Tomoyuki Shimabukuro

Cytokine-mediated antitumor effect of bacillus Calmette-Guérin on tumor cells in vitro.

Intravesical instillation therapy of bacillus Calmette-Guérin (BCG) is a useful modality for recurrent superficial transitional-cell carcinoma (TCC) of the urinary bladder. The mechanism of BCG effect has not yet been well characterized. BCG was tested in vitro for cytokine-mediated antiproliferative activity against T24 and KK47 cells (cell lines established from human TCC of the urinary bladder), and ACHN cells (cell line established from human renal cell carcinoma) using a modified human tumor clonogenic assay. Continuous exposure of cells to BCG at concentrations of more than 5 μg/ml in the presence of peripheral blood mononuclear cells (PBMC) consisting of a mixture of 5×104 monocytes/dish and 5×105 lymphocytes/dish, obtained from healthy donors, significantly inhibited colony formation of T24 and ACHN cells in comparison with growth inhibition in the absence of PBMC (P<0.05). Slightly inhibited colony formation was observed with KK47 cells under the same conditions. At the same time various cytokines were measured in supernatants when BCG and the same conditioned PBMC were co-cultured. Tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) were detected at markedly high levels at 24 h, and interferon γ (IFNγ) was detected at 120 h. IL-2 and macrophage-colony-stimulating factor were not detected. Neutralizing anti-TNFα monoclonal antibody significantly reduced the anti-proliferative activity of ACHN cells, and anti-IFNγ antibody reduced that of T24 cells. The results obtained suggest that cytokines mediated by BCG play an important role in the antitumor activity of BCG and that the sensitivity of bladder cancer cells to the cytokines induced by BCG may differ considerably.

Hyperbaric oxygen therapy for control of intractable cyclophosphamide-induced hemorrhagic cystitis

We report a case of intractable hemorrhagic cystitis due to cyclophosphamide therapy for Wegeners granulomatosis. Conservative treatment, including bladder irrigation with physiological saline and instillation of prostaglandin F2 alpha, failed to totally control hemorrhage. We then used hyperbaric oxygen at an absolute pressure of 2 atm, 5 days a week for 8 consecutive weeks. The bleeding ceased completely by the end of treatment and the patient remained free of hematuria thereafter. No side effect was noted during the course of therapy. In future, this form of therapy can offer a safe alternative in the treatment of cyclophosphamide-induced hemorrhagic cystitis.

9p21 index as estimated by dual-color fluorescence in situ hybridization is useful to predict urothelial carcinoma recurrence in bladder washing cytology

Recent studies have shown that chromosome 9p21 locus is frequently deleted in the early stages of urothelial carcinogenesis. To study the predictive value of the 9p21 aberrations in recurrence of urothelial carcinoma of the urinary bladder, we applied dual-color fluorescence in situ hybridization for 9p21 and chromosome 9 centromere to the bladder washing cytology samples that were obtained from the patients with urothelial carcinoma of the urinary bladder treated by transurethral resection. For the evaluation, the 9p21 index was defined as the ratio of the mean number of 9p21 signals per nucleus for that of the chromosome 9 centromere signals per nucleus in each of the bladder washing cytology samples. The 9p21 index values of the bladder washing cytology samples with no (G0) cytologic atypia were significantly higher than those of the bladder washing cytology samples with moderate (G2) (P < .01) and severe (G3) (P < .001) cytologic atypia, but the index values did not statistically differ from those of the bladder washing cytology samples with mild (G1) cytologic atypia. Recurrence-free survival in the patients with a low 9p21 index value (<0.9) was significantly poorer in comparison with the patients with a high 9p21 index value (>0.9). Furthermore, 2 patients of bladder washing cytology G1 with a low 9p21 index value recurred much sooner than the other patients of the bladder washing cytology G1 category. These findings indicate that a decreased 9p21 index value is associated with recurrence of urothelial carcinoma of the urinary bladder, and the 9p21 index may be useful as a marker to identify patients with elevated risk of recurrence of urothelial carcinoma of the urinary bladder.

Direct and indirect effects of recombinant human granulocyte-colony stimulating factor on in vitro colony formation of human bladder cancer cells

Although the present experimental use of recombinant human granulocyte-colony-stimulating factor (rG-CSF) has been proven to alleviate the myelosuppression induced by antitumor chemotherapy, it is also believed to stimulate growth of some nonhematopoietic tumor cells. We investigated both the direct and indirect effects of rG-CSF on in vitro colony formation of human bladder cancer cell lines using a modified human tumor clonogenic assay. Peripheral blood mononuclear cells (PBMC) were used as feeder cells (a mixture of 5×104 monocytes/dish and 5×105 lymphocytes/dish obtained from healthy donors). Human bladder cancer cell lines KK-47, TCCSUP and T24, all derived from human transitional-cell carcinomas, were incubated continuously with various concentrations of rG-CSF ranging from 0.01 ng/ml to 10 ng/ml both with and without PBMC for 7–21 days. The concentrations of rG-CSF used were chosen as being in the range of achievable serum concentrations in patients treated with rG-CSF. At the end of incubation, colonies were counted under an inverted phase-contrast microscope, and an increase in the number of colonies in comparison with the control was used to evaluate the effects of rG-CSF. Results were expressed as a percentage of controls. rG-CSF in the upper layer at concentrations ranging from 0.1 ng/ml to 10 ng/ml stimulated the colony formation of all the cancer cell lines tested in the absence of PBMC in the feeder layer, whereas cells with PBMC in the feeder layer were significantly stimulated more than those without PBMC in the feeder layer (P<0.05) up to a certain concentration, which varied from cell line to cell line. At higher concentrations of rG-CSF, no further stimulation but, on the contrary, a decrease in colony formation was observed in cells with PBMC in the feeder layer in all the cell lines tested. Colony formation in KK-47 and T24 cell lines was significantly inhibited at 5 ng/ml and/or 10 ng/ml rG-CSF compared with cells without PBMC in the feeder layer. Our results suggest that rG-CSF may have both direct and indirect stimulatory effects on the growth of human bladder cancer cell lines in vitro. The results obtained also raise the possibility of adverse effects of rG-CSF in bladder cancer patients whose malignant cells may be directly and indirectly stimulated by this factor while it is being used clinically to alleviate the myelosuppression induced by antitumor chemotherapy.

Combination of hemoglobin, alkaline phosphatase, and age predicts optimal docetaxel regimen for patients with castration-resistant prostate cancer

BackgroundWe aimed to find the prognostic factors predicting overall survival (OS) in patients with castration-resistant prostate cancer (CRPC) who had docetaxel (DTX) chemotherapy, and to construct a model predicting the optimum number of cycles of DTX.MethodsA total of 279 CRPC patients who received DTX (≥50xa0mg/m2) every 3–4xa0weeks were studied retrospectively. Prognostic factors predicting treatment cycles as well as OS were analyzed, and a risk table for predicting treatment cycles was constructed.ResultsThe longer treatment group (>10 cycles) had a significantly longer OS than the standard treatment group (pxa0<xa00.0001). Multivariate analysis demonstrated that a decrease ofxa0≥50xa0% in prostate-specific antigen (PSA), serum markers at the start of DTX therapy [PSA, alkaline phosphatase (ALP), and C-reactive protein (CRP)], and the number of DTX courses were independent predictors of OS. The risk table employing the combination of three factors [ALP (cut-off 189xa0IU/L), hemoglobin (11.3xa0g/dL), and age (65xa0years) at the start of DTX therapy], and scoring based on the hazard ratio of each risk factor (ALP 4, hemoglobin 2, age 3) could effectively predict the probability of the length of DTX therapy, with lower score (0–6) predictingxa0>10 cycles, and higher score (7–9) predicting ≤5 cycles (pxa0<xa00.0001). No significant difference was found regarding grade 3/4 adverse events between the two groups.ConclusionA model using three factors prior to chemotherapy may be beneficial for deciding the duration of DTX therapy in patients with CRPC.

Optimal conditions of fixation for immunohistochemical staining of proliferating cell nuclear antigen in tumour cells and its cell cycle related immunohistochemical expression

Abstract. Comparison of the results of immunohistochemical expression, such as proliferating cell nuclear antigen (PCNA) in archival material of tumours, with the clinical course is extremely valuable in determining the biological malignant potential of newly detected tumours. To obtain stable and reproducible results of immunohistochemical expression of the PCNA of tumours, we studied the optimal conditions of fNation, processing and staining of samples using animal‐implanted MBT‐2 cells derived from chemical‐induced mouse bladder carcinoma and PC10, a monoclonal antibody for PCNA. The intensity of staining and PCNA positive rates were stable and reproducible when resected specimens from the tumours were covered with gauze wetted with physiological saline at room temperature before fixation for less than 12 h, fixation in formaldehyde was less than 48 h, and paraffin‐embedded sections were dried for less than 1 h. The most clear staining of PCNA positive nuclei was observed when 10% neutral buffered formaldehyde was used as a fixative. The PCNA positive rates obtained under these conditions was compared with the bromodeoxyuridine (BrdUrd) labelling indices. Although the average PCNA positive rate was significantly higher than the BrdUrd labelling index (P≪0.01), a significant correlation between PCNA positive rates and BrdUrd labelling indices was observed. In order to study the cell cycle related expression of PCNA, Ehrlich ascites tumour cells were separated by centrifugal elutriation. PCNA positive nuclei were observed in all phases of the cell cycle including G,. Occurrence of PCNA positive G1 cells was expected at a half‐life of the PCNA‐protein of 20 h and a tumour cell doubling time of about 24h. Thus, the percentage of PCNA positive nuclei in a conventionally paraffin‐embedded specimen of a tumour reflects both the growth fraction and the doubling time of the tumour and it may be a useful parameter of the biological malignant potential of tumours.

Granulocyte colony-stimulating factor may promote proliferation of human bladder cancer cells mediated by basic fibroblast growth factor.

Objectives: To investigate whether granulocyte colony-stimulating factor (G-CSF) promotes the proliferation of two bladder cancer cell lines, and to assess the mechanism of tumor proliferation in terms of cytokine expression. Material and Methods: The proliferation of two bladder cancer cell lines derived from transitional cell carcinoma (KK-47 and T-24) was assessed by using the double-layer soft agarose colony assay in combination with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Seven cytokines were measured in the culture supernatant. Expression of granulocyte colony-stimulating factor (G-CSF) receptor and fibroblast growth factor (FGF) receptor mRNA was studied by means of reverse transcriptase polymerase chain reaction (RT-PCR). Results: Recombinant human G-CSF (rhG-CSF) caused greater induction of the proliferation of KK-47 cells in the presence than in the absence of peripheral blood mononuclear cells (PBMCs) and its effect was dose-dependent. In contrast, rhG-CSF did not stimulate the proliferation of T-24 cells. Among several cytokines measured, only basic FGF was elevated in cultures of KK-47 cells with or without PBMCs. The basic FGF level was significantly increased by rhG-CSF stimulation in a dose-dependent manner. Specific PCR products for the G-CSF and FGF receptors were observed in KK-47 cells as well as PBMC, while no G-CSF receptor was detected in T-24 cells. Conclusion: rhG-CSF may promote the proliferation of KK-47 cells, probably via an increase in basic FGF production.

Expression of thymidine phosphorylase in human superficial bladder cancer

Abstract Background:u2002 The purpose of the present paper was to investigate the expression level of thymidine phosphorylase (TPase) in superficial bladder cancer tissues obtained by transurethral resection, and determine whether its expression correlates with tumor recurrence.

Tumor-infiltrating lymphocytes derived from human renal cell carcinoma : Clonal analysis of its characteristics

Aim:u2003 To assess the characteristics of activated tumor‐infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4‐ CD8+ TIL‐clone derived from human renal cell carcinoma (RCC).

Expression of Proliferating Cell Nuclear Antigen and Deoxyribonucleic Acid Value in Renal Cell Carcinoma: Correlation with Different Histopathological Parameters and Patient Survival

Forty-six human renal cell carcinoma tissues obtained from radical nephrectomy, fixed in 10% formaldehyde solution and embedded in paraffin for histopathological examination were used for immunohistochemical staining of proliferating cell nuclear antigen (PCNA) using a monoclonal antibody PC10, and 37 of the 46 were also used for flow cytometric DNA ploidy analysis. PCNA-positive rates were compared with different histopathological parameters and patient survival. The relationships between the DNA ploidy and PCNA-positive rates and patient survival were also determined. Statistically significant differences in PCNA-positive rates were observed in different histopathological grades and stages of renal cell carcinoma. No relationship was observed between the PCNA-positive rate and DNA ploidy. For all cases analyzed, patients whose tumors had PCNA-positive rates of less than 10% survived statistically longer than those with tumors with PCNA-positive rates of 10% or more (p < 0.02). When patients were stratified by histopathological stage-I and grade-1 tumors, however, there was no significant difference between the PCNA-positive rate and survival. No difference in survival was observed according to DNA ploidy. The PCNA-positive rates showed a close relation to the different histopathological grades and stages of renal cell carcinoma. For all cases analyzed, high PCNA-positive rates showed poor prognosis. But for patients with histopathological stage-I and grade-1 tumors, the PCNA-positive rates and DNA ploidy did not provide independent prognostic information.