Shimeru Kamihira

Selective cytotoxic mechanism of GTP-14564, a novel tyrosine kinase inhibitor in leukemia cells expressing a constitutively active Fms-like tyrosine kinase 3 (FLT3)

The receptor tyrosine kinase FLT3 is constitutively activated by an internal tandem duplication (ITD) mutation within the juxtamembrane domain in 20–30% of patients with acute myeloid leukemia. In this study, we identified GTP-14564 as a specific kinase inhibitor for ITD-FLT3 and investigated the molecular basis of its specificity. GTP-14564 inhibited the growth of interleukin-3-independent Ba/F3 expressing ITD-FLT3 at 1 μm, whereas a 30-fold higher concentration of GTP-14564 was required to inhibit FLT3 ligand-dependent growth of Ba/F3 expressing wild type FLT3 (wt-FLT3). However, this inhibitor suppressed the kinase activities of wt-FLT3 and ITD-FLT3 equally, suggesting that the signaling pathways for proliferation differ between wt-FLT3 and ITD-FLT3. Analysis of downstream targets of FLT3 using GTP-14564 revealed STAT5 activation to be essential for growth signaling of ITD-FLT3. In contrast, wt-FLT3 appeared to mainly use the MAPK pathway rather than the STAT5 pathway to transmit a proliferative signal. Further analysis demonstrated that the first two tyrosines in an ITD were critical for STAT5 activation and growth induction but that all of the tyrosines in the juxtamembrane region were dispensable in terms of the proliferation signals of wt-FLT3. These results indicate that an ITD mutation in FLT3 elicits an aberrant STAT5 activation that results in increased sensitivity to GTP-14564. Thus, FLT3-targeted inhibition is an attractive approach, with the potential for selective cytotoxicity, to the treatment of ITD-FLT3-positive acute myeloid leukemia.

An Alternative Transcript Derived from the Trio Locus Encodes a Guanosine Nucleotide Exchange Factor with Mouse Cell-transforming Potential

By screening cDNA expression libraries derived from fresh leukemic cells of adult T-cell leukemia for the potential to transform murine fibroblasts, NIH3T3, we have identified a novel transforming gene, designated Tgat. Expression of Tgat in NIH3T3 resulted in the loss of contact inhibition, increase of saturation density, anchorage-independent growth in a semisolid medium, tumorigenicity in nude mice, and increased invasiveness. Sequence comparison revealed that an alternative RNA splicing of the Trio gene was involved in the generation of Tgat. The Tgat cDNA encoded a protein product consisting of the Rho-guanosine nucleotide exchange factor (GEF) domain of a multifunctional protein, TRIO, and a unique C-terminal 15-amino acid sequence, which were derived from the exons 38-46 of the Trio gene and a novel exon located downstream of its last exon (exon 58), respectively. A Tgat mutant cDNA lacking the C-terminal coding region preserved Rho-GEF activity but lost the transforming potential, indicating an indispensable role of the unique sequence. On the other hand, treatment of Tgat-transformed NIH3T3 cells with Y-27632, a pharmacological inhibitor of Rho-associated kinase, abrogated their transforming phenotypes, suggesting the coinvolvement of Rho-GEF activity. Thus, alternative RNA splicing, resulting in the fusion protein with the Rho-GEF domain and the unique 15 amino acids, is the mechanism generating the novel oncogene, Tgat.

Clinical relevance of survivin as a biomarker in neoplasms, especially in adult T-Cell Leukemias and acute Leukemias

Survivin has been identified as one of the top 4 transcripts among 3.5 million human transcriptomes uniformly upregulated in cancer tissues but not in normal tissues. Therefore, we quantitatively determined the messenger RNA (mRNA) expression profile for survivin by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) technique in 113 patients with leukemias, such as adult T-cell leukemia (ATL), acute lymphoid leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia in crisis, and chronic lymphocytic leukemia (CLL), and in 25 cell lines, including 7 ATL cell lines and 15 solid-tumor cell lines. Furthermore, we examined whether the plasma level of survivin protein as measured by enzyme-linked immunosorbent assay (ELISA) substituted for mRNA expression by PCR quantification. Gene expression was quantitatively confirmed to be up-regulated in approximately 90% of ATL and acute leukemia cases and in all of the cell lines tested, whereas it was down-regulated in almost all cases of CLL. Furthermore, with respect to the interpretation of the gene expression findings, attention was paid to standardization with a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the real-time PCR quantification, because the variability in GAPDH expression among the different cell types was significant. GAPDH expression was relatively low in ATL cells and high in ALL and AML cells. The rates of increase in the levels of survivin protein in the plasma of ATL patients and in the supernatants from in vitro cultures of solid-tumor cell lines were low compared with rates of increase of the mRNA and protein level in the cells, suggesting that the protein levels in plasma do not always reflect survivin expression in tumor cells. Our findings indicate the potential clinical relevance of survivin quantified by real-time PCR but not for the protein level in plasma as determined by ELISA, especially in cases of ATL and acute leukemias.

Clinical and Oncologic Implications in Epigenetic Down-Regulation of CD26/ Dipeptidyl Peptidase IV in Adult T-Cell Leukemia Cells

CD26/dipeptidyl peptidase IV (DPPIV), a T-cell-activation antigen, is a 110-kD type II surface glycoprotein expressed on various types of normal cells. CD26/DPPIV is considered a multifunction housekeeping protein. Malignant cells often show altered CD26/DPPIV expression or no CD26/DPPIV expression, thus suggesting a useful marker for assessing some T-cell malignancies. In this study, cell surface protein and messenger RNA expression profiles for CD26/DPPIV were examined in 49 patients with adult T-cell leukemia (ATL), 10 carriers of human T-lymphotropic virus I (HTLV-I), and 4 HTLV-I-infected cell lines to assess the utility of CD26/DPPIV expression as a useful molecular marker for ATL pathology. In contrast to normal lymphocytes, ATL cells and HTLV-I-infected cell lines apparently down-regulated or completely lost the CD26/DPPIV antigen. Furthermore, the positive rate and antigen density for CD26/DPPIV in ATL cells gradually declined along with the advancement of ATL stage. Analysis of genomic DNA and the CD26/DPPIV transcript showed that CD26- ATL cells possessed faintly detected transcripts of the gene that were aberrantly methylated at the CpG islands within the promoter region in parallel with the advancement of ATL, a finding supported by a rescue experiment for transcript reexpression using 5-azacytidine as demethylation agent. Moreover, there was no relationship between loss of CD26/DPPIV and HTLV-I tax expression. These results indicate that ATL cells down-regulate CD26 antigens by means of epigenetic machinery and that this antigen abnormality is a useful molecular marker for the pathology of ATL.

Minilaparotomy May Be Independently Associated with Reduction in Inflammatory Responses after Resection for Colorectal Cancer

Objectives: A minilaparotomy approach (skin incision less than 7 cm) to resection of colon cancer is technically feasible, but objective data supporting its benefit are scarce. The aim of this study was to clarify whether minilaparotomy is independently associated with a reduction in the acute inflammatory response after resection of colorectal cancer. Design: Thirty-one patients who underwent surgical resection of colorectal cancer using minilaparotomy or conventional laparotomy were included in this nonrandomized prospective study. Inflammatory responses were evaluated with serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels. Results: In both the minilaparotomy and conventional laparotomy groups, serum IL-6 and CRP levels significantly increased 24 h after the operation (1POD) compared to preoperative levels (p < 0.0001 and p < 0.0001, respectively). Median serum levels of IL-6 and CRP in the minilaparotomy group were significantly lower at 1POD versus the conventional group (p = 0.0066 and p = 0.0033, respectively). Multivariate analyses showed that a smaller increase in serum IL-6 or CRP levels at 1POD [less than 75th percentile (112.9 or 10.6 mg/ml, respectively)] was independently related to only minilaparotomy. Conclusions: These data in this nonrandomized trial suggest that minilaparotomy may be independently associated with reduced inflammatory responses in colorectal cancer resection.

Automation of bone marrow aspirate examination using the XE‐2100 automated hematology analyzer

Attempts to analyze bone marrow aspirates have been reported with the use of several automated blood cell counters, but sufficient accuracy in examination is not acquired yet. Major problems have included difficulties in correctly differentiating various immature cells and interference by lipid in bone marrow aspirates. The goal of this study was to solve these problems to attain more accurate assessment of bone marrow aspirates with automated blood cell counters.