Barbara A. Spengler

Specifically altered metaphase chromosomes in antifolate-resistant Chinese hamster cells that overproduce dihydrofolate reductase

Abstract Specific karyotype alterations are described which occur in 16 different antifolate-resistant Chinese hamster sublines with increases in target enzyme dihydrofolate reductase activity ranging from 2.9- to 281-fold. Sublines were selected with either methotrexate or methasquin and exhibit a complex set of relationship between increase in resistance and increase in target enzyme activity. Drug resistant sublines generally contain one or the other of two types of abnormally banding chromosome regions as revealed by the Giemsa-banding method. In those cell lines with less than a 50-fold increase in dihydrofolate reductase activity there are long segments with distinctly abnormal band patterns. In sublines with greater than 85-fold increases in enzyme activity, cells contain a long, homogeneously staining region (HSR). HSRs comprise 2 to 5% of the total metaphase chromosome complement. The specific, abnormally banding regions are a consistent feature of the antifolate-resistant sublines. There is usually 1 per cell, preferentially located on the long arm of a chromosome #2. Results of experiments with cloned, HSR-containing cells maintained for about 2 years in antifolate-free growth medium indicate a quantitative relationship between length of the HSR and levels of drug resistance and dihydrofolate reductase activity; there was a parallel decline in these three parameters with time in culture.

Neurotransmitter-synthesizing enzymes in 14 human neuroblastoma cell lines

Fourteen human neuroblastoma cell lines were studied for expression and regulation of neurotransmitter-synthesizing enzymes. All cell lines contained enzyme activities of adrenergic and/or cholinergic neurons and 13 expressed activities for both. None contained enzymes for serotonergic or G AB Aergic neurons. Enzyme activity was characteristic for a given cell line. Enzyme activity in cell lines was sensitive to growth phase, culture medium, and concentration of fetal bovine serum.

Sorcin (V19), a soluble acidic calcium-binding protein overproduced in multidrug-resistant cells: Identification of the protein by anti-sorcin antibody

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.

Reverse transformation of multidrug-resistant cells

Spontaneously transformed Chinese hamster lung cells with high levels of resistance (≈ 100-fold to 70,000-fold) to actinomycin D, daunorubicin, or vincristine exhibit morphology and growth patterns characteristic of normal cellsin vitro and reduced tumorigenicityin vivo. These reverse transformed, multidrug-resistant cells amplify and highly overexpress one or more genes encoding P-glycoprotein. Similarly, hydrocarbon-induced mouse sarcoma cells selected with actinomycin D, vincristine, or ethidium bromide developed high levels of resistance associated with reduced drug accumulation and suppression of malignancy. To determine whether human tumor cells would undergo similar changes and whether reverse transformation reflected an altered state of differentiation, nine multidrug-resistant sublines were selected with four agents from human neuroblastoma cells with well defined pathways of differentiation. Those five with resistance levels above about 125-fold showed a reduced tumor frequency as compared to control cells. All resistant sublines showed altered differentiation. The changes in transformation phenotype appear to be intrinsic and not the result of altered immunogenicity. Two additional consequences of high level multidrug resistance have been observed: change in ganglioside composition in the Chinese hamster cells, manifested as a block in higher ganglioside biosynthesis and/or a relative increase in GM3, and increase in epidermal growth factor receptor in all three cell systems. A tentative hypothesis links ganglioside and growth factor receptor changes to the change in transformation phenotype. The basis of the reverse transformation phenomenon is not known, but the major alterations in expression of P-glycoprotein, gangliosides, and the epidermal growth factor receptor implicate, in some way, the plasma membrane.

The fine structure of continuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC

SummaryCell cultures of the continuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC at exponential and stationary growth phase have been examined by electron microscopy. At the level of fine structure these cells did not show typical neuronal differentiation such as extensive granular endoplasmic reticulum or neurites with microtubules and neurofilaments. Instead they were characterized by abundant free ribosomes, moderate Golgi complexes, and usually scant granular endoplasmic reticulum, features similar to the fine structure of early normal embryonic autonomic neurons. However, in several respects appearance of differentiated features of the neuroblastoma cells did not follow the pattern observed for normal neurons, suggesting noncoordinate, expression of neuronal phenotypic properties. First, an occasional neuroblastoma cell had as extensive granular endoplasmic reticulum as would be found at later stages in normal developing neurons. Second, the cellular processes of these neuroblastoma cells did not have the fine structure of developing or mature axons in vivo. Third, few dense core vesicles were found in SK-N-SH and SK-N-BE(2), though these organelles are numerous in early normal adrenergic neurons and the adrenergic character of these two lines is apparent from other studies that have demonstrated expression of neurotransmitter synthesizing enzymes (SK-N-MC is cholinergic). The fine structural characterization of these continuous human neuroblastoma cell lines will allow this parameter to be utilized with other approaches in future experimental studies.

Homogeneously Staining Regions and Double Minute Chromosomes, Prevalent Cytogenetic Abnormalities of Human Neuroblastoma Cells

Publisher Summary This chapter focuses on in vitro cultivation of human neuroblastoma cells and presents several morphological and biochemical characteristics of human neuroblastoma cells in culture. Both the human and mouse neuroblastoma cell lines have morphological, biochemical, and electrophysiological attributes of neuronal cells. The chapter describes an earlier study in which large double-minute chromosomes (DMs) and ring forms, such as observed in methotrexate-resistant cells with amplified dihydrofolate reductase genes and in several human neuroblastoma cell lines, were observed in a small number of cases. Although a large number of human tumors with DMs have been described in the literature, the relative proportion of tumors or tumor cell lines characterized by DM-containing cells is low. Various studies of antifolate-resistant rodent cell lines have demonstrated that both homogeneously staining regions and DMs in those cells are cytological indicators of amplified dihydrofolate reductase genes.

Multidrug resistance in human neuroblastoma cells

Neuroblastoma remains a significant problem in pediatric oncology. Recently a multidrug-resistance gene that may cause cells to become resistant to various chemotherapeutic agents has been cloned. The gene encodes the high-molecular-weight plasma membrane protein known as P-glycoprotein. To study the expression of this gene in cells exhibiting the multidrug-resistant phenotype, a panel of sublines selected with several different natural product drugs was established. The drug-sensitive parental BE(2)-C cells were clonally isolated from the human neuroblastoma SK-N-BE(2) line and exhibit a 150-fold increase in the copy number of the N-myc protooncogene. Sublines were selected by stepwise increases in the concentration of actinomycin-D, doxorubicin, vincristine, or colchicine. Gene amplification was assessed using Southern analysis, and RNA levels were determined by Northern and dot-blot analysis. Western blotting was used to determine protein levels. N-myc amplification and expression were simultaneously determined to assess possible alterations associated with development of multidrug resistance. Amplified P-glycoprotein-encoding genes were not seen in control lines but were clearly present in those that had undergone exposure to each of the chemical agents. Similarly, steady-state messenger RNA and protein levels were greatly increased in the drug-resistant sublines. We conclude that human neuroblastoma cells can acquire the multidrug-resistant phenotype after exposure to various chemotherapeutic agents.

Assignment of human nerve growth factor receptor gene to chromosome 17 and regulation of receptor expression in somatic cell hybrids

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 transacting regulatory signals.

Two cell-surface markers for human chromosome 1 in interspecies hybrids

Monoclonal antibodies AbAJ9 and AbT87 define two distinct human cell-surface antigen systems (1, 2). Both antibodies react with a wide variety of cultured human cell types but not with rodent cell lines. AbAJ9 identifies a glycoprotein of 140,000 mol wt and AbT87 identifies a glycoprotein of 60,000 mol wt. The genetic control of antigen expression was studied in rodent-human somatic cell hybrids containing different subsets of the entire human chromosome complement, using an immune rosetting assay for serological analysis and hybrid selection. The presence of human chromosome 1 was found to be both necessary and sufficient for expression of antigens AJ9 and T87 in hybrid cells. Two independent gene loci on chromosome 1, designated MSK-1 for AJ9 and MSK-2 for T87, control the expression of these antigens. We have assigned MSK-1 to region 1 cen-p22 and MSK-2 to region 1 q32-qter, using hybrids containing only fragments of human chromosome 1.

Three human cell surface antigen systems determined by genes on chromosome 12.

The mouse monoclonal antibodies AbA123, AbA127, AbK152, AbM68, and AbV1 were derived after immunization with cultured human tumor cells or melanocytes. Antibodies AbA123, AbA127, and AbK152 recognize human cell surface antigens expressed on most cultured human cells and show an identical pattern when tested on a panel of 47 human cell lines. They recognize at least two different epitopes on the same glycoprotein complex, designated A123/A127, which consists of 30,000- and 40,000-mol-wt glycopeptides. Antigens M68 and V1 are also expressed on most cultured human cell types but show distinct patterns of distribution on the cell line panel. The antigens defined by AbM68 and AbV1 have the characteristics of glycolipids. They are heat stable, and immunoprecipitation of metabolically labeled cell lysates did not yield any detectable components when analyzed by SDS-polyacrylamide gel electrophoresis. Serological typing of a panel of 23 independently derived mouse-human and Chinese hamster-human somatic cell hybrids showed unequivocally that the expression of cell surface antigens A123/A127, M68, and V1 segregates with human chromosome 12. The analysis of hybrids containing karyotypically defined deletions of chromosome 12 permitted the assignment of the loci determining the expression of antigens A123/A127 and V1 to region 12cen-qter, and the locus determining the expression of antigen M68 to region 12cen-pter. These antigens can be distinguished from the cell surface molecules previously assigned to chromosome 12 and thus represent new assignments to this chromosome.