Expression of the avian c-erb B (EGF receptor) protooncogene during estrogen-promoted oviduct growth

Expression of cellular erb B protooncogene messenger RNAs has been analyzed in the oviducts of immature chicks during estrogen-promoted growth. Hybridization of oviduct total cellular RNA with viral-derived erb B oncogene probes demonstrated significant expression of c-erb B mRNA in oviduct cells of untreated chicks. Daily administration of estrogen (diethylstilbestrol) to chicks results in marked oviduct growth but did not appreciably affect expression levels of c-erb B messenger RNA in oviducts after 2, 4 or 6 days of treatment. Withdrawal of chicks from estrogen treatment resulted in termination of oviduct growth. However, c-erb B messenger RNAs were detectable in the nonproliferative tissue at 5 days after hormone withdrawal. Readministration of diethylstilbestrol, progesterone or diethylstilbestrol plus progesterone to hormone-withdrawn birds (secondary stimulation) also did not affect c-erb B messenger RNA levels in the oviduct. These results demonstrate significant expression of the cellular erb B (epidermal growth factor receptor) gene in the avian oviduct. However, EGF receptor messenger RNA synthesis is not modulated in the oviduct by steroid hormones.     

Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.     

Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.     

Functional interaction between the two zinc finger domains of the v-erb A oncoprotein.

The v-erb A oncogene of avian erythroblastosis virus is a mutated and virally transduced copy of a host cell gene encoding a thyroid hormone receptor. The protein expressed by the v-erb A oncogene binds to DNA and acts as a dominant negative inhibitor of both the thyroid hormone receptor and the closely related retinoic acid receptor. The v-erb A protein has sustained two amino acid alterations within its DNA-binding domain relative to that of c-erb A, one of which, at serine 61, is known to be important for v-erb A function in the neoplastic cell. We report here that the second alteration, at threonine 78, also plays an important, although more indirect, role: alteration of the sequence at threonine 78 such that it resembles that of c-erb A can act as an intragenic suppressor and can partially restore function to a v-erb A protein rendered defective due to a mutation at position 61. Threonine 78 lies within the D-box of the v-erb A protein, a region thought to mediate receptor-receptor dimerizations, and is not in physical proximity to the serine at position 61. It therefore appears that an indirect interaction occurs between these two sites and that this interaction is crucial for v-erb A function.     

c－erbB2／neu扩增诱导人前列腺癌细胞的转移能力

用突变的ｃ－ｅｒｂＢ２／ｎｅｕ癌基因转染人类ＰＣ３细胞系,使转化克隆Ｎ３５具有癌转移能力,在接种到裸鼠后能向肺、骨髓等远处转移．各项实验指标证实,外源性的ｎｅｕ基因已整合至人类细胞的染色质ＤＮＡ中,并获得基因扩增和高表达．结果提示ｎｅｕ癌基因与肿瘤的发展和转移能力的形成有密切的关系．     

Molecular cloning and characterization of the chicken DNA locus related to the oncogene erbB of avian erythroblastosis virus.

Chicken cell DNA contains sequences which are homologous to the avian erythroblastosis virus oncogene v-erb. These cellular sequences (c-erb) have been isolated from a library of chicken cell DNA fragments generated by partial digestion with AluI and HaeIII and shown to be shared by at least two loci in the chicken DNA. One of them, denoted c-erbB, contains approximately 1.8 kilobase pairs of chicken DNA homologous to the 3' part of the v-erb oncogene (v-erbB). Restriction mapping studies show that the c-erbB DNA sequences homologous to v-erbB are distributed among six EcoRI fragments located in a single genomic region. Heteroduplexes between v-erbB in viral RNA and cloned c-erbB DNA show that the chicken DNA sequences homologous to v-erbB are interrupted by 11 DNA sequences not present in the v-erb oncogene. We conclude from our data that the c-erbB locus might represent the cellular progenitor for the v-erbB domain of the v-erb oncogene.     

Recognition of rat liver and kidney nuclear T3 receptors by an antibody against c-erb A peptide

It has been reported that c-erb A encodes nuclear T3 receptors (NT3R). Based on the sequence of c-erb A cDNA, we synthesized a polypeptide consisting of 15 amino acids, the sequence of which has high homology between c-erb A alpha 1 and beta. The antibody against this c-erb A peptide not only immunoprecipitated rat liver and kidney NT3R but also inhibited T3 binding to NT3R. In a displacement study, the inhibition of [125I]T3-binding by the antibody was parallel to that by T3 in terms of the concentration of the competitor added in the incubation mixture. Scatchard analysis revealed that the antibody decreased the value for the association constant in a dose dependent manner. The antibody did not bind T3 itself. The results show that the antibody against c-erb A peptide recognizes rat liver and kidney NT3R and that the sequence encoding this peptide, the closest carboxyl-terminal of c-erb A may be critical or at least closely related to the hormone binding.     

Binding of 3,5,3'-triiodothyronine (T3) and its analogs to the in vitro translational products of c-erbA protooncogenes: differences in the affinity of the alpha- and beta-forms for the acetic acid analog and failure of the human testis and kidney alpha-2 products to bind T3

We have compared the affinities for T3 and the T3 analog binding characteristics of the in vitro translational products of seven c-erbA cDNAs (chicken c-erbA alpha; human placental c-erbA beta; rat c-erbA beta-1; rat c-erbA alpha-1; rat c-erbA alpha-2; human testis c-erbA alpha-2; and human kidney c-erbA alpha-2). Four of these (chicken c-erbA alpha, human placental c-erbA beta, rat c-erbA beta-1, rat c-erbA alpha-1) bound T3 with high affinity as previously described. When compared under identical conditions of synthesis and [125I]T3 binding, there was no significant difference between the affinity of the chicken c-erb A alpha-1 and the human c-erbA beta but in a more limited series the affinity of rat c-erbA beta-1 for T3 was 4.6-fold higher than that of the rat c-erbA alpha-1. In vitro translational products of the beta-probes showed a characteristic 2.2-fold higher triiodothyroacetic acid/T3 ratio than did the products of the alpha-probes, regardless of the species of origin of the probe. As previously established, the rat c-erbA alpha-2 product did not bind T3. However, in contrast to two published reports, the human testis and kidney alpha-2 probe products also failed to bind T3. These findings indicate that highly conserved C-terminal 37-40 residues are important for high affinity T3 binding by proteins encoded by the c-erb A family of genes.     

Structure and inducible regulation of the human c-erb B2/neu promoter

Overexpression of the p185 product of the c-erb B2/neu gene is correlated with cell transformation and tumorigenesis. To study expression of this gene, a 1548-base pair fragment (-1571 to -24 bp relative to the ATG initiator codon) of the human c-erb B2/neu 5'-noncoding region was isolated, sequenced, and analyzed with respect to basal and inducible activity using the luciferase expression vector pSVOAL delta 5'. This fragment contained an Alu repetitive element which was confirmed in two independent clones. Basal activity of the 1548-base pair fragment was equivalent to the epidermal growth factor receptor promoter region and 32 and 16% as active as the Herpes simplex thymidine kinase and Rous sarcoma virus promoters, respectively. Induction of luciferase activity was observed in response to epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, dibutyryl cAMP, and retinoic acid. Additive and synergistic responses with more than 30-fold increases were observed after treatment with combinations of inducing agents, indicating complex regulation of this gene. These results show that the promoter region of the c-erb B2/neu gene contains sequences that dictate regulatory responses to several environmental signals.     

Recognition of a 56 kDa protein in partially purified rat hepatic nuclear thyroid hormone receptor by anti-human c-erb A beta antibody.

Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.     

A sensitive colorimetric detection of virus DNA and oncogene

Advantage of cloning probe DNA fragment in phage M13 DNA was taken to provide a larger single stranded DNA as a hybridization probe. High level of direct enzyme labels was introduced via the M13 DNA moiety as well as probe DNA. A highly sensitive colorimetric detection of virus DNA and oncogene was developed.     

Evidence for the presence of protein kinases which stimulate phosphorylation of c-erb A protein in rat kidney nuclei.

Protein kinases were separated from rat kidney nuclear extract by hydroxylapatite column chromatography. Five (I-V) different protein kinases were isolated when histone was used as a substrate. Two (I and III) of them stimulated phosphorylation of c-erb A-beta protein (50 kDa) expressed in Escherichia coli. The c-erb A product has an activity of high affinity T3 binding. One (I) of the kinases was dependent on cyclic adenosine 3',5'-monophosphate (cyclic AMP). The other kinase (III) was not dependent on cyclic nucleotides. The latter kinase was eluted from hydroxylapatite column with 0.05 M PO4 at pH 7.4. The sedimentation coefficient(s) estimated by continuous sucrose density gradient centrifugation was approximately 6.0 Km values for ATP were estimated by double reciprocal analyses, which gave 110.0 microM in the protein kinase I (in the presence of 10(-6) M cyclic AMP) and 25 microM in the protein kinase III, respectively. The data showed that 1.0 mol phosphate was incorporated into 80 mol of c-erb A protein (50 kDa) either in the presence of protein kinase I (with 10(-6) M cyclic AMP) or in the presence of protein kinase III. These results suggested that there are protein kinases for c-erb A protein, whose functional properties are similar to those of nuclear T3 receptor, in rat kidney nuclei.     

Quantitative and qualitative immunohistochemical detection of myc and src oncogene proteins in normal, nodule, and neoplastic rat liver

This study examined the possibility of using an immunohistochemical technique to detect the expression of myc and src oncogene proteins (ops) in livers of male Sprague-Dawley rats after treatment with the carcinogen diethylnitrosamine (with or without phenobarbital promotion) or untreated. We found that the majority of nodules and tumors from these livers stained for myc and src ops, indicating that myc and src expression did occur in these structures. These results were expected, since myc and src expression has been previously observed by others using different techniques. However, in our study, myc and src op staining was also noted in normal liver areas from rats in any of the four treatment groups (DENA, DENA + PB, PB alone, or untreated). The staining pattern of normal liver was different for each oncogene probe but was consistent within the four groups. In most cases, oncogene expression of normal liver occurred at sites of abnormal (but non-neoplastic) hepatocytes. The method reported here used both a qualitative technique of op expression analysis and a quantitative method using a Zeiss computer-driven image analysis system.     

Isolation of a transforming sequence from a human bladder carcinoma cell line

We have isolated the component of human bladder carcinoma cell DNA that is able to transform mouse fibroblasts. The oncogenic sequence was isolated initially from a lambda phage genomic library made from DNA of a transfected mouse cell carrying the human oncogene. A subcloned insert of 6.6 kb that carried transforming activity was amplified in the plasmid vector pBR322. The subcloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has failed so far to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human tumor cell lines derived from colonic and lung carcinoma and from neuroblastoma. In contrast, the EJ bladder oncogene appears closely related to one that is active in the human T24 bladder carcinoma cell line. The oncogene appears to have undergone little, if any, amplification in several bladder carcinoma cell lines.     

Single base mutation in the hormone binding domain of the thyroid hormone receptor beta gene in generalised thyroid hormone resistance demonstrated by single stranded conformation polymorphism analysis.

Thyroid hormone resistance is a syndrome of considerable clinical heterogeneity. Three mutations in the c-erb A beta gene encoding the human beta thyroid hormone receptor have been described in different kindreds. We report here, in a family affected with peripheral thyroid hormone resistance, a unique point mutation in the ligand binding domain of the c-erb A beta gene resulting in histidine replacement of an arginine residue at position 438. The region in which the mutation occurred was identified by single stranded conformation polymorphism analysis and confirmed by subcloning and sequencing of the mutant alleles from each of the affected members. Binding of tri-iodothyronine to isolated nuclei from family members was normal suggesting the mechanism of thyroid hormone resistance in this family is not mediated by abnormal binding of ligand and receptor.     

Oncogene activation induces metabolic transformation resulting in insulin-independence in human breast cancer cells

Normal breast epithelial cells require insulin and EGF for growth in serum-free media. We previously demonstrated that over expression of breast cancer oncogenes transforms MCF10A cells to an insulin-independent phenotype. Additionally, most breast cancer cell lines are insulin-independent for growth. In this study, we investigated the mechanism by which oncogene over expression transforms MCF10A cells to an insulin-independent phenotype. Analysis of the effects of various concentrations of insulin and/or IGF-I on proliferation of MCF10A cells demonstrated that some of the effects of insulin were independent from those of IGF-I, suggesting that oncogene over expression drives a true insulin-independent proliferative phenotype. To test this hypothesis, we examined metabolic functions of insulin signaling in insulin-dependent and insulin-independent cells. HER2 over expression in MCF10A cells resulted in glucose uptake in the absence of insulin at a rate equal to insulin-induced glucose uptake in non-transduced cells. We found that a diverse set of oncogenes induced the same result. To gain insight into how HER2 oncogene signaling affected increased insulin-independent glucose uptake we compared HER2-regulated gene expression signatures in MCF10A and HER2 over expressing MCF10A cells by differential analysis of time series gene expression data from cells treated with a HER2 inhibitor. This analysis identified genes specifically regulated by the HER2 oncogene, including VAMP8 and PHGDH, which have known functions in glucose uptake and processing of glycolytic intermediates, respectively. Moreover, these genes specifically implicated in HER2 oncogene-driven transformation are commonly altered in human breast cancer cells. These results highlight the diversity of oncogene effects on cell regulatory pathways and the importance of oncogene-driven metabolic transformation in breast cancer.