Promoter region of the human Harvey ras proto-oncogene: similarity to the EGF receptor proto-oncogene promoter.

Regulation of transcription of members of the ras gene family undoubtably plays an important role in controlling cellular growth. Examination of this level of regulation requires identification of the promoter regions of the ras proto-oncogenes. Four major transcriptional start sites were detected in the human Harvey ras 1 proto-oncogene. The promoter region contains neither a TATA box nor a CAAT box in their characteristic upstream positions, has an extremely high G+C content (80 percent), and contains multiple GC boxes including seven CCGCCC repeats and three repeats of the inverted complement, GGGCGG. This region has strong promoter activity when placed upstream from the chloramphenicol acetyl transferase gene and transfected into monkey CV1 cells. In these ways the Harvey ras 1 proto-oncogene promoter resembles the promoter of the gene encoding the epidermal growth factor (EGF) receptor. The similarity between the two proto-oncogene promoters may be relevant to the mechanism by which the expression of such "growth control" genes is regulated.

Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter.

Members of the ras gene family encode proteins that when overproduced or mutated can transform immortalized mammalian cells. It is therefore important to understand the mechanisms by which the ras genes are regulated. The promoter region of the human Harvey ras proto-oncogene c-Ha-ras1 initiates RNA transcription at multiple sites and contains repeated copies of the hexanucleotide GGGCGG and its inverted complement CCGCCC, referred to as GC boxes. These GC boxes consist of sequences identical to those found in the SV40 early promoter, where the human cellular transcriptional factor Sp1 binds. Footprinting analysis with deoxyribonuclease I was used to show that Sp1 binds to six GC box sequences within the c-Ha-ras1 promoter. An in vivo transfection assay showed competition between the 21-base pair repeats of the SV40 promoter and the c-Ha-ras1 promoter for common regulatory factors. In this system the presence of Sp1 is apparently required for c-Ha-ras1 transcription. Analysis of deletions of the c-Ha-ras1 promoter region by means of a transient expression assay revealed that the three Sp1 binding sites closest to the RNA start sites were sufficient for full transcriptional activity.

Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells.

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).

Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene.

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 x 10(5) EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 10(7) focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.

Elevated epidermal growth factor receptor gene copy number and expression in a squamous carcinoma cell line.

The human epidermal growth factor (EGF) receptor is known to be homologous to the v-erb B oncogene protein of the avian erythroblastosis virus. Overexpression of the EGF receptor gene in A431 epidermoid carcinoma cells is due to gene amplification. In this study, a variety of squamous cell carcinomas were examined and one, SCC-15, contained high levels of the EGF receptor as determined by immunoprecipitation via an EGF receptor-specific polyclonal antibody. Using a cloned EGF receptor complementary DNA as a probe, the level of EGF receptor RNA was found to be elevated four-fold in SCC-15 relative to normal cultured keratinocytes. When the same probe was used to identify EGF receptor gene fragments on a genomic DNA blot, the SCC-15 cell line was shown to possess an EGF receptor gene copy number amplified four to five times. Gene amplification results in the enhancement in the level of the EGF receptor in several carcinomas and could be responsible for the appearance of the transformed phenotype in these cells.

Pancreatic gastrin stimulates islet differentiation of transforming growth factor alpha-induced ductular precursor cells.

Gastrin is transiently expressed in fetal islets during a critical period of their development from protodifferentiated islet precursors in fetal pancreatic ducts. To examine the possible role of gastrin as an islet cell growth factor, postnatal islet growth was studied in transgenic mice which overexpress gastrin and TGF alpha in their pancreas. Overexpression of a TGF alpha transgene causes metaplastic ductules containing numerous insulin expressing cells that resemble protodifferentiated precursors of the fetal pancreas. However, islet mass of the TGF alpha transgenic mice was not increased. Pancreatic overexpression of gastrin from a chimeric insulin/gastrin transgene transcribed from the insulin promoter markedly decreased the TGF alpha-stimulated increase in pancreatic duct mass. Furthermore, pancreatic coexpression of both gastrin and TGF alpha significantly increased islet mass in mice expressing both transgenes. These findings indicate that TGF alpha and gastrin can act synergistically to stimulate islet growth, although neither peptide alone is sufficient. Islet growth may possibly be stimulated through gastrin promoting the differentiation of insulin-positive cells in the TGF alpha-induced metaplastic ducts. This transgenic study suggests that islet neogenesis can be reactivated in the ductular epithelium of the adult pancreas by local expression of two growth factors, gastrin and TGF alpha.

Modulation of epidermal growth factor receptor proto-oncogene transcription by a promoter site sensitive to S1 nuclease.

The epidermal growth factor (EGF) receptor is the functional target of the mitogen EGF and the cellular homolog of the avian erythroblastosis virus erbB oncogene product. Regulation of expression of the proto-oncogene encoding the EGF receptor can be elucidated by studying the structure and function of the gene promoter outside the confines of the cell. Previously, we reported the isolation of the human EGF receptor gene promoter. The promoter is highly GC rich, contains no TATA or CAAT box, and has multiple transcription start sites. An S1 nuclease-sensitive site has now been found 80 to 110 base pairs (bp) upstream from the major in vivo transcription initiation site. Two sets of direct repeat sequences were found in this area; both conform to the motif TCCTCCTCC. When deletion mutations were made in this region of the promoter by using either Bal 31 exonuclease or S1 nuclease, we found that in vivo activity dropped three- to fivefold, on the basis of transient-transfection analysis. Examination of nuclear protein binding to normal and mutated promoter DNAs by gel retardation analysis and DNase I footprinting revealed that two specific factors bind to the direct repeat region but cannot bind to the S1 nuclease-mutated promoter. One of the specific factors is the transcription factor Sp1. The results suggest that these nuclear trans-acting factors interact with the S1 nuclease-sensitive region of the EGF receptor gene promoter and either directly or indirectly stimulate transcription.

Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia.

The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.

Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efficacy, potency, and toxicity.

A common form of multidrug resistance in human cancer results from expression of the MDR1 gene which encodes a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice which express this multidrug transporter in their bone marrow cells and demonstrated that peripheral WBC of these animals provide a rapid and reliable system for assessing the bioactivity of agents that reverse multidrug resistance. Immunocytochemical analysis of bone marrow smears suggests that the activation of the MDR1 transgene has probably occurred at a very early stage of bone marrow differentiation since most bone marrow cells express the transporter. Expression of this transgene in bone marrow produces about 10-fold resistance to leukopenia induced by taxol compared to normal bone marrow. Chemosensitization of MDR1 mice to daunomycin and taxol, measured by a fall in WBC, is detectable at a dose as low as 0.01 mg/kg R-verapamil. A dose of 0.5 mg/kg R-verapamil reduces the WBC by nearly 50%. Chemosensitization of MDR-transgenic mice with 5 mg/kg R-verapamil, which is highly effective in reversing MDR and readily tolerated by mice, necessitates a reduction of the maximum tolerated dose of most chemotherapeutic agents by only 20%. In addition, detailed histopathological examination shows that treatment of mice with chemotherapeutic drugs and R-verapamil does not change the organ-related toxicity pattern but only moderately accentuates inherent toxic side effects of the chemotherapeutic agents. We conclude that MDR1-transgenic mice represent a valid model for evaluating efficacy, potency, and toxicity associated with chemotherapy and chemosensitization of multidrug-resistant cells in animals.

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA.

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.

Characterization of five members of the actin gene family in the sea urchin.

Hybridization of an actin cDNA clone (pSA38) to restriction enzyme digests of Strongylocentrotus purpuratus DNA indicates that the sea urchin genome contains at least five different actin genes. A sea urchin genomic clone library was screened for recombinants which hydridize to pSA38 and four genomic clones were isolated. Restriction maps were generated which indicate that three of these recombinants contain different actin genes, and that the fourth may be an allele to one of these. The restriction maps suggest that one clone contains two linked actin genes. This fact, which was confirmed by heteroduplex analysis, indicates that the actin gene family may be clustered. The linked genes are oriented in the same direction and spaced about 8.0 kilobases apart. In heteroduplexes between genomic clones two intervening sequences were seen. Significant homology is confined to the actin coding region and does not include any flanking sequence. Southern blot analysis reveals that repetitive DNA sequences are found in the region of the actin genes.

'Northern Cross' hybridization for rapid identification of exon-containing restriction fragments.

The Northern Cross method allows direct comparison of restriction digests of cDNA and genomic clones to RNA populations by a specialized form of hybridization. This technique is based on the use of Northern and Southern blotting techniques and requires the use of two nylon membranes of differing chemical characteristics. A nylon membrane containing permanently affixed, electrophoretically fractionated RNAs is contact-hybridized at a right angle to a second, chemically different nylon membrane containing transiently bound, fractionated labeled DNA fragments. RNA and DNA bands possessing homology will hybridize where they cross, forming an autoradiographically detectable spot. This Northern Cross procedure proportionately represents the amounts of different RNAs derived from a particular sequence in a manner similar to what would have been observed in a Northern blot. This method, which can be used in the analysis of even relatively rare RNA species, permits rapid and fairly inexpensive identification of exon-containing fragments or determination of the relationship between related, multiple RNA species.

Epidermal growth factor receptor regulation and function.

The epidermal growth factor (EGF) receptor is a transmembrane, cell-surface glycoprotein that mediates the mitogenic action of a family of ligands, including EGF and transforming growth factor alpha (TGF alpha). Perturbation of this signal transduction pathway by exposure to excess ligand, by overproduction of the normal EGF receptor, or by the presence of specific mutated forms of this receptor can result in dramatic alterations in cellular phenotype, including malignant transformation. Overstimulation of normal cells is avoided by precise control of the synthesis and degradation of EGF receptors. Regulation occurs at multiple levels, including transcriptional control. A number of DNA-binding proteins have now been identified which positively and negatively modulate EGF receptor gene transcription.

Transgenic animals in biomedical research.

The advent of transgenic technology, in which foreign genetic information is stably introduced into the mammalian germ line, has dramatically enhanced our basic knowledge of physiologic and pathologic processes. Transgenic animals created by these genetic manipulations are being used to provide insights into gene regulation, development, pathogenesis, and the treatment of disease. Furthermore, transgenic biotechnology holds great promise for the creation of genetically superior livestock and the industrial production of precious pharmaceuticals. It is evident now that the study and use of transgenic animals will significantly improve the human condition.

Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

Methylation status of epidermal growth factor receptor gene in lung carcinoma cells.

The methylation status of the epidermal growth factor receptor (EGFR) gene was compared in cell lines from four major types of lung carcinoma, small cell lung carcinoma (SCLC), large cell lung carcinoma, squamous cell carcinoma and adenocarcinoma, in order to examine whether DNA methylation is responsible for the suppression of EGFR gene in SCLC cells. Southern blot analysis revealed that the structural region of the EGFR gene is methylated to various degrees regardless of the expression of EGF receptor on the surface. An 8-kilobase EcoRI fragment which contains the EGFR gene promoter region is readily digested with various methylation-sensitive restriction enzymes in all types of cells, indicating that the EGFR gene 5' region is barely methylated. Thus, a mechanism other than DNA methylation appears to control EGFR gene expression and the lack of EGFR gene expression in SCLC cells may be caused by a paucity of some transcription regulatory factor(s).

Molecular insights into breast cancer from transgenic mouse models.

We desperately need to know more of the biological details of the onset and progression of breast cancer. The disease is of startlingly high incidence (approaching 1 in 9 women), our current therapies for the disease are inadequate once it has metastasized, and the disease is characterized by excessive morbidity and mortality. Most of the growth and differentiation of the mammary gland occurs relatively late in life: during sexual maturation, and then cyclically during pregnancy and lactation. Normal as well as malignant growth is regulated by endocrine hormones as well as by local tissue factors, such as polypeptide growth factors. Cancer seems to progress as hyperplastic ductal or lobular epithelial growth, acquiring progressive genetic changes (including those of oncogenes and tumor suppressor genes) leading to clonal outgrowths of progressively more malignant cells. The nature of proliferative controls and the relevant genetic changes are the subjects of the current review.

Rapid development of hepatic tumors in transforming growth factor alpha transgenic mice associated with increased cell proliferation in precancerous hepatocellular lesions initiated by N-nitrosodiethylamine and promoted by phenobarbital.

The carcinogenic and tumor-promoting effects of human transforming growth factor alpha (TGF-alpha) overexpression were examined in a two-stage chemical carcinogenesis protocol using TGF-alpha transgenic mouse line MT42. Male MT42 and CD-1 mice received a single i.p. injection of 5 mg N-nitrosodiethylamine (DEN)/kg body wt at 15 days of age, and were placed on a diet containing 0.05% of phenobarbital (PB) from 4 weeks of age for 35 weeks. DEN-, PB-treated and saline-injected animals in each strain were used as controls. A total of three sequential sacrifices (at 10, 23 and 37 experimental weeks) was performed. Hepatocellular carcinomas (HCCs) developed earlier at high incidence (100%) after 23 experimental weeks in MT42 mice receiving DEN/PB, while CD-1 mice had a 40% incidence of HCCs only after week 37. HCCs also developed in the DEN-initiated MT42 mice at 80% incidence after week 23, but no HCCs were observed in the DEN-initiated CD-1 mice. PB induced preneoplastic foci (67%), adenomas (33%) and HCCs (33%) after 37 weeks in MT42 mice, but no lesions were found in CD-1 mice. Thus, the carcinogenic response to DEN and/or PB was accelerated in the MT42 transgenic mice. Furthermore, PB promotion was observed from week 10 in MT42 mice and week 23 in CD-1 mice. Thus, the promoting effect of PB was also accelerated in the MT42 transgenic mice. Proliferating cell nuclear antigen (PCNA) labeling indices of hepatocellular foci and adenomas in DEN- or DEN/PB-treated MT42 mice were significantly higher than those of CD-1 mice. TGF-alpha expression determined by immunohistochemistry revealed higher levels in these lesions than in hepatocytes of surrounding parenchyma of MT42 transgenic mice. In conclusion, TGF-alpha transgenic mice clearly demonstrated enhanced sensitivity to the development of hepatocellular carcinoma in the DEN initiation and PB promotion regime, possibly through a mechanism of increased hepatocyte proliferation in precancerous lesions (foci and adenomas), driven by high expression of the mitogen TGF-alpha in these lesions.