obtained under various conditions. The value of k increased with powder concentration but became constant beyond the concentration representing the solubility of Ca(OH)2. A linear inverse relationship between k and temperature was found, and from this relationship the activation energy required for the death of B. subtilis spores in the heated shell powder slurry could be determined.

Animals↗

PubMed2002

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells.

Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.

Antigens, CD34↗

PubMed2002

Hepatocellular carcinoma caused by loss of heterozygosity in Lkb1 gene knockout mice.

Germline mutations of the LKB1 gene are associated with Peutz-Jeghers syndrome, which is characterized by mucocutaneous pigmentation and gastrointestinal hamartoma with an increased risk of cancer development. To investigate the role of LKB1 in vivo, we have recently constructed Lkb1 gene knockout mice. Because of Lkb1 gene haploinsufficiency, the heterozygous Lkb1 mice develop gastrointestinal polyps of which the histological characteristics resemble those of the Peutz-Jeghers syndrome hamartomas. Here we demonstrate that the Lkb1 (+/-) mice develop hepatocellular carcinomas (HCCs). In Lkb1 (+/-) mice >50 weeks of age, >70% of the male mice developed HCCs, whereas only 20% of the females had HCCs, showing a sex difference in the susceptibility. Histological examinations revealed various types of HCCs, such as "trabecular," "clear cell," "pseudoglandular," and "sarcomatous" types, which were strikingly similar to those found in human HCCs. Western blotting and PCR analyses showed loss of Lkb1 heterozygosity in all of the HCC tissues examined, indicating a tumor suppressor role of LKB1 in the mouse liver. These results suggest that lack of LKB1 is a novel mechanism for HCC development. Thus, the Lkb1 (+/-) knockout mutant should be an important and useful model for human HCC.

AMP-Activated Protein Kinases↗

PubMed2002

Gastrointestinal hamartomatous polyposis in Lkb1 heterozygous knockout mice.

Peutz-Jeghers syndrome (PJS) is a hereditary disorder characterized by gastrointestinal hamartomatous polyposis associated with mucocutaneous pigmentation. Germ-line mutations of the gene encoding LKB1 (STK11), a serine/threonine kinase, are identified in most PJS patients. To investigate the role of LKB1 in the PJS phenotypes, we introduced a germ-line mutation in the mouse Lkb1 gene by homologous recombination in mouse embryonic stem cells. In most Lkb1 (+/-) mice >20 weeks of age, hamartomatous polyps developed in the glandular stomach, often in the pyloric region. Small intestinal hamartomas also developed in approximately one-third of the Lkb1 (+/-) mice >50 weeks of age. A genomic PCR and sequence analysis showed that all hamartomas retained both the wild-type and targeted Lkb1 alleles, indicating that allelic loss of the wild-type Lkb1 was not the cause of polyp formation. Moreover, the LKB1 protein level was not reduced in hamartomatous polyps compared with that in the Lkb1 (+/-) normal gastric mucosa. In addition, the remaining allele showed neither missense mutations in the coding sequence nor produced truncated LKB1 in the hamartoma. Taken together, these data suggest that the wild-type Lkb1 is expressed in the hamartoma at the haploid amount. Accordingly, the gastrointestinal hamartomas appear to develop because of the Lkb1 haploinsufficiency. Although additional genetic events may be critical in hamartoma and adenocarcinoma development, these data strongly suggest that the initiation of polyposis is not the result of loss of heterozygosity in Lkb1.

AMP-Activated Protein Kinases↗