bluuebunny/crossref_metadata_2025 huggingface dataset:
Transfection of a Rat Cytochrome P450b cDNA Into C3H10T1/2CL8 Mouse Embryo Fibroblasts
Authors: Hansen Suzanne K. , Ross Jeffrey A. , Siegfried Jill M. , Leavitt Sharon , Rudo Kenneth , Langenbach Robert , Nesnow Stephen
Published: February 2007

A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418‐resistance selectable marker. This bifunctional plasmid (pJRSL 100) was transfected into the C3H 10T1/2CL8 mouse embryo fibroblast cell line. G418‐resistant clones were selected and tested for enhanced sensitivity to the carcinogen 2‐acetylaminofluorene (2‐AAF), a compound that does not normally induce cytotoxicity or morphological transformation in these cells. One subclone, 19P450b‐4, exhibited an increased cytotoxic response to 2‐AAF compared to the parental C3H10T1/2CL8 cells. DNA analyses of this subclone showed increased number of copies of the cytochrome P450b and the appearance of unique restriction fragment bands relative to parental and control transfected cells. This subclone also exhibited increased levels of mRNA complementary to the P450b cDNA. Metabolism studies of 2‐AAF in this subclone demonstrated an increase in the C‐hydroxylated metabolites 1‐, 3‐, 5/9‐, and 7‐hydroxy‐AAF compared with parental C3H 10T1/2CL8 cells. The results indicate that C3H 10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfection may enhance the usefulness of the C3H 10T1/2CL8 cells in studies on chemically induced cytotoxicity and morphological transformation.
Solubilization of SV40 Plasma‐Membrane‐Associated Large Tumor Antigen Using Single‐Phase Concentrations of 1‐Butanol
Authors: Maxwell Steve A. , Santos Myriam , Wong Connie , Rasmussen Graciela , Butel Janet S.
Published: February 2007

The nature of the interaction of the simian virus 40 (SV40) transforming protein, large tumor antigen (T‐ag), with the plasma membrane of transformed cells is not well understood. We report here that SV40 plasma‐membrane‐associated large tumor antigen (pmT‐ag) can be solubilized by using single‐phase concentrations of 1‐butanol. Purified plasma membranes from SV40‐transformed mouse cells yielded T‐ag when treated with 2.5% butanol; solubilization of T‐ag from the purified membranes in butanol was temperature dependent, with approximately 10‐fold more T‐ag extracted at 37°C than at 22°C; and application of 2.5% butanol to mKSA cells after cellular surface proteins had been radiolabeled with 125I resulted in the release of iodinated T‐ag. Butanol‐extracted pmT‐ag coprecipitated with p53 and several cellular proteins ranging in size from 35 to 60 kDa. One cellular component migrated at a mobility similar to that of tubulin (56 kDa), and a monoclonal antibody against the a subunit of tubulin coprecipitated T‐ag. Immunoblotting of proteins immunoprecipitated with monoclonal antibodies against T‐ag or p53 from butanol extracts with a monoclonal antibody against the β subunit of tubulin revealed specific coprecipitation of tubulin with T‐ag and p53. This suggests that complexes composed of tubulin, T‐ag, and p53 exist in butanol extracts. Control experiments eliminated the possibility of an artifactual association of tubulin with T‐ag and p53 induced by butanol. Two‐dimensional gel analyses revealed that 2.5% butanol at 37°C extracted a subset of membrane‐associated proteins and some cytosolic proteins, as well as a number of proteins that were not soluble in either high salt or detergent. Thus, the butanol extraction conditions employed in this study recovered a species of pmT‐ag that appears to complex with tubulin. As butanol reportedly is less deleterious to native protein structures than other agents, including high salts and detergents, this extraction procedure may be useful for studying the structure and function of other membrane‐associated proteins.
Insulin‐Induced Phosphorylation of the Beta‐4 Integrin Subunit Expressed on Murine Metastatic Carcinoma Cells
Authors: Falcioni Rita , Perrotti Nicola , Piaggio Giulia , Kennel Stephen K. , Sacchi Ada
Published: February 2007

A tumor surface protein (TSP‐180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS‐PAGE under reducing conditions, TSP‐180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP‐180 complex are glycosylated and are labeled by lactoperoxidase‐catalyzed radioiodination of viable cells. The mouse TSP‐180 complex described here is homologous to the human integrin α6 β4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the β4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP‐180 (MoAb 135–13C) stimulates cell growth in vitro and induces phosphorylation of the 204‐kDa protein. We now report that insulin increases the phosphorylation of the 204‐kDa protein 30‐fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulinlike growth factor (IGF‐I) and platelet‐derived growth factor have no effect. The effect of insulin and of IGF‐I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF‐I each induced a fivefold increase in the phosphorylation of their respective receptor β subunits. In order to test if phosphorylation of the 204‐kDa protein was induced by direct binding of growth factors to TSP‐180 and to identify growth factor receptors on line 1 cells, affinity cross‐linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF‐I both bind to a 135‐kDa protein that corresponds to the insulin and IGF‐I receptor α subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175‐kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204‐kDa protein purified from insulinstimulated cells demonstrated that this β4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that β4 integrin molecule is a target for phosphorylation through an indirect receptor‐mediated mechanism.
Isolation and characterization of kirsten murine sarcoma virus‐transformed mouse keratinocytes resistant to transforming growth factor beta
Authors: Sipes Nancy J. , Lyons Russette M. , Moses Harold L.
Published: February 2007

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sar coma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor‐β (TGF‐β). When MK and KC cells were maintained in the presence of TGF‐β, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF‐β resistance, the TGF‐β‐resistant cells (KCR cells) were compared with TGF‐β‐sensitive KC cells with regard to growth properties, TGF‐β1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF‐β1 concentrations up to 2 nM, which was 500‐fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF‐β1, we observed differences in affinity cross‐linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c‐Ki‐ras and v‐Ki‐ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF‐α and TGF‐β, mRNA was similar in MK, KC, and KCR cells. The expression of proliferation‐associated genes, such as c‐myc and MGSA/c‐gro/kc, which were markedly decreased by TGF‐β1, in the MK and KC cells, was not altered by TGF‐β1 in the KCR cells. The data suggest that the loss of TGF‐β1 responsiveness in the KCR cells was due to an alteration in the TGF‐β receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.
Development of an in vitro model of tumor progression using v‐raf and v‐raf/v‐myc transformed rat liver epithelial cells: Correlation of tumorigenicity with the downregulation of specific proteins
Authors: Worland Peter J. , Hampton Lori L. , Thorgeirsson Snorri S. , Huggett Anthony C.
Published: February 2007

A series of clonal cell lines were derived from rat liver epithelial cells after being infected with a defective retrovirus containing either v‐raf (3611‐MSV) or v‐raf/v‐myc(J2) together with a helper virus. These clones exhibited a different morphology from the regular cuboid shape of the control cells, infected only with the helper virus. All of the infected cell lines contained at least one full length copy of appropriate proviral DNA and expressed comparable levels of v‐raf mRNA, although only the cells transformed with the v‐raf/v‐myc combination were capable of anchorage‐independent growth in soft agar. All of the clones except the controls formed tumors in nude mice but with markedly different latency periods and growth rates. Thus, these cell lines represent an in vitro model for tumor progression. Two‐dimensional polyacrylamide electrophoresis was used to investigate changes in cellular protein expression related to malignant conversion. The expression of three proteins of pl/Mr × 10−3 5.9‐7.2/205 (RP1), 6.5‐7.5/160 (RP2) and 4.0/85 (RP3) consistently matched the transformed phenotype. In particular the expression of RP1 and RP2 correlated with the relative tumorigenicity of the cell lines. Rat liver epithelial cell lines transformed by other protocols that did not involve v‐raf also showed downregulation of these three polypeptides. Crude fractionation studies determined RP1 to be soluble and RP2 and RP3 to be membrane associated. RP2 was shown to be a glycoprotein containing mannose and galactose residues. These three proteins are consistent markers for the tumorigenic potential of rat liver epithelial cells.
Chromosome site‐specific immunohistochemical detection of DNA adducts in N‐acetoxy‐2‐acetylaminofluorene‐exposed chinese hamster ovary cells
Authors: Olivero Ofelia A. , Huitfeldt Henrik , Poirier Miriam C.
Published: February 2007

In these studies a polyclonal antiserum elicited against a carcinogen‐DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N‐(deoxyguanosin‐8‐yl)‐2‐aminofluorene (dG‐C8‐AF) in Chinese hamster ovary (CHO) cells exposed to the direct‐acting carcinogen N‐acetoxy‐2‐acetylaminofluorene (N‐Ac‐AAF) was accomplished by indirect immunofluorescence with an anti‐G‐C8‐AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti‐BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N‐Ac‐AAF for 0.5 h during early S phase, the chromosomal pattern of dG‐C8‐AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N‐Ac‐AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.
Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts
Authors: Hyatt Susannah L. , Klauck Theresa , Jaken Susan
Published: February 2007

Transformed cells differ from normal cells in that they fail to respond to normal signals for regulation of growth and differentiation. This disordered signal transduction probably contributes to maintenance of the transformed phenotype. Several lines of evidence suggest that changes in the Ca2+‐ and phospholipid‐dependent protein kinase, protein kinase C (PKC), may be important for transformation. To determine the role of PKC in transformation, we compared the levels and subcellular distribution of total phorbol ester receptors and PKC in normal and SV40‐transformed rat embryo fibroblasts (REF52 cells). We also used our α‐PKC (Type 3)‐specific monoclonal antibodies to compare α‐PKC content and regulation. We found no differences in quantity or subcellular distribution of PKC in 100,000 × g soluble and pelletable fractions. Downmodulation, which represents a feedback loop for limiting PKC activity, occurs to the same extent in both cell types. A major difference between the normal and transformed cells was revealed by immunofluorescence of α‐PKC. In normal cells, α‐PKC is tightly associated with the cytoskeleton and appears to be organized into focal contacts because it colocalizes with talin. In contrast, in SV40‐REF52 cells, α‐PKC is not tightly associated with the cytoskeleton and does not colocalize with talin. The difference in subcellular localizations correlates with a loss of two α‐PKC‐binding proteins in the transformed cells. These results indicate that inappropriate subcellular location of α‐PKC may contribute to maintenance of the transformed phenotype.
Infection of rat liver epithelial cells with v‐Ha‐ras: Correlation between oncogene expression, gap junctional communication, and tumorigenicity
Authors: De Feijter Adriaan W. , Ray Jean S. , Weghorst Chris M. , Klaunig James E. , Goodman Jay I. , Chang Chia C. , Ruch Randall J. , Trosko James E.
Published: February 2007

The role of the v‐Ha‐ras oncogene in tumorigenesis in an in vitro/in vivo model system was studied by investigating the expression of the Ha‐ras gene, gap junctional intercellular communication, and tumorigenicity as endpoints. Infection of a Fischer 344 rat liver epithelial cell line (WB 344) with a retrovirus containing the v‐Ha‐ras oncogene resulted in altered cell morphology and decreased contact sensitivity. Gap junctional intercellular communication in v‐Ha‐ras infected WB cells (WBHa‐ras), assessed by fluorescence redistribution after photobleaching (FRAP), microinjection/dye transfer, and scrape‐loading/dye transfer techniques, was markedly decreased compared with the level in control WB cells. Injection of 107 WBHa‐ras cells into the portal vein of male F344 rats caused multiple focal hepatic lesions within 1 and 2 wk, merging to large invading tumors after 3 and 4 wk. Examination of the methylation pattern of the Ha‐ras gene in WBHa‐ras and control WB cells showed that the infected Ha‐ras gene was relatively hypomethylated in comparison to the normal cellular Ha‐ras gene, indicating a greater potential for expression. There was an increased level of Ha‐ras mRNA in hepatomas as compared with both adjacent nontumor liver tissue and liver tissue obtained from normal animals. Three cell lines derived from three different primary hepatic tumors induced by an injection of WBHa‐ras cells in a F344 rat displayed similar growth characteristics, levels of gap junctional communication, and methylation patterns as the original WBHa‐ras cells. The results of these studies have established a strong positive correlation between expression of the Ha‐ras oncogene, reduced gap junctional intercellular communication, decreased contact sensitivity, and tumorigenicity of the v‐Ha‐ras‐infected rat liver epithelial cells.
Tissue‐specific activating mutations of Ha‐ and Ki‐ras oncogenes in skin, lung, and liver tumors induced in mice following transplacental exposure to DMBA
Authors: Loktionov Alexandre , Hollstein Monica , Martel Nicole , Galendo Dominique , Cabral J. Ricardo P. , Tomatis Lorenzo , Yamasaki Hiroshi
Published: February 2007

Transplacental carcinogenesis represents a good model in which to study the involvement of tissue‐specific oncogene activation in carcinogenesis because a single exposure to a carcinogen induces tumors at various sites. We tested tumors of the skin, liver, and lung produced in mice after transplacental 7, 12‐dimethylbenz[a]‐anthracene (DMBA) exposure for possible activation of ras genes. Xbal restriction fragment‐length polymorphism analysis has shown that exposure to DMBA in utero may result in appearance of A → T transversion at the second position of codon 61 of Ha‐ras oncogene in skin and liver tumors but not in lung tumors. Moreover, DNA samples isolated from spontaneous and DMBA‐induced lung and liver tumors were analyzed for mutations at the same position of Ki‐ras oncogene using differential hybridization with specific oligonucleotides. Among five spontaneous lung tumors, three cases of A → G transition, and one case of A → T transversion were found, whereas four of ten lung tumors of DMBA‐treated animals were positive for A → T mutation. No Ki‐ras mutation was detected in one spontaneous and four DMBA‐induced hepatomas. In two cases, we revealed Ki‐ras A → T mutation in the lung tumor and Ha‐ras mutation in the liver tumor taken from the same animal. These results indicate first that DMBA treatment may induce A → T mutation at the second position of codon 61 both in Ha‐ras and in Ki‐ras and, second, that the role of different activated oncogenes in carcinogenesis may differ, depending on the tissue in which the tumor develops.
Activation of c‐Ki‐ras not detectable in adenomas or adenocarcinomas arising in rat pancreas
Authors: Schaeffer Barbara K. , Zurlo Joanne , Longnecker Daniel S.
Published: February 2007

The objective of this study was to determine whether activation of c‐Ki‐ras occurs in carcinogen‐induced rat pancreatic tumors. DNAs from 27 samples, which included adenomas, carcinomas in situ, and adenocarcinomas arising in azaserine‐treated rats and corn oil‐gavaged rats along with a nafenopin‐induced rat pancreatic adenocarcinoma were examined for mutation of c‐Ki‐ras at codons 12, 13, and 61 by using the polymerase chain reaction. Our results indicate that activation of c‐Ki‐ras is not a common event during pancreatic carcinogenesis in the rat.
Point mutation in codons 12 and 61 of the ha‐ras gene in rat urinary bladder carcinomas induced by N‐[4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl]formamide
Authors: Masui Tsuneo , Mann Angela M. , Garland Emily M. , Okamura Takehiko , Johansson Patrik L. , Cohen Samuel M.
Published: February 2007

Male F344 rats were fed N‐[4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl]formamide (FANFT) for up to 4 wk, then were given the basal diets (Prolab 3200 or AIN‐76A) with or without 5% sodium saccharin for up to 100 wk. Eleven transitional cell carcinomas (TCCs), one undifferentiated carcinoma, and two sarcomas of the urinary bladder were examined for the expression of ras gene product, p21, by immunohistochemical staining and western blot analysis. Point mutation in codons 12 or 61 of the Ha‐ras genes amplified by polymerase chain reaction was examined by a slot‐blot screening procedure using allele‐specific oligonucleotide probes. Immunohistochemical staining showed enhanced immunoreactivity with the antibody to ras p21 in seven TCCs and one undifferentiated carcinoma. Western blot analysis showed faster migration of the p21 band in 6 of 11 TCCs. Oligonucleotide hybridization revealed the point mutation in codon 12 of Ha‐ras gene (GGA → GTA in 1 TCC) and in codon 61 (CAA → CGA in 5 TCCs and CAA → CTA in 1 TCC). Two mutations in codons 12 and 61 coexisted in one tumor, which were found to be present in different Ha‐ras alleles. The incidence of Ha‐ras gene mutations were similar in groups treated with (3 of 6) or without (3 of 8) sodium saccharin. These results suggest the involvement of activated Ha‐ras gene in rat urinary bladder carcinogenesis induced by FANFT.
DNA sequence analysis of gamma radiation—induced deletions and insertions at the APRT locus of hamster cells
Authors: Miles Carol , Sargent Geoffrey , Phear Geraldine , Meuth Mark
Published: February 2007

Gamma radiation–induced gene rearrangements at the Chinese hamster ovary cell locus coding for the purine salvage enzyme adenine phosphoribosyl transferase (APRT) consist of both simple deletions and more complex alterations that are presumably the result of multiple strand breaks. To characterize these mutations at the DNA sequence level, fragments altered by deletion and insertion mutations were obtained by cloning in λ phage vectors or by using the polymerase chain reaction. The radiation‐induced deletions characterized here eliminate 3–4 kb and have at least one breakpoint in an AT‐rich region or near short direct or inverted repeats. Insertions involve small fragments (102 and 456 bp) of repetitive DNA that appear to be related to B2 (short interspersed repetitive) and long interspersed repeat families. The novel fragments bear little resemblance to each other or to sequences at the integration sites, and their introduction is accompanied by a small target site deletion.
Deletion mapping of tumor promotion–susceptibility gene pro1 implicates an RNA polymerase III transcription unit
Authors: Garrity Robert R. , Hegamyer Glenn A. , Seed John L. , Colburn Nancy H.
Published: February 2007

The murine gene pro1 has been cloned from JB6 epidermal cell lines that are sensitive to neoplastic transformation by tumor promoters. Insensitive JB6 variants acquire susceptibility to neoplastic transformation by tumor promoters when transfected with pro1. The repetitive nature of pro1 was indicated by sequence and Southern analysis. In contrast, northern analysis of RNA from promotion‐sensitive cells revealed the presence of a small pro1‐hybridizing transcript. Strand‐specific RNA probes implicated an RNA polymerase III (RNAPIII) coding domain in pro1 as the source of this hybridization signal. Ribonuclease protection of gel‐purified pro1 RNA from JB6 variant cell lines identified a 130‐nucleotide transcript. The size of this transcript is compatible with in vitro RNAPIII transcription of pro1. Deletion mapping of pro1 by exonuclease III demonstrated that the biologically active domain included the RNAPIII transcription unit. RNA probes map pro1 RNA within the activity domain. These results delineate an activity domain of 597 nucleotides and suggest that a small RNA is the product of promotion‐sensitivity gene pro1.
Amplification and expression of the c‐erb B‐2/neu proto‐oncogene in human bladder cancer
Authors: Zhau Haiyen E. , Zhang Xin , Von Eschenbach Andrew C. , Scorsone Kathleen , Babain R. Joseph , Ro Jae Y. , Hung Mien‐Chie
Published: February 2007

The c‐erb B‐2/neu gene encodes a cell‐surface glycoprotein with extensive homology to, but distinct from, the epidermal growth‐factor receptor. In this study, we compared the c‐erb B‐2/neu gene amplification and expression of tissue specimens obtained from the bladders of normal controls and patients with high‐grade transitional cell bladder carcinoma. Southern blot analysis of DNAs from 24 patients and 5 controls showed 2 cases of c‐erb B‐2/neu gene amplification in patients and none in controls. Western blot analysis demonstrated that c‐erb B‐2/neu was expressed in 67.6% (23/34) of patient specimens but in none of the controls (0/5). This finding agreed with the result of immunohistochemical staining, which showed that tissue from 74.3% (26/35) of the patients and none of the controls (0/7) showed positive immunofluorescence staining. This is the first report suggesting that c‐erb B‐2/neu gene amplification and expression may be associated with human bladder carcinogenesis.
Aberrant expression of gap junction gene in primary human hepatocellular carcinomas: Increased expression of cardiac‐type gap junction gene connexin 43
Authors: Oyamada Masahito , Krutovskikh Vladimir A. , Mesnil Marc , Partensky Christian , Berger Françoise , Yamasaki Hiroshi
Published: February 2007

The expression of connexin 32 (the major liver gap junction protein) and connexin 43 (the major cardiac gap junction protein) was examined in six surgically removed human hepatocellular carcinoma tissues and the surrounding nontumorous livers using specific rat connexin probes. No decrease in connexin 32 mRNA expression was found in carcinomas compared with the surrounding nontumorous tissue. Morphometrical analysis also showed that in most of the carcinomas the number of gap junction spots stained with connexin 32 antibody was not less than that in the surrounding livers. These results are in striking contrast to the significant reductions in connexin 32 mRNA and protein expression observed in rat primary liver tumors induced by chemicals. On the other hand, all of the six human hepatocellular carcinomas exhibited elevated levels of connexin 43 mRNA, which was expressed at a very low level in the surrounding nontumorous livers. These carcinomas exhibited no detectable amplification of the connexin 43 gene. The present study suggests that gap junctional intercellular communication is altered in human hepatocellular carcinomas by molecular mechanisms different from those in rat hepatocarcinogenesis.
Ki‐ras activation and expression in transformed mouse lung cell lines
Authors: Pan Y. H. Laurie , Nuzum Edwin O. , Hanson Laurie A. , Beer David G.
Published: February 2007

The Ki‐ras proto‐oncogene is activated by specific point mutations and is the transforming gene often identified in rodent and human lung tumors. An in vitro model to aid in the study of the consequences of Ki‐ras activation and expression in mouse lung is needed. Accordingly, we have examined cell lines derived from chemically induced mouse lung tumors as well as spontaneous transformants of untreated mouse lung epithelial cells. The specific Ki‐ras‐activating gene mutations and the level of mRNA expression were examined for each cell line. Polymerase chain reaction and oligonucleotide hybridization were used to demonstrate that five of seven transformed lung cell lines contain codon 61 Ki‐ras‐activating mutations, resulting in an arginine substitution for wild‐type glutamine. One transformed line contained this activating mutation and had also lost, or contained an altered, wild‐type codon 61 Ki‐ras allele. No codon 12 Ki‐ras mutations were observed. Two transformed and two nontransformed epithelial lung cell lines contained only the wild‐type codon 12 and 61 Ki‐ras alleles. Northern blot analysis demonstrated that the Ki‐ras mRNA was present in all the cell lines and was overexpressed in some, but not all, of the transformed lung cell lines. Those transformed lines with the highest levels of Ki‐ras mRNA also expressed more H4‐histone mRNA, suggesting that these cells have a greater proliferation rate. The level of Ki‐ras mRNA increased during the proliferation of the nontransformed lung cells but then decreased upon reaching confluency. In contrast, the level of Ki‐ras mRNA in the transformed lung cells was high during both growth and confluency, suggesting a potential defect in the regulation of Ki‐ras in these cells. These lung cell lines will help provide a better understanding of the regulation of both the Ki‐ras proto‐oncogene and oncogene in the lung.
Elevated expression of secondary, but not early, responding genes to phorbol ester tumor promoters in papillomas and carcinomas of mouse skin
Authors: Hashimoto Yu , Tajima Osamu , Hashiba Hiroki , Nose Kiyoshi , Kuroki Toshio
Published: February 2007

A single topical treatment of mouse skin with the potent tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) results in transient inductions of a variety of genes. Based on the time courses of their inductions, these genes can be classified into two main groups: “early” response genes whose mRNA expression reaches a maximum 0.5‐2h after TPA treatment and “secondary” response genes whose mRNA expression is maximal 4 h or more after treatment. The nuclear oncogenes c‐fos, c‐myc, and c‐jun belong to the early response group, whereas the metallothionein, osteopontin, and urokinase genes belong to the secondary response group. The steady‐state expressions of these early and secondary response genes are all very low in normal skin, except that of c‐jun, which is relatively high. Steady‐state levels of expression and inducibility of these genes by TPA were not altered in initiated skin or in apparently normal skin during tumor promotion. We examined the expressions of these genes in papillomas and carcinomas produced by two‐stage (initiator‐promoter) and three‐stage (initiator‐promoter‐initiator) protocols in mouse skin. Steady‐state expression of the early responding nuclear oncogenes in papillomas and carcinomas was found to remain at the same low level as in normal skin. However, all the secondary responding genes were found to be expressed constitutively at high levels in these tumors. Elevated expressions of the genes for transforming growth factor α and β were also observed in papillomas and to varying extents in carcinomas. These observations suggest that the regulatory machinery for transcription by the protein kinase C‐me‐diated pathway through nuclear oncogenes is altered during the processes of tumor promotion and progression. The genes whose expression is elevated may be associated directly or indirectly with tumor promotion and progression.
Effects of ionizing radiation on expression of genes encoding cytoskeletal elements: Kinetics and dose effects
Authors: Woloschak Gayle E. , Shearin‐Jones Pocahontas , Chang‐Liu Chin‐Mei
Published: February 2007

We examined the modulation in expression of genes encoding three cytoskeletal elements (β‐actin, γ‐actin, and α‐tubulin) in Syrian hamster embryo (SHE) cells following exposure to ionizing radiations. Early‐passage SHE cells were irradiated in plateau phase with various low doses (12‐200 cGy) of neutrons, γ‐rays, or x‐rays. RNA samples were prepared from cells at different times postexposure and were analyzed for levels of specific transcripts by northern blots. The results revealed that α‐tubulin was induced by both high‐linear energy of transfer (LET) (neutrons) and low‐LET (γ‐rays and x‐rays) radiations with similar kinetics. The peak in α‐tubulin mRNA accumulation occurred between 1 and 3 h postexposure; for γ‐actin mRNA, accumulation was similarly induced. For both γ‐actin and α‐tubulin, the higher the dose during the first hour postexposure (up to 200 cGy γ‐rays), the greater the level of mRNA induction. In contrast, mRNA specific for β‐actin showed decreased accumulation during the first hour following radiation exposure, and remained low up to 3 h postexposure. These results document the differential modulation of genes specific for cytoskeletal elements following radiation exposure. In addition, they demonstrate a decrease in the ratio of β‐actin:γ‐actin mRNA within the first 3 h following γ‐ray exposure. These changes in mRNA accumulation are similar to those reported in some transformed cell lines and in cells treated with tumor promoters, which suggests a role for changes in actin‐ and tubulin‐mRNA expression in radiation‐mediated transformation.
Activated Ki‐ras proto‐oncogene in spontaneously transformed and chemical tumor‐derived cell lines related to the mouse lung alveologenic carcinoma
Authors: Leigh Don A. , Ferguson Vicki , Bentel Jacqueline M. , Miller Juliet O. , Smith Garry J.
Published: February 2007

An in vitro cell model of mouse lung alveologenic carcinoma consisting of preneoplastic nonmalignant cells, spontaneously transformed cells, and urethane‐induced malignant cells was analyzed for phenotypic and genotypic changes associated with the transition to neoplasia. The polymerase chain reaction (PCR) was used to amplify the cDNA derived from the c‐Ki‐ras mRNA corresponding to exons 1 and 2 of the proto‐oncogene. This approach allowed analysis of the gene transcription product rather than potentially unexpressed DNA. Direct sequencing of the PCR product identified a common single point mutation, alone or together with wild‐type mRNA for c‐Ki‐ras, in all of the malignant clones. An A → G transition in the second base position of codon 61 was common to spontaneously malignant and chemical tumor‐derived cell lines and to lines selected for lung metastatic behavior. The absence of the mutation in the nonmalignant cells suggests that the Ki‐ras mutation may be a factor in onset or maintenance of the malignant phenotype and, at least in vitro, may be a late event in the transformation process.
Detection of loss of heterozygosity in the p53 gene in renal cell carcinoma and bladder cancer using the polymerase chain reaction
Authors: Oka Koji , Ishikawa Jiro , Bruner Janet M. , Takahashi Rei , Saya Hideyuki
Published: February 2007

The human p53 gene, a putative tumor suppressor gene, has a polymorphism in amino acid residue 72. We recently developed a method of detecting codon 72 polymorphism in this gene by digestion of polymerase chain reaction–amplified DNA using an allele‐specific restriction endonuclease. This polymorphism allows the identification of loss of heterozygosity for the coding region of the p53 gene in limited tissue samples in a short time without using radioactive materials. We examined 33 patients with renal cell carcinoma and 29 with bladder cancer; heterozygosity in the p53 gene was lost in 60% (6 of 10 cases) and 73% (8 of 11 cases) of the renal and bladder tumors, respectively. Additionally, the assay's sensitivity could be improved by using DNA extracted from frozen sections of the tumors. Because the proportions of tumor cells and nontumor cells could be assessed by microscopic evaluation of the frozen sections, we were able to minimize contamination from nontumor cells, which occasionally causes false readings of retained heterozygosity. This simple and sensitive method for detecting loss of heterozygosity in the p53 gene makes it possible to rapidly screen a large number of tissue samples and has the potential to be a useful diagnostic tool for a wide variety of human neoplasms.
Transformation of Human Breast Epithelial Cells by c‐Ha‐ras Oncogene
Authors: Basolo Fulvio , Elliott James , Tait Larry , Chen Xiao Qin , Maloney Terry , Russo Irma H. , Pauley Robert , Momiki S. , Caamano Jorge , Klein‐Szanto Andres J. P. , Koszalka Maria , Russo Jose
Published: February 2007

To determine whether the c‐Ha‐ras oncogene plays a role in the initiation of mammary carcinogenesis, an immortalized human breast epithelial cell line, MCF‐10A, was transfected with the plasmid vector pHo6T1 containing the T24 Ha‐ras oncogene and the aminoglycoside phosphotransferase gene, which confers resistance to geneticin. Transfected cells exhibited an altered pattern of growth and tridimensional morphology in collagen gel. They also exhibited anchorage‐independent growth and loss of requirement for hormones and epidermal growth factor; in addition, they expressed invasiveness and increased collagenolytic activity in an in vitro system and became tumorigenic in irradiated nude mice, all properties indicative of malignant transformation. Transformed cells contained the mutated c‐Ha‐ras oncogene and expressed the p21 mutated protein. These data indicate that the c‐Ha‐ras oncogene is capable of inducing malignant phenotypes in immortalized human breast epithelial cells.
Expression of ras genes in rainbow trout liver
Authors: Mangold Kathy , Chang Yung‐Jin , Mathews Catherine , Marien Koenraad , Hendricks Jerry , Bailey George
Published: February 2007

Members of the ras gene family have been studied in a variety of species. Two ras genes expressed in the normal liver of rainbow trout, ras‐1 and ras‐2, as well as a portion of a genomic ras‐1 allele, are described for the first time in this report. Over 500 bp of trout ras‐1 and at least 300 bp of trout ras‐2 genes expressed in normal liver have been sequenced; DNA homology to the human ras genes ranges from 76.8% to 87.1%. The base changes resulting from over 400 million years of evolutionary divergence between the species were primarily silent, with few changes in protein sequence. The partial DNA sequence of the genomic ras‐1 allele has 86.8% homology to the first two exons of human c‐Ha‐ras, and its intron has several conserved sequences characteristic of vertebrate intron‐exon junctions. However, the predicted amino acid sequence of trout ras‐1 differs at only one of the first 172 amino acid residues from human c‐Ki‐ras with the alternate exon 4b. Since trout ras‐1 differs at 17 and 18 residues from the human c‐N‐ras and c‐Ha‐ras proteins, respectively, over this region, we conclude that trout ras‐1 is a c‐Ki‐ras gene. The highly conserved nature of this gene suggests that the ras p21 protein has identical functions in normal and neoplastic cells among higher and lower vertebrates.
Structural analysis of the mouse c‐HA‐ras gene promoter
Authors: Plumb Mark , Telliez Jean‐Baptiste , Fee Frances , Daubersies Pierre , Bailleul Bernard , Balmain Allan
Published: February 2007

Previous studies have demonstrated that the mouse c‐Harvey ras proto‐oncogene (c‐Ha‐ras,) promoter sequences are GC rich and contain several potential transcription factor SP1 binding sites. We investigated the endonuclease hypersensitivity of this region in nuclei in vitro and whole mouse tissues in vivo and identified a very strong, ubiquitous hypersensitive site covering the proximal promoter sequences. Footprint protection studies using nuclear extracts from various cell types including fibroblasts, erythroid cells, and both normal and transformed epithelial cells revealed a consistent protein‐binding pattern. Five protein binding sites were observed, four of which correlated with potential SP1 binding sites. Competition experiments using an oligonucleotide corresponding to a consensus SP1 binding site confirmed that these sequences were indeed bound by the SP1 (or SP1‐like) trans‐acting factor. In addition, no differences were observed between the footprint patterns obtained using extracts from cells of different lineages or between normal and transformed epithelial cells carrying activated ras genes. The controlling elements responsible for differential c‐Ha‐ras transcription between cell types or at different stages of carcinogenesis therefore probably lie in other regions of the gene.
Dietary N‐3 fatty acids do not affect induction of Ha‐ras mutations in mammary glands of NMU‐treated rats
Authors: Ronai Zeev , Lau Yoyen , Cohen Leonard A.
Published: February 2007

The purpose of this study was to determine whether dietary n‐3 polyunsaturates (PUFA) can affect the frequency of N‐nitrosomethylurea (NMU)‐induced Ha‐ras mutations in virgin female F344 rat mammary glands. Two groups of 15 rats each were fed isocaloric diets containing either 23% w/w corn oil or corn oil plus menhaden oil (1:1) at starting 14 d before NMU administration (day 50 of age) and continuing for 13 wk. Mammary gland samples were taken serially at 3, 5, 9, and 13 wk post‐NMU treatment. Total cellular DNA was isolated and analyzed by a newly devised enriched amplification procedure that involves predigestion of normal Ha‐ras alleles at codon 12, amplification of the mutant alleles, and restriction fragment length polymorphism analysis. Selective amplification enabled the detection of Ha‐ras mutations as early as 3 wk post–NMU treatment. Approximately 40‐50% of all glands and 75% of all rats tested had the Ha‐ras codon 12 mutation. No significant differences were found between the two dietary groups at any time point, indicating that the mammary tumor‐inhibiting effect of n‐3 PUFA is probably not exerted at the level of the Ha‐ras activation step in NMU tumorigenesis.
Alterations in DMA‐restriction enzyme interactions by O4‐alkyldeoxythymidines
Authors: Richardson Frank C , Richardson Katherine K.
Published: February 2007

O4‐Alkyldeoxythymidines have been extensively studied for their ability to cause mutations and to induce cancer. Since these adducts can change DNA conformation, they may also have a more immediate effect of altering DNA‐protein interactions. To address this issue, the effects of these adducts on restriction enzyme activity were examined. Oligodeoxyribonucleosides containing O4‐ethyldeoxythymidine (O4‐EtdT) or O4‐methyldeoxythymidine (O4‐MedT) at a unique site within the sequence 5′‐GAATGGATCCTAATGAGATC‐3′ were constructed by automated DNA synthesis. This sequence contains the recognition site for various restriction enzymes. These oligomers were annealed to various complementary strands and digested with restriction enzymes: BamHI or BstI (GGATCC); Sau3A, Ndell, or Mbol (GATC); Dpnl (GmATC); and BstYl, Mfll, or Xholl (PuGATCPy). Analysis of the digests demonstrated that the presence of either O4‐EtdT or O4‐MedT abolished the ability of Xholl, Mbol, Mfll, or Ndell to cut at the restriction site. Dpnl failed to cut any of the oligomers. BamHI, Sau3A, Bstl, and BstYI exhibited alterations in cutting specificity depending upon the oligomers used. These results demonstrated that O4‐alkyldeoxythymidine adducts alter DNA‐restriction enzyme interactions in a protein‐ and sequence‐dependent manner. Because of the importance of natural methylation in genetic regulation, it is possible that aberrant methylation in the form of DNA adducts could also alter protein‐DNA interactions in cells exposed to DNA‐modifying agents.
Suppression of adenovirus type 5 E1A‐mediated transformation and expression of the transformed phenotype by caffeic acid phenethyl ester (CAPE)
Authors: Su Zao‐Zhong , Grunberger Dezider , Fisher Paul B.
Published: February 2007

Viral transformation and DNA‐transfection assays were employed to investigate the differential toxic effect of caffeic acid phenethyl ester (CAPE), an extract of the honeybee hive product propolis, on adenovirus type 5 (Ad5)‐transformed cloned rat embryo fibroblast (CREF) cells. CAPE inhibited, in a dose‐dependent manner, both de novo and carcinogen‐enhanced transformation of CREF cells by H5hr1, the cold‐sensitive (cs) host‐range mutant of Ad5. CAPE had a selective inhibitory effect on Ad5‐induced transformation when a wild‐type (wt) Ad5 E1A gene or a es Ad5 E1A gene (at 37°C, but not at 32°C) was cotransfected into CREF cells with a dominant‐acting bacterial hygromycin‐resistance gene. A requirement for the expression of Ad5 E1A‐encoded mRNAs and transforming proteins and sensitivity to CAPE was demonstrated using CREF cells stably transformed by a cs Ad5 E1A gene and an Ad5 E1A gene under the transcriptional control of a mouse mammary tumor virus promoter. To distinguish between the effects of the two Ad5 E1A‐encoded proteins of 289 amino acids (aa) and 243 aa, CREF cells were stably transformed with cDNAs encoding either the 13S or the 12S E1A mRNA. CREF cells expressing the 13S E1A‐encoded 289‐aa protein were more sensitive to the growth‐suppressing effect of CAPE than cells producing only the 12S E1A‐encoded 243‐aa protein. However, the growth‐suppressing and toxic effects of CAPE were greatest in cells expressing both E1A‐encoded transforming proteins. Analysis of the effect of CAPE on E1A and β‐actin gene expression in wt and cs E1A and H5hr1‐transformed CREF cells indicated that low levels of CAPE, which were growth suppressive, did not selectively suppress E1A expression. These results demonstrated that cellular changes induced in CREF cells by the 13S E1A‐encoded 289‐aa protein of Ad5, when expressed alone or in combination with the 125 E1A‐encoded 243‐aa protein, rendered transformed cells sensitive to the growth‐suppressing and toxic effects of CAPE.
Possible involvement of c‐myc but not ras genes in hepatocellular carcinomas developing after spontaneous hepatitis in LEC rats
Authors: Fujimoto Yoshinori , Ishizaka Yukihito , Tahira Tomoko , Sone Hideko , Takahashi Hidetoshi , Enomoto Katsuhiko , Mori Michio , Sugimura Takashi , Nagao Minako
Published: February 2007

LEC (Long‐Evans with a cinnamon‐like coat color) rats develop hepatocellular carcinomas (HCCs) spontaneously. We examined mutations of codons 12, 13, and 61 of the Ha‐ras, Ki‐ras, and N‐ras genes in four HCCs by the polymerase chain reaction (PCR)‐single‐stranded DNA direct sequencing method. No ras gene mutations were observed, suggesting that ras activation is not involved in spontaneous hepatocarcinogenesis in LEC rats. The expression of mRNAs for c‐myc, Ha‐ras, c‐raf, and the protein phosphatase 2Aα gene (PP‐2Aα) was also examined in the four HCCs by northern blot analysis. Three of the four HCCs had c‐myc expression levels approximately 30‐fold higher than that in the liver of control Long‐Evans rats with an agouti coat color (LEA), a sibling line of LEC rats, while the remaining HCC had an expression level sevenfold higher than that of control. In contrast, the expression levels of the Ha‐ras, c‐raf, and PP‐2Aα genes were the same as those in the livers of control rats. Studies of c‐myc expression and mitotic index in five other HCCs, two hyperplastic nodules, and two nontumorous portions of livers of HCC‐bearing LEC rats that had chronic‐phase hepatitis suggested that the high level of c‐myc gene expression was not due only to increased cell proliferation but might possibly be more integrally involved in hepatocarcinogenesis.
Activating point mutations of the gsp oncogene in human thyroid adenomas
Authors: O'Sullivan Catriona , Barton Claire M. , Staddon Susan L. , Brown Christopher L. , Lemoine Nicholas R.
Published: February 2007

The gene for the alpha polypeptide chain (αs) of the heterotrimeric G protein Gs can be activated to the putative oncogene gsp by specific point mutations at codons 201 and 227. Such mutations have been reported in 40% of human growth hormone‐secreting pituitary adenomas and in a single autonomously functioning thyroid adenoma. We examined an archival series of 45 differentiated human thyroid tumors by polymerase chain reaction amplification and oligonucleotide hybridization to identify point mutations at each of the affected codons. Successful amplification was achieved in 38 cases, and activating mutations were identified in 5 of 13 (38%) autonomously functioning adenomas, but in none of 16 nonfunctioning adenomas, six papillary carcinomas, or three follicular carcinomas. Our results confirm that the gsp oncogene is involved in the pathogenesis of autonomously functioning tumors but do not support a role in other thyroid tumors.
Analysis of ras DNA sequences in rat renal cell carcinoma
Authors: Recio Leslie , Lane Steven C. , Ginsler Julie , Walker Cheryl
Published: February 2007

The DNA sequences for Ha‐, Ki‐, and N‐ras were determined in six cell lines derived from independent rat hereditary renal cell carcinomas (RCC). Genomic regions encompassing codons 12, 13, and 61 of Ha‐ras, Ki‐ras, and N‐ras, and codon 117 of Ha‐ras were PCR amplified and directly sequenced. The DNA sequences of Ha‐ras and Ki‐ras were normal in all lines tested, as were the codon 12 and 61 sequences of N‐ras. However, DNA sequence variations that could code for amino acid substitutions were observed in codons 13, 14, and 18 of N‐ras in all the lines. The codon 13 Gly→Val alteration observed was consistent with activating N‐ras mutations previously reported. When normal kidney DNA from rats with the hereditary tumor syndrome was sequenced, the same N‐ras sequence variations observed in the tumor lines were found. DNA from outbred Long‐Evans and inbred Fischer rats also had the altered N‐ras sequences. The variant N‐ras sequence was not observed in PCR‐amplified N‐ras cDNA from the RCC lines. Thus, tumor‐associated activation of ras oncogene appears to be an infrequent event in spontaneous rat RCC. In addition, these data indicate that rats contain an N‐ras DNA polymorphism that appears to be a species‐specific anomaly.