Relationship between production of epidermal growth factor receptors,gene amplification,and chromosome 7 translocation in variant A431 cells

Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant A431 human epidermoid carcinoma cell clones reported to contain different amounts of EGF binding sites. The amount of EGF receptor protein, quantitated by immunoaffinity chromatography, and EFG receptor mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene amplification. This correlation existed in variants selected for reduced EGF receptors and in revenants from those variants with increased EGF receptors. There was also a correlation between the frequency of translocation of chromosome 7, containing the EGF receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 cell EFG receptor protein and determining growth responses.     

Relationship between production of epidermal growth factor receptors, gene amplification, and chromosome 7 translocation in variant A431 cells

Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant A431 human epidermoid carcinoma cell clones reported to contain different amounts of EGF binding sites. The amount of EGF receptor protein, quantitated by immunoaffinity chromatography, and EGF receptor mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene amplification. This correlation existed in variants selected for reduced EGF receptors and in revertants from those variants with increased EGF receptors. There was also a correlation between the frequency of translocation of chromosome 7, containing the EGF receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 cell EGF receptor protein and determining growth responses.     

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.     

Variant of A431 cells isolated by ricin A-conjugated monoclonal antibody directed to EGF receptor: Phosphorylation of EGF receptor and phosphatidylinositol

A monoclonal antibody specific for human EGF receptors was cross-linked to subunit A of toxic ricin. Using this conjugate, we isolated a variant of A431 cells, designated C1-B7, with approximately 40 times less EGF binding capacity. Unlike the parental cells, the C1-B7 variant was resistant to EGF-induced suppression of cell growth. The EGF receptors retained in this variant were of high-affinity type and susceptible to EGF-induced autophosphorylation. Membrane prepared from C1-B7 cells was highly phosphorylated in the presence of 2 M [^{– 32}P]-ATP, primarily on the lipid components shown as phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This same level of lipid phosphorylation was observed on A431 membrane only in the presence of higher ATP concentrations. After addition of EGF to A431 membrane, phosphatidylinositol phosphorylation was significantly decreased with a concomitant increase in EGF-dependent protein phosphorylation. Thus, the EGF-dependent receptor-mediated protein phosphorylation precedes phosphatidylinositol phosphorylation. These observations support the idea that the growth inhibitory effect of EGF on A431 cells is caused by high ATP consumption due to the EGF-induced protein phosphorylation and reduction of phosphatidylinositol turnover.     

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.     

Unique chromosomal location of amplified EGF receptor genes in EGF receptor-hyperproducing tumor cell line NA

The epidermal growth factor receptor (EGFR) gene was analyzed by in situ hybridization using a squamous cell carcinoma line NA, which has high numbers of EGF receptors and carries a 20-fold amplification of EGFR genes. NA cells are pseudotriploid (mode of chromosome number is 69) and have three copies of an apparently normal chromosome 7 together with several aberrant chromosomes. Strong hybridization signals were observed in the abnormal banding region of one of the aberrant chromosomes, MH1, which has no structural homology to chromosome 7. This MH1 chromosome was lost in NA-derived variant lines that possess reduced numbers of EGF receptors. These results are in contrast to previous findings that EGFR gene amplification is associated with structural alterations of the short arm of chromosome 7 and provide new evidence in regard to the location of the amplified EGFR gene in tumor cells.     

Physical and genetic localization of quinonoid dihydropteridine reductase gene (QDPR) on short arm of chromosome 4

A portion of a cDNA clone corresponding to the 3 end of the human quinonoid dihydropteridine reductase (QDPR) mRNA was used as a probe to physically map the QDPRgene by analysis of somatic cell hybrid lines. The provisional assignment of QDPRto chromosome 4, based on expression of the human enzyme in hybrids, was confirmed. The gene was further regionally localized on the short arm to 4p16.14p15.1. This physical localization places QDPRin the same area of the genome that contains the defect causing Huntington's disease (HD). The QDPRprobe revealed a restriction fragment length polymorphism with the enzyme BanII, permitting determination of its genetic proximity to D4S10,an anonymous DNA marker tightly linked to HD. QDPRis only loosely linked to D4S10,excluding any primary role for the gene in HD.     

Heparin binding epidermal growth factor in renal ischaemia/reperfusion injury

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding‐epidermal growth factor (HB‐EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB‐EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK‐2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB‐EGF protein excretion was studied after renal transplantation. Finally, HB‐EGF KO and WT mice were subjected to IRI to study the role of HB‐EGF in renal injury. HB‐EGF mRNA was significantly up‐regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI‐166 reduces macrophage accumulation and interstitial α‐SMA in the early phase of IRI in rats. In vitro, PKI‐166 causes a marked reduction in HB‐EGF‐induced cellular proliferation. Urinary HB‐EGF is increased after transplantation compared with control urines from healthy subjects. HB‐EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB‐EGF mRNA and protein in vivo and in vitro. Absence of HB‐EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB‐EGF. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Chromosomal mapping of the human interleukin-1 receptor antagonist gene (IL-1RN) and isolation of specific YAC clones

Using a panel of somatic rodent-human cell hybrids, we show that the interleukin-1 receptor antagonist gene (IL-1RN) maps to the long arm of human chromosome 2. Linkage studies permitted the regional localization of this gene to band q14-21. This is the same region in which the IL-1 and IL-1 genes are localized. Three yeast artificial chromosome (YAC) clones containing the IL-1RN gene were isolated, and these will be used for further characterization of this chromosome 2 region.     

Epidermal growth factor modulates cell attachment to hyaluronic acid by the cell surface glycoprotein CD44

Cell adhesion to and migration through extracellular matrices (ECM) are critical events in tumor invasion and metastasis. Previous work by us had demonstrated that signaling of epidermal growth factor receptor (EGFR) confers an oncogenic phenotype on NR6 cells and that these cells when transfected with holo EGFR demonstrate greater motility and invasiveness than cells carrying a carboxy-terminal truncated EGFR. Recently, a cell surface glycoprotein, CD44, has been implicated in cell-ECM adhesion involved in tumor cell migration, signal transduction, and metastasis. We investigated whether EGF regulates cellular interactions with ECM components, and in particular, hyaluronate, by modulating CD44 expression. In vitro cell attachment assays on hyaluronate-coated plates demonstrated similar basal level of binding -33%) for murine NR6 parental cells devoid of endogenous EGFR (P) or expressing wild-type EGFR (WT), while a time-dependent increase in binding was observed in WT cells stimulated with EGF. Additionally, utilizing monoclonal antibody blocking assays, CD44, but not EGFR, was shown to be directly involved in this attachment. Both WT and P cells possessed equivalent 95 kDa bands on immunoblots, corresponding to CD44. The existence of CD44 mRNA was verified by RT-PCR using synthetic oligonucleotides in which a 1.1 kb cDNA was detected in both cell lines and confirmed by DNA sequencing. After 24-h exposure to exogenous EGF, an increase in CD44 protein and mRNA expression was found in WT cells, but not in P cells, supporting the contention that a functional EGFR signaling pathway is required for CD44 regulation. Thus, EGF stimulates cell binding to hyaluronate in vitro by regulating CD44 expression.     

Expression of the receptor for epidermal growth factor correlates with increased dosage of chromosome 7 in malignant melanoma

Epidermal growth factor (EGF) receptor is expressed selectively by human melanoma cells which show the presence of an extra copy of chromosome 7. None of the cells of benign pigmented lesions (nevi) or radial growth phase (nonmetastatic) primary melanoma expressed EGF receptor and none of these cells showed an extra copy of chromosome 7. The results indicate that a single extra dose of a gene (for EGF receptor) may provide a selective advantage to cells in the late stages of tumorigenesis.     

Increased binding capacity of receptors for the epidermal growth factor in benign thyroid nodules and thyroid malignancies

Summary To determine the role of epidermal growth factor (EGF) receptors in thyroid tumorigenesis, EGF binding was compared in membranes from malignant and from benign thyroid tumors. Surgical specimens were obtained from 28 patients with thyroid carcinomas (3 papillary, 13 follicular, 6 undifferentiated, and 6 medullary carcinomas) and from 30 patients with benign thyroid tumors (15 scintigraphically functional and 15 nonfunctional nodules). In 30 cases normal tissue adjacent to the tumor was also obtained. EGF binding was seen to be increased not only in thyroid carcinomas but also in benign thyroid tumors, particularly in functional thyroid adenomas. The highest EGF binding was found in undifferentiated carcinomas. A direct comparison of the EGF binding characteristics in tumor and adjacent normal thyroid tissue revealed that the increased binding of EGF is due mainly to an increase in the number of binding sites rather than an alteration in receptor affinities. EGF binding capacities were 18.4±16.7 fmol/mg protein in thyroid carcinomas and 10.5±5.2 fmol/mg in the corresponding normal tissue (P<0.05, K _d 0.84±0.26 nM, n=11). In autonomously functioning thyroid adenomas binding capacities were 14.2±8.2 fmol/mg in the nodules and 8.9±4.8 fmol/mg in normal tissue (P < 0.01, K _d 0.73±0.62 nM, n = 15). In conclusion, EGF receptor levels are increased not only in malignant thyroid tumors but also in well-differentiated benign thyroid nodules. The data indicate that an increased expression of EGF receptors, although likely to be important in the regulation of thyroid growth in vivo, is not by itself associated with malignant cell transformation and loss of differentiated function.Abbreviations EGF epidermal growth factor - EGFr epidermal growth factor receptor - TGF- transforming growth factor- Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

Human Luteinizing hormone-releasing hormone gene (LHRH) is located on short arm of chromosome 8 (region 8p11.2 → p21)

Luteinizing hormone-releasing hormone (LHRH) is synthesized by hypothalamic neurons and affects the release of gonadotropic hormones from the anterior pituitary gland. A cDNA clone encoding the human LHRH precursor molecule was used to assign theLHRH gene to a human chromosome by in situ hybridization and Southern blot analysis. Metaphase spreads from two normal individuals were hybridized with³H -labeled LHRH-specific sequence. Of 120 cells analyzed, 33 had silver grains over the p11.2 p21 bands of chromosome 8. No other chromosomal site was labeled above background, indicating the presence of a single site for LHRH sequences in the human genome. Independent confirmation for this location of the humanLHRH gene on chromosome 8p was provided by analysis of DNA from human × Chinese hamster somatic cell hybrids. DNA samples were digested with EcoRI, blotted, and hybridized with the³²P-labeled human LHRH precursor cDNA probe. The single 11.5-kb human-specific band was detected only in hybrids containing human chromosome 8. Also, hybridization was observed in DNA from hybrids in which a portion of human chromosome 8 (region 8pter 8q21) had been spontaneously translocated onto a Chinese hamster chromosome.Presented in part at the 8th Human Gene Mapping Conference, Helsinki, August 1985.     

A Novel Splice Variant of FR4 Predominantly Expressed in CD4+CD25+ Regulatory T Cells#

Folate receptor 4 (FR4) is recently found as a lymphoid tissue specific protein. In this study, we have identified an alternative splicing variant of the FR4 gene from murine splenocytes, termed FR4v, which is almost identical to FR4 cDNA sequence except with the retained 108 bp intron 3 between exon 3 and 4 of FR4 gene. FR4v mRNA encodes a larger protein than FR4 and is constitutively expressed on CD4⁺CD25⁺ regulatory T cell (Treg) membrane via a GPI anchor mechanism. Whether FR4v plays a redundant or unique functional role in Tregs should be investigated further in the future.     

Culture of A-431 human epidermoid carcinoma cells in serum-free medium: Effect of culture conditions on the binding of [125I]-epidermal growth factor

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [³H]-thymidine incorporation of A-431 cells in serum-free medium. A-431 cells have a high number of receptors for epidermal growth factor (EGF); they bind and rapidly internalize EGF. Nevertheless, EGF did not stimulate either the growth or the [³H]-thymidine incorporation of these cells. Analyses of [¹²⁵I]-EGF binding data indicated that A-431 cells grown in the presence of calf serum had about 3.2–3.9 × 10⁶ specific, saturable EGF receptor sites on their surface. Linear Scatchard plots indicated a single class of noninteracting receptors with an apparent equilibrium dissociation constant of about 2.8 × 10^?9 M. The average number of receptors of A-431 cells maintained in DME supplemented with only fetuin, insulin, and transferrin for several months was significantly less, 1.54 × 10⁶, than that of A-431 stock cells cultured in the same medium for 2 days only (2.68 × 10⁶). The apparent dissociation constants for the same cell populations were, however, similar, 4.5 × 10^?9 M and 4.1 × 10^?9 M, respectively. Stimulation of growth by oleic acid resulted in about 20% decrease in the average number of receptor sites, with an increase in the apparent equilibrium dissociation constant.     

Mouse immune interferon (IFN-γ) gene is on chromosome 10

A cDNA clone for mouse immune interferon has been used to map the mouse interferon gene (Ifg)to a specific chromosome. This clone, which contains a 638-bp insert, detects an 18-kb HindIII fragment of mouse DNA. The presence of the mouse Ifgtgene in cell hybrids and its chromosomal location were determined by assaying cell hybrid DNA for the presence of the 18-kb HindIII fragment by Southern filter hybridization. Under the hybridization conditions used, Chinese hamster DNA did not hybridize to the cDNA probe. The segregation of mouse chromosomes in cell hybrids indicated that Ifgis located on chromosome 10. Previously, we have mapped immune interferon to the p12.05 qter region of chromosome 12 in humans (1). This region of chromosome 12 also contains the genes for peptidase B and citrate synthase. The homologous genes in mouse are also located on chromosome 10, suggesting that these genes comprise a conserved linkage group.     

Direct mapping of the human TATA box-binding protein (TBP) gene to 6q27 by fluorescencein situ hybridization

Summary TBP (TATA box-binding protein) participates in the expression of eukaryotic genes transcribed by RNA polymerases I, II, and III. Molecular cloning of human TBP revealed that the N-terminal region contains a polymorphic (CAG)_n repeat. We report here the direct localization of human TBP gene to chromosome 6q2705qter region by fluorescencein situ hybridization, using the cDNA clone with or without the (CAG)_n repeat as a probe.     

Characterization of site-specific antibodies to the erbB gene product and EGF receptor: inhibition of tyrosine kinase activity

Site-specific antibodies were generated against the erbB protein and epidermal growth factor (EGF) receptor by immunizing rabbits with a synthetic peptide corresponding to amino acid residues 285-296 of the predicted AEV-H erbB protein sequence. This peptide region lies within the tyrosine kinase domain of erbB and EGF receptor. Antibodies directed against this region readily identified native and denatured forms of the erbB gene product and EGF receptor as demonstrated by immuneprecipitation and immunoblot analysis. The anti-peptide antibody immuneprecipitated a functional EGF binding receptor molecule. Scatchard analysis demonstrated a KD for 125I-labeled EGF binding of 40 nM, a value consistent with that of detergent solubilized EGF receptor. Immuneprecipitates, though able to bind EGF, were unable to transfer phosphate from gamma-labeled ATP in a standard phosphorylation reaction. In detergent solubilized extracts of crude A431 microsomes, the anti-peptide antibody inhibited in a dose dependent manner the autophosphorylation of EGF receptor as well as receptor mediated phosphorylation of exogenously added substrates. In addition, this anti-peptide antibody reduced the overall level of tyrosine kinase activity present in microsomes prepared from AEV-transformed erythroblasts. This site-specific antisera should be useful for understanding the role of EGF receptor and erbB tyrosine kinase activity and their link with cell proliferation.     

Human interstitial retinol-binding protein (IRBP): cloning,partial sequence,and chromosomal localization

A cloned 2184-bp cDNA coding for human interstitial retinol-binding protein (IRBP) has been isolated and sequenced. The probe hybridized to a 5.2-kb poly(A) RNA from human retinas. Nineteen tryptic peptides (363 amino acids) sequenced and purified from bovine IRBP could be aligned with 86–88% homology to the translated sequence. Two segments approximately 200 amino acids long were found to have a 41% residue identity,suggesting an internal duplication event. This cloned cDNA was used to probe DNA samples from a panel of 29 rodent-human somatic cell hybrids, mapping the structural gene for IRBP to chromosome 10. In situ hybridization suggested a regional localization near the centromere (p11.2q11.2), although a secondary site of hybridization at q2425 was also observed.