The human breast carcinoma cell line SW 613-S: an experimental system to study tumor heterogeneity in relation to c-myc amplification, growth factor production and other markers (review)

Amplification of the c-myc gene has been frequently reported in breast carcinomas. However the precise function of the c-myc protein is still unknown and the nature of the selective advantage offered to a cell by an overexpression of such a protein is unclear. We are addressing this question using the SW 613-S human breast carcinoma cell line as a model system. This cell line harbours an amplified c-myc gene and a mutated c-Ki-ras gene. By various criteria the amplified c-myc gene of SW613-S cells appears undistinguishable from a normal human c-myc gene. The SW613-S cell line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. DM-containing cells are progressively lost upon in vitro cultivation but are selected for during in vivo growth, as tumors in nude mice, or by cultivating the cells in a chemically defined, serum-free medium or under conditions preventing anchorage. Clones with different levels of amplification and different chromosomal localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice, whereas those with a low level are not. Introduction of c-myc gene copies by transfection confers tumorigenicity to the nontumorigenic clones, indicating that a high level of amplification of the c-myc gene contributes to the tumorigenic phenotype of SW 613-S cells. Tumorigenic clones grow unattached, are able to proliferate in a chemically defined medium, and produce high levels of several growth factors (e.g. TGF-alpha, IGF2). Nontumorigenic clones are more dependent upon anchorage for growth, show a restricted growth in defined medium, and produce low or undetectable level of the growth factors tested. We have identified several genes, besides c-myc, the expression level of which is markedly different in the two types of clones. TGF-alpha, IGF2, PDGF-A, int-2, cytokeratins K8 and K18 and ferritin H chain are overexpressed in tumorigenic clones. In contrast, c-erbB1 (EGF receptor), c-jun, vimentin and p53 are expressed at a higher level in the nontumorigenic clones. Finally the major histocompatibility class I antigens, ferritin L chain, TGF-beta and c-Ki-ras, are examples of genes expressed at the same level in both types of clones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.     

Interferon induced increases in c-myc expression in a human breast carcinoma cell line.

Previous studies from our laboratory have indicated that interferon-gamma inhibits the growth of a human breast carcinoma cell line, MDA 468. We measured steady state levels of c-myc mRNA to determine if the antiproliferative effect of IFN-gamma was mediated by changes in the expression of this proto-oncogene. C-myc message levels increased after 24 hours of IFN treatment, peaked at 48 hours, but remained elevated through 96 hours of treatment. The increase in c-myc mRNA was observed with as little as 10 U/ml of IFN-gamma. The stability of mRNA was enhanced in interferon treated cells. These data suggest that IFN-gamma increased expression of c-myc mRNA, but decreased cell growth, and point out the complexities of the relationship of the expression of this protooncogene to cell growth.     

Amphiregulin contributes to the transformed phenotype of human hepatocellular carcinoma cells

Hepatocellular carcinoma is a major cause of cancer-related deaths. Current treatments are not effective, and the identification of relevant pathways and novel therapeutic targets are much needed. Increasing evidences point to the activation of the epidermal growth factor receptor (EGFR) as an important mechanism in the development of hepatocarcinoma. We previously described that amphiregulin (AR), a ligand of the EGFR, is not expressed in healthy liver but is up-regulated during chronic liver injury, the background on which most liver tumors develop. Now, we have studied the expression and role of AR in human hepatocarcinoma. AR expression and function was studied in human liver tumors and cell lines. AR is expressed in human hepatocellular carcinoma tissues and cell lines and behaves as a mitogenic and antiapoptotic growth factor for hepatocarcinoma cells. We provide several lines of evidence, including AR silencing by small interfering RNAs and inhibition of amphiregulin by neutralizing antibodies, showing the existence of an AR-mediated autocrine loop that contributes to the transformed phenotype. Indeed, interference with endogenous AR production resulted in reduced constitutive EGFR signaling, inhibition of cell proliferation, anchorage-independent growth, and enhanced apoptosis. Moreover, knockdown of AR potentiated transforming growth factor-beta and doxorubicin-induced apoptosis. Conversely, overexpression of AR in SK-Hep1 cells enhanced their proliferation rate, anchorage-independent growth, drug resistance, and in vivo tumorigenic potential. These observations suggest that AR is involved in the acquisition of neoplastic traits in the liver and thus constitutes a novel therapeutic target in human hepatocarcinoma.     

Differential characteristics of two new tumorigenic cell lines of human breast carcinoma origin

Permanent human tumor cell lines are an important tool for the study of breast cancer. Two new breast cancer cell lines (BrCa-MZ-01 and BrCa-MZ-02) were isolated from a solid tumor and a pleural effusion, respectively. One cell line was established from a medullary carcinoma, the other from a ductal carcinoma. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. Intermediate filament and cytokeratin typing showed a clear predominance of the simple-epithelial cytokeratins CK 8, CK 18 and CK 19, although the expression was reduced in comparison to the hormone receptor-positive reference cell lines MCF-7 and ZR-75-1. Both cell lines produced slow-growing tumors after subcutaneous (s.c.) transplantation of 1 × 10⁷ viable tumor cells into nude mice. The cell line BrCa-MZ-01 expresses the estrogen and progesterone receptor, whereas the cell line BrCa-MZ-02 remains negative. Both cell lines are positive for secretion of platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), whereas interleukin-6 (IL-6) is only secreted by the cell line BrCa-MZ-02. Int. J. Cancer 77:415–423, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of tumorigenic sub-lines from a poorly tumorigenic human colon-adenocarcinoma cell line

The interaction of tumor cells with extracellular-matrix components is suspected to play an important role in tumorigenesis induction. The tumorigenicity of a poorly tumorigenic human colon-adenocarcinoma cell line (BCS-TC2) was induced by co-injection with Matrigel. A new cell sub-line, BCS-TC2.1, was isolated and established from these tumors. Implantation of these cells in nude mice in the absence of Matrigel-generated tumors which allowed the establishment of another tumorigenic cell sub-line, BCS-TC2.2. Matrigel and laminin, but not collagens, promote the tumorigenicity of BCS-TC2 cells, probably due to specific interactions of a pre-existing minor cell sub-population with laminin, which facilitate the initial growth of these cells in vivo. Cytogenetic analysis reveals that both sub-lines originate from the parental one, but a new marker in chromosome 9 is observed. These sub-lines present a lower degree of differentiation, as deduced from the lower CEA content, 5′-nucleotidase and alkaline-phosphatase activities. No variation is observed in the mRNA and protein expression of the 67-kDa laminin-binding protein. However, an increase in β₁ integrins and a parallel decrease in β₄ integrin were detected. Thus, the new sub-lines, compared to the parental cells, present karyotypic and phenotypic differences such as the expression of a distinctive integrin pattern. This system represents a useful model for understanding the development and progression of tumorigenicity in cancer cells. © 1996 Wiley-Liss, Inc.     

DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

Background  

DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.     

RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.     

Over-expression and amplification of the c-myc gene in human urothelial carcinoma

To understand the mechanisms underlying increased expression of Myc protein in human urinary bladder cancer, expression of c-myc mRNA and the copy number of the c-myc gene were determined. Expression of mRNA was measured by quantitative RT-PCR in 40 urothelial carcinomas and in 18 histologically normal mucosae. Mean expression in tumors was significantly increased (3.23+/-2.63 AU vs. 1.90+/-0.95 AU, p < 0.023) and exceeded the highest level in normal mucosa in 15 (37.5%) tumors. The c-myc gene copy number was higher than in leukocytes and normal bladder mucosa in 14 of 40 tumors, but only 3 among these showed a more than 4-fold increase indicative of gene amplification. Most, but not all, tumors with elevated expression displayed an increased gene copy number (p < 0.0001). In line with other studies of the protein level, no significant association either of c-myc mRNA over-expression or of increased gene copy number with tumor stage or grade was observed. The data indicate that elevated mRNA expression as a consequence of increases in c-myc gene copy number often underlies Myc protein over-expression in bladder cancer. This increase may be a consequence of, most frequently, chromosome 8q gain and, occasionally, gene amplification, while in some tumors deregulation of mRNA expression occurs without evident changes in the c-myc gene copy number.     

Development of monoclonal antibodies to the human breast carcinoma cell line PMC42

In an attempt to identify antigens expressed during breast differentiation, three murine monoclonal antibodies, CIBr2, CIBr7, and CIBr18, were produced against the human pleomorphic breast carcinoma cell line PMC42. All three monoclonal antibodies reacted with previously undescribed antigenic determinants on the PMC42 cell line. Antibody CIBr18 reacted only with the immunizing cell line PMC42, whereas antibodies CIBr2 and CIBr7 showed minimal reactivity toward a panel of 34 human leukemia- and solid tumor-derived cell lines. The antigenic determinants detected by the three antibodies were distinct, and each showed variable expression in PMC42 monolayer and organoid cultures. The heterogeneity of staining seen on PMC42 cultures may reflect the fact that this cell line contains up to eight morphologically distinct cell types. Antigen expression correlated with cell type in some instances, whereas in other instances phenotypic subdivision within a cell type was apparent. Antigens recognized by antibodies CIBr7 and CIBr18 were characterized biochemically. In Western blotting, antibody CIBr7 identified a single band of an apparent molecular weight of 38,000 within PMC42 cell lysates. Sodium dodecyl sulfate-polyacrylamide gel analysis of polypeptides immunoprecipitated by antibody CIBr18 from [35S]methionine-labeled PMC42 cell lysates identified two glycoproteins of apparent molecular weights of 115,000 and 120,000, respectively. No biochemical data for the CIBr2 antigen are yet available. All three antigens were detected in human mammary epithelium and some non-breast tissues. The expression of these antigens in normal and neoplastic mammary epithelia is discussed in terms of antigen heterogeneity and changes in antigen expression upon conversion to the malignant state.     

Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene.

Two unique human signet ring cell gastric carcinoma cell lines (designated HSC-39 and HSC-40A) were established in vitro from the ascites of a 54-year-old male patient. Both cell lines were biologically quite similar, grew in vitro in suspension with a population doubling time of 28-30 h, and had cytological features of mucinous epithelial tumor cells. They formed colonies in soft agar, with a cloning efficiency of 0.8-1.0%. Ultrastructurally, numerous granules were observed in the cytoplasm, suggesting secretory activity. The frequent presence of desmosome and the tight junction at the cell boundary certifies the epithelial origin of the lines. Immunocytochemistry and radioimmunoassay showed production of tumor marker antigens (carcinoembryonic antigen, CA 19-9, and sialyl-Lex-i) and gastrin in both lines. These lines were transplantable in athymic BALB/c nude mice. The histopathology of each line growing in athymic BALB/c nude mice was similar to that of the original tumor. The karyotype of the cells was highly aberrant with structural and numerical changes. The presence of numerous double minute chromosomes and loss of the 13 chromosome and Y-chromosome characterize these lines. In addition, the amplified c-myc oncogene (16-32-fold) was found in both cell lines and original ascitic tumor cells. Overexpression of the c-myc mRNA was noted. These cell lines may be a useful tool, providing both in vivo and in vitro systems for further studies of the biology and therapy of human signet ring cell (or Borrmann's type IV carcinoma) gastric carcinoma.     

Establishment of a peripheral T-cell lymphoma cell line showing amplification of the c-myc oncogene

A new T-cell lymphoma cell line, designated T34, was established from freshly isolated lymph node tumor cells of a patient with non-Hodgkin's diffuse large cell lymphoma. The T34 cells, as well as the parental lymphoma cells, showed mature helper/inducer immunophenotypes in that they formed spontaneous sheep erythrocyte rosettes and reacted with OKT-3 and OKT-4 monoclonal antibodies. They were negative for OKT-6, OKT-8, terminal deoxynucleotidyl transferase, WT-1, and HLA-DR antigens. Molecular analysis revealed that the T34 cells contained 8- to 16-fold amplified c-myc DNA. The same genetic change was observed in parental lymphoma cells, indicating that the c-myc amplification had occurred in vivo. There was no gross rearrangement of the c-myc DNA. The c-myc gene of the T34 cell line was actively transcribed into normal-sized c-myc mRNA. Cytogenetic analysis showed that both the T34 and the parental lymphoma cells had a near-triploid karyotype with multiple structural chromosome changes. The terminal end of the long arm of chromosome No. 8, the chromosomal locus of single-copy c-myc, was elongated (8q+ chromosome), perhaps reflecting the site of c-myc amplification. These data suggested that amplification of the c-myc oncogene played some role in progression and proliferation of this peripheral T-cell neoplasm.     

A cell line from a human ovarian carcinoma with amplification of the K-ras gene

We have recently reported that ascitic cells from a human ovarian carcinoma have a 10- to 20-fold K-ras amplification and that the level of such amplification did not change over a 9-month period during which the patient received chemotherapy and underwent clinical progression. Here we describe an ovarian tumor cell line (HOC-8) which has been derived from that tumor and which also shows a similar level of K-ras amplification. The amounts of K-ras specific mRNA and the Mr 21,000 protein encoded by the amplified gene are correspondingly elevated. karyotypic analysis revealed no detectable double minute chromosomes but did show an abnormal banding region on chromosome 6. This cell line may represent a useful model to investigate the significance of the K-ras gene product for the pathogenesis of human tumors.     

In vivo amplification and rearrangement of c-myc oncogene in human breast tumors

We have studied the genomic organization of cellular myc (c-myc) proto-oncogene in 48 human primary breast tumors. Two types of alterations (amplification and rearrangement) were observed in 27 (56%) of the tumors studied. The c-myc proto-oncogene appeared to be amplified 2- to 15-fold in the DNA of 20 tumors (41%). Non-germ line c-myc-related fragments (rearrangements) of variable size were detected in 7 primary breast tumors (6 malignant, 1 benign); 4 of these tumors presented both rearrangement and amplification, and the other 3 presented rearrangement only. The majority of the tumors analyzed were invasive ductal adenocarcinomas; 58% of these showed c-myc locus genetic alterations. Although the c-myc alterations described here do not appear to correlate with the aggressive behavior of primary breast tumors, they seem to be associated with development of breast carcinoma.