Jun Nagayama

Hypomethylation Status of CpG Sites at the Promoter Region and Overexpression of the Human MDR1 Gene in Acute Myeloid Leukemias

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. Moreover, overexpression of the MDR1 gene has been shown to be associated closely with clinical outcome in various hematological malignancies, including acute myeloid leukemia (AML). However, the precise mechanism underlying overexpression of the MDR1 gene during acquisition of drug resistance remains unclear. We recently described an inverse correlation between the methylation status of CpG sites at the promoter region and expression of the MDR1 gene in malignant cell lines. In this study, we expanded this analysis to 42 clinical AML samples. We adapted a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for gene expression and a quantitative PCR after digestion by Hpa II for methylation status of the MDR1 gene. We observed a statistically significant inverse correlation between methylation and MDR1 expression in clinical samples. The hypomethylation status of the MDR1 promoter region might be a necessary condition for MDR1 gene overexpression and establishment of P-glycoprotein-mediated multidrug resistance in AML patients.

Increased expression of multidrug resistance-associated proteins in bladder cancer during clinical course and drug resistance to doxorubicin

Overexpression of the P‐glycoprotein/multidrug resistance 1 (MDR1) and multidrug resistance protein 1 (MRP1) gene is closely associated with the clinical outcome of various malignancies, and it is involved in responses to some anticancer chemotherapeutic agents including doxorubicin. Six human MRP subfamily members (MRP2‐7) with structural similarities to MRP1 have been identified. Recently, the relationships between MRP2 and MRP3 expression levels of some cancer cells and drug sensitivity to doxorubicin have been reported, but the relationship between the clinical samples and drug sensitivity remains unclear. We determined the expressions of the MDR1, MRP1, MRP2 and MRP3 gene in bladder cancer during the clinical course and sought to learn whether the expression was correlated with drug responses to doxorubicin. Doxorubicin, used in chemotherapeutic treatment including intravesical and systemic chemotherapy, is an important anticancer agent for the treatment of bladder cancer. We used quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis for our study, and the sensitivity to doxorubicin in bladder cancer was determined using the in vitro succinate dehydrogenase inhibition test. Using 47 clinical samples of bladder cancer, we confirmed the significant correlation of MDR1, MRP1 and MRP3 mRNA levels with resistance to doxorubicin. We showed that the expression of MDR1, MRP1, MRP2 and MRP3 in recurrent tumors and residual tumors after chemotherapeutic treatment was higher than that in untreated primary tumors. In particular, the MDR1 expression in residual tumors was 5.7‐fold higher than that in untreated primary tumors.

Alu-associated interstitial deletions and chromosomal re-arrangement in 2 human multidrug-resistant cell lines.

Previous studies have shown that gene re‐arrangements play a significant role in tumorigenesis. Gene re‐arrangements involving the human multidrug resistance‐1 (MDR1) gene have been identified as a mechanism for MDR1 over‐expression in human malignant cells. In 2 multidrug‐resistant human cancer sublines with high levels of MDR1 and P‐glycoprotein (MCF7/TX400 and S48‐3s/Adr10), hybrid mRNAs containing sequences from MDR1 and an unrelated gene have previously been identified. To characterize and determine the site of the re‐arrangements resulting in generation of hybrid mRNAs, we first constructed a λ phage library extending over a contiguous genomic region of 100 kb and containing the region upstream of MDR1. In MCF7/TX400 cells, homologous recombination was observed involving an Alu repeat 80 kb upstream of the MDR1 gene, with a 79 bp intra‐Alu deletion flanked by χ‐like sequences at the re‐arrangement junction. By contrast, non‐homologous recombination was observed in S48‐3s/Adr10 cells with Alu repeats near the junction sequence. While the specific features of the breakpoints appear to be different, Alu repeats might be involved in both gene re‐arrangements. The gene re‐arrangements at or near the Alu sequence should be regarded as potentially involved in the transcriptional activation of human MDR1. Int. J. Cancer 86:506–511, 2000.

Anaplastic large cell lymphoma in leukemic presentation: a case report and a review of the literature.

It is extremely rare that a patient with anaplastic large cell lymphoma (ALCL) demonstrates circulating lymphoma cells. A 10-year-old Japanese boy was presented with high-grade fever and cough. The physical examination revealed marked hepatosplenomegaly with ascites and lymphadenopathy in the cervical and periauricular areas. The white cell count was 26.2x10(9)/L with 95% of abnormal lymphoid cells, which were small to medium-sized with a high nucleus/cytoplasm ratio, basophilic cytoplasm, condensed nuclear chromatins, and 1 or 2 distinct nucleoli, hemoglobin 6.4 g/dL, and platelet 0.9x10(9)/L. A flow cytometric analysis of abnormal cells in both the peripheral blood and bone marrow samples was strongly positive for CD30 on their cell membranes. Karyogram and fluorescent in situ hybridization showed abnormal cells to have a characteristic chromosomal translocation, t(2;5)(p23;q35). Reverse transcriptase-polymerase chain reaction of peripheral blood cell-derived mRNA also indicated the fusion gene product of anaplastic lymphoma kinase and nucleophosmin. Subsequently, the patient was diagnosed to have ALCL with a rare clinical feature of a peripheral leukemic presentation, and his disease revealed to be refractory to chemotherapy. On the basis of the 11 childhood cases of ALCL with leukemic presentation so far published and reviewed herein, the prognosis is very poor.

Bone marrow transplantation in children with severe aplastic anemia using a conditioning regimen containing 3 Gy of total body irradiation, cyclophosphamide with or without antithymocyte globulin

Abstract:  We have employed the 3 Gy toal body irradiation (TBI) containing conditioning regimen to bone marrow transplantation (BMT) for severe aplastic anemia (SAA) in pediatric patients irrespective of donor type since March 1986. The outcome of BMT for 17 SAA patients is favorable. Eight patients received BMT from human leukocyte antigen matched‐related donors (MRD) and nine received BMT from alternative donors. The conditioning regimen consisted of 3‐Gy TBI and cyclophosphamide of 200 mg/kg in the BMT from MRD. In the case of BMT from alternative donor, antithymocyte globulin 10 mg/kg was added to the regimen. Fifteen of 17 patients (88%) engrafted on median of day 18 (range, 11–26) and all 13 evaluable patients showed complete donor chimerism by median 30 (range, 13–47) days after BMT. Fourteen patients have survived with a median follow‐up of 67 (range, 2–228) months and the probability of survival was 81.9% (95% CI, 63.3–100%). No late complications including second malignancies caused by TBI have been observed and all three female patients have regular menstruation. In conclusion, TBI of 3 Gy appears to be an appropriate dose regarding to ensure engraftment and avoid the risk of late adverse event for SAA patients.

Case of epilepsy with speech disorders: EEG changes and magnetoencephalographic analysis

We report a 6-year-old girl who developed speech and oromotor deficits during the course of epilepsy. Before the onset of the speech disorders, EEG showed spikes over the bilateral frontotemporal region (predominantly on the left side) and occasionally over the bilateral central region. At the onset of the speech disorders, EEG showed background activity mixed with high-voltage slow waves over diffuse regions, predominantly over the left side, and multifocal spikes and slow spike and wave (SW) complexes. Magnetoencephalography (MEG) showed three dimensionally that the calculated foci of temporal spikes were especially located beneath the somatosensory area for the right thumb. The focal EEG abnormalities over the left temporal region and the abnormal background activity were considered to have become serious and to have caused the speech and oromotor deficits. Antiepileptic drug (AED) therapy led to marked improvement of EEG, and oral corticosteroid administration had a beneficial effect on the speech disorders. Some investigators believe that benign childhood epilepsy with centrotemporal spikes (BECTS) and acquired epileptic aphasia represent a spectrum. Our patient had some of the clinical manifestations and EEG findings of these two epileptic syndromes. Therefore, we suggest that the condition of our patient lies between these two syndromes