Koh Yamamoto

Positive Modulation of IL-12 Signaling by Sphingosine Kinase 2 Associating with the IL-12 Receptor β1 Cytoplasmic Region

IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rβ1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rβ1, while the regions including the carboxyl terminus and Box II in the IL-12Rβ1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-γ production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-γ production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rβ1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.

GATA-3 suppresses IFN-γ promoter activity independently of binding to cis-regulatory elements

The regulatory mechanism by which GATA‐3 suppresses IFN‐γ gene expression was investigated. A reduction of GATA‐3 using RNA interference technology enhanced, whereas overexpression of GATA‐3 suppressed IFN‐γ mRNA expression. IL‐4 expression was reciprocally affected by GATA‐3. GATA‐3‐mediated down‐regulation of IFN‐γ was achieved through the inhibition of its promoter/enhancer activity. Two GATA elements located in the cis‐regulatory elements did not contribute to the suppression of IFN‐γ promoter activity, even though they behaved as binding sites for GATA‐3. The effect of GATA‐3 on IFN‐γ promoter was lost upon removal of the region encompassing −257 to −172. Among several transcription factors putatively interacting with this region, Stat4, which enhanced IFN‐γ promoter activity, was down‐regulated by GATA‐3 at gene transcription level. Although GATA‐3 has the capacity to interact with the cis‐regulatory elements, it suppresses IFN‐γ gene transcription via down‐regulation of Stat4.

Fusion of MLL and MSF in adult de novo acute myelomonocytic leukemia (M4) with t(11;17)(q23;q25).

TheMLL gene at chromosome band 11q23 is frequently rearranged and fused to partner genes in acute leukemias. Previously, theMSF gene, also calledAF17q25, has been cloned as a fusion partner of theMLL gene in therapy-related or infant acute myelogenous leukemias with t(11;17)(q23;q25). MSF belongs to the septin family of proteins, which includes other MLL fusion partners, hCDCrel1 and Septin 6, and has also been implicated in the pathogenesis of human ovarian tumor and murine T-cell lymphoma. We describe here a 64-year-old man with de novo acute myelomonocytic leukemia (French-American-British subtype M4) with t(11;17)(q23;q25). His leukemia was successfully induced into a first remission, which, however, lasted only briefly. A second remission was never attained, and the patient died of sepsis 16 months after the diagnosis of leukemia. Examination of his leukemic cells at diagnosis revealed anMLL gene rearrangement, by Southern blotting, and anMLL-MSF fusion transcript, by the reverse transcriptase polymerase chain reaction (RT-PCR) method. Sequence analysis of the RT-PCR product further revealed thatMLL exon 5 was fused in-frame toMSF exon 3. Further clinical and molecular analyses of acute leukemias with theMLL-MSF transcript may shed more light on the clinical characteristics and molecular mechanisms of the MLL-septin type leukemias.

Aberrant expression of the LHX4 LIM-homeobox gene caused by t(1;14)(q25;q32) in chronic myelogenous leukemia in biphenotypic blast crisis

Chromosomal aberrations observed in addition to the Philadelphia chromosome in chronic myelogenous leukemia (CML) are likely to be involved in disease progression to the blast crisis. We describe here a t(1;14)(q25;q32) as an additional chromosomal aberration in a patient with CML in biphenotypic blast crisis. By use of long‐distance inverse polymerase chain reaction (PCR), we cloned the chromosomal breakpoint and revealed that the immunoglobulin heavy chain gene is fused near its Eμ enhancer region to the 5′ region of the LHX4 LIM‐homeobox gene, whose expression is restricted to the central nervous system. By use of quantitative real‐time reverse‐transcription PCR, we found that the LHX4 mRNA is expressed at high levels in the patients leukemic cells and in an acute lymphoblastic leukemia (ALL) cell line. The aberrant expression of the LHX4 gene by the t(1;14)(q25;q32) has very recently been reported in a case of ALL, thus, representing a rare, but recurrent genetic abnormality of possible importance in leukemogenesis.

T-cell prolymphocytic leukemia with der(11)t(1;11)(q21;q23) and ATM deficiency

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell malignancy and is similar to a mature T-cell leukemia seen in some patients with ataxia telangiectasia, which is a recessive hereditary chromosomal instability syndrome caused by mutations of the ataxia telangiectasia mutated (ATM) gene located on 11q23. Intriguingly, recent studies have strongly implicated ATM in the pathogenesis of T-PLL as a tumor suppressor gene, because biallelic inactivation of ATM is frequently observed in this disease; however, translocations involving 11q23 have rarely been reported in T-PLL. We report here a case of T-PLL with der(11)t(1;11)(q21;q23). Southern blot analysis did not reveal any abnormality of ATM, nor of MLL, which is also located on 11q23 and is involved in t(1;11)(q21;q23) in acute myelomonocytic leukemia. Northern blot analysis further showed that the ATM transcript of normal size is expressed in the leukemic cells at a level higher than that in normal peripheral blood lymphocytes. Western blot analysis, however, revealed that expression of ATM in the leukemic cells is much lower than that in normal lymphocytes. These results imply that translation of the ATM transcript is impaired or that the ATM protein is highly unstable in the leukemic cells, thus suggesting the presence of nucleotide changes in both alleles.

Clonal identification of Burkitt's lymphoma arising from lymphocyte-predominant Hodgkin's disease

The occurrence of large cell lymphomas subsequent to, or concurrent with, lymphocyte predominant Hodgkins disease (LPHD) is a well‐documented phenomenon. We present a case of Burkitts lymphoma of the bladder, occurring after the successful treatment of LPHD of a cervical lymph node. To evaluate the clonal relationship of the two tumours, we amplified the complementarity‐determining‐region 3 of two samples from paraffin‐embedded slides, using the polymerase chain reaction (PCR). The sequences of the PCR products showed 96% homology to each other. These results indicate that the malignant clone of Burkitts lymphoma arose from the corresponding LPHD.

Two M‐components in a single cell lineage in a patient with a dual isotype secretory B‐cell tumour

We describe a case of Waldenströms macroglobulinaemia with two M‐components (IgM and IgG) with the same λ light chain. Southern blot analysis of bone marrow cells showed rearrangements of immunoglobulin heavy and λ light chain genes.

Using biopsy materials to detect mutations in gastrointestinal tumors.

Because endoscopic examinations are a crucial first step in the diagnosis of carcinomas, we have analyzed point mutations of the c-K-ras and p53 genes using biopsied samples. There is no previous report of such an approach, using only biopsied materials. We analyzed point mutations of the c-K-ras and p53 genes using 60 colorectal and 31 gastric tumor biopsy specimens. None of the gastric and seven of the colorectal tumors had mutations of the c-K-ras gene. p53 gene mutations were detected in six gastric carcinomas [two out of 11 intramucosal carcinomas (18.2%) and four out of 11 invasive carcinomas (36.4%)]. Eight of 15 invasive colorectal carcinomas (53.3%) showed point mutations in the p53 gene. Although point mutation frequencies in this study were relatively low when compared with previous reports in which surgical samples were used, our study shows that biopsied specimens are adequate and useful for analysis of gene point mutations.

Cross-reacting Material-positive Hemophilia A Diagnosed in a Patient with a Spontaneous Thigh Hemorrhage

A 53-year-old man, who had been diagnosed with mild hemophilia A (HA) at 35 years of age, was hospitalized with a thigh hematoma. His bleeding continued despite the administration of recombinant factor VIII (FVIII). The results of an FVIII/von Willebrand factor binding assay were normal. The patients FVIII coagulant activity (FVIII:C) was low, but his FVIII antigen levels were within the normal limits, suggesting FVIII protein dysfunction. The FVIII:C measurements obtained by one-stage clotting and chromogenic assays were different. An FVIII gene analysis revealed a missense mutation p.Ser308Leu, which is rare in Japan. This case highlights that gene analyses and chromogenic assays are necessary to interpret the discrepancies between FVIII:C and the bleeding phenotype of patients with mild HA.