Biomedical subjects
Publications and source records attributed to B A Spengler.
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Previous studies of the human neuroblastoma cell line SK-N-SH had demonstrated the presence of and phenotypic interconversion (transdifferentiation) between two morphologically and biochemically distinct cell types: N (neuroblastic) cells with properties of noradrenergic neurons and S (substrate-adherent) cells with properties of melanocytes. Current studies have sought to test the generality of these findings among other cultured human neuroblastoma cell lines and to define further the S-cell phenotype and that of a newly identified, morphologically intermediate, I-type cell. Morphologically homogeneous populations (clonal sublines or subpopulations) of N, S, and I cells were isolated from five additional neuroblastoma cell lines and analyzed biochemically for neuronal, glial, and melanocytic marker enzyme activities and norepinephrine uptake. Immunoblot techniques were used to detect intermediate filament proteins (neurofilament protein, vimentin, glial fibrillary acidic protein) and fibronectin. All N-type cells exhibited neuronal marker enzyme activities, specific uptake of norepinephrine, and presence of one or more neurofilament proteins. S-type cells generally lacked neuronal characteristics but contained, instead, tyrosinase activity (a melanocytic marker enzyme), vimentin, and fibronectin. This combination of attributes is suggestive of a multipotent embryonal precursor cell of the neural crest. I-type cells differentially expressed both S- and N-cell properties and could represent either a stem cell or an intermediate in the transdifferentiation process. Studies of the biological significance of human neuroblastoma cell transdifferentiation and the molecular mechanisms underlying this process may be of relevance to the biological and clinical behavior of this tumor in the patient.
Six independently derived multidrug-resistant Chinese hamster cell lines selected with vincristine, daunorubicin, actinomycin D, or colchicine were probed by in situ hybridization techniques with the cloned cDNA, p5L-18, to chromosomally localize known or presumed amplified P-glycoprotein genes. One or two clusters of amplified genes were demonstrable in all of the highly resistant sublines and were localized to homogeneously staining regions and/or abnormally banding regions, gene amplification-associated cytogenetic abnormalities, on eight different chromosomes. Analysis of trypsin-Giemsa banded karyotypes revealed additional, multiple chromosomal rearrangements that were apparently nonspecific. Mapping studies localized the native P-glycoprotein gene(s) to the region q23 to 31 (most probably band 26) on the long arm of chromosome 1 of normal Chinese hamster bone marrow fibroblasts and normal chromosome 1 homologues in resistant cells. Southern blot analysis of restriction endonuclease fragments indicated the amplification of one or both of (at least) two wild-type nonallelic genes in four of the lines and the presence in one line (DC-3F/DMM XX) of a unique 5.0-kilobase BamHI fragment resulting from a recombinational event during amplification. Comparison with the cytogenetic data indicated no correlation between restriction patterns generated by EcoRI, HindIII, PstI, or BamHI and chromosomal location of amplified genes. However, the only sublines in which the homogeneously staining region or abnormally banding region is positioned at 1q26 (at or near the site of the native gene) exhibit either alterations in gene structure (DC-3F/DM XX) or in regulation of gene expression (DC-3F/AD X), suggesting a process more complex than simply amplification of the gene in loco.
Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.
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Two morphologically distinct types of cells have been observed in cultures of human neuroblastoma cells. One, designated N-type, is composed of small neuroblast-like cells; the other, designated S-type, is composed of large, substrate-adherent cells. To obtain additional information on the nature of these two phenotypes, we have investigated the collagen biosynthesis in several clones of human neuroblastoma cells with N-type, S-type, or mixed morphology, using acrylamide gel electrophoresis, ion exchange chromatography, and Northern and Southern blots. A direct correlation between the proportion of cells with S-type morphology and the amount of collagen secreted was observed, with the largest amount of collagen being produced by clones composed exclusively of S-type cells. Type I trimer and type III collagens were the two major isotypes secreted by these cells. The absence of secretion of the alpha 2(I) collagen chain was associated with absence of RNA coding for this protein chain. The pro-alpha 2 gene of type I collagen was found highly methylated in these cells. Our data indicate that the presence of S-type cells in neuroblastoma cultures represents a differentiation of these neural crest-derived neuroblastic cells into glial cells such as Schwann cells.
Sorcin (soluble resistance-related calcium-binding protein), an acidic (pI = 5.7) protein (Mr approximately 20 kDa) previously designated V19, was originally identified in cells selected for high levels of resistance to vincristine. Two-dimensional gel electrophoresis and/or Western blot techniques now show sorcin to be overproduced in cells selected for resistance to actinomycin D (QUA/ADj), colchicine (CHRC5), and adriamycin (BE(2)-C/ADR). Not all cell lines selected for resistance to these drugs overproduced sorcin; e.g. cells of an independently selected actinomycin D-resistant subline of QUA, QUA/ADsx, did not contain increased amounts of sorcin. Sorcin was purified by preparative gel electrophoresis from QUA/ADj cells and used to generate specific antiserum in chickens. By Western blot analyses the antiserum was shown to recognize sorcin in QUA/ADj and in vincristine-resistant mouse and Chinese hamster lung, colchicine-resistant Chinese hamster ovary, and adriamycin-resistant human neuroblastoma lines. Low level expression of the protein was detectable in control, drug-sensitive cells. Direct binding assays with 45Ca2+ showed that sorcin was a calcium-binding protein. QUA/ADj cells contained increased numbers of double minute chromosomes (DMs), cytogenetic indicators of gene amplification. As found for two other multidrug-resistant sublines, sorcin overproduction in QUA/ADj cells may be the result of amplification of the sorcin-encoding gene. The overproduction of this protein in multidrug-resistant cells of various species implies that sorcin plays a role in expression of the resistant phenotype.
At least five gene classes are amplified in the multidrug-resistant CHO cell line CHRC5. Protein products have been identified for two classes; class 2 codes for the large membrane P-glycoprotein, whereas class 4 encodes the small cytoplasmic calcium-binding protein sorcin (V19). By DNA analysis we have shown previously that these five genes are linked in two groups: class 1 + 2 + 3; and class 4 + 5. By use of in situ hybridization with complementary DNAs derived from the resistant cell line we demonstrate here that genes from both linkage groups are amplified and situated together in each of two different chromosomal regions of the resistant Chinese hamster cell line. The positions of the amplicons correspond to cytogenetically identified homogeneously staining regions in an altered 7q+ chromosome and in a rearranged Z-7 [t(3;4)] chromosome. The native genes were mapped both in the CHRC5 line and in a normal diploid Chinese hamster cell strain, CHNF 86. We confirm the position of the class 2 gene on 1q26 and we show that class 4 and 5 genes are located in the same region of 1q. We conclude that the gene classes 2, 4, and 5 are closely juxtaposed in the normal Chinese hamster genome and comprise one amplicon in resistant cells. Our results are compatible with the hypothesis that multidrug resistance is due to overexpression of P-glycoprotein genes and that the other genes amplified in the CHRC5 line are coamplified because they happen to lie close to the P-glycoprotein genes.
Human neuroblastoma cells growing in culture offer a unique opportunity to study proliferating human cells with a neuronal phenotype. We have previously identified several neuroblastoma cell lines which show spontaneous conversion (N/S interconversion) between two morphologically distinct cell types: neuroblastic (N-type) cells and variant, substrate-adherent (S-type) cells resembling cultured glial or mesenchymal cells. In the present study, we have used molecular markers to confirm the neuronal phenotype of N-type cells and to demonstrate that S-type cells have a nonneuronal phenotype. Furthermore, we have used these markers, including a series of cell surface differentiation antigens, to compare S-type neuroblastoma cells with a wide range of cultured epithelial, mesenchymal, and neuroectodermal cells. The results suggest that N/S interconversion represents an ordered transition between two neuroectodermal differentiation programs rather than random phenotypic instability of cultured cells; S-type variant cells show a molecular phenotype most closely resembling the phenotype of cultured ectomesenchymal cells; and in vitro variant formation of human neuroblastomas may provide an experimental model for the observed in vivo transition of some malignant neuroblastomas into benign ganglioneuromas.
Human malignant melanoma cells express specific chondroitin sulfate proteoglycans (mel-CSPG) on the surface, both in vivo and in vitro. Melanocytes in normal skin show no detectable mel-CSPG but can be induced to express the antigen when cultured in the presence of cholera toxin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Most other cell types do not express mel-CSPG either in vivo or in vitro. A study was designed to examine regulatory signals controlling mel-CSPG expression. The gene encoding mel-CSPG was mapped to human chromosome 15, and this chromosome was introduced into rodent cells derived from distinct differentiation lineages. Three types of mel-CSPG--expressing hybrids were found: (i) hybrids derived from human melanomas; (ii) hybrids derived from human cells that do not express mel-CSPG; and (iii) hybrids derived from human cells expressing mel-CSPG that are antigen-negative but that are induced to express mel-CSPG when cultured on extracellular matrix instead of plastic surfaces. Thus, mel-CSPG expression can be controlled both through intrinsic signals, provided by the differentiation program of the rodent fusion partner, and through extrinsic signals, provided by specific cell-matrix interactions.
Nerve growth factor (NGF) is a polypeptide hormone which plays a central role in the development and growth of sympathetic and sensory neurons. The effects of NGF on target cells are mediated by a specific cell surface structure, nerve growth factor receptor (NGFr), which has been identified in human cells as a 75,000-mol-wt glycoprotein. We have used a monoclonal antibody to human NGFr to study cell-surface expression of the receptor on a panel of mouse-human neuroblastoma hybrids, and the serological typing results permit assignment of the gene coding for NGFr (NGFR) to chromosome 17q21-qter. In addition to mouse-human neuroblastoma hybrids, human NGFr was also detected on hybrids derived from fusions between mouse L-cell fibroblasts and human neuroblastoma and melanoma cells. Furthermore, induction of human NGFr expression was observed in hybrids derived from NGFr- human kidney epithelial cells and mouse L cells, but not in hybrids derived from human kidney epithelial cells and mouse RAG kidney carcinoma cells. These results suggest that cell-surface expression of human NGFr is controlled by trans-acting regulatory signals.
We have previously assigned several genes controlling expression of cell-surface antigens to human chromosomes 1 and 12. In the present study, we characterize three additional cell-surface antigens determined by these chromosomes. Two monoclonal antibodies, AbSR75 and AbMG6, define antigens expressed on a wide range of cultured human cells. In contrast, AbK66 defines an antigen with a more restricted distribution which is expressed on normal and malignant human epithelial cells but not on neuroectoderm-derived cells or hematopoietic cells. Normal adult fibroblasts are K66-, whereas fetal fibroblasts are K66+. Serological analysis of rodent-human hybrid cells permitted the assignment of MSK31, controlling SR75, and MSK32, controlling K66, to chromosome 1, whereas MSK27, controlling MG6, maps to chromosome 12. Analysis of hybrids containing only deleted copies of chromosome 1 or chromosome 12 established regional assignments for MSK31, MSK32, and MSK27 and also for the previously defined genes MSK1, MSK4, and MSK7. The MSK1 gene, coding for a 140,000 mol wt cell surface glycoprotein, was found to map to the same band (1p22) as the NRAS protooncogene and the gene encoding the beta-subunit of nerve growth factor (NGFB).
Multidrug-resistant sublines of Chinese hamster lung and mouse tumor cells selected for high levels of resistance to vincristine or actinomycin D have increased numbers of epidermal growth factor (EGF) receptors compared to control cells. Evidence for this increase was found in six of six resistant cell lines with the use of receptor binding or immunoprecipitation techniques. Levels of 125I-labeled EGF binding to intact actinomycin D-resistant cells derived from DC-3F or CLM-7 Chinese hamster lines are increased 3- to 10-fold compared to controls. Scatchard analysis of these data suggests that increased binding is a result of increased receptor number rather than altered affinity of receptor for ligand. Affinity-labeling and immunoprecipitation studies confirmed the finding of increased receptor amount in resistant hamster and mouse cells. Multidrug-resistant variants of DC-3F cells overproduce a plasma membrane glycoprotein (gp150-180) with several physicochemical properties that resemble those of EGF receptor. However, electrophoretic transfer blots with a polyclonal antibody to gp150-180 show that EGF receptor and gp150-180 are probably different molecules. Resistant cells described in this report manifest a normalized phenotype compared to transformed, tumorigenic, drug-sensitive parental cells. EGF receptor increase in resistant variants may be associated with this reverse transformation.
The chromosomal locations of the genes for the beta subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).