bluuebunny/crossref_metadata_2025 huggingface dataset:
Filled Polysilsesquioxanes: A New Approach to Chemical Sensing
Authors: Castaldo Anna , Massera Ettore , Quercia Luigi , Di Francia Girolamo
Published: February 2007

Summary: In an attempt to investigate new polymeric materials as constituents of an e‐nose we focus our attention on a new emerging class of versatile three‐dimensional polyhedral silicon polymers, called polysilsesquioxanes, containing nanosized inorganic cages. Such hybrid amphiphilic materials offer exceptional opportunities to create composites with singular properties. In particular we found that the polyhedral organosilsesquioxane (POSS) cages covalently attached to the polymer backbone as side‐chain act as an “internal” filler with a nanometric homogeneous dispersion. We show how it is possible to fabricate sensing devices based on selected POSS matrix and, by a suitable choice of other “external” home‐made fillers, (e.g. graphite, copper, silicon, zinc and their alloys) obtained by mechanical milling the response of the resulting composites towards different classes of compounds can be tuned. In particular we fabricated a new high sensitive relative humidity device, exhibiting a fivefold response change for relative humidity changing in the range 0% to 100%. This behaviour can not be modelled on the basis of the matrix swelling operating mechanism. Rather, the combined effect of the matrix and the filler has to be invoked in order to explain the formation of nanopores inside the material that are responsible of the porous behavior of our sensors.
Gas Sensor Devices Obtained by Ink‐jet Printing of Polyaniline Suspensions
Authors: Loffredo F. , Burrasca G. , Quercia L. , Sala D. Della
Published: February 2007

We report on the fabrication process of a sensing device obtained by ink‐jet printing of polyaniline suspension on alumina substrates. We optimized the inkjet parameters (amplitude and duration of jetting impulse, jetting frequency, substrate velocity) to obtain thin polyaniline lines as sensitive layers and we analyzed the morphology of PANI lines on substrate. Device response towards ammonia are also discussed and compared with reference device obtained by spin‐coating.
Halogen Free Flame Retardant Agents for Polypropylene in Wire Coating Process
Authors: Maio Luciano Di , Acierno Domenico , Martino Daniela Di
Published: February 2007

the processing behaviour and physical properties of polypropylene/flame retardant blends were investigated in order to evaluate their use in the wire‐coating process. In particular, the need of flame retardancy, suitable melt processability and good mechanical properties of blends was taken into account. Three types of halogen free flame retardants were chosen and were melt blended to the polypropylene matrix. Blends properties were analysed by rheological, mechanical, and thermal characterization including flame retardancy tests. The wire coating process was also performed by a laboratory wire coating apparatus. The results demonstrated that some of the blends are suitable for the purpose of flame retardancy of electrical household wires as an alternative to PVC.
Effect of Solvent (Plasticizers) on PVC Degradation
Authors: Zaikov G. E. , Artsis M. I.
Published: February 2007

Both plasticized (semi‐rigid and flexible) PVC materials as well as PVC in solutions, the rate of their thermal degradation and effective stabilization are caused by essentially different fundamental phenomena in comparison to aging of PVC in absence of the solvent. Both structure and macromolecular dynamics render the significant influence on its stability, i.e. chemical nature of the solvent (plasticizer), its basicity, specific and non‐specific solvation, degree of PVC in a solution (solubility), segmental mobility of macromolecules, thermodynamic properties of the solvent (plasticizer), formation of associates, aggregates, etc.

The chemical stabilization of PVC plays a less significant role. The effect of above factors on stability (behavior) of semi‐rigid and flexible PVC will be done on quantitative level.

It will be described effect of “echo”‐type of stabilization on the stability of PVC in the presence of plasticizers. If we would like to have stable material from PVC we should make stabilization of plasticizers as more reactive chemical compounds.
TGFα stimulates growth of skin papillomas by autocrine and paracrine mechanisms but does not cause neoplastic progression
Authors: Finzi Eric , Kilkenny Anne , Strickland James E. , Balaschak Mike , Bringman Timothy , Derynck Rik , Aaronson Stuart , Yuspa Stuart H.
Published: February 2007

To investigate the role of transforming growth factor α (TGFα) in tumor development, we introduced the human TGFα (hTGFα) cDNA into cultured primary mouse epidermal cells or papilloma cells using a replication‐defective retroviral vector and analyzed skin grafts constructed with such cells. Expression of the exogenous gene was confirmed by detection of hTGFα mRNA by northern RNA blot analysis, and the secreted hTGFα was measured by ELISA of culture supernatants. Tumor cells expressing hTGFα produced benign tumors (papillomas), which were 1.5‐ to 2‐fold larger than tumors of parental cells when tested as skin grafts on nude mice. Grafts of normal cells that expressed hTGFα produced normal skin. When mixtures of parental tumor cells and normal mouse keratinocytes were grafted to nude mice, papilloma formation was suppressed and tumors that did form were small. Grafts of hTGFα‐producing papilloma cells combined with either normal epidermal cells or hTGFα‐producing epidermal cells yielded large tumors. Mixed grafts containing keratinocytes expressing hTGFα and parental papilloma cells also produced large tumors. While the tumor size was substantially increased by hTGFα expression, the tumors that developed in all groups were histologically benign and reached a stable size 4–5 wk after grafting. These results indicate that expression of hTGFα by either tumor cells (autocrine) or adjoining normal cells (paracrine) can stimulate tumor growth, particularly when tumor growth is suppressed by normal tissue. However, expression of this growth factor did not appear to influence tumor progression directly.
Transforming activity of DNA from rat liver tumors induced by the carcinogen methyl(acetoxymethyl)nitrosamine
Authors: Goyette Michele , Dolan Maureen , Kaufmann William , Kaufman David , Shank Peter R. , Fausto Nelson
Published: February 2007

Altered c‐Ha‐ras genes have been frequently detected in the DNA of spontaneous or chemically induced mouse liver tumors. To determine if ras gene mutation is a frequent event during liver carcinogenesis in rats, we examined the transforming activity of DNA from liver tumors that developed in rats injected with methyl(acetoxymethyl)nitrosamine (DMN‐OAc) after a partial hepatectomy. Three weeks after the injection of DMN‐OAc, rats were fed a diet contaiing phenobarbital. This carcinogen acts only on replicating liver cells. Six of eight tumor DNAs induced the transformation of NIH 3T3 cells. The transforming activity was stable upon a second round of transfection, and the transformants were tumorigenic in nude mice. Southern blot analysis of transformant DNAs showed that the transforming activity was not due to the acquisition of a ras (Ha, Ki, or N), neu, myc, A‐raf, v‐raf, erbA, or erbB gene of rat origin. Several transformants' restriction enzyme sensitivity was analyzed, and their activity indicated that similar transforming sequences were present in at least two tumors and that one tumor contained two different transforming sequences. These results suggest that during hepatocarcinogenesis induced in rats by DMN‐OAc, alterations in the ras gene family occur infrequently or not at all and that several different genes (which are not homologous to common oncogenes) become activated and are capable of transforming NIH 3T3 cells.
Comparison of P+ ‐active and ‐inactive pro‐1 homologues from human nasopharyngeal carcinoma cells
Authors: Dowjat W. Karol , Ya Cao , Nagashima Kunio , Sakai Ayako , Colburn Nancy H.
Published: February 2007

Eleven pro‐1 homologous clones from human nasopharyngeal carcinoma cell line CNE2 were studied with respect to their ability to transfer sensitivity to tumor promoter‐induced neoplastic transformation (P+ activity), their molecular structure, and their homology to both mouse pro‐1 and each other. Restriction mapping and Southern analysis of CNE2 pro‐1 homologous clones revealed three structural classes, all of which showed P+ activity. The approximate limits of mouse pro‐1 hybridizing sequences within CNE2 clones from each structural class were identified, and some of these minimum homologous sequences were tested for P+ activity by transfection into P+ cells. For all classes, a strong association between P+ activity and pro‐1 homology was observed. This finding implies that, for CNE2 clones to be P+‐active, structural homology to mouse pro‐1 is required. The minimum P+‐active sequence thus far identified was a 2.6‐kbp EcoRI‐Sstl fragment. One clone was inactive, even though it was indistinguishable from active clones of the same class by restriction mapping, Southern analysis, and electron microscope examination of heteroduplexes formed by an active and an inactive clone. This raises the possibility that discrete changes, involving a few nucleotides rather than gross rearrangement, may determine the P+ activation of these pro‐1‐homologous sequences.
Timing of proto‐oncogene replication: A possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens
Authors: Doggett Norman A. , Cordeiro‐Stone Marila , Chae Chi‐Bom , Kaufman David G.
Published: February 2007

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence‐induced arrest of proliferation followed by treatment with 2 μg/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1‐ or 2‐h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot‐blot apparatus, and hybridized with various 32P‐labeled probes. Ha‐ras was among the first genes replicated at the onset of the S phase. The myc proto‐oncogene replicated later but within the first hour of the S phase. The replication of Ki‐ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0–2 h) and the myb proto‐oncogene replicated in mid‐S phase (2–4 h). An immunoglobulin VH sequence and the β‐globin gene replicated late in 10T1/2 cells, 4–6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto‐oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of protooncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.
Specific viral oncogenes cause differential effects on cell‐to‐cell communication, relevant to the suppression of the transformed phenotype by normal cells
Authors: Bignami Margherita , Rosa Silvia , Falcone Germana , Tató Franco , Katoh Fumitaka , Yamasaki Hiroshi
Published: February 2007

We have studied growth regulation in mixed cultures of normal and oncogene‐transformed 3T3 cells. The NIH 3T3 cells transformed by myc, src, and ras showed comparable cloning efficiency in semisolid medium. However, when they were plated on plastic with an excess of normal mouse embryo fibroblasts, BALB/c 3T3 CIA31–1–1, ras‐ and src‐transformed cells were able to form distinct foci on the layer of density‐arrested normal cells, whereas myc‐transformed cells lacked this ability. In order to determine whether suppression or expression of the transformed phenotype could be correlated with the ability of the different cell populations to communicate, gap‐junctional intercellular communication (IC) was measured by the Lucifer yellow dye transfer assay in coculture of normal and transformed cells. The dye was observed to spread from BALB/c 3T3 to myc‐NIH 3T3 cells, indicating the presence of IC between these two cell types. In contrast no passage of Lucifer yellow was observed between src‐NIH 3T3 or ras‐NIH 3T3 and BALB/c 3T3. Addition of a phorbol ester tumor promoter, phorbol‐12,13‐didecanoate, efficiently rescued proliferation and focus formation by myc‐transformed cells. The tumor promoter was able to inhibit IC in BALB/c 3T3 cells, although this response greatly varied among the different oncogene transformed clones. Tumorigenicity in nude mice strongly correlated with growth behavior in vitro: myc‐transformed cells were either nontumorigenic or slowly tumorigenic, and src‐ and ras‐transformed cells were highly tumorigenic. These data suggest an important role of IC in modulating abnormal growth behavior in vitro and in vivo.
Tumorigenic and molecular characterization of novel phorbol ester‐resistant and ‐sensitive lines of mice
Authors: Cope Frederick O. , Wagner Frederic J. , Conway Toni , Burns Pamela D. , Johnston Donna , Murphy Paula , Triggs Gloria , Wille John J.
Published: February 2007

Two outbred lines of CD‐1 mice were developed using males and females in an initiation (dimethyl‐benz[a]anthracene; DMBA), promotion (12‐O‐tetradecanoylphorbol‐13‐acetate; TPA) challenge, posttumorigenesis breeding protocol. Our results indicate that the phorbol ester‐resistive (PESTI) line developed tumors at a rate 4.1 times faster than the CD‐1 parental line, while the phorbol ester‐resistant (PERTI) line developed tumors at a rate 36 times slower than the CD‐1 parents. The average number of tumors per mouse reached levels of 27.5 at 12 wk in the PESTI line, 0.1 at 16 wk in the PERTI line, and 6.7 at 16 wk in the CD‐1 line. Biochemical tests showed that the PESTI line had both a high basal level and an enhanced epidermal ornithine decarboxylase (E.C. 4.1.1.17) response to TPA, the latter being nine times that of the PERTI line at their maximum dosages. An autoradiographic analysis of in vivo epidermal cell protein phosphorylation indicated marked differences in basal protein phosphorylation profiles (with high phosphate incorporation, PERTI, 112.7, 95.5, 64.4, 40.8, 18.6, 17.4, and 12.3 kDa; PESTI, 64.4, 40.8, 31.8, and 12.3 kDa) as well as TPA‐dependent changes in these profiles (difference from basal levels, PERTI, 31.8 and 12.8 kDa; PESTI, 139.6, 126.3, 37.2, and 18.6 kDa). These heterogeneous profiles indicate strong genetic segregation of these protein kinase C target substrates. This dermatologically based selection and breeding process also resulted in cosegregating genetic changes in TPA binding constants (Kd) for brain protein kinase C, which were reversed for cytosolic and plasma membrane fractions in the PESTI (cytosolic, 0.50 nM; plasma membrane, 1.89 nM) and PERTI (cytosolic, 2.02 nM; plasma membrane, 0.51 nM) mice. Moreover, TPA‐inducible phosphorylation of 43 and 110 kDa proteins in vivo in the brains of only the PERTI line mice suggests that the PESTI line has either high levels of endogenous phosphorylation or reduced expression of these proteins. In contrast, in vitro screening of brain cytosolic proteins indicated that both lines have a 120 kDa protein whose phosphorylation can be induced by TPA while the PESTI line has a 14 kDa protein whose phosphorylation regulatory controls differ from the PERTI line. Thus, these lines of mice provide experimental systems for defining the skin tumorigenesis process and possibly extending initiationpromotion observations and concepts to other tissues.
Malignant conversion of murine squamous papilloma cell lines by transfection with the fos oncogene
Authors: Greenhalgh David A. , Yuspa Stuart H.
Published: February 2007

Murine papilloma cell lines 308 and SP‐1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated crasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma‐derived c‐myc (minus exon 1), adenovirus 5 E1A, FBJ v‐fos or a human c‐fosl FBJ v‐fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418‐selected cell lines and in the derived tumors. Unlike the E1A‐ and myc‐containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express γ‐glutamyl transpeptidase in the tumors. Since both 308 and SP‐1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous‐activated c‐rasHa gene to convert these cells to malignancy. However, since γ‐glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated crasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.
Interaction of an ultimate carcinogen, benzo[a]pyrene diol epoxide, with nucleosomal core particles: Apparent lack of protection of DNA by histone proteins
Authors: Macleod Michael C. , Smith Bettye , Lew Linda Kao
Published: February 2007

The binding of chemical carcinogens to nuclear macromolecules, especially to DNA, is thought to be central to the initiation of carcinogenesis. Previous studies of the interactions of one such ultimate carcinogen, benzo[a]pyrene diol epoxide (BPDE‐I) with nuclei, chromatin and purified DNA, demonstrated that although some BPDE‐I‐DNA interactions were altered in chromatin, covalent binding to chromatin DNA at saturating chromatin concentrations was quantitatively the same as binding to purified DNA. We have now extended these studies to include the basic subunit of chromatin, the nucleosomal core particle. Association constants for BPDE‐I and a nonreactive analogue were determined by absorbance and fluorescence spectroscopy using either core particles or purified DNA and were found to be lower, by a factor of 30, for core particles. One of the major pathways of interaction of BPDE‐I with DNA is the catalysis of BPDE‐I hydrolysis by the exocyclic amino group of deoxyguanosine in native DNA. This detoxification reaction is inhibited about 30‐fold in core particles compared with DNA, consistent with the hypothesis that intercalation is important in this catalytic reaction. In contrast to these findings, at DNA concentrations that allow maximal binding, similar amounts of BPDE‐I are bound covalently to either free DNA or the DNA contained in core particles. This finding suggests that the interaction of DNA with histones to form the subunit structure of chromatin does not significantly protect DNA from damage by this ultimate carcinogen. The pattern of DNA adducts formed with core particle DNA shows a subtle shift toward the pattern seen with denatured DNA.
Chromatin structure and transcriptional regulation of human papillomavirus type 18 DNA in hela cells
Authors: Rösl Frank , Westphal Eva‐Maria , Hausen Harald Zur
Published: February 2007

Mapping analysis of the nucleosomal organization of integrated human papillomavirus type 18(HPV 18) DNA in HeLa cells reveals a very prominent nuclease‐hypersensitive site within the viral noncoding regulatory region that harbors transcriptional control sequences and coincides with most of the 5′ ends of the cytoplasmic early mRNAs. Moreover, it is shown that the conserved coamplified 5′ cellular flank, common to all HPV18 copies in HeLa cells and located close to the virus‐cell integration site, also contains several distinct hypersensitive sites, accessible not only to DNase I but also to restriction enzymes. Nuclear run‐on analysis in isolated HeLa nuclei demonstrates the occurrence of nascent transcripts covering the cellular flank (the late and the viral noncoding regulatory region), indicating that a cellular promoter, marked by the hypersensitive sites, cooperates with the viral control region in generating the HPV18 transcripts.

Cycloheximide treatment of HeLa cells results in a reduction of the cytoplasmic steady‐state level of the 3.5‐kb mRNA corresponding to the viral E6, E7, and parts of the E1 open reading frames (ORFs), whereas the expression of the 1.6‐kb transcript corresponding only to the E6 and E7 ORFs is not influenced. Nuclear run‐on analysis carried out after the cycloheximide chase reveals that the distribution of nascent transcripts spanning the viral E6, E7, and parts of the E1 region is substantially decreased. In contrast to this finding, an even, pronounced increase of the elongation rate of those transcripts, which cover the cellular flank, the late and the viral noncoding regulatory region was noted indicating a different involvement of regulatory factors in the activity of both promoters.
Induction of metallothionein mRNA by tumor promoters in mouse skin and its constitutive expression in papillomas
Authors: Hashiba Hiroki , Hosoi Junichi , Karasawa Mika , Yamada Shuhei , Nose Kiyoshi , Kuroki Toshio
Published: February 2007

A single topical application of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was found to induce mRNA of a metallothionein (MT) gene or genes in the skin of Sencar mice, and papillomas produced by repeated applications of TPA were shown to have elevated levels of MT mRNA. Induction of MT mRNA was maximal 4–8 h after application of TPA and returned to the control level 24 h later. A dose‐dependent increase of MT mRNA was observed with doses of TPA of 1–5 μg. Of the other promoters tested, phorbol‐12, 13‐didecanoate, mezerein, and the ionophore A23187 also induced MT mRNA, but 4‐O‐methyl‐TPA and benzoyl peroxide did not. Phorbol and 4α‐phorbol‐12, 13‐didecanoate, which are not promoters, also did not induce MT mRNA. Retinoic acid and 1α, 25‐dihydroxyvitamin D3, inhibitors of tumor promotion, did not induce MT mRNA themselves or inhibit the induction of MT mRNA by TPA. In C57BL/6 promotion‐resistant mice, TPA caused only slight induction of MT mRNA. These data suggest a correlation between induction of MT mRNA and epidermal hyperplasia. The constitutive elevation of MT mRNA levels in papillomas may be due to the loss, during the process of tumor promotion, of some mechanism regulating MT gene expression.
Mutations at codon 61 of the Ha‐ras proto‐oncogene in precancerous liver lesions of the B6C3F1 mouse
Authors: Buchmann Albrecht , Mahr Johanna , Bauer‐Hofmann Richard , Schwarz Michael
Published: February 2007

Liver tumors of the B6C3F1 mouse frequently contain mutations at specific sites of codon 61 of the Ha‐ras proto‐oncogene. To address whether these mutations occur early or late during carcinogenesis, we analyzed mutations in the Ha‐ras gene in small precancerous liver lesions of the B6C3F1 mouse. For this purpose, 10‐μm frozen liver sections were prepared and stained for glucose‐6‐phosphatase activity. Using punching cannuli, we then took small tissue samples of approximately 5–30 μg from enzyme‐deficient liver lesions and from normal parts of the liver. These tissue samples were analyzed for mutations in the Ha‐ras gene by in vitro amplification of DNA via the polymerase chain reaction combined with selective oligonucleotide hybridization. By this approach we were able to analyze mutations in the Ha‐ras gene within lesions with diameters of less than 0.5 mm. Our results demonstrate that approximately 15% of the glucose‐6‐phosphatase‐negative lesions that occurred 24–28 wk after a single injection of diethylnitrosamine contain either C → A transversions at the first base or A → G transitions and A → T transversions at the second base of codon 61 of the Ha‐ras gene. The same types of mutations, although with a somewhat higher frequency (33%), were found in liver tumors taken 68 wk after diethylnitrosamine treatment. These findings demonstrate that Ha‐ras mutations can be detected even in very small precancerous liver lesions, suggesting that these mutations may be an early, perhaps even the first, critical event during murine hepatocarcinogenesis.
Glutathione and glutathione S‐transferases in clones of cultured rat liver epithelial cells that express varying activity of γ‐glutamyl transpeptidase
Authors: Tsao Ming‐Sound , Duong Minh , Batist Gerald
Published: February 2007

In rat chemical hepatocarcinogenesis models, the hepatocytes in the preneoplastic/neoplastic nodules characteristically demonstrate common biochemical changes including significant and often marked elevation in the cellular glutathione (GSH) content and in the activities of the enzymes γ‐ glutamyl transpeptidase (GGT) and glutathione S‐transferase (GST). Such consistent and concomitant biochemical changes may signify a common regulatory mechanism in the expression of these enzymes. We have utilized a panel of clonally derived rat liver epithelial cell lines that express varying activities of GGT to study the quantitative correlation between these three cellular components of the phase II drug metabolizing enzyme system. The results indicate that in confluent cultures, cells with high GGT activities have significantly higher cellular GSH content, and a linear correlation exists between the glutathione content and the logarithm of the GGT activity. In contrast, the basal activities of GST and GGT were not coordinately regulated. However, most of the chemical carcinogen‐treated cell lines, regardless of their GGT activity, expressed higher GST activity than the normal parental rat liver epithelial cells. The basal expressions of both the Yb and Yp subunits of GST were also not correlated with the relative expression of GGT. Since GGT may play an important role in supplying the cells with the basic constituents for the synthesis of GSH and since GSH is an important cellular molecule in the protection of cells from toxic electrophiles, enhancement of GGT activity in preneoplastic/neoplastic nodules of chemical carcinogen‐treated rats may represent a necessary biochemical adaptation for the induction of the “resistant” phenotype of these hepatocytes.
Activation of ras oncogenes in chemically transformed BALB/MK‐2 mouse keratinocytes
Authors: Weissman Bernard E. , Arboleda Marie J. , Kimoto Kathleen A. , Macdonald Marsha J. , Ochiai Ruth , Der Channing J.
Published: February 2007

BALB/MK‐2 cells are an epidermal growth factor (EGF)‐dependent cell line derived from BALB/c mouse epidermis that can undergo terminal differentiation under appropriate conditions. Previous studies have shown that transformation of these cells by retroviral oncogenes relieves the EGF requirement while blocking the terminal differentiation program. In this report we show that BALB/MK‐2 cells are sensitive to transformation by the chemical carcinogens dimethylbenz[a]anthracene (DMBA), 3‐methylcholanthrene (MCA), and N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG). BALB/MK‐2 cells transformed by these carcinogens proliferate in the absence of EGF and do not undergo terminal differentiation in response to calcium. However, the cells retain their anchorage growth dependence and are nontumorigenic in nude mice. NIH 3T3 transfection analysis showed that the endogenous Ha‐ras gene had been activated in both DMBA‐ and MNNG‐transformed cells and the Ki‐ras gene had been activated in the MCA‐transformed cells. Additionally, non‐ras transforming activity was detected in some MNNG‐transformed BALB/MK‐2 cells. Thus, the BALB/MK‐2 cell line provides a reproducible in vitro assay system for chemical transformation of epithelial cells and for identification of oncogene activations associated with changes in growth control and differentiation.
A Human Endonuclease Incises Ultraviolet‐Irradiated DNA at Cytosines and Oxidized DNA at Thymines
Authors: Gallagher Patricia E. , Weiss Randi B. , Brent Thomas P. , Duker Nahum J.
Published: February 2007

Both ultraviolet irradiation and oxidation of DNA produce a variety of pyrimidine base damages. A human endonuclease recognizes such altered bases on these DNA substrates. This human endonuclease incises ultraviolet‐irradiated DNA exclusively at sites of photochemically modified cytosines. The precise sites of incision by the human enzyme were determined by DNA sequencing. Chemically oxidized DNA was incised exclusively at thymine loci. The degree of enzymic cleavage at cytosine photoproducts was identical at each site. However, the extent of incision at selected oxidized thymine residues varied within the DNA sequence. These results indicate that the distribution of thymine oxidative modifications is influenced by the neighboring DNA bases.
Oncogene Expression in Cell Lines Derived From Rat Tracheal Implants Exposed In Vivo to 7,12‐Dimethylbenz[a]anthracene
Authors: Cosma Greg N. , Marchok Ann C. , Garte Seymour J.
Published: February 2007

Expression of four oncogenes and two keratin genes was determined in rat tracheal epithelial cell lines derived from tracheal implants exposed in vivo to 7,12‐dimethylbenz[a]anthracene. Cell lines were grouped into four stages of neoplastic progression based on phenotypic markers in order to correlate oncogene expression with stage of malignancy. Northern analysis of RNA revealed a significantly enhanced expression of the c‐myc oncogene in the most tumorigenic or tumor‐derived cell lines, whereas preneoplastic cells expressed approximately fivefold less transcript. Southern analysis of tracheal cell DNA did not demonstrate amplification of the c‐myc gene in any of the positive cell lines. In contrast to c‐myc, other oncogenes such as ras and fos were expressed in all cell lines, as well as in control cell cultures, to a similar extent. Patterns of differentiation were examined in these epithelial cell lines by determining the expression of two distinct keratin genes, KA‐1 and KB‐2. Both malignant and preneoplastic cells expressed the KB‐2 gene at variably high levels, whereas the expression of the KA‐1 keratin was barely detectable in any of the cell lines. The stage‐specific expression of the c‐myc oncogene in these tracheal cell lines suggests a correlation between the regulation of certain oncogenes and neoplastic progression in this model of respiratory carcinogenesis.
Role of TGF‐Beta in Normal Differentiation and Oncogenesis in Rat Liver
Authors: Nagy Peter , Evarts Ritva P. , McMahon James B. , Thorgeirsson Snorri S.
Published: February 2007

Transforming growth factor‐β1 (TGF‐β1) is capable of eliciting a myriad of biological responses associated with cellular proliferation, as well as effects unrelated to the control of cell growth. We examined the possible role of TGF‐β1 in the differentiation of rat liver epithelial (RLE) cells in vitro and studied the cellular distribution of TGF‐β1 transcripts and protein during in vivo differentiation of oval cells. Furthermore, we followed the cellular distribution of TGF‐β1 transcripts and protein during chemical hepatocarcinogenesis. By using in situ hybridization and immunohistochemical techniques, we showed that both TGF‐β1 transcripts and protein are localized in nonparenchymal cells in normal liver, are expressed in oval cells during very early stages of hepatocytic differentiation in vivo, and are exclusively expressed in the nontumorous mesenchymal cell compartment during hepatocarcinogenesis. Furthermore, we showed that TGF‐β1 is capable of inducing differentiation of RLE cells in vitro consistent with early stages of hepatocytic lineage differentiation. Our data indicate that the RLE cells similar to the oval cells in vivo may be an epithelial progenitor cell for hepatocytic cell lineage in adult mammalian liver.
Increased levels of phorbin, c‐myc, and ornithine decarboxylase RNAs in human colon cancer
Authors: Guillem Jose G. , Levy Miriam F. , Hsieh Ling Ling , Johnson Mark D. , Logerfo Paul , Forde Kenneth A. , Weinstein I. Bernard
Published: February 2007

Our previous work on protein kinase C (PKC) and colon cancer has shown altered levels of PKC activity in human colon tumors, as well as activation of PKC by colon tumor promoters such as bile acids. To understand further the role of PKC in colon carcinogenesis, we analyzed the expression of phorbin, a gene induced by PKC activation, in a series of different stages of human colon tumors. As shown by northern blot analyses of poly (A)+ RNA, higher levels of phorbin RNA were seen in 26 colon tumor samples than in their adjacent normal colonic mucosa. There also appeared to be a correlation between the abundance of phorbin RNA in the tumors and the extent of invasion (tumor‐to‐normal tissue phorbin RNA ratio = 4.2, 8.0, and 11.9 for Dukes' A, B, and C, respectively). Phorbin RNA was also abundant in a human colon cancer line (HT29). We also examined the expression of other mitogen‐responsive genes (c‐myc, ODC, and β‐actin) in a set of 19 colon tumor samples. All tumors displayed significant (mean 3.8‐fold) increases in the level of c‐myc RNA compared with their adjacent normal colonic mucosa. About 47% and 16% of these tumor samples also showed increased levels of ODC (mean 3.1‐fold) and β‐actin (mean 1.6‐fold) RNA, respectively. The increased levels of c‐myc, ODC, and β‐actin RNA did not correlate with the extent of tumor invasion. Taken together, these results demonstrate that human colon tumors usually display increased levels of both phorbin and c‐myc RNAs. The marked increases in phorbin RNA suggest that this could serve as a useful biomarker in studies on human colon cancer.
Novel phenotype of C3H 10T1/2 fibroblasts cotransfected with the c‐Ha‐ras and adenovirus 5 E1A oncogenes
Authors: Davenport Elizabeth A. , Taparowsky Elizabeth J.
Published: February 2007

C3H 10T1/2 fibroblasts are converted to fully transformed phenotype following coexpression of an activated c‐Ha‐ras gene and either a constitutively expressed viral or cellular myc gene. In this report, we examined whether the early region 1A (E1A) of adenovirus 5, which synergizes with ras to convert primary embryonic cells to a transformed phenotype, can synergize with ras to transform the established mouse embryonic cell line, C3H 10T1/2. We demonstrate that coexpression of ras and E1A generated a transformed phenotype that could be scored by colony assays and by soft agarose assays but not by standard focus assays. The ras‐E1A‐transformed phenotype relies on sequences present in conserved regions 1 and 2 of the E1A proteins and, in part, on information encoded by the extreme carboxy terminus of E1A. The contrast between the transformed phenotypes generated following the transfection of C3H 10T1/2 cells with either ras and myc or ras and E1A suggests that myc and E1A cooperate with ras to transform C3H 10T1/2 cells by mechanisms that can be distinguished using this established cell line as a model system.
Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli. III: The cyclic nitrosamine N‐nitrosopyrrolidine is a complex mutagen
Authors: Zielenska Maria , Ahmed Alia , Glickman Barry W.
Published: February 2007

The mutational specificity of the cyclic nitrosamine N‐nitrosopyrrolidine (NPYR) was determined through the DNA sequence characterization of 33 lacl−d mutations of Escherichia coli. Base substitution was the predominant class of mutation induced (91%). The majority of these (64%) occurred at GC base pairs, in accordance with the predicted significance of NPYR‐derived guanine adducts. In addition, this nitrosamine efficiently produced other kinds of base substitution events as 11 of the 33 mutations occurred at AT base pairs. Deletion, frameshift, and duplication events were also recovered. The complexity of the NPYR mutational spectrum appears to be consistent with the suggestion that this compound acts through both direct and indirect mutational pathways.
Chemical induction of thymomas in AKR mice: Interaction of chemical carcinogens and endogenous murine leukemia viruses. Comparison of N‐methyl‐N‐nitrosourea and methyl methanesulphonate
Authors: Warren William , Clark Jeremy P. , Gardner Emily , Harris Gilmour , Cooper Colin S. , Lawley Philip D.
Published: February 2007

The time course of development of thymic lymphoma, which occurs spontaneously in mice of the AKR strain, is accelerated by the methylating agents N‐methyl‐N‐nitrosourea (MNU) and methyl methanesulphonate (MMS). Since MNU is a potent mutagen inducing G → A transition mutations and MMS a relatively weak mutagen, it was of interest to examine the genetic alterations associated with each class of the chemically induced tumors and to compare these alterations with those found in the spontaneous tumors. The same spectrum of genetic alterations was found for MMS‐induced and spontaneous thymomas. Both showed rearrangements of c‐myc and Pim‐1 genes that appeared to result from integration of recombinant mink cytopathic focus‐forming (MCF) proviruses but failed to reveal evidence for activation of ras oncogenes, either by DNA transfection experiments or by hybridization of DNA to specific oligonucleotide probes. Some alterations in c‐myc and Pim‐1 genes were also found in MNU‐induced tumors, but, mainly, these involved integration of ecotropic‐like rather than recombinant MCF viruses. Furthermore, MNU‐induced tumors frequently (in 24% of thymomas) contained G → A transition mutations, activating the Ki‐ras oncogene at codon 12 position 2. Another feature that distinguishes the MNU‐induced tumors from those occurring in untreated and MMS‐treated mice was the consistently high level of c‐myc mRNA that occurred in the absence of c‐myc gene rearrangement. Taken together, the data indicate that the mechanisms of development of tumors following treatment with MNU and MMS are distinct, and that the effect of MMS is probably to speed up the process of viral leukemogenesis.
Enhancement of adenovirus transformation of cloned rat embryo fibroblast cells by gamma irradiation
Authors: Su Zao‐Zhong , Zhang Peiquan , Geard Charles , Fisher Paul B.
Published: February 2007

We have analyzed the effect of γ irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host‐range cold‐sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1–6 Gy of γ irradiation immediately prior to viral infection resulted in dose‐dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1–6 Gy of γ radiation alone resulted in a similar dose‐dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of γ irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of γ irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single‐cell colony transfer assay and in situ hybridization with a 32P‐labeled Ad5 DNA probe, we found that γ irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA‐filter hybridization (Southern blot analysis) in H5hr1‐transformed CREF clones isolated from untreated and γ‐irradiated cultures indicates that γ irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that γ irradiation enhancement of adenovirus transformation was a consequence of radiation‐induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral‐DNA integration in γ‐irradiated cells.
Plasma membrane—mediated nuclear uptake and chromatin binding of insulin in tumor cell lines
Authors: Rakowicz‐Szulczynska Ewa M. , Otwiaska Danuta , Koprowski Hilary
Published: February 2007

Analysis of different cellular fractions after incubation of SW 948 and SW 707 colorectal carcinoma cells or WM 266‐4 melanoma cells with 125I‐insulin revealed the nondegraded hormone in the chromatin of these cells. Nuclear 1251‐insulin was bound to specific fragments of EcoRI‐, Haelll‐, and Hincll‐digested chromatin. A 45‐kDa chromatin protein species that binds 125I‐insulin was identified. Actinomycin D and cycloheximide inhibited the insulin‐stimulated expression of chromatin receptors. Uptake of 125I‐insulin by isolated nuclei occurred only in the presence of plasma membranes. Thus, at least some effects of insulin on target cells can be explained by direct gene regulation instead of “second messenger” action.
Ha‐ras oncogene mutations in cell lines derived from rat tracheal implants exposed in vivo to 7,12‐dimethylbenz[a]anthracene
Authors: Cosma Greg N. , Wirgin Isaac I. , Marchok Ann C. , Garte Seymour J.
Published: February 2007

The frequency of Ha‐ras mutations was determined as a function of neoplastic progression in cell lines derived from rat tracheal implants exposed in vivo to 7,12‐dimethylbenz[a]anthracene. Restriction fragment‐length polymorphism (RFLP) analysis revealed an A → T transversion in the second base of codon 61 in 2 of 11 cell lines. One of the positive cell lines was tumorigenic, but the other was neither tumorigenic nor anchorage independent, thus indicating a lack of correlation between neoplastic stage and ras mutation. Densitometry analysis of the RFLP bands indicated that approximately 50% of the cells within these two heterogeneous populations contained the mutation. Direct sequence analysis of polymerase chain reaction—amplified DNA confirmed these results and did not reveal any other mutations in this region of the Ha‐ras gene.
Changes in rasT24 expression do not induce changes in c‐jun, jun‐B, or jun‐D RNA levels in rat liver epithelial cells
Authors: Krizman David B. , Lebovitz Russell M. , Lieberman Michael W.
Published: February 2007

We used a series of rat liver epithelial (RLE) cell lines that carry a zinc‐regulatable metallothionein/rasT24 fusion gene (MTrasT24) to investigate the relation of ras oncogene expression to steady‐state RNA levels of the jun family of genes. In these cells, steady‐state RNA levels of c‐jun, jun‐B, and jun‐D were unrelated to rasT24 RNA levels or the phenotypic changes induced by the ras oncogene. Steady‐state levels of the three jun mRNAs varied among different rasT24 transformed clones, and, although some clones exhibited concomitant induction of rasT24 and jun mRNAs, other clones exhibited no such correlation. We conclude that the effects of rasT24 in transformed RLE cells do not appear to be mediated by c‐jun, jun‐B, or jun‐D and that studies examining only a single transformed clone may give misleading results with respect to the role of various oncogenes in the transformation process.
Polyoma DNA replication dependent upon growth condition of SEWA sarcoma cells
Authors: Robinson Richard , Ronai Zeev
Published: February 2007

Extrachromosomal replication of viral DNA sequences has been observed in transformed as well as in normal cells following “stress”‐inducing treatments. To explore the effect of growth conditions on the ability to support such replication, we analyzed SEWA sarcoma cells that grew subcutaneously or as ascites tumors in vivo as well as cell lines that were established from each of these tumors. The replicative form of polyoma DNA sequences was observed in SEWA tumors grown in ascites fluids but not in cells maintained as solid tumors. Polyoma DNA replication was found in ascites‐derived cells that were adapted to grow in culture, only when the cultured cells are stimulated with UV irradiation. Immunoprecipitation of T antigens enabled detection of large T antigen only in the ascites‐derived cells. The mechanisms that may regulate this phenomenon and the possible role large T may play in different growth conditions of SEWA cells are discussed.
Loss of a tumor suppressor gene function is correlated with downregulation of chondrocyte‐specific collagen expression in syrian hamster embryo cells
Authors: Cizdziel Paul E. , Hosoi Junichi , Montgomery Jeffry C. , Wiseman Roger W. , Barrett J. Carl
Published: February 2007

We previously described the isolation of closely related, preneoplastic Syrian hamster cell lines that have retained (supB+) or lost (supB−) the ability to suppress the anchorage‐independent growth and tumorigenicity of a sarcoma cell line (BP6T) in cell hybrids. In this report, we have used differential cDNA screening to clone several genes that are expressed in supB+ cells and downregulated in supB− cells. The nontumorigenic supB+ and supB− variants are advantageous for differential cDNA cloning because multiple independent cell lines differing in their tumor suppressor activity have been isolated. Differentially expressed cDNAs were isolated and placed into one of four groups based on DNA cross‐hybridization. Representative cDNAs from Groups I and II, which were expressed at relatively high levels in two independently derived supB+ cell lines (DES4 and 10W) and downregulated in the supB− and tumor cell lines, were sequenced. The DNA and predicted amino acid sequences of these genes were found to be highly homologous to the chondrocyte‐specific collagens type II and type IX. In contrast to the chondrocyte‐specific collagens, another collagen isoform, collagen type I, was expressed at similar levels in both supB+ and supB− cells. These results suggest that carcinogen‐induced immortalization selected for chondrocyte‐like cell lines from the mixed embryo cell population. As these cells progressed toward tumorigenicity, the ability to express the chondrocyte differentiation markers was lost concomitantly with the ability to suppress the tumorigenicity of the BP6T sarcoma cell line. These results are consistent with the hypothesis that the supB+ tumor suppressor gene is involved in the regulation of differentiation. The identification of genes regulated by this suppressor gene may aid in its isolation.