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)     

High c-myc amplification level contributes to the tumorigenic phenotype of the human breast carcinoma cell line SW 613-S

The c-myc gene is amplified in the SW 613-S cell line which was established from a human breast carcinoma. This 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. Clones with different levels of amplification and different cytological 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 were highly tumorigenic in nude mice whereas those with a low level were not. Introduction of c-myc gene copies by transfection into the cells of several non-tumorigenic clones restored the tumorigenic phenotype. Our results indicate that a high level of amplification of the c-myc gene is a requirement for the tumorigenicity of SW 613-S cells in animals.     

The role of transforming growth factor alpha production and ErbB-2 overexpression in induction of tumorigenicity of lung epithelial cells.

Over-expression of erbB-2 is associated with shortened survival of patients with lung adenocarcinomas. We demonstrated that human lung epithelial cells, overexpressing erbB-2, formed tumours in nude mice only when high levels of transforming growth factor (TGF)-alpha were produced (E6T cells). To define the role that TGF-alpha production played in induction of tumorigenicity, a non-tumorigenic TGF-alpha-negative clone of ErbB-2 overexpressing cells (E2 cells) was transfected with an expression vector for TGF-alpha (E2alpha cells). Transfected clones produced TGF-alpha at 11-25% of the level produced by the E6T cell line. Tumorigenic E6T cells transfected with a TGF-alpha antisense vector (E6TA cells) expressed only 6% of the TGF-alpha level of the parental cells. Clones of E6T, E6TA, E2 and E2alpha were inoculated into athymic nude mice to measure tumorigenic potential. E6T cells formed tumours with a 70% efficiency. E2, E6TA and E2alpha cells failed to form tumours. The levels of EGFR were similar in non-tumorigenic E2 and tumorigenic E6T cells but higher in E2alpha and E6TA cells, and ErbB-2 were greatly overexpressed in an E2alpha clone. In vitro, ErbB-2 co-immunoprecipitated with EGFR in lysates of unstimulated E6T and E2alpha TGF-alpha-producing cells, indicating that the lower TGF-alpha levels were sufficient to induce in vitro heterodimerization. These studies suggest that induction of the tumorigenic phenotype depends on achieving a threshold level of TGF-alpha sufficient to activate downstream signalling by ErbB-2 containing active heterodimers.     

An in vitro fibroblast model system to study myc-driven tumour progression.

We have utilized fibroblast cell lines to investigate myc-mediated cell transformation and tumour progression. Deregulated myc alleles were introduced into the rat fibroblast cell line, Rat-1, and a partially-transformed derivative of this cell line termed Rat-1 (PT). A human c-myc gene coupled to the Moloney murine leukemia virus long terminal repeat was introduced into both cell lines by transfection. The avian MC29 v-myc gene was introduced into the Rat-1 cell line by retroviral infection using a Moloney murine leukemia recombinant retrovirus. For both cell lines, the introduction of exogenous myc genes resulted in an increased degree of transformation. For the non-tumorigenic Rat-1 cell line, this also resulted in the acquisition of tumorigenicity, while for the Rat-1 (PT) cell line the degree of tumorigenicity was increased. Various clones were isolated and, for both human c-myc and avian v-myc, the level of myc expression correlated with the degree of transformation and the tumorigenic potential of the cell lines. In addition, both these parameters could be increased by passaging through syngeneic recipients. Our data show that tumour progression may be driven by the deregulated expression of myc genes; that transformation and tumorigenicity correlate with the level of exogenous myc expression; that additional events involving both in vitro and in vivo selection are involved in this process; and that myc expression may increase the cells' metastatic capacity.     

Overexpression of c-K-ras, c-N-ras and transforming growth factor {beta} co-segregate with tumorigenicity in morphologically transformed C3H 10T1/2 cell lines

Morphologic transformation and tumorigenicity are separate cellularphenotypes in transformed 1OT1/2 cells. We have investigatedthe levels of expression of genes for c-myc, c-H-ras, c-K-ras,c-N-ras, TGFß and Rb in 42 morphologically transformed1OT1/2 cell lines, in an attempt to define the molecular mechanisntsgoverning morphologic transformation and tumorigenicity in the1OT1/2 cell system. The 101 1/2 cell lines investigated generallyoverexpressed mRNAs for c-myc, c-H-ras, and TGFß relativeto the levels expressed by wild- type 10T1 cells (levels ofexpression >1.5-fold that of wild- type 1011/2 cells). Incontrast, only half of these cell lines overexpressed mRNAsfor c-N-ras and/or Rb relative to wild- type 1OT1/2 cells, andonly 25%; overexpressed c-K-ras mRNA. The mean levels of mRNAexpression for each of c-K-ras, c-N-ras and TGFß genesin tumorigenic cell lines were significantly greater than themean levels of expression in non-tumorigenic cell lines, suggestingan association between twnorigenicity and the levels of expressionof these specific genes. In contrast, levels of expression forc-myc, c-H-ras and Rb genes were not correlated with tumorigenicity.Cell lines that coexpressed high levels of c-K-ras, c-N-rasand TGFß genes were likely to be tumorigenic (11/12cell lines were tumorigenic), whereas cell lines that coexpressedlow levels of these genes were unlikely to be tumorigenlc (1/10cell lines were tumorigenic). High expression of TGFßwas sufficient for tumorigenicity in the absence of high levelsof expression of c-K-ras and c-N-ras (5/5 cell lines were tumorigenic).Elevated expression of either c-K-ras or c-N-ras alone was Insufficientfor tumorlgenlclty, however, coordinate overexpresslon of bothc-K-ras and c-ras was associated with tumorigenicity irrespectiveof the expression status for TGFß (13/15 cell lineswere tuinorigenic). These results suggest that overexpresslonof c-ras, c-H-ras and TGFß are conunonly associatedwith, and possibly mechanistically related to, the process ofmorphologic transformation in 1OTI/2 cells. In addition, theseresults suggest that progression from morphologic transformationto tumorigenicity in 1011/2 cell lines is frequently accompaniedby overexpression of c-K-ras and c-N-ras, and by enhancementof the level of overexpresslon of TGFß     

Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes on in vitro transformation of human mammary epithelial cells.

MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-alpha (TGF-alpha) gene in MCF-10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF-10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-10A Ha-ras and MCF-10A TGF-alpha cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-10A TGF-alpha/c-erbB-2 (MCF-10A TE) and 10 MCF-10A Ha-ras/c-erbB-2 (MCF-10A HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-10A TE and MCF-10A HE clones expressed a 10-fold to 40-fold increase in p185 erbB-2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF-alpha compared with MCF-10A cells. Moreover, both MCF-10A TE and MCF-10A HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-10A Ha-ras, MCF-10A c-erbB-2, or MCF-10A TGF-alpha clones. However, neither MCF-10A TE nor MCF-10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erbB-2, and TGF-alpha genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.     

Elevated expression of an exogenous c-myc gene is insufficient for transformation and tumorigenic conversion of established fibroblasts

Two established rat fibroblast lines, differing only by their number of generations in culture, show dramatically different responses to the elevated c-myc expression delivered by an efficient murine c-myc retrovirus vector. Thus, a late passage (60 generation) FR3T3 line acquires a transformed and tumorigenic phenotype upon introduction of this activated c-myc gene as indicated by its altered morphology, high efficiency of focus formation, soft agar clonability, saturation density in monolayer culture, and short latency of tumorigenicity in syngeneic hosts. Remarkably, none of these characteristics, except for an increased refractility in monolayers and an epidermal growth factor (EGF)-dependent agar clonability, were observed in a variety of early passage (10 generation) FR3T3 c-myc clones. BALB/c A31 fibroblasts transfected with this c-myc retroviral vector behaved essentially the same as the FR3T3 early line except for their inability to grow in suspension in response to EGF. However, transformation and tumorigenic conversion of each of these three fibroblast lines was achieved by an activated ras oncogene. Hence, elevated c-myc expression is insufficient for transformation of established fibroblasts but depends upon other acquired cooperating functions which are not necessary for ras induced transformation. We also demonstrate that endogenous c-myc expression remains unaffected even in clones expressing a 100-fold excess of exogenous c-myc RNAs demonstrating that c-myc autoregulation is not operative in these cells.     

Characterization of novel cell lines established from three human oral squamous cell carcinomas

Human oral squamous cell carcinoma cell lines (KOSCC-11, -25A, -25B, -25C, -25D, -25E, -33A, and -33B) were established by explantation culture from these oral squamous cell carcinomas. The histopathology of the primary tumors, in vitro growth characteristics, epithelial origin, in vitro anchorage-independency, in vivo tumorigenicity, the frequency of human papillomavirus (HPV) infections, and the status of proto-oncogenes, tumor suppressor genes, DNA mismatch repair genes, and microsatellite instability were investigated in the cell lines. KOSCC-11 is a well-differentiated oral squamous cell carcinoma (OSCC) derived from mandibular gingiva. KOSCC-25A, -25B, -25C, -25D, and -25E cell lines were derived from the same OSCC. KOSCC-33A and -33B were established from the same tumor that originated from the maxillary sinus. All tumor lines studied grew as monolayers and showed: i) epithelial origin by the presence of desmosome and keratin; ii) in vitro anchorage-independent growth ability; and iii) tumorigenic potential in nude mice. The cancer cell lines did not contain HPV DNA and did not express viral genes. Northern blot analysis revealed: i) overexpression of EGFR in four cell lines, ii) overexpression of c-H-ras in four cell lines, iii) overexpression of c-myc in three cell lines, iv) decreased expression of TGF-alpha in seven cell lines, and v) decreased expression of c-jun in five cancer cell lines compared with normal human oral keratinocytes. In all KOSCC cell lines and their corresponding tumor tissues, mutations were identified in highly-conserved functional regions of the p53 gene. The KOSCC-11 cell line contained a frameshift mutation and the other cell lines harbored an identical p53 mutation at codon 175 from CGC (Arg) to CTC (Leu). In five cell lines, a significant reduction of p21WAF1/Cip1 protein was evident. Cancer cell lines expressed higher level of Rb protein than normal human oral keratinocytes. DCC, a tumor suppressor gene, was not detected in KOSCC-25C. The KOSCC-33A cell line displayed microsatellite instability and showed a loss of hMSH2 expression. These well-characterized human OSCC cell lines should serve as useful tools for understanding the biological characteristics of oral cancer.     

Immediate early genes induced by H-Ras in thyroid cells

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5(Ras)) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5(MEK/Rac)) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5(Ras), however, FRTL-5(MEK/Rac) cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5(Ras) cells and differentiated and non-tumorigenic FRTL-5(MEK/Rac) cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells.     

Genetically modified tumour vaccines

Two genes, the gene coding for IL-2 and the gene encoding the CD80 molecule, were inserted into murine sarcoma MC12 cells. Tumorigenicity of a variety of cell clones with different expression of the inserted genes was assessed. Most of the genetically manipulated MC12 cell clones were less tumorigenic than the parental MC12 cell population. Tumorigenicity of the clones declined with increasing production of IL-2 as well as with the increasing expression of the CD80 molecule. When the tumorigenicity of the clones carrying an inserted IL-2 gene was compared with that of the clones carrying an inserted CD80 gene, it was found that the insertion of the IL-2 gene suppresses tumorigenicity more efficiently than insertion of the CD80 gene. Admixture of the IL-2-producing MC12 clones to the tumorigenic CD80(+) MC12 cell doses could completely inhibit the tumorigenicity of the CD80(+) cells. Insertion of the CD80 gene into sarcoma cells substantially enhanced the adhesive interaction between the MC12 sarcoma and syngeneic T lymphocytes.