bluuebunny/crossref_metadata_2025 huggingface dataset:
Microwave‐Assisted Free Radical Copolymerizations of Styrene and Methyl Methacrylate
Authors: Stange Heiner , Greiner Andreas
Published: February 2007

Free radical copolymerizations of styrene and MMA were performed in toluene and DMF as solvents using different peroxide initiators with and without microwave irradiation. A general trend showed significant solvent dependence of monomer conversion rate only for copolymerizations initiated by tert‐butyl perbenzoate in DMF compared to toluene but nearly no impact for copolymerizations initiated by dicumyl peroxide and dibenzoyl peroxide. Significant variations in copolymer composition were not observed for reactions under conventional heating compared to microwave irradiation. It was concluded that enhanced initiator decomposition under microwave irradiation in DMF accelerate polymerization of styrene and MMA by formation of larger amount of initiator radicals.

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The Thermoviscoelastic Constitutive Equation of PP and PA Blends and Its Rate Temperature Equivalency at High Strain Rates
Authors: Shaoqiu Shi , Bing Yu , Lili Wang
Published: February 2007

By using Instron 1342 testing system and an improved SHPB technique, PP/PA blends are tested at a wide range of strain rates from 10−4 up to 103 s−1 and at a temperature range of 25–80 °C. Their mechanical responses are shown to be sensitive both to the strain rate and temperature. Based on the experimental data of 113 blends at a wide range of strain rates from 10−4 up to 103 s−1 and at a temperature range of 25–80 °C, it is shown that the mechanical behavior of this PP/PA blends can be described with ZWT thermoviscoelastic constitutive equation. The corresponding thermoviscoelastic parameters for 113 blends are obtained. The predicted theoretical results coincide quite well with the experimental data.The experimental results also reveal that the rate/time‐temperature equivalence relation is shown for PP/PA blends. Raising the temperature is equivalent to the increasing of time (the decreasing of strain rate). Conversely, decreasing the temperature is equivalent to the decreasing of time (the increasing of strain rate). Through introducing a dimensionless parameter $Z = {{\dot \varepsilon \eta _0 } \over E}\exp \left( {{A \over {kT}}} \right)$, two characteristic parameters: strain rate $\dot \varepsilon$ and temperature T are put together to this unified parameter Z. From experimental results, A/k is fitted, A/k = 8.595 °C, and a unified curve characterizing the rate/time –temperature equivalence relation is obtained.
Effect of Molecular Weight on Enthalpy Relaxation in Syndiotactic Poly(Methyl‐Methacrylate)
Authors: Sciucca Stefania Della , Penco Maurizio , Sartore Luciana
Published: February 2007

The structural relaxation behaviour of narrow fractions (Mw/Mn < 1.1) of syndiotactic poly(methyl methacrylate) with molecular masses ranging from 2,000 to 200,000 Daltons have been studied by DSC with two classical procedures, namely: the rate of cooling and the isothermal approaches. The apparent activation energy (Δh*) of enthalpy relaxation was evaluated from the dependence of the glass transition temperature on the cooling rate while a comparison of the apparent relaxation rates was appraised from the enthalpy loss by annealing the different samples at the same level of undercooling (Ta = Tg − 10 °C). As expected, the increase of molecular weights gives rise to both a continuous increase of Δh* and a decrease of the apparent isothermal relaxation rate. More interestingly, both Δh* and the apparent isothermal relaxation rate showed abrupt changes around the syndiotactic PMMA entanglement mass (Me
).
Synthesis and Characterization of Conductive Polypyrrole/Montmorillonite Nanocomposites via One‐pot Emulsion Polymerization
Authors: Peighambardoust S. J. , Pourabbas Behzad
Published: February 2007

A series of electrically conductive polypyrrole/clay nanocomposites were synthesized in this work by using one‐pot emulsion oxidative polymerization of pyrrole in the presence of unmodified clay and using DBSNa as the surfactant. The effect of surfactant on the morphological and electrical properties of PPy also were investigated and discussed in some extent. Electrical conductivity of the samples was measured by using samples in which the conductive materials was sandwiched between two Ni electrodes at room temperature. PPy/MMT nanocomposites were characterized by using XRD, TEM, TGA and DSC means of investigation. Intercalated structures were determined for the nanocomposites as confirmed by XRD and TEM studies. Electrical conductivity of the nanocomposites was measured to be dependent to the clay content, and the methods of preparation. Measurement also showed that polymerization of pyrrole monomers pre‐intercalated between the clay gallery spaces of the clay led to higher conductivity for the nanocomposite in the same level of clay content. Thermal property measurements showed a lower thermal decomposition rate for the PPy/MMT nanocomposites with respect to the PPy.
Study on the Effect of Uniaxial Elongational Flow on Polyamide Based Nanocomposites
Authors: Garofalo Emilia , Russo Giovanna Maria , Di Maio Luciano , Incarnato Loredana
Published: February 2007

This work focuses on the study of uniaxial elongational flow and its effects on morphology and stiffness of polyamide‐6 based nanocomposites prepared by melt compounding. The elongational flow characterization was realized by converging flow method and fiber spinning technique. During the haul‐off tests, fibers of the neat polyamide‐6 and the hybrids (at 3 and 6 wt% of silicate) were collected at different draw ratios. Mechanical properties of the produced fibers were investigated and correlated to their nanostructure through analytical techniques sensitive to different aspects of morphology, such as DSC and TEM analysis. Rheological results, obtained with a capillary rheometer, indicate that the shear viscosity decreases with the silicate loading, while the extentional viscosity increases. Moreover, the presence of the silicate in polymer matrix leads to enhancements of draw‐down force and reduction of the breaking draw ratio. In hybrid fibers an enhanced degree of exfoliation of the filler was observed upon drawing. Moreover, DSC analyses suggest that the crystalline structure of the fibers is the result of two opposite effects: the presence of the silicate which stabilizes the γ form and the drawing which promotes the α crystal phase. The degree of silicate exfoliation and the amount of the different crystal phases strongly affect the tensile properties of the fibers.
PMMA Based Nanocomposites Filled with Modified CaCO3 Nanoparticles
Authors: Avella Maurizio , Errico Maria Emanuela , Gentile Gennaro
Published: February 2007

PMMA based nanocomposites filled with calcium carbonate nanoparticles (CaCO3) have been prepared by in situ polymerization approach. In order to improve inorganic nanofillers/polymer compatibility, PBA chains have been grafted onto CaCO3 nanoparticle surface. Morphological analysis performed on nanocomposite fractured surfaces has revealed that the CaCO3 modification induces homogeneous and fine dispersion of nanoparticles into PMMA as well as strong interfacial adhesion between the two phases. Mechanical tests have shown that both unmodified and modified CaCO3 are responsible for an increase of the Young's Modulus, whereas only PBA‐grafted nanoparticles allow to keep unchanged impact strength, strongly deteriorated by adding unmodified CaCO3. Finally, the presence of CaCO3 nanoparticles significantly improves the abrasion resistance of PMMA also modifying its wear mechanism.
Thermal Properties of E‐beam Cured Epoxy/Thermoplastic Matrices for Advanced Composite Materials
Authors: Alessi S. , Dispenza C. , Spadaro G.
Published: February 2007

The aim of this work is to investigate the possibility to improve the thermal behaviour of epoxy based systems, cured by ionizing radiation, in order to produce matrices for advanced carbon fibres composites. Blends of two epoxy monomers, difunctional and trifunctional, have been polymerized by e‐beam irradiation and the dynamic mechanical thermal properties have been investigated. The increase of the concentration of the trifunctional epoxy monomer in the blend causes a marked increase of the Tg, but strongly decreases the reactivity. Subsequently, blends of the same epoxy monomers with a high Tg thermoplastic toughening agent have been considered and their reactivity and the thermal properties of the obtained materials have been also studied. The experimental results show that the thermoplastic component increases the reactivity of the difunctional/trifunctional blends, without a significant decrease of the glass transition temperature which still satisfies the requirements for advanced composite materials.
Mechanical and Thermal Analysis of Composites Based on Rubbers From Used Tires
Authors: Acierno Domenico , Ciccarelli Ireneo , Romano Vittorio , Russo Pietro
Published: February 2007

Summary: Reuse and recycling of tires at the end of their useful life is an environmental challenge owing to the huge volumes discarded all over the world. This paper tries to give a scientific contribution toward the mechanical recycling of elastomers derived from pre treatment of tires to produce anti‐shock tiles and heat‐insulating panels for civil applications. Thermal and mechanical results herein reported satisfactorily justify the interest devoted by now to this topic.
Deposition of Super‐Hydrophobic and Oleophobic Fluorocarbon Films in Radio Frequency Glow Discharges
Authors: Laguardia L. , Ricci D. , Vassallo E. , Cremona A. , Mesto E. , Grezzi F. , Dellera F.
Published: February 2007

Fluorocarbon films using a monomer, 1H, 1H, 2H‐perfluoro–1‐dodecene were deposited in a continuous radio frequency (RF) glow discharge, the process was carried out in a parallel‐plate RF discharge onto stainless steel reactor in order to produce coating with a water‐and oil–repellent surface. Fourier‐Transform Infrared spectroscopy (FT‐IR) and X‐ray Photoelectron Spectroscopy (XPS) revealed that the films obtained contain mainly perfluoromethylene (CF2) species. Film wettability was tested using water and hydrocarbon liquids for contact angle measurements, furthermore surface energy was also calculated. Oil‐repellency was found to increase as the amount of CF2 species increases in the film structure. Film morphology was studied by Atomic Force Microscopy (AFM), films showing an usual morphology from that typical of Plasma Polymerised Fluorocarbon (PPFC) films. The combination of the low surface energy coating and the surface morphology produces materials which are both water and oil repellency.
Methylsiloxane Oligomers with Oxyalkyl Fragments in the Side Chain
Authors: Mukbaniani O. , Zaikov G. , Tatrishvili T. , Titvinidze G. , Mukbaniani N.
Published: February 2007

The dehydrocondensation reactions of α,ω–bis(trimethylsiloxy)methylhydridesiloxane with saturated primary n‐alcohols in the presence of anhydrous powder‐like potassium hydroxide or platinum on the carbon (Pt/C‐5%) at 1:30 ratio of initial compounds, at various temperature (40–60 °C) was carried out, and methylsiloxane oligomers with n‐alkyloxy substituted groups in the side chain were obtained. It was shown that completely dehydrocondensation of all active SiH groups do not take place. Dehydrocondensation reaction order, activation energy and rate constants were found. The synthesized oligomers were characterized by 1H, 13C NMR, Cosy and FTIR spectra data. Gel‐permeation chromatography, differential scanning calorimetric, thermogravimetric and wide‐angle X‐ray investigations of synthesized oligomers were carried out.
Molecular analysis of mutations induced in human cells by N‐ethyl‐N‐nitrosourea
Authors: Eckert Kristin A. , Ingle Caroline A. , Klinedinst Donna K. , Drinkwater Norman R.
Published: February 2007

Mutational activation of cellular proto‐oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the on P‐tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N‐ethyl‐N‐nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein‐Barr virus (EBV) and is stably maintained as a plasmid in EBV‐transformed human lymphoblastoid cells. Populations of plasmid‐bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7–8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV‐tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC→Ã transitions and 17% were AT→GC transitions. In addition, the number of AT→TA (20%) and AT→CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O2‐ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT→TA transversion mutations in the activation of cellular proto‐oncogenes by ENU.
Phenobarbital specifically reduces gap junction protein mRNA level in rat liver
Authors: Mesnil Marc , Fitzgerald D. James , Yamasaki Hiroshi
Published: February 2007

The gene expression of liver major gap junction (GJ) protein was studied in rats systemically administered phenobarbital, a rat liver tumor promoter. Using a GJ protein cDNA and northern blot analysis, the level of GJ protein mRNA in liver was observed to be markedly reduced at 4 and 11 wk of phenobarbital exposure (0.1% in drinking water). However, the level of GJ protein mRNA was not altered in kidney at 11 wk of exposure. In liver, phenobarbital did not induce expression of the neoplasm‐associated marker genes glutathione S‐transferase (placental form) and γ‐glutamyltranspeptidase, while in kidney the observed expression of these genes was not changed. These in vivo results indicate that phenobarbital reduces GJ protein gene expression specifically in rat liver without altering expression of genes often altered during liver carcinogenesis, and they support assigning a role for the impairment of gap junctional intercellular communication in phenobarbital‐mediated liver tumor promotion.
Activated Ki‐ras genes in bladder epithelial cell lines transformed by treatment of primary mouse bladder explant cultures with 7,12‐dimethylbenz[a]anthracene
Authors: Brookes Peter , Cooper Colin S. , Ellis Moira V. , Warren William , Gardner Emily , Summerhayes Ian C.
Published: February 2007

DNA from five lines of transformed bladder epithelial cells derived from cultures of primary cells that had been treated with 7,12‐dimethylbenz[a]anthracene (DMBA) can transform NIH 3T3 mouse fibroblasts in DNA transfection experiments. Southern analysis of DNA from NIH 3T3 primary and secondary transformants established that four of the DMBA‐transformed cell lines contained activated cellular Ki‐ras, while the remaining cell line contained a transforming gene that is unrelated to Ki‐ras, N‐ras, and Ha‐ras. The point mutations responsible for Ki‐ras activation were detected using oligonucleotide probes following selective amplification of Ki‐ras specific sequences using the polymerase chain reaction. The results showed that activation of Ki‐ras invariably involved a GC → AT transition mutation of the first position of codon 12. Surprisingly, a Ki‐ras gene that was activated by a GC → AT transition mutation at the same position was also detected in a single transformed bladder urothelial cell line derived from control cultures of mouse bladder cells. Together, our results indicate that Ki‐ras activation in the DMBA‐transformed bladder cell lines may not be a direct consequence of interaction of activated DMBA metabolites with the Ki‐ras gene.
Modified responsiveness of v‐Ha‐ras‐transfected rat fibroblasts to growth factors and a tumor promoter
Authors: Huang Ming , Kamata Nobuyuki , Nose Kiyoshi , Kuroki Toshio
Published: February 2007

The correlation of the phenotypic changes of v‐Ha‐ras transfected cells with the expression of p21ras and the modified responses to growth factors and a tumor promoter were examined. Transfection of the v‐Ha‐ras gene together with the neomycin‐resistance gene into 208F rat fibroblasts yielded transformed clones characterized by morphological changes, anchorage‐independent growth, and tumorigenicity in nude mice. The degrees of these biological alterations were parallel with the expression of mRNA and protein of the ras gene. In rastransformed cells, anchorage‐independent growth was stimulated by epidermal growth factor (EGF), insulin, bombesin, and fibroblast growth factor, whereas in the parental 208F cells, anchorage‐independent growth was observed only in the presence of EGF, and there were many fewer EGF‐induced colonies than those in the ras‐transformed clones. A tumor promoter, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) also augmented anchorage‐independent growth of ras‐transformed cells and induced morphological changes in monolayer cultures without altering the expression of the ras gene or phosphorylation of the p21ras protein. Retinoic acid inhibited the TPA‐induced anchorage‐independent growth. These results showed a good correlation of the expression of p21ras with the phenotypic changes and the increased sensitivity of the p21ras‐expressing cells to the stimulation of growth factors and tumor promoter.
Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral harvey ras
Authors: Amstad Paul , Reddel Roger R. , Pfeifer Andrea , Malan‐Shibley Louise , Mark George E. , Harris Curtis C.
Published: February 2007

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v‐Ha‐ras‐containing retrovirus, Zip‐ras, was generated by inserting the coding region of the v‐Ha‐ras oncogene into the Zip‐NeoSV(X) [Cepko et al., Cell 37:1053–1062, 1984] retroviral vector. Amphotrophic Zip‐ras retrovirus was used to infect an SV40 large T antigen‐positive immortalized cell line, BEAS‐2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip‐ras—infected BEAS‐2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip‐NeoSV(X)—infected BEAS‐2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v‐Ha‐ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v‐Ha‐ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174–1179, 1985] is consistent with the hypothesis that the “immortalization” step is rate‐limiting in in vitro human epithelial cell carcinogenesis.
Isolation and characterization of the 5′ flanking region of the mouse C‐harvey‐ras gene
Authors: Brown Ken , Bailleul Bernard , Ramsden Martin , Krumlauf Frances Fee, Robb , Balmain Allan
Published: February 2007

The complete 5′ flanking region of the murine c‐Ha‐ras gene was cloned and sequenced. An untranslated exon (–1) was identified and the promoter region of the gene located. Like the rat and human homologues, the murine promoter is GC rich and contains several GC boxes together with a CAAT element, but lacks a TATA box, an arrangement similar to that found in many housekeeping genes. From primer extension studies, the gene was shown to have three transcriptional start sites, whose positions differ from those previously found for the human gene. No alterations in these start sites were detected between the normal gene and activated Haras genes from mouse skin tumors. A region of strong homology between mouse, rat, and human Ha‐ras genes exists within the large intron separating exon (‐1) from the first coding exon. In addition, from chloramphenicol acetyltransferase assays, the upstream region has promoter activity which appears to be enhanced by the inclusion of sequences within this intron.
Malignant progression of papilloma‐derived keratinocytes: Differential effects of the ras, neu, and p53 oncogenes
Authors: Dotto Gian Paolo , O'connell John , Patskan George , Conti Claudio , Ariza Aurelio , Slaga Thomas J.
Published: February 2007

The p117 keratinocyte cell line was derived in culture from chemically induced mouse papillomas. The benignly transformed nature of these cells was demonstrated by their ability to re‐form benign papillomas when grafted back onto the animal. Retroviral vectors were used to introduce into the p117 cells three distinct oncogenes: v‐Ha‐ras, p53, and neu. All three oncogenes were able to induce tumorigenic conversion of the p117 keratinocytes when assayed by subcutaneous injection into nude mice. However, grafting the oncogene‐transformed cells onto the back of the mouse revealed important differences in the ability of the three oncogenes to induce a fully malignant phenotype. While the ras‐transformed papilloma cells formed aggressive carcinomas, p53 and neu transformation yielded an intermediate phenotype, with formation of large exophytic tumors, not yet invasive but with highly dysplastic features remarkably similar to those of in situ carcinomas. These findings establish a homologous, genetically modifiable cell system in which various stages of malignant transformation can be directly compared.
Neoplastic transformation and lineage switching of rat liver epithelial cells by retrovirus‐associated oncogenes
Authors: Garfield Susan , Huber Brian E. , Nagy Peter , Cordingley Michael G. , Thorgeirsson Snorri S.
Published: February 2007

Tumors produced by a chemically transformed rat liver epithelial (RLE) cell line and its single cell‐derived clonal subpopulations demonstrate wide‐ranging morphological presentations including carcinomas, sarcomas, “mixed epithelial‐mesenchymal” tumors, and undifferentiated tumors [Am J Pathol 127:168–181, 1987]. To address the question of heterogeneity of tumors derived from transformed RLE cells, we have used recombinant retroviruses containing the following transforming oncogenes: v‐raf (3611‐MSV), v‐raf/v‐myc (J2), v‐myc (J5), and v‐Ha‐ras (pRNR16). All of the oncogenes, with the exception of v‐myc (J5), were efficient transforming agents in the RLE cells. Tumors derived from the v‐raf‐ and, to a lesser extent, those from v‐Ha‐ras—transformed RLE cells showed mixed epithelial‐mesenchymal morphology, whereas the combination of v‐raf/v‐myc (J2) consistently produced differentiated trabecular carcinomas. These data suggest that the lineage commitment of the RLE cells can be perturbed by a single transforming oncogene and that different tumor types derived from these cells may reflect the expression of a selective oncogene or a combination of oncogenes.
Activation of transcription as a general mechanism of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin action
Authors: Fisher Joan M. , Jones Keith W. , Whitlock James P.
Published: February 2007

We studied the response to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) of mouse hepatoma cells that contain a single, integrated copy of a chimeric gene under the control of a dioxin‐responsive DNA domain, which was originally associated with the cytochrome P450IA1 gene. Our findings indicate that TCDD increases the RNA polymerase II‐catalyzed transcription rate of the chimeric gene and that the transcripts are initiated at the correct promoter. Therefore, the dioxin‐responsive DNA operates as a bona fide transcriptional enhancer. Other studies imply that the Ah receptor mediates the transcriptional response to TCDD. Our results indicate that the Ah receptor‐dependent, dioxin‐responsive enhancer can activate transcription when in a regulatory context and in a chromosomal location different from those of the cytochrome P450IA1 gene. Therefore, in principle, the receptor‐enhancer system represents a mechanism by which numerous genes can respond to aromatic hydrocarbons in the environment.
Cotransfection of plasmids with ras and myc oncogenes to diploid cells derived from rodent fetuses: Alteration of neoplastic transformation frequency depending on the gestation period
Authors: Yagi Takashi , Sasayama Satoshi , Sasai Hitoshi , Kakunaga Takeo
Published: February 2007

To analyze the mechanism of neoplastic transformation of rodent diploid cells by ras and myc oncogenes, human EJ c‐Ha‐ras and mouse c‐myc second and third exons promoted by SV40 promoter were connected to pSV2neo and pSV2gpt, respectively. Mouse and rat primary fetal cells cotransfected with both genes formed transformed and nontransformed colonies in a medium containing G418 and myc ophenolic acid (MPA). The proportion of transformed colonies in the total G418/MPA‐resistant colonies decreased dependent on the stage of the gestation period of rat fetuses from which primary cells had been obtained. Analysis of randomly isolated colonies showed that the transformed colonies had a high copy number and high amount of expression of the introduced genes, were anchorage independent, and were tumorigenic in nude mice. On the other hand, the nontransformed colonies had a low copy number, low amount of expression, and no tumorigenicity. This contrast indicated not only that the activated Ha‐ras and myc oncogenes had been integrated, but also that the amplification or overexpression (or both) of these genes was required for the rodent diploid cells to be transformed. We conclude that early‐stage rat fetal cells might have endogenous factors that promote cell transformation. Alternatively, late‐stage cells might have factors that suppress cell transformation by activated Ha‐ras and myc oncogenes.
Spontaneous progression of hyperplastic outgrowths of the D1 lineage to mammary tumors: Expression of mouse mammary tumor virus and cellular proto‐oncogenes
Authors: Knepper Janice E. , Kittrell Frances S. , Medina Daniel , Butel Janet S.
Published: February 2007

Mammary cancer in mice is characterized by progression through defined stages of preneoplasia, with the most common preneoplastic stage being the hyperplastic alveolar nodule (HAN). We determined the relative levels of RNA expression of various cellular proto‐oncogenes and endogenous mouse mammary tumor virus genes in outgrowths and tumors of three sublines of the transplantable D1 HAN preneoplastic outgrowth line. The three sublines differed in relative tumor‐producing capabilities. Subline D1B produced a high incidence of tumors with short latency periods, whereas sublines D1C and D1D produced low incidences of tumors with long latency periods. No consistent alteration in proto‐oncogene expression correlated with relative tumorigenicity, although tumors frequently contained higher levels of one or more proto‐oncogene transcripts as compared with preneoplastic tissue. Slightly elevated (2‐ to 6‐fold) levels of different oncogene transcripts were detected in 13 of 17 tumors as compared with outgrowth tissue, including abl (2 tumors), fps (5 tumors), Ha‐ras (6 tumors), and Ki‐ras (8 tumors). One tumor contained 45 times more Ki‐ras‐specific RNA than outgrowth tissue because of a comparable amplification of Ki‐ras DNA sequences. Elevated levels of Ha‐ras occurred more frequently in tumors of a high‐incidence subline than in a less‐aggressive subline (5/10 vs 1/7), but this difference was not statistically significant. However, consistent changes in MMTV expression accompanied progression from preneoplastic tissues to mammary tumors. All 17 tumors displayed reduced levels of the MMTV‐specific long terminal repeat (LTR) transcript (1.6 kb) as compared with HAN tissue; tumors with moderate levels of LTR transcript expressed the 3.8‐kb envelope message as well, one not detected in HANs. Expression of the LTR transcript is apparently influenced by factors in addition to the methylation status of endogenous mouse mammary tumor virus genes, which was similar in outgrowths and tumors. As the survey of representative proto‐oncogenes failed to identify a uniform change between HAN and tumors, it is likely that other genes are involved in tumor progression in the mammary gland.
Preferential modification of GC boxes by benzo[a]pyrene‐7,8‐diol‐9,10‐epoxide
Authors: Kootstra Arend , Lew Linda Kao , Nairn Rodney S. , Macleod Michael C.
Published: February 2007

The distribution of binding sites for the ultimate carcinogen anti‐benzo[a]pyrene‐7,8‐diol‐9, 10‐epoxide(BPDE‐l) in the 5′ region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3′ and 5′ flanking regions was inserted into the BamHl site of the multiple cloning site of pGEM so that the T7 promoter was 5′ to the aprt gene. In vitro transcription of BPDE‐l‐modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC‐box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE‐I DNA binding sites in the 5′ flanking region of the aprt gene, we found that the DNA region containing the GC‐box consensus sequences was indeed a hot spot for BPDE‐I modification.
Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa
Authors: Koi Minoru , Morita Hiroyuki , Yamada Hideto , Satoh Hitoshi , Barrett J. Carl , Oshimura Mitsuo
Published: February 2007

We examined the ability of human chromosome 11 derived from normal fibroblast cells to suppress the tumorigenicity of SiHa cells, a human cervical tumor cell line. Using DNA transfertion, the human chromosome was tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic G418), transferred to mouse A9 cells by cell hybridization and microcell transfer techniques, and then transferred to SiHa cells by microcell transfer. These procedures resulted in the appearance of 15 independent, G418‐resistant clones, 5 of which had one or two extra copies of an intact human chromosome 11. In situ chromosomal hybridization of these clones with the pSV2neo plasmid revealed the presence of a neo‐tagged human chromosome 11 in all of the five SiHa‐microcell hybrids. Two SiHa‐microcell hybrids that contained a single copy of neo‐tagged human chromosome 12 were also isolated by the same methods. The tumorigenicities of SiHa clones with one or two extra copies of chromosome 11 (SiHa‐11) were suppressed; four of the five SiHa‐11 clones formed no tumors in nude mice, whereas both parental SiHa cells and SiHa cells with an extra chromosome 12 formed tumors within 30 d. One SiHa‐11 cell clone formed a single tumor 90 d after injection. This rare tumor had lost one copy of chromosome 11 and rapidly formed tumors when reinjected. These results indicate that the introduction of a single copy of normal human chromosome 11, but not chromosome 12, suppresses the tumorigenicity of SiHa cells, indicating the presence on human chromosome 11 of a putative tumor‐suppressor gene (or genes) for human cervical tumors.
Sequential trisomization of chromosomes 6 and 7 in mouse skin premalignant lesions
Authors: Aldaz C. Marcelo , Trono David , Larcher Fernando , Slaga Thomas J. , Conti Claudio J.
Published: February 2007

Using a direct cytogenetic technique, we identified a nonrandom trisomy of chromosome 6 in 12 of 12 aneuploid mouse skin papillomas and in 10 of 11 squamous cell carcinomas induced by chemical carcinogenesis. The second most common abnormality observed was trisomy of chromosome 7 found in most dysplastic papillomas and in 10 of 11 carcinomas. The two trisomies were the only abnormalities found in all aneuploid papillomas and in several carcinomas. Mutation at codon 61 of the Ha‐ras gene, which resides on chromosome 7, was also a common feature of the tumors sampled.

Extensive homology exists between mouse chromosome 6 and human chromosome 7, the trisomy of which was recently suggested as a primary cytogenetic event in several human epithelial cancers. We propose a multistep model of tumor progression in which a sequence of specific nonrandom chromosomal abnormalities appear to be required for malignant transformation.
Activation of the cellular harvey ras gene in mouse skin tumors initiated with urethane
Authors: Bonham Keith , Embry Travis , Gibson David , Jaffe Debbie R. , Roberts Robin A. , Cress Anne E. , Bowden G. Tim
Published: February 2007

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c‐Ha‐ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha‐ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha‐ras gene was shown to be activated by a specific A → T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha‐ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.
Expression of c‐raf‐1 and A‐raf‐1 during regeneration of rat liver following surgical partial hepatectomy
Authors: Silverman Jeffrey A. , Zurlo Joanne , Watson Michael A. , Yager James D.
Published: February 2007

The major objective of this study was to investigate the expression of members of the raf family of protooncogenes during rat liver regeneration. The steady‐state level of expression of both c‐raf‐1 and A‐raf‐1 increased three‐ to fivefold 18–24 h following partial hepatectomy, and it returned to basal levels by 72 h. Expression of c‐myc and Ha‐ras mRNA was increased at 3 and 18–24 h, respectively, confirming previous reports. Increased steady‐state levels of c‐raf‐1, A‐raf‐1, and Ha‐ras mRNA were also detected in hepatocytes isolated from rat liver 24 h after partial hepatectomy. Thus, elevated expression of the raf genes closely correlated with that of Ha‐ras, beginning at 12 h and reaching maximal levels during the first peak of DNA synthesis following partial hepatectomy.
Interleukin‐1 alpha gene intron containing variable repeat region coding for the SP1 transcription factor recognition sequence is polymorphic
Authors: Haugen Aage , Mann Dean , Murray Christine , Weston Ainsley , Willey James C.
Published: February 2007

Interleukin‐1 alpha (IL‐1α) is a cytokine produced by a number of cell types including macrophages, fibroblasts, keratinocytes, and mesangial cells. We were interested in identifying a DNA restriction fragment length polymorphism (RFLP) for the IL‐1α gene for use in studies of genetic alteration in various human cancers. Human genomic DNA from 32 unrelated individuals was digested with various restriction enzymes, alone and in combination, and subjected to Southern blot analysis. Hybridization to 32P‐labeled IL‐1α cDNA revealed an insertion deletion‐type polymorphic pattern. After digestion with Rsal, insertion‐deletion‐type polymorphic bands with sizes of 3.4 kb, 3.1 kb, and 2.8 kb and one invariant band of 0.8 kb were observed. These three alleles, designated A1, A2, and A3, had relative frequencies of 0.18, 0.06, and 0.78 with heterozygosity observed in 38% of the unrelated individuals studied. Evaluation of nine related individuals for this Rsal polymorphism was consistent with a Mendelian inheritance.

Comparison of restriction patterns following Southern analysis of DNA digested with several different enzymes showed that the polymorphic region resides within the sixth intron. Furthermore, this RFLP results from a variable length region containing multiple copies of a recognition sequence for SP1, an imperfect copy of viral enhancer elements, and an inverse and complementary sequence of the glucocorticoid receptor binding site. The identified polymorphism may be of value in analyses of chromosome 2 and may help to elucidate mechanisms by which IL‐1α transcription is regulated.
Activation of the c‐Ha‐ras‐1 proto‐oncogene by methylation in vitro with α‐acetoxy‐N‐nitrosodimethylamine
Authors: Hirani‐Hojatti Samin , Milligan Jamie R. , Kovnat Asya , Brown Ken , Archer Michael C.
Published: February 2007

α‐Acetoxy‐N‐nitrosodimethylamine, an activated derivative of the carcinogen N‐nitrosodimethylamine, methylated in vitro a plasmid containing the human c‐Ha‐ras‐1 proto‐oncogene, resulting in the generation of a transforming oncogene, assayed by transfection into NIH 3T3 cells. The resulting transformed cells were tumorigenic and metastatic in immune‐deprived mice. Further transfection using tumor DNA led to the formation of three secondary NIH 3T3 transformants. DNA from these secondary transformants contained human ras gene sequences. Two of the three secondary transformants contained G→A mutations at guanine 35 in codon 12, and the third secondary transformant retained the wild‐type sequence at codons 12 and 61. For the latter, the activating mutation was not determined. These results demonstrate that a simple methylating agent can activate a normal human ras proto‐oncogene to a transforming oncogene.
Potential role of the human Ha‐ras oncogene in the inhibition of gap junctional intercellular communication
Authors: El‐Fouly Mohamed H. , Trosko James E. , Chang Chia‐Cheng , Warren Stephen T.
Published: February 2007

The modulation of gap junctional intercellular communication (GJIC) plays an important role during tumor promotion. Several tumor‐promoting agents are known to inhibit this form of cellular coupling. In addition, tumor cells and cells expressing certain oncogenic products have been shown to exhibit inhibited or reduced GJIC. The Ha‐ras oncogene is expressed in a wide variety of human tumors from different tissues. Its p21 product is a membrane‐bound polypeptide, the function of which is not fully characterized. We tested the effects of the expression of the human c‐Ha‐ras‐1 oncogene, derived from the EJ/T4 bladder carcinoma cell line, on the ability of the Chinese hamster V79 cells to conduct gap Junctional communication. The Junctional competence was studied by two different methods, the scrape‐loading/dye transfer technique and the metabolic cooperation assay. The results indicate a strong correlation between the expression of p21 ras protein and the inhibition of gap Junctional function. Assuming that reversible inhibition of intercellular communication plays a role during tumor promotion and stable inhibition during the tumor progression phase of carcinogenesis, our data suggest that, while chemical tumor promoters and the ras oncogenes might work by different biochemical mechanisms, they both affect a critical cellular function; namely, GJIC.
Permanent conversion of mouse and human cells transformed by activated ras or raf genes to apparently normal cells by treatment with the antibiotic azatyrosine
Authors: Shindo‐Okada Nobuko , Makabe Osamu , Nagahara Hikaru , Nishimura Susumu
Published: February 2007

Azatyrosine [L‐sβ‐(5‐hydroxy‐2‐pyridyl)‐alanine], an antibiotic isolated from Streptomyces chibanensis, inhibited the growth of NIH 3T3 cells transformed by the activated human c‐Ha‐ras gene but did not significantly inhibit the growth of normal NIH 3T3 cells. Surprisingly, upon treatment with azatyrosine most of the transformed cells apparently became normal. These apparently normal cells, named revertant cells, grew in the presence of azatyrosine and stopped growing when they reached confluency, and their normal phenotype persisted during prolonged culture in the absence of azatyrosine. The revertant cells did not grow in soft agar and scarcely proliferated in nude mice.

The human c‐Ha‐ras gene present in transformed NIH 3T3 cells was still present in the revertant cells and was expressed to the same extent as in the original transformed cells, producing the same amount of activated p21.

Treatment with azatyrosine caused similar conversion of NIH 3T3 cells transformed by activated c‐Ki‐ras, N‐ras, or c‐raf to apparently normal cells, but NIH 3T3 cells transformed by hst or ret were not exclusively converted by azatyrosine. Human pancreatic adenocarcinoma cells, which are known to contain an amplified activated c‐Ki‐ras gene and an amplified c‐myc gene, were also converted to flat and giant revertant cells by treatment with azatyrosine.
Spontaneous Ha‐ras Gene Activation in Cultured Primary Murine Keratinocytes: Consequences of Ha‐ras Gene Activation in Malignant Conversion and Malignant Progression
Authors: Greenhalgh David A. , Welty David J. , Strickland James E. , Yuspa Stuart H.
Published: February 2007

The activation of the c‐Ha‐ras gene and its contribution to the tumorigenic phenotype were examined in cultured mouse keratinocytes and squamous tumors using transfection into NIH 3T3 cells and nucleic acid hybridization. When normal keratinocytes were cultured in medium with 0.05 mM Ca2+ (low Ca2+ medium), many cells died within 2–3 wk, while others formed rapidly growing foci that could be subcultured. These rapidly growing cells produced benign tumors when grafted to nude mice and possessed a heterozygous mutation in the c‐Ha‐ras gene with an A‐>T transversion in codon 61. Fibroblast‐conditioned low Ca2+ medium prevented cell death, focus formation, c‐Ha‐ras gene mutation, and tumorigenicity. Thus, suboptimal culture conditions favored a spontaneous mutation in codon 61 of the c‐Ha‐ras gene of keratinocytes. When a v‐Ha‐ras gene was introduced into normal keratinocytes by a replication‐defective retrovirus, the recipient cells produced papillo‐mas in vivo, and after 2 mo, 60% of the tumors converted to squamous cell carcinomas. None of the 22 converted tumors had an endogenous c‐Ha‐ras gene mutation at codon 61. However, the A‐>T transversion mutation developed when these carcinoma cells were cultured in low Ca2+ medium but not in fibroblast‐conditioned medium. Cells with both an exogenous v‐Ha‐ras and an activated c‐Ha61‐ras gene produced undifferentiated, rapidly lethal carcinomas, while cells with only v‐Ha‐ras maintained the squamous carcinoma phenotype. Undifferentiated carcinomas also developed when the v‐Ha‐ras gene was introduced into papilloma cells with a chemically induced endogenous c‐Ha61‐ras gene mutation. These results suggest that mutation in the c‐Ha‐ras gene can contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis, and gene dosage may determine the phenotype expressed.
DNA Damage Stimulates Human Cell Transformation by Integrative But Not Episomal Epstein‐Barr Virus‐Derived Plasmid
Authors: Vos Jean‐Michel H. , Wauthier Eliane L. , Hanawalt Philip C.
Published: February 2007

Previous work has demonstrated that ultraviolet (UV) irradiation of SV40‐based plasmids can strikingly enhance the frequency of stable transformation of human cells. In this study we compared the effect of UV‐induced DNA damage on transformation mediated by integrative versus autonomously replicating plasmids derived from human Epstein‐Barr virus (EBV). We report that transfection of human fibroblasts with UV‐irradiated integrative EBV‐based plasmid results in enhanced transformation. However, transfection of UV‐damaged episomal EBV‐based constructs into the same human cell line does not enhance transformation; in fact, the extrachromosomal status of the plasmid is maintained irrespective of the UV dose to the plasmid. We conclude that enhanced transformation of human cells by damaged DNA requires its chromosomal integration.
Identification of a Transformation‐Sensitive Nuclear Protein From Normal Human Fibroblasts That Specifically Interacts With Minute Virus of Mice DNA and Correlates With Cell Resistance to the Parvovirus
Authors: Avalosse Bernard L. , Barrijal Saïd , Chen Yong Q. , Cassiman Jean‐Jacques , Rommelaere Jean
Published: February 2007

Normal human fibroblasts (MRC‐5, KMS‐6) were compared to transformed derivatives induced by SV40 (MRC‐5V1) or γ rays (KMST‐6) for the expression of nuclear proteins that interact with the genome of minute virus of mice (MVMp), using the southwestern blot technique. A protein of 100–104 kDa apparent molecular weight was found to form a specific complex with MVMp DNA and to have an especially high affinity for the 3' terminal portion of the viral genome. This protein (p102) was differentially expressed by normal and transformed cells, i.e., its availability or DNA binding activity (or both) was much reduced in the transformants. A high level of p102 cosegregated with resistance to MVMp in cell hybrids between normal human and transformed mouse fibroblasts. Taken altogether these data suggest that the p102 protein may be a candidate for a transformation‐sensitive cellular marker and for a negative regulator of parvovirus replication.
Early Events in Methyl Methanesulfonate Enhancement of Adenovirus Transformation of Cloned Rat Embryo Fibroblast Cells
Authors: Su Zao‐Zhong , Fisher Paul B.
Published: February 2007

Pretreatment of a cloned rat embryo fibroblast (CREF) cell line with methyl methanesulfonate (MMS) prior to infection with a specific host‐range and cold‐sensitive type 5 adenovirus mutant (H5hr1), results in a unique carcinogen enhancement of transformation (CET) phenotype (Carcinogenesis 8:967, 1987). By using low‐density clonal assays and in situ hybridization techniques with 32P‐labeled type 5 adenovirus (Ad5) probes, we demonstrated that 5–10 d following infection the proportion of CREF colonies containing H5hr1 DNA and RNA is increased two‐to threefold as a result of pretreatment with MMS. Twenty‐five days following infection of CREF cells, Ad5 DNA assays showed that both solvent and MMS‐pretreated CREF colonies no longer contained detectable levels of viral DNA or RNA. Analysis of free viral DNA by the Hirt procedure suggested that more free viral DNA persisted in MMS‐pretreated H5hr1‐infected CREF cells than in solvent‐pretreated H5hr1‐infected CREF cells. The relative amount of free viral DNA in both types of cultures was directly related to the multiplicity of H5hr1 infection and decreased with time following infection. As observed using in situ hybridization techniques, by 25 d after infection no free viral DNA was detected in either MMS‐or solvent‐pretreated H5hr1‐infected CREF cells. By using a protein synthesis inhibitor (cycloheximide) and an RNA transcription inhibitor (actinomycin D), it was further demonstrated that the ability of MMS to induce a unique CET in CREF cells following infection with H5hr1 was dependent on the synthesis of new protein and RNA. In contrast, inhibition of protein and RNA synthesis did not alter the de novo rate of H5hr1 transformation of CREF cells. Temporal kinetic studies indicated that the ability of MMS to enhance H5hr1 transformation of CREF cells and to increase the percentage of CREF colonies containing Ad5 genetic information is regulated in a strict temporal manner. The results of the present investigation suggest that the ability of MMS to enhance H5hr1 transformation of CREF cells is dependent on the induction of new protein(s) in CREF cells, and enhancement is associated with an increase in the proportion of cells in the infected CREF cell population that initially contain Ad5 DNA/RNA.