Gene amplification in human cells may involve interchromosomal transposition and persistence of the original DNA region.

In tumor cells in vivo and in vitro the amplification of large DNA sequences is a spontaneous and frequently occurring genetic event. We have used human cells to study independent events leading to a low level of amplification of a single copy of an integrated plasmid. Fluorescence in situ hybridization, chromosome banding, and chromosome painting revealed that the new amplified DNA sequences can become located on chromosomes that are totally unrelated to the chromosome that harbors the original DNA sequences, indicating that the transposition of amplified DNA sequences is interchromosomal. In cells containing amplified DNA sequences the integrated single-copy plasmid remained at its original location. The unit of amplification contained a DNA fragment of at least a 800 kb and the same fragment was also present in the parental single-copy cell clone. The data suggest that a doubling of the DNA region at the original location precedes or is coupled to gene amplification.     

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.     

Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line.

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.     

DNA content and chromosome number of a heteroploid human tumour cell line.

In this paper investigations concerning the relation between variability of chromosome number and variability of DNA content within the cells of a tumour stemline are reported. A highly heteroploid human tumour cell line was used, which was derived from a chondrosarcoma. Flow cytometrical and scanning cytophotometrical measurements confirmed the heteroploid nature of the original cell line and of several subclones. Measurement of the DNA content per metaphase showed a linear relation between chromosome number and DNA content of heteroploid cells. This finding is discussed with regard to its implications for the mechanism of heteroploidy in tumour cells.     

Stable transfection of the oestrogen receptor gene into a human osteosarcoma cell line

The oestrogen receptor (ER) gene was introduced into an ER-negative osteoblast-like osteosarcoma cell line HTB 96 by transfection. A number of clones were isolated which expressed ER at levels of up to 70 fmol/mg cytosol protein as determined by immunoassay. Scatchard analysis of the binding of [3H]17 beta-oestradiol in cytosols demonstrated the presence of high affinity binding sites, with a dissociation constant of 0.08-0.13 nM at 4 degrees C. High levels of a 3 kb ER mRNA are produced by the clones, which have gene copy numbers ranging from 2 to greater than 10. Functional receptor activity has been demonstrated by co-transfection of a plasmid containing the chloramphenicol acetyl transferase (CAT) gene linked to an oestrogen response element. Induction of CAT activity is observed in the presence of added oestradiol and is concentration-dependent. The transfected ER is also able to affect endogenous cellular function as several ER-positive clones, but not HTB 96 cells, are growth inhibited by oestradiol in the concentration range 10(-9)-10(-7) M. These effects on growth are not induced by other classes of steroids and are reversible by antioestrogens. No endogenous genes have yet been identified which are oestrogen-regulated in ER-transfected clones.     

7-Hydroxymethotrexate formation in a human lymphoblastic cell line

7-Hydroxymethotrexate is a major metabolite of methotrexate in patients undergoing chemotherapy with this drug. It has now been demonstrated that when cultures of CCRF-CEM, a human lymphoblastic cell line, are incubated in the presence of 100 microM methotrexate, significant quantities of 7-hydroxymethotrexate are formed. This finding is relevant to clinical use of the drug since it is now clear that during the treatment of acute lymphocytic leukemia conversion of methotrexate to the relatively inactive 7-hydroxy derivative probably occurs not only in the liver but also in the target cells.     

DNA content and chromosome number of a heteroploid human tumour cell line

Summary In this paper investigations concerning the relation between variability of chromosome number and variability of DNA content within the cells of a tumour stemline are reported. A highly heteroploid human tumour cell line was used, which was derived from a chondrosarcoma.Flow cytometrical and scanning cytophotometrical measurements confirmed the heteroploid nature of the original cell line and of several subclones. Measurement of the DNA content per metaphase showed a linear relation between chromosome number and DNA content of heteroploid cells. This finding is discussed with regard to its implications for the mechanism of heteroploidy in tumour cells.Supported by grant no. 28-394 of the Praeventiefonds, 's-Gravenhage, The Netherlands     

Gene expression modulation is associated with gene amplification, supernumerary chromosomes and chromosome loss in antimony-resistant Leishmania infantum

Antimonials remain the first line drug against the protozoan parasite Leishmania but their efficacy is threatened by resistance. We carried out a RNA expression profiling analysis comparing an antimony-sensitive and -resistant (Sb2000.1) strain of Leishmania infantum using whole-genome 70-mer oligonucleotide microarrays. Several genes were differentially expressed between the two strains, several of which were found to be physically linked in the genome. MRPA, an ATP-binding cassette (ABC) gene known to be involved in antimony resistance, was overexpressed in the antimony-resistant mutant along with three other tandemly linked genes on chromosome 23. This four gene locus was flanked by 1.4 kb repeated sequences from which an extrachromosomal circular amplicon was generated in the resistant cells. Interestingly, gene expression modulation of entire chromosomes occurred in the antimony-resistant mutant. Southern blots analyses and comparative genomic hybridizations revealed that this was either due to the presence of supernumerary chromosomes or to the loss of one chromosome. Leishmania parasites with haploid chromosomes were viable. Changes in copy number for some of these chromosomes were confirmed in another antimony-resistant strain. Selection of a partial revertant line correlated antimomy resistance levels and the copy number of aneuploid chromosomes, suggesting a putative link between aneuploidy and drug resistance in Leishmania.     

Characterization of newly established human osteosarcoma cell line,LM-1

Summary The characteristics of Cell Line LM-1, established from a human osteosarcoma, have been studied extensively. The cell produced both bone-specific and placental-like alkaline phosphatases when treated with hydrocortisone 21-phosphate; they had specific membrane antigens that reacted with sera from osteosarcoma patients. Injection of LM-1 cells into newborn hamsters treated with antilymphocyte serum produced nodular tumors. The characteristics of LM-1 suggest that this tumor cell line has unique features that may be useful in a variety of studies of human and animal osteosarcoma. This research is supported in part by USPHS Grants HD-09938 and CA-25746.     

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.     

Gene amplification in methotrexate-resistant mouse cells. III. Interrelationships between chromosome changes and DNA sequence amplification or loss

The amplified DNA sequences within a single subline of methotrexate-resistant mouse cells have a complex intracellular location, some being associated with “double minute” chromosomes, and some with normal-sized chromosomes. However, within a cell line and probably within a cell their internal structure is stable and homogeneous. These results, and those from the previous paper (Bostock &; Tyler-Smith, 1981), suggest that gene amplification in these cells has two distinct stages. Firstly, an amplifiable unit is generated by a process involving DNA rearrangement. Secondly, the unit increases in number but remains unchanged in structure.     

Retroviral expression of the human IL-2 gene in a murine T cell line results in cell growth autonomy and tumorigenicity.

In mature T lymphocytes (T cells) the regulated expression of the genes for interleukin-2 (IL-2) and its receptor (IL-2R) constitutes an essential part in controlling the cell growth. Evidence has been provided which suggests the involvement of an aberrant function of the IL-2 system in developing T cell neoplasms, particularly the adult T cell leukemia/lymphoma (ATL). As an approach to examine the extent of the IL-2 system contribution to T cell neoplasms, we created the experimental conditions wherein both IL-2 and IL-2R are expressed constitutively in a murine T cell line. We made use of a retroviral vector to infect an IL-2-dependent CTLL-2 line and lead to the expression of human IL-2. Here, we show that the virus-infected cells not only proliferate in vitro in the absence of exogenously supplied IL-2 under certain conditions, but also develop tumors (lymphomas) in nude and syngeneic mice.