Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line

We are exploring the origin and function of amplified DNA sequences associated with double minutes (DMs) in a spontaneously transformed derivative of mouse 3T3 cells. Toward that goal, we have constructed a cDNA library using RNA from these cells and have isolated cDNA clones representing sequences that are amplified and overexpressed in these 3T3-DM cells. From results of Northern- and Southern-blot analyses, we conclude that these cDNAs represent two distinct genes, which we have designated mdm-1and mdm-2.Using DNAs from a panel of Chinese hamster-mouse somatic cell hybrids together with in situ hybridization protocols for gene mapping studies, we have found that these DM-associated, amplified DNA sequences originate from mouse chromosome 10, region C1–C3. Sequences homologous to mdm-1and mdm-2are present in the genomes of several species examined, including that of man.     

N-myc amplified in retinoblastoma cell line FMC-RB1

A recently described retinoblastoma cell line, FMC-RB1, showed a 16-fold N-myc oncogene amplification. The patient from whom the cell line was obtained died from an aggressive disease. It is suggested that N-myc amplification may be an adverse prognostic indicator.     

Binding of EGF peptide and EGF receptor antibodies and its fragments in different tumor models

Binding of epidermal growth factor (EGF) to the EGF receptor is known to trigger a number of biological responses in the target cells including EGF receptor phosphorylation and stimulation of DNA synthesis leading to cell proliferation. Agents that bind to the EGF receptors could have a significant role in the therapy of tumors that express increased numbers of receptors by blocking the stimulatory effect of EGF. Different monoclonal antibodies (MAbs) directed to the EGF receptor have been generated that inhibit EGF binding and do not induce activation of the receptor tyrosine kinase. When there is sufficient uptake these antibodies can be used for immunotherapy and, after labeling with an appropriate radionuclide, also for radioimmunotherapy. For evaluation of a ligand as a therapeutic agent, it is necessary to investigate its binding characteristics in tumor cells and experimental tumors in vivo. Because the effectiveness of the antitumor activity of the MAb is dependent upon the amount of receptors in the tissue and the penetration of the MAb into the tissue, the receptor density, biokinetics, and tumor distribution of the MAb or its fragments were evaluated in different tumor models. The results of the experimental studies with tumor cell spheroids and different xenotransplanted human tumors have shown that the uptake and distribution in the tumor tissue is dependent on the molecular weight of the ligand. The correlation between the uptake of the substances and the receptor density is an indication for a noninvasive scintigraphic characterization of human tumors using radiolabeled compounds with specific binding to the tumor receptor and for selection of an optimal therapeutic regimen or radionuclide targeting of the tumor.     

Simultaneous analysis of chromosomal aneusomy and 5-bromodeoxyuridine incorporation in MCF-7 breast tumor cell line.

We describe a new method to determine simultaneously both proliferative status and chromosome copy number within individual interphase cells. The MCF-7 human breast cancer cell line was used as a model system to characterize proliferative activity in karyotypically defined cell subpopulations. Cells were labeled with bromodeoxyuridine (BrdU) in vitro and incorporation was monitored with IU4 mouse anti-BrdU. Biotinylated, digoxigenin-labeled, or acetylamino-fluorene-conjugated repetitive sequence DNA probes were hybridized to target interphase nuclei. Three-color fluorescence labeling allowed simultaneous detection of two chromosome pairs and designation of cells undergoing DNA synthesis. This technique may also be used for simultaneous characterization of proliferative and karyotypic heterogeneity in primary human tumors.     

Cloning and chromosomal location of human genes inducible by type I interferon

When cells are treated with interferon several new proteins are induced. We have isolated by differential screening two cDNA clones corresponding to human genes inducible by IFN-, termed IFI-4 and IFI-54K. The accumulation of the corresponding mRNA was followed as a function of either IFN dose or of time. The IFI-4 and IFI-54K genes, as well as two previously isolated IFN-inducible genes, namely the IFI-56K and low-molecular-weight 2–5A synthetase, were localized on the human chromosomes. Using cloned probes on Southern blots of DNA from a panel of rodent-human somatic cell hybrids, we have assigned the IFI-4 gene to chromosome 1 and the gene coding for the low-molecular-weight 2–5A synthetase to chromosome 12. We also showed that the IFI-54K and IFI-56K genes, unlike most of the IFN-inducible genes, are syntenic. They are both located on chromosome 10. In addition, evidence is given for the presence of a pseudogene homologous to IFI-56K on chromosome 13.     

Rearrangements of chromosomal regions containing ribosomal RNA genes and centromeric heterochromatin in the human melanoma cell line MeWo

A chromosomal examination of cells from the earliest available passage of the human melanoma cell line MeWo revealed the presence of seven hypodiploid cell types that shared common complex marker chromosomes. Two of the cell types had long homogeneously staining regions (HSR) by Q-banding on three different chromosomes. Distamycin A/DAPI staining and silver staining for active nucleolar organizing regions (NOR) confirmed that the HSR were derived from chromosome #15. All HSR-containing cells had 4-9 pairs of large NOR distributed along the length of each HSR, with all acrocentric chromosomes being negative. The HSR-lacking cells differed primarily with respect to the morphology of the short arm of one #13 chromosome and NOR activity. One cell type had four chromosomes with active NOR, whereas all other cell types had a single active NOR on one #13. One of these cell types had a satellited #8 with NOR. Cells from three other MeWo cultures at higher passages were examined. Two of these contained both hypodiploid and hypotetraploid cells, some of which had satellited X chromosomes or satellited #3 chromosomes with active NOR. The majority of the new chromosomal rearrangements in cells from the later cultures involved the NOR-containing regions, many of which were associated with the distamycin A/DAPI-positive centromeric heterochromatin from chromosome #15. These results indicate that the chromosomal instability in the MeWo cultures is mainly limited to sequences containing active NOR and centromeric heterochromatin from chromosomes #13 and #15. This may be due to a selective pressure to increase the number of active NOR in the MeWo cells. If this is so, it would appear that amplification of active NOR occurs more readily than the activation of the many silent NOR present in these cells.     

A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.     

EGF receptor variant III as a target antigen for tumor immunotherapy

The EGF receptor (EGFR) is the first tyrosine kinase receptor ever cloned and remains at the forefront of targeted therapies against cancer. Currently, there are four US FDA-approved drugs and several more in Phase III studies that target the EGFR. These drugs, while resulting in some dramatic remissions, have not resulted in strong nor consistent improvements in survival. EGFR variant III (EGFRvIII) is the most common variant of the EGFR and is present in many different cancer types but not in normal tissue. It results from the fusion of exon 1 to exon 8 of the EGFR gene, which results in a novel glycine at the junction. This mutant receptor is constitutively active in these tumors and can lead directly to cancer phenotypes due to its oncogenic properties. EGFRvIII is an attractive target antigen for cancer immunotherapy because it is not expressed in normal tissue and because cells producing EGFRvIII have an enhanced capacity for dysregulated growth, survival, invasion and angiogenesis. In this review, we will discuss preclinical and clinical data from studies using EGFRvIII as the target antigen for immunotherapy, with a focus on the potential for greatly improved survival for patients diagnosed with glioblastoma multiforme.     

Laryngeal granular cell tumor; rare location

Granular cell tumors are benign subcutaneous or submucosal lesions of neurogenic origin. In this case study one patient was diagnosed and treated successfully with complete surgical resection of a laryngeal granular cell tumor that was originated from the left arytenoid region that very rare location. There is no evidence of recurrence 2 years after surgery. Granular cell tumors should be considered in the differential diagnosis of laryngeal masses, particularly in the posterior glottis.     

Instability of immunoglobulin genes in S107 cell line

Somatic mutation occurs frequently in rearranged and expressed immunoglobulin variable region genes in vivo. In contrast, V region hypermutation seldom occurs in antibody-forming cells in culture. The S107 mouse myeloma cell line is one of the few cell lines that has been observed to generate V region mutations frequently and spontaneously in vitro. Detailed examination reveals that both the S107 tumor and the cell line derived from it contain and express a duplicated heavy-chain gene. In culture, only one of the two heavy-chain genes undergoes both V and C region mutation, and variants with complex phenotypes and genotypes arise as a result of mutation and segregation of these duplicated genes.     

The action of epidermal growth factor (EGF) is limited to specific phases of the cell cycle in an EGF dependent colonic cell line

In the presence of epidermal growth factor (EGF) a human colon cell line, LIM 1215, proliferates in serum-free medium. Under these culture conditions the cells are dependent on the presence of EGF for both proliferation and survival. In order to study the action of growth factors at different stages of the LIM 1215 cell cycle, pure populations of G1, S and G2/M cells were obtained by cell sorting after supravital staining of the DNA with Hoechst 33342. Conditions were established for Hoechst 33342 staining which produced satisfactory DNA histograms and greater than 80% survival of cells. The kinetics of passage for sorted S or G2/M cells into G1 were not affected by EGF or fetal calf serum. After sorting there appeared to be a 4 h delay before the cells proceeded in the cell cycle. Sorted S cells entered G2 over an 8 h period and maintained this same transition period from G2 into G1. If EGF or serum was present, these cells then re-entered the cell cycle after a variable delay and in an asynchronous manner. EGF was applied to S phase and G2/M phase LIM 1215 cells for periods of 2-10 h at various times after replating in serum-free conditions. Cells in S phase only responded to EGF as they passed from G2/M into G1. Exposure to EGF in S phase resulted in little growth stimulus once the cells returned to G1. For cells in G2/M phase, EGF was required immediately to give the maximum stimulus for re-entering the cell cycle. If the EGF was delayed for more than 8 h, the cells did not re-enter the cycle within the following 20 h. Exposure to EGF for less than 2 h failed to stimulate proliferation. These results indicate that EGF must be present as cells enter G1 from mitosis. Once the cells have entered G1, EGF is required for a 10 h period for a large number of cells to re-enter the cycle from G1.     

T cell receptor genes in rheumatoid arthritis

Conclusion Rheumatoid arthritis is an oft debilitating chronic disease of an autoimmune nature. Although the putative antigen remains unknown, the recent elucidation of the structure and functional relationships of the trimolecular complex governing the immune response to antigen, facilitates the development of novel approaches to the treatment of RA and enhances our understanding of the etiopathogenesis of this disorder and autoimmunity in general. The characterization of immune active T cell populations, particularly at inflammatory sites such as the synovium, with respect to their expressed and genomic TCR repetoire, may permit the identification of crucial genetic components which contribute either directly or indirectly to the susceptibility and/or the development of RA.Abbreviations CIA collagen-induced arthritis - IL-2 interleukin-2 - MHC major histocompatibility complex - RA rheumatoid arthiritis - RFLP restriction fragment length polymorphism - TCR T cell receptor - RR relative risk     

The G401 cell line, utilized for studies of chromosomal changes in Wilms' tumor, is derived from a rhabdoid tumor of the kidney.

The histology, ultrastructure, and messenger RNA expression of heterotransplants derived from the G401 cell line (American Type Culture Collection) have been characterized by comparison with Wilms' and rhabdoid tumors of the kidney. This analysis illustrates that the properties of G401 heterotransplant were consistent with a rhabdoid phenotype rather than that of a Wilms' tumor. The G401 cell line has been utilized in recent experiments to demonstrate the central role of chromosome 11 in Wilms' tumor. However, the present results suggest that these experiments may be more relevant to define the involvement of chromosome 11 in rhabdoid tumor of the kidney, a malignancy distinct from Wilms' tumor. This is clinically relevant since the rhabdoid tumor of the kidney is very aggressive and associated with an extremely poor prognosis.     

Caveolae participate in tumor necrosis factor receptor 1 signaling and internalization in a human endothelial cell line

Caveolae are abundant in endothelial cells (ECs) in situ but markedly diminished in cultured cells, making it difficult to assess their role in cytokine signaling. We report here that the human EC line EA.hy926 retains an abundant caveolar system in culture. Tumor necrosis factor (TNF) receptor 1 (TNFR1/CD120a) was enriched in caveolae and co-immunoprecipitated with caveolin-1 from caveolae isolated from these cells. To further investigate the role(s) of caveolae in TNF signaling in ECs, cells were treated with methyl-beta-cyclodextrin to disrupt caveolae. Methyl-beta-cyclodextrin did not alter total cell surface expression of TNFR1 or TNF-induced degradation of IkappaBalpha, a measure of nuclear factor-kappaB activation, but it did inhibit TNF-induced phosphorylation of Akt, a measure of phosphatidylinositol-3 kinase activation. Serum-induced phosphorylation of AKT was unaffected. Treatment with TNF induced disappearance of TNFR1 from caveolae and dissociation from caveolin-1 within 5 minutes. In contrast to transferrin receptor, internalized TNFR1 did not co-localize with clathrin, except possibly in the Golgi, at any time point examined. By 60 minutes of treatment with TNF, TNFR1 appeared in endosomes. We conclude that caveolae function in ECs to allow TNFR1 to activate phosphatidylinositol-3 kinase and Akt, perhaps through receptor cross talk, and that ligand-induced internalization and trafficking of TNFR1 to endosomes may originate directly from this compartment.     

Differentially expressed genes in giant cell tumor of bone

Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.     

Genetic exchange in Trypanosoma brucei brucei: variable chromosomal location of housekeeping genes in different trypanosome stocks.

A Trypanosoma brucei brucei clone from West Africa was crossed with another T. b. brucei clone from the East African kiboko group. This group is defined by characteristic isoenzyme patterns and kinetoplast DNA maxicircle polymorphisms, and is associated with a wild animal-tsetse transmission cycle. Three types of clone were isolated from the cross, 2 of which were hybrid. The hybrids were heterozygotic at 7 loci where the parents were homozygotic and the hybrids also had molecular karyotypes different from those of both parents. Both molecular karyotypes had an extra non-parental band, which was shown to have a different origin in the 2 sets of clones by Southern analysis with various housekeeping gene probes. This analysis also revealed that although the GPI and PGK genes reside on the same chromosome in parent J10, they are on different chromosomes in parent 196. Hybridisation of PFG blots carrying a variety of other trypanosome stocks confirmed that the GPI gene is not always in the same linkage group as the PGK gene cluster. Given that genetic exchange in trypanosomes involves meiosis, such differences in gene linkage will give rise to progeny with incorrect gene dosage, i.e., certain crosses will be partially infertile. This incipient speciation may explain why natural populations of T. brucei spp. are observed not to be in a randomly mating equilibrium.