Development and validation of an UPLC-Q/TOF-MS assay for the quantitation of neopanaxadiol in beagle dog plasma: Application to a pharmacokinetic study.

Neopanaxadiol (NPD), the main panaxadiol constituent of Panax ginseng C. A. Meyer (Araliaceae), has been regarded as the active component for the treatment of Alzheimer's disease. However, few references are available about pharmacokinetic evaluation for NPD. Accordingly, a rapid and sensitive method for quantitative analysis of NPD in beagle dog plasma based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. Analytes were extracted from plasma by liquid-liquid extraction and chromatographic separation was achieved on an Agilent Zorbax Stable Bond C18 column. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions both at m/z 461.4 → 425.4 for NPD and internal standard of panaxadiol. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for multitude sample determination. After oral intake, NPD was slowly absorbed and eliminated from circulatory blood system and corresponding plasma exposure was low. Application of this quantitative method will yield the first pharmacokinetic profile after oral administration of NPD to beagle dog. The information obtained here will be useful to understand the pharmacological effects of NPD.

Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells.

The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin(-) Sca-1 (+) c-Kit (+) (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1(-/-), Pim2(-/-), Pim3(-/-) single knockout (KO) mice. HSCs from Pim1(-/-) KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2(-/-) or Pim3(-/-) KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34(-) HSCs was reduced by approximately 28-fold in Pim1(-/-) KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.

The Pim protein kinases regulate energy metabolism and cell growth.

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMPATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5'-cap-dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.

A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma.

The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-LBL/T-ALL) being highly sensitive. Incubation of pre-T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27(Kip1), apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre-T-LBL cells. In immunodeficient mice carrying subcutaneous pre-T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL.

Regulation of Skp2 levels by the Pim-1 protein kinase.

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF(Skp2) ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser(64) and Ser(72), we have identified Thr(417) as a unique Pim-1 phosphorylation target. Phosphorylation of Thr(417) controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.

Retroviral insertional mutagenesis identifies genes that collaborate with NUP98-HOXD13 during leukemic transformation.

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

NUP98-HOX translocations lead to myelodysplastic syndrome in mice and men.

The myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute myeloid leukemia (AML). A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene, most commonly leading to fusions of NUP98 with abd-b group HOX genes, including HOXD13. We used vav regulatory elements to direct expression of a NUP98-HOXD13 (NHD13) fusion gene in hematopoietic tissues. NHD13 transgenic mice faithfully recapitulate all the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia and apoptosis, and transformation to acute leukemia. The MDS that develops in NHD13 transgenic mice is highly lethal; within 14 months, 90% of the mice died of either leukemic transformation or severe anemia and leukopenia due to progressive MDS. These mice provide a preclinical model that can be used for the evaluation of MDS therapy and biology.

[Dynamic changes of microtubule in parthenogenetic and in vitro fertilized preimplantation embryos in mouse.].

In this study we detected dynamic changes and function of beta-tubulin, a subtype of microtubule, during the first cleavage period in mouse parthenogenetic and in vitro fertilized embryos. Firstly, we compared the developmental potential of in vitro fertilized, parthenogenetic, and in vivo fertilized embryos in culture. Then, the dynamic changes of beta-tubulin and nucleus in parthenogenetic and in vitro fertilized preimplantation embryos were detected by immunofluorescence and confocal microscopy to analyze the role of microtubules in meiotic division and embryonic development. The results indicated that the development rate of in vivo fertilized embryos was significantly higher than that of in vitro fertilized or parthenogenetic embryos (P<0.05). However, there was no significant difference in developmental potential between in vitro fertilized and parthenogenetic embryos. During in vitro fertilization, oocyte was activated when sperm entered it. Oocyte resumed the second meiotic division. Condensed maternal chromosomes aligning at the equator of the spindle were pulled to the spindle poles by kinetochore microtubules in anaphase. Furthermore, in telophase, there were microtubules between the two sets of decondensed maternal chromosomes. One set formed the second polar body (Pb(2)), which was extruded to the perivitelline space. The other set formed female pronucleus. Meanwhile, 5-8 h after fertilization, sperm chromatin condensed and decondensed to form male pronucleus. Microtubule composed mesosome and cytaster remodeling around male and female pronuclei to form long microtubules, which pull the pronuclei to get close. During 4-6 h parthenogenetic activation, SrCl(2) activated oocytes to resume meiosis. As a consequence, sister chromatids were pulled to spindle poles. Cytochalasin B, which was applied in the medium, inhibited the extrusion of Pb(2). Two haploid pronuclei in the cytoplasm were connected by microtubules. Compared with that in in vitro fertilization, oocyte is easier to be activated in parthenogenetic activation. Chemical activation is more efficient than sperm penetration in in vitro fertilization as indicated by earlier and better remodeling of the microtubules.

OLIG2 (BHLHB1), a bHLH transcription factor, contributes to leukemogenesis in concert with LMO1.

OLIG2 (originally designated BHLHB1) encodes a transcription factor that contains the basic helix-loop-helix motif. Although expression of OLIG2 is normally restricted to neural tissues, overexpression of OLIG2 has been shown in patients with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL). In the current study, we found that overexpression of OLIG2 was not only found in oligodendroglioma samples and normal neural tissue but also in a wide spectrum of malignant cell lines including leukemia, non-small cell lung carcinoma, melanoma, and breast cancer cell lines. To investigate whether enforced expression of OLIG2 is oncogenic, we generated transgenic mice that overexpressed OLIG2 in the thymus. Ectopic OLIG2 expression in the thymus was only weakly oncogenic as only 2 of 85 mice developed pre-T LBL. However, almost 60% of transgenic mice that overexpressed both OLIG2 and LMO1 developed pre-T LBL with large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2/LMO1 mice revealed up-regulation of Notch1 as well as Deltex1 (Dtx1) and pre T-cell antigen receptor alpha (Ptcra), two genes that are considered to be downstream of Notch1. Of note, we found mutations in the Notch1 heterodimerization or proline-, glutamic acid-, serine-, and threonine-rich domain in three of six primary thymic tumors. In addition, growth of leukemic cell lines established from OLIG2/LMO1 transgenic mice was suppressed by a gamma-secretase inhibitor, suggesting that Notch1 up-regulation is important for the proliferation of OLIG2-LMO1 leukemic cells.

Leukemic transformation.

Leukemia results from the uncontrolled accumulation of primitive, poorly differentiated blood cells, and is a consequence of the accumulation of mutations in hematopoietic precursor cells. These mutations include point mutations (single base pair insertions, deletions, or substitutions), gross chromosomal rearrangements such as deletions, insertions, amplifications, and translocations, and epigenetic changes. It seems likely that mutations affecting at least two pathways are required for the development of leukemia. One of these pathways regulates cell accumulation; the second regulates hematopoietic differentiation. Molecularly targeted therapy, which interrupts functions of the leukemogenic proteins generated by mutations, has been developed and shown to be effective for several forms of malignancy. Therefore, it is our hope and belief that a clearer understanding of the mechanism(s) that underlie leukemic transformation will lead to effective new therapies for this dreaded disease.

Somatic cell mutation in pediatric patients undergoing allogeneic bone marrow transplantation.

In order to examine whether bone marrow transplantation (BMT) has genotoxic effects in vivo, mutant frequencies (Mfs) at the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus were evaluated. Thirty-seven pediatric patients who had received allogeneic BMT for various hematologic or immunologic disorders were enrolled. Nine out of the 37 patients (24.3%) were found to have Hprt-Mfs exceeding the 99% confidence limits calculated from observation of healthy controls. Among factors including gender, primary disease of the patient, donor-recipient histocompatibility relationship, age of donor, and total body irradiation as conditioning regimen, none was associated with an increased Hprt-Mf. In three patients who had chimerism in their peripheral blood after BMT, Hprt mutant clones turned out to be of donor- or recipient-origin. Mfs at the T-cell receptor (TCR) locus were examined in 28 patients. Four patients (14.3%) were found to have increased TCR-Mfs. However, there were not any patients who showed elevation of both Hprt-and TCR-Mfs. These data, taken together, suggest that BMT may cause genotoxicity in vivo in some patients.

The BH3 mimetic ABT-737 induces cancer cell senescence.

ABT-737, a small molecule cell-permeable Bcl-2 antagonist that acts by mimicking BH3 proteins, induces apoptotic cell death in multiple cancer types. However, when incubated with this agent many solid tumor cell lines do not undergo apoptosis. The current study reveals a novel mechanism whereby ABT-737 when added to apoptosis-resistant cancer cells has profound biologic effects. In PV-10 cells, a renal cell carcinoma that does not die after ABT-737 treatment, this agent induces a two-fold change in the transcription of nearly 430 genes. Many of these induced mRNA changes are in secreted proteins, IL-6, IL-8, and IL-11 and chemokines CXCL2 and CXCL5, or genes associated with an "inflammatory" phenotype. Strikingly, these gene changes are highly similar to those changes previously identified in cellular senescence. Brief exposure of apoptosis-resistant renal, lung and prostate cancer cell lines to ABT-737, although not capable of inducing cell death, causes the induction of senescence-associated ß-galactosidase and inhibition of cell growth consistent with the induction of cellular senescence. Evidence indicates that the induction of senescence occurs as a result of reactive oxygen species elevation followed by low-level activation of the caspase cascade, insufficient to induce apoptosis, but sufficient to lead to minor DNA damage and increases in p53, p21, IL-6 and 8 proteins. By overexpression of a dominant-negative p53 protein, we show that ABT-737-induced cellular senescence is p53-dependent. Thus, in multiple cancer types in which ABT-737 is incapable of causing cell death, ABT-737 may have additional cellular activities that make its use as an anticancer agent highly attractive.

p53-dependent induction of prostate cancer cell senescence by the PIM1 protein kinase.

The PIM family of serine threonine protein kinases plays an important role in regulating both the growth and transformation of malignant cells. However, in a cell line-dependent manner, overexpression of PIM1 can inhibit cell and tumor growth. In 22Rv1 human prostate cells, but not in Du145 or RWPE-2, PIM1 overexpression was associated with marked increases in cellular senescence, as shown by changes in the levels of beta-galactosidase (SA-beta-Gal), p21, interleukin (IL)-6 and IL-8 mRNA and protein. During early cell passages, PIM1 induced cellular polyploidy. As the passage number increased, markers of DNA damage, including the level of gammaH2AX and CHK2 phosphorylation, were seen. Coincident with these DNA damage markers, the level of p53 protein and genes transcriptionally activated by p53, such as p21, TP53INP1, and DDIT4, increased. In these 22Rv1 cells, the induction of p53 protein was associated not only with senescence but also with a significant level of apoptosis. The importance of the p53 pathway to PIM1-driven cellular senescence was further shown by the observation that expression of dominant-negative p53 or shRNA targeting p21 blocked the PIM1-induced changes in the DNA damage response and increases in SA-beta-Gal activity. Likewise, in a subcutaneous tumor model, PIM1-induced senescence was rescued when the p53-p21 pathways are inactivated. Based on these results, PIM1 will have its most profound effects on tumorigenesis in situations where the senescence response is inactivated.

The high-mobility group A1a/signal transducer and activator of transcription-3 axis: an achilles heel for hematopoietic malignancies?

Although HMGA1 (high-mobility group A1; formerly HMG-I/Y) is an oncogene that is widely overexpressed in aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. HMGA1 encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. To determine how HMGA1 leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Here, we show that the STAT3 gene, which encodes the signaling molecule signal transducer and activator of transcription 3 (STAT3), is a critical downstream target of HMGA1a. STAT3 mRNA and protein are up-regulated in fibroblasts overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a in fibroblasts. HMGA1a also binds directly to a conserved region of the STAT3 promoter in vivo in human leukemia cells by chromatin immunoprecipitation and activates transcription of the STAT3 promoter in transfection experiments. To determine if this pathway contributes to HMGA1-mediated transformation, we investigated STAT3 expression in our HMGA1a transgenic mice, all of which developed aggressive lymphoid malignancy. STAT3 expression was increased in the leukemia cells from our transgenics but not in control cells. Blocking STAT3 function induced apoptosis in the transgenic leukemia cells but not in controls. In primary human leukemia samples, there was a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function in human leukemia or lymphoma cells led to decreased cellular motility and foci formation. Our results show that the HMGA1a-STAT3 axis is a potential Achilles heel that could be exploited therapeutically in hematopoietic and other malignancies overexpressing HMGA1a.

Notch1 mutations are important for leukemic transformation in murine models of precursor-T leukemia/lymphoma.

NOTCH1 is frequently mutated in human precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). In the current study, we found that 13 of 19 cell lines and 29 of 49 primary tumors from SCL/LMO1, OLIG2/LMO1, OLIG2, LMO1, NUP98/HOXD13, and p27(-/-)/SMAD3(+/-) mice had Notch1 mutations in either the heterodimerization (HD) or the glutamic acid/serine/threonine (PEST) domain but not both. Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks did not have Notch1 mutations, whereas thymocytes from clinically healthy SCL/LMO1 mice aged 8 to 12 weeks did have Notch1 mutations and formed tumors upon transplantation into nude mice. Remarkably, all of the HD domain mutations that we identified were single-base substitutions, whereas all of the PEST domain mutations were insertions or deletions, half of which mapped to 1 of 2 mutational "hot spots." Taken together, these findings indicate that Notch1 mutations are very frequent events that are acquired relatively early in the process of leukemic transformation and are important for leukemic cell growth.

NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia.

The myelodysplastic syndromes (MDSs) are a group of clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis and dysplasia. A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene. Using a NUP98-HOXD13 fusion gene, we have developed a mouse model that faithfully recapitulates all of the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia, and apoptosis, and transformation to acute leukemia. The MDS that develops in NUP98-HOXD13 transgenic mice is uniformly fatal. Within 14 months, all of the mice died of either leukemic transformation or severe anemia and leucopenia as a result of progressive MDS. The NUP98-HOXD13 fusion gene inhibits megakaryocytic differentiation and increases apoptosis in the bone marrow, suggesting a mechanism leading to ineffective hematopoiesis in the presence of a hypercellular bone marrow. These mice provide an accurate preclinical model that can be used for the evaluation of MDS therapy and biology.

Distinct mechanisms lead to HPRT gene mutations in leukemic cells.

Leukemias are considered malignant clonal disorders arising from the accumulation of mutations in hematopoietic cells; the majority of these mutations are thought to be acquired somatically. Measurement of mutation frequency (Mf) at the hypoxanthine phosphoribosyltransferase (HPRT) locus has been developed as a method for estimating genomic instability. We investigated the Mf in 16 leukemic cell lines to determine whether these cell lines showed evidence of genomic instability. Although some leukemic cell lines had markedly elevated Mfs, the Mfs at the HPRT locus in leukemic cell lines were not always higher than those of B-lymphoblastoid cell lines and T lymphocytes from normal individuals. We were able to identify the HPRT mutation for 159 of 160 individual HPRT mutants. The HPRT mutations were characterized at a molecular level and classified as either gross chromosomal rearrangements (GCRs) or point mutations, such as single-nucleotide substitutions, insertions, or deletions. With rare exceptions, individual leukemic cell lines showed either point mutations or GCR, but not both. Of note, all the cell lines that primarily showed point mutations are known to be defective in mismatch repair machinery.

Serial granulocyte transfusions as a treatment for sepsis due to multidrug-resistant Pseudomonas aeruginosa in a neutropenic patient.

The emergence of multidrug-resistant Pseudomonas aeruginosa (MRPA) has become a major clinical problem. We successfully treated MRPA sepsis in a neutropenic patient undergoing peripheral blood stem cell transplantation with serial granulocyte transfusions. Granulocyte transfusion should be considered as a treatment for severe infection in patients with neutropenia.

A successful liver transplantation for refractory hepatic veno-occlusive disease originating from cord blood transplantation.

An 11-month-old boy with acute lymphoblastic leukemia (ALL) underwent umbilical cord blood transplantation (CBT) from an unrelated donor after a first complete remission. Despite the prophylactic use of low molecular weight heparin, prostaglandin E1 and ursodeoxycholic acid, hepatic veno-occlusive disease (VOD) occurred on the 29th day after CBT. Furthermore, neither defibrotide nor antithrombin-III improved the hepatic coma and coagulopathy due to the hepatic VOD. On the 42nd day after CBT, he underwent living related liver transplantation (LRLT) with a left lateral segment graft from his father. He received tacrolimus for the prevention of rejection and graft-vs.-host disease (GVHD) and also received aggressive antifungal and antiviral prophylaxis. Although he showed signs of acute rejection on postoperative days 5 and 10, the postoperative course was uneventful in general. At present, 17 months after LRLT, the patient shows stable liver function and no signs of either GVHD or a relapse of ALL. In conclusion, LRLT can be seen as a feasible option for the treatment of a hepatic VOD after CBT, though aggressive prophylaxis for infection and the anticipation of acute rejection are of importance.