bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractPrevious 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

AbstractNormal 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

AbstractPretreatment 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.
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

AbstractA 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

AbstractThe 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

AbstractA 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

AbstractBALB/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

AbstractA 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

AbstractIn 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

AbstractTransformed 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

AbstractThe 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.