PubMed Health⌕ Search

Biomedical subjects

R Hanada

Publications and source records attributed to R Hanada.

At least 37 records · Page 2Linked to original sources
PubMed2000

Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia.

Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.

Acute Disease↗
PubMed2000

Allelic imbalance on chromosome 18 in neuroblastoma.

We previously demonstrated that chromosome 18 is frequently deleted in neuroblastoma. To further elucidate the role of chromosome 18 deletions in the development of neuroblastomas we examined 82 cases of neuroblastomas for allelic imbalance (AI) at 17 loci on chromosome 18 to define the common region of AI in neuroblastoma. AI at one or more loci on chromosome 18 was detected in 18/82 (22%) cases. AI on 18q was detected in 17/82 (21%) cases, whereas AI on 18p was detected in 4/82 (5%) cases. There was a distinct common region of AI at 18q21.1 between the D18S363 and D18S858 loci. In addition, cases 16 and 53, which did not show AI at 18q21.1, showed AI at 18pter-q12.3 between the D18S52 and D18S36 loci, indicating that another common region of AI may exist on chromosome 18. AI on chromosome 18 did not significantly correlate with any clinicopathological findings of patients with neuroblastoma. The common region of AI at 18q21.1 includes the DCC gene but not the Smad2 and Smad4 genes. However, our previous studies together with the present study indicated that the incidence of DCC mutation is much less than that of AI at 18q21.1 in neuroblastoma. These results indicate that novel tumour suppressor genes involved in the development of neuroblastoma are present at 18q21.1, and possibly at 18pter-q12.3.

Blotting, Southern↗
PubMed2000

Long-term follow-up of childhood acute lymphoblastic leukemia in Tokyo Children's Cancer Study Group 1981-1995.

The objectives were as follows: Firstly, to estimate the overall probability of event-free survival (EFS) and isolated CNS relapse in the studies for children with acute lymphoblastic leukemia (ALL) during the 1980s and 1990s. Secondly, to report the EFS according to presenting features and lineage. Thirdly, to evaluate the treatment results re-classified by the risks of NCI criteria. Four consecutive protocol studies were performed in the Tokyo Children's Cancer Study Group: L81-10 protocol (1981-1984, 189 patients), L84-11 (1984-1989, 484 patents), L89-12 (1989-1992, 418 patients) and L92-13 (1992-1995, 347 patients). Overall EFS at 5 years in each protocol was 56.5 +/- 3.8(1 s.e.)%, 71.0 +/- 2.1%, 67.8 +/- 2.3%, and 63.4 +/- 2.7%, respectively. The cumulative isolated CNS relapse rate at 5 years was 8.1 +/- 2.1%, 3.5 +/- 0.9%, 3.6 +/- 1.0%, 1.0 +/- 0.6. The EFS in SR/HR (standard risk/high risk) according to the NCI criteria in B-precursor ALL at 5 years was 61.9 +/- 4.3%/41.4 +/- 7.4% (lineage was not confirmed.), 72.5 +/- 2.6%/63.4 +/- 5.0%, 77.4 +/- 2.7%/56.3 +/- 4.7%, and 67.8 +/- 3.4%/56.7 +/- 5.4% in each protocol. Also EFSs according to NCI SR/HR at 5 years of T-ALL in protocols L84-11, L89-12 and L92-13 were 55.6 +/- 16.6%/60.9 +/- 10.1%, 72.7 +/- 13.4%/51.6 +/- 9.1%, and 77.1 +/- 14.4%/53.6/10.1%, respectively. The truncation of maintenance therapy to 6 months resulted in a decreased EFS in L92-13, particularly due to an increase of bone marrow relapse after cessation of therapy in SR and HR. The NCI risk criteria work properly even in the patients treated by different intensities, so that it makes the comparison possible among the patients in various groups. The overall EFSs in childhood ALL improved in 1980s, but it seemed stable or decreased in 1990s. The short maintenance therapy resulted in poor outcome in SR on the L92-13 protocol. Many of these late relapsers were effectively rescued and overall survival remained at a high level. The proportion of patients who received cranial irradiation reduced without any increase of the CNS events.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Tissue-specific expression of the uncoupling protein family in streptozotocin-induced diabetic rats.

The vulnerability of streptozotocin (STZ)-induced diabetic rats to cold stress has been established. One of the elements controlling body temperature is thermogenesis, in which uncoupling protein (UCP) is known to play an important role. We have examined UCP2 and UCP3 expressions in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle (MSL) during the acute and chronic phases of STZ-induced diabetes in rats. The long-term effect and the effect of insulin treatment thereafter were also unexplored previously and are examined in this study. In the acute phase of diabetes (2.5 days after STZ injection), UCP2 gene expression in BAT, WAT, and MSL, and UCP3 expression in the muscle were significantly increased. In the chronic phase of diabetes (21 days after STZ injection), UCP2 and UCP3 expression in the MSL were restored to the control levels without insulin supplementation. UCP2 in BAT and WAT remained high in the chronic phase, whereas UCP3 expression in BAT and WAT, which did not change in the acute phase, was significantly decreased. Insulin supplementation restored UCP2 expression in BAT and WAT, but over-corrected UCP3 in WAT above the control and did not affect UCP3 expression in BAT. Insulin supplementation depressed UCP3 expression in the MSL below control. These results indicate that the effects of STZ-induced diabetes on UCPs gene expression are tissue-specific as well as dependent on the duration of diabetes.

Adipose Tissue↗
PubMed2000

Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood.

PURPOSE: We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS: Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS: The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION: Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high-risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course.

Adolescent↗
PubMed2000

The p73 gene is less involved in the development but involved in the progression of neuroblastoma.

We performed expression, mutation, loss of heterozygosity (LOH) and fluorescence in situ hybridization (FISH) analyses of the p73 gene in neuroblastomas (NBs). Reverse transcription-polymerase chain reaction (RT-PCR) using primers which can detect both the p73alpha and p73beta transcripts was performed on 30 fresh NBs and 22 NB cell lines. Aberrant expression of the p73 gene was found in 4 (25%) of 16 primary tumors found by mass screening and in 10 (71.4%) of 14 primary tumors found clinically. The rates of expression in these two types of tumors were significantly different (p=0.026, Fisher's exact test). The incidence of aberrant expression of the p73 gene was significantly higher in stage IV patients than in stages I, II, III plus IVS patients (p=0.0236, Fisher's exact test). No homozygous deletions or rearrangements of the p73 gene were found in any samples examined. In addition to the polymorphism in exon 2, a silent mutation (codon 336 GCC/GCT) was found in one primary tumor. LOH of the p73 gene was detected in 5 (15%) of 33 primary NBs using PCR-LOH analysis. FISH analysis showed that all 17 NB cell lines used in this study revealed allelic loss of the p73 gene, while most of them expressed the p73 gene. These results suggest that the p73 gene is not monoallelically expressed in NB. We conclude that the p73 gene is less involved in the development but involved in the progression of neuroblastoma.

DNA Primers↗
PubMed2000

Reduced or absent expression and codon 201Gly/Arg polymorphism of DCC gene in rhabdomyosarcoma and Ewing's sarcoma/PNET family.

Genetic alterations occurring in various chromosomes have been described in many human tumors. The DCC gene was originally identified in colorectal cancer and was reported as a tumor suppressor gene that might be related to tumor metastasis. We investigated 10 cell lines and 15 fresh tumors of childhood rhabdomyosarcoma, 7 cell lines of Ewing's sarcoma, and 4 cell lines of primitive neuroectodermal tumor (PNET) for the expression and mutation of DCC gene by RT-PCR analysis and PCR-single stranded conformation polymorphism (SSCP) analysis. Twenty-five pairs of primers were used for PCR-SSCP. Six of ten (60%) cell lines of rhabdomyosarcoma and 3 of 7 (43%) cell lines of Ewing's sarcoma showed reduced or absent expression of DCC gene. There was no mobility shift within 24 exons by SSCP analysis, although 3 types of polymorphism were found at codon 201 in exon 3. Direct sequencing of different bands showed types I, II, and I/II representative of codon 201Gly, codon 201Arg, and codon 201Gly/Arg, respectively. The proportion of type I between fresh rhabdomyosarcoma and normal controls was not significant. Our results suggested that the inactivation of DCC gene may play a role in the pathogenesis of a subset of rhabdomyosarcoma and Ewing's sarcoma.

Adolescent↗
PubMed1999

Characterization of orexin-A and orexin-B in the microdissected rat brain nuclei and their contents in two obese rat models.

Orexin-A and orexin-B (also known as hypocretins) are newly discovered hypothalamic peptides that stimulate food intake. Using separate radioimmunoassays for these rat orexins, we determined their distributions in microdissected nuclei of the diencephalon and brainstem which have accumulations of orexin fibers. High orexin contents (orexin-A: between 250 and 350 fmol/mg protein and orexin-B: between 650 and 900 fmol/mg protein) were present in the lateral hypothalamus; ventromedial hypothalamic, paraventricular thalamic and dorsal raphe nuclei; periaqueductal central gray and locus coeruleus. Moderate orexin contents (orexin-A: between 100 and 250 fmol/mg protein and orexin-B: between 300 and 500 fmol/mg protein) were found in the median eminence; suprachiasmatic, paraventricular hypothalamic, arcuate and supraoptic nuclei; substantia nigra and the nucleus of the solitary tract. Mature orexin-A and -B peptides were the major endogenous orexin molecules in these nuclei. The orexin-A and -B contents in the brains of obese Zucker rats that have disrupted leptin receptor were significantly higher than in their lean littermates, but in Otsuka Long-Evans Tokushima Fatty rats that have disrupted cholecystokinin type-A receptor the contents were similar to those of the controls. The widespread orexin distributions in the nuclei of diencephalon and brainstem suggest that orexins serve as neuromodulators, neurotransmitters, or both, in a wide variety of neural networks that regulate the autonomic and neuroendocrine systems.

Animals↗
PubMed1999

Four novel mutations of the Fanconi anemia group A gene (FAA) in Japanese patients.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by pancytopenia, predisposition to cancers, and a diverse variety of congenital malformations. At least eight complementation groups, A through H, have been described. Recently, the FA-A gene (FAA) has been isolated, and a large number of distinct mutations reported in ethnically diverse FA-A patients. Here, we report on the mutation analysis of five FA patients by single-strand conformation polymorphism. Out of five patients, at least three were found to have mutations in the FAA gene. The first patient was a compound heterozygote with a 1-bp deletion and a single-base substitution. The second patient had a heterozygous 2-bp deletion, which introduces a premature termination codon, and the third patient had a heterozygous splice donor site mutation in intron 27.

Base Sequence↗
PubMed1999

Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia.

We investigated the alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia (T-ALL) and T-ALL cell lines by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Mutations of the p53 gene were found in three of 57 (5%) patients at diagnosis, one of 14 (7%) patients at relapse and in 12 of 18 (67%) cell lines. In these 12 cell lines, four had more than two mutations of the p53 gene. The p53 mutations were found in four of five cell lines whose original fresh leukemic cells were simultaneously examined original fresh leukemic cells. However, only one of the four fresh leukemic cells had the same mutation. All patients with p53 mutations in the course of disease died. Mutations of the p21 gene were not identified in 71 fresh samples and in 18 cell lines. N-RAS mutations were found in two of 57 (4%) fresh T-ALL patients at diagnosis, and four of 18 cell lines (22%), whereas no mutations were detected in any samples at relapse. Alterations of the p16 gene were found in 18 of 47 (38%) patients at diagnosis and in seven of 14 (50%) at relapse. These differences were not statistically significant. There were no differences in the frequency of alteration of the p16 and p15 genes between event-free patients and the remaining patients. Furthermore, we found the methylation of p16 gene in three of seven patients lacking homozygous deletions, suggesting higher frequency of p16 inactivation than previous reports in T-ALL. Interestingly, we found that one allele is inactivated by methylation and another allele had nonsense mutation in one cell line (KOPT-KI), resulting in loss of protein expression of p16. This type of p16 inactivation has not been so far reported in leukemia. We conclude that, (1) p53 mutations are infrequent at diagnosis but tend to be associated with poor clinical outcome; (2) RAS and p21 mutations may not be involved in the pathogenesis of T-ALL; (3) not only frequent alterations of p16 and p15 genes but also methylation of p16 gene are involved in initiating the leukemogenesis of T-ALLs, and (4) these 5 genes are independently involved in T-ALL.

Adolescent↗
PubMed1999

Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children.

We examined mRNA expression and internal tandem duplication of the Fms-like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic PCR followed by sequencing. By RT-PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B-precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B-ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Acute Disease↗
PubMed1999

[Two cases of hearing disorder following general anesthesia].

Hearing impairment is not often considered as a potential complication of general anesthesia, despite several reports of post-operative sensorineural hearing loss. These disorders have occurred after otological as well as cardiobypass surgery. We experienced two patients both of whom had undergone orthopedic surgery. In both cases the patients experienced bilateral reversible hearing impairment after general anesthesia with nitrous oxide. It is likely that a change in the middle ear pressure as a result of Eustachian tube dysfunction may have caused transient conductive hearing loss added to sensorineural hearing disorder. After these cases we interviewed a series of 115 patients who had undergone general anesthesia to assess the extent of this problem. Contrary to our expectation, 7 patients complained of ear fullness or autophony after inhalation of nitrous oxide, although these symptons diminished within 24 hours. It is important to be aware of the possibility of hearing impairment when nitrous oxide is used especially if the patient has a history of a previous middle ear disease.

Aged↗
PubMed1999

Clinical and biological aspects of acute lymphoblastic leukemia in 62 infants: retrospective analysis of the Tokyo Children's Cancer Study Group.

BACKGROUND: As a first step to formulate a new treatment strategy for refractory acute lymphoblastic leukemia (ALL) in infants, clinical results and immunophenotypic and cytogenetic data were analyzed and compared with those from overseas. METHODS: There were 62 infants with ALL who were treated between 1977 and 1995 at 30 institutions affiliated with the Tokyo Children's Cancer Study Group. Clinical and laboratory data obtained from these infants (all under 1 year of age) were retrospectively studied. RESULTS: The morphological diagnoses were FAB-L1 for 51 patients (82.2%) and FAB-L2 for 11 patients (17.8%). Hepatomegaly and splenomegaly were found in 40 (70.0%) and 40 patients (68.3%), respectively. The mean (+/- SEM) leukocyte count at diagnosis was 205,900 +/- 35,700/microL. The involvement of the central nervous system was evident in nine of 36 patients who were subjected to lumbar puncture, while three of these nine patients were free of neurological symptoms at diagnosis. Thirty-one patients (55.4%) were CD10 negative and 14 (25.0%) were CD10 positive. Thirty-one of 47 patients (65.9%) exhibited chromosomal abnormalities, including 28 patients (59.6%) with 11q23 abnormalities. Rearrangements in the MLL gene were found in nine of 13 infants (69.2%) examined. Translocation of 11q23 and/or MLL gene rearrangement (11q23/MLL) was significantly associated with the absence of the CD10 antigen. Hyperleukocytosis of more than 50,000/microL and 11q23/MLL gene rearrangements were related to a poor prognosis. The probability of an event-free survival in 62 infants was 13.1 +/- 4.8% at 48 months. CONCLUSIONS: New therapeutic strategies and large-scale cooperative prospective trials are needed to improve the prognosis of ALL in infants.

Chromosomes, Human, Pair 11↗
PubMed1999

Biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia.

The purpose of this study was to investigate the biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia (ALL). We carried out in vitro tests with a 4-day culture and a methyl-thiazol-tetrazolium assay on bone marrow samples from 209 children newly diagnosed with ALL. After testing the resistance of leukemic cells to 14 drugs, we classified the patients into two groups according to their sensitivity to three drugs (prednisolone, L-asparaginase, and vincristine) used in remission induction therapy. The three-drug resistant group (RR: sensitive to no drugs or to one drug) correlated with both short-term and long-term treatment failure. Three-year event-free survival (95% confidence interval) for the sensitive group (SS: sensitive to two or three drugs) was 0.813 (0.773-0.853) and that of the RR group was 0.616 (0.569-0.669) (P = 0.0001). Univariate analysis showed that Philadelphia-chromosome (Ph1) positivity and immunophenotype of mixed lineage were also prognostic factors in the 209 patients. The prognosis of the SS/RR drug resistance profile within 14 Ph1 patients was marginally significant (P = 0.062). Multivariate Cox regression analysis showed that Ph1 was an overwhelmingly adverse factor in event-free survival, with a relative hazard of 5.37 (2.57-11.21, P < 0.0001), followed by RR, with a relative hazard of 2.98 (1.69-5.25, P = 0.0001). Furthermore, we clarified the characteristics of the RR group by examination of the pattern of drug resistance to other drugs in comparison with the SS group. The leukemic cells of RR patients were more resistant than those of SS patients (P < 0.0001) to all the drugs tested, with resistance ratios of 1.6 to 13.1 (mean 3.4). In conclusion, in vitro three-drug resistance at the initial stage is an important independent predictor of treatment failure for both induction response and long-term outcome in childhood ALL.

Adolescent↗