Charles W. Taylor

Two-dimensional electrophoresis of proteins of citric acid nuclei prepared with aid of a Tissumizer®☆

Abstract Nuclei prepared from normal rat liver and Novikoff hepatoma ascites cells with the aid of a Tissumizer® in media containing 0.5 and 5 % citric acid were compared on the basis of electron microscopic appearance, DNA, RNA and protein content. Electron microscopy revealed better preservation of the nucleolar and nuclear morphology in the nuclei isolated in 0.5 % citric acid than in nuclei isolated in 5 % citric acid. Moreover, losses of protein and DNA from liver nuclei prepared by the sucrose-Ca 2+ procedure were significantly less in nuclei treated with 0.5 % citric acid than in nuclei treated with 5 % citric acid. The preservation of nuclear morphology and the retention of the majority of types of nuclear protein were significantly better with the procedure using 0.5 % citric acid than with the procedure using 5 % citric acid. The 5 % citric acid treatment was found to alter nuclear morphology and extract specific nuclear proteins, as demonstrated by two-dimensional polyacrylamide gel electrophoresis of the proteins.

Two-dimensional polyacrylamide gel electrophoresis of acid extractable nuclear proteins of normal rat liver and Novikoff hepatoma ascites cells

Abstract The nuclear proteins of normal rat liver and Novikoff hepatoma nuclei were extracted with 0.4 N H 2 SO 4 and subjected to two-dimensional polyacrylamide gel electrophoresis. A total of 98 protein components were found in the liver extract and 111 components in the tumor extract. A comparison of the patterns obtained revealed 11 qualitative differences and 5 quantitative differences.

Inhibition of colon tumor cell growth by 8-chloro-cAMP is dependent upon its conversion to 8-chloro-adenosine

Recent interest in site-selective cAMP analogs has focused on the role of 8-chloro-adenosine (8-Cl- adenosine) In the inhibition of tumor cell growth by 8-chloro-cAMP (8-Cl-cAMP) (Van Lookeren Campagne, et al. Cancer Res 1991; 51: 1600–5). We have evaluated 8-Cl-cAMP and 8-Cl-adenosine for their growth inhibitory activity against two human colon adenocarcinoma cell lines, HCT116 and FET. Because these cell lines have been adapted to grow in chemically defined medium we were able to evaluate the effect of serum on 8-Cl-cAMPs growth inhibitory activity. In addition, cells grown in serum-free medium were tested for their sensitivity to 8-Cl-cAMP, serum-activated 8-Cl-cAMP and 8-Cl-adenosine. IC50 values, determined by measuring cell growth using a MTT colorimetric assay, showed that ‘serum activation’ of 8-Cl-cAMP was required to achieve inhibition of HCT116 (IC50 = 1.3 ± 0.1 (μM) and FET (IC50 = 2.0 ± 0.1 μM) cell growth. IC50 values were not reached at the highest concentrations tested (IC50 > 500 μM) in the absence of serum, permitting us to conclude that 8-Cl-cAMP does not have growth Inhibitory activity between 1.0 and 500 μM doses. HCT116 and FET cells grown in media containing serum and in the presence of 8-Cl-adenosine had IC50 values of 0.6 ± 0.1 and 0.9 ± 0.2 μM, respectively. HCT116 and FET cells grown in chemically defined medium containing 8-Cl-adenosine exhibited IC50 values of 1.0 ± 0.1 and 3.1 μM, respectively. Reversed-phase HPLC analysis showed an 11.4 ± 0.7% conversion of 8-Cl-cAMP to 8-Cl-adenosine in 1 h at 37°C In the presence of 10% fetal bovine serum (FBS). Analysis of the continued conversion of 8-Cl-cAMP after 72 h in media containing 10% FBS revealed that 69.5 ± 0.7% of the 8-Cl-cAMP was converted to 8-Cl-adenosine. These results strongly support the conclusion that enzymatic conversion of 8-Cl-cAMP to 8-Cl-adenosine occurs in the presence of serum and that 8-Cl-adenosine is the active inhibitory compound.

Sensitivity of nuclear c-myc levels and induction to differentiation-inducing agents in human colon tumor cell lines

Six human colon tumor cell lines were analyzed for their constitutive levels of the c-myc protein. The nuclear proto-oncogene, c-myc, was detected as an expressed product in all of the human colon tumor cell lines analyzed. The poorly differentiated cell lines HCT116, RKO and C showed c-myc levels that averaged 2-fold greater than their well-differentiated counterparts, i.e., GEO, CBS and FET. When c-myc levels and responses to serum induction were analyzed in the presence of inducers of differentiation, i.e., dimethylformamide, retinoic acid, sodium butyrate and TGF-beta, distinct patterns of sensitivity and resistance emerged. Nuclear c-myc levels were reduced in all the colon cell phenotypes treated with dimethylformamide or sodium butyrate. Only the well-differentiated human colon tumor cell lines were responsive to transforming growth factor-beta. Only one of the human colon tumor cell lines (GEO) responded to retinoic acid. Increased levels of c-myc protein were found to correlate well with greater growth rates and with poor differentiation class. Similarly, a parallel sensitivity to down-regulation of c-myc levels and attenuation of c-myc induction curves for inducers of differentiation were observed in growth sensitive human colon tumor cell lines.

Differences in chromatin proteins of growing and non-growing tissues.

Abstract The non-histone chromatin proteins of growing and non-growing tissues were compared by two-dimensional polyacrylamide gel electrophoresis. The tissues studied were normal rat liver, regenerating rat liver, thioacetamide-treated rat liver, normal rat kidney, Novikoff hepatoma and Walker 256 carcinosarcoma. Although most of the protein components were common to all of the tissues studied, the densities and sizes of spots C18, CP, C21, C25, CQ, CR, CS and CT were greater in the growing tissues than in the non-growing tissues, including the thioacetamide-treated liver. In the latter, the increased densities and sizes of spots C18, C21 and CQ are presumably related to the markedly increased nucleolar size rather than to cell division. Accordingly the increases in sizes and densities of spots C25, CP, CR, CS and CT are apparently of importance to the growth processes of normal and tumor tissues. The number of tissue specific proteins was small compared with the number of proteins in this fraction and includes BP and CBL for normal liver, BJ′ for kidney and CG′, CH′ and CP

Chapter 18 The Isolation of Nuclei with Citric Acid and the Analysis of Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis

for the tumors.

Comparison of immunoelectrophoretic techniques for the analysis of cytosol antigens

Publisher Summary This chapter discusses the isolation of nuclei with citric acid and the analysis of proteins by two-dimensional polyacrylamide gel electrophoresis. The recent studies compare the acid-soluble nuclear protein complement obtained from rat liver nuclei isolated in sucrose medium containing 3.3 m M Ca 2+ and those isolated in 0.025 M citric acid. The citric acid procedure was chosen for the comparison studies on the 0.4 N sulfuric acid extracts and the chromatin fractions in normal and tumor cell nuclei. The absence of contaminating outer nuclear envelope elements, the low pH (2.5) of the 0.025 M citric acid medium, and the ease of cytoplasmic tag removal from the tumor cell nuclei were the reasons for the use of a citric acid procedure. One way to determine the quality of nuclear isolation products is light microscope examination of the preparation. In addition, electron microscopic evaluation provides information that cannot be obtained by light microscopy. Another level of study is evaluation of the proteins in the preparation. The application of high resolution and high sensitivity two-dimensional polyacrylamide gel electrophoresis provides further evaluation of nuclear isolation methods. This approach can supplement cytoplasmic enzyme assays which would, however, not detect noncatalytic cytoplasmic contaminants or nuclear protein losses. With two-dimensional methods, the protein constituents of nuclei and nucleoli can be evaluated at various stages of nuclear isolations and nuclear fractionations.

Characterization and reactivity of a monoclonal antibody that recognizes a 76 kilodalton human colon tumor antigen

Crossed immunoelectrophoresis was employed in the analysis of cytosol antigens of a human colon adenocarcinoma cell line. Other immunoelectrophoretic techniques - crossed immunoelectrophoresis in the presence of Ampholine (incorporation of Ampholine in the first dimension electrophoresis gel), crossed immunotachophoresis and crossed immunoelectrofocusing - were also investigated ad compared with crossed immunoelectrphoresis in an attempt to select the optimal immunoelectrophoretic system for the analysis of cytosol antigens. The results of these comparisons showed that each technique offered its own advantages. The maximum number of immunoprecipitin peaks were detected by crossed immunoelectrophoresis. Both crossed immunoelectrophoresis in the presence of Ampholine and crossed immunotachophoresis provided the greatest resolution of electrophoretically similar antigens. Crossed immunoelectrofocusing provided IP values for these antigens. It was concluded that these techniques, when employed in combination, provided a more complete analysis of the complex cytosol antigenic mixture and may be useful when employed in combination to other antigen-antibody systems.

Identification of Cytosolic Antigens from GW-39 Adenocarcinoma Cells by Crossed Immunoelectrophoresis and Immunofluorescence

A monoclonal antibody (MAb 76) was produced by immunizing mice with a soluble cytoplasmic protein fraction from a human adenocarcinoma of the colon. MAb 76 showed specific immunoreactivity against a 76 kDa protein in immunoblot studies using total colon tumor cytosol proteins. Immunoprecipitation of phosphorylated cytosolic protein products with MAb 76 and subsequent analysis on SDS containing polyacrylamide gels revealed a single 38 kDa band, indicating that the 76 kDa antigen is associated with a 38 kDa phosphoprotein species. Indirect immunofluorescence analysis of primary tumor specimens and human colon tumor cell lines showed positive immunoreactivity with 6/7 human colon adenocarcinoma tissues and 15/18 human colon tumor cell lines. MAb 76 was unreactive with normal colon, liver and lung specimens from human, mouse and hamster. The epitope-bearing monomer detected by MAb 76 is immunologically conserved in a high percentage of colon tumor cells and tissues and may represent a cellular product that is characteristic of the transformed colon cell phenotype.

Characterization of two monoclonal antibodies that recognize high molecular weight colon antigens

Rabbits were immunized with a cytosolic fraction prepared from human adenocarcinoma cells of the colon (GW-39). The antibodies obtained were analyzed using crossed immunoelectrophoresis and were found to precipitate 23 distinct antigens in the cytosolic fraction from colon tumor cells. After preabsorption with human serum and plasma as well as acetone powders prepared from 2 normal human tissues and 4 normal hamster tissues, 1 major immunodominant cytosol antigen (CA-3) and two less intense immunoprecipitin peaks (CA-1 and CA-5) remained detectable by the crossed immunoelectrophoretic method. The preabsorptions with normal tissues were sufficiently complete to remove anti-carcinoembryonic antigen antibodies and showed that antigens CA-1, CA-3 and CA-5 are immunologically distinct from carcinoembryonic antigen. Indirect immunofluorescence localization studies with preabsorbed anti-cytosol antibodies and FITC-conjugated second antibody showed that these antigens were expressed in the cytoplasm and at the cell surface in several human colon tumor cell lines, their subclones and in primary colon tumor specimens.