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.     

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.     

c-K-ras overexpression is characteristic for metastases derived from a methylcholanthrene-induced fibrosarcoma

We investigated the relationship between the activation of the c-myc and c-K-ras proto-oncogenes and the acquisition of metastatic potential in a methylcholanthrene-induced BALB/c fibrosarcoma. The murine fibrosarcoma GR9 was originally induced in BALB/c mice following exposure to the carcinogenic chemical 3-methylcholanthrene. To induce spontaneous metastasis, we used two tumor cell clones (B9 and G2) known to differ in their metastatic potential, local tumor growth, H-2 class I expression and sensitivity to natural killer (NK) cells. The metastatic nodes were obtained from the lung, liver and kidney. The results showed: (1) amplification of the c-myc proto-oncogene in original tumor clones as well as in all metastatic nodes; (2) mRNA overexpression without amplification of the K-ras proto-oncogene in the metastatic cells, regardless of their anatomical location; (3) no c-K-ras point mutations at codons 12 and 61, and (4) in general, a statistically significantly reduced in vitro sensitivity of metastatic tumor cells to NK cells as compared with the tumor clones used to induce them (p<0.05). These results therefore suggest that overexpressed c-K-ras mRNA is important during tumor progression, perhaps rendering metastatic tumor cells more resistant to lysis by NK cells.     

N-myc and c-myc oncogenes amplification in medulloblastomas. Evidence of particularly aggressive behavior of a tumor with c-myc amplification

N-myc and c-myc amplification was investigated in 27 medulloblastomas. DNA was extracted from 19 formalin fixed and paraffin embedded tumors and from fresh frozen tumor tissue in 8 other cases. The results showed no evidence of amplification of N-myc oncogene and only 1 case had a 27 fold amplification of c-myc. Cytogenetically, this neoplasm presented numerous double minute chromosomes (DMs). Moreover, it had an unusual rapidly aggressive course with massive cerebrospinal fluid dissemination unresponsive to intrathecal chemotherapy. Our results indicate a low incidence of N-myc and c-myc gene amplification in medulloblastomas, suggesting that the oncogenic mechanism in these neoplasms is not closely related to DNA gene amplification. C-myc amplification, although not frequently observed, may however provide a growth advantage for medulloblastoma cells in vivo, favoring their rapid dissemination. Medulloblastomas with c-myc activation may represent a subgroup of tumors with a more aggressive behavior.     

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM‐carried genes remains to be clarified. The human colorectal cancer cell line NCI‐H716 and human malignant primitive neuroectodermal tumor cell line SK‐PN‐DW are known to contain many DMs. To examine the amplification of DM‐carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI‐H716 and SK‐PN‐DW tumor cells. We identified the amplification regions and the DM‐carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM‐carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM‐carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM‐carried genes led to a decrease in the number of DMs and reduced the amplification of the DM‐carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM‐carried genes resulted in a decrease in cell proliferation and invasion ability.     

Amplification, expression and localization of the c-myc gene in BSp73 rat tumor cell lines

Metastatic (ASML 14-1, ASmv) and non metastatic (AS17-4) cell lines of the rat BSp73 pancreatic adenocarcinoma were investigated for amplification and expression of oncogene DNA. The c-myc gene was amplified, but only in one metastatic variant, ASML. The degree of amplification (3.5-fold) increased after prolongued in vitro cultivation (17.5-fold). All three tumor cell lines expressed c-myc and ras mRNA. Ras expression was at the same level as in rat liver. C-myc expression was considerably above the level in rat liver, but also differed considerably between the metastatic variants. In the metastatic ASML cells the c-myc gene was localized by in situ hybridization on a marker chromosome derived from chromosome 7. The karyotypes of the metastatic variants are different and have no common marker chromosomes. Our results obtained with the BSp73 tumor model do not support a role of the c-myc gene in the metastatic process.     

The role of chromosome 15 in murine leukemogenesis. II. Relationship between tumorigenicity and the dosage of lymphoma vs. normal-parent-derived chromosomes 15 in somatic cell hybrids

Somatic cell hybrids were generated between an MCF-virus-induced 15-trisomic T-cell lymphoma of AKR origin with a proviral insertion near the c-myc locus, and normal diploid fibroblasts or lymphocytes of CBAT6T6 origin. Three lymphoma/fibroblast fusions were performed. Six independently-derived clones from 2 fusions were tested for tumorigenicity. Three of the 6 clones were weakly malignant (take incidence 20% below), and 3 were strongly malignant (take incidence over 80%). All 3 lymphoma/lymphocyte hybrids and 6 derived clones were strongly malignant. All hybrids contained a nearly complete chromosomal complement of both parental cells. This was confirmed at the molecular level by determining the ratio of germ-line (G) vs. rearranged (R) myc-carrying Eco RI fragments that showed the expected 1.9-2.7:1 proportion. Malignant segregants selected from the weakly malignant lymphoma/fibroblast hybrids by in vivo inoculation showed changed 15-chromosome ratios. Four out of the 6 clones showed amplification of the lymphoma-derived 15-chromosome that carries the R-myc fragment and a concomitant decrease in the average number of the G-myc-carrying chromosomes. This was deduced from the fact that the G:R ratio was between 2 and 3:1 in the in vitro hybrids but became inverted (1:2-3) in the tumors. Two tumors showed no amplification of R-myc. G-myc was decreased. One of these tumors showed a change in the G:R ratio from 2.5:1.0 to 1.2:1.0, while the other was essentially unchanged (1.9:1.0 in the in vitro clone and 2.2:1.0 in the derived tumor). These findings support the notion that both the amplification of the lymphoma-derived 15-chromosome with the retrovirally rearranged c-myc carrying fragment and/or the loss of the G-myc-carrying 15-chr can contribute to the tumorigenic potential of the hybrids.     

Down-regulation of MHC class I antigens is not a general mechanism for the increased tumorigenicity caused by c-myc amplification

Previous studies have shown that increased expression of oncogenes from the myc-family can down-regulate the level of MHC class I antigens in tumor cells. This has suggested a mechanism by which amplification/overexpression of myc-genes may contribute to the malignancy development of certain tumors. Earlier published data from the murine SEWA tumor, a polyomavirus-induced osteosarcoma, have correlated the degree of tumorigenicity of different sublines to their level of c-myc amplification. Here I present a quantitative and qualitative analysis of MHC class I antigens from five sublines of this tumor system. No differences could be found, between sublines with different degrees of tumorigenicity, regarding MHC class I antigen expression. Thus, in the SEWA tumor, the enhancement of the tumorigenicity caused by c-myc amplification is not mediated through down-regulation of MHC class I antigens.     

Amplification of the c-myc oncogene during progression of radiation-induced rat skin tumors

Evaluation of a large panel of radiation-induced rat skin tumors of diverse size and histological type revealed a correlation between c-myc copy number and tumor size. Both the frequency and degree of c-myc gene amplification were increased in large compared to small carcinomas, but none of the sarcomas examined showed c-myc amplification. Serial biopsies of individual tumors exhibited similar trends of increasing c-myc copy number in later biopsies. In one regressing tumor, the c-myc gene copy number paralleled the growth rate of the tumor during growth and regression. The average time required from tumor appearance to significant gene amplification was close to the average period between tumor appearance and the onset of rapid growth. The data suggest that, rather than being a target gene for the direct early effects of ionizing radiation, c-myc functions as a late-stage progression-related oncogene in this model system.     

Differentiation of F9 cells is independent of c-myc expression.

The level of c-myc expression rapidly decreases during in vitro induced differentiation of mouse F9 embryonic teratocarcinoma to endoderm cells, raising the question of whether down regulation of c-myc expression is a part of the mechanism regulating differentiation. We have investigated the effect of enforced increases or decreases in c-myc RNA expression in F9 cells on growth and differentiation. The enforced expression of c-myc RNA in clones transfected with sense c-myc did not inhibit their terminal differentiation. Dramatically decreased c-myc RNA expression in antisense c-myc transfected clones also did not substantially alter the differentiation pathway, although the transformed cells withdraw from the cell cycle slightly earlier than control cells during the differentiation induction. These results suggest that the mechanism controlling differentiation operates independently of the level of c-myc RNA expression in F9 teratocarcinoma cells.     

Evidence for selection of homogeneously staining regions in a human melanoma cell line

Cytogenetic analysis of tumor cells from a human malignant melanoma was performed on both the primary tumor colony-forming cells and a cell line (HA-A) established subsequently from the clonogenic population. Chromosome-banding analysis demonstrated identical karyotypic alterations in both the tumor colony-forming cells and the HA-A cell line, documenting their origin from a common precursor. The most distinctive chromosome alterations shared between tumor colony-forming cells and the HA-A cell line were double-minute bodies (DMs) and a homogeneously staining region (HSR) on chromosome 7 at band p22. This represents the first observation of DMs or HSRs in cells from a human malignant melanoma. The frequency of HSR-bearing cells observed in the original tumor was less than 1%, while DM-containing cells were present in greater than 90% of all cells examined. In contrast, serial chromosome harvests at early passage of the HA-A cell line revealed positive selection for HSR-bearing cells with concomitant loss of DM-containing cells after increasing time in vitro. Following the ninth serial passage in vitro, HSRs were observed in 100% of cells with DMs no longer observed in the HA-A cell line. The finding of an HSR-bearing marker in the original tumor sample supports the view that HSRs are not an artifact of in vitro monolayer growth. However, our results demonstrate that the frequency of HSR-bearing cells within established cell lines may reflect in vitro selection and therefore not accurately reflect the frequency of this population in vivo.     

A new myeloblastic leukemia cell line with double minute chromosomes. Induction of methotrexate resistance and dihydrofolate reductase gene amplification.

To test the relationship between DMs and drug resistance in newly established AML cell lines, KY821, and its clone KY821A3, the latter had lost DMs during cloning, were cultured in increasing concentrations of MTX. KY821 became resistant against 2 x 10(-4) M MTX, whereas KY821A3 did against 2 x 10(-5) M MTX in a same period. Enhanced enzyme activities of DHFR were correspondent to the increased DMs numbers and DHFR gene amplification in both resistant clones. The amplified DHFR gene was located on DMs by in situ hybridization. These data indicated that the presence of DMs in KY821 would facilitate the acquisition of drug resistance.     

Frequent incidence of double minute chromosomes in cancers, with special up-to-date reference to leukemia

Double minute chromosomes (DMs) are small chromatin bodies consisting of gene amplification in an extrachromosomal location. Although found in an variety of human tumor cells, their presence in hematologic malignancies is rare and their role in leukemogenesis is controversial. However, they are thought to be involved in tumorigenesis and in drug resistance, representing a mechanism for upregulated oncogene expression generally associated with a poor prognosis. The presence of DMs has been associated with a rapid disease course, low response rate, and short survival. Little knowledge is, however, available on DMs in leukemias. To elucidate this issue, a web-based search for all types of articles published was initiated using MEDLINE/PubMed, the Mitelman database and other pertinent references on websites. We found that DMs have the highest frequency in adrenal carcinoma (28.6%), and lowest rate noted as 2.6% for large intestine. The large Mitelman database and other web based pertinent reports provide novel knowledge of DMs and their association in the wide field of cancers.