bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic Layered Silicate as a Carrier for Liquid Ingredients for the Rubber‐ and Tire Industry
Authors: Meledina Liudmila , Kandyrin Kirill , Knee Christopher S. , Zaikov Guennadi
Published: February 2007

We propose the use of novel materials based on synthetic calcium silicate hydrate (CSH) for the rubber‐ and tire industry. We found that the synthetic silicate belongs to the family of layered calcium silicate hydrates. Due to its layered structure and inert surface it easily adsorbs liquid components of rubber compounds, such as bis(triethoxysilylpropyl) tetrasulfide (TESPT) and liquid polysulfide (LP). Then, in processing, the CSH can smoothly release these components. The water, trapped in the material's gallery layers, plays the role of a catalyst in the silane coupling on silica's surface. In addition, we used this silicate as a component in promoter systems in order to improve adhesion stability between a brass‐coated steel cord and rubber. The application of the calcium silicate hydrate as an inert substrate for the promoter system allowed the development of materials that have comparable adhesion strength with most commercial promoters.
Activation of cytochrome P450IA1 gene expression by 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin in wild‐type and high‐activity variant mouse hepatoma cells
Authors: Hirst Margaret A. , Jones Keith W. , Whitlock James P.
Published: February 2007

We analyzed the function of a DNA domain located upstream of the cytochrome P450IA1 gene in wild‐type (Hepa 1c1c7) mouse hepatoma cells and in high‐activity variant (HAV) cells that overtranscribe the gene in response to the inducer 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Transfection experiments indicated that both wild‐type and HAV DNA confer responsiveness to TCDD upon the bacterial chloramphenicol acetyltransferase (CAT) gene. However, the level of CAT activity was four‐ to fivefold higher when the hybrid genes were expressed in the HAV cells. These findings imply that an alteration in a trans‐acting function confers the HAV phenotype. Studies of mRNA accumulation imply that TCDD acts by enhancing the rate of mRNA initiation rather than by removing a block in mRNA elongation. We found that both wild‐type and HAV cells used the same transcriptional promoter as that described previously for the cytochrome P450IA1 gene in C57BL/6 mouse liver. Both cell types exhibited superinduction of cytochrome P450IA1 gene expression in response to TCDD plus cycloheximide.
Expression Pattern of the Genes for Different Members of the Heat‐Shock Protein 70 Family, Ornithine Decarboxylase, and c‐Ha‐ras During the Early Stages of Hepatocarcinogenesis
Authors: Tacchini Lorenza , Schiaffonati Luisa , Rappocciolo Emilia , Cairo Gaetano , Bernelli‐Zazzera Aldo
Published: February 2007

Northern blot analysis with specific probes indicates that, in the liver of normothermic rats under the carcinogenic regimen of Solt and Farber (Nature 263:701, 1976), diethylnitrosamine (DEN), 2‐acetylaminofluorene (AAF), and partial hepatectomy (PH), either alone or in combination, do not induce the expression of the heat‐shock protein 70 (hsp70) gene family. On the contrary, the inducibility of hsp70 mRNAs in the liver of heat‐shocked animals is maintained throughout the treatment, as it is in nodules that are found in the liver 2 weeks posttreatment. The steady‐state level of the constitutive hsp70‐related cognate protein (hsc73) mRNA, which is known to be particularly high in fast‐growing cells, increases above the normal level only during liver regeneration, stays at a high level up to 8 days after hepatectomy, independently of any associated exposure to carcinogenic chemicals, and is practically unchanged in the nodules. Ornithine decarboxylase mRNA increases only in livers of normothermic rats at 16 h of regeneration. The expression of the c‐Ha‐ras oncogene increases slightly, but steadily, during the carcinogenic treatment and persists at a high level when other changes have subsided.
Differential Effects of Chromium(VI) on Constitutive and Inducible Gene Expression in Chick Embryo Liver In Vivo and Correlation With Chromium(VI)‐Induced DNA Damage
Authors: Hamilton Joshua W. , Wetterhahn Karen E.
Published: February 2007

The effect of DNA damage induced by the carcinogen chromium(VI) on the function of DNA as a template for transcription of constitutive and inducible genes was examined in chick embryo liver in vivo. Changes in gene expression, determined using solution hybridization and northern blot analyses to measure steady‐state mRNA levels and a nuclear run‐off assay to measure gene transcription rates, were compared to chromium‐DNA binding and to chromium(VI)‐induced DNA damage as previously measured by DNA alkaline elution. Chromium(VI) treatment had little or no effect on either the steady‐state mRNA levels or the transcription rates of the constitutively expressed genes for albumin, conalbumin (avian transferrin), or B‐actin. In contrast, chromium(VI) treatment had significant but opposite effects on the basal and drug‐inducible expression of 5‐aminolevulinate synthase and cytochrome PB1 P450. The changes in steady‐state expression of these two inducible genes were similar to the changes in transcription rate, indicating that the effects of chromium were principally transcriptional. Chromium(VI) treatment increased the basal expression of both inducible genes four‐ to fivefold at maximum, and the time course of this effect was similar to the time course for chromium(VI)‐induced DNA damage and repair. In contrast, chromium(VI) pretreatment suppressed by 60–70% at maximum the subsequent induction of these genes by glutethimide, a phenobarbital analog, and the time course of this effect also corresponded to that of chromium(VI)‐induced DNA damage and repair. The time courses of the changes in expression of these genes were bimodal, with the second peak corresponding closely to that of chromium(VI)‐induced DNA cross‐links. However, the first peak occurred during a period when no DNA cross‐links or strand breaks were detectable by alkaline elution, although significant levels of chromium were bound to DNA. This suggests that chromium(VI), like cisplatin, may initially produce a DNA monoadduct that subsequently leads to DNA cross‐link formation and that both types of chromium(VI)‐induced lesions have a significant effect on the expression of targeted genes.
Altered levels of endogenous retrovirus‐like sequence (VL30) RNA during mouse epidermal cell carcinogenesis
Authors: Han Kyung‐An , Rothberg Paul , Kulesz‐Martin Molly
Published: February 2007

Gene expression during mouse keratinocyte carcinogenesis was examined in a clonal cell model. Tumor cells from three separate initiated cell lineages were compared with their nontumorigenic precursors and with the progenitor cell strain prior to treatment with 7,12‐dimethylbenz[a]anthracene (DMBA). The steady‐state levels of VL30 RNA in normal and papilloma cells were regulated by extracellular Ca2+ (which controls proliferation and differentiation in normal epidermal keratinocytes) and culture density. In contrast, steady‐state levels of VL30 RNA were not regulated by these factors in the squamous cell carcinoma or the anaplastic carcinoma cells. VL30 expression was Ca2+ dependent in the initiated cell precursors within each tumor cell lineage, suggesting that the loss of response to extracellular Ca2+ was associated with the malignant conversion stage of carcinogenesis. No differences between normal and tumor cells were found in the cellular RNA levels of five additional proto‐oncogenes. The mouse epidermal cell model should provide a means for direct assessment of a potential functional role of VL30 sequences in cancer development.
A single mutation in one of the core elements of moloney murine leukemia virus reduced binding of a 42‐kDa T lymphoma cell nuclear factor but did not affect lymphomagenesis
Authors: Case Roger S. , Khang Yong‐Ho , Kumar Abhay , Yuen Pick‐Hoong
Published: February 2007

The genetic determinant responsible for virulence in Moloney murine leukemia virus (MoMuLV) induced T‐cell lymphomagenesis has recently been mapped [J Virol 63:471–480, 1989] by homologous genomic fragment exchange between MoMuLV and MoMuLV‐TB to the Clal/Xbal at the 3' end of the genome. This region of MoMuLV and MoMuLV‐TB differs in 11 nucleotides. Of these 11 nucleotide differences, 9 are distributed within the two CORE, the two distal NF1, and the two GRE/LVa elements of the enhancer. Since both the CORE binding sites of MoMuLV‐TB are mutated with respect to those of MoMuLV, we compared nuclear proteins of a thymusbone marrow cell line and a T‐lymphoma cell line (EMT), which bind to the wild‐type and mutant CORE binding sites. Using both the bandshift assay and southwestern analysis with labeled synthetic deoxyoligonucleotides, we showed that a 42‐kDa protein from TB and EMT cells bound specifically to the MoMuLV CORE element. The T SSC transversion of nucleotide 6 of the CORE consensus, TGTGGT/CTAA, significantly reduced binding of the 42‐kDa TB and EMT cell factors. However, the transversion of nucleotide 3 from T →C had little effect on the binding of the 42‐kDa protein to the CORE element. In addition, the 42‐kDa protein bound weakly to the CCAAT element of MoMuLV. A recombinant virus, NwtTB‐6, was generated by introducing the two CORE mutations of MoMuLV‐TB into the MoMuLV genome. Although the latency period of NwtTB‐6 in the induction of lymphoma was not significantly different from that of MoMuLV, preliminary findings suggest that the lymphoma induced by NwtTB‐6 may be more widely distributed.
Partial characterization of epidermal protein kinase C in mice sensitive or resistant to phorbol ester
Authors: Hirabayashi Naoki , Warren Barbour S. , Wang Xiao‐Jing , Petersen‐Marht Svend , Beltran Linda , Davis Melika M. , Ashendel Curtis L. , Digiovanni John
Published: February 2007

The present study has characterized several aspects of the mouse epidermal protein kinase C (PKC) system and compared phorbol ester‐sensitive and‐resistant mice. Protein immunoblots of partially purified epidermal PKC preparations from SENCAR and C57BL/6 mice indicated the presence of the γ‐, β‐, and α‐isozymes of PKC in both strains. Hydroxylapatite chromatography profiles of epidermal PKC isozymes from SENCAR and C57BL/6 mice revealed three major peaks of PKC activity eluting in fractions similar to those observed in chromatograms of brain tissue and corresponding to PKC‐γ, ‐β, and ‐α. Further analyses of hydroxylapatite chromatography fractions revealed that PKC‐γ and ‐β were present in approximately similar proportions and were much more abundant than PKC‐α. This distribution of epidermal PKC isozymes was similar in both strains. After a single topical application of 3.4 nmol 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to SENCAR mouse epidermis, total PKC activity in the cytosol fraction decreased rapidly to about 50% of control within 15 min and was accompanied by an increase (∼150% of control) of PKC activity in the membrane fraction. At 4 h, PKC activities were significantly lower than the control levels and remained downregulated through 96 h with a maximal decrease (to ∼25–30% of the control) in both cytosol and membrane fractions at 48 h. PKC activity returned to control levels by 168 h. Ca++/phospholipid‐independent kinase activity was the same as control levels at 15 min, 1 h, and 4 h after TPA treatment but was elevated above control levels at 24 h, 48 h, and 96 h, and by 168 h returned essentially to control levels. No differences were found in the magnitude or kinetics of TPA‐induced translocation and downregulation of total PKC or appearance of Ca++/phospholipid‐independent kinase activity between SENCAR, DBA/2, and C57BL/6 mice. Scatchard analyses using a two binding site model revealed that the apparent Kd and Bmax values for binding of PDBu to epidermal cytosol and membrane fractions were similar between SSIn, SENCAR, DBA/2, and C57BL/6 mice. The present results demonstrate for the first time that mouse epidermis contains significant amounts of the three major PKC isozymes that are present in brain, especially PKC‐γ. In addition, topical application of a promoting dose of TPA did not lead to complete loss of PKC activity in either the membrane or cytosol fractions of mouse epidermis. In conclusion, no differences were observed between phorbol ester‐sensitive and ‐resistant mice in any aspect of epidermal PKC examined.
Relationship between chemically induced ha‐ras mutation and transformation of BALB/c 3T3 cells: Evidence for chemical‐specific activation and cell type—specific recruitment of oncogene in transformation
Authors: Nakazawa Hisayoshi , Aguelon Anne‐Marie , Yamasaki Hiroshi
Published: February 2007

BALB/c 3T3 cells were exposed to 7, 12‐dimethylbenz[a]anthracene (DMBA) and resultant transformed foci were analyzed for the presence of A182 → T mutation at codon 61 of Ha‐ras (a mutation found in many DMBA‐induced animal tumors). None of the 30 independently cloned transformed cell lines contained such a mutation. In order to see whether DMBA is able to induce this mutation in BALB/c 3T3 cells, we developed a method sensitive enough to detect this specific mutation at the frequency of 10–6. Employing this assay, we found time‐and dose‐dependent induction by DMBA of Ha‐ras A182 → T mutation in BALB/c 3Tc cells; for example, 2 wk after exposure to 100 μg/mL DMBA, 1.4 in 1 × 104 cells contained this specific mutation. On the other hand, other agents that also induce BALB/c 3T3 cell transformation, such as 3‐methylcholanthrene (MCA), 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), N‐methyl‐N'‐nitro‐N‐nitrosoguanidine (MNNG), or ultraviolet light, did not induce the mutation at detectable frequency (< 10–6). These results suggest that DMBA efficiently induces Ha‐ras mutation in BALB/c 3T3 cells but that this mutation is not recruited in the process of cell transformation. A hypothesis of carcinogen‐specific mutation of Ha‐ras gene and its tissue (cell type)‐specific recruitment in carcinogenesis is proposed.
Nickel mutagenesis: Alteration of the MuSVts 110 thermosensitive splicing phenotype by a nickel‐induced duplication of the 3′ splice site
Authors: Chiocca Susanna M. , Sterner Deborah A. , Biggart Neal W. , Murphy Edwin C.
Published: February 2007

We investigated DNA damage caused by carcinogenic metals in a murine sarcoma virus (MuSV)‐based mutagenicity assay in which mutations targeted to v‐mos expression can be selected. Nickel chloride treatment of NRK cells (termed 6m2 cells) infected with MuSVts110, a retrovirus conditionally defective in viral RNA splicing and cell transformation, caused the outgrowth of transformed “revertants” with changes in the MuSVts110 RNA splicing phenotype. Cadmium and chromium treatment of 6m2 cells resulted in the selection of a second class of revertants with what appeared to be frameshift mutations allowing the translation of a readthrough gag‐mos protein. In both classes of metal‐induced revertants, viral gene expression was distinct from that observed in revertants arising in untreated 6m2 cultures, arguing that metal treatment did not simply enhance the rate of spontaneous reversion. In one representative nickel revertant line the operative nickel‐induced mutation affecting MuSVts110 RNA splicing was a duplication of 70 bases surrounding the 3′ splice site. The effect of this mutation was to direct splicing to the most downstream of the duplicated 3′ sites and concomitantly relax its characteristic thermosensitivity. These data establish the mutagenic potential of nickel and provide the first example of a defined nickel‐induced mutation in a mammalian gene.
Expression of retinoic acid α and β receptor genes in liver and hepatocellular carcinoma
Authors: Sever Cordelia E. , Locker Joseph
Published: February 2007

cDNA probes for human retinoic acid receptors α and β (RARα and RARβ) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RARβ mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RARβ mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RARα mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RARα or RARβ mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low α‐fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of α‐fetoprotein were markedly stimulated by RA.
Role of growth hormone in the regulation of the c‐myc gene during progression of sex‐differentiated rat liver carcinogenesis in the resistant hepatocyte model
Authors: Hällström I. Porsch , Gustafsson Jan‐Åke , Blanck Agneta
Published: February 2007

In this study, we extended the previous observations of a growth hormone‐regulated sex difference in hepatic c‐myc expression in the resistant hepatocyte model during the selection/promotion phase, when sex differences in growth rate of enzyme‐altered foci are first identified, to studies of the regulation of this gene during later stages of hepatocarcinogenesis. The expression of the c‐myc gene was studied in preneoplastic nodules, hepatocellular carcinomas, and the corresponding surrounding livers of male and female Wistar rats treated according to the resistant hepatocyte model. In males, nodules isolated 8 and 11‐12 mo after initiation and hepatocellular carcinomas exhibited a 2.5‐ to threefold higher c‐myc expression than the surrounding liver. In females, no increase in c‐myc expression was observed in nodules 8 mo after initiation, while nodules isolated after 11‐14 mo and tumors snowed a twofold and threefold increase, respectively, when compared with the surrounding tissue. Increased transcription of the c‐myc gene was observed in nuclei from male nodules isolated 11 mo after initiation compared with nuclei from the surrounding liver. The difference in transcription between male nodules and surrounding tissue is similar for the first and second exon of the gene. Continuous infusion of growth hormone to nodule‐bearing male rats 8 and 11 mo after initiation decreased c‐myc expression in the surrounding tissue and downregulated the expression in 8‐mo nodules to the level in the surrounding liver. No significant decrease in response to growth hormone treatment was seen in 11‐mo nodules. In hypophysectomized nodule‐bearing males, nodular c‐myc remained upregulated. Taken together, the data showed that the sex difference in c‐myc expression was maintained during a large part of the progression period. Furthermore, the loss of growth hormone regulation of the c‐myc gene in advanced male nodules indicated an escape from normal regulatory mechanisms during progression. These findings might reflect a role for the c‐myc gene in sex‐differentiated rat liver carcinogenesis.
Ki‐ras mutations in spontaneous and chemically induced renal tumors of the rat
Authors: Ohgaki Hiroko , Kleihues Paul , Hard Gordon C.
Published: February 2007

A high frequency of point mutations at codon 12 of the Ki‐ras gene has previously been reported for rat kidney mesenchymal tumors induced by methylating N‐nitroso compounds. In this study, we analyzed renal tumors with divergent histogenesis, i.e., mesenchymal tumors (sarcomas), cortical epithelial tumors (carcinomas), and embryonal tumors (nephroblastomas). Renal mesenchymal tumors and carcinomas were induced in juvenile or young adult Wistar rats by a single dose of N‐nitrosodimethylamine (NDMA) while nephroblastomas were induced in Nb hooded rats by a single transplacental dose of N‐nitrosoethylurea (NEU). Nephroblastomas developing spontaneously in WAB/Not rats were also examined. Amplification of Ki‐ras sequences from formalin‐fixed, paraffin‐embedded tissue by the polymerase chain reaction was followed by direct DNA sequencing. GGT GAT point mutations at codon 12 of the Ki‐ras gene were found in 9 of 12 (75%) renal mesenchymal tumors and in 9 of 12 (75%) cortical epithelial tumors induced by NDMA. Even higher incidences were observed in nephroblastomas (8/8; 100%) induced by NEU and in spontaneous nephroblastomas (10/11; 91%). These results indicate that Ki‐ras mutations are frequent events during the development of kidney tumors irrespective of their histogenesis and suggest that they may play an important role in renal carcinogenesis in rats. These data further indicate that mutational activation of Ki‐ras proto‐oncogenes in carcinogen‐induced rat kidney tumors occurs in a tissue‐specific, rather than cell‐specific, manner.
Correlation of increased levels of Ha‐ras T24 protein with extent of loss of gap junction function in rat liver epithelial cells
Authors: de Feijter Adriaan W. , Trosko James E. , Krizman David B. , Lebovitz Russell M. , Lieberman Michael W.
Published: February 2007

Although it is known that cells transformed by ras and other oncogenes show reduced gap junction function, to date there has been no investigation of the quantitative relationship between intracellular levels of ras oncoprotein and loss of cell‐cell communication. Using the rat liver epithelial cell line MTR6, which carries a zinc‐inducible metallothionein ras T24 (MT ras T24) fusion gene, we showed a direct correlation between the accumulation of ras T24 protein and the loss of dye transfer as measured by interactive laser cytometry. After stimulation with zinc sulfate, changes in both parameters were rapid and measurable by 24 h. Similarly, there was a dose‐response relationship between loss of gap junction function and increase in ras T24 protein. Northern analysis of two gap junction proteins (connexins 43 and 32) showed no differences between cells that expressed high levels of ras and control cells. These data demonstrate that the degree of loss of gap junction function is dependent on the amount of increase in ras T24 protein levels, but the mechanism by which these changes are effected remains unclear. © 1992 Wiley‐Liss, Inc.
Rare frequency of activation of the ki‐ras gene in rat colon tumors induced by heterocyclic amines: Possible alternative mechanisms of human colon carcinogenesis
Authors: Kakiuchi Hideki , Ushijima Toshikazu , Ochiai Masako , Sugimura Takashi , Nagao Minako , Imai Kozo , Yachi Akira , Ito Nobuyuki
Published: February 2007

Heterocyclic amines present in cooked foods are known to produce colon tumors in F344 rats at a high incidence, indicating the possibility of involvement of ras gene activation in colon carcinogenesis in rats as in humans. We examined mutations at codons 12, 13, and 61 of the Ki‐ras, Ha‐ras, and N‐ras genes by polymerase chain reaction—direct sequencing in seven colon tumors in F344 rats induced by 2‐amino‐6‐methyldipyrido‐[1,2‐a:3′,2′‐d]imidazole (Glu‐P‐1), 11 induced by 2‐amino‐3‐methylimidazo[4,5‐f]quinoline, and nine induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine. A Ki‐ras gene mutation (G → T at the second position in codon 12) was found in one Glu‐P‐1—induced colon adenocarcinoma. None of the other 26 tumors had mutations in any of these three ras family genes. These results indicate that in rats, colon carcinogenesis induced by heterocyclic amines may be induced by alterations of other oncogenes or tumor suppressor genes. We think this experimental system using carcinogens to which humans are exposed is a good model for studying alterations of other genes in human colon tumors in which no Ki‐ras alterations are observed. © 1993 Wiley‐Liss, Inc.
No point mutation of ha‐ras or p53 genes expressed in preneoplastic‐to‐neoplastic progression as modeled in mouse JB6 cell variants
Authors: Sun Yi , Nakamura Kazuki , Hegamyer Glenn , Dong Zigang , Colburn Nancy
Published: February 2007

Alterations of the p53 gene have been found in many human and animal tumors, and ras mutations have been seen somewhat less frequently. Loss of p53 tumor suppressor activity has been implicated in multistage tumor promotion/progression after initiation by Ha‐ras or Ki‐ras activation. Whether p53 or ras alterations occur during promotion of neoplastic transformation in JB6 cells has not been reported. Using a series of mouse JB6 variants representing different stages of progression toward the tumor‐cell phenotype, we investigated whether mutational activation of Ha‐ras and mutational inactivation and altered expression of the p53 gene occurred during progression. We report here that neither point mutations of the Ha‐ras or p53 genes nor p53 structural alterations were involved in this process. Although there was no significant difference in steady‐state levels of p53 mRNA, an elevated level of immunoprecipitable p53 protein was observed in a subset of transformed cells. We also report the detection of a second 2.2‐kb p53‐hybridizing transcript. This transcript was not translated into p53 protein and may be a nuclear precursor of the mature message. Both p53‐hybridizing transcripts were downregulated by prolonged 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) treatment (24 h) regardless of the stage of progression. We conclude from this study that in JB6 variants (1) mutational activation of Ha‐ras and inactivation of p53are unlikely to be involved in preneoplastic progression; (2) increased amounts of p53 protein may be involved in a subset of transformed cells, possibly reflecting a longer half‐life, as demonstrated in other systems; and (3) late downregulation of p53 mRNA but not protein expression may be a secondary response in the TPA‐mediated signal transduction pathway. © 1993 Wiley‐Liss, Inc.
Genetic analysis of carcinogen enhancement of type 5 adenovirus transformation of cloned fischer rat embryo fibroblast cells
Authors: Su Zao‐Zhong , Shen Ruoqian , Young C. S. H. , Fisher Paul B.
Published: February 2007

Pretreatment of CREF cells with methyl methanesulfonate (MMS) before infection with the host‐range cold‐sensitive type 5 adenovirus (Ad5) mutant H5hr1 results in a dose‐dependent carcinogen enhancement of viral transformation (CET). The properties of CET observed with H5hr1, which include both an MMS dose‐dependent enhancement in the number of transformed foci and an increase in transformation frequency after correction for cell toxicity, are not observed in carcinogen‐pretreated wild‐type (wt) Ad5 (H5wt)‐infected CREF cells. This study was conducted to determine the role of the viral E1A and E1B transforming genes of H5hr1 in mediating the unique CET phenotype of H5hr1. Coinfection of MMS‐pretreated CREF cells with H5wt or H5sub309 (which displays a wt Ad5 phenotype) and H5hr1 resulted in a suppression of the unique CET phenotype that was directly related to the multiplicity of infection with wt Ad5. Suppression of the unique H5hr1 CET phenotype was also apparent in MMS‐pretreated CREF cells coinfected with H5hr1 and an Ad5 mutant expressing either a wt 13S E1A‐encoded 289 amino‐acid (aa) protein and an intact wt E 1B gene or a wt 13S E1A‐encoded 289‐aa protein and a 22S E1B‐encoded 495‐aa protein. In contrast, the unique H5hr1 CET phenotype was not suppressed in MMS‐pretreated CREF cells coinfected with H5hr1 and Ad5 or Ad2 mutants expressing either a wt 12S E1A‐encoded 243‐aa protein and both wt E1B gene products or an intact wt E1A gene and a wt E1B 13S‐encoded 175‐aa protein. That genetic changes in both the E1A and E1B viral regions of H5hr1 were required to induce the unique CET phenotype was also indicated by the inability of a recombinant Ad5 containing the 0—4.5 map‐unit region of H5hr1 and the 4.5–100 map‐unit region of H5sub309 to display the H5hr1 unique CET phenotype. Direct confirmation of the requirement for both gene regions of H5hr1 to mediate its unique CET was obtained by generating CREF cells stably expressing a wt Ad5 E1A 13S‐encoded 289‐aa protein and a wt E1B 22S‐encoded 495‐aa protein. In these CREF transformants (which displayed a CREF‐like morphology), transformation by H5hr1 was not reduced, but the unique CET phenotype after MMS pretreatment was eliminated. These results suggest that alterations in both the 13S‐encoded E1A and 22S‐encoded E1B gene products of H5hr1 contribute to its unique CET.
Association of tumorigenic and nontumorigenic (immunogenic) variants in a mouse t‐cell lymphoma with two distinct p53 mutations
Authors: Bergel Michael , Hochman Jacob , Siwarski David , Huppi Konrad , Bhatia Kishor , Gutierrez Marina
Published: February 2007

An in vitro model system for xenogenization has been developed in which an immunogenic, nonmalignant phenotype was selected from a highly malignant T‐cell line (S49). We showed by single‐strand conformation polymorphism and DNA sequence analysis that specific point mutations in the p53 tumor suppressor gene correlated with a change from a tumorigenic to a nontumorigenic (immunogenic) phenotype. Specifically, we found that the highly malignant S49 cell line T‐60 contains an Arg→Gln substitution at residue 246 in exon 7 of p53. In contrast, nontumorigenic (immunogenic) variants (T‐25‐Adh and Rev‐1) exhibited a Gly→Ser substitution at residue 242 of p53. In two subsequent tumorigenic revertants derived from Rev‐1, we again found the Arg→Gln substitution at residue 246 that was found initially in the T‐60 cells. Thus, mutation at residue 246 of p53 was associated with a highly malignant phenotype, whereas a novel mutation at residue 242 of p53 appeared to be associated with a nonmalignant phenotype and may have actually protected the host through immunization. We conclude that mutation of residue 242 may represent a new class of permissive (nonmalignant) mutations in the mouse that are analogous to the Li‐Fraumeni mutation in humans. © 1993 Wiley‐Liss, Inc.
Analysis of calcium‐dependent protein kinase C isoforms in the early stages of diethylnitrosamine‐induced rat hepatocarcinogenesis
Authors: Perletti Gian Paolo , Piccinini Francesco , La Porta Caterina A. M. , Comolli Roberto
Published: February 2007

The profiles of the calcium‐dependent protein kinase C (PKC) isozymes α, β, and γ were examined in subcellular fractions from Fischer 344 rat liver during the early stages (48 h, 96 h, 7 d, and 60 d) of diethylnitrosamine (DEN)‐induced carcinogenesis, using the Solt‐Farber “resistant hepatocyte” model (DEN‐2‐acetylaminofluorenepartial hepatectomy; DEN‐AAF‐PH), and then related to the presence of focal or nodular m̀‐glutamyl transpeptidase (GGT)—positive morphologic changes in the liver. After DEAE and hydroxyapatite column chromatograaphy, two peaks, immunologically identified as PKC‐α and ‐β isoforms, were detected in the liver of normal (α/β ratio=4.0) and treated rats. In DEN‐AAF‐PH hepatocarcinogenesis an increase in PKC‐α expression was found after PH ( + 43 ± 19% at 48 h, α/β ratio=5.1; + 125 ± 25% at 96 h, α/β ratio=4.8), whereas the PKC‐β isoform appeared less significantly modified (+ 11 ± 3% at 48 h and + 89 ± 17% at 96 h). Seven and 60 days after PH, a marked increase in the PKC‐α ( + 96 ± 20% and + 150 ± 48%, respectively) and PKC‐β isoforms (+ 158 ± 41%, α/β ratio=3.1 and + 130 ± 26%, α/β ratio=4.4, respectively), occurred along with the appearance of GGT‐positive altered hepatic foci and nodules in the liver sections. Sham hepatectomy caused PKC‐α and ‐β isoform activities similar to those of normal controls. In contrast, saline‐AAF‐PH‐treated rats had downregulation of PKC‐α after PH (α/β ratio=1.8 at 96 h), possibly due to the mitoinhibitory effect of the carcinogen AAF on normal uninitiated hepatocytes. Immunohistochemical analysis with monoclonal antibodies to PKC‐α and ‐β revealed diffuse positive cytoplasmic signals in GGT‐positive foci and nodules in rat liver. Taken together, these preliminary results, using the Solt‐Farber model of liver carcinogenesis, suggest a role for PKC in tumor promotion. They also suggest that the PKC‐α isoform may play a specific role in clonal expansion of DEN‐initiated hepatocytes after PH.
Mutations and expression of the p53 gene in rat bladder carcinomas and cell lines
Authors: Asamoto Makoto , Mann Angela M. , Macatee Timothy L. , Cohen Samuel M.
Published: February 2007

Abnormalities of the p53 gene are frequently observed in human tumors, including urinary bladder carcinoma, suggesting that p53 plays an important role in human carcinogenesis. However, its role in rat bladder carcinogenesis is unclear. We investigated p53 gene mutations and expression in rat urinary bladder carcinogenesis in vivo and in Vitro. Fifteen urothelial cell lines, including six untransformed (nontumorigenic) ones, six transformed (tumorigenic) in vitro, and three derived from tumors induced in vivo, were examined for p53 expression by immunochemical analysis and for p53 mutations; in addition, 81 rat bladders were analyzed immunohistochemically for p53 expression, and 23 rat bladder tumors were analyzed for p53 mutations. Four cell lines had mutations in the p53 gene. Two of these were missense point mutations, and the other two were splicing mutations. On the other hand, no mutations were found in the bladder tumors induced in rats. By immunoprecipitation with PAb240, which is supposed to be specific for mutant p53, we detected mutations in three of the cell lines; PAb240 did not react with wild‐type p53. However, in all cell lines and in growing populations of primary cultured bladder urothelial cells, p53 expression was detected immunohistochemically or by western blotting using PAb240 or PAb 421 monoclonal antibodies. In a high percentage of transitional cell carcinomas, wild‐type p53 expression was detected by immunohistochemical analysis with PAb240. These results suggest that p53 gene mutations may not occur frequently in rat bladder carcinogenesis in vivo but may occur in vitro and that p53 overexpression detected immunohistochemically is common and may be related to cell proliferation rather than to the presence of mutations in rat bladder carcinogenesis. © 1994 Wiley‐Liss, Inc.
Association of elevated levels of protein kinase C‐ζ mrna and protein with murine B‐lymphocytic neoplasia
Authors: Goodnight Joanne , Mischak Harald , Mushinski J. Frederic
Published: February 2007

Expression of mRNA for protein kinase C(PKC)‐α, ‐β, ‐γ, ‐δ, ‐ϵ, ‐ζ, and ‐η has been shown, by polymerase chain reaction‐generated isozyme‐specific probes, to be cell‐type‐ and differentiation‐stage‐specific in mouse hemopoietic cells. Recently, we cloned a 2.2‐kb mouse PKC‐ζ cDNA. In this study, we used the nearly full‐length cDNA PKC‐ζ probe to demonstrate that expression of PKC‐ζ was significantly elevated in lymphocytic neoplasms at both the mRNA and protein levels. Normal brain, kidney, and liver contain 2.4‐ and 4.4‐kb mRNAs, whereas normal lymphoid organs (spleen, thymus, and lymph nodes) express barely detectable amounts of PKC‐ζ. These vanishingly small levels of PKC‐ζ mRNA did not increase when polyclonal spleen B‐cell proliferation and differentiation were induced in vivo with anti—immunoglobulin D antiserum or in vitro with lipopolysaccharide. In contrast, 2.4‐kb transcripts of PKC‐ζ are abundant in virtually all neoplastic B‐lymphocytic cell lines. Furthermore, additional transcripts of a novel size, about 7 and 8 kb, were found in several mature B‐cell lymphomas and plasma cell tumors. Western blot analysis of protein extracts from normal B cells and hemopoietic tumors confirmed that these quantitative differences in PKC‐ζ mRNA also exist at the protein level. That is, only trace amounts of PKC‐ζ protein were detectable in pro‐B cells and pre‐B cells, but abundant amounts of this isoform were found in protein extracts from most B‐cell lymphomas and plasma cell tumors. These findings suggest that this atypical member of the PKC multigene family participates in the multistep process of malignant transformation of lymphocytes.
Lack of concordant p53 mutations in some paired primary and metastatic mouse squamous cell carcinomas induced by chemical carcinogenesis
Authors: Zhang Shi‐Yu , Bauer Brett , Mitsunaga Shin‐Ichiro , Goodrow Tamra L. , Klein‐Szanto Andres J. P.
Published: February 2007

We studied the frequency and pattern of p53 mutations in 16 mouse skin primary squamous carcinomas induced by chemical carcinogens and their 19 matched metastases. The molecular changes were analyzed by polymerase chain reaction‐single‐strand conformation polymorphism and subsequent direct sequencing analysis. Eleven mutations of the p53 gene were detected in a total of eight primary tumors, and 10 mutations were detected in nine metastases. Only four pairs had identical mutations in primary and paired metastatic tumors. Eight mutations in six pairs were detected in primary tumors but not in their metastases, and four mutations from three matched pairs were detected in metastases but not in primary tumors. The four pairs that contained the same mutations in both the primary and secondary tumors had lymph‐node metastases, and all mutations occurred in exon 8. Conversely, five of six pairs with p53 mutations only in primary tumors had lung metastases and only one of the mutations occurred in exon 8. None of the mutations found only in metastases were located in exon 8. These data indicate that p53 mutations are prevalent in lymph‐node metastases and infrequent in lung metastases of mouse skin tumors and that primary tumors with exon 8 mutations may be more likely to metastasize to the lymph nodes. © 1995 Wiley‐Liss Inc.
Differential mutagenicity and cytotoxicity of (±)‐benzo[a]pyrene‐trans‐7,8‐dihydrodiol and (±)‐anti‐benzo[a]pyrene‐trans‐7,8‐dihydrodiol‐9, 10‐epoxide in genetically engineered human fibroblasts
Authors: Quan Taihao , Reiners John J. , Culp Sandra J. , Richter Patricia , States J. Christopher
Published: February 2007

DNA repair–deficient (xeroderma pigmentosum group A (XPA)) and DNA repair‐proficient (normal) human skin fibroblasts were genetically engineered by transformation with a controllable human cytochrome P450 (CYP)1A1 expression vector. Induction of CYP1A1 enabled these cells to metabolize (±)‐benzo[a]pyrene‐trans‐7,8‐dihydrodiol (BPD) into a potent cytotoxicant and mutagen. The XPA cells were more susceptible than the normal cells to the cytotoxic effects of both CYP1A1‐metabolized BPD and exogenously supplied (±)‐antibenzo[a]pyrene‐trans‐7,8‐dihydrodiol‐9, 10‐epoxide (BPDE). Furthermore, the differential cytotoxicity between XPA and normal cells induced by CYP1A1‐metabolized BPD was 8.4‐fold greater than that induced by exogenously supplied BPDE. The two cell lines had similar CYP1A1 activities, suggesting that a difference in metabolic potential was not the cause of the differential response to BPD. At comparable cytotoxicity in both XPA and normal cells, BPD treatment induced more mutants and more DNA adducts than BPDE treatment did. At similar levels of DNA adducts in XPA cells, the levels of cytotoxicity induced by CYP1A1‐metabolized BPD and exogenously supplied BPDE were similar, but CYP1A1‐metabolized BPD induced a threefold higher hypoxanthine phosphoribosyltransferase mutation frequency. In contrast, at similar levels of adducts in CYP1A1‐expressing normal cells, BPD induced less cytotoxicity and a lower mutation frequency. DNA adducts were identified and quantified by 32P‐postlabeling analyses. The principal adduct formed by both CYP1A1‐metabolized BPD and exogenously supplied BPDE was 10‐β‐(deoxyguanosin‐N2‐yl)‐7β,8α,9α‐trihydroxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene, indicating that the differential effects of BPD‐ and BPDE‐induced adducts were not due to a difference in the types of adducts formed. The results of these studies suggest that CYP1A1‐metabolized BPD may form adducts preferentially in transcriptionally active genes or that the intracellular concentration of BPDE may influence the balance between cytotoxicity and mutagenicity (or both). © 1995 Wiley‐Liss Inc.
Expression of β1 integrin receptors in transformed mouse epidermal keratinocytes: Upregulation of α5β1 in spindle carcinoma cells
Authors: Gomez Manuel , Cano Amparo
Published: February 2007

The adhesive properties and the expression of extracellular matrix receptors of the β1‐integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One‐ and two‐dimensional analyses of the immunoprecipitates obtained with anti‐β1‐ and specific anti‐α‐integrin subunits showed qualitative and quantitative changes in the expression of β1 integrins by the different cell lines. The polyvalent α3β1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor α5β1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of α5β1 integrin in spindle cells appeared organized in cell‐substratum contact areas on spread cells. In addition, high and homogenous expression of α5β1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of α5β1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor α2β1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of α2β1 was correlated with the increased adhesion to collagen type I and Collagen type IV exhibited by the tumorigenic cell lines. © 1995 Wiley‐Liss Inc.
Contribution of O6‐methylguanine‐DNA methyltransferase to monofunctional alkylating‐agent resistance in human brain tumor—derived cell lines
Authors: Bobola Michael S. , Blank A. , Berger Mitchel S. , Silber John R.
Published: February 2007

The DNA repair protein O6‐methylguanine‐DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma‐ and gliomaderived cell lines differ in sensitivity to the methylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), as shown by their 28‐fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6‐benzylguanine (O6‐BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3‐ to 30‐fold reductions in LD10 among the lines, and varies up to 20‐fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20‐fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6‐BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N‐ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial. © 1995 Wiley‐Liss, Inc.
Analysis of v‐Ha‐ras and v‐fos oncogene transduction into a mouse epidermal cell line with “initiated” phenotype in culture but normal skin phenotype in vivo
Authors: Ueda Masato , Kawamura Hideki , Sutter Christian , Glick Adam , Yuspa Stuart H. , Strickland James E.
Published: February 2007

Cell line SCR722 was derived from adult SENCAR mouse epidermal cells initiated in culture by treatment with the carcinogen N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and selection for foci proliferating in medium with calcium levels that induce terminal differentiation in normal cells. Expansion of one of these foci and two additional cell clonings produced cell line SCR722, which was near‐tetraploid and formed normal skin when grafted to athymic nude mouse hosts. However, unlike normal keratinocytes, SCR722 cells fail to suppress papilloma formation when grafted along with papilloma cell line SP‐1. For optimum growth in culture, SCR722 cells required fibroblast‐conditioned medium and 0.5 mM Ca2+. SCR722 cells had a wild‐type c‐Ha‐ras gene but had lost their requirement for conditioned medium in culture and produced dysplastic papillomas in grafts when transduced with the v‐Ha‐ras gene. SCR722 cells stably expressing the v‐fos gene produced normal epidermis in grafts, but when these cells were transduced with the v‐Ha‐ras gene, they produced carcinomas. Clones with greater expression of the transfected v‐fos gene had a more invasive phenotype in vivo. These results indicate that carcinogen treatment of epithelial cells can result in an altered but nontumorigenic phenotype that may be at risk for becoming a more advanced neoplastic state with additional genetic alterations. © 1995 Wiley‐Liss. Inc.
Overexpression of MDM‐2 mRNA and mutation of the p53 tumor suppressor gene in bladder carcinoma cell lines
Authors: Cheng Ya‐Tse , Li Yu‐Ling , Wu Jeng Dau , Long Shih‐Bin , Tzai Tzong‐Shin , Tzeng Ching‐Cherng , Lai Ming‐Derg
Published: February 2007

To investigate the importance of oncogenes and tumor suppressor genes in bladder carcinogenesis, we determined the status of the expression of the MDM‐2 and p53 genes and genetic alterations in the p53 gene in five bladder carcinoma cell lines and one kidney urothelial carcinoma cell line. Overexpression of MDM‐2 mRNA was observed in three bladder carcinoma cell lines, J82, SCaBER, and BFTC‐905. Amplification of the MDM‐2 gene was not detected in any of the six cell lines by Southern analysis. The deletion in the p53 gene was observed in J82, and point mutation was detected in J82 and BFTC‐909, the kidney urothelial carcinoma cell line. In contrast, no mutations were found in codons 12, 13, and 61 in the Ha‐ras and Ki‐ras genes in these six cell lines. These results indicate that alterations in the p53‐regulated pathway are important in bladder carcinogenesis. © 1995 Wiley‐Liss, Inc.
p53 gene mutational spectra in hepatocellular carcinomas induced by 2‐acetylaminofluorene and N‐nitroso‐2‐acetylaminofluorene in rats
Authors: Ho Yuan‐Soon , Cheng Hui‐Teng , Wang Ying‐Jan , Lin Jen‐Kun
Published: February 2007

In this study, the mutation frequencies of the p53 gene in rat hepatocellular carcinomas (HCCs) induced by N‐nitroso‐2‐acetylaminofluorene (NO‐AAF) and 2‐acetylaminofluorene (AAF) were 19.23% (20 of 104) and 31.1% (33 of 106), respectively. Four noteworthy features of the mutation spectrums of the p53 gene in HCCs induced by both NO‐AAF and AAF were observed: (i) There was preferential clustering of mutations at exons 5–8 in both the NO‐AAF or AAF groups, (ii) Nearly all the mutations (98%) induced by NO‐AAF and AAF were point mutations, (iii) A high frequency of the p53 mutations were transition mutations, and the ratios of transition to transversion in the NO‐AAF and the AAF group were 13:6 and 21:12, respectively. Almost all the mutations were G→A transitions and guanosine was the major target base. (iv) The frequency and base location of p53 mutations were significantly associated with cancer cell differentiation. In poorly differentiated HCCs (58 individual tumor samples), mutations were detected in 24 of 58 samples (41.1%) and clustered mostly in exons 7 and 8 (19 of 24 samples), whereas in well‐differentiated HCCs (105 individual tumor samples), the incidence of mutations was low (one of 10 in the AAF group, 17 of 95 in the NO‐AAF group), and the mutations were located in exon 5 (11 of 18). The biological significance of these different mutational spectra among p53 genes deserves further investigation. © 1995 Wiley‐Liss, Inc.