Role of interleukin 6 in the growth of myeloma-derived cell lines.

The role of interleukin 6 (IL-6) in the growth of five multiple myeloma-derived cell lines was characterized. The U266 and RPMI 8226 cell lines demonstrated increased DNA synthesis when cultured with exogenous IL-6, expressed IL-6 cell surface receptors (IL-6Rs) and expressed mRNA for IL-6R. However, these cells did not secrete detectable IL-6 protein, and a neutralizing antibody to IL-6 did not inhibit their growth. Three other myeloma-derived cell lines ARH-77, IM-9 and HS-Sultan did not respond to exogenous IL-6, secrete IL-6 or express cell surface IL-6Rs. The IL-6 responsive cell lines bore late B-cell surface antigens (Ags), CD38 and PCA-1, whereas those lines which were non-IL-6 responsive strongly expressed B1 (CD20) and B4 (CD19) Ags, representing earlier stages in B-cell differentiation. Finally, the two IL-6 responsive cell lines did not express Epstein-Barr virus (EBV) proteins; in contrast, EBV encoded proteins typically expressed during latency could be detected in the three non-IL-6 responsive lines, confirming infection with virus. These studies clarify the heterogeneity observed in the myeloma cell line phenotype and biology and suggest that the U266 and RPMI 8226 cell lines, which express IL-6 cell surface receptors and are IL-6 responsive, may be useful for further study of IL-6 signal transduction in and related IL-6 mediated growth of myeloma in vivo. In contrast, those cell lines which are IL-6-independent provide a model for further study of EBV transformation and IL-6-dependent growth mechanisms in malignancy.     

A recognition site on synthetic helical oligonucleotides for monoclonal anti-native DNA autoantibody.

The binding site in native DNA for a murine monoclonal anti-DNA autoantibody was investigated by measurements of competitive binding of a series of synthetic helical oligonucleotides. The antibody bound to a (dG-dC)3 or (dG-dC)4 core in the center of a base-paired octadecanucleotide. Reactions of analogues containing modifications or substitutions at specific sites indicated that the antibody bound to portions of cytosine and guanine in the major groove, a limited region of the backbone, and the 2-amino group of one guanine in the minor groove. For these interactions to occur, the antibody combining site would straddle the backbone of one of the helical strands of DNA.     

Hydrolytic stability of pneumococcal group 6 (type 6A and 6B) capsular polysaccharides.

The hydrolyses of the immunologically cross-reactive and constitutionally isomeric group 6 pneumococcal polysaccharides, types 6A and 6B, were investigated by 31P nuclear magnetic resonance spectroscopy, gel filtration through Sepharose 4B, reducing-sugar analysis, and rocket immunoelectrophoresis. Phosphorus nuclear magnetic resonance spectroscopy showed that cleavage of the repeating-unit phosphodiester linkages at pH 10, 60 degrees C was considerably faster (greater than 10(3) ) for the type 6A than the type 6B polysaccharide. Under these reaction conditions, 31P nuclear magnetic resonance kinetic measurements showed that the Na+ form of the type 6A polysaccharide underwent phosphodiester-linkage hydrolysis two times slower than the corresponding Ca+2 form; a stoichiometrically excess amount of Ca+2 caused a 30-fold enhancement of the latter hydrolysis rate. The spectroscopic characterization of phosphorus-containing end groups resulting from hydrolysis of the type 6A polymer provided additional mechanistic information. Heating the type 6A and 6B polysaccharides at 56 degrees C for various times led to gel filtration coefficients of distribution (Kd values) which indicated that the type 6A material underwent size reductions considerably faster than did the type 6B antigen; these increased Kd values qualitatively correlated with the loss of immunochemical reactivity measured by rocket immunoelectrophoresis. The application of a statistical theory to the depolymerization of the type 6A and 6B polysaccharides was consistent with random bond cleavage, as evidenced by the calculated versus measured gel filtration patterns. Although the molecular changes causing the size reductions were not fully elaborated, it was established that the acetal linkages of the type 6A and 6B polysaccharides were comparatively resistant to hydrolysis and that depolymerization by hydrolysis of the phosphodiester linkage was a major factor only in the type 6A structure. It was concluded that the hydrolytic stability of the type 6B antigen would favor its use in the polyvalent pneumococcal vaccine rather than the cross-reactive, but comparatively unstable, type 6A polysaccharide, if all other factors are equal.     

Is there any correlation between restriction fragment length polymorphism of the L-MYC gene and metastasis of human nonsmall cell lung cancer?

A potential molecular marker associated with cancer susceptibility as well as metastasis, prognosis and adverse survival, is the L-myc gene. The studies of lung cancer patients from different populations have yielded controversial results. We studied 64 nonsmall cell lung cancer (NSCLC) patients and 37 healthy controls of Turkish origin for L-myc gene polymorphism. Our aim was to test the hypothesis that there was association between L-myc S allele in NSCLC and predisposition to the disease and TNM stage indicating tumor size, node classification and metastasis. Polymerase chain reaction restriction fragment length polymorphism and agarose gel electrophoresis were used to determine the L-myc oncogene genotypes. We found no significant difference, both in the distribution of the LL, LS and SS genotypes and in the allelic frequencies, between the patient group and the control group; that is, the frequencies of L-myc alleles were, L and S, 0.59 and 0.41, 0.60 and 0.40, respectively. Our data between the patient group and the control group; that is, the frequencies of L-myc alleles were, L and S, 0.59 and 0.41, 0.60 and 0.40, respectively. Our data concerning age, sex, size of tumors, histological type of tumors showed no significant association with L-myc genotype. However, a higher frequency of L-myc S allele in the squamous cell carcinoma compared to other histological groups was found, although this difference was not statistically significant. No association was found between the L-myc RFLP and increased risk of metastasis either to the lymph nodes or to other organs. Our results suggested that L-myc gene polymorphism was not a suitable prognostic marker of metastatic development in Turkish NSCLC patients.     

Zebrafish: a genetic approach in studying hematopoiesis

The zebrafish (Danio rerio) has emerged in recent years as an exciting animal model system for studying vertebrate organ development and, in particular, the development of the hematopoietic system. The combined advantages of developmental biology and genetic screens for mutations in zebrafish have provided insights into early events in hematopoiesis and identified several genes required for normal blood development in vertebrates. As a result of the large-scale mutagenesis screens for developmental mutants, several zebrafish mutants with defects in blood development have been recovered. This review discusses how these blood mutations in zebrafish have given new perspectives on hematopoietic development.     

SPRED1 deletion confers resistance to MAPK inhibition in melanoma

Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAF^V600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAF^V600E inhibition in melanoma.     

Site‐directed zebrafish transgenesis into single landing sites with the phiC31 integrase system

Background: Linear DNA‐based and Tol2‐mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long‐term transgene stability unpredictable with variable expression. Targeted, site‐directed transgene integration into pre‐determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site‐directed transgenesis. Results: We report the implementation of the phiC31 integrase‐mediated attP/attB recombination for site‐directed zebrafish transgenics of attB‐containing transgene vectors into single genomic attP landing sites. We generated Tol2‐based single‐insertion attP transgenic lines and established their performance in phiC31 integrase‐catalyzed integration of an attB‐containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue‐specific transgene applications as well as long‐term stability of phiC31‐mediated transgenes. Conclusions: Our results establish phiC31 integrase‐controlled site‐directed transgenesis into single, genomic attP sites as space‐, time‐, and labor‐efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community. Developmental Dynamics 242:949–963, 2013. © 2013 Wiley Periodicals, Inc.     

Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

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