c-erbB-2 anti-sense phosphorothioate oligodeoxynucleotides inhibit growth and serum-induced cell spreading of p185c-erbB-2-overexpressing ovarian carcinoma cells

Overexpression of the c-erbB-2 proto-oncogene product (p185^c-erbB-2) occurs frequently ihd different types of human cancer and is correlated with a significantly decreased survival in ovarian cancer patients. The effect of c-erbB-2 anti-sense phosphorothioate oligodeoxynucleotides (S-ODNs) was examined on the ovarian cancer cell line SK-OV-3. p185^c-erbB-2 levels were specifically reduced by a single-dose application of 5 μM c-erbB-2 anti-sense S-ODNs. This was accompanied by a 60% inhibition of anchorage-dependent cell growth. More strikingly, c-erbB-2 anti-sense S-ODNs almost completely abrogated serum-induced cell spreading. A control of complementary sense oligodeoxynucleotides did not show significant inhibitory effects on cell growth or on cell spreading. The inhibition of cell spreading was imitated by a monoclonal antibody (9G6) targeting the extracellular domain of p185^c-erbB-2 and by the tyrosine kinase inhibitor erbstatin. The inhibitory activity of these 2 compounds was lost after a few hours, while the inhibition of serum-induced cell spreading by anti-sense S-ODNs was still present after 24 hr. Our results show that c-erbB-2 anti-sense S-ODNs effectively inhibit the mitogenic and spreading activity of p 185^c-erbB-2 in ovarian cancer cells. Thus, anti-sense strategies have the potential of providing new strategies for the therapy of ovarian cancer.     

Transfection with a CRIPTO anti-sense plasmid suppresses endogenous CRIPTO expression and inhibits transformation in a human embryonal carcinoma cell line

CRIPTO is a member of the epidermal growth factor (EGF) gene family originally isolated from undifferentiated human NTERA2 clone D1 (NT2D1) multipotent embryonal carcinoma cells. Retinoic acid (RA) treatment of NT2D1 cells leads to a neuronal differentiation program and to concomitant loss of CRIPTO mRNA expression. To assess the role of CRIPTO in the control of NT2D1 cell growth or differentiation, these cells were treated with 3 anti-sense oligodeoxynucleotides complementary to the 5′ end of the human CRIPTO mRNA. A dose-dependent inhibition of monolayer and soft agar growth was observed with each of these CRIPTO anti-sense oligodeoxynucleotides but not with a control oligodeoxynucleotide of random sequence or with the 3 corresponding CRIPTO sense oligodeoxynucleotides. In addition, NT2D1 cells were transfected with a recombinant expression vector containing a 918-bp coding fragment of the human CRIPTO cDNA in the 3′ to 5′ orientation. NT2D1 CRIPTO anti-sense transfectants exhibited a significantly reduced endogenous CRIPTO mRNA and protein, a 4-to 5-fold decrease in growth rate in monolayer and a 50–70% reduction in cloning efficiency in soft agar as compared with NT2D1 parental cells or with NT2D1 cells transfected with a plasmid containing the neomycin-resistance gene alone (NT2D1 neo cells). Finally, we examined the expression of immunophenotypic markers that are modulated during the differentiation of NT2D1 cells following RA treatment. The globoseries stage-specific embryonic antigen-3 recognized by the monoclonal antibody (MAb) SSEA-3 was expressed in 60% of undifferentiated parental NT2D1 or NT2D1 neo cells and in only 20% of NT2D1 CRIPTO anti-sense transfectants, whereas it was down-regulated in all cell lines following RA treatment. A neuroectodermal antigen recognized by the A2B5 MAb, which was not expressed in parental NT2D1, in NT2D1 neo or in CRIPTO anti-sense NT2D1 cells, was induced by RA treatment in all cell lines. Taken together, our results show that inhibition of endogenous CRIPTO expression in human embryonal carcinoma cells interferes with both transformation and differentiation. © 1996 Wiley-Liss, Inc.     

Identification of endogenous inhibitory growth factors from a human colon carcinoma cell line

A line of human colon carcinoma cells, designated MOSER, was established which synthesized tumor-inhibitory factor (TIF) and transforming growth factor (TGF) activity. Both activities were found in serum-free conditioned medium and in cell extracts. The activities coelute on Bio-Gel P-10 in acetic acid, but can be completely separated by reverse-phase high-pressure liquid chromatography. The TIF and TGF activities were acid and heat stable and were sensitive to trypsin and dithiothreitol. MOSER cell TIF prevented the anchorage-independent growth of the more differentiated colon carcinoma cell lines tested but did not affect the less differentiated lines. Using anchorage-dependent growth conditions, the effect of TIF appeared to be noncytotoxic and partially reversible. Purified TGF stimulated the growth of normal rat kidney fibroblasts and the slow-growing CBS colon carcinoma cell line but did not stimulate MOSER cell growth. MOSER cells contain both positive (TGF) and negative (TIF) factors with relative concentrations that may be important parameters in the regulation of cell growth.     

Autocrine secretion of a colorectum-derived growth factor by HT-29 human colon carcinoma cell line

The human colon cancer cell line HT-29 produces a growth factor (CRDGF; Mr = 25,000) which inhibits EGF binding to a wide variety of different normal and tumoral cell types in culture. Scatchard analysis of EGF binding shows that CRDGF induces a decrease in EGF receptor affinity. In contrast, EGF binding to any of the human colorectal cancer cell lines tested, i.e., HT-29, HT-29 (clone D4), HRT-18 or CAL-14, remains unaltered in the presence of exogenous CRDGF. However, the inhibitory effect of CRDGF becomes apparent on HT-29 cells after overnight exposure of these to suramin (at 37 degrees C). A short exposure to suramin (1 hr at 4 degrees C) or a mild acid washing of HT-29 cells can partially restore the inhibitory activity of CRDGF. These observations suggest that the action of suramin results in an unmasking of substantial levels of CRDGF receptors on HT-29 cells. Scatchard analysis of EGF binding on suramin-treated HT-29 cells shows that CRDGF inhibits EGF binding by decreasing EGF receptor affinity, as previously observed with the non-colonic cell types. A similar unmasking of CRDGF receptors is observed when the other colorectal cell lines are exposed to suramin. These results provide evidence for a model in which the colorectal cell lines have the property of secreting a unique growth factor that binds to its receptor by an autocrine mechanism.     

Genes upregulated in a metastasizing human colon carcinoma cell line

Differential gene expression between the metastatic human colon cancer cell line HT29p and its nonmetastatic counterpart HT29-MTX was revealed by suppression subtractive hybridization. Fifty-eight individual genes showed increased mRNA levels in HT29p cells. Only 15 of these genes had been related to transformation in previous studies; the majority of genes are new candidates encoding proteins relevant for the metastatic process. Cancer profiling arrays as well as in situ hybridization study revealed that at least some of the genes obtained in the SSH screen are also differentially expressed in human tumors.     

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.     

Characterization of the inhibitory effects of transforming growth factor-beta on a human colon carcinoma cell line

The effects of transforming growth factor-beta (TGF beta) on a human colon carcinoma cell line (MOSER) were investigated. TGF beta, at low concentrations (between 0.1 and 1.0 ng/ml), inhibited the proliferation of MOSER cells both in monolayer culture and soft agarose, in a dose-dependent manner. MOSER cells adapted to growth in chemically defined serum-free medium were more sensitive to the inhibitory effects of TGF beta than cells maintained in serum-supplemented medium. Morphological changes in MOSER cells, observed with TGF beta, were similar to those seen with the chemical differentiation agent N,N-dimethylformamide. Also in similarity to the effects of N,N-dimethylformamide, TGF beta induced a time- and concentration-dependent increase in soluble extracellular fibronectin. Binding studies with [125I]TGF beta revealed a relatively low number of binding sites on MOSER cells (13%) compared with mouse embryo fibroblastic (AKR-2B) cells. Thus far, other colon carcinoma cell lines, some displaying TGF beta receptors, have been reported to be unresponsive to TGF beta. This study is therefore the first to demonstrate a TGF beta-responsive colon carcinoma cell line.     

Phenotypic instability of drug sensitivity in a human colon carcinoma cell line

Colon cancer is one of the tumors most refractory to treatment by chemotherapy, and this may be due to inherent phenotypic instability of such tumor cells with respect to the biochemical determinants of drug sensitivity. To test this hypothesis, a clonal human colon carcinoma cell line, clone A, was passaged in culture in the absence of selection conditions or mutagens. During this time, sensitivity to several drugs was examined, and was found to decrease 4-fold during 30 weeks of culture. Five randomly selected subclones, having never been exposed to drug or mutagen, displayed a range of sensitivities to etoposide (50% inhibitory concentrations ranging from 1.5 to 4.9 microM) and to vincristine (9-fold range), but all had the same sensitivity to methotrexate. With time these sensitivities also changed, and subsequent subclones were chosen from the lines with highest and lowest drug sensitivity. Again a wide range of phenotypes was observed. Sensitivity to vincristine ranged 14-fold and to doxorubicin 3-fold. Several biochemical determinants of drug sensitivity had a broad range of expression between cell lines. Cellular accumulation of [3H]vincristine, as well as expression of multidrug resistance protein P170 and glutathione transferase activity all varied significantly between subclonal lines. This suggests that some human colon tumors may be phenotypically unstable with respect to drug sensitivity, and this could contribute to clinical resistance to chemotherapeutic compounds.     

Quinone-induced DNA single strand breaks in a human colon carcinoma cell line

It has been demonstrated that synthetic quinones, such as menadione, cause DNA damage in different cell systems, possibly being mediated by free radicals generated during redox cycling. It has been suggested that the damage caused could be related to tumor induction in different sites. To our knowledge it has not yet been demonstrated that the natural quinones, vitamin K1 and K2, exert the same activity. Using a colon carcinoma cell line, HT-29, we examined the extent of DNA damage induced by menadione, vitamin K1 and K2. Menadione caused significant DNA damage at low concentrations (25-200 microM) with a linear correlation of r = 0.95. In the presence of dicoumarol, a DT-diaphorase inhibitor, the damage was detected at concentrations five times lower indicating that free radicals generated during the redox cycling play a key role. Neither vitamin K1, incorporated in micelles, nor K2 caused detectable single strand breaks with respect to the controls either in the presence or in absence of dicoumarol. Our results demonstrate that, despite their redox cycling properties, the natural forms of vitamin K do not cause DNA damage in HT-29 cells as menadione does in the experimental conditions used.      

人端粒酶催化亚基反义核酸对子宫内膜癌细胞的抑制作用

目的 探讨人端粒酶催化亚基(hTERT)基因反义寡核苷酸(AODN)对子宫内膜癌细胞系HEC-1A的生长抑制作用及作用机理。方法 采用四甲基偶氮唑蓝(MTT)法检测AODN对细胞增殖能力及细胞生长的影响;逆转录聚合酶链反应技术(RT-PCR)、定量端粒酶重复扩增法(TRAP)、Westerm blot蛋白免疫印迹法和凋亡相关酶活性测定检测反义核酸对hTERT基因转录、蛋白表达、细胞端粒酶活性以及凋亡相关酶活性的变化。结果 低浓度hTERT基因AODN能够下调HEC-1A细胞HTERT、mRNA含量,抑制细胞hTERT蛋白表达,下调端粒酶活性,并且激活凋亡相关酶Caspase-1和Caspase-3活性;其对细胞的生长抑制作用有明显的时效性和剂量依赖性。结论 hTERT基冈反义核酸能够抑制子宫内膜癌细胞的增殖能力,有望成为内膜癌治疗的新方向。     

Formation, growth and morphology of multicellular tumor spheroids from a human colon carcinoma cell line (LoVo)

LoVo human colon carcinoma cells cultured by a liquid overlay technique form and grow as multicellular tumor spheroids. The growth curve of LoVo spheroids exhibits Gompertzian growth kinetics, i.e., exponential growth for 10 days, followed by exponential retardation in the rate of growth. Doubling time in the exponential growth phase is longer than in monolayer cultures (5 days for LoVo spheroids vs. 37 h for monolayers). When LoVo spheroids reach a diameter of about 300 microns, a necrotic core appears in their center and continuously increases with spheroid growth. The cell ultrastructure and organization in spheroids closely resemble those of the same cells when grown as tumors in vivo or as monolayer, i.e. intestinal epithelium, desmosomes, intracytoplasmic lumina and acinar structures. Individual cells from spheroids can be obtained by trypsinization and assayed for colony formation. LoVo spheroids provide a model which can be readily manipulated and appears to be suitable for evaluation of anticancer drugs. A comparison of LoVo spheroids exposed to doxorubicin with the same cells grown in monolayers emphasized the role of cell organization in determining drug resistance.     

Cytogenetic and phenotypic analysis of a human colon carcinoma cell line resistant to mitoxantrone

A human colon carcinoma cell line selected for a 21-fold resistance to mitoxantrone was cross-resistant to the anthracycline, doxorubicin, but not to the anthracene, bisantrene. A 2-fold resistance was observed with vinblastine, another drug associated with multidrug resistance. Net intracellular mitoxantrone and doxorubicin accumulation were decreased at 1 h for all dose levels in the resistant cell line compared to the sensitive cell line. Although the resistant cells were more resistant to mitoxantrone than doxorubicin, the net accumulation of mitoxantrone was only 19% less than the sensitive cell line; whereas doxorubicin accumulation was decreased by 49%. No significant difference between the sensitive and resistant cell lines was observed in the initial accumulation of mitoxantrone; however, the efflux of mitoxantrone was increased in the resistant cell line. Verapamil did not overcome the resistance to mitoxantrone and did not increase the net accumulation of drug. No alterations in the electrophoretic mobility of membrane proteins were observed. Using immunoblotting techniques, the resistant cell line did not express P-glycoprotein which is frequently observed for cells resistant to anthracycline antibiotics. Cytogenetic analysis showed a putative homogenously staining region on the short arm of chromosome 7 in the resistant cell line. The limited cross-resistant phenotype, lack of verapamil reversal, nondetection of P-glycoprotein, and cytogenetic evidence of gene amplification suggests the involvement of a novel drug-resistant gene associated with resistance to mitoxantrone.     

Sodium butyrate alters the response of a human colon carcinoma cell line to transforming growth factor-beta 1.

The effects of sodium butyrate (NaB) on the response of the RCA human colon carcinoma cell line to transforming growth factor-beta 1 (TGF-beta 1) were examined. NaB induced differentiation, as judged by an increase in cellular alkaline phosphatase, in the RCA cells and this differentiation was accompanied by a decreased growth rate. TGF-beta 1 did not significantly alter the growth or state of differentiation of the RCA cells. The growth rate of cells treated simultaneously with NaB and TGF-beta 1 was similar to that of control untreated cells while the alkaline phosphatase levels remained comparable to cells treated with NaB. Addition of TGF-beta 1 to cells grown in the presence of NaB resulted in a stimulation of growth. Cells pretreated with TGF-beta 1 remained sensitive to the growth inhibitory and differentiation inducing effects of NaB. These results suggest that NaB may alter the expression of proteins responsible for a stimulatory signal response to TGF-beta 1 in RCA cells.     

神经降压素对人结肠癌细胞株生长调控及其机制

目的　研究神经降压素对人结肠癌细胞株的生长调控作用及其的信号传导途径。方法　以不同浓度的神经降压素处理人低分化结肠癌细胞系Clone A细胞,用MTT法观察细胞的生长情况;分别应用液体闪烁测量法、γ-闪烁记数仪测定细胞内三磷酸肌醇(IP3)及环磷酸腺苷(c AMP)含量。结果　不同浓度的神经降压素(10 - 9～10 - 5m ol/ L)能显著地促进Clone A细胞的生长(P<0 .0 1) ,且呈剂量依赖关系(r=0 .94 2 ,P<0 .0 1) ,这种促生长作用可以被神经降压素受体拮抗剂所阻断。神经降压素能显著促进Clone A细胞内IP3、c AMP含量增加,这些作用也可以被神经降压素受体拮抗剂所抑制。结论　神经降压素能促进Clone A细胞生长,且可被神经降压素受体拮抗剂所抑制,其受体后信息传递途径,可能是通过磷酸肌醇和腺苷环化酶途径。     

Characterization of a colon carcinoma cell line for tumor immunotherapy

Chessboard vaccination (i.d. injection of various mixtures of mitomycin-treated fresh cells of the DE-TA colon carcinoma cell line and Vibrio cholerae neuraminidase (VCN] had a beneficial effect on recurrence and survival in Duke C patients. To standardize this kind of immunotherapy the following parameters of the DE-TA cell have been evaluated:--Karyotype (46 chromosomes, deletions in chromosome 8; 17); doubling time 24 hr; expression of CEA.--Stability of membrane antigens characterized by 9 different monoclonal antibodies over more than 40 cell culture passages.--Homogeneity of cell culture as evaluated by limiting dilution cloning at different culture passages and evaluation of expression of membrane antigens. Immunogenicity of lyophilized cells compared to cultured fresh cells by counting the number of specific antibody secreting clones after fusing spleen cells of immunized mice with SP-2/0-Ag14 mouse myeloma. As this characterization as well as safety tests (lack of infectious particles, tumorigenicity in nude mice) revealed no apparent risks, lyophilized DE-TA cells will be used as a standardized stable cell preparation for clinical trials.     

Establishment and characterization of a novel neuroendocrine carcinoma cell line derived from a human ascending colon tumor

The incidence of rare neuroendocrine tumors (NET) is rapidly increasing. Neuroendocrine carcinoma (NEC) is a NET with poorly differentiated histological features, high proliferative properties and associated poor prognoses. As these carcinomas are so rare and, thus, affect only a small number of patients allowing for few cell lines to be derived from patient biopsies, the histological, immunohistochemical, and clinical characteristics associated with colorectal NEC and NEC in other organs have yet to be clearly defined. Herein, we describe the establishment of a novel NEC cell line (SS‐2) derived from a tumor resection of the ascending colon from a 59‐year‐old Japanese woman. The histological, electron microscopic and immunohistochemical features of chromogranin A (CgA) as well as confirmation of synaptophysin positivity in this tumor were typical of those commonly observed in surgically resected colorectal NEC. Further, the Ki‐67 labeling index of the resected tumor was >20% and, thus, the tumor was diagnosed as an NEC of the ascending colon. The SS‐2 cell line maintained characteristic features to those of the resected tumor, which were further retained following implantation into subcutaneous tissues of nude mice. Additionally, when SS‐2 cells were seeded into ultra‐low attachment plates, they formed spheres that expressed higher levels of the cancer stem cell (CSC) marker CD133 compared to SS‐2 cells cultured under adherent conditions. SS‐2 cells may, therefore, contribute to the current knowledge on midgut NEC biological function while providing a novel platform for examining the effects of colorectal NEC drugs, including CSC.     

Patterns of growth and differentiation in the colon carcinoma cell line LIM 1863.

The LIM 1863 colon carcinoma cell line grows in the form of morphologically and functionally organized organoids. Cells are arranged around a central lumen with a brush border and nuclei are polarized to the periphery. The organoids contain 3 morphological cell types (columnar, goblet and caveolated cells). By agar cloning it has been possible to isolate 29 subclones of the cell line, all of which display the same phenotype and percentage of morphological cell types as the parent line. Cell-sorting experiments showed that precursor cells of LIM 1863 cultures could express either mucin (large-intestinal-mucus antigen) or a brush-border enzyme (sucrase-isomaltase). Proliferating cells were predominantly found near the outer periphery of organoids with cell maturation towards the internal lumen. Dead cells were continuously shed from the organoids but terminal non-cycling cells were not observed within the organoids. The organoid structure was calcium-dependent and promoted cell survival. Suspension cultures of disaggregated cells could be grown in medium containing less than 100 microM calcium. No decrease in differentiation antigens was observed in the low-calcium cultures, although polarization of the cells was lost. The organoid cultures formed by this cell line represent a unique in vitro model for colonic crypt growth and organization.