Yoshiko Shimizu

Genetic analysis of hyperproduction of epidermal growth factor receptors in human epidermoid carcinoma A431 cells

Human epidermoid carcinoma A431 cells, possessing an extraordinarily high number of epidermal growth factor (EGF) receptors (1), were found to be hypotetraploid in their chromosome constitution and to contain two copies of intact chromosome 7 and two types of the translocation chromosomes involving chromosome 7 (M4 and M14) as well as several other rearranged chromosomes. The A431 cells were fused with mouse A9 cells, which lack EGF receptors (2) and are deficient in hypoxanthine phosphoribosyltransferase (3), and the human-mouse cell hybrid (AA series) were selected in HAT/ouabain medium (3, 4). The expression of high EGF binding ability was correlated with the presence of human translocation chromosome M4. AA hybrid clones that contained intact human chromosome 7 but not the marker chromosome M4 expressed only ordinary levels of EGF receptors. The EGF receptors expressed in the AA hybrids were proven to be of human nature by immunoprecipitation of the receptors cross-linked with [125I]EGF. These observations and our previous gene assignment of the EGF receptor to human chromosome 7 (2, 5) suggest that the marker chromosome M4 may carry an alteration(s) in the gene(s) involved in EGF receptor biosynthesis.

Protein kinase C phosphorylates DNA topoisomerase I

The induction of mammalian cell proliferation requires the expression of a specific set of genes. Tumor promoters stimulate cell growth by activating the Ca2+ and phospholipid‐dependent protein kinase, protein kinase C (PKC). DNA topoisomerase I, a nuclear enzyme involved in transcription, was phosphorylated by activated PKC in vitro. Phosphorylation by PKC stimulated the DNA relaxation activity of topoisomerase I two‐ to three‐fold. Therefore, DNA topoisomerase I is a substrate for PKC‐mediated activation by phosphorylation and may serve as a nuclear target of mitogenic signals generated by tumor promoters in vivo.

Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of humanmouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p+ (7qter>7p22::Xq21>Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-q21.

Nicotinamide inhibits adipocyte differentiation of 3T3-L1 cells

3T3-L1 cells are a clone of Swiss/3T3 fibroblasts that spontaneously differentiate into adipocytes with a high frequency when in a resting state [1-4]. The differentiation process normally takes several weeks after the cells have become confluent, but it may be speeded up by treating the cells with the inducers l-methyl-3-isobutylxanthine (MIX) and dexamethasone (Dex) [5] or others [2,3,6-8]. During differentiation of the adipocytes a number of morphological and physiological changes occur. The 3T3-LI cells change from spindleshaped fibroblast cells to larger, spherical cells and accumulate large triglyceride droplets [1-4]. During differentiation almost all the enzymes involved in de novo fatty acid [6-10] and triacylglycerol [11] synthesis increase coordinately and the synthesis of >60 cellular proteins is greatly increased [12,13]. The differentiating cells acquire sensitivity to physiological concentrations of insulin via an increased number of receptors which have a higher binding capacity than preadipocyte insulin receptors [5]. The cells also acquire a catecholamine-sensitive adenylate cyclase system involved in the lipolytic response to catecholamines [14]. Poly(ADP)ribosylation also changes during adipocyte conversion: the activity of poly(ADP-ribose) synthetase drops sharply upon the induction of differentiation by MIX, Dex and insulin, continues to decrease for several hours, and then, concurrently with the appearance of phenotypic expression, increases to a level higher than that in pre-adipocytes [15]. This transient reduction in poly(ADP)ribosylation activity has been postulated as an early essential event in differentiation [15] which reflects changes in chromatin structure [16,17]. In a separate study, involvement of poly(ADP-ribose) synthetase in insulin-stimulated cell growth has been suggested [ 18]. Here, we examined the effect of nicotinamide, which is known to be a strong inhibitor of poly(ADP-ribose) synthetase [19,201, on the adipocyte differentiation of 3T3-L1 cells. Our results indicated that in fact nicotinamide inhibits differentiation and that this inhibition is correlated with the prevention of inducer-stimulated cell growth. Studies using nicotinamide analogues further support the previous proposition [15] that poly(ADP)ribosylation is crucial in triggering the adipocyte differentiation program.

Genetics of cell-surface receptors for bioactive polypeptides: A variant of mouse BALBc/3T3 fibroblasts possessing altered insulin-binding ability

An insulin-nonresponsive variant was isolated from mutagenized mouse BALBc/3T3 fibroblasts. Selection was based on the insulins mitogenic action upon quiescent cells and subsequent arrest at mitosis by vinblastine sulfate to remove insulin-responsive cells. Among four surviving colonies, one, designated IN-2, exhibited no binding for [125I] insulin at 2× 10−10 M and at 4° C. The binding ability, however, recovered substantially at 15° C and increased with higher temperature and at higher ligand concentrations. The binding profiles, Scatchard plot analysis, and the dissociation kinetics indicated that the receptors expressed on IN-2 cells possess lower affinity than the parental 3T3 cells. The IN-2 cells were negative for stimulating effects of insulin on 2-deoxyglucose uptake, thymidine incorporation, and cell growth. The IN-2 cells were also negative for cross-reactivity to antibodies which react with insulin receptors on 3T3 cells and for the susceptibility to a cytotoxic chimeric insulin which was cross-linked to diphtheria toxin fragment A. This negative response of IN-2 cells can be attributed to a deficiency in “high-affinity receptors” for insulin. The insulin bound to the “low-affinity binding sites” of IN-2 cells, however, undergoes internalization and intracellular degradation. Therefore, such processing by itself does not account for insulins mitogenic action.

A cytotoxic epidermal growth factor cross-linked to diphtheria toxin A-fragment

In order to better understand the molecular and genetic mechanisms of cellular responses to bioactive poiypeptides, such as peptide hormones and growth factors, through interaction with specific receptors [l-3] we have been undertaking an approach using the strategy of mutagenesis [4,5]. We have devised a new selection procedure to isolate insulin receptordeficient mutants by using a cytotoxic insulin crosslinked to diphtheria toxin A-fragment and proposed a possible mechanism of cell killing [6,7]. Recently, this technique has enabled us to isolate several variants from mouse Swissl3T3 fibroblasts which have lost insulin binding ability and insulin responsiveness ([8], submitted). Here, we have appfied the crosslinking method to a potent mitogen, epidermal growth factor (EGF, Mr 6045) [2]. Fragment A of diphtheria toxin (DTa; Mr 21 150) [9] was coupled via a disulfide bond to a cystamine~erivatized EGF. The crosslinked molecules were purified by Biogel P-10 column chromatography. The purified EGF-DTa conjugate was shown to inhibit the binding of ‘251-labeled EGF to cell surface receptors. It revealed strong cytotoxicity to human epidermoid carcinoma cells, A43 1, possessing a high number of EGF receptors while it was ineffective to mouse NR-6 cells which are EGF receptor-deficient variants from Swiss/3T3 fibroblasts. These data strongly suggest that the EGF-DTa conjugate delivers catalytically active toxin A-fragment into A431 cells through EGF receptor-mediated endocytosis and inactives elongation factor 2 by ADP-ribosylation,

Phosphorylation of DNA topoisomerase I is increased during the response of mammalian cells to mitogenic stimuli.

DNA topoisomerase I is phosphorylated after mitogenic stimulation of 3T3-L1 mouse fibroblasts by 12-O-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester tumor promoter. In vivo labeling with [32P]orthophosphate and immunoprecipitation with an anti-DNA topoisomerase I antibody has demonstrated an increase in the phosphorylation of DNA topoisomerase I in Swiss/3T3 mouse fibroblasts treated with epidermal growth factor (EGF) and H35 rat hepatoma cells treated with insulin. The only phosphorylated form of DNA topoisomerase I observed was the 100-kDa protein Digestion of DNA topoisomerase I with trypsin revealed two phosphopeptides. In addition, VT-1, a non-responsive genetic variant of 3T3-L1, and the DNA topoisomerase I inhibitor camptothecin were used to further study TPA-induced DNA topoisomerase I phosphorylation. Our results indicate that the phosphorylation of DNA topoisomerase I may be an ubiquitous response of cultured mammalian cells to mitogenic agents, even in the absence of DNA replication.

Cell genetic evidence of correlation of intracellular translocation of protein kinase C(PKC) and PKC-mediated phosphorylation of 80-kDa protein with mitogenic action of tumor promoters

Recently, we isolated a series of 3T3-L1 cell variants that are unable to respond to mitogenic stimulation by the tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA). Since protein kinase C (PKC) is the major receptor for TPA and appears to play a key role in cellular proliferation, we have examined the distribution of PKC in the parental 3T3-L1 cells and the variant VT-1 cells. PKC was located predominantly in the cytosol of growth-arrested confluent 3T3-L1 cells, and upon TPA treatment it was rapidly translocated into the plasma membrane. In contrast, PKC was located predominantly in the plasma membrane of confluent VT-1 variant cells and was no longer activated by TPA. Two-dimensional gel analysis showed that a Mr 80,000 acidic protein (80-kDa protein) was rapidly phosphorylated in 3T3-L1 cells upon TPA treatment, whereas phosphorylation of this protein was barely detected in VT-1 cells. In growing cultures, the majority of PKC was found in the plasma membrane of both cell lines, and no change occurred upon TPA treatment. Hydroxyapatite column chromatography revealed the presence of α-type PKC as the major component in both cell lines. These results suggest that the intracellular translocation of α-type PKC and the PKC-mediated phosphorylation of the 80-kDa protein may be involved in the mechanism of mitogenic signal transfer.

Insulin and epidermal growth factor stimulate poly ADP-ribosylation

Abstract Treatment of contact-inhibited BALBc 3T3 fibroblasts with insulin or epidermal growth factor elicited a considerable increase in the capacity of poly (ADP-ribose) synthesis. Stimulation of the poly ADP-ribosylation was observed after long-term hormone treatment (18 hours) but not after short-term treatment (1 hour) and it appeared to be correlated with the cells entry into DNA synthesis. Several cellular components were found to be enhanced in poly ADP-ribosylation. The data suggest a possible role for poly ADP-ribosylation in mitogenic action of polypeptide hormones and growth factors.

Genetics of receptors for bioactive polypeptides: Expression of the human EGF receptor gene and internalization and processing of the receptor-bound EGF in human-mouse cell hybrids

We previously postulated that the structural gene for epidermal growth factor (EGF) receptor is located on human chromosome 7 (1, 2). In this study, EGF receptor and certain postreceptor functions were further analyzed in a unique cell hybrid line, C2B5, that retains only one human chromosome of an X;7 translocation besides a nearly complete mouse parental genome. Kinetics and Scatchard analysis of [125I]EGF binding to the C2B5 hybrid cells indicated that they carry a single class of EGF receptors with a dissociation constant of 4×10−10 M. The receptors expressed in the hybrids are proven to be immunologically of human nature. The human EGF receptors now embedded in essentially mouse plasma membrane are subject to “down regulation” mediated by the ligand EGF. Analysis of the cell-bound EGF indicated that internalization and processing take place in the human-mouse cell hybrids. The degradation of EGF appears to be through a lysosomal pathway since it was substantially delayed or inhibited by lysosomotropic agents.