Analysis of interleukin-6-induced signaling in murine hybridoma cell lines

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

Download Full-text

Interleukin-6 prevents dexamethasone-induced myeloma cell death

Blood ◽

10.1182/blood.v84.9.3063.bloodjournal8493063 ◽

1994 ◽

Vol 84 (9) ◽

pp. 3063-3070 ◽

Cited By ~ 12

Author(s):

J Hardin ◽

S MacLeod ◽

I Grigorieva ◽

R Chang ◽

B Barlogie ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Interleukin 6 ◽

Nuclear Translocation ◽

Myeloma Cell ◽

Concomitant Treatment ◽

Multiple Myeloma Cell ◽

Mediated Effects ◽

Human Multiple Myeloma ◽

Polymerase Chain

The effects of dexamethasone on the growth of four human multiple myeloma cell lines were studied. In addition, the effects on the expression of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) genes were investigated by the use of reverse-transcriptase polymerase chain reaction. Dexamethasone (Dex) concentrations of 10(-7) to 10(-6) mol/L inhibited IL-6 gene expression in three of four cell lines studied, whereas the higher concentration of the hormone inhibited also IL-6R gene expression. Dex effects were modulated through the glucocorticoid receptor (GR). Dex treatment resulted in killing of sensitive cells associated with DNA fragmentation, which could be reversed by concomitant treatment with IL-6. The reversal of Dex-mediated effects by IL-6 did not result from an inhibition of GR function as measured by receptor nuclear translocation or Dex-regulated reporter gene function. These results indicate that blockage of the IL-6 signaling pathway is essential for effective myeloma cell kill by Dex.

Download Full-text

Requirements of src family kinase activity associated with CD45 for myeloma cell proliferation by interleukin-6

Blood ◽

10.1182/blood.v99.6.2172 ◽

2002 ◽

Vol 99 (6) ◽

pp. 2172-2178 ◽

Cited By ~ 57

Author(s):

Hideaki Ishikawa ◽

Naohiro Tsuyama ◽

Saeid Abroun ◽

Shangqin Liu ◽

Fu-Jun Li ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Lines ◽

Interleukin 6 ◽

Kinase Inhibitor ◽

Myeloma Cell ◽

Src Family Kinases ◽

Src Family Kinase ◽

Receptor Complexes ◽

Intracellular Signals ◽

Cellular Context

Abstract Specific intracellular signals mediated by interleukin-6 (IL-6) receptor complexes, such as signal transducer and activator of transcription 3 (STAT 3) and extracellular signal–regulated kinase (ERK) 1/2, are considered to be responsible for inducing a variety of cellular responses. In multiple myeloma, IL-6 only enhanced the proliferation of CD45+ tumor cells that harbored the IL-6–independent activation of src family kinases even though STAT3 and ERK1/2 could be activated in response to IL-6 in both CD45+ and CD45− cells. Furthermore, the IL-6–induced proliferation of CD45+ U266 myeloma cells was significantly suppressed by Lyn-specific antisense oligodeoxynucleotides or a selective src kinase inhibitor. These results indicate that the activation of both STAT3 and ERK1/2 is not enough for IL-6–induced proliferation of myeloma cell lines that require src family kinase activation independent of IL-6 stimulation. Thus, the activation of the src family kinases associated with CD45 expression is a prerequisite for the proliferation of myeloma cell lines by IL-6. We propose a mechanism for IL-6–induced cell proliferation that is strictly dependent upon the cellular context in myelomas.

Download Full-text

Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2443-2447.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2443-2447

Author(s):

C B Siegall ◽

R P Nordan ◽

D J FitzGerald ◽

I Pastan

Keyword(s):

Cell Lines ◽

Interleukin 6 ◽

Hepatoma Cell ◽

Chimeric Protein ◽

Peptide Bond ◽

Myeloma Cell ◽

Mrna Levels ◽

Pseudomonas Exotoxin ◽

Chain Reactions ◽

Hepatoma Cell Lines

IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6. IL6 receptor mRNA levels were measured by polymerase chain reactions. When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors. However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively. RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate. These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40.

Download Full-text

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato

Plant Disease ◽

10.1094/pdis-07-20-1551-re ◽

2021 ◽

Vol 105 (4) ◽

pp. 832-839

Author(s):

Wanqin He ◽

Deqing Huang ◽

Jiayu Wu ◽

Xue Li ◽

Yajuan Qian ◽

...

Keyword(s):

Sweet Potato ◽

Cell Lines ◽

Immunosorbent Assay ◽

Root Rot ◽

Enzyme Linked Immunosorbent Assay ◽

Dickeya Dadantii ◽

Crude Extracts ◽

Tissue Print ◽

Hybridoma Cell Lines ◽

Dot Elisa

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

Download Full-text