Marian B. Meyers

Increased synthesis of a low molecular weight protein in vincristine-resistant cells.

Abstract A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells ( DC-3F VCRd-5 ) has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of DC-3F VCRd-5 , and only trace amounts were detected in control DC-3F cells. A similar protein (M r = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in DC-3F VCRd-5 cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome.

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.

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.

Purification of deoxycytidine kinases from two p815 murine neoplasms and their separation from deoxyguanosine kinase.

Abstract Deoxycytidine kinase, which catalyzes the phosphorylation of deoxycytidine and 1-β d -arabinofuranosylcytosine (Ara-C) at the 5′-position, has been extracted and extensively purified from a murine neoplasm P815, either sensitive (P815) or resistant (P815/ Ara-C) to 1-β- d -arabinofuranosylcytosine. Gel filtration and ion-exchange chromatography were used to accomplish the purification. The purified enzyme exhibited a single band upon disc electrophoresis. During the extraction procedure an enzyme catalyzing the phosphorylation of deoxyguanosine and deoxyadenosine was successfully separated for the first time from deoxycytidine kinase. The K m values and turnover numbers with deoxycytidine as phosphate acceptor for the kinase from P815 cells sensitive to 1-β- d arabinofuranosylcytosine and that from P815 cells resistant to the drug are 9.3 μ m , 4.7 × 10 6 /min and 15.4 μ m , 8.0 × 10 4 /min, respectively.

Conformational changes in erythrocyte membranes by prostaglandins as measured by circular dichroism

Abstract Membranes were prepared from fresh, washed human erythrocytes by hemolysis and washing with 5 m m sodium phosphate buffer (pH 7.4). The mean residue ellipticity, [θ], of erythrocyte membrane circular dichroism was altered by prostaglandin E 1 or prostaglandin F 2α at 37 °C when observed from 250 nm to 190 nm. The decrease in negativity of [θ] with 10 −6 m prostaglandin E 1 was 12.7% at 222 nm and 17.7% at 208 nm, and with 10 −6 m prostaglandin F 2α 22.5% and 34.2%, respectively ( P −5 m to 3 × 10 −12 m was evident. A persistent alteration of [θ] with prostaglandin was observed at 37 °C. Transient change of [θ] occurred at 25 °C with prostaglandin. No change of [θ] was observed at 15 or 20 °C. Buffer or palmitic acid were without effect on membrane [θ]. Phosphatidyl inositol or methyl arachidonate caused an increase in negativity of membrane spectra. The observed alterations of membrane [θ] did not arise from changes in light scattering as the OD 700 –OD 200 of membranes was not changed by prostaglandin. Effects of prostaglandin were not dependent on light path length. The prostaglandin E 1 antagonist, 7-oxa-13-prostynoic acid, at 10 −7 m produced no change of [θ] of membrane spectra and prevented the otherwise demonstrable effects of 10 −10 m prostaglandin E 1 on [θ]. The decrease in negativity of [θ] at 222 nm is indicative of a decrease in ellipticity of membrane protein. These studies suggest that prostaglandins may act by inducing a conformational change in membrane protein.

Crosstalk between epidermal growth factor receptor and P-glycoprotein in actinomycin D-resistant Chinese hamster lung cells

Multidrug-resistant cells can manifest an increase in epidermal growth factor (EGF) receptor number along with increased P-glycoprotein (Pgp) synthesis. An interrelationship of the two membrane proteins in actinomycin D-resistant Chinese hamster lung cells (DC-3F/AD X) in terms of the effect of EGF on Pgp phosphorylation was investigated. EGF was not a mitogen for the resistant cells, nor was it mitogenic for DC-3F, the parental drug-sensitive line. Brief treatment of DC-3F/AD X cells with EGF resulted in a 30-50% decrease in the level of Pgp phosphorylation, and treatment of the cells with okadaic acid, a specific inhibitor of protein phosphatases-1 and -2A (PP1 and 2A), increased Pgp phosphorylation. Okadaic acid also increased phosphorylation of Pgp in plasma membranes isolated from DC-3F/AD X cells by 30-40%. Protein phosphatase activity in extracts of cells grown in EGF-containing medium was greater by 30% than that of cells grown in standard medium, and okadaic acid inhibited the increases. The results suggested that EGF activated PP1 and PP2A in DC-3F/AD X cells and that Pgp was a substrate for the phosphatases. The properties of Pgp may be modulated by the signalling system transduced by ligand-activated EGF receptor.

Association of sorcin with drug resistance in L1210 cells

SummaryL1210 sublines independently selected for resistance to teniposide (VM-26), etoposide (VP-16), doxorubicin (DOX), dactinomycin (DACT), or vincristine (VCR) express an anionic, 22-kDa protein that is not observed in extracts of parental L1210 cells. Antibody raised against sorcin, an acidic calcium-binding protein overproduced in many other cells resistant to these agents, cross-reacts with the 22-kDa polypeptide. The levels of the 22-kDa protein (sorcin) increase with the relative levels of drug resistance of the L1210 sublines. The appearance of sorcin in these various sublines further supports the notion that the overproduction of this protein is related to the general phenomenon of multidrug resistance rather than to specific drug resistance and that selection for resistance to teniposide produces L1210 sublines with multidrug resistance.