Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component.

A serum-free medium supplemented with a few growth factors was devised to grow lymphocyte hybridomas. The medium was developed with the hybridoma line MPC11-BL, a fusion product between a mouse plasmacytoma cell line (MPC11TG70na3) and mouse (BALB/c) spleen cells. In the process of developing the medium, ethanolamine was found to be an essential growth factor for the hybridoma. Phosphoethanolamine at 10-fold higher concentration could substitute for ethanolamine. Long-term cultivation of the cells was achieved in the defined medium supplemented with insulin, transferrin, ethanolamine, and selenium. The defined medium supported the growth of various other mouse hybridoma cell lines, mostly at a rate comparable to that observed in a serum-containing medium. After one-step ammonium sulfate precipitation of the spent medium, more than 95% of the protein recovered was immunoglobulin as shown by NaDodSO4/polyacrylamide gel electrophoresis.     

Tumor promoters: from chemicals to inflammatory proteins

Purpose

The classic two-stage chemical carcinogenesis in rodents is not directly linked to multistage carcinogenesis in humans. In light of our findings that tumor necrosis factor-α (TNF-α) is an endogenous tumor promoter and that TNF-α-inducing protein (Tipα) of Helicobacter pylori stimulates progression of cancer and epithelial–mesenchymal transition, we think it is necessary to re-examine the concept of tumor promoter, from chemicals to inflammatory proteins.

Topics and results

This paper begins with “inflammation,” discovered by Virchow, studies of Yamagiwa and Tsutsui, and briefly reviews numerous topics, such as (1) the classic concept of tumor promoter, (2) tumor promotion on mouse skin induced by protein kinase C activators and okadaic acid class compounds, (3) organ specificity of tumor promoters, presenting numerous tumor promoters in various organs, (4) unique tumor promotion induced by inhibitors of protein phosphatases 1 and 2A in mouse skin, rat glandular stomach, and rat liver, (5) the significant role of TNF-α in tumor-promoting inflammation, (6) progression induced by Tipα of H. pylori, and (7) enhancement of cancer treatment efficacy with the combination of anticancer drugs and green tea catechins, to inhibit tumor-promoting inflammation.

Conclusion

Human cancer development involves both durable genetic changes caused by carcinogens and proinflammatory cytokines, and simultaneous inflammation in progression induced by proinflammatory cytokines and chemokines.     

Hepatitis B and C Viruses Infection, Lifestyle and Genetic Polymorphisms as Risk Factors for Hepatocellular Carcinoma in Haimen, China

A case‐control study was carried out to investigate the impact of factors including virus infection, aflatoxin B1, microcystins, smoking/drinking and dietary habits as well as genetic polymorphisms of aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1), on susceptibility to hepatocellular carcinoma (HCC) in Haimen, China. A total of 248 patients with HCC and 248 sex‐, age‐ and residence‐matched population‐based controls were recruited into the study. Virus infection, and ALDH2 and CYP2E1 gene polymorphisms were assessed in 134 paired cases and controls. By univariate analysis, hepatitis B virus (HBV) infection (odds ratio [OR]=9.75; 95% confidence interval [CI] =4.71–20.2), history of intravenous injection (OR=1.50; 95%CI=1.02–2.22), average income (OR=0.63; 95% CI=0.43–0.92), frequent intake of foods rich in protein, e.g., egg (OR=0.6; 95% CI=0.42–0.87), chicken (OR=0.53; 95% CI=0.35–0.79), pork (OR=0.67; 95% CI=0.46–0.98) and fresh fish (OR=0.58; 95% CI=0.39–0.87) significantly differed between cases and controls. However, peanut intake (OR=0.66; 95% CI=0.43–1.01), source of drinking water, including tap (OR=1.33; 95% CI=0.81–2.20), deep well (OR=0.94; 95% CI=0.56–1.55), shallow well (OR=0.85; 95% CI=0.55‐–1.30), river (OR=0.95; 95% CI=0.65–1.38), ditch (OR=1.09; 95% CI=0.76–1.55) and pond water (OR=1.0; 95% CI=0.14–7.10) were not significantly associated with risk. Univariate analysis also indicated that the 1–1 genotype of ALDH2 (OR=1.38; 95% CI=0.86–2.23) as well as the Pst1‐ and Rsa1‐digested c1/c1 genotype of CYP2E1 (OR=1.36; 95% CI=0.81–2.28), was slightly more frequent in the case group. On multivariate analysis, HBV infection (OR=13.9; 95% CI=5.78–33.6) and history of intravenous injection (OR=2.72; 95% CI=1.24–6.00) were still associated with significantly increased risk of HCC, while frequent intake of fresh fish (OR=0.32; 95% CI=0.12–0.86) decreased this risk. These findings suggest that whereas peanut intake, water sources as well as genetic polymorphisms in ALDH2 and CYP2E1 do not significantly correlate with the risk of HCC, HBV infection is a main risk factor, and dietary items rich in protein, especially fresh fish, might protect against the risk of HCC in Haimen, China.     

Anti-cancer effects of morphine through inhibition of tumour necrosis factor-{alpha} release and mRNA expression

Morphine is mainly used to relieve pain in the terminal stageof cancer patients. We found that morphine has inhibitory effectson growth of various human cancer cell lines, with IC₅₀ from2.7 to 8.8 mM, and BALB/3T3 cells, with IC₅₀ of 1.5 mM. Althoughthe IC₅₀ values were relatively high, we decided to study themechanisms of anti-carcinogenic effects of morphine. Morphineinhibited activation of protein kinase C induced by teleocidin,one of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumourpromoters (IC₅₀, 1 mM). Based on our previous evidence thattumour necrosis factor-     

Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells

Insulin-like growth factor binding proteins (IGFBPs) are secreted into the extra-cellular matrix and inhibit cell growth through IGF-dependent and -independent mechanisms. In this study, we investigated the role of IGFBP-6, a relatively unexplored member of the IGFBP family, in the proliferation of non-small cell lung cancer (NSCLC) cells. Infection of NSCLC cell lines in vitro with an adenovirus expressing human IGFBP-6 under the control of a CMV promoter (Ad5CMV-BP6) reduced NSCLC cell number through activation of programmed cell death, as shown by cell staining with Hoechst 33342 or DNA end-labeling with bromodeoxyuridine triphosphate. The growth regulatory effect of IGFBP-6 was investigated in vivo by intratumoral injection of Ad5CMV-BP6 in NSCLC xenografts established in nu/nu mice. A single injection of Ad5CMV-BP6 reduced the size of NSCLC xenografts by 45%. These findings indicate that IGFBP-6 is a potent inducer of programmed cell death in cancer cells and support investigations into IGFBP-6 as a potential target in cancer therapeutics.     

hnRNP B1 protein may be a possible prognostic factor in squamous cell carcinoma of the lung

Heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is an RNA-binding protein that is required for the maturation of mRNA precursor. It was previously reported that hnRNP A2/B1 was overexpressed at the early clinical stage of lung cancer, and that hnRNP B1 protein, a splicing variant of hnRNP A2 mRNA, was elevated in lung cancer tissues. In this study, we applied the immunohistochemical method, using anti-hnRNP B1 antibody to analyze the usefulness of the hnRNP B1 antibody as a prognostic marker and also as a marker useful for early detection. A total of 206 specimens were examined. Histological examination revealed this protein to be positive in 79 (71.2%) of 111 squamous cell carcinomas and in 45 (64.3%) of 70 adenocarcinomas, respectively. This protein was also expressed in 24 (63.2%) of 38 roentgenographically occult carcinomas and in seven (63.6%) of 11 dysplastic lesions. These findings suggest the possible participation of this protein in early carcinogenesis. Furthermore, the survival curve of the squamous cell carcinoma patients with hnRNP B1 overexpresseion showed a better prognosis compared with that of the patients without hnRNP B1 expression (P=0.014), whereas in adenocarcinoma patients, there was no such a difference between them (P=0.889). These findings indicate that hnRNP B1 could be a useful marker for the early detection of bronchogenic squamous cell carcinoma and that it may be a prognostic factor in this cell type.     

Disaccharide Esters Screened for Inhibition of Tumor Necrosis Factor-α Release Are New Anti-cancer Agents

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine playing a part in various pathological states. Non-toxic inhibitors of TNF-α release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF-α release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12-dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF-α release with synthetic disaccharide esters, such as 6,6′-di-0-alkanoyl-α,α-trehaloses (6,6′-diester-trehaloses), 4,4′-di-0-alkanoyl-α,α-trehaloses (4,4′-diester-trehaloses) and 6,6′-diamino-6,6′-dideoxy-N, N′-dial-kanoyl-α,α-trehaloses (6,6′-diamide-trehaloses) bearing fatty acids of various chain lengths, and n- dodecyl-β-D-maltoside as a disaccharide monoester. 6,6′-Diester-trehaloses and 4,4′-diester-treha-loses of C₈ to C₁₄ fatty acids, 6,6′-diamide-trehaloses of C₈ to C₁₄ fatty acids, and n-dodecyl-β-D-maltoside all inhibited TNF-α release in a dose-dependent manner. The IC₅₀ values are 7.4-14.8 μM for 6,6′-diester-trehaloses (C₈ to C₁₂), 14.6-21.6 μM 4,4′-diester-trehaloses (C₈ to C₁₂), 2.9-15.0 μM for 6,6′-diamide-trehaloses (C₈ to C₁₄) and 23 μM for dodecyl-β-D-maltoside. Both 6,6′-di-O-octanoyl-α,α-trehalose (C₈, designated as SS555) and n-dodecyl-β-D-maltoside (C₁₂) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor-bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n-dodecyl-β-D-maltoside. Moreover, SS555 inhibited TNF-α gene expression mediated through inhibition of AP-1 activation, but not NF-αB activation. This paper reports that diester-trehaloses of C₈ to C₁₂ fatty acids and mimics of disaccharide monoesters such as n-dodecyl-β-D-maltoside appear to be potential cancer-preventive agents of a new type.     

Preliminary Evaluation Study of a Prototype Hollow Fiber Membrane for the Continuous Membrane Autotransfusion System

A totally new autotransfusion system has been developed utilizing a hollow fiber membrane, based upon plasmapheresis technology. Prior to fabricating the system, it was essential to evaluate the basic performance characteristics of the filter, which was designed particularly for the new system. The objective of this study was to prove or disprove that such a system would be available using this filter. An in vitro study was conducted on the filter using bovine blood. The result of the study showed that the filter could process 2–20% of hematocrit blood at a flow rate greater than 250 ml/min of inlet blood continuously. Moreover, it could concentrate 5–20% hematocrit blood to hematocrit percentages greater than 40% by a single passage through the filter. These results seemed to prove that a rapid, continuous, and compact autotransfusion system could be developed using this filter.