Yoshiki Katsumi

Restoration of p53 Pathway by Nutlin-3 Induces Cell Cycle Arrest and Apoptosis in Human Rhabdomyosarcoma Cells

Purpose: Seventy to eighty percent of rhabdomyosarcoma (RMS) tumors retain wild-type p53. The tumor suppressor p53 plays a central role in inducing cell cycle arrest or apoptosis in response to various stresses. p53 protein levels are regulated by MDM2 through ubiquitin-dependent degradation. In this study, we evaluated whether nutlin-3, a recently developed small-molecule antagonist of MDM2, has an effect on p53-dependent cell cycle arrest and apoptosis in cultured human RMS cell lines. Experimental Design: Five RMS cell lines with different p53 statuses and MDM2 expression levels were treated with nutlin-3. Gene expression patterns, cell viability, cell cycle, and apoptosis after nutlin-3 treatment, and antitumor activity of combination treatment with vincristine or actinomycin D were assessed. Results: Significant p53 activation was observed in wild-type p53 cell lines after nutlin-3 treatment. p53 activation led to cell cycle arrest in parallel with increased p21 expression. Furthermore, these cell lines underwent p53-dependent apoptosis, concomitant with elevation of proapoptotic genes and activation of caspase-3. The effect of nutlin-3 was almost the same in terms of half maximal inhibitory concentration and apoptosis whether or not MDM2 was overexpressed. Nutlin-3 did not induce either cell cycle arrest or apoptosis in p53 mutant cell lines. A combination of vincristine or actinomycin D with nutlin-3 enhanced the antitumor activity in RMS cell lines with wild-type p53. Conclusions: Nutlin-3 effectively restored p53 function in both normal MDM2 expression and MDM2 overexpression RMS cell lines with wild-type p53. p53 restoration therapy is a potential therapeutic strategy for refractory RMS with wild-type p53.

Circulating Methylated-DCR2 Gene in Serum as an Indicator of Prognosis and Therapeutic Efficacy in Patients with MYCN Nonamplified Neuroblastoma

Background:MYCN amplification (MNA) in neuroblastoma is a strong indicator of poor prognosis. However, some MYCN nonamplified (non-MNA) cases show poor outcomes, and examining the status of the gene requires an operation, which may have surgical complications. Therefore, a new marker is needed to identify cases of non-MNA neuroblastomas with poor prognoses using less risky procedures. Aberrant hypermethylation of the DCR2 promoter has recently been associated with rapidly progressing neuroblastoma. We aimed to develop a noninvasive DCR2 methylation assay for patients with neuroblastoma using serum DNA, which predominantly originates from tumor-released DNA. Methods: Using DNA-based real-time PCR, we simultaneously quantified a methylated-DCR2 specific sequence (M) and a reference sequence (R) located in the promoter region in serum DNA, and evaluated DCR2 methylation status as M/R ratios in 86 patients with neuroblastoma. Results: Serum DCR2 M/R ratios were strongly correlated with those in the tumor (r = 0.67; P = 0.002). DCR2 methylation was associated with stage both in the whole neuroblastoma group and in the non-MNA group (P < 0.001), and DCR2-methylated patients showed significantly poorer 5-year event-free survival in the whole neuroblastoma group (43% versus 84%; P < 0.001), especially in the non-MNA group (12% versus 96%;P < 0.001). Among five DCR2-methylated patients whose clinical courses were followed, serum M/R ratios were close to 0 in the patients in remission, whereas the ratios increased in patients who relapsed. Conclusions: Detection of methylated-DCR2 in serum DNA has promise as a noninvasive assay for predicting prognosis and therapeutic efficacy in neuroblastoma, especially in non-MNA cases. Furthermore, it might be a sensitive marker of tumor recurrence in DCR2-methylated cases.

Sensitivity of Malignant Rhabdoid Tumor cell lines to PD 0332991 is inversely correlated with p16 expression

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC(50) values 0.01 to 0.6 μM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r=0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p<0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.

The incidence and direct medical cost of hospitalization due to rotavirus gastroenteritis in Kyoto, Japan, as estimated from a retrospective hospital study

Anticipating imminent licensure of rotavirus vaccine for use in Japan, we estimated the incidence of rotavirus hospitalization and calculated the direct medical cost associated with rotavirus hospitalization in a hospital that provided virtually exclusive pediatric beds to the local community adjacent to the northern outskirts of metropolitan Kyoto, Japan. For a 2 year period between September, 2008 and August, 2010, there were 103 hospitalizations due to acute gastroenteritis among children less than 5 years of age. Stool specimens from 77 (75%) of the 103 hospitalized patients were tested for rotavirus antigen, and 46 (60%) were positive. The proportion of rotavirus positives was 65% in the peak-season months (January-June) and 17% in the off-season months (July-December). By extrapolating the test results to those patients with acute gastroenteritis who were not tested, 13 additional cases were estimated to be rotavirus positive. Assuming that all patients with rotavirus gastroenteritis less than 5 years of age in the catchment (5532 according to the 2005 census) were admitted to this hospital, the annual incidence of rotavirus hospitalization was estimated to be 4.1 (testing-unadjusted)-5.3 (adjusted) per 1000 child-years. Thus, it was estimated that one child in 48 or one child in 37 born in this area would be hospitalized due to rotavirus gastroenteritis by the age of 5 years. The incidence of rotavirus hospitalization was similar to the rate in Ise city (4.9 per 1000 child-years), also in central Japan, and lower than the rate in Honjo city in northern Japan (13 per 1000 child-years). Nevertheless, the burden of rotavirus hospitalization was substantial, and the total direct medical cost was estimated to be 6.6 billion Japanese Yen (US

Trastuzumab Activates Allogeneic or Autologous Antibody-Dependent Cellular Cytotoxicity against Malignant Rhabdoid Tumor Cells and Interleukin-2 Augments the Cytotoxicity

57 million). While economic analysis and comparisons with alternative preventive procedures may be necessary, this study provides the policymakers and pediatricians with further evidence that is necessary to decide whether to introduce rotavirus vaccines into the routine childhood immunization schedule in Japan.

Effect of a Single Inhalation of Laninamivir Octanoate in Children With Influenza

Purpose: Malignant rhabdoid tumor (MRT) is an early childhood cancer with poor prognosis. Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor-2 (HER-2), has been shown to be effective against breast cancer and other cancers. We investigated the effect of trastuzumab on MRT cell lines. Experimental Design: We examined expression of HER-2 on four MRT cell lines and two tumor tissues by indirect immunofluorescence, flow cytometry, and immunohistochemistry. The effect of trastuzumab against MRT cells was examined by cell growth assay. To observe the antibody-dependent cellular cytotoxicity of effector cells, we examined the cytotoxicity of trastuzumab in combination with allogeneic or autologous human peripheral blood mononuclear cells with and without IL-2 using the chromium release assay. Results: All four MRT cell lines and both MRT tissues expressed HER-2 protein. Trastuzumab alone did not reduce the viability of the MRT cell lines. On the other hand, the cytotoxicity of trastuzumab against each of the MRT cell lines was significantly increased by the presence of allogeneic and autologous peripheral blood mononuclear cells (P < 0.01). There was a strong correlation coefficient (r = 0.825) between HER-2 expression and the cytotoxicity enhanced by trastuzumab. Moreover, trastuzumab in combination with peripheral blood mononuclear cells augmented by interleukin-2 (IL-2) was significantly more cytotoxic than trastuzumab alone or IL-2 alone (P < 0.01). Conclusions: Our results indicate that (1) trastuzumab can exert antitumor effects on MRT cells by using the antibody-dependent cellular cytotoxicity of effector cells and (2) IL-2 can enhance the cytotoxicity of trastuzumab against MRT cells.

Preoperative analysis of 11q loss using circulating tumor-released DNA in serum: A novel diagnostic tool for therapy stratification of neuroblastoma

OBJECTIVE: The purpose of this study was to compare the efficiency and safety of a new neuraminidase inhibitor, laninamivir octanoate (LO), with zanamivir (ZN) in pediatric patients with influenza. METHODS: One hundred twelve pediatric patients ≤15 years, diagnosed with a rapid diagnostic test as having influenza from January to May 2011, were randomly assigned to the LO group or the ZN group, and their parents were asked to complete a questionnaire during the recovery at home. The LO group was instructed to inhale LO once (20 or 40 mg depending on age), and the ZN group was instructed to inhale ZN (20 mg) twice daily for 5 days. RESULTS: The LO group (n = 55) and the ZN group (n = 57) were well balanced. Finally, 44 patients in the LO group and 41 patients in the ZN group could be evaluated. Median times to fever resolution after initial treatment were 36 hours in the LO group and 37 hours in the ZN group. No differences were observed between the 2 groups with respect to the frequencies of asthmatic symptoms, pneumonia, gastrointestinal symptoms, or abnormal behaviors. Six younger children could not inhale LO well for technical reasons. CONCLUSIONS: Our data suggest that the efficiency and safety of LO are the same as those of ZN in pediatric patients with influenza but that LO may be more convenient than ZN because it requires only a single inhalation. However, younger patients may not inhale LO efficiently.

A NOXA/MCL‐1 Imbalance Underlies Chemoresistance of Malignant Rhabdoid Tumor Cells

Allelic deletion of the long arm of chromosome 11 (11q loss) is closely associated with the prognosis of neuroblastoma (NB). Here we examined 11q loss using tumor-released DNA fragments in the sera of 24 cases. The allelic intensity score of a panel of polymorphic markers in 11q23 in serum DNA was significantly different between the 11q loss-positive group and the11q loss-negative group. The 11q loss-positive and -negative groups did not overlap when a cut-off value of 0.5 was chosen for the allelic intensity score. Our serum-based 11q loss analysis could predict the allelic status of 11q in tumors.

Incomplete Inhalation of Laninamivir Octanoate in Children with Influenza

Malignant rhabdoid tumor (MRT) is a rare aggressive pediatric cancer characterized by inactivation of SNF5, a core subunit of SWI/SNF complexes. Previously, we showed that SNF5 contributes to transcriptional activation of NOXA, a pro‐apoptotic protein that binds and inhibits the anti‐apoptotic protein MCL‐1. In this study, we found that NOXA expression was downregulated in MRT cell lines as well as in clinical MRT samples and that ectopically expressed NOXA bound MCL‐1 and increased the sensitivity of MRT cell lines to doxorubicin (DOX) by promoting apoptosis. Consistent with this finding, knockdown of MCL‐1 in MRT cell lines induced apoptosis and increased DOX sensitivity in MRT cells, and the MCL‐1 inhibitor TW‐37 synergized with DOX to induce MRT cell death. Our results suggest that modulation of the NOXA/MCL‐1 pathway may be a potential strategy for the treatment of patients with MRT. J. Cell. Physiol. 231: 1932–1940, 2016.

Saffold cardiovirus infection in a 2-year-old boy with acute pancreatitis.

Background: Laninamivir octanoate (LO), an inhaled neuraminidase inhibitor, has been used against influenza in Japan. However, children who cannot inhale well might not receive the full dose. We examined the relation between the residual amounts of LO in the device after its use and the time to fever resolution in children with influenza. Methods: The subjects of the study were 161 children (4-15 years) who had been diagnosed as having influenza from 2011-2014 and who had demonstrated an ability to properly use an inhalation training device. After its use, the LO device was returned to the authors and the residual amount of LO was measured. After flu symptoms had resolved, the patients’ parents reported the time to fever resolution in a questionnaire. Results: The percentage of the residual LO was not significantly correlated with the time to fever resolution or with patient age. The percentage of residual LO tended to be higher and the time to fever resolution tended to be longer in 4-6 year-olds than in older children. In the 4-6 year group, seven patients were inhaling steroids. These patients tended to inhale more of the LO dose and tended to have a shorter time to fever resolution than the other patients. Conclusions: Physicians need to be aware that some 4-6 year-old influenza patients will not be able to inhale the full dose of LO. Influenza patients, even young ones, who are also inhaling steroids for other conditions are better able to inhale LO.