Fludarabine induces growth arrest and apoptosis of cytokine- or alloantigen-stimulated peripheral blood mononuclear cells, and decreases production of Th1 cytokines via inhibition of nuclear factor kappaB.

Fludarabine is a purine analog that has demonstrated significant activity in B-cell malignancies, including CLL. Fludarabine also possesses an immunosuppressive effect and is being used to prevent GVHD in hematopoietic stem cell transplantation. However, the molecular mechanism by which fludarabine inhibits immunoreaction remains to be fully elucidated. This study found that fludarabine inhibited tumor necrosis factor alpha (TNF-alpha)-stimulated degradation of IkappaBalpha, resulting in blockade of nuclear translocation of nuclear factor kappaB (NF-kappaB) in Jurkat T cells, as measured by western blot analysis and immunocytochemistry. The ability of fludarabine to inhibit NF-kappaB was further confirmed by electrophoretic mobility shift assay. We also found that fludarabine induced growth arrest and apoptosis of alloreactive and TNF-alpha-stimulated PBMCs. In addition, fludarabine inhibited TNF-alpha-stimulated production of IL-2 and IFN-gamma, which play important roles in the onset of GVHD, in Jurkat cells. Taken together, fludarabine is useful for management of immune diseases, including GVHD, through inactivation of NF-kappaB.

Fludarabine induces apoptosis of human T-cell leukemia virus type 1-infected T cells via inhibition of the nuclear factor-kappaB signal pathway.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease in which the human T-cell lymphotropic virus type I (HTLV-I) has been recognized as the etiologic agent. Fludarabine is a purine analog that has demonstrated significant activity in B-cell malignancies, including chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma. This study explored the effects of fludarabine on HTLV-1-infected T cells (MT-1, -2, -4 and HUT102). Fludarabine induced growth arrest and apoptosis of these cells, as measured by 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell cycle analysis and annexin V staining. Moreover, exposure of HTLV-1-infected T cells to fludarabine decreased the levels of X-inhibitor of apoptosis protein in conjunction with inhibition of nuclear factor kappaB (NF-kappaB)/DNA-binding activity, as measured by Western blot analysis and electrophoretic mobility shift and reporter gene assays, respectively. Further studies found that fludarabine accumulated NF-kappaB and inhibitory subunit of NF-kappaB in cytosole in conjunction with downregulation of NF-kappaB in nucleus, suggesting that fludarabine blocked nuclear translocation of NF-kappaB. Taken together, fludarabine may be useful for treatment of individuals with ATL and other types of cancer in which NF-kappaB plays a role.

Thrombomodulin blocks calcineurin inhibitor-induced vascular permeability via inhibition of Src/VE-cadherin axis.

Recombinant human soluble thrombomodulin (rTM) counteracted capillary leakage and alleviated edema in individuals with sinusoidal obstruction syndrome and engraftment syndrome after hematopoietic stem cell transplantation. We previously showed that rTM increased levels of antiapoptotic protein Mcl-1 and protected endothelial cells from calcineurin inhibitor cyclosporine A (CsA)-induced apoptosis. However, the molecular mechanisms by which rTM enhances barrier function in vascular endothelial cells remain unknown. Here we show that exposure of vascular endothelial EA.hy926 cells to CsA induced phosphorylation of Src/vascular endothelial cadherin (VE-cadherin) and translocation of VE-cadherin from cell surface to cytoplasm, resulting in an increase in vascular permeability. In addition, CsA increased production of inflammatory cytokines, including interleukin (IL)-1ß and IL-6, associated with an increase in nuclear levels of nuclear factor-κB (NF-κB) which also enhanced vascular permeability. Importantly, the fourth and fifth regions of epidermal growth factor-like domain of TM (TME45) attenuated CsA-induced p-Src/VE-cadherin and vascular permeability in parallel with a decrease in nuclear levels of NF-κB and cytokine production in EA.hy926 cells. In conclusion, TM, especially TME45, maintains vascular integrity, at least in part, via Src signaling.

The fifth epidermal growth factor-like region of thrombomodulin exerts cytoprotective function and prevents SOS in a murine model.

The present study found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) possesses the cytoprotective function in association with an increase in levels of anti-apoptotic myeloid cell leukemia-1 protein in an activated protein C-independent manner in human umbilical vein endothelial cells (HUVECs). Importantly, TME5 counteracted calcineurin inhibitor-induced vascular permeability and successfully prevented monocrotaline-induced sinusoidal obstruction syndrome (SOS) in a murine model. Taken together, TME5 may be useful for preventing or treating lethal complications that develop after hematopoietic stem cell transplantation such as SOS and thrombotic microangiopathy in which endothelial cell damage has a role.

Down-Regulation of prostate-specific antigen expression by ligands for peroxisome proliferator-activated receptor gamma in human prostate cancer.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Recent studies found that ligand-activated PPARgamma regulated differentiation and clonal growth of several types of cancer cells, including prostate cancer, suggesting that PPARgamma could be a tumor suppressor. Troglitazone was a widely used antidiabetic drug that activates PPARgamma. Recently, we reported that this agent had antiprostate cancer effects in vitro and in vivo. In this study, we administered troglitazone for over 1.5 years to an individual with occult recurrent prostate cancer. Using the prostate-specific antigen (PSA) levels as a surrogate marker of the disease, the oral administration of troglitazone (600-800 mg/day) reduced the increase velocity of PSA levels, suggesting clinical efficacy of troglitazone in prostate cancer. PSA promoter/ enhancer reporter assays showed that the PPARgamma ligands troglitazone (10(-5) M), pioglitazone (10(-5) M), or 15-deoxy-delta12,14-prostaglandin J2 (10(-5) M) down-regulated androgen-stimulated reporter gene activity in LNCaP cells, a prostate cancer cell line. The PSA promoter contains androgen receptor response elements (AREs). Reporter gene studies showed that troglitazone inhibited androgen activation of the AREs in the PSA regulatory region. Consistent with inhibition of gene expression, 2 days of incubation of LNCaP with troglitazone dramatically suppressed PSA protein expression without suppressing AR expression, suggesting that troglitazone inhibited ARE activation by a mechanism other than down-regulation of expression of the AR. Taken together, ligands of PPARgamma may be a useful therapeutic approach for the treatment of prostate cancer and may be acting, in part, by inhibiting transactivation of androgen-responsive genes.

Mutational analysis of the peroxisome proliferator-activated receptor gamma gene in human malignancies.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in adipocyte differentiation and is expressed in many human malignancies, including those from prostate, breast, as well as colon. It regulates differentiation and/or cell growth of these cells. However, expression of this nuclear hormone receptor in other types of cancer, especially in hematological malignancies, remains to be fully elucidated. The PPARgamma gene has been mapped to chromosome band 3p25, where chromosomal abnormalities are observed in a variety of human malignancies. Furthermore, a recent study revealed that the PPARgamma gene is functionally mutated in sporadic colon cancer cells. Therefore, PPARgamma could be an important tumor suppressor gene. This prompted us to investigate the expression and mutational status of the PPARgamma gene in cancers of a variety of tissues. A total of 159 samples were interrogated for their expression of PPARgamma as measured by reverse transcription-polymerase chain reaction and/or Western blot analysis. In each of the samples, expression of PPARgamma was detectable. In addition, a total of 397 clinical samples and cell lines including colon, prostate, breast and lung cancers, and leukemias were analyzed for mutations of the PPARgamma gene by either reverse transcription-polymerase chain reaction-single-strand conformation polymorphism or polymerase chain reaction-single-strand conformation polymorphism analysis. No abnormalities were detectable in any of the human malignancies. On the other hand, shifted bands were easily detectable when using positive controls, which harbored the same sequence alterations reported previously in colon cancer cells. Taken together, PPARgamma is expressed in a variety of cancers, and mutation of the PPARgamma gene is a very rare event in human malignancies.

Comprehensive mutational analysis of primary and relapse acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

Prevention of human T cell lymphotropic virus type I infection in Japanese macaques by passive immunization.

Prophylaxis against human T cell lymphotropic virus type I (HTLV-I) is of primary importance for the eradication of adult T cell leukemia and other diseases associated with this virus. Hyperimmune globulin (H-IgG) prepared from healthy blood donors with high antibody titers for HTLV-I was evaluated for its prophylactic effect against HTLV-I in Japanese macaques (Macaca fuscata). Normal IgG (N-IgG) prepared from seronegative healthy blood donors was used as control. Both preparations contained 50 mg/ml IgG and H-IgG had a neutralizing antibody titer of 1:7100 by vesicular stomatitis virus (HTLV-I) pseudotype neutralization assay. Two macaques were infused with 2 ml/kg N-IgG and three macaques were immunized with 2-0.5 ml/kg H-IgG. They were immediately challenged by inoculation of 8 x 10(6)/kg cells from an HTLV-I-producing rabbit lymphoid cell line (Ra-1). Another macaque was immunized with 1 ml/kg H-IgG 24h after inoculation of 8 x 10(6)/kg Ra-1 cells. HTLV-I infection, as determined by seroconversion and verified by polymerase chain reaction, occurred in both of the N-IgG-injected macaques but in none of the four H-IgG-injected macaques. These results demonstrate the protective efficacy of H-IgG against HTLV-I infection in a primate model and provide an experimental basis for passive immunization trials in humans.

Analysis of the Smad2 gene in hematological malignancies.

A total of 34 leukemia and lymphoma samples (17 clinical samples and 17 cell lines) were analyzed for mutations of the Smad2 gene by reverse transcriptase-polymerase chain reaction single strand conformation polymorphism (RT-PCR-SSCP) analysis. Nine of the 34 samples had 18q chromosomal abnormalities. No shifted bands were detected in any of the hematological malignancies. Our results suggest that resistance to cell growth inhibitory effects of TGF-beta in hematological malignancies is not due to alterations of the Smad2 gene.

Thrombomodulin alleviates murine GVHD in association with an increase in the proportion of regulatory T cells in the spleen.

Recombinant human soluble thrombomodulin (rTM), a potent anticoagulant, has been used for the treatment of disseminated intravascular coagulation in Japan since 2008. Interestingly, rTM possesses anti-inflammatory and cytoprotective functions. This study examined whether rTM alleviates GVHD in a murine hematopoietic SCT (HSCT) model. Use of rTM significantly improved the survival of mice on day 28 of transplantation (survival rate 70% in rTM - treated mice vs 35% in control, P<0.05) in association with a significant decrease in plasma levels of IL-6, IFN-γ and high-mobility group B1 DNA-binding protein on day 7 of HSCT. Intriguingly, the proportion of regulatory T cells in the spleen was significantly increased in rTM-treated mice on day 7 of transplantation compared with control diluent-treated mice. In addition, elevated plasma levels of TM and fibrin/fibrinogen degradation product were noted in HSCT-recipient mice, suggesting coagulopathy caused by endothelial cell damage in this GVHD model. The use of rTM potently decreased these levels. Importantly, rTM did not hamper the anti-GVL and engraftment of hematopoietic cells. Taken together, the use of rTM may prevent GVHD and serve as a potential therapeutic strategy to improve clinical outcomes in individuals who receive HSCT.

The novel function of CD82 and its impact on BCL2L12 via AKT/STAT5 signal pathway in acute myelogenous leukemia cells.

The aim of this study was to explore the biological functions of a tetraspanin family protein CD82 expressed aberrantly in chemotherapy-resistant CD34(+)/CD38(-) acute myelogenous leukemia (AML) cells. Microarray analysis of patient-isolated CD34(+)/CD38(-) AML cells revealed that the levels of anti-apoptotic protein BCL2L12 were downregulated after CD82 depletion by specific short hairpin RNA (shRNA). Western blot analysis indicated that BCL2L12 was aberrantly expressed in patient-isolated AML cells and AML cell lines. Furthermore, CD82 blockade by a specific antibody downregulated BCL2L12 in parallel with dephosphorylation of signal transducer and activator of transcription 5 (STAT5) and AKT, whereas pharmacological inhibition of STAT5 and AKT activation decreased BCL2L12 expression in leukemia cells. In addition, shRNA-mediated downregulation of BCL2L12 increased the levels of cleaved caspase-3 and suppressed proliferation of leukemia cells, impairing their engraftment in immunodeficient mice. Taken together, our results indicate that CD82 regulated BCL2L12 expression via STAT5A and AKT signaling and stimulated proliferation and engrafting of leukemia cells, suggesting that CD82 and BCL2L12 may be promising therapeutic targets in AML.

Mutation analysis of the DNA-damage checkpoint gene CHK2 in myelodysplastic syndromes and acute myeloid leukemias.

Checkpoint genes code for a family of proteins which sense DNA damage in eukaryotic cells. They play an important role in the control of the cell cycle. The human CHK2 is a homolog of the yeast G(2) checkpoint kinases known as CDS1 and RAD53. The CHK2 may be a tumor suppressor gene because it was found to be mutated in some individuals with the Li-Fraumeni syndrome. These cases had a normal, non-mutated p53 gene. We performed a mutational analysis of the CHK2 gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) in 41 bone marrow samples from individuals with myelodysplastic syndrome (MDS) and 41 samples of acute myeloid leukemias (AML). We found a novel G to C transversion resulting in a change from Ala to Gly at codon 507 of CHK2 in one MDS sample, but normal cells from this individual did not have the abnormality. In addition, we demonstrated a previously described polymorphism at codon 84 (A to G at nucleotide 252) of exon 1 of CHK2 in three of 41 MDS and three of 41 AML patients. The presence of a CHK2 mutation in MDS highlights the importance of alterations of cell cycle checkpoint genes in this disease.

Simple detection of the 5S ribosomal RNA of Pneumocystis carinii using in situ hybridisation.

AIMS: To investigate the effectiveness of digoxigenin incorporated double stranded DNA probes produced by the polymerase chain reaction (PCR), for the detection of Pneumocystis carinii, using in situ hybridisation (ISH). METHODS: Formalin fixed, paraffin wax embedded sections of 26 human lung samples from 11 patients with P carinii pneumonia (PCP), and 15 with various types of fungal and viral pneumonia, were obtained during necropsy or transbronchial lung biopsy. Three additional PCP induced rat lung samples were also tested. PCR probes were prepared using the digoxigenin labelling mixture, and they were amplified from the DNA of a PCP induced rat lung after administration of dexamethasone, on the basis that 5S ribosomal RNA sequences are identical in human and rat P carinii. ISH was performed using this probe, and visualised using the digoxigenin nucleic acid detection kit. An immunohistochemical study using anti-human Pneumocystis monoclonal antibody was also carried out in parallel. RESULTS: ISH positively stained eight (of eight) lung necropsy specimens from patients with PCP, three (of three) transbronchial lung biopsy specimens from patients with PCP, and none of the three PCP induced rat lung specimens. In contrast, none of the specimens from patients with pneumonia caused by Aspergillus sp (n = 5), Candida sp (n = 4), Cryptococcus sp (n = 2), mucormycosis (n = 2), or cytomegalovirus (n = 2) were positive on ISH or immunohistochemistry. CONCLUSIONS: Using a digoxigenin labelled PCR probe for the entire 5S rRNA sequence in conjunction with conventional staining, ISH is highly reactive and specific for the diagnosis of PCP.

Analysis of the CHK2 gene in lymphoid malignancies.

The CHK2 gene encodes a protein kinase that is important for the regulation of cell cycle arrest after DNA damage. CHK2 acts downstream of ataxia teleangiecstasia mutated (ATM), modulates the function of p53 and may help mediate cell cycle arrest at G2/M by phosphorylation of Cdc25C. Recently, the human homolog of the checkpoint kinase Cds1 (CHK2) has been suggested to be a tumor suppressor gene. Heterozygous germline mutations have been reported in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype, and in sporadic colon cancer. LFS is associated with the development of lymphoid malignancies, especially childhood ALL. Therefore, we analyzed the DNA from 143 lymphoid malignancies to determine whether they had mutations of the CHK2 gene. The 14 exons of CHK2 were studied by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing of aberrantly migrating bands. One missense mutation changing serine to phenylalanine (codon 428) in an evolutionarily highly conserved domain was found in a non-Hodgkin's aggressive lymphoma. Another point mutation in the non-coding region was identified in one of adult T-cell leukemias (ATL) samples. This result suggests that mutation of the CHK2 gene may rarely be involved in the development of selected lymphomas.

In situ detection of Aspergillus 18S ribosomal RNA in invasive pulmonary aspergillosis.

OBJECTS: We attempted to evaluate the usefulness of in situ hybridization (ISH) in the specific diagnosis of Aspergillus pulmonary infection. METHODS: We used an ISH technique using a multiple digoxigenin-incorporating probe, which was constructed by means of the polymerase chain reaction (PCR) from the 18S ribosomal RNA of Aspergillus fumigatus. MATERIALS: We studied twelve formalin-fixed, paraffin-embedded lung tissue sections from autopsy-confirmed invasive pulmonary aspergillosis (IPA) (5 acute myelocytic leukemias, 2 acute lymphocytic leukemias, 2 chronic myelocytic leukemias, 1 adult T-cell leukemia, 1 non-Hodgkin's lymphoma and 1 chronic obstructive pulmonary disease.), and 18 sections from other pulmonary infections as control. RESULTS: ISH using the probe and a low-viscosity hybridization buffer solution (LV) positively stained hyphal elements in 12 of 12 autopsy lung tissue specimens from subjects with IPA, while ISH using the probe and a high viscosity hybridization buffer solution (HV) positively stained the hyphal elements in 6 of 12. Specifically, ISH (LV) demonstrates hyphal elements of Aspergillus spp. in the center of Aspergillus abscess. While, ISH (HV) can detect hyphal elements located in the periphery of a suppurative abscess as well as those in the blood vessel. Conversely, ISH did not show positive results for any of the autopsy tissue specimens from subjects with other fungal pneumonia infections (Candida n=5, Mucor n=2, Cryptococcus n=2, and Pseudallescheria n=1), Pneumocystis carinii pneumonia (n=5), and cytomegalovirus pneumonia (n=3). Dual staining by means of ISH and immunohistochemistry (IHC) using anti-neutrophil elastase (NE) and anti-CD68 monoclonal antibodies showed that NE positive cells were localized at the edge of the radial growth of the organism, but CD68 positive cells were located around the center of the abscess. The accumulation of NE positive cells was rarely seen in half of the cases (6/12). In contrast, CD68 positive cells were routinely present in the center of the abscess (12/12). CONCLUSION: ISH in conjunction with IHC is a useful tool for differentiating Aspergillus spp. from other fungal genera in tissue sections from patients with IPA and may have a certain role in the evaluation of the interactions between organisms and recruiting inflammatory cells.

Imatinib causes epigenetic alterations of PTEN gene via upregulation of DNA methyltransferases and polycomb group proteins.

We have recently reported the possible imatinib-resistant mechanism; long-term exposure of leukemia cells to imatinib downregulated levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) via hypermethylation of its promoter region (Leukemia 2010; 24: 1631). The present study explored the molecular mechanisms by which imatinib caused methylation on the promoter region of this tumor suppressor gene in leukemia cells. Real-time reverse transcription PCR found that long-term exposure of chronic eosinophilic leukemia EOL-1 cells expressing FIP1L1/platelet-derived growth factor receptor-α to imatinib induced expression of DNA methyltransferase 3A (DNMT3A) and histone-methyltransferase enhancer of zeste homolog 2 (EZH2), a family of polycomb group, thereby increasing methylation of the gene. Immunoprecipitation assay found the increased complex formation of DNMT3A and EZH2 proteins in these cells. Moreover, chromatin immunoprecipitation assay showed that amounts of both DNMT3A and EZH2 proteins bound around the promoter region of PTEN gene were increased in EOL-1 cells after exposure to imatinib. Furthermore, we found that levels of DNMT3A and EZH2 were strikingly increased in leukemia cells isolated from individuals with chronic myelogenous leukemia (n=1) and Philadelphia chromosome-positive acute lymphoblastic leukemia (n=2), who relapsed after treatment with imatinib compared with those isolated at their initial presentation. Taken together, imatinib could cause drug-resistance via recruitment of polycomb gene complex to the promoter region of the PTEN and downregulation of this gene's transcripts in leukemia patients.