Umihiko Sawada

Effect of Tween-80 on cell killing by etoposide in human lung adenocarcinoma cells.

Purpose: The non-ionic detergent Tween-80, a surface-active agent, has been shown to modulate the cytocidal effect of certain antitumor agents. In the present study, we sought to determine whether or not Tween-80 could enhance the antitumor effect of etoposide (VP16) in human lung cancer cells in vitro. Methods: Survival fractions were measured by growth inhibiton assays of PC14, H69, KB, and PC14/CDDP (the corresponding cisplatin-resistant subline of PC14) cells. An in vitro clonogenic assay of PC14 and PC14/CDDP cells was undertaken after incubation for 10–12 days in RPMI-1640 medium with 20% fetal calf serum and 1.72% methyl cellulose, plus continuous exposure to VP16 with Tween-80. We also investigated the direct toxicity of Tween-80 to PC14 and PC14/CDDP cells using a clonal assay. The intracellular accumulation of VP16 was further analyzed using [3H]VP16 in PC14, PC14/CDDP, A549, KB and H69 cells, and compared with that of daunorubicin (DNR), a hydrophilic anticancer agent, using [3H]DNR in PC14, A549 and KB cells. Results: It was found that PC14/CDDP had collateral sensitivity to VP16 and Tween-80 markedly enhanced the killing effect of VP16 not only of PC14 cells but also of PC14/CDDP cells while exerting little cytotoxic effect. Moreover, Tween-80 increased the intracellular accumulation of VP16 in PC14, PC14/CDDP and A549 cells, and not in KB and H69 cells. Tween-80 did not increase the intracellular DNR levels in PC14, A549 and KB cells. Conclusions: Tween-80 was shown to potentiate the cytotoxicity of VP16 against several human lung adenocarcinoma cells by increasing the accumulation of VP16 in vitro. Tween-80-mediated sensitization of lung adenocarcinoma cells to VP16 is considered to be related to both the characteristics of the cell membrane in adenocarcinoma cells and the lipotropic properties of VP16. These results suggest that this combination might have the potential to improve the therapeutic index of VP16 in human lung adenocarcinoma.

Non-ionic detergent Tween 80 modulates VP-16 resistance in classical multidrug resistant K562 cells via enhancement of VP-16 influx.

The non-ionic detergent Tween 80, which is used as a solvent for lipophilic drugs such as VP-16 and Taxotere, was found to reverse VP-16 resistance of the P-glycoprotein-associated multidrug resistance phenotype via increasing VP-16 influx. In adriamycin-resistant human chronic myelogenous leukemia K562 cells (K562/ADM), which overexpress mdr1 mRNA, the accumulation of VP-16 was only about 10% that in wild-type K562 cells. Tween 80 enhanced VP-16 accumulation in K562/ADM cells but did not influence VP-16 accumulation in parental K562 cells. VP-16 efflux was rapid and similar in both sensitive and resistant cell lines and was not blocked by Tween 80 or verapamil. Under glucose-free conditions, VP-16 accumulation in K562/ADM cells was only half of that in K562 cells. Tween 80 increased VP-16 accumulation in K562/ADM cells in glucose-free medium. In growth inhibition assay, Tween 80 reversed K562/ADM sensitivity to VP-16 without cell damage. Taken together, Tween 80 reverses VP-16 sensitivity in multidrug-resistant K562 cells by increasing influx, which is considered to be the primary mechanism of VP-16 resistance in K562/ADM cells.

Expression of a multidrug-resistance gene in human malignant lymphoma and related disorders

The expression of mdr1 gene was measured to determine whether it plays a role in clinical resistance to chemotherapy of human malignant lymphomas. mdr1 expression was found in 4 of 9 cases resistant to chemotherapy. Expression of mdr1 was not detectable in any of 7 chemotherapy-sensitive tumors. The 2 cases of reactive lymphadenitis and the 3 samples of normal mononuclear cells did not show any expression of mdr1 gene, either. These results indicate that expression of the mdr1 gene is not always detectable in cases of malignant lymphoma resistant to chemotherapy, but the detectable expression of mdr1 gene may predict clinical resistance to chemotherapy.

Treatment of high-risk peripheral T-cell lymphomas other than anaplastic large-cell lymphoma with a dose-intensified CHOP regimen followed by high-dose chemotherapy. A single institution study.

We investigated the efficacy of a dose-intensified double-CHOP regimen followed by high-dose chemotherapy with or without peripheral blood stem cell transplantation (PBSCT) in 11 patients with four types of peripheral T-cell lymphoma (PTCL). Three of the 4 patients with unspecified PTCL (PTCLu) achieved complete response (CR); 1 patient relapsed and 1 died of secondary leukemia after consolidation therapy. All angioimmunoblastic T-cell lymphoma (AILT) and subcutaneous panniculitis-like T-cell lymphoma (SPTCL) patients achieved CR; 5 of 6 have remained disease free for more than 3 years. The patient with hepatosplenic lymphoma did not achieve CR even after PBSCT and underwent allogenic bone marrow transplantation (allo-BMT). Thus, our regimen appears to be effective for high-risk AILT and SPTCL. However, allo-BMT should be considered for high-risk of PTCLu and hepatosplenic T-cell lymphoma.

Azacitidine induces demethylation of p16INK4a and inhibits growth in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is one of the peripheral T-cell malignant neoplasms strongly associated with human T-cell leukemia virus type-I (HTLV-I). Although the viral transactivator protein Tax has been proposed to play a critical role in leukemogeneis, additional cellular events are required for the development of ATL. One of the genetic events of the disease is inactivation of tumor suppressor genes. The CDKN2A locus on chromosome 9p encodes 2 cell cycle regulatory proteins, p14ARF and p16INK4a, which share exon 2 using different reading frames. The p14ARF and p16INK4a genes have been implicated as tumor suppressor genes by their frequent mutation, deletion or promoter hypermethylation in a variety of human tumors. In this report, we describe the expression status of p14ARF and p16INK4a in 9 ATL cell lines (MT1, MT2, OKM3T, F6T, K3T, Oh13T, S1T, Su9T01 and HUT102). By reverse transcription polymerase chain reaction (RT-PCR), expression of p14ARF was not detected in one cell line (OKM3T), while expression of p16INK4a was not detected in 6 cell lines (OKM3T, MT1, MT2, Oh13T, S1T and Su9T01). In the OKM3T cell line, the shared exon 2 of the p14ARF/p16INK4a gene was deleted; however, the p16INK4a gene, was epigenetically inactivated in 5 other cells lines. In primary tumor cells obtained from ATL patients, p14ARF expression was absent in 6 of the 11 samples. We confirmed the methylation of the p16INK4a gene in MT1 and MT2 cells using the methylation-specific PCR (MSP) method. Treatment with 2.0 µM of Azacitidine (AZA), a demethylating agent, for 72 h restored p16INK4a transcript expression and induced growth inhibition in MT2 cells. Our results demonstrate that p16INK4a is epigenetically silenced in ATL. AZA offers a potential new therapeutic approach to improve the poor outcomes associated with ATL.

Quality of care associated with number of cases seen and self-reports of clinical competence for Japanese physicians-in-training in internal medicine

BackgroundThe extent of clinical exposure needed to ensure quality care has not been well determined during internal medicine training. We aimed to determine the association between clinical exposure (number of cases seen), self- reports of clinical competence, and type of institution (predictor variables) and quality of care (outcome variable) as measured by clinical vignettes.MethodsCross-sectional study using univariate and multivariate linear analyses in 11 teaching hospitals in Japan. Participants were physicians-in-training in internal medicine departments. Main outcome measure was standardized t-scores (quality of care) derived from responses to five clinical vignettes.ResultsOf the 375 eligible participants, 263 (70.1%) completed the vignettes. Most were in their first (57.8%) and second year (28.5%) of training; on average, the participants were 1.8 years (range = 1–8) after graduation. Two thirds of the participants (68.8%) worked in university-affiliated teaching hospitals. The median number of cases seen was 210 (range = 10–11400). Greater exposure to cases (p = 0.0005), higher self-reports of clinical competence (p = 0.0095), and type of institution (p < 0.0001) were significantly associated with higher quality of care, using a multivariate linear model and adjusting for the remaining factors. Quality of care rapidly increased for the first 100 to 200 cases seen and tapered thereafter.ConclusionThe amount of clinical exposure and levels of self-reports of clinical competence, not years after graduation, were positively associated with quality of care, adjusting for the remaining factors. The learning curve tapered after about 200 cases.

Pure Red Cell Aplasia and Myelofibrosis in B-cell Neoplasm

We describe an unusual case of B-cell neoplasm accompanied by pure red cell aplasia (PRCA) and myelofibrosis in a 67-year-old male presenting with severe anaemia. A few unclassified, myeloperoxidase-negative blastoid cells were seen on bone marrow aspiration, and erythroid cell hypoplasia and myelofibrosis on bone marrow biopsy. An autoimmune PRCA was suspected, as serum CH50, C3 and C4 levels were consistently low. Ciclosporin was effective in treating the anaemia, but anaemia returned when the drug was discontinued. Thirteen months later, the patient was admitted with pleural effusion and ascites that contained monoclonal CD19+ CD20+ immature blast cells with a complex karyotype, thought to be neoplastic B-cells. The unclassified blastoid cells seen earlier may therefore have been from the same origin. The patient deteriorated rapidly and died. Only one case of non-Hodgkins lymphoma with PRCA and myelofibrosis has been reported previously. We discuss the possibility that dysregulated T-cells induced by neoplastic B-cells may have given rise to concomitant PRCA and myelofibrosis.

Purging in autologous hematopoietic stem cell transplantation using adenosine triphosphate (ATP) and 4-hydroperoxycyclophosphamide (4-HC)

In this study, we show potent in vitro purging induced by adenosine triphosphate (ATP) for leukemic cells. The treatment of murine L1210 leukemic cells with 2mM of ATP in vitro for 3h was able to reduce the number of leukemic clonogenic cells by about one order of magnitude presumably by changing the permeability of the leukemic cell membrane. Furthermore, the incubation of L1210 cells with ATP (2mM) and low dose 4-hydroperoxycyclophosphamide (4-HC, 2 microg/ml) for 3h resulted in at least a four-log reduction of clonogenic L1210 cells. Only a slight degree of toxicity to pluripotent hematopoietic stem cells (CFU-S) was observed in both treatment protocols. To determine the efficacy of pharmacological purging by ATP, we designed a murine system to mimic the conditions expected in the clinical setting of autologous transplantation using simulated partial remission marrow (SPRM) which was prepared by mixing normal marrow cells and L1210 cells at a ratio of 9:1. After the SPRM cells were incubated in vitro at a concentration of 1 x 10(6)/ml with both ATP (2mM) and low dose 4-HC (2 microg/ml) for 3h, 5 x 10(4) of the cells were then injected into lethally irradiated 9 weeks male BDF1 mice. All the mice given untreated-SPRM died of leukemia by day 27, whereas none of the recipients transplanted treated-grafts had died by day 70, thus suggesting that the combination use of ATP and 4-HC may be a potentially effective way to purge leukemic cells in autologous stem cell transplantation. The mechanism of the selective killing of leukemic cells is assumed that 4-HC is effectively incorporated into leukemic cells by increasing the permeability of the cell membrane by ATP. Taken together, this simple and rapid procedure is able to purge leukemic cells from autologous bone marrow grafts.

A case of anaplastic large cell (Ki-1) lymphoma of B-cell phenotype, occurring in Waldenström's macroglobulinemia.

A case of anaplastic large cell (Ki‐1) lymphoma of B‐cell lineage occurred in a 59‐year‐old male with Waldenström’s macroglobulinemia. Immunostaining of the lymphoma cells showed sporadic positivity for IgM and occasional positivity for κ chain. This immunoglobulin specificity is the same as that of plasmacytoid lymphocytes in the bone marrow; therefore anaplastic transformation of Waldenström’s macroglobulinemia was strongly suggested. This seems to be the first reported case of anaplastic large cell lymphoma, confirmed by CD30 expression, arising in Waldenström’s macroglobulinemia.

Safety and efficacy of high-dose cyclophosphamide, etoposide and ranimustine regimen followed by autologous peripheral blood stem cell transplant for patients with diffuse large B-cell lymphoma.

Abstract We retrospectively evaluated the safety and efficacy of high-dose chemotherapy consisting of cyclophosphamide, etoposide and ranimustine (CEM) with autologous peripheral blood stem cell transplant (PBSCT) in 55 adult patients with relapsed or high-risk de novo diffuse large B-cell lymphoma (DLBCL) or DLBCL associated with follicular lymphoma. This included 36 patients in the upfront setting in their first complete remission. The median follow-up of 42 patients surviving at the time of the analysis was 52 months (range 1–159). Relapse or disease progression after PBSCT was a frequent cause of death, but no therapy-related mortality associated with PBSCT was observed. The 5-year overall survival and progression-free survival were 70.6% (95% confidence interval [CI], 54.0–82.1) and 57.0% (95% CI, 39.5–71.2), respectively. Chronic renal impairment, therapy-related myelodysplastic syndrome and prostate cancer were the major late complications. The CEM regimen is a tolerable, effective conditioning regimen for autologous PBSCT for DLBCL, with no therapy-related mortality observed.