bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutational specificities of n‐nitrosamines in a host‐mediated assay: comparison with direct‐acting n‐nitroso compounds in vitro and an approach to deducing the nature of ultimate mutagens in vivo
Authors: Liu Yu‐Xin , Guttenplan Joseph B.
Published: February 2007

The mutational activities and specificities of several N‐nitrosamines in Salmonella recovered from mouse liver in the host‐mediated assay (HMA) were compared with the specificities of related direct‐acting N‐nitroso compounds in vitro. The specificities of the direct‐acting methyl, ethyl, propyl, and 2‐hydroxypropyl compounds were all different and presumably are attributable to the DNA adducts resulting from the corresponding alkyldiazonium or carbonium ions. Introduction of a 2‐hydroxyl group greatly influenced the mutational specificity. The 2‐oxopropyl compound showed the same specificity as the methyl compound. This result is consistent with one of the known breakdown pathways of the oxopropyl diazonium ion (or related reactive species), which leads to a methyl diazonium ion. The N‐nitrosodialkylnitrosamines N‐nitrosodimethylamine (NDMA), N‐nitrosodiethylamine (NDEA), and N‐nitrosodipropylamine (NDPA), which all require metabolic activation, showed specificities in the HMA similar to those of their direct‐acting counterparts. The cyclic nitrosamine N‐nitro‐sopyrrolidine was weakly active in the HMA, although its direct‐acting derivative was a potent mutagen in vitro. The results for NDMA and NDEA were consistent with most previous studies of the metabolism of these compounds in vivo. However, NDPA can yield methylating, and probably hydroxypropylating, species in addition to propyldiazonium ion. As the specificity of NDPA was similar to that of a propylating agent, NDPA appears to lead to genotoxic products in the mouse liver mainly by direct α‐hydroxylation. The initial results described here indicate that mutational specificity in the HMA can be used to deduce metabolic pathways leading to genotoxic products when the appropriate proximate mutagens are available as standards. Furthermore, we observed a reasonable correlation between potency in the HMA and hepatocarcinogenesis. © 1992 Wiley‐Liss, Inc.
Partial suppression of tumorigenicity in a human lung cancer cell line transfected with Krev‐1
Authors: Caamano Jorge , Dirado Marion , Lizasa Toshihiko , Momiki Shigeru , Fernandes Elma , Ashendel Curt , Noda Makoto , Klein‐Szanto Andres J. P.
Published: February 2007

A human non‐small‐cell lung carcinoma cell line, Calu‐6 (from an anaplastic carcinoma), was transfected with the Ki‐ras‐related anti‐oncogene Krev‐1. Several transfectant lines were obtained that showed a reduced tumorigenicity in nude mice with respect to the parental and control transfected cell lines. This decrease was approximately 50% in tumor incidence at 4 wk after subcutaneous inoculation of the transfected cells. In addition, the volume of the Calu‐6 revertant‐derived tumors was three to 10 times smaller than that of the equivalent tumors produced by inoculation of the control cell line transfected with the neomycin‐resistance gene. Krev‐1‐transfected cells that exhibited reduced tumorigenicity expressed Krev‐1 mRNA and had variable numbers of copies of the Krev‐1 gene. Moreover, Krev‐1 ‐transfected cells exhibited a more differentiated squamous epithelial morphology than the parental and control cell lines did. Moderately elevated levels of protein kinase C activity were detected in some revertant clones. Such activity correlated with the level of expression of Krev‐1 mRNA in most cases. In summary, Krev‐1 induced important morphological and biological changes in transfected Calu‐6 cells that we interpreted as partial reversion of the malignant phenotype. © 1992 Wiley‐Liss, Inc.
Growth and transformation suppressor genes for BHK syrian hamster cells on human chromosomes 1 and 11
Authors: Annab Lois A. , Dong Jin‐Tang , Futreal P. Andrew , Satoh Hitoshi , Oshimura Mitsuo , Barrett J. Carl
Published: February 2007

To map putative tumor suppressor genes for the near‐diploid baby hamster kidney fibrosarcoma cell line BHK, we transferred five different normal human chromosomes (1, 3, 7, 11, and 12) into these tumor cells by microcell‐mediated chromosome transfer. Transfer of human chromosome 1 into BHK cells resulted in suppression of cell growth both on plastic and in soft agar, indicating that chromosome 1 has a generalized effect on cell growth and thereby suppresses anchorage‐independent growth. Selection against cells with an intact chromosome 1 was observed. In contrast, the introduction of chromosome 11 into BHK cells resulted in suppression of anchorage independence but not growth on plastic. Most chromosome‐11 growth‐suppressed BHK hybrids retained intact copies of human chromosome 11. Tumorigenic derivatives of chromosome 11 hybrids had lost this chromosome. Transfer of human chromosome 3, 7, or 12 into BHK cells did not correlate with growth suppression of BHK cells on plastic or in soft agar. Thus, we conclude that genes that suppress BHK‐cell growth in general or in agar reside on human chromosomes 1 and 11, respectively. © 1992 Wiley‐Liss. Inc.
Lack of mutations of the p53 tumor suppressor gene in hepatocellular carcinomas induced in rats by a peroxisome proliferator
Authors: Smith Martin L. , Yeleswarapu Lakshmi , Lombardi Benito , Shinozuka Hisashi
Published: February 2007

Immunohistochemical, immunoblotting, and DNA‐sequencing analyses were performed on hepatocellular carcinomas induced in rats chronically fed BR931, a peroxisome proliferator, to determine whether the tumors carried mutations or other alterations of the p53 gene. None were detected. Inactivation of this tumor suppressor gene does not appear, therefore, to be involved in the carcinogenicity of BR931, a nongenotoxic chemical hepatocarcinogen.
Low frequency of codon 61 Ha‐ras mutations and lack of keratin 13 expression in 7,12‐dimethylbenz[a]‐anthracene—induced hamster skin tumors
Authors: Robles Ana I. , Gimenez‐Conti Irma B. , Roop Dennis , Slaga Thomas J. , Conti Claudio J.
Published: February 2007

Alterations in the pattern of keratin expression are a common feature of skin‐tumor development. In this study, we investigated whether the loss of epidermal keratin 1 (K1) and its replacement by mucosal keratin 13 (K 13) is unique to mouse skin tumors induced by 7,12‐dimethylbenz[a]anthracene (DMBA) and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), since it has been reported that human epidermal tumors do not exhibit aberrant expression of K13. With that purpose, we analyzed the keratin profiles of 16 DMBA‐induced hamster skin tumors using monospecific antibodies against K1 and K13. Although all the tumors expressed K1, they also showed an overall tendency towards loss of this keratin; furthermore, none of the tumors expressed K13. Previous studies have suggested that the induction of K13 in mouse skin is related to the mutation of the Ha‐ras gene by the initiating agent DMBA, a mutation consistently found in murine DMBA/TPA‐induced tumors and rarely found in human skin tumors. Therefore, we also evaluated the tumors for the presence of codon‐61 mutations by direct sequencing of DNA extracted from paraffin‐embedded tissue sections. Only three tumors showed an A→T transversion in the second nucleotide of Ha‐ras codon 61. However, presence of the mutation did not correlate with K1 staining. Although hamster skin tumors were induced by the same initiator as were mouse skin tumors, hamster skin tumors did not show the same keratin profile. Moreover, their immunohistochemical expression of K1 and K13 and their codon 61 sequences resembled that of their human counterparts. These results suggest that the aberrant expression of K13 may be unique to murine skin. Furthermore, although codon 61 Ha‐ras mutation appears to be related to keratin alterations in the mouse model, this mutation is not sufficient to produce the same biochemical changes in other species.
Application of denaturing gradient gel blots to detect p53 mutations in X‐ray—transformed mouse C3H 10T1/2 clones
Authors: Krolewski Bozena , Little John B.
Published: February 2007

The denaturing‐gradient gel electrophoresis (DGGE) blot technique is a highly sensitive method developed to detect sequence differences (e.g., single base changes and small deletions) in genomic DNA. We used this method to screen for mutations within the p53 locus of independent X‐ray—transformed C3H 10T1/2 cell clones. Genomic DNA from 29 transformed clones was digested with Haell and Hinfl endonucleases, electrophoresed on denaturing gradient gels, electroblotted onto nylon membranes, and hybridized to a radioactive p53 cDNA probe. Changes in the melting pattern of p53 were observed in DNAs from 9 of 29 X‐ray—transformed clones examined. No changes were found in untransformed, wild‐type 10T1/2 cell clones. These results indicate that X‐ray—induced p53 mutations may contribute to cell transformation.
Novel variants of CD44 arising from alternative splicing: Changes in the CD44 alternative splicing pattern of MCF‐7 breast carcinoma cells treated with hyaluronidase
Authors: Tanabe Kenneth K. , Nishi Toru , Saya Hideyuki
Published: February 2007

CD44 is a cell‐surface glycoprotein postulated to play a role in a variety of biological processes, including lymphocyte homing and tumor‐cell metastasis. Several isoforms of CD44 have been identified in human cells, and the genesis of some of these isoforms has been attributed to alternative splicing. In the study presented here we amplified three novel transcript variants of CD44 from human cell lines using a reverse transcriptase‐polymerase chain reaction strategy. Two of the novel isoforms differed from previously described CD44 isoforms as a result of alternative splicing that occurred at previously reported splice junctions. The third novel CD44 isoform was generated from a previously unreported alternative splice junction near the 5′ end of the open reading frame. Southern blot analysis of genomic DNA revealed that these novel isoforms and all of the previously described CD44 isoforms arose from alternative splicing. The capability of cells to modify their CD44 alternative splicing pattern was demonstrated in MCF‐7 cells, which altered their CD44‐isoform expression pattern in response to treatment with hyaluronidase. A better understanding of mechanisms regulating CD44 alternative splicing may provide insights into diverse processes, including tumor‐cell metastasis and lymphocyte homing.
Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures
Authors: Fischer Susan M. , Lee Marilyn L. , Maldve Regina E. , Morris Rebecca J. , Trono David , Burow David L. , Butler Andrew P. , Pavone Amy , Warren Barbour
Published: February 2007

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c‐jun and c‐fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD‐1 mice and from discarded adult human skin were grown in primary culture in a high‐calcium serum‐free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKCα, PKCβ, and PKCδ; no PKCγ, PKCϵ, or PKCζ were detected. In fresh MEKs, PKCα, PKCβ, PKCΔ, and PKCζ were observed, but not PKCγ or PKCζ. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKCγ was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA‐induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c‐jun and c‐fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c‐jun, c‐fos, and ODC mRNA in HEKs. However, c‐jun and c‐fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.
High expression of cytochrome P450 2a‐5 (coumarin 7‐hydroxylase) in mouse hepatomas
Authors: Kobliakov Valery , Kulikova Ludmila , Samoilov Dimitri , Lang Matti A.
Published: February 2007

A high level of Cyp2a‐5 was found in spontaneous and transplanted mouse hepatomas compared with normal liver. Increased expression of Cyp2a‐5 was associated with an increase in coumarin 7‐hydroxylation, a marker activity of Cyp2a‐5, and the corresponding mRNA, suggesting that regulation of Cyp2a‐5 in hepatomas is pretranslational. In contrast, the total P450 content and arylhydrocarbon hydroxylase and amidopyrene demethylase activities decreased. Pyrazole, a strong inducer of Cyp2a‐5 in normal mouse livers, also increases this isozyme in hepatomas. A parallel increase in the corresponding mRNA suggests that pyrazole, like the formation of hepatomas, affects the regulation of Cyp2a‐5 pretranslationally.
Preferential alteration of inducible gene expression in vivo by carcinogens that induce bulky DNA lesions
Authors: Hamilton Joshua W. , Louis Claudine A. , Doherty Kristen A. , Hunt Steven R. , Reed Michael J. , Treadwell Melinda D.
Published: February 2007

Our laboratory is interested in whether chemical carcinogen—induced DNA damage is nonrandomly distributed in the genome, i.e., “targeted,” at the level of individual genes. To examine this, we have been investigating whether carcinogen treatment in vivo differentially alters the expression of specific genes. In this study, we examined the effects of four model carcinogens that induce bulky lesions in DNA—benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1), 7,12‐dimethylbenz[a]anthracene (DMBA), and 2‐acetylaminofluorene (AAF)—on the steady‐state mRNA expression of several constitutive and drug‐inducible genes in vivo. We specifically tested the hypothesis that carcinogen‐induced DNA damage is preferentially targeted to inducible genes relative to constitutively expressed genes using the chick embryo as a simple in vivo test system. In summary, the four carcinogens had no effect on the steady‐state mRNA expression of constitutively expressed β‐actin, transferrin, or albumin genes over a 24‐h period after a single dose of each carcinogen. In contrast, each of these same treatments significantly altered the mRNA expression of two glutethimide‐inducible genes, ALA synthase and CYP2H1. Both the basal expression of these genes and their drug‐inducible expression was altered. B[a]P and AFB1 had similar effects on expression of the two inducible genes and caused similar levels of covalent adducts in total DNA, even though the administered doses differed by 30‐fold. B[a]P metabolism, B[a]P binding to DNA, and the basal expression of CYP2H1 were similar in liver and lung. However, B[a]P significantly altered basal CYP2H1 mRNA expression in liver, a tissue in which this gene is highly inducible by glutethimide, and had no effect on basal CYP2H1 mRNA expression in lung, a tissue in which this gene is not drug‐inducible. These data support the hypothesis that inducible gene expression is a target for carcinogen‐induced DNA damage in vivo. © 1993 Wiley‐Liss, Inc.
Activation of distinct multidrug‐resistance (P‐glycoprotein) genes during rat liver regeneration and hepatocarcinogenesis
Authors: Teeter Larry D. , Estes Marc , Chan John Y. , Becker Frederick F. , Kuo M. Tien , Atassi Hammad , Sell Stewart
Published: February 2007

The multidrug transporter P‐glycoproteins are encoded by three multidrug‐resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2. Only the first two genes are functionally related to multidrug resistance. Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported. In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration. In this communication, we report isolating three gene‐specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols. Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2‐acetylaminofluorene. Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy. Nevertheless, the general patterns of tissue‐specific expression of these three mdr genes were similar in rats and mice. These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.
Characterization of a nuclear factor, papilloma enhancer binding factor‐1, that binds the long control region of human papillomavirus type 16 and contributes to enhancer activity
Authors: Cuthill Scott , Sibbet Gary J. , Campo M. Saveria
Published: February 2007

Human papillomavirus type‐16 (HPV‐16) is an epitheliotropic DNA tumor virus associated with the development of cervical carcinoma. The expression of HPV‐16 early genes is driven by a cell‐type—specific enhancer located in the long control region of the viral genome. We identified an element within the HPV‐16 minimal enhancer that has enhancer activity and binds a nuclear factor, designated papillomavirus enhancer binding factor‐1 (PEF‐1). The element (called FP‐F by us and fp5e by Gloss et al. (EMBO J 6:3735–3743, 1987)) was originally identified as a footprint by DNase I protection experiments. The PEF‐1 binding site is centered around a CCAAT box—like CCAAC element. Introduction of A→T transversions into the CCAAC element of fp5e abolished binding of PEF‐1 and concomitantly abolished enhancer function. fp5e resembles binding sites for the transcriptional activators CTF/NF‐1 and AP‐2; however, we showed that neither of these factors interacted with this element. PEF‐1 has an apparent molecular weight of about 110 kDa, and we propose that it is a novel factor involved in the transcriptional activation of HPV‐16 gene expression.
Gene expression screening for specific genes associated with mouse mammary tumor development
Authors: Zhang Lixin , Medina Daniel
Published: February 2007

The preneoplastic hyperplastic alveolar nodule is a frequent and well‐characterized precursor to mammary tumors in the mouse. Although the biological characteristics of the preneoplastic state have been understood for many years, the molecular alterations associated with preneoplasia and tumorigenicity are unknown. We applied the technique of differential display of mRNA to two closely matched cell populations of mammary preneoplasias that differed only in their tumorigenic potential. Two mRNAs were isolated that were overexpressed only in tumorigenic preneoplasias and in tumors and not in normal pregnant mammary gland or in nontumorigenic preneoplasias. Partial nucleotide sequencing indicated that one of the mRNAs had not yet been described, whereas the second mRNA was highly homologous to a relatively uncharacterized gene termed pT‐2. These results illustrate the utility of the differential display method for isolating and identifying uniquely expressed genes from tissues maintained in the microenvironment where tumors arise naturally.
Mutational specificities of environmental carcinogens in the lacl Gene of Escherichia coli VII: The host‐mediated assay and its comparison with in vitro mutagenesis induced by 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone
Authors: Jiao Jianli , Glickman Barry W. , Zielenska Maria , Guttenplan Joseph B. , Xin Liu‐Yu , Anderson Marshall W.
Published: February 2007

To investigate the influence of different types of metabolic activation (9,000 x g supernatant (S9) activation vs. a host‐mediated approach) on 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK)‐induced mutational specificity, we determined by DNA sequencing the distribution of forward mutations recovered in the N‐terminal region of the lacl gene of Escherichia coli. After activation with the S9 liver fraction from rats treated with Aroclor 1254, a diverse spectrum of mutations was recovered, with 55% of the events being G:C→A:T transitions. In contrast, after the host‐mediated assay in mice, G:C→A:T transitions accounted for over 94% of the mutations recovered. Generally, NNK metabolism can proceed through two distinct pathways, involving either α‐methyl or methylene hydroxylation. These two pathways produce different distributions of DNA damage. The difference in the mutational spectra we observed thus likely reflects the difference in the contributions of each pathway under the two different treatment conditions.
Effect of gastrin‐releasing peptide on the pancreatic tumor cell line (capan)
Authors: Avis Ingalill , Cuttitta Frank , Treston Anthony M. , Mulshine James L. , Jett Marti , Kasprzyk Philip G. , Maneckjee Rhoda
Published: February 2007

Gastrin‐releasing peptide (GRP) has previously been shown to be an autocrine growth factor for small cell lung cancer, and our objective in the study presented here was to determine whether GRP has a similar role in pancreatic cancer. Using 125I‐GRP, we demonstrated binding to specific, saturable, high‐affinity sites (Kd=1 nM; Bmax=245 fmol/mg protein) in membrane preparations from the pancreatic tumor cell line Capan. The receptors were found to be biologically active. In whole cells, a GRP analogue bound to these receptors and stimulated rapid transfer of tritium from the tritiated lipid inositol pool to inositol triphosphates. Exogenous GRP addition stimulated incorporation of [3H]thymidine into DNA 20–60%. This stimulatory effect was blocked by the addition of a monoclonal antibody that complexed specifically with the receptor‐binding portion of the peptide. In addition, the monoclonal antibody inhibited the growth of Capan cells in an in vitro growth assay without exogenous peptide. Bombesin receptor—specific antagonists also inhibited growth in a similar fashion. These data suggest that paracrine production of GRP may be important in pancreatic tumor growth, or that low‐levels of a GRP‐like peptide may play an autocrine role in this tumor. © 1993 Wiley‐Liss, Inc.
Dose‐ and time‐dependent expression of transforming growth factor‐β1 mRNA and protein in mouse epidermis and papillomas after repeated topical application of benzo[a]pyrene
Authors: Sherman James H. , Miller Marian L. , Albert Roy E. , Baxter C. Stuart
Published: February 2007

Topical weekly application of 64 μg of benzo[a]pyrene (BAP) for 4 wk induced transforming growth factor (TGF)‐β1 mRNA in the epidermis of Swiss (ICR) mice, with a maximum at 6–12 h after the last treatment. The increase in TGF‐β1 mRNA concentration was accompanied by an increase in immunohistochemically detectable intracellularly localized TGF‐β1 protein in the suprabasal epidermis and by the appearance of extracellularly localized TGF‐β1 in the basal layers. A dose rate of 16 μg/wk for 4 wk was unable to induce the same response. In contrast, after 20 weekly topical applications of 16 or 64 μg of BAP, an increase in TGF‐β1 mRNA concentration and the appearance of extracellularly localized protein in the epidermis were observed. These changes in TGF‐β1 expression were paralleled by changes in epidermal morphology. A similar group of animals treated with 4 μg of BAP/wk for 20 wk did not respond differently from untreated controls. Papillomas resulting from treatment with 16 or 64 μg of BAP/wk for 28 wk stained for intracellularly localized TGF‐β1 predominantly in the differentiating and nondividing layers. Papillomas stained for extracellularly localized TGF‐β1 solely in the less differentiated and dividing cells. These results suggest that tumorigenesis by BAP involves the induction of cumulative changes in epidermal TGF‐β1 mRNA and protein concentrations as well as alterations in skin morphology associated with a tumor‐promotion process.
Further analysis of c‐Ha‐ras mutations in papillomas initiated by several polycyclic aromatic hydrocarbons and papillomas from uninitiated, promoter‐treated skin in sencar mice
Authors: Digiovanni John , Beltrán Linda , Rupp Amy , Gill Rosalynn D. , Harvey Ronald G.
Published: February 2007

In this study we analyzed the mutations in c‐Ha‐ras from skin papillomas initiated with benzo[a]pyrene (B[a]P), 7‐methylbenz[a]anthracene (7‐MBA), and 10‐fluoro‐7‐methylbenz[a]anthracene (10‐F‐7‐MBA) and from papillomas induced by treatment with tumor promoter alone. Among the papillomas induced by treatment with tumor promoter alone, 56% (nine of 16) had mutations in c‐Ha‐ras. These mutations were found primarily in codon 61 and included both A182→T and A182→G mutations. In addition, one promoter‐induced tumor had a G35→A mutation in codon 12, and one had a G37°C mutation in codon 13. The other promoter‐induced papillomas did not have detectable mutations in codons 12, 13, or 61. Most of the B[a]P‐initiated papillomas (77%; 10 of 13) did not have detectable mutations in c‐Ha‐ras codons 12, 13, or 61. However, three of these B[a]P‐initiated papillomas had c‐Ha‐ras codon 13 mutations; one had a G37°C transversion and two had G38→ T transversions. Most of the 7‐MBA—initiated tumors and all of the 10‐F‐7‐MBA—initiated tumors had an activated c‐Ha‐ras gene [nine of 10 (90%) and 11 of 11 (100%), respectively]. These mutations were almost exclusively A182→ T transversions in codon 61 except for two 7‐MBA—initiated papillomas that had G37°C transversions in codon 13. The results suggest that more than one mechanism may contribute to activation of c‐Ha‐ras by polycyclic aromatic hydrocarbons (PAHs) in mouse skin. Furthermore, the absence of c‐Ha‐ras mutations in most B[a]P‐initiated papillomas, as well as in a significant fraction of those induced by tumor promoter alone, suggests that there may be other molecular targets involved in tumor initiation by PAHs in mouse skin.
Absence of MDM‐2 gene amplification in experimentally induced tumors regardless of p53 status
Authors: Saez Guillermo T. , Oliva Maria R. , Mangues Ramon , Pellicer Angel
Published: February 2007

To assess the generality of the hypothesis that murine double‐minute‐2 (MDM‐2) gene amplification complements the absence of p53 mutation during tumor development, we analyzed 143 murine tumors induced by a variety of carcinogenic agents in two different mouse strains. Only three of 143 tumors showed p53 genetic alterations and none showed MDM‐2 amplification, indicating the existence of alternative pathways that permit tumor cells to bypass p53–MDM‐2 control. © 1994 Wiley‐Liss, Inc.
Allele loss and point mutation in codons 12 and 61 of the c‐Ha‐ras oncogene in carcinogen‐transformed human breast epithelial cells
Authors: Zhang Pei‐Li , Calaf Gloria , Russo Jose
Published: February 2007

There is significant evidence that the ras oncogene plays a role in experimental mammary carcinogenesis; the evidence in human breast cancer, however, is more limited. We induced the expression of transformation phenotypes in the human breast epithelial cell line MCF‐10F with the chemical carcinogens 7,12‐dimethylbenz[a]‐anthracene, N‐methyl‐N‐nitrosourea, N‐methyl‐N‐nitro‐N′‐nitrosoguanidine, and benzo[a]pyrene. This work was designed to clarify whether chemically induced neoplastic transformation correlates with alterations in the ras gene. MCF‐10F cells have two c‐Ha‐ras alleles, identified by 1.0‐kb and 1.2‐kb restriction fragments. Treatment with carcinogens resulted in the loss of one of the alleles (1.0 kb). Polymerase chain reaction–amplified DNA from all carcinogen‐treated cells was analyzed for point mutations in c‐Ha‐ras at codons 12 and 61. All of the carcinogens induced a mutation of the remaining allele at the first position of codon 12 (GGCÀC). Another frequent mutation occurred at the first position of codon 61 (CAG←GAG). The changes in c‐Ha‐ras were associated with the emergence of colony formation in agar‐methocel, but no specific changes in this gene correlated with the emergence of invasiveness or tumorigenesis, indicating that other genes may be involved in the process. © 1994 Wiley‐Liss, Inc.
Screening for p53 mutations in c3h/he mouse liver tumors derived spontaneously or induced with diethylnitrosamine or phenobarbitone
Authors: Rumsby Paul C. , Davies Margaret J. , Evans John G.
Published: February 2007

Distinct differences have been described in the development of C3H/He mouse liver tumors induced by the genotoxic carcinogen diethylnitrosamine (DEN) and by the nongenotoxic agent phenobarbitone (PB) in terms of pathology and the frequency of mutation at codon 61 of the Ha‐ras oncogene. To further define the mechanisms involved, we screened the tumor suppressor gene p53 for mutations in exons 5, 7, and 8 using polymerase chain reaction (PCR)–single‐strand conformation polymorphism (SSCP) analysis. Nearly all the mutations so far described have been found within these three exons. In this study a total of six spontaneous tumors, eight tumors induced by PB, 14 tumors induced by DEN, and five samples of normal liver tissue were screened, and no mutations were found in any of the tumors examined. The positive control, the plasmid LTRp53cG (val), had a point mutation in exon 5 that was detected by PCR‐SSCP. Since many of the tumors were late‐stage hepatocellular carcinomas, we concluded that mutations in exons 5, 7, and 8 of the p53 gene do not play an important role in the development of chemically induced liver tumors in the C3H/He mouse. © 1994 Wiley‐Liss, Inc.
The effects of bile acids on phospholipase c activity in extracts of normal human colon mucosa and primary colon tumors
Authors: Nomoto Koji , Morotomi Masami , Miyake Masami , Xu Ding‐Bang , Logerfo Paul P. , Bernard Weinstein I.
Published: February 2007

Phospholipase C (PLC) activity and its response to stimulation by bile acids was assayed in cellular extracts from 16 primary human colon tumors of various Duke′s stages and paired adjacent normal mucosal samples. In the absence of bile acid, there was negligible degradation of phosphatidylinositol (PI) 1‐stearoyl‐2‐[14C]‐arachiodonoyl by tumor or normal tissue, but the addition of deoxycholic acid (DCA) or taurocholic acid (TCA) resulted in concentration‐dependent and time‐dependent stimulation of diacylglycerol (DAG) formation at optimal concentrations of 2 mM DCA and 4 mM TCA. Triton X‐100 (0.125–1.0%) inhibited rather than enhanced the PI‐degrading activity of these extracts, indicating that the stimulatory effects of DCA and TCA were not simply due to a detergent effect. Under the same assay conditions there was only a small amount of labeled monoacylglycerol or free arachidonic acid produced by extracts incubated in the absence or presence of DCA or TCA. No major differences in DAG production from PI were seen between paired samples of normal colon mucosa and primary colon tumors, in assays done in the presence of 2 mM TCA. Extracts from tumors in the distal part of the colon had higher activity than those from the proximal colon. This was also true for the extent of release of free arachidonic acid from labeled PI. Under the same conditions, labeled phosphatidylcholine or phosphatidylethanolamine did not serve as substrates for the colon mucosa or tumor extracts. Nor was there significant hydrolysis of the labeled DAG (1‐stearoyl‐2‐14C‐arachidonoylglycerol) by normal colon mucosa or tumor extracts, in the absence or presence of DCA or TCA. On the other hand, a low level of DAG lipase activity was detected in the presence of Triton X‐100. These findings provide the first evidence that normal human colon mucosa and primary colon tumors contain a PI‐specific PLC activity that is markedly stimulated by bile acids. Our results also suggest that bile acids may enhance colon carcinogenesis by acting on this enzyme system, thereby influencing signal transduction pathways in the target cells. © 1994 Wiley‐Liss, Inc.
In vitro growth suppression and morphological change in a human renal cell carcinoma cell line by the introduction of normal chromosome 3 via microcell fusion
Authors: Yoshida Mitsuaki A. , Shimizu Motoyuki , Ikeuchi Tatsuro , Tonomura Akira , Yokota Jun , Oshimura Mitsuo
Published: February 2007

Cytogenetic and molecular studies of human renal cell carcinoma (RCC) have suggested that the genetic and functional losses of one or more putative tumor suppressor genes on the short arm of chromosome 3 play a crucial role in the development of this disease. To examine whether the introduction of chromosome 3 has any effects on the biology of RCC cells, we introduced either chromosome 3, 7, or 11 from normal human fibroblasts into a newly established human RCC cell line with loss of heterozygosity for 3p, via microcell‐mediated chromosome transfer. Microcell hybrids containing an introduced, intact chromosome 3 showed a significant reduction in in vitro growth rate and saturation density together with morphological alteration; these properties were not altered in microcell hybrids containing an introduced chromosome 7 or 11. During long‐term cultivation, one of the clones that had lost the introduced chromosome 3 showed growth properties and morphology similar to those of the parental cell lines. Thus, our findings provide additional evidence for the presence of a putative tumor suppressor gene or genes on normal chromosome 3p and indicate that the gene is a dominant, negative growth regulator whose loss promotes progressive features of the neoplastic phenotype. © 1994 Wiley‐Liss, Inc.
Focal transgene expression associated with papilloma development in v‐Ha‐ras–transgenic TG.AC mice
Authors: Hansen Laura A. , Tennant Raymond
Published: February 2007

The homozygous transgenic mouse line TG.AC contains a v‐Ha‐ras transgene and rapidly develops epidermal papillomas in response to either wounding or treatment with tumor promoters such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). The transgenic v‐Ha‐ras protein product was detected in all papillomas removed from TPA‐treated TG.AC mice but not in vehicle‐or TPA‐treated TG.AC skin without tumors. In situ hybridization demonstrated that focal expression of the transgene was limited to regions of papilloma development and further localized the expression of the transgene message to the epidermal component of the papillomas, with the strongest signal in the basal epidermoid cells. Cellular proliferation, as indicated by immunohistochemical staining for proliferating‐cell nuclear antigen (PCNA), was similarly localized primarily to basal epidermoid cells and, to a lesser extent, stratum spinosum cells in all papillomas analyzed. Cells that stained positively for PCNA were much more common in the papillomas than in the surrounding, normal‐appearing skin. The focal nature of papilloma development was also evidenced by protein kinase C activity and hyperplasia after TPA treatment. As early as 18 d after the start of TPA treatment, focal hyperplasias associated with the follicular epidermis were observed in TG.AC but not nontransgenic FVB/N skin; these hyperplasias were assumed to be the precursors of the epidermal papillomas. To explain the development of transgene‐expressing tumors from apparently transgene‐negative, normal‐appearing skin, we hypothesize that the papillomas arise from the clonal expansion of focal areas of epidermal cells that overexpress the transgene. We also propose that the TG.AC line is an excellent model for studying very early events in papillomagenesis. © 1994 Wiley‐Liss, Inc.
Infrequent transforming mutations in the transmembrane domain of the neu oncogene in spontaneous rat schwannomas
Authors: Perantoni Alan O. , Turusov Vladimir S. , Buzard Gregory S. , Rice Jerry M.
Published: February 2007

Ethylnitrosourea (ENU) given transplacentally to rats induces schwannomas of the cranial, spinal, and peripheral nerves, with a high frequency of mutations in the neu proto‐oncogene. To establish the requirement for such mutations in tumorigenesis of the Schwann cell, spontaneous schwannomas from BD‐VI rats were evaluated for transforming mutations in the transmembrane domain of the protein encoded by the neu protooncogene. While all five schwannomas induced transplacentally with ENU were shown to contain T←A transversions in base 2012 of neu by selective oligonucleotide hybridization and dideoxy sequencing of polymerase chain reaction–amplified products from paraffin sections, only one of nine spontaneous schwannomas from untreated rats had the same mutation. Examination of tumors for mutations in codon 12 of Ki‐ras revealed normal alleles. Therefore, the high frequency of mutations in neu in ENU‐induced tumors may be directly attributable to the carcinogen or to the period of development at which exposure occurred, and transforming mutations of the transmembrane domain of neu are not required for tumorigenesis of the Schwann cell. © 1994 Wiley‐Liss, Inc.
A role for myn in Ha‐ras transformation of mouse 10T1/2 fibroblasts
Authors: Taylor William R. , Wright Jim A.
Published: February 2007

The human max protein has been shown to form heterodimers with myc family proteins. The ability of myc+max heterodimers to act as sequence‐specific transcriptional activators appears to be essential for the oncogenic activity of myc. max (also called myn in murine cells) can homodimerize to form a transcriptionally inactive complex. We previously showed that in mouse 10T1/2 cells, a combination of activated ras, c‐myc, and a mutant form of p53 can cooperate in the induction of cellular transformation and metastasis. In the study presented here, we tested the hypothesis that expression of the myn gene may play a role in this cooperative process. Analysis of myn mRNA in these cell lines revealed the presence of a major 2.0‐kb RNA species. This message and the 21‐ and 22‐kDa myn polypeptides it encodes were significantly over expressed in cells transformed by activated ras a lone, by ras in combination with c‐myc or mutant p53, or by ras plus myc plus mutant p53, in comparison with untransformed parental 10T1/2 cells. We also found that induction of ras expression in a cell line harboring an inducible ras gene was accompanied by an increase in myn mRNA expression. Interestingly, cotransfection of 10T1/2 cells with ras and myn inhibited cellular transformation in a focus‐forming assay when compared with transfection with ras alone. These results suggest a role for ras in the regulation of myn gene expression and suggest a model of oncogene cooperativity in which the relative levels of myc and myn gene expression can influence ras transformation of mouse 10T1/2 cells. © 1994 Wiley‐Liss, Inc.
Expression and transcriptional activity of Ap‐1, CRE, and URE binding proteins in B16 mouse melanoma subclones
Authors: Rutberg Susan E. , Goldstein Ira M. , Yang Yang M. , Stackpole Christopher W. , Ronai Zeev
Published: February 2007

The expression and DNA binding activity of members of the activating protein‐1 (AP‐1) and activating transcription factor (ATF) families of transcription factors were analyzed in sham and ultraviolet (UV)‐irradiated subclones of the B16 mouse melanoma cell system. The four subclones we used represent sequential stages in the development and progression of malignant melanoma and exhibit differences in growth and metastatic potential. Western blot analysis revealed differential expression of some AP‐1 (c‐jun, jun‐B, and jun‐D) and ATF (43‐ and 47‐kDa cyclic AMP‐responsive element binding protein (CREB) family members) in the different subclones; while c‐jun expression was noted in the subclones with the greater malignant potential, jun‐D was expressed in those with the lesser malignant potential. Furthermore, a delicate balance between the two forms of CREB was noted; the 47‐kDa CREB appeared, when expressed exclusively, in subclones that exhibit a greater malignant potential. Electrophoretic mobility shift assays using AP‐1, CRE, and UV‐responsive element (URE) consensus sequences indicated that distinct complexes were formed with extracts from each of the four subclones. The complexes were competitively inhibited by each of the target sequences used, suggesting that “cross‐talk”occurs between some AP‐1 and ATF family members in this cell system. Moreover, a multimer of the URE sequence, cloned upstream of a chloramphenicol acetyltransferase reporter gene, was transcriptionally active and responsive to UV irradiation in two of the four subclones. UV‐related transcriptional activation was directly correlated with the expression of a 43‐kDa CREB. Together, these observations identify members of AP‐1 and CREB families whose expression and activities correlate with the malignant potential of subclones that represent different stages in melanoma development and progression. © 1994 Wiley‐Liss, Inc.
Positive and negative regulations of albumin gene expression by retinoids in human hepatoma cell lines
Authors: Yamada Yasuhiro , Shidoji Yoshihiro , Fukutomi Yasushi , Ishikawa Takashi , Kaneko Takashi , Nakagama Hitoshi , Imawari Michio , Moriwaki Hisataka , Muto Yasutoshi
Published: February 2007

All‐trans‐3, 7, 11, 15‐tetramethyl‐2, 4, 6, 10, 14‐hexadecapentaenoic acid (designated “acyclic retinoid”) induced upregulation of the albumin gene expression at its transcriptional level, whereas all‐trans‐retinoic acid (RA) induced downregulation of the expression in both PLC/PKF/5 and HuH7 human hepatoma cell lines. These up and downregulations of the albumin gene expression coordinated with high and low levels of mRNA for hepatocyte nuclear factor‐1 (HNF‐1), which is one of the most potent transcription factors for the albumin gene, implying that retinoids may regulate albumin gene expression through HNF‐1 expression in opposite ways. The PLC/PRF/5 and HuH7 hepatoma cell lines expressed retinoid × receptor‐α (RXRα) mRNA, whose expression was constitutive. Acyclic retinoid and all‐trans‐RA both induced upregulation of retinoic acid receptor‐β (RARβ), and both suppressed cell proliferation‐related phenotypic expressions by the α‐fetoprotein gene and the c‐myc oncogene. 9‐cis‐RA, whose receptor is known to be RXRα, also induced upregulation of albumin and HNF‐1 expression. These results suggest that acyclic retinoid may act through both RXRα and RARβ, whereas all‐trans‐RA conveys only RARβ‐mediated functions, at least in these two hepatoma cell lines. © 1994 Wiley‐Liss, Inc.
The role of 12 cDNA‐expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene trans‐7, 8‐dihydrodiol
Authors: Shou Magang , Korzekwa Kenneth R. , Crespi Charles L , Gonzalez Frank J. , Gelboin Harry V.
Published: February 2007

The potent carcinogen benzo[a]pyrene (B[a]P) and its metabolite B[a]P trans‐7, 8‐dihydrodiol (7, 8‐diol) require metabolic activation by the microsomal cytochrome P450s (P450s) to exert several adverse biological effects, including binding to DNA, toxicity, mutagenicity, and carcinogenicity. In the study reported here, we defined the role of each of 12 individual cDNA‐expressed cytochrome P450s in the metabolism of B[a]P and 7, 8‐diol. Human P450s 1A1 and 1A2 were expressed in the absence or presence of epoxide hydrolase (EH) in a human lymphoblastoid cell line, and six human and five rodent and rabbit P450s were expressed from cDNA with vaccinia virus vectors in the hepatoma cell line Hep G2. B[a]P metabolism resulted in nine metabolites (three diols, three quinones, and three phenols), which were separated, identified, and quantitated by high‐pressure liquid chromatography. In the human lymphoblastoid cells, human 1A1 metabolized B[a]P at a rate 4.5 times greater than that for 1A2. EH was shown to be directly involved in B[a]P activation, since increasing the amount of EH resulted in less 7‐hydroxybenzo[a]pyrene and more 7, 8‐diol formation. Of the human P450s expressed with the vaccinia virus vectors in Hep G2 cells, 1A2 and 2C9 showed the highest activity and 2B6 showed moderate activity for B[a]P metabolism. Mouse 1A1 had activity 40 times higher than any human, rabbit, or rodent P450s, indicating the potential pitfalls of extrapolating P450 activity across species. Metabolism of the 7, 8‐diol resulted in six metabolites (four tetrols and two triols). In the lymphoblastoid cells, human 1A1 was shown to be 4.2 times more active than 1A2 for 7, 8‐diol metabolism. Among human P450s expressed from vaccinia virus, 1A2, 2E1, and 2C9 gave the highest activity, and 2C8 and 3A4 showed moderate activity for 7, 8‐diol metabolism to the diol epoxides. Again, mouse 1A1 was much more active than any other P450. These studies, in which we determined the capacity of individual P450 in the metabolism and activation of B[a]P and 7, 8‐diol, may thus lead to a better understanding of how P450s control the detoxification and activation of polycyclic aromatic hydrocarbons. © 1994 Wiley‐Liss, Inc.
Ultraviolet b light‐induced mutagenesis of p53 hotspot codons 248 and 249 in human skin fibroblasts
Authors: Amstad Paul , Hussain S. Perwez , Cerutti Peter
Published: February 2007

Mutations in the p53 tumor suppressor gene are detected in approximately half of non‐melanoma skin cancers. The type of base‐pair changes observed strongly suggests solar radiation as the causative mutagen. Mutations are distributed nonrandomly and form moderate hotspots. We studied the capacity of ultraviolet B light (UVB, 280–320 nm) to induce base‐pair changes into the p53 exon 7 sequence extending from nt 14067 to 14075 in human skin fibroblasts. This sequence contains hotspot codon 248. UVB induced mostly C←A and G←T transversions. The base‐pair change with the highest relative abundance was C←A in the first position of codon 250 (CCC←ACC), followed by (in diminishing relative abundance) G←T in the third position of codon 249 (AGG←AG1), C←A in the first position of codon 248 (CGG←AGG), and C←A in the third position of codon 247 (AAC←AAA). The C←T transition in the third position of codon 247 (AAC←AAT) occurred with moderate efficiency. These base‐pair changes are compatible with pyrimidine photodimers as premutagenic lesions, but they could also form opposite 8‐hydroxyguanine, which is the major oxidation product of guanine. No evidence was obtained for the presence of tandem double CC←TT transitions in the untranscribed strand at codons 247/248 and 250. The relative abundance of mutations induced by UVB in the p53 sequence extending from codon 247 to 250 in human fibroblasts does not correlate with mutations observed in the DNA from non‐melanoma skin cancer. This lack of correlation suggests that the mutability of this p53 sequence at the DNA level plays only a minor role in the pathogenesis of non‐melanoma skin cancer in humans. © 1994 Wiley‐Liss, Inc.