Oral squamous cell carcinoma arising in a patient with Werner syndrome.

Werner syndrome (WS) is an autosomal recessive disorder characterized by physical signs and symptoms, including premature aging and scleroderma-like skin changes. The gene responsible for WS is the WRN gene. A significant proportion of WS-related malignant tumours are non-epithelial types, and the incidence of oral squamous cell carcinoma (SCC) is rare. A case of oral SCC of the lower alveolus and gingiva arising in a 63-year-old woman with WS is reported here. Biopsy confirmed moderately differentiated SCC. Surgical resection was performed and there was no recurrence or metastasis at the 3-year follow-up. Mutation analysis using next-generation sequencing, detected no mutations in the genes encoding the molecules strongly involved in the development of oral SCC, such as TP53 or PIK3CA. No obvious mutations were detected. Based on the results of the study, the results of mutation analysis suggest that this case might be genetically different from the common mechanisms of SCC in the oral cavity.

Efficacy and safety of the dipeptidyl peptidase-4 inhibitor sitagliptin compared with α-glucosidase inhibitor in Japanese patients with type 2 diabetes inadequately controlled on sulfonylurea alone (SUCCESS-2): a multicenter, randomized, open-label, non-inferiority trial.

We assessed the efficacy and safety of sitagliptin compared with α-glucosidase inhibitor (αGI) in 120 of Japanese patients with type 2 diabetes mellitus (T2DM) inadequately controlled on stable ≤2 mg/day glimepiride alone [mean hemoglobin A1c (HbA1c) 7.7%] by the randomized, active-controlled, non-inferiority trial. Patients were randomly assigned to receive additional sitagliptin or αGI for 24 weeks. The primary endpoint was change in HbA1c from baseline to week 12. After 12 weeks, sitagliptin reduced HbA1c by -0.44% (p < 0.001) relative to αGI. At 24 weeks, the reduction was almost identical between the groups (-0.091%, p = 0.47). Gastrointestinal disorders were more common with αGI than with sitagliptin, but only minor hypoglycaemia occurred in both groups at similar frequency. These data suggested that sitagliptin was not inferior to αGI for reduction of HbA1c in Japanese T2DM patients receiving glimepiride alone, and well tolerated with minimum risk of gastrointestinal symptoms and hypoglycaemia.

Observational 6-month open-label study of Japanese type 2 diabetes patients switching from NPH insulin to insulin detemir in basal-bolus regimen: 23rd article of the Japan Diabetes Clinical Data Management Study Group (JDDM23).

OBJECTIVE: Glycaemic control is critical to prevent diabetic complications and mortality. This 6-month, open-label, observational study assessed the efficacy and safety of switching Japanese patients with type 2 diabetes from neutral protamine Hagedorn (NPH) insulin to insulin detemir. METHODS: Patients with type 2 diabetes (n = 126) receiving basal-bolus insulin therapy with NPH insulin plus rapid-acting insulin analogues were recruited. NPH insulin was replaced with insulin detemir for 6 months. Glycosylated haemoglobin (HbA(1c)), fasting plasma glucose (FPG), daily glucose levels and hypoglycaemia were monitored. Nocturnal quality of life was assessed by insulin therapy related quality of life at night questionnaire. RESULTS: HbA(1c), FPG and body weight were all significantly reduced after treatment with insulin detemir for 6 months, without increasing severe hypoglycaemia. Insulin dose increased significantly over the same time. There were significant improvements in overall nocturnal quality of life, as well as well-being. CONCLUSIONS: Treatment with insulin detemir for 6 months resulted in substantial benefits, including reduced HbA(1c), FPG and body weight, and improvements in nocturnal quality of life, without increasing hypoglycaemia.

Shocks and curvature dynamics: A test of global kinetic faceting in crystals

We investigate the microscopic mechanisms underlying the dynamical faceting of crystals. Partially faceted crystal shapes of CCl4 are formed from a melt contained in a Bridgman apparatus and pressure is used to control growth which is observed using optical microscopy. In contrast to predictions of models in which the local interfacial motion is greatest where the step density is the highest, the loss of rough orientations is observed to occur via a local decrease in curvature which results in the formation of discontinuities-shocks-in the surface of the growth forms, a feature predicted by a recent theory of kinetic faceting.

Myelodysplastic syndrome following treatment of malignant melanoma with vincristine, ACNU, and dacarbazine.

Therapy-related myelodysplastic syndrome is a rare adverse effect in melanoma patients elicited by chemotherapy. We report a case of myelodysplastic syndrome following treatment of malignant melanoma with alkylating agents. Peripheral blood showed a remarkable suppression of three cell lineages, and the bone marrow was slightly hypercellular. However, no morphological abnormalities were detected in the peripheral blood or the bone marrow, and chromosomal analysis was normal.

Establishment and characterization of human immature teratoma cell line (TES-1).

A new human immature teratoma cell line, TES-1, was established from a surgical specimen from a 12-year old male with third ventricular immature teratoma. TES-1 shows polygonal morphology rich in neurites, and proliferated as adherent monolayer, with an approximate population doubling time of 48 hours. Electron microscopic analysis revealed the presence of swollen rough endoplasmic reticulum, and prominent lipid droplets, lysosomes and microfilaments. The chromosome numbers were between 41 and 160 (mode 78), including abnormal karyotypes 1p-, 5q-, 12p+ and 17p+ (G-band analysis). Hetero-transplantation of TES-1 into BALB/c nude mice produced no visible tumors. Multipotential differentiation was not induced in TES-1 monolayer culture, but significant neuron specific enolase activity was expressed in both extracellular (by RIA method) and intracellular fractions (by immunohistochemical method), suggesting the differentiation toward neurocytes. This cell line provides a useful in vitro model for the pathophysiological analysis of immature teratoma.

A plasma cell leukemia patient showing bialleic 14q translocations: t(2;14) and t(11;14).

We report here an IgG/lambda-type plasma cell leukemia patient showing bialleic 14q32 translocations. All immunoglobulins were suppressed in this patient, but a small amount of monoclonal IgG was detected by immunoelectrophoresis. Two cells of six peripheral blood mononuclear cells showed 46,XY,t(2;14)(q11;q32), i(8)(q10), t(11;14)(q13;q32), del(12)(q13.1) by karyotypic analysis. We confirmed the juxtaposition of IgH and PRAD1/Cyclin D1 genes by fluorescent in situ hybridization and overexpression of the PRAD1/Cyclin D1 gene, but Southern analysis showed no bcl-1 rearrangement. We analyzed the t(2;14)(q11;q32) using DNA fragments derived from childhood B-chronic lymphocytic leukemia cases bearing t(2;14)(p13;q32). Southern and Northern analyses demonstrated no alteration of these genes, indicating that this t(2;14) was different from that of childhood B-chronic lymphocytic leukemia. At the IgH loci, Southern analysis showed two rearranged bands and one germ-line band of JH. Cmicro was deleted on one rearranged allele but remained on the other, suggesting that the chromosome translocation occurred after productive class switch recombination on the Cmicro deleted allele.

Establishment and characterization of a CD95 (Fas/Apo-1)-negative myeloma cell line.

Although expression of CD95 (Fas/Apo-1) on myeloma cells has been reported, its significance is not clearly understood. We established a myeloma cell line, KHM-11ad (11ad), from a parental cell line, KHM-11, by collecting cells adhered to a plastic dish. KHM-11 cells have been reported to be positive for CD45 and CD95 (Fas/Apo1), and negative for a myelomonocytic antigen, CD13. Interestingly, CD95 was not detected in 11ad. Expression of CD45 was also significantly decreased in 11ad cells while expression of CD13 was detected in these cells. The growth rate of 11ad cells was 1.7 times lower than that of KHM-11 cells. Analysis of adhesion molecules showed that expression of VLA4 and CD44 was significantly suppressed in 11ad. The IC50 of melphalan (L-PAM) for 11ad cells was 50 times higher than that for KHM-11, indicating that 11ad is significantly refractory to L-PAM than KHM-11 cells. Induction of apoptosis by doxorubicin and cycloheximide was suppressed in 11ad cells compared with those in KHM-11 cells. Western blot analysis for Bcl-2 family of proteins showed that Bax was expressed at a 2.2 times lower level in 11ad cells than in KHM-11 cells while there was no difference in expression of Bcl-2, Bcl-Xs nor Bcl-XL. These results suggest that CD95-negative myeloma cells may have characteristics as follows: (1) slow proliferation; (2) low sensitivity to apoptosis; (3) low expression of VLA4, CD44 and Bax. Although these intraclonal variations were based on the findings of cell lines, these may reflect similar variations in vivo. The 11ad line may be a suitable model for analyzing intraclonal variation of myeloma cells.

Endonuclease activity and hydrogen peroxide-induced cytotoxicity in human glioblastoma and glioma cell lines.

Hydrogen peroxide (H2O2) induced internucleosomal DNA cleavage in human myelogenous leukemic cell lines (HL-60, ML-1, THP-1, U-937), but not in human glioblastoma (T98G, U87MG) and glioma (KG1C) cell lines. However, H2O2 produced apoptotic cells, characterized by cell shrinkage, nuclear fragmentation and chromatin condensation in glioblastoma and glioma cell lines. Autodigestion experiments revealed that the major endonucleases, present in all leukemic, glioblastoma and glioma cell lines, were divalent cation-independent endonuclease(s). The endonudease(s) present in the lysates of all these cells were activated at acidic, but not at neutral pH. The results suggest that the endonuclease activity might be differently regulated between leukemic and glioma cell lines.

Detection of inducible nitric oxide synthase (iNOS) mRNA by RT-PCR in ATL patients and HTLV-I infected cell lines: clinical features and apoptosis by NOS inhibitor.

Various tumors have been reported to express an inducible form of nitric oxide synthase (iNOS), and nitric oxide (NO) may affect the clinicopathological features of these tumors. Previously, Burkitt's lymphoma and Epstein-Barr virus (EBV)-infected cells were shown to express iNOS constitutively at a low level. We analyzed iNOS expression by the reverse transcriptase-polymerase reaction method (RT-PCR) in eight HTLV-I-infected cell lines (five were ATL-derived lines and there were in vitro transformed lines), nine ATL patients (three were chronic, two were acute, and four were lymphoma type), and an HTLV-I-negative T cell line (CEM). In four ATL derived and in all three in vitro transformed cell lines, iNOS was expressed constitutively, but it was not expressed in CEM cells. Four out of nine ATL patients also showed iNOS expression. The expression of iNOS was found in all subtypes of ATL. Three of four iNOS-positive patients had infiltration of ATL cells to organs such as skin, lung, or liver. In NOS inhibitor (NG-monomethyl-L-arginine: L-NMMA)-containing medium, an iNOS-positive ATL cell line (K3T) showed growth inhibition and DNA ladder. Although only a limited number of patients was analyzed, our results suggest that NO may be involved in the invasive character of ATL cells. The NOS inhibitor can induce apoptosis in an ATL cell line, as it does in EBV-infected cell lines.

Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects.

We analysed PRAD1/cyclin D1 expression in 20 patients with plasma cell malignancy by Northern analysis. 6/17 multiple myeloma patients and 3/3 plasma cell leukaemia patients showed PRAD1/cyclin D1 expression. This incidence appeared to be higher than the expected incidence based on previous studies. Southern analysis did not show rearrangement of the bcl-1 region. Although there was no statistical difference, the PRAD1/cyclin D1 negative group showed a 1-year survival of 81.8%, 3-year survival of 45.5% and 5-year survival of 22.7%, and those for the PRAD1/cyclin D1 positive group were 63.5%, 16.9% and 16.9%, respectively. Further study is required to determine whether PRAD1/cyclin D1 expression is a prognostic factor.

Effect of glutathione-modulating compounds on platinum compounds-induced cytotoxicity in human glioma cell lines.

The relation between the effect of glutathione(GSH)-modulating compounds and platinum compounds (Cisplatin, Nedaplatin)-induced cytotoxicity was investigated. Pretreatment of human glioblastoma (T98G, U87MG) and glioma (KG1C) cell lines with L-buthionine-[S,R]-sulfoximine, which decrease the intracellular GSH concentration, remarkably increased their sensitivity against platinum compounds, whereas pretreatment with N-acetyl-L-cysteine, which increase the intracellular GSH concentration, only marginally protected the cells from the cytotoxic effect of platinum compounds. The results suggest that platinum compounds-induced cytotoxicity can be modified by GSH-modulating compounds in glioblastoma and glioma cell lines.

Expression of Bcl-2 family of proteins in fresh myeloma cells.

Members of the Bcl-2 family of proteins, Bcl-2, Bcl-X(L), Bcl-Xs and Bax, are considered to play important roles in the regulation of apoptosis and drug resistance. To understand the significance of these proteins in fresh human myeloma cells, expression of Bcl-2 family of proteins was analyzed by Western blotting in 17 cases with multiple myeloma (MM) and three cases with plasma cell leukemia (PCL). Bcl-2 and Bcl-X(L) were found in 12 and nine samples, respectively. All PCL cases showed co-expression of Bcl-2 and Bcl-X(L). Analysis of MM cases showed that Bcl-2 was preferentially expressed in samples from cases with early clinical stage while Bcl-X(L) tended to be expressed in samples from cases at advanced clinical stage. Bcl-X(L) was significantly expressed in tumor cells from cases with extramedullar lesions. There was no correlation between the expression levels of Bcl-2 or Bcl-X(L) and preceding chemotherapy. Expression of Bax was found in only one patient who had pleural effusion caused by invasion of myeloma cells and a high serum LDH level. Survival analysis revealed that there was no statistical significance in expression of Bcl-2 or Bcl-X(L) although Bcl-X(L) tended to be expressed in cases with poor prognosis. These findings indicate that expression of Bcl-2 family of proteins is heterogeneously regulated in fresh myeloma cells. Expression of Bcl-X(L) and Bcl-2 may correlate with extramedullar invasion and early stage of the disease, respectively. Absence of Bax in myeloma cells may contribute to low sensitivity of myeloma cells to anti-cancer agents since Bax is reported to mediate cytotoxicity of some anti-cancer drugs.

Multiple myeloma associated with serum amino acid disturbance and high output cardiac failure.

We experienced a plasma cell leukemia (PCL) patient complicated with high output cardiac failure (HOCF), proved as his elevated cardiac index and pulmonary artery wedge pressure and decreased systemic vascular resistance index in a hemodynamic study. We found no possible causes of HOCF. Interestingly, HOCF was improved as PCL responded to intensive chemotherapy. On the other hand, he showed consciousness disturbance, and had frequent attacks of generalized seizure. His electroencephalogram showed slow waves, and a spike and wave complex. Hyperammonemia and abnormal amino acid distribution were also found. This abnormal serum amino acid distribution, especially elevated glycine level, was different from that seen in chronic liver failure, and he had no hepatic disease. After intensive chemotherapy, the serum ammonia level and glycine level decreased. In this patient, PCL seemed to be responsible for HOCF, hyperammonemia, and abnormal amino acid distribution. We experienced two more cases of multiple myeloma (MM) with HOCF, hyperammonemia, abnormal serum amino acid distribution, and consciousness disturbance of unknown origin. Those two cases showed slow waves in the electroencephalogram. Improvement was seen in their HOCF, hyperammonemia, and abnormal amino acid levels after chemotherapy. The possibility of MM as a cause of HOCF is discussed.

Establishment of a monoclonal antibody to human myeloma cell: relation to chemotherapy and extramedullar infiltration.

Resistance of myeloma cells to melphalan (L-PAM) is a serious problem. To investigate mechanisms of drug resistance, we generated a monoclonal antibody, clone O3, to melphalan-resistant myeloma cells, KHM-11R. Western blot analysis showed that molecular weight of O3 antigen was approximately 90 kDa. Expression of O3 antigen was approximately two times higher in KHM-11R than in parental melphalan sensitive cell line, KHM-11. O3 was preferentially expressed in plasma cell, B-cell, and monocytic cell lines, but not in T-cell lines. Analysis of bone marrow samples from myeloma patients revealed that 13 of 23 samples expressed O3 antigen at various levels, and that O3 antigen expression in patients correlate with preceding chemotherapy, advanced clinical stage and extramedullar invasion of myeloma cells. Furthermore, patients expressing O3 antigen at the time of diagnosis tended to have poor prognosis. The investigation of O3 antigen in myeloma cells will be useful to reveal the pathophysiology of extramedullar invasion and the mechanism of cell killing by melphalan.

Hyperdiploid myeloma cell as an indicator of poor prognosis and drug refractoriness.

Although almost 40% of patients with multiple myeloma respond to initial chemotherapy, myeloma with no response to initial chemotherapy remains a serious problem. To understand the characteristics of drug-refractoriness of myeloma, fresh tumor cells from 13 untreated myeloma patients were fixed and stained with anti-human immunoglobulins and propidium iodide for subsequent flow cytometric analysis of DNA content. More than 10% of myeloma cells were hyperdiploid in eight cases (hyperdiploid + cases) while less than 10% of myeloma cells were hyperdiploid in five cases (hyperdiploid - cases). The proportion of hyperdiploid cells among all myeloma cells was highly correlated with incidence of myeloma cells with morphologically abnormal nuclei such as those with multiple-nuclei or convoluted nuclei (P = 0.001). Among the eight hyperdiploid + cases, two (2/8) showed good response to subsequent chemotherapy while four of five hyperdiploid - cases (4/5) responded well. Cases with poor response had more hyperdiploid myeloma cells (average 25.7% of all myeloma cells) than sensitive cases (average 6.8%), suggesting a contribution of hyperdiploid myeloma cells to primary drug resistance (P = 0.065). The 3 year survival rate of hyperdiploid+cases was 0% while that of the control group was 41.9%. These results suggest that myeloma cells with abnormal nuclear morphology may show hyperdiploidy and poor response to chemotherapy.

Effect of methionine depletion on growth and apoptosis in various tumor cell lines.

Sodium ascorbate, sodium 5,6-benzylidene-L-ascorbate (SBA), gallic acid and caffeic acid induced apoptotic cell death in human myelogenous leukemic cell lines, and stimulated oxidation of methionine into methionine sulfoxide in the culture medium. When various tumor cell lines were cultured in methionine-free medium, their growth was nearly terminated at G1 phase of the cell cycle, producing much smaller number of apoptotic cells. Addition of methionine sulfoxide to the methionine-free medium did not stimulate the apoptosis induction. These data suggest that induction of apoptosis by ascorbates, gallate or by caffeate cannot be simply explained by methionine oxidation.

Effect of glutathione-modulating compounds on hydrogen-peroxide-induced cytotoxicity in human glioblastoma and glioma cell lines.

The relation between the intracellular glutathione (GSH) concentration and hydrogen-peroxide(H2O2)-induced cytotoxicity was investigated. The intracellular GSH concentration in human glioblastoma (T98G, U87MG) and glioma (KG1C) cell lines was one or two orders of magnitude higher than that in a human myelogenous leukemic cell line (HL-60), which showed higher sensitivity to H2O2. Pretreatment of these cell lines with L-buthionine-[S,R]-sulfoximine, which significantly reduced the intracellular GSH concentration, increased their sensitivity against H2O2, whereas pretreatment with N-acetyl-L-cysteine, which did not significantly change the intracellular GSH concentration, only marginally protected the cells from the cytotoxic effect of H2O2. The results suggest that drug sensitivity of tumor cells can be modified by glutathione-modulating compounds.