Lynn C. Yeoman

Heterogeneity of human colon carcinoma.

SummaryIn order to better understand colon cancer, a model system reflecting the heterogenous nature of this disease was developed and used in the development of new cytotoxic and non-cytotoxic therapeutic approaches. A large bank of colon carcinoma cell lines was established from primary human colon carcinomas and grouped based on their tumorigenicity in athymic mice, their growth rates in soft agarose and in tissue culture, and their secreted levels of carcinoembryonic antigen. These cell lines were later characterized based on cell surface proteins and antigens detected with antisera raised against a differentiated colon carcinoma cell line. Although these biochemical markers correlated with the biological classification of these cell lines, there was still extensive heterogeneity within each group in all properties examined. This colon carcinoma cell system was used to study natural vs. selected resistance to the anticancer drug mitomycin C (MMC). The differing IC50 values in vitro were reflected in the inhibition by MMC of xenograft growth in athymic mice. A new, more readily bioactivatable analogue of MMC was tried and shown to be more active in vitro and in vivo, suggesting that rapid efflux of the drug before activation may be important in examining causes of resistance to MMC. Another approach to the treatment of colon cancer is the use of non-cytotoxic agents such as growth factors and differentiation agents to restore normal growth to the malignant cells. We have isolated and characterized two types of polypeptides from colon carcinoma cells and conditioned medium from these cells. The first, transforming growth factors (TGFs) confer a transformed phenotype on non-transformed fibroblasts while the second, tumor inhibitory factors (TIFs), inhibits the anchorage independent growth of transformed cells. The fact that extracts of colon carcinoma cells contain both activities suggests that the heterogeneity of the cell lines could be due to different levels of TGFs and TIFs produced. The effectiveness of differentiation agents to restore normal growth control using a transformed mouse embryo cell line was examined. Treatment of these cells with differentiation agents restored normal growth control to these cells. An increased synthesis of TGFs resulted from these treatments. Therefore, differentiation agents may be useful in non-cytotoxic treatment. The use of this model system for human colon carcinoma will hopefully lead to more effective drugs for the treatment of colon cancer in man.

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.

Different epidermal growth factor growth responses and receptor levels in human colon carcinoma cell lines

The growth response to epidermal growth factor (EGF) and the numbers and types of EGF receptors were studied in three human colon tumor cell lines from each of two groups of cell lines that differ markedly in their growth properties and extent of differentiation. Aggressively growing and poorly differentiated colon cells (group I) did not respond to EGF alone, while less aggressively growing and more differentiated cells (group III) responded with increased growth when EGF was added to their chemically defined, serum-free medium. The average number of EGF receptors (EGF-R) measured at the surface of group III cell lines by radioligand binding assays, was eight-fold higher than that measured for group I cell lines. These observations provide evidence for possible autocrine mechanisms that maintain available EGF-R levels in more differentiated group III colon tumor cells and down-regulate EGF-R levels in group I colon tumor cells.

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Abstract Highly acidic phosphoprotein B23 ( 37 5.1 ; M.W. x 10 3 / pI ) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.

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.

An improved procedure for the ‘dot immunobinding’ analysis of hybridoma supernatants

The recently introduced dot immunobinding assay is well suited as a rapid and sensitive procedure for the analysis of those hybridoma clones that are producers of a specific antibody. We present a modification of the dot immunobinding assay which utilizes a single nitrocellulose sheet for up to 96 assays. By using a single nitrocellulose sheet, sample manipulation is greatly reduced, reaction conditions can be better standardized and a comparison of background reactivities is provided. Results are presented which demonstrate the effectiveness of this modified dot immunobinding assay.

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.

Identification of a tumor inhibitory factor in rat ascites fluid

A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.

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