Hiroko Hiraoka

Yamaguchi University, Yamaguti, Yamaguchi, Japan

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Publications (23)27.38 Total impact

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    ABSTRACT: The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.
    Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire 07/2015; 79(3):201-9. · 0.85 Impact Factor
  • Y. Ema · M. Igase · Y. Takeda · T. Yanase · S. Umeki · H. Hiraoka · M. Okuda · T. Mizuno
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    ABSTRACT: The cyclin-dependent kinase (CDK) inhibitor, flavopiridol, was tested as a potential new cancer therapeutic agent to treat canine lymphoma by examining its effect on cell growth of canine lymphoma cell lines in vitro. Flavopiridol induced profound cell death in all eight lymphoma cell lines at 400 nM, and in all cases cell death was due to apoptosis. Apoptosis was inhibited by caspase inhibitor, despite the variable sensitivities between cell lines. Analysis of the mechanism of flavopiridol-induced apoptosis showed that Rb phosphorylation was inhibited, possibly due to CDK4 or CDK6 inhibition. There was also decreased expression of Rb protein and anti-apoptotic proteins, Mcl-1 and XIAP, possibly through transcriptional regulation by inhibition of CDK7 or CDK9 activation. Canine lymphoma cell line-xenotransplanted mice were then treated with flavopiridol and profound tumour shrinkage was observed. This study describes a new therapeutic approach using flavopiridol for canine lymphoma treatment.
    Veterinary and Comparative Oncology 01/2015; DOI:10.1111/vco.12130 · 1.45 Impact Factor
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    ABSTRACT: The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.
    PLoS ONE 09/2013; 8(9):e73555. DOI:10.1371/journal.pone.0073555 · 3.23 Impact Factor
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    ABSTRACT: Five novel, canine lymphoma cell lines (Ema, CLC, CLK, Nody-1 and UL-1) were established from dogs suffering from lymphoma and characterized in vitro and in vivo. All cell lines, except CLC, were characterized with T-cell phenotypes, by flow cytometric analysis and polymerase chain reaction for antigen receptor rearrangement. Cell proliferation rates and transcriptional levels of MYC, PTEN, KIT, and FLT3 varied between each cell line. Intraperitoneal xenotransplantation of Ema, CLC, Nody-1 and UL-1 lymphoma cell lines into NOD/SCID mice induced ascites, intraperitoneal tumors and severe infiltration of lymphoma cells into the pancreas and mesentery. Establishment of novel canine lymphoma cell lines with different characteristics is critical for elucidating the pathophysiology of canine lymphoma and improving current therapies.
    Journal of Veterinary Medical Science 11/2012; 75(4). DOI:10.1292/jvms.12-0448 · 0.88 Impact Factor
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    ABSTRACT: Two dogs, emigrated from Zambia and China to Japan, were diagnosed with Ehrlichia canis infection. Both cases had thrombocytopenia, non-regenerative anemia, and hypergloblinemia with polyclonal gammopathy. Case 1 had ataxia of the hind limbs. Severe meningitis was revealed by magnetic resonance imaging examination. Intracytoplasmic inclusions were observed in mononuclear cells of cerebrospinal fluid. Case 2 had a history of bilateral epistaxis, and severe pancytopenia was noticed in complete blood count. Diagnosis was finally achieved by nested polymerase chain reaction and sequence analysis. Thus, even in non-endemic areas, E. canis infection should be included in the differential diagnosis of clinically ill dogs that emigrated from endemic areas.
    Journal of Veterinary Medical Science 12/2011; 74(6):775-8. DOI:10.1292/jvms.11-0401 · 0.88 Impact Factor
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    ABSTRACT: Second malignancies are frequent complications in human patients with chronic lymphocytic leukemia (CLL). However, the clinical details and outcome of this phenomenon were unclear in their canine counterparts. Here, we report a dog with high-grade lymphoma concurrent with T-cell CLL. A 10-year-old male golden retriever presented with lymphadenopathies. The lymph nodes contained large-sized lymphocytes, raising suspicion of high-grade lymphoma. Meanwhile, small lymphocytic lymphocytosis in the peripheral blood was consistent with CLL. Interestingly, molecular biological analyses revealed that CLL cells were of the T-cell type, whereas lymphoma cells were of the B-cell type. Chemotherapy using the L-VCA short protocol was effective for 155 days, but the dog died on day 194 after diagnosis, despite rescue therapies.
    Journal of Veterinary Medical Science 12/2011; 74(5):677-80. DOI:10.1292/jvms.11-0453 · 0.88 Impact Factor
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    ABSTRACT: A 4-year-old, intact male Shiba dog was referred to Yamaguchi University Animal Medical Center, Yamaguchi, Japan, for the following complaints: anorexia, lethargy, intermittent fever, gingival bleeding and abdominal purpura. The dog presented with persistent neutropenia. Histopathological examination of a bone marrow sample revealed round to oval structures that resembled Hepatozoon micromerozoites and formed a "wheel-spoke" pattern. Furthermore, mature neutrophils were observed around these structures. PCR and sequencing using bone marrow aspirate confirmed Hepatozoon canis (H. canis) infection. These findings suggest that the neutropenia observed in this case was associated with osteomyelitis due to H. canis infection. This is the first report of neutropenia associated with H. canis infection. H. canis infection can be included in the differential diagnosis in canine cases of neutropenia in areas where the disease is endemic.
    Journal of Veterinary Medical Science 06/2011; 73(10):1389-93. DOI:10.1292/jvms.11-0202 · 0.88 Impact Factor
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    ABSTRACT: Prekallikrein (PK) deficiency is an uncommon disorder in dogs. In this report, we describe a case of a dog that was referred for neurological defects and had a prolonged activated partial thromboplastin time (aPTT) and normal prothrombin time (PT) with no hemostatic defects. By using human PK-deficient plasma, the dog was diagnosed to have PK deficiency. The nucleotide sequence of normal canine PK cDNA was determined and compared with the genomic sequences of PK in the affected dog. The comparison revealed that the dog had a point mutation in exon 8 that leads to an amino acid substitution in the fourth apple domain of PK. This is the first report showing a point mutation of PK in a dog with PK deficiency.
    Journal of Veterinary Medical Science 01/2011; 73(1):107-11. DOI:10.1292/jvms.10-0207 · 0.88 Impact Factor
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    ABSTRACT: We performed continuous renal replacement therapy (CRRT) on clinically healthy dogs to evaluate the effects of CRRT on hemodynamics. Heart rate, arterial blood pressure, and central venous pressure of the dogs (n=6) were recorded during the procedure, which was performed under general anesthesia. Throughout the CRRT, heart rate and arterial blood pressure were stable. Central venous pressure increased after CRRT termination but returned to the basal level within 30 min. In this study, hemodynamic alterations, including hypotension, hypertension, and arrhythmias, were not observed during CRRT. These observations suggest that the CRRT protocol used in the present study can be safely applied to clinical cases with acute renal failure.
    Journal of Veterinary Medical Science 12/2009; 72(4):493-7. DOI:10.1292/jvms.09-0463 · 0.88 Impact Factor
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    ABSTRACT: We performed continuous renal replacement therapy (CRRT) in clinically healthy dogs (n=7) to evaluate the utility of nafamostat mesilate (NM) as an anticoagulant. In 3 of the 7 dogs, CRRT had to be discontinued before the target duration due to coagulation in the extracorporeal circuit, into which NM was administered constantly at the rate of 2.0-6.0 mg/kg per hour. The rate of administration of NM was greater than the recommended dose of NM in humans. Further, all the dogs suffered vomiting during CRRT with NM infusion. We therefore recommend that NM is not used as an anticoagulant during CRRT in dogs.
    Journal of Veterinary Medical Science 12/2009; 72(3):363-7. DOI:10.1292/jvms.09-0369 · 0.88 Impact Factor
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    ABSTRACT: The fragile histidine triad (FHIT) gene is a tumor-associated gene, and aberrant FHIT gene and protein expression have been described in many types of human tumors. Furthermore, it has been reported that FHIT gene inactivation is induced by hypermethylation of 5' CpG islands in the gene or by genomic deletion around the open reading frame (ORF). In this study, we explored the aberrations in the canine FHIT gene and Fhit protein expression and assessed the methylation status and genomic deletions by using 5 canine lymphoma cell lines. We found that the decrease in the expression of the Fhit protein in canine lymphoma cell lines was similar to that in human tumors. The expression of the wild-type FHIT transcript was reduced in all 5 cell lines. However, we could not confirm the involvement of aberrant methylation events in the 5' CpG islands of the canine FHIT gene. We were able to identify homozygous or heterozygous deletions in the canine FHIT genes in all 5 cell lines. Moreover, a widespread genomic deletion of the FHIT gene, which included the ORF region, was detected in 1 cell line. In the present study, we detected aberrations in the FHIT gene and Fhit protein expression in all 5 canine lymphoma cell lines, and this phenomenon might be an important factor in promoting canine lymphoma.
    Journal of Veterinary Medical Science 07/2009; 71(6):769-77. DOI:10.1292/jvms.71.769 · 0.88 Impact Factor
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    ABSTRACT: A fragile histidine triad (FHIT) gene has been studied as a tumor-associated gene in humans. The aberrant FHIT gene and its protein expression have been reported in many types of human cancers. The present study explored the canine FHIT gene structure and its protein expression in the peripheral blood mononuclear cells of healthy dogs by RT-PCR, RACE and immunoblot analysis. The obtained canine FHIT gene contained nine small exons and was located on canine chromosome 20. Furthermore, we identified an alternative splicing form of the FHIT transcript. The deduced amino acid sequence was well conserved between species, and anti-human Fhit antibody could be used to detect the canine Fhit protein. These findings will be useful for future research.
    Journal of Veterinary Medical Science 06/2009; 71(5):645-9. DOI:10.1292/jvms.71.645 · 0.88 Impact Factor
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    ABSTRACT: We evaluated the usefulness of polymerase chain reaction for antigen receptor gene rearrangement analysis (PARR) of endoscopic biopsy specimens for diagnosis of canine alimentary lymphoma. Two endoscopic biopsy specimens were obtained from each lesion in 78 dogs with gastrointestinal symptoms. One specimen was histopathologically examined by a pathologist, and the other was analyzed by PARR. All samples were categorized into three groups [lymphoma (n=4), adenocarcinoma (n=5) and enteritis groups (n=69)] based on the histopathological diagnosis. In the lymphoma group, one case was IgH major-positive, and three cases were TCRgamma-positive, representing clonal expansion of B- and T-cells, respectively. PARR produced negative results for all cases in the adenocarcinoma group. In the enteritis group, six cases were TCRgamma-positive. Two of the six TCRgamma-positive enteritis cases were cytologically diagnosed as lymphoma by fine needle aspiration during a laparotomy. In the enteritis group, the survival times were compared between the TCRgamma-positive and TCRgamma-negative cases. The overall survival time of the TCRgamma-positive enteritis cases was significantly shorter than that of the TCRgamma-negative enteritis cases according to a log-rank test (p<0.0001). With regard to other factors, such as age, clinical signs and the serum albumin concentration, there were no significant differences between the TCRgamma-positive and TCRgamma-negative enteritis cases. In conclusion, PARR is capable of detecting alimentary lymphoma and latent alimentary lymphoma, which cannot be histopathologically diagnosed using endoscopic biopsy specimens. Furthermore, a TCRgamma-positive result in PARR may imply a poor prognosis.
    Journal of Veterinary Medical Science 05/2009; 71(5):555-9. DOI:10.1292/jvms.71.555 · 0.88 Impact Factor
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    ABSTRACT: PCR for antigen receptor gene rearrangement analysis (PARR) is a new diagnostic method for lymphoid neoplasia. In PARR using formalin-fixed paraffin-embedded tissues (PARR-FFPE), control DNA amplification was successful in only three of five samples. The formalin fixation times of the three samples were shorter than those of the others. Analysis of the formalin fixation time and DNA amplification controls suggested that a formalin fixation time of less than one week is appropriate. Additionally, application of single strand conformation polymorphism (SSCP) for PARR provided clearer results than conventional PARR in 16 unfixed tissues and three FFPE tissues. These results show that PARR-FFPE is viable for tissues with an appropriate formalin fixation time and that application of FFPE and SSCP for PARR are useful for diagnosis and retrospective study of canine lymphoid neoplasia.
    Journal of Veterinary Medical Science 05/2009; 71(4):535-8. DOI:10.1292/jvms.71.535 · 0.88 Impact Factor
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    ABSTRACT: An 11-year-old Shih Tzu presented with crusting and erythema, mainly on the abdomen and the root of the tail. Based on histopathological findings, blood examinations and necropsy findings, the condition was diagnosed as superficial necrolytic dermatitis associated with a glucagon-secreting extrapancreatic neuroendocrine tumour. Gross necropsy revealed tumour invasion into the spleen, liver, adrenal glands and mesenteric lymph nodes. Immunohistochemical analysis of the neoplastic cells revealed that the tumour was a glucagonoma, consistent with earlier findings of persistent glucagonaemia and hypoaminoacidaemia.
    Veterinary Dermatology 03/2009; 20(1):72-9. DOI:10.1111/j.1365-3164.2008.00729.x · 1.99 Impact Factor
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    ABSTRACT: An eleven-year-old female pug was referred to Yamaguchi University Animal Hospital for evaluation of anemia and thrombocytopenia. The cytological examination of the peripheral blood showed some giant monocytic lineage blast cells. A few granulocytes and platelets had dysplastic features. On day 7, in addition to increasing the monocytic lineage cells, the dysplastic features of the blood had also increased compared to the initial examination. We performed bone marrow aspiration upon her death. The bone marrow revealed dysplastic features in all three hematopoietic cell lines, and an increase in the monocytic cell line. Based on the features of the bone marrow and the peripheral blood, this case was confirmed to be myelodysplastic syndrome--Chronic myelomonocytic leukaemia (MDS-CMML).
    Journal of Veterinary Medical Science 07/2007; 69(6):665-8. DOI:10.1292/jvms.69.665 · 0.88 Impact Factor
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    ABSTRACT: Ticks removed from 1136 dogs and 134 cats all over Japan were examined for Borrelia infection by PCR and sequencing. The 5S-23S rDNA intergenic spacer of Borrelia was detected from two Ixodes persulcatus ticks from two dogs and two unidentified Ixodes spp. from another two dogs in Hokkaido, and two Ixodes granulatus ticks from two cats in Okinawa. Additional 2 I. granulatus from the same cats also showed positive. Sequence analysis of the PCR products revealed that the one from Hokkaido was similar to B. garinii, the three from Hokkaido to B. tanukii, and the four from Okinawa to a novel Borrelia sp. closely related to B. valaisiana. The data was confirmed by analysis of the flagellin gene sequence. Infected ticks carried by companion animals can be introduced into the human environment.
    Veterinary Parasitology 04/2007; 144(1-2):188-92. DOI:10.1016/j.vetpar.2005.05.073 · 2.55 Impact Factor
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    ABSTRACT: Growth arrest and DNA damage-inducible protein 45 (GADD45) plays an important role in suppressing multistep carcinogenesis. In this report, we describe the isolation of the complete wild-type feline GADD45 cDNA from feline tissues. Expression of feline GADD45 mRNA was detected in the liver, spleen, kidney, lung, and testis. The predicted amino acid sequences encoded by the full-length feline GADD45 cDNA display sequence homology with those from other vertebrates, and as in the case of human GADD45, cell growth suppression was observed by ectopic expression of feline GADD45. However, no mutations were detected by sequence analysis of feline GADD45 in several feline lymphoma cell lines, indicating that the GADD45 mutation might be uncommon in feline oncogenesis.
    Journal of Veterinary Medical Science 04/2006; 68(3):297-301. DOI:10.1292/jvms.68.297 · 0.88 Impact Factor
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    ABSTRACT: Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.
    Veterinary Parasitology 04/2006; 136(2):147-54. DOI:10.1016/j.vetpar.2005.10.021 · 2.55 Impact Factor
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    ABSTRACT: DNA from ticks recovered from 1137 dogs and 133 cats from all over Japan were examined for Rickettsia infection by citrate synthase gene (gltA)-based PCR and partial nucleotide sequencing. A total of 91 dog tick samples and 18 cat tick samples showed a single band of the appropriate size in the nested PCR. Sequence analysis was successfully performed on 102 samples. DNA of Rickettsia japonica or closely related Rickettsia spp. strains were detected from 38 ticks in 16 prefectures mainly in western Japan. The other 33, detected from 13 prefectures including Hokkaido and Okinawa, were found to be Rickettsia helvetica or closely related strains. A total of 29 DNA that showed highest homology with Rickettsia akari or closely related strains were detected in 19 prefectures, widespread throughout Japan. Rickettsia canada-like DNA was detected from Haemaphysalis sp. removed from a dog in Fukuoka, and ;Candidatus Rickettsia tarasevichiae'-like DNA was from Ixodes sp. removed from a dog in Hokkaido.
    Journal of Veterinary Medical Science 01/2006; 67(12):1217-22. DOI:10.1292/jvms.67.1217 · 0.88 Impact Factor

Publication Stats

105 Citations
27.38 Total Impact Points


  • 2005–2015
    • Yamaguchi University
      • • Joint Faculty of Veterinary Medicine
      • • Faculty of Agriculture
      Yamaguti, Yamaguchi, Japan
  • 2013
    • Japan Animal Referral Medical Center
      Kawasaki Si, Kanagawa, Japan
  • 2007
    • Azabu University
      Sagamihara, Kanagawa, Japan