Additional gene therapy with Ad5CMV-p53 enhanced the efficacy of radiotherapy in human prostate cancer cells.

PURPOSE: The aim of this study was to investigate the efficacy of combination therapy of ionizing radiation (IR) and adenoviral p53 gene therapy and to evaluate its molecular mechanisms. METHODS AND MATERIALS: Two human prostate cancer cell lines, DU145 and PC-3 cells, containing different types of p53 gene mutations, were investigated. The recombinant adenovirus vector containing the wild-type p53 gene (Ad5CMV-p53) was used for this study. Cells were irradiated (in 0, 2, 4, and 6 Gy, 300 cGy/min) and after 12 h of irradiation, the cells were infected with various doses of Ad5CMV-p53 (0-40 multiplicity of infection [MOI]). Cytotoxicity was determined by clonogenic assay. The molecular mechanisms were evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), apoptotic cell detection, and cell cycle analysis. RESULTS: The cell growth inhibition in DU145 (p53-mutated) cells by IR was strongly enhanced by additional Ad5CMV-p53 infection in a viral dose-dependent manner. In DU145 cells, IR alone induced minimal p53 mRNA expression. However, IR combined with Ad5CMV-p53 infection stimulated significant increase in p53 mRNA expression supplemented with Bax and p21 mRNA expressions. In PC-3 (p53-null), IR induced Bax and p21 mRNA expression, while the combination effects were observed in p53, Bax, and p21 mRNA expression. Apoptotic cell deaths were rarely observed after IR alone (DU145: 3%, PC-3: 5%). However, after combination therapy, the proportion of apoptotic cells greatly increased (sevenfold in DU145 cells, and twice in PC-3 cells). G1 cell cycle arrest was observed after Ad5CMV-p53 infection and the combination in both cell lines. CONCLUSION: In this study, we demonstrated that the combination of IR and Ad5CMV-p53 gene therapy resulted in remarkable synergistic effects in human prostate cancer cells. This combination therapy could be one of the optimal treatment strategies for radioresistant prostate cancer.

[First documentation of transfusion-associated babesiosis in Japan].

A 40-year-old man received blood transfusion in December 1998 because of gastric bleeding from a peptic ulcer. One month later, he developed febrile hemolytic anemia. Administration of high doses of glucocorticoid significantly reduced the hemolysis, but did not cure the disease. To investigate the cause of the hemolysis, the patient was transferred to our hospital in May 1999. Giemsa-stained peripheral blood smears showed Babesia parasites in the red blood cells (RBC), and PCR analysis confirmed the presence of Babesia microti DNA. The parasitemia disappeared hematologically after 2 weeks of quinine and clindamycin therapy. However, parasite DNA was still detectable in the RBC. Although treatment with oral atovaquone was given for 2 weeks, parasitemia and febrile hemolysis recurred within a month after the last treatment. Fortunately, complete remission was obtained after a second 12-week course of therapy with quinine and clindamycin. PCR analysis revealed asymptomatic Babesia infection in one of eight samples from the original blood donor. The initial steroid therapy given to the patient without an accurate diagnosis seemed to have delayed augmentation of the specific antibodies (IgG) against Babesia microti, thus prolonging the parasitemia after the initial acute stage of babesiosis.

T-cell-specific expression of kinase-defective Eph-family receptor protein, EphB6 in normal as well as transformed hematopoietic cells.

Although most kinase-defective growth factor receptor proteins are associated with pathogenic conditions, a kinase-defective Eph-family receptor protein, EphB6, is expressed in normal human tissues. We generated monoclonal antibodies specific for human EphB6 to characterize its expression on human hematopoietic cells. A very small population of normal human peripheral white blood cells (0.57 +/- 0.07%, n = 12) expressed EphB6. The EphB6-positive cells were CD2+, CD7+, CD3+ and CD4+ or CD8+ lymphocytes, but they did not express CD19 or CD11b. In human bone marrow, only 1.5 +/- 0.19% of lymphocytes expressed EphB6. Compared with the expression in peripheral lymphocytes, prominent expression of EphB6 protein was demonstrated in CD4+CD8+ double-positive mouse thymocytes. The T-cell lineage-specific expression was strictly conserved in human leukemia/lymphoma cells. Among T-cell-derived leukemia cells, the expression level of EphB6 seemed to decrease with maturation of the cells. These results suggest that EphB6 expression is regulated in T-cell development.

Feasibility of high-dose chemotherapy without stem cell support as a first-line treatment for non-Hodgkin's aggressive lymphoma: a pilot study.

A regimen which incorporates cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) is the standard treatment for patients with non-Hodgkin's lymphoma (NHL), but it has not been effective in patients with aggressive NHL who are at high risk. The aim of the present trial was to investigate the feasibility of high-dose chemotherapy (HDC) without stem cell support as a first-line treatment. The primary endpoint was a complete remission rate. The second endpoint was survival. Fourteen patients with aggressive NHL entered the study and were treated according to the K93 protocol (3 cycles of CHOP, high-dose etoposide and ifosfamide, and high-dose methotrexate) Eleven patients (79%) achieved complete remission (CR) and two (14%) achieved partial remission (PR). Overall survival (OS) after five years was 79%. The actuarial five year disease free survival (DFS) for the eleven cases of CR was 75%. During chemotherapy, grade IV hematologic toxicity was observed in all patients and grade IV non-hematologic toxicity in only one patient, who experienced oral ulcers. Peripheral blood stem cell (PBSC) apheresis was performed in eight cases. One harvesting was enough to provide an adequate number of CD34+ cells for the subsequent PBSC transplantation (PBSCT). In conclusion our study confirmed the efficacy of the K93 protocol in obtaining a good response (CR + PR) rate and a very good DFS rate in most cases of aggressive NHL, with acceptable toxicity. This regimen may improve the outcome in cases of aggressive NHL without stem cell support. It seems worthwhile to conduct a randomized controlled study comparing the K93 protocol with the standard CHOP regimen.

Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction.

smg p25A is a small G protein which has been suggested to regulate neurotransmitter release from the synapses. We investigated here the ultrastructural localization of this small G protein in the rat neuromuscular junction by an immunoperoxidase method. The results showed that smg p25A was distributed non-uniformly on the presynaptic plasma membrane and among the synaptic vesicles with the focal accumulation on the discrete presynaptic sites which corresponded to the active zones, the regions of the presynaptic plasma membrane specialized for the exocytosis of the synaptic vesicles. This unique distribution of smg p25A suggests that it plays an important role in the attachment and fusion of the synaptic vesicles with the active zones.