bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of interleukin‐1α mRNA expression in mouse epidermis by tumor promoters and antagonists
Authors: Lee Wha Young , Fischer Susan M. , Butler Andrew P. , Locniskar Mary F.
Published: February 2007

Increased expression of interleukin‐1 (IL‐1) in skin elicits a variety of responses, including inflammation and epidermal hyperplasia, which are also characteristic events elicited by tumor promoters. The goal of this study was to investigate whether various classes of tumor promoters increase expression of IL‐1α and whether phorbol ester—induced IL‐1α expression can be blocked by antitumor promoters. Northern analysis of mRNA isolated from the dorsal skins of SENCAR mice treated with 1 μg of 4β‐12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) showed that a single application of TPA produced a significant increase in IL‐1α mRNA at 6 h that decreased by 24 h after treatment. Two treatments of TPA at 48‐h intervals induced, at 6 h, twice as much IL‐1α mRNA as one treatment. Of the other promoters tested, anthralin (22.6 μg), mezerein (2 μg), calcium ionophore A23187 (120 μg), and benzoyl peroxide (20 mg) induced IL‐1α mRNA with different kinetics and to different extents. On the other hand, the non‐tumor promoting phorbol ester analogue 4α‐12‐O‐tetradecanoylphorbol‐13‐acetate had little effect on the expression of IL‐1α mRNA. The effects of various antitumor promoters on TPA‐induced IL‐1α mRNA expression were also assessed. Fluocinolone acetonide, mepacrine, and 5,8,11,14‐eicosatetraynoic acid were the most effective inhibitors, and each produced about 80% inhibition. Other antitumor promoters such as retinoic acid, N‐tosyl‐l‐phenylalanine chloromethyl ketone, and butylated hydroxytoluene inhibited approximately 35%, 65%, and 50% of TPA‐induced IL‐1α mRNA expression; respectively. Therefore, this study suggests a possible role of IL‐1α in the promotion stage of skin carcinogenesis.
Localization of a novel chromosome 7 locus that suppresses development of N‐methyl‐N‐nitrosourea—induced murine thymic lymphomas
Authors: Angel Joe M. , Morizot Donald C. , Richie Ellen R.
Published: February 2007

N‐Methyl‐N‐nitrosourea (MNU) is a potent carcinogen that causes the development of murine thymic lymphomas. MNU‐induced tumor incidence varies considerably among different inbred mouse strains. In particular, the AKR strain is highly susceptible, whereas the C57L strain is highly resistant to MNU‐induced lymphoma formation. Crosses between AKR and C57L mice were established to investigate the genetic basis for the differential susceptibility of these inbred strains. A strong association between MNU‐induced lymphoma development and coat color was observed in (AKR x C57)F2 and AKR x (AKR x C57)F1 progeny such that albino mice developed a higher tumor incidence than nonalbino animals. These data suggest that a locus on chromosome 7 influences tumor development. Analysis of four additional polymorphic loci (D7Rp2, Fes, Hbb, and Int‐2) on chromosome 7 in AKR x (AKR x C57)F1 backcross mice revealed a significant linkage between high tumor incidence and homozygous inheritance of AKR alleles at the albino (tyrosinase) and Hbb loci. Thus, inheritance of at least one C57L allele at the albino or Hbb loci was associated with protection against MNU‐induced lymphoma development. There was no association between tumor incidence and genotype at the D7Rp2, Fes, or Int‐2 loci. Taken together, the data suggest that whereas C57L mice contain a dominant tumor suppressor gene on chromosome 7, in the AKR strain both alleles at this locus are defective resulting in enhanced susceptibility to MNU‐induced lymphomagenesis.
Carcinogen—induced diploid hepatocytes: Sensitive target cells for transformation by mutated c‐Ha‐ras oncogene
Authors: Höhne Martin W. , Zieroth Siegfried , Veser Ulrich , Kahl Georg F. , Schwarz Leslie R.
Published: February 2007

Sequential treatment of partially (two‐thirds) hepatectomized rats with diethylnitrosamine and 2‐acetylaminofluorene induces the emergence of diploid hepatocytes in rat liver. These carcinogen‐induced diploid cell populations are thought to contain the progenitors of hepatocellular carcinoma (HCC), i.e., initiated, cells. In the study presented here, we addressed the question of whether putative mutations in carcinogen‐induced diploid hepatocytes can cooperate with activated oncogenes in the process of transformation in vitro. Both carcinogenesis in vivo and transformation in vitro have been shown to be multistep processes requiring at least two independent transforming events. Diploid and polyploid rat hepatocytes were isolated by centrifugal elutriation. The purity of the elutriated fractions was 88 ± 3% in the diploid fraction and 84 ± 3% in the polyploid fraction. Hepatocytes from both the elutriated cell fractions and, for comparison, hepatocytes from untreated rats were transfected by electroporation with oncogene expression vectors containing the mutated human T24 c‐Ha‐ras gene and of the N‐myc gene. Transient expression of transfected DNA was similar in both hepatocyte populations. No cell lines could be established by using the N‐myc vector. In contrast, the carcinogen‐induced diploid hepatocytes, but not polyploid hepatocytes, could be converted by transfection with the ras vector into permanent anchorage‐independent growing cell lines with hepatocyte‐like morphology and differentiation. These cell lines expressed the myc proto‐oncogene and transforming growth factor‐α constitutively. Thus, carcinogeninduced diploid hepatocytes are sensitive to tranformation by the ras oncogene, suggesting cooperation between putative preexisting mutations in the diploid cells and the ras oncogene product in hepatocellular transformation.
Expression of fibroblast growth factors in ultraviolet radiation—induced corneal tumors and corneal tumor cell lines from Monodelphis domestica
Authors: Sabourin Carol L. K. , Kusewitt Donna F. , Applegate Lee A. , Budge Cindy L. , Ley Ronald D.
Published: February 2007

Chronic exposure of the gray, short‐tailed opossum, Monodelphis domestica, to ultraviolet radiation (UVR) induces highly vascularized mesenchymal tumors of the cornea. Cell lines derived from these UVR‐induced corneal tumors and the corneal tumors themselves were examined for the presence of mRNA coding for basic and acidic fibroblast growth factors (FGF), transforming growth factors‐β and ‐α (TGF‐β and TGF‐α), epidermal growth factor (EGF), and tumor necrosis factor‐α (TNF‐α). Basic FGF was expressed in the cell lines derived from corneal tumors and in the corneal tumors. Expression of basic FGF was high in one corneal tumor. Transcripts for acidic FGF were detected only in the corneal tumor cell lines, not in primary tumors. TGF‐β expression was detected in the corneal tumors and tumor‐derived cell lines. TGF‐α, EGF, and TNF‐α transcripts were not detectable in any opossum material; however, homologous gene sequences for TGF‐α and EGF were detected on Southern blots of opossum genomic DNA. Southern blot analysis revealed no evidence of amplification or rearrangement of the genes for basic FGF or acidic FGF in the UVR‐induced corneal tumor that expressed high levels of basic FGF. Opossum basic FGF, which stimulated the proliferation of fetal bovine heart endothelial cells, was purified by heparin affinity chromatography from a UVR‐induced corneal tumor and a corneal tumor cell line. Immunoblotting of opossum basic FGF from a corneal tumor cell line using antiserum to bovine basic FGF showed two prominent immunoreactive bands of 17.5 and 18.5 kDa. Expression of basic FGF and acidic FGF may play a role in the development and progression of UVR‐induced corneal tumors in M. domestica.
Dinitropyrene metabolism, DNA adduct formation, and DNA amplification in an SV40‐transformed chinese hamster embryo cell line
Authors: Neft Robin E. , Roe Amy L. , Smith Beverly A. , Beland Frederick A.
Published: February 2007

The environmental pollutants 1,6‐dinitropyrene (1,6‐DNP) and 1,8‐dinitropyrene (1,8‐DNP) are strongly carcinogenic in a number of animal models. These DNPs are metabolized by nitroreduction to N‐hydroxy arylamine derivatives that either directly or after acetylation bind to cellular DNA. In the experiments reported here, we examined whether DNA adduct formation by 1,6‐DNP and 1,8‐DNP was associated with amplification of specific DNA sequences, a process that may be causally related to tumorigenesis. CO60 cells, an SV40‐transformed Chinese hamster embryo cell line, were incubated with 2.5 or 50 ng/mL [4,5,9,10‐3H]1,6‐DNP for 5 h. Highpressure liquid chromatographic analysis of organic extracts of the medium indicated the presence of 1‐acetylamino‐6‐nitropyrene, suggesting that these cells are capable of nitroreduction and acetylation. 32P‐Postlabeling analysis of DNA isolated from cells exposed to 1.0 or 2.5 ng/mL 1,6‐DNP revealed dose‐related formation of N‐(deoxyguanosin‐8‐yl)‐1‐amino‐6‐nitropyrene. A similar adduct, presumably N‐(deoxyguanosin‐8‐yl)‐1‐amino‐8‐nitropyrene, was detected after incubations with 1,8‐DNP. DNA isolated from analogous experiments was slot‐blotted onto nylon membranes and hybridized with 32P‐labeled SV40, c‐fos, or β‐actin DNA probes. β‐Actin was not amplified and c‐fos was amplified only a small amount; however, there was dose‐related amplification of SV40 sequences, whose levels were in some instances approximately 20 times that observed in solvent‐treated controls. These data indicate that DNA adduct formation by 1,6‐DNP and 1,8‐DNP is associated with the amplification of certain DNA sequences, a response that may be related to the tumorigenic potential of these compounds.
Negative regulation of mitogen‐stimulated, anchorage‐independent cell growth by a tumor‐suppressor gene function
Authors: Afshari Cynthia A. , Barrett J. Carl
Published: February 2007

Immortal, nontumorigenic cell lines of Syrian hamster embryo (SHE) cells with different tumor‐suppressing activity were isolated. Subclones from the parental cells were isolated that either had retained (supB+) or lost (supB−) the ability to suppress tumorigenicity after hybridization with tumor cells. The growth properties of these cells were studied to determine how this tumor‐suppressor gene function influences cell growth. When the cells were grown on plastic, their growth properties were similar, and neither cell type grew in soft agar containing 10% serum, which supported the growth of tumorigenic cells. However, in agar supplemented with growth factors and 10% serum, supB− cells formed colonies whereas supB+ cells did not. Efficient growth (colony‐forming efficiencies greater than 20%) of supB− cells was obtained in agar supplemented with serum and a combination of epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), and insulin (EPI) or with serum and basic fibroblast growth factor (BFGF). The effect of EPI and bFGF together was additive. supB+ cells failed to grow under any of these conditions, suggesting that the suppressor gene function blocked the growth response of the cells to multiple growth factors when the cells were suspended in agar. In SupB− cells, transforming growth factor‐β1 and retinoic acid inhibited anchorage‐independent growth response to EPI but not the growth response to bFGF. These observations are consistent with the hypothesis that bFGF stimulates the growth of supB− cells by a signal transduction pathway that differs from the pathway stimulated by EGF or PDGF. Thus, this suppressor gene function may regulate anchorage‐independent growth at some common point in signal transduction for multiple mitogens.
Detection of p53 gene mutations in rat hepatocellular carcinoma cell lines by denaturing gradient gel electrophoresis
Authors: Fukuda Ikue , Ogawa Katsuhiro
Published: February 2007

Structural alterations of the p53 gene were investigated in chemically induced rat hepatocellular carcinomas (HCCs), hyperplastic hepatic nodules (HPNs), and cell lines derived from rat neoplastic and normal liver cells. The mutations were detected by GC‐clamped denaturing gradient gel electrophoresis using DNA that had been amplified from p53 mRNA by the reverse transcriptase—polymerase chain reaction. This method enabled us to find single‐base changes within the p53 gene without using radioisotopes. The presence of mutations was subsequently confirmed by DNA sequencing. No mutations were detected in six primary HCCs and 12 HPNs induced by the Solt and Farber regimen (Nature 236:701–703, 1976), suggesting that p53 gene mutations do not play a major role in rat hepatic carcinogenesis. However, five of seven HCC cell lines and one of two cell lines derived from normal liver cells had the mutated p53 gene and had lost the normal p53 gene. Five cell lines had a G→T transversion at various codons, whereas one line had a 21‐base deletion in exon 5. Therefore, we conclude that p53 gene mutations may occur in vitro during establishment of the cell lines or may be derived from very small populations within the primary tumors.
A sensitive restriction fragment length polymorphism method to detect CAA → AAA mutations at codon 61 of Ha‐ras
Authors: Shinder Gayle A. , Manam Sujata , Nichols Warren W.
Published: February 2007

A rapid and sensitive assay was developed to detect CAA → AAA mutations at codon 61 of Ha‐ras. The region surrounding codon 61 was amplified by the polymerase chain reaction (PCR) using one primer containing a mismatch at the second position of codon 60. Using this primer creates an Msel restriction enzyme site if codon 61 carries the C·G → A·T transversion. An aliquot of the second PCR primer was 5′‐end‐labeled with 32P to increase the sensitivity of detection of the PCR product. After cleavage with Msel, DNA was electrophoresed on a nondenaturing polyacrylamide gel, and the products were visualized by autoradiography. The sensitivity of this assay was such that the mutation could be detected when present in only one of 200 alleles. DNA samples from spontaneous Crl:CD‐1(ICR)BR mouse liver tumors were analyzed using this method. Nine of 38 samples contained the mutation, and in one of those nine, the mutation had not been previously detected by either direct sequencing of tumor DNA or by sequencing the DNA from NIH 3T3 cells transfected with the tumor DNA.
ras gene activation and aberrant expression of keratin K13 in ultraviolet B radiation—induced epidermal neoplasias of mouse skin
Authors: Sutter Christian , Winter Hermelita , Schweizer Jürgen , Strickland Paul T. , Mukhtar Hasan , Agarwal Rajesh
Published: February 2007

Both papillomas and squamous cell carcinomas (SCC) induced in mouse epidermis by initiation with 7,12‐dimethylbenz[a]anthracene (DMBA) and promotion with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) exhibit aberrant expression of a type I keratin, K13, that is normally characteristic of terminal differentiation of internal stratified epithelia. There is evidence that the aberrant expression of K13 depends on the presence of an activated ras gene in mouse epidermal keratinocytes (Sutter et al., Mol Carcinog 4:467–476, 1991). To assess the general validity of this hypothesis, we investigated both aberrant K13 expression and activation of each of the three members of the ras gene family in epidermal tumors induced in four different mouse strains (SKH‐1hr, SENCAR, BALB/c, and C3H/He) by chronic irradiation with ultraviolet (UV) B. The tumor collection comprised nine papillomas and 30 well or poorly differentiated SCC. Aberrant K13 expression occurred in only five of 39 tumors and was restricted to SCC of both types. This indicates that aberrant K13 expression in UV‐induced epidermal tumors was intrinsically different from that in chemically induced tumors. Polymerase chain reaction analysis of the tumors for different point mutations in codons 12, 13, and 61 of the Ha‐ras and Ki‐ras genes and in codon 61 of the N‐ras gene revealed that only one of the well differentiated tumors from a SKH‐1hr mouse exhibited a GGA → GAA mutation in codon 12 of the Ha‐ras gene. Although this tumor was also positive for aberrant K13 expression, such a correlation could not be made for the remaining K13‐expressing tumors. This indicates that the activation of one of the members of the ras gene family is not a general prerequisite for the aberrant expression of K13 in mouse epidermal keratinocytes. © 1993 Wiley‐Liss, Inc.
Alterations of the Rb gene and its association with ki‐ras activation and p53 inactivation in endometrial adenocarcinoma
Authors: Enomoto Takayuki , Nomura Taisei , Fujita Masami , Inoue Masaki , Nakazawa‐Miyamoto Aiko , Tanizawa Osamu
Published: February 2007

Alterations of the retinoblastoma (Rb) gene were evaluated in nine primary endometrial adenocarcinomas that we had previously analyzed for the presence of Ki‐ras activations and p53 alterations and in three endometrial carcinoma cell lines. Loss of mRNA expression in the Rb gene was detected in two of the 12 tumors. Internal deletions of Rb cDNA were observed in two tumors; one was a deletion of exon 21 in a primary carcinoma, and the other was a deletion of exon 8 in one allele in one cell line. Loss of heterozygosity of the Rb gene, which was detectable by polymorphisms in introns 1 and 17, was analyzed using polymerase chain reaction‐restriction fragment length polymorphism analysis in 29 endometrial carcinomas. Of 13 heterozygous cases, two cases (15%) showed loss of heterozygosity. We therefore suggest that alteration of the Rb gene, as well as activation of the Ki‐ras gene and alterations of the p53 gene, plays a significant role in the etiology of endometrial adenocarcinoma.
Loss of heterozygosity: The most frequent cause of recessive phenotype expression at the heterozygous human adenine phosphoribosyltransferase locus
Authors: Zhu Yuan , Tischfield Jay A. , Stambrook Peter J.
Published: February 2007

Expression of a recessive phenotype can occur by a number of different mechanisms, such as chromosomal deletion, recombination, and intragenic frameshift mutation or base substitution. To examine the contribution of different mutational events, we isolated and characterized a human fibroblast cell line heterozygous at the adenine phosphoribosyltransferase (APRT) locus. Cells that subsequently lost APRT activity were selected, cloned, and analyzed for the mechanisms contributing to the loss of APRT activity. Loss of APRT activity occurred at a rate of 7.8 x 10‐5 per allele per cell generation. Molecular analysis of DNA from 21 independent APRT‐ clones demonstrated that 62% of mutants had lost the functional allele and that the rest had incurred intragenic mutations. Loss of the functional allele was frequently accompanied by loss of the proximal marker D16S77 but not the more distant proximal marker D16S4, indicating that a high frequency of mitotic recombination or deletion occurred at the region between D16S77 and D16S4 on chromosome 16. Loss of APRT activity in the remaining 38% of the clones was predominantly due to point mutations. These data demonstrate that the mechanisms for loss of heterozygosity at the APRT locus are similar to those found in retinoblastoma and other tumors. The autosomal location of the APRT gene and the ease with which its phenotype can be selected make this gene useful for modeling mutational events at loci important to carcinogenesis.
Mutagenic specificity of the (+)anti‐diol epoxide of dibenz[a,j]anthracene in the supF gene of an Escherichia coli plasmid
Authors: Gill Rosalynn D. , Digiovanni John , Cortez Cecilia , Harvey Ronald G. , Rodriguez Henry , Loechler Edward L.
Published: February 2007

This study was designed to examine the mutagenic specificity of (+)anti‐dibenz[a,j]anthracene 3,4‐diol‐1,2‐epoxide ((+)anti‐DB[a,j]A‐DE) in SOS‐induced repair‐proficient Escherichia coli ES87 (Δpro‐lac, strA)/F′(pro+, laclQ, laclam26, lacZΔM 15). The plasmid pUB3, which contains the mutation target gene, supF, was modified with (+)anti‐DB[a,j]A‐DE in vitro (two to five adducts/plasmid) and then transformed into bacteria by electroporation. The spontaneous mutation frequency for unmodified pUB3 in uninduced cells was about 2 x 10‐6 and for SOS‐induced cells, about 8 x 10‐6. The spontaneous supF‐ mutations were primarily insertions, deletions, and frameshifts. The mutation frequency for (+)anti‐DB[a,j]A‐DE‐modified pUB3 was about 8 x 10‐6 and about 32 x 10‐6 for uninduced cells and SOS‐induced cells, respectively. (+)anti‐DB[a,j]A‐DE induced primarily point mutations in supF in SOS‐induced cells. GC→ transitions were the major mutations observed in SOS‐induced cells (37%). GC→T́ (21%) and GC→C̀ (8.6%) transversion mutations were also observed, whereas mutations at AT base pairs were rare (1.9%). Furthermore, a large number of tandem GC/GC→Ã/AT transition mutations were also observed (about 15% of all mutations in SOS‐induced cells). Taken together, single and tandem GC→Ã mutations accounted for slightly over half (about 51%) of the mutations observed in SOS‐induced cells. These results demonstrated that (+)anti‐DB[a,j]A‐DE was mutagenic in repair‐proficient E. coli; however, unlike other polycyclic aromatic hydrocarbons that induce primarily transversion mutations, (+)anti‐DB[a,j]A‐DE caused mostly GC→Ã transitions.
Modulation of pp60v‐src and pp60c‐src expression in rous sarcoma virus—transformed hamster fibroblasts transfected with activated N‐ras
Authors: Topol Lilia Z. , Gutierrez Maria L. , Blair Donald G. , Kisseljova Natalya P. , Musatkina Elena A. , Shtutman Michael S. , Zakamaldina Tatyana Z. , Tatosyan Alexander G. , Deichman Galina I.
Published: February 2007

Three phenotypically different hamster cell lines transformed with Rous sarcoma virus (RSV) were transfected with plasmid DNA containing an activated N‐ras oncogene, and nine clones expressing various levels of p21N‐ras were characterized. We examined the effects of p21N‐ras on expression and kinase activity of resident src proteins by using a variety of assays that allowed us to discriminate between viral and cellular src proteins. In eight clones with a 10‐ to 20‐fold increase in p21N‐ras levels relative to the endogenous protein, we observed a marked reduction in the synthesis and kinase activity of p60v‐src. This decrease correlated with transcriptional downregulation of RSV genomic and v‐src subgenomic mRNAs. In the same cells, we found a concomitant accumulation of p60c‐src and, accordingly, an increase in its protein kinase activity without an apparent increase in c‐src mRNA levels. Therefore, modulation of viral and cellular src proteins in cells overexpressing p21N‐ras appeared to result from two distinct effects: a downregulation of long terminal repeat—driven transcription and a more complex interaction with cellular effectors that control the stability of p60c‐src. Such modulation also seemed to depend on the levels of p21N‐ras and, possibly, on host‐cell factors, since it was not observed in the third cell line, in which the relative increase in p21N‐ras was only 2.5‐fold to fivefold.
Comparison of pulmonary O6‐methylguanine dna adduct levels and Ki‐ras activation in lung tumors from resistant and susceptible mouse strains
Authors: Devereux Theodora R. , White Catherine M. , Hegi Monika E. , Patel Arti C. , Anderson Marshall W. , Maronpot Robert R. , Foley Julie F. , Greenwell Arnold , Belinsky Steven A.
Published: February 2007

The role of O6‐methylguanine (O6MG) DNA adduct formation and persistence in the formation of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK)—induced lung tumors from resistant C57BL/6 and susceptible A/J mice was investigated. In addition, the frequencies of pulmonary tumor formation and Ki‐ras activation were defined in C57BL/6 mice treated with NNK or vinyl carbamate (VC), and the role of the p53 gene in pulmonary carcinogenesis in these resistant mice was examined. One day after treatment with 100 mg/kg NNK, O6MG adduct concentrations were twofold to eightfold higher in Clara cells and type II cells than in small cells or whole lungs from both mouse strains. The concentrations of O6MG in isolated cells decreased at a similar rate in the two strains of mice. Lung tumors were detected by 27 mo of age in 18% of the C57BL/6 mice after a single 100 mg/kg dose of NNK and in 46% of these mice after a single 60 mg/kg dose of VC. In contrast, the tumor incidence in untreated C57BL/6 mice was 4%. Only one of 22 lung tumors from C57BL/6 mice treated with NNK contained an activated Ki‐ras gene that was associated with an O6MG DNA adduct, whereas previous studies detected activated Ki‐ras oncogenes in most of the NNK‐induced lung tumors analyzed from susceptible A/J and resistant C3H mice. The small differences in formation and persistence of the O6MG adduct in whole lung or isolated lung cells from A/J and C57BL/6 strains do not account for the differences in either susceptibility for tumor formation or activation of the Ki‐ras gene between these strains. In contrast to the low number of NNK‐induced tumors with Ki‐ras mutations in the resistant mice, 11 of 20 lung tumors from VC‐treated mice contained activated Ki‐ras genes. Neither p53 tumor suppressor gene mutations nor overexpression of the p53 protein were detected in spontaneous or chemically induced lung tumors in C57BL/6 mice. Thus, although Ki‐ras activation was detected in some tumors, pathways independent of ras activation and p53 inactivation also appear to be involved in lung tumorigenesis in this resistant mouse strain.
Low frequency of retinoblastoma gene alterations in rat hepatocellular carcinomas
Authors: Smith Martin L. , Lombardi Benito , Chandar Nalini
Published: February 2007

Abnormalities of the retinoblastoma (Rb) gene have been reported in some human cancers, including hepatocellular carcinomas (HCCs). We examined by Southern blotting the status of the Rb gene in HCCs induced in rats in four experimental models. A low frequency of Rb gene alterations, detected as novel hybridizing bands unique to each tumor, was observed. Expression of the Rb protein product was examined in the HCCs and in seven established rat hepatoma cell lines studied. It appears, therefore, that alterations in the structure or expression of the Rb gene do occur but probably do not contribute in a major way to hepatocarcinogenesis in the rat. © 1993 Wiley‐Liss, Inc.
Restoration of gap‐junctional intercellular communication in a communication‐deficient rat liver cell mutant by transfection with connexin 43 cDNA
Authors: Jou Yuh‐Shan , Matesic Diane , Dupont Emmanuel , Lu Shu‐Chen , Rupp Heather L. , Madhukar Burra V. , Oh Saw Yin , Trosko James E. , Chang Chia‐Cheng
Published: February 2007

To study the biochemical basis of gap‐junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC‐deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control‐vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46‐kDa phosphorylated Cx43 protein that was prominent in the parental GJIC‐competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46‐ and 44‐kDa proteins disappeared, whereas the approximately 42‐kDa proteins remained with increasing intensity, indicating that the higher molecular‐weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC. © 1993 Wiley‐Liss, Inc.
Carbon tetrachloride induction of rapid changes in liver nuclear protein factors capable of sequence‐specific binding to regulatory elements in the long terminal repeat of polytropic‐class endogenous murine leukemia virus—related proviruses
Authors: Wang Tzu‐Hao , Henley Donald C. , Yang Den Mei , Ch'Ang Lan‐Yang , Yang Wen K. , Hoyt Peter R.
Published: February 2007

Treatment of mice with hepatic carcinogens, including CCl4, has been shown to rapidly enhance the transcription of endogenous murine leukemia virus—related proviral sequences in the liver. To understand the mechanism for this transcriptional stimulation, we used nuclear protein preparations from mouse livers to perform DNase I protection analyses and identified nuclear protein binding on approximately 20 individual sequences within the regulatory regions of the long terminal repeat (LTR) of a polytropic‐class endogenous provirus clone. From 3 to 144 h after treatment with CCl4, the livers of FVB/N mice were analyzed for specific nuclear protein binding to the LTR DNA. Three to nine hours after CCI4 treatment, decreased protection was seen at potential regulatory cis‐elements throughout the LTR, including specific sites within the putative negative regulatory element (located 5′ of the consensus enhancer sequences) and the 3′ terminal portion of the polytropic class‐specific enhancer‐like inserted sequence element and around the CCAA(C/T) box in the promoter region. In addition, by 3–6 h after treatment, a transient increase in protection activity for the transcription initiation site occurred. The loss of cis‐element protection expanded to other binding sites and became most marked by 48 h after treatment. As the regenerating liver recovered, the nuclear protein binding activities for these LTR sequences also recovered, but protection at the TATAA and transcription initiation sites remained deprotected at 144 h after treatment. Nuclear protein protection of other sites, particularly in the conserved LTR enhancer sequences, was minimally affected by CCl4 treatment. Three nuclear protein binding sites that showed rapid CCI4‐induced kinetic changes were homologous to the consensus sequence for the binding of the transcription factor families MEF‐2, HNF‐1, and C/EBP. The complex kinetic changes in factors that may contribute to the rapid and transient induction of endogenous retroviral gene expression in the liver after CCI4 exposure are discussed.
Constitutive ap‐1 dna binding and transactivating ability of malignant but not benign mouse epidermal cells
Authors: Domann Frederick E. , Levy John P. , Finch Joanne S. , Tim Bowden G.
Published: February 2007

The mouse epidermal cell line 308 contains an activated Ha‐ras gene and forms benign papillomas when transplanted to the skin of athymic nude mice. A radiation‐associated malignant variant of this cell line, 308‐10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in malignant conversion of 308‐10Gy5, we assessed the activator protein‐1 (AP‐1) binding and transactivating ability of 308 and 308‐10Gy5. In nuclear protein extracts of 308, AP‐1 sequence‐specific binding to an oligonucleotide containing a single high‐affinity AP‐1 binding site was induced by the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate, as determined by gel shift analysis. Nuclear extracts of 308‐10Gy5 bound to the AP‐1 oligonucleotide without treatment with tumor promoters. Not only was sequence‐specific AP‐1 DNA binding constitutively active in malignant versus benign tumor cells, but so was transactivation of a unique AP‐1‐responsive chloramphenicol acetyltransferase reporter construct, pTiCTaK. Constitutive transactivation of this AP‐1–responsive reporter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady‐state transcript levels of the tumor‐associated AP‐1–responsive genes stromelysin, urokinase‐type plasminogen activator, c‐jun, and c‐fos were higher in malignant 308‐10Gy5 cells than in benign 308 cells. These results suggest that acquisition of constitutive AP‐1 DNA binding and transactivation can result in sustained deregulation of gene expression. While malignant progression in keratinocytes is probably not due solely to the acquisition of constitutive cellular AP‐1 activity, the effect of deregulated expression of AP‐1–regulated genes, especially basement membrane–degrading enzymes, may be functionally related to malignant conversion. © 1994 Wiley‐Liss, Inc.
2‐hydroxyamino‐1‐methyl‐6‐phenylimidazo [4,5‐b] pyridine induction of recombinational mutations in mammalian cell lines as detected by dna fingerprinting
Authors: Kitazawa Tomoji , Kominami Ryo , Tanaka Ryuichi , Wakabayashi Keiji , Nagao Minako
Published: February 2007

2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) is the most abundant mutagenic heterocyclic amine in cooked foods. Two mouse tumor cell lines, BMT11 and FM3A, were exposed to its proximate form, 2‐hydroxyamino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (N‐OH‐PhIP). Fifty‐six subclones of BMT11 and 39 subclones of FM3A were isolated and analyzed by DNA fingerprinting. Treatment with 10–20 μM N‐OH‐PhIP gave rise to extra bands or shifted bands, but treatment without N‐OH‐PhIP did not. This suggests that mutations resulting from recombination were induced. The mutation frequencies were 21–53% and 22–35% for BMT11 and FM3A, respectively. These findings suggest that PhIP induces recombinational mutations. © 1994 Wiley‐Liss, Inc.
Combined effects of human papillomavirus‐18 and n‐methyl‐n‐nitro‐n‐nitrosoguanidine on the transformation of normal human oral keratinocytes
Authors: Shin Ki‐Hyuk , Min Byung‐Moo , Cherrick Henry M. , Park No‐Hee
Published: February 2007

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV‐18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c‐myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a “high‐risk” HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco′s modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV‐18 sequences; (d) transcribed substantially more HPV‐18 E6/E7, transforming growth factor‐α, and c‐myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild‐type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a “high‐risk” HPV and chemical carcinogens. © 1994 Wiley‐Liss, Inc.
p53 mutations in primary human lung tumors and their metastases
Authors: Reichel Martin B. , Ohgaki Hiroko , Petersen Iver , Kleihues Paul
Published: February 2007

In a total of 26 primary human lung tumors and 60 metastases derived from them, exons 5–8 of the p53 tumor suppressor gene were analyzed by single‐strand conformation polymorphism and subsequent direct DNA sequencing of amplified DNA. Mutational inactivation of the p53 gene was identified in four of five squamous cell carcinomas, three of nine adenocarcinomas, and two of nine small‐cell carcinomas, the overall incidence being 35%. Point mutations occurred at a similar incidence in exons 5–8, with a preference for G←T transversions. In seven of nine cases (78%), mutations were identical in the primary tumor and all of its metastases, indicating that in lung tumors, p53 mutations usually precede metastasis and that hematogenic and lymphogenic dissemination of tumor cells to other tissues is not associated with a selection against p53 inactivation. In one case, a kidney metastasis had the same mutation as the primary squamous cell carcinoma, whereas a liver metastasis had no mutation, indicating heterogeneity of the primary lung neoplasm and selective metastasis of mutated and nonmutated tumor cells to kidney and liver, respectively. Only in one liver metastasis was a mutation identified that was neither present in the primary lung tumor nor in a kidney metastasis, suggesting that occasionally p53 mutations occur after metastatic spread. © 1994 Wiley‐Liss, Inc.
Assignment of two human epidermal squamous cell carcinoma cell lines to more than one complementation group for the immortal phenotype
Authors: Berry Isabella J. , Burns Julie E. , Parkinson E. Kenneth
Published: February 2007

Two human cell lines derived from squamous cell carcinomas (SCCs) of the epidermis, SCC‐12 clone F and SCC‐13 clone Y, were made to be independent of the Swiss 3T3 feeder layer to perform somatic‐cell genetic experiments. We fused these SCC lines with normal human fibroblasts, and all resulting hybrids senesced after completing 12–17 population doublings, suggesting that in part, immortalization of the keratinocyte during SCC development results from the loss of gene function. We also tested whether these two SCC lines mapped to known complementation groups for immortality by fusing them with representatives of groups A (GM847), B (HeLa), and C (143B), but most of these hybrids were indistinguishable from those derived from homotypic crosses set up as immortal hybrid controls. As reported by others, fusions of cell lines from different complementation groups–143B (group C) × HeLa (group B) or GM847 (group A) × HeLa (group B)–resulted in predominantly senescent hybrids. Our results confirmed and extended previous observations by others that the phenomenon of senescence is dominant to that of immortality, but they did not allow us to assign either of the SCC lines we studied to a complementation group for immortality. © 1994 Wiley‐Liss, Inc.
Recovery from ultraviolet‐induced growth arrest of primary rat hepatocytes by p53 antisense oligonucleotide treatment
Authors: Tsuji Kunihiko , Ogawa Katsuhiro
Published: February 2007

Recent evidence suggests that wild‐type p53 prevents cell‐cycle progression after DNA damage, which may provide a sufficient period for the cells to repair the genetic lesions that may otherwise lead to cell death or cellular transformation. We tested whether this hypothesis is generally applicable to parenchymal cells of internal organs. When primary neonatal rat hepatocytes were exposed to a nonlethal dose of ultraviolet light, actinomycin D, or mitomycin C, most cells expressed abundant p53 with an abnormally extended half life in their nuclei, and their growth was arrested despite treatment with growth factors (epidermal growth factor and insulin). When DNA‐damaged cells were treated with p53‐antisense oligonucleotides, p53 expression was significantly suppressed, and an appreciable fraction of the cells entered S phase. However, when damaged cells were administered p53‐sense or retinoblastoma susceptibility gene‐antisense oligonucleotides, there was no recovery from growth arrest. The data strongly suggest that p53 is a component of at least one signal transduction pathway leading to growth arrest in DNA‐damaged cells. © 1994 Wiley‐Liss, Inc.
In vitro and in vivo accelertration of the neoplastic phenotype of a low‐tumorigenicity rat bladder carcinoma cell line by transfected transforming growth factor‐α
Authors: Kawamata Hitoshi , Kameyama Shuji , Oyasu Ryoichi
Published: February 2007

We conducted an experiment to determine whether expression of transforming growth factor‐α (TGF‐α) enhances tumorigenicity in a low‐tumorigenicity rat bladder carcinoma cell line and whether it is sufficient to induce a tumorigenic phenotype in a nontumorigenic rat bladder cell line. D44c cells (which are nontumorigenic) were derived from a minute nodule from a bladder treated with N‐methyl‐N‐nitrosourea (MNU); G1‐200 cl‐17 cells (which have low tumorigenicity) were isolated from D44c cells exposed to MNU in vitro. Neither cell line expressed TGF‐α mRNA. The cells were contransfected with pSV2neo and pSRα‐rTGF‐α. The latter plasmid contains the rat TGF‐αcDNA under the transcriptional control of the SRα promoter. In the low‐tumorigenicity G1‐200 cl‐17 cells, the expression of TGF‐α mRNA and the subsequent synthesis of TGF‐α protein activated epidermal growth factor receptors (EGFRs) and markedly enhanced tumorigenicity in nude mice (i.e., shortened the latency period before tumor appearance, accelerated the rate of growth, and increased the size of the tumors) as well as anchorage‐independent growth in vitro. In nontumorigenic D44c cells, however, transfected TGF‐α did not induce either anchorage‐independent growth or tumorigenicity in nude mice, in spite of overexpression of EGFR mRNA and the constitutive expression of c‐jun and junB mRNA. These results suggest that the increased signal transduction mediated by TGF‐α enhanced tumorigenicity in a cell that was already tumorigenic but was not sufficient to induce tumorigenicity in a nontumorigenic cell. © 1994 Wiley‐Liss, Inc.
Mutations in a shuttle vector exposed to activated mitomycin C
Authors: Srikanth Nadadur S. , Mudipalli Anuradha , Maccubbin Alexander E. , Gurtoo Hira L
Published: February 2007

The cytotoxicity of the potent antibiotic and antitumor agent mitomycin C (MMC) is due to its irreversible binding to DNA. Alkylating species generated by bioreductive activation of MMC are known to cause monoadducts and cross‐links in DNA by specifically binding to guanine residues. To gain insight into how these lesions lead to base‐ and sequence‐specific mutations, shuttle vector pSP189 was treated with MMC chemically reduced by treatment with sodium borohydride, replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence. The MMC‐induced mutations were predominantly base substitutions. Eighty‐four percent of the base substitutions were transversions, with G:C←T:A the major transversion. Single base deletions were the other major mutational event, and 77% of these were G:C deletions. Base positions 115, 123, and 163 were mutational hot spots based on the frequency of independent mutations. Identification of a single MMC adduct (presumed to be a modified G on the basis of its Rf value) and clustering of MMC‐induced mutations at three GC‐rich areas (nt 100–123, 152–163, and 168–176) suggested that the mutational spectrum we found was due to binding of MMC to guanine on either strand of the plasmid DNA. © 1994 Wiley‐Liss, Inc.
Inhibition of plasmid reporter gene expression in cho cells by DNA adducts of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline and 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5–b]pyridine
Authors: Fan Liju , Snyderwine Elizabeth G.
Published: February 2007

2‐Amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) and 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b)]pyridine (PhIP) are two members of a family of carcinogenic heterocyclic amines (HAs) found in cooked meats that form DNA adducts after activation to N‐acetoxy derivatives. The ability of IQ‐ and PhlP‐DNA adducts to inhibit gene expression was investigated using a human growth hormone (hGH) reporter gene in a pUC12‐based mammalian expression vector under the control of either the herpes simplex virus‐1 thymidine kinase promoter or the human immunodeficiency virus‐1 long terminal repeat. The plasmids were treated in vitro with 0, 5, 10, or 40 μM N‐hydroxy‐IQ or N‐hydroxy‐PhIP in the presence of a 10‐fold molar excess of acetic anhydride to generate the N‐acetoxy derivatives in situ. The adduct levels in the plasmids were quantitated by the 32P‐postlabeling method. The adducted (and control) plasmids were each transfected into repair‐deficient or ‐proficient Chinese hamster ovary cells, and expression of hGH was measured by immunoassay of growth hormone secreted into the cell medium. The results showed that IQ‐ and PhlP‐DNA adducts inhibited gene expression in both plasmids and that the degree of inhibition of hGH production was proportional to the levels of IQ‐ and PhlP‐DNA adducts. The degree of inhibition, however, was independent of the promoter, despite the differences in the strengths of the two promoters to drive hGH production. Repair capacity influenced the extent of inhibition of gene expression by HA adducts since, in general, fewer adducts were needed to inhibit reporter gene expression in repair‐deficient cells than in repair‐proficient cells. In both cell lines, DNA adducts of PhIP appeared to be more potent in inhibiting hGH expression than adducts of IQ. Whether alteration of gene expression by HA adducts plays a role in the carcinogenicity of these compounds deserves further study. © 1994 Wiley‐Liss, Inc.
Absence of p53 mutations and various frequencies of ki‐ras exon 1 mutations in rat hepatic tumors induced by different carcinogens
Authors: Tokusashi Yoshihiko , Fukuda Ikue , Ogawa Katsuhiro
Published: February 2007

Mutations of p53 and Ki‐ras exon 1 were investigated in rat hepatic lesions induced by four kinds of hepatocarcinogenic protocols: continuous feeding of 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐Me‐DAB), daily intraperitoneal injection of aflatoxin B, (AFB,), and the Soft and Farber regimen (Nature 236:701–703, 1976), in which diethylnitrosamine (DEN) or nitrosomethylurea (NMU) was used as initiating agents. DNA from microdissected tissue sections was amplified by the polymerase chain reaction (PCR) directly using primers for p53 exons 5–8 and Ki‐ras exon 1 and analyzed for mutations by denaturing gradient gel electrophoresis (DGGE) or constant denaturant gel electrophoresis (CDGE). One or both of the p53 PCR primers were located within introns to prevent amplifying the p53 processed pseudogenes. In a total of 59 hepatocellular carcinomas (HCCs), no p53 aberrations were detected, indicating that p53 mutations are not very important in rat hepatic carcinogenesis. On the other hand, Ki‐ras codon 12 mutations were found at low frequency in HCCs, hyperplastic foci, and cholangiof ibroses induced by 3′‐Me‐DAB and by AFB! but not in the lesions induced by the Solt and Farber regimen. Although Ki‐ras codon 12 mutations are generally infrequent in rat hepatic tumors, their incidence thus appears to vary depending on the carcinogen used for their induction. © 1994 Wiley‐Liss, Inc.