bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐1α gene expression and localization of interleukin‐1α protein during tumor promotion
Authors: Oberyszyn Tatiana M. , Sabourin Carol L. K. , Bijur Gautam N. , Oberyszyn Andrew S. , Boros Laszlo G. , Robertson Fredika M.
Published: February 2007

Treatment of the dorsal epidermis of SENCAR mice with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced a time‐ and dose‐dependent stimulation of interleukin‐1α (IL‐1α) gene expression. Levels of IL‐1α mRNA were elevated as early as 15 min and peaked at 3–4 h after a single application of TPA (2 μg or 10 μg). IL‐1α gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12‐dimethylbenz[a]anthracene (DMBA) and subsequent twice‐weekly application of 2 μg TPA. IL‐1α‐immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA‐TPA for 1–22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL‐1α‐immunoreactive protein. DMBA treatment alone did not induce IL‐1α gene expression. Injection of IL‐1α‐specific antibodies (50 μg) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 μg or 10 μg) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti—IL‐1α antibodies inhibited incorporation of [methyl‐3H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL‐1α is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL‐1α primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL‐1α may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.
Activation of immediate‐early gene expression by peroxisome proliferators in vitro
Authors: Ledwith Brian J. , Manam Sujata , Troilo Philip , Galloway Sheila M. , Nichols Warren W. , Joslyn Dennis J.
Published: February 2007

The hepatocarcinogenicity of peroxisome proliferators (PPs) in rodents has been attributed both to oxidative DNA damage resulting from excessive leakage of peroxisomal H2O2 and to increased hepatocellular replication that may be independent of peroxisome proliferation. Because of the growing association between tumor promotion and alterations in growth‐regulatory signal transduction pathways, we investigated whether PPs can modulate these pathways in a mouse liver epithelial cell line, BNL‐CL.2. We tested two PPs that differ markedly in rodent tumorigenicity for their ability to activate immediate‐early proto‐oncogene expression. 4‐Chloro‐6‐(2,3‐xylidino)‐2‐pyrimidinylthioacetic acid (Wy‐14643), a highly tumorigenic PP, was an exceptionally strong inducer of c‐fos expression. In contrast, diethylhexyl phthalate (DEHP), a weakly tumorigenic PP, was a very poor inducer of c‐fos expression. Wy‐14643 was also stronger than DEHP in stimulating c‐jun expression, whereas both PPs were fairly strong inducers of jun‐B and jun‐D. The induction of fos and jun expression by Wy‐14643 was specifically inhibited by the protein kinase C inhibitor 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperizine dihydrochloride (H‐7). DEHP‐induced gene expression was strongly inhibited by H‐7, but was also partially inhibited by an inhibitor of protein kinase A. The activation of fos and jun gene expression by PPs was independent of peroxisome proliferation since it was an immediately‐early response not requiring protein synthesis and since the cell lines used in this study do not undergo peroxisome proliferation. Our results raise the possibility that the carcinogenicity of PPs may be due, in part, to epigenetic modulation of growth‐regulatory signal transduction pathways. © 1993 Wiley‐Liss, Inc.
Mutations of the p53 tumor suppressor gene in neoplasms of the human nervous system
Authors: Ohgaki Hiroko , Eibl Robert H. , Reichel Martin B. , Mariani Luigi , Petersen Iver , Höll Thomas , Wiestler Otmar D. , Kleihues Paul , Schwab Manfred , Gehring Manuela
Published: February 2007

A variety of neoplasms of the human nervous system were analyzed for the presence of mutations in the p53 tumor suppressor gene. DNA was extracted from frozen or formalin‐fixed, paraffin‐embedded material. Single‐strand conformation polymorphism (SSCP) analysis for exons 5–8 was followed by direct DNA sequencing. Mutations leading to an amino acid change were found in three of 11 (27%) low‐grade (World Health Organization (WHO) Grade II) astrocytomas. They were located in codon 183 (TCA → TGA) of exon 5, codon 237 (ATG → ATA) of exon 7, and codon 273 (CGT → CAT) of exon 8. In one of these cases, the sequence indicated loss of the wild‐type allele. Of 12 juvenile pilocytic astrocytomas (WHO Grade I), none contained a p53 mutation, suggesting a different molecular basis for this childhood neoplasm. Except for a mutation in one of seven (14%) meningeal hemangiopericytomas (codon 238; TGT → TTT, Cys → Phe), no mutations were observed in exons 5–8 of the p53 gene in any of the following tumors of the nervous system and its coverings: 13 schwannomas, 12 central neurocytomas, 22 meningiomas, 10 choroid plexus papillomas and carcinomas, and 30 neuroblastomas of the sympathetic nervous system. These and published data support the view that p53 mutations are frequently involved both in low‐grade and progressive (anaplastic) astrocytomas, including glioblastomas multiforme. Oligodendrogliomas, medulloblastomas, meningiomas, and hemangiopericytomas rarely (<15%) show p53 mutations in exons 5–8, whereas none of the remaining nervous system neoplasms revealed evidence of an involvement of the p53 gene in their development.
Ki‐ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice
Authors: Mass Marc J. , Ross Jeffrey A. , Nesnow Stephen , Jeffers Anita J. , Nelson Garret , Galati Anthony J. , Stoner Gary D.
Published: February 2007

Strain A/J mice received intraperitoneal injections of benz[j]aceanthrylene (B[j]A) or benzo[a]pyrene (B[a]P). At 24, 48, and 72 h, lung tissues were removed for analysis of B[a]P‐ or B[j]A‐derived DNA adduct formation during the first 3 d of exposure. One group of mice exposed to these hydrocarbons was kept for 8 mo to determine lung tumor multiplicity, the occurrence of mutations in codons 12 and 61 of the Ki‐ras gene in the tumors that arose, the relationship between Ki‐ras oncogene mutations in tumors, and the presence and quantity of genomic DNA adducts. The major DNA adduct in the lungs of mice exposed to B[a]P was N2‐(10β‐[ + B,7α, 9α‐trihydroxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene]yl)‐deoxyguanosine (BPDE‐l‐dGuo) arising from bay‐region diolepoxide activation of B[a]P and was consistent with the occurrence of tumors with mutations GGT → TGT (56%), GGT → GTT (25%), and GGT → GAT (19%) in codon 12, all involving mutations of a guanine. B[j]A, a demethylated analogue of 3‐methylcholanthrene (3‐MCA) with an unsaturated cyclopenta ring, produced 16‐ to 60‐fold more tumors at equivalent doses than did B[a]P; the mutations in tumors were GGT → TGT (4%), GGT → GTT (30%), and GGT → CGT (65%). Analysis of adduction patterns in DNA suggested that B[j]A was activated to form DNA‐binding derivatives in A/J mouse lungs primarily at the cyclopenta ring even though B[j]A contains a bay region. As reported in the published literature, the mutation spectrum induced by 3‐MCA in Ki‐ras codon 12 of mouse cells is similar to that of B[a]P but not to that of its close relative B[j]A. In contrast to B[j]A, 3‐MCA is activated mostly via a bay‐region diol‐epoxide since its cyclopenta ring is saturated and not easily epoxidated. Therefore, we propose that the GGT → CGT mutations produced by B[j]A in Ki‐ras codon 12 were mostly the result of cyclopentaring‐derived adducts.
Ki‐ras mRNA regulation in untransformed mouse lung cells
Authors: Li Jia , Beer David G.
Published: February 2007

Although the Ki‐ras gene is an often‐observed transforming gene in lung tumors, little is understood of the factors that regulate the expression of the normal gene in lung cells. Therefore, we used untransformed mouse lung epithelial cells to determine the effect of serum, growth factors, and cell confluence on the regulation of Ki‐ras mRNA expression. In subconfluent cells synchronized by 24‐h serum deprivation, the refeeding of media containing serum resulted in the expression of both Ki‐ras and H4 histone mRNA. No change in the expression of either gene was observed in cells refed with media alone. In confluent cell cultures, the refeeding of media with serum had no effect on the expression of these genes, suggesting that cell‐density—dependent mechanisms can override the serum‐induced stimuli for Ki‐ras and H4 histone mRNA expression. Confluent cells expressed low but detectable Ki‐ras mRNA levels consistent with constitutive expression of this gene independent of its role in mitogenic stimuli. EGF (10 ng/mL) and TGF‐α (10 ng/mL) were found to induce transient increases in Ki‐ras mRNA but large increases in H4 histone mRNA levels. Similarly, 48‐h‐conditioned media obtained from two transformed mouse lung cells containing activated Ki‐ras genes and overexpressing Ki‐ras mRNA were observed to increase both Ki‐ras and H4 histone mRNA in the untransformed mouse lung cells. The expression of these genes in mouse lung cells therefore appeared to be linked to stimuli provided by specific growth factors as well as by autocrine factors elaborated by lung tumor cells. The studies reported here provide insight into the regulation of the Ki‐ras mRNA in untransformed lung cells and the factors that may contribute to the elevated levels of Ki‐ras mRNA often observed in transformed lung cells.
ras mutations in 2‐amino‐3‐methylimidazo‐[4,5‐f]quinoline—induced tumors in the CDF1 mouse
Authors: Herzog Christopher R. , Schut Herman A. J. , You Ming , Maronpot Robert R.
Published: February 2007

2‐Amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) is a very potent mutagen that is carcinogenic in rodents and nonhuman primates. IQ‐induced CDF1 mouse lung and liver tumors were examined for activated Ki‐ras and Ha‐ras genes, respectively. Polymerase chain reaction (PCR)—amplified target DNAs were analyzed for mutations of codons 12, 13, and 61 by single‐strand conformation polymorphism (SSCP) and direct sequencing methods. All mutations were localized to codon 61 of the ras genes. Forty‐nine of 54 lung tumors induced by IQ possessed activating Ki‐ras mutations, as did 20 of 26 lung tumors from the vehicle‐treated animals; 80% and 75% of these mutations, respectively, were A → T transversions of the second nucleotide redundant. One lung adenoma from the IQ‐treated group contained a tandem duplication of the sequence corresponding to codons 50–57 of the Ki‐ras gene (unpublished observations). In addition, seven of 34 IQ‐induced liver tumors harbored activating Ha‐ras mutations: five were C → A (G → T) transversions at the first nucleotide, and two were A → T transversions at the second nucleotide of codon 61. None of the 15 liver tumors collected from the vehicle‐treated mice possessed Ha‐ras mutations in codon 12, 13, or 61. These data indicate that IQ induces Ha‐ras gene activation in CDF1 mouse liver tumors. The mechanism of lung tumor induction by IQ, however, is obscured by the high frequency of Ki‐ras A → T mutations observed in both the IQ‐induced and spontaneous lung tumors. The different ras mutational spectra in lung and liver tumors may suggest either that two different pathways of IQ metabolism exist in these organs or that IQ contributes to CDF1 lung tumorigenesis by a mechanism other than its direct interaction with the Ki‐ras gene.
Dependence of induction of osteocalcin gene expression on the presence of wild‐type p53 in a murine osteosarcoma cell line
Authors: Chandar Nalini , Campbell Phil , Novak Josef , Smith Martin , Smith Martin
Published: February 2007

The p53 gene undergoes rearrangement in a high percentage of osteosarcomas, resulting in loss of its expression. A p53‐null murine osteosarcoma cell line F6 was transfected with either a wild‐type or a mutant p53 gene. Stably transfected cell lines were obtained, and their differentiation capabilities were compared in vitro with the parental cell line. Alkaline phosphatase and osteocalcin expression were measured as early and late differentiation markers, respectively. Induction of alkaline phosphatase expression was not affected by the presence of either p53 gene, whereas osteocalcin expression was seen in cells containing the wild‐type p53 gene but not in the parental p53‐null or mutant‐expressing cell lines. That the induction of osteocalcin was intrinsically dependent on the presence of wild‐type p53 was also indicated by the use of a temperature‐sensitive Val 135 p53 mutant at 32°C; predominant expression of p53 in the wild‐type conformation resulted in osteocalcin expression. While the wild‐type p53 gene could suppress tumor formation in vivo, the tumors expressing the mutant p53 gene grew two to three times as large as the tumors that did not express p53. Therefore, the absence of end‐point differentiation in bone due to p53 rearrangements may contribute to the maintenance of the tumorigenic phenotype in osteosarcomas.
Mutations of the p53 tumor suppressor gene and the ras gene family in intrahepatic cholangiocellular carcinomas in Japan and Thailand
Authors: Kiba Takayoshi , Tsuda Hitoshi , Hirohashi Setsuo , Inoue Shuji , Sugimura Takashi , Pairojkul Chawalit
Published: February 2007

The incidence and pattern of mutations of the ras oncogenes and the p53 tumor suppressor gene have been shown to differ among different cancer types and even among the same cancer types with different etiological backgrounds. For example, in a previous study we showed that not only the etiology but also the incidence of point mutation of the c‐Ki‐ras oncogene in cholangiocellular carcinomas (CCCs) differ between Japanese and Thai patients. In the study presented here, we examined the incidence of mutations in the ras gene family and the p53 gene in CCCs of both Japanese and Thai patients by single‐strand conformation polymorphism and direct sequencing analyses and compared the pattern of p53 mutation between these two CCC groups. Although the incidence of ras mutation differed markedly between Japanese (seven of 12, 58%) and Thai (two of 26, 8%) cases, the incidence of p53 mutation was similar: four of 12 (33%) and nine of 26 (35%), respectively. Except for one case in which deletion‐insertion was detected in the second exon of the N‐ras gene, all ras mutations occurred at codon 12 or 13 of the c‐Ki‐ras gene. All p53 mutations but one were detected in a highly conserved region, and the predominant form of the mutations was G:C→A:T transition at CpG sites in both Japanese and Thai cases, similar to that reported for colorectal cancers. Therefore, in contrast to the ras oncogenes, mutation of the p53 gene was frequently involved in the development of CCCs in both Japanese and Thai patients, irrespective of any difference in etiology.
Transforming growth factor‐β1 expression in Syrian hamster cheek pouch carcinogenesis
Authors: Zenklusen Jean C. , Stockman Sharon L. , Fischer Susan M. , Conti Claudio J. , Gimenez‐Conti Irma B.
Published: February 2007

The expression pattern of transforming growth factor‐β1 (TGF‐β1) during the stages of complete carcinogenesis in the hamster cheek pouch model was studied. The right cheek pouches of 18 male hamsters were treated with 0.5% 7,12‐dimethylbenz[a]anthracene (DMBA) for 16 wk. TGF‐β1 was detected immunohistochemically in the resulting samples with two different polyclonal monospecific antibodies that recognize intracellular and extracellular forms of TGF‐β1. In the normal cheek pouch, extracellular protein stained the corium strongly, but the reaction was not evenly distributed. As treatment progressed, the reaction increased in both area and intensity; the peak was reached at 8 wk. Intracellular TGF‐β1 expression followed a similar pattern, with a peak at 4 wk of treatment. The results of northern blot analysis were concordant with the immunohistochemical results. Overexpression of TGF‐β1 was also observed in the malignant tumors, but only the extracellular form of the protein was present; intracellular TGF‐β1 was not detected in these tumors. The expression of TGF‐β1 in this carcinogenesis model seems to have two formal stages, the first being an overexpression step as a reaction to the uncontrolled growth and the second being one in which tumors have no internal expression of TGF‐β1 but in which external protein accumulates in the surrounding stroma. A possible explanation of this paradox may be that TGF‐β1 has functions other than its growth‐repressing activity. © 1994 Wiley‐Liss, Inc.
Expression of myc, fos, and ha‐ras in the livers of furan‐treated f344 rats and b6c3f1 mice
Authors: Butterworth Byron E. , Sprankle Catherine S. , Goldsworthy Susan M. , Wilson Daniel M. , Goldsworthy Thomas L.
Published: February 2007

Furan administered by gavage for 2 yr has been reported to induce hepatocellular carcinomas in male and female B6C3F1 mice and in male but not female F344 rats. Chronic exposure studies in our laboratory using bioassay conditions showed extensive hepatocellular toxicity and sustained increases in regenerative cell proliferation after 1,3, and 6 wk of treatment in male and female rats and male mice. Altered expression of growth‐control genes associated with this hyperproliferative state may enhance the susceptibility of these genes to mutation or may provide a selective growth advantage to preneoplastic cells. Quantitative northern blot analysis of mRNA was used to examine the expression of the oncogenes myc, fos, and Ha‐ras in the livers of animals treated with furan. In male rats, a single administration of 30 mg/kg furan produced necrosis and a subsequent wave of cell proliferation 48 h after treatment and induced transient peaks in the expression of myc, fos, and Ha‐ras 6–24 h after treatment. In male rat liver from our cell proliferation studies, only a slight increase in myc expression was seen at the end of week 1 of treatment. However, beginning at week 3 and increasing at week 6, up to a 15‐fold increase over control values was observed in the expression of myc in the treated animals. The only other notable increase in expression observed in any animals from the cell proliferation study was a threefold increase in myc at week 6 in treated female rats. The absence of an increase in Ha‐ras expression in the male mouse liver suggests that the unique patternof Ha‐ras mutations previously reported in furan‐induced mouse liver tumors is not due to increased mutational susceptibility related to overexpression of this gene. The lack of sustained expression of myc, fos, and Ha‐ras in rapidly proliferating liver suggests that continuous expression of these genes is not necessary to maintain increased rates of cell replication. The large increase in myc expression in male but not female rats suggests an adaptive change that may be related to the sex‐specific incidence of furan‐induced hepatocellular carcinomas in rats. © 1994 Wiley‐Liss, Inc.
Dna polymerase arrest by adducted trivalent chromium
Authors: Bridgewater Laura C. , Manning Francis C. R. , Woo Elizabeth S. , Patierno Steven R.
Published: February 2007

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase‐arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium‐treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose‐dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium‐induced polymerase arrest. Interstrand crosslinking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low‐level, dose‐dependent interstrand cross‐linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium‐mediated interstrand DNA‐DNA cross‐links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium‐mediated DNA‐DNA cross‐links is a potential mechanism of chromium‐induced genotoxicity in vivo. © 1994 Wiley‐Liss, Inc.
Alterations in the methylation status and expression of the raf oncogene in phenobarbital‐induced and spontaneous B6C3F1 mouse liver tumors
Authors: Ray Jean S. , Harbison Margaret L. , McClain R. Michael , Goodman Jay I.
Published: February 2007

The liver tumor‐prone B6C3F1 mouse (C57BL/6 ♂ × C3H/He♀), in conjunction with the more susceptible C3H/He paternal strain and the resistant C57BL/6 maternal strain, is an excellent model for studying the mechanisms involved in carcinogenesis. The study reported here indicated that the B6C3F1 mouse inherited a maternal raf allele containing a methylated site not present in the paternal allele. Seven days after partial hepatectomy or after administration of a promoting dose of phenobarbital (PB) for 14 d; raf in B6C3F1 mouse liver was hypomethylated. The additional methylated site in the allele inherited from C57BL/6 was not maintained. The methylation status of raf in the liver of the C57BL/6 mouse was not affected by PB treatment. This indicates that the B6C3F1 mouse is less capable of maintaining methylation of raf than the C57BL/6 strain is. In both PB‐induced and spontaneous B6C3F1 liver tumors, raf was hypomethylated in a nonrandom fashion. The level of raf mRNA increased in seven of 10 PB‐induced tumors but in only one of five spontaneous tumors, whereas the level of Ha‐ras mRNA increased in nine of 10 PB‐induced tumors and in four of five spontaneous tumors. The results of our investigation (a) support the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism involved in tumorigenesis, (b) support the notion that PB promotes liver tumors that develop along a pathway different from that leading to spontaneous tumors, and (c) indicate that differences in DNA methylation between C57BL/6 and B6C3F1 mice could, in part, account for the unusually high tendency of the latter strain to develop liver tumors. © 1994 Wiley‐Liss, Inc.
Infrequent p53 mutations in 7,12‐dimethylbenz[a]anthracene–induced mammary tumors in BALB/c and p53 hemizygous mice
Authors: Jerry D. Joseph , Butel Janet S. , Donehower Lawrence A. , Paulson Emily J. , Cochran Charles , Wiseman Roger W. , Medina Daniel
Published: February 2007

We conducted experiments to determine if p53 alterations, which are frequent in human breast cancers, were also common in murine mammary tumors. In 13 mammary tumors from 7,12‐dimethylbenz[a]anthracene (DMBA)‐treated BALB/c mice were immunohistochemically analyzed for overexpression of p53; p53 protein was not detectable. Three of the tumors were established as cell lines in vitro. p53 protein was rarely detected at passage 4 in these lines but was overexpressed by passage 8 in two of them. The p53 nucleotide sequence was shown to be wild type in one primary mammary tumor and in the two p53‐overexpressing cell lines. One cell line that overexpressed p53 in vitro was implanted into BALB/c mice. The resulting tumors retained the wild‐type p53 nucleotide sequence but no longer expressed detectable levels of p53 protein, suggesting that the overexpression of wild‐type p53 was related to in vitro culture conditions. The effect of DMBA on mammary‐tumor development was also tested in mice rendered hemizygous for p53. These mice and wild‐type littermate controls had no differences in susceptibility to induction of mammary tumors by oral administration of DMBA. Furthermore, Southern blot hybridizations detected no gross alterations in the wild‐type p53 allele in mammary tumors from the p53‐deficient mice. Point mutation of the wild‐type p53 allele was also infrequent in the DMBA‐induced mammary tumors from hemizygous p53 mice; it occured in only one of seven tumors. Thus, the p53 gene is apparently not a primary target for genetic alterations in DMBA‐induced mammary tumors. Next, we examined mammary tumors derived from D1 and D2 transplantable hyperplastic alveolar nodule (HAN) outgrowths, which rapidly form tumors containing Ha‐ras mutations after DMBA treatment. As ras and p53 mutants can cooperate in transformation, we examined whether D1 and D2 HAN outgrowths have p53 mutations. Unlike in the DMBA‐induced primary mammary tumors, nuclear p53 accumulation was observed frequently (10 of 14) in tumors that arose from D1 and D2 HAN outgrowths. Direct sequencing of the entire coding region of the p53 cDNA from six D1 and D2 tumors confirmed that the sequence was wild type. Although wild‐type p53 was retained in both DMBA‐induced mammary tumors and mammary tumors derived from D1 and D2 preneoplastic outgrowths, wild‐type p53 overexpression was detected only in D1 and D2 tumors. Therefore, D1 and D2 tumors appear to arise by a pathway in which p53 expression is altered, whereas DMBA induction affects a different pathway that does not require such alteration. © 1994 Wiley‐Liss, Inc.
Inhibition of hepatitis b virus surface antigen gene expression in carcinogen‐induced liver tumors from transgenic mice
Authors: Farza Hend , Dragani Tommaso A. , Metzler Thomas , Manenti Giacomo , Tiollais Pierre , Porta Giuseppe Della , Pourcel Christine
Published: February 2007

We previously showed that hepatitis B surface antigen (HBsAg)‐producing transgenic mice were more sensitive to hepatocarcinogens than their normal littermates were. We have now investigated the regulation of hepatitis B virus (HBV) gene expression in carcinogen‐induced liver tumors of HBV‐carrier transgenic mice and in three cell lines derived from tumor samples. Transcription of the S gene was repressed in 17 tumors even though they had normal levels of liver‐specific mRNAs such as albumin and transferrin. Three hepatoma cell lines, derived from independent tumor samples, were analyzed for their capacity to express the S gene after transfection of cloned DNA. Although they no longer expressed the endogenous S gene, they were still able to express it from transfected viral DNA both transiently and stably. The loss of HBsAg expression in tumors and in the cell lines was accompanied by de novo methylation of the S region, which is a way to permanently repress gene expression. Our data confirm in an animal model previous observations of S‐gene expression in human hepatocarcinoma and suggest a role for its downregulation in tumor progression. © 1994 Wiley‐Liss, Inc.
Characterization of cell lines derived from formaldehyde‐induced nasal tumors in rats
Authors: Bermudez Edilberto , Chen Zhuchu , Gross Elizabeth A. , Walker Cheryl L. , Recio Leslie , Pluta Linda , Morgan Kevin T.
Published: February 2007

Cell lines derived from formaldehyde‐induced nasal tumors in Fischer 344 rats were established. All of the lines were found to be epithelial and aneuploid and to express keratin, transforming growth factor‐α, and epidermal growth factor receptor transcripts. Two of four lines were tumorigenic upon injection into nude mice, and these lines also contained point mutations in the p53 suppressor gene. The data indicate that these lines possess characteristics that make them a valuable tool for the study of chemically induced respiratory tract carcinogenesis. © 1994 Wiley‐Liss, Inc.
Altered expression of transforming growth factor‐β1 mrna and protein in mouse skin carcinogenesis
Authors: Patamalai Benjamas , Burow David L. , Gimenez‐Conti Irma , Zenklusen Jean Claude , Conti Claudio J. , Klein‐Szanto Andres J. P. , Fischer Susan M.
Published: February 2007

Transforming growth factor (TGF)‐β1, whose gene is located on mouse chromosome 7, has been proposed to be involved in skin carcinogenesis. In the study presented here, we demonstrated that single topical treatments with different types of tumor promoters, i.e., the protein kinase C activator 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA, 2 μg); the non–protein kinase C activators anthralin (22.6 μg), benzoyl peroxide (20 mg), and cumene hydroperoxide (1.2 mg); the first‐stage tumor promoters 4‐O‐methyl‐TPA (500 μg) and A23187 (166 μg); and the second‐stage tumor promoter mezerein (2 μg) produced transient induction of TGF‐β1 mRNA in SSIN (inbred SENCAR) mouse skin. The time of maximum induction varied from 3 to 12 h; the relative extent of induction was ranked as cumene hydroperoxide > benzoyl peroxide > anthralin > TPA > 4‐O‐methyl‐TPA > mezerein > A23187. These findings suggested that TGF‐β1 mRNA induction is a common response of skin to several types of complete and stage‐specific promoters; however, the extent of induction did not correlate with the reported hyperplastic activity of single applications of these promoters. We also demonstrated that TGF‐β1 mRNA expression in papillomas of SENCAR mice generally correlated with expression levels of cyclin D1, another gene on chromosome 7, and with stage of tumor progression. TGF‐β1 mRNA expression was constitutively elevated in most squamous cell carcinomas from either initiation‐promotion or complete carcinogenesis protocols. Cell lines established from carcinomas also overexpressed TGF‐β1 mRNA. Immunohistochemical staining of tissue sections of normal and TPA‐treated skin revealed the presence of extracellular TGF‐β1 protein in the dermis and intracellular TGF‐β1 protein in the epidermis, especially in the suprabasal layers. The staining patterns of papillomas varied, with 62 ± 13% of the tissue showing strong intracellular staining but only 25 ± 8% of the connective tissue staining for extracellular TGF‐β1. Variable staining patterns were also found in carcinomas; some areas stained heavily for both the intracellular and extracellular forms of TGF‐β1. Overall, 28 ± 6% of the tissue of the 12 analyzed carcinomas stained for the intracellular form and 18 ± 5% for the extracellular from of TGF‐β1. © 1994 Wiley‐Liss, Inc.
Epidermal expression of transforming growth factor‐α in transgenic mice: Induction of spontaneous and 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced papillomas via a mechanism independent of Ha‐ras activation or overexpression
Authors: Wang Xiao‐Jing , Greenhalgh David A. , Eckhardt Joshua N. , Rothnagel Joseph A. , Roop Dennis R.
Published: February 2007

To assess the requirements for papilloma formation in transgenic mice that over express transforming growth facto‐α (TGF‐α) in the epidermis (HK1.TGFα), we tested the sensitivity of HK1 TGFα mice to tumor promotion with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and analyzed the resultant papillomas for synergic c‐Ha‐ras activation and overexpression. We observed that HK1.TGFα mice were highly sensitive to TPA promotion, exhibition multiple papillomas as early as the third week of treatment. After 60 wk of promotion, malignant conversion was not observed and tumors regressed upon removal of the TPA promotion stimulus. Most of the TPA‐induced papillomas did not have detectable c‐Ha‐ras mutations at codons 12, 13, or 61, but three papillomes arising after long‐term TPA promotion (5–7 mo) exhibited c‐Ha‐ras activation at codon 61 (A←T and A←G). Conversely, spontaneous papillomas arising without TPA promotion, including persisting autonomous pipiliomas, were all negative for activating c‐Ha‐ras mutations. Both spontaneous and TPA‐induced HK1.TGFα pipiliomas expressed c‐Ha‐ras message levels similarto those in normal, nontransgenic epidermis or HK1 TGFα nyperpiastic epidermis. These data demonstrate that TGF‐α overexpression can be an initiating event for TPA promotion, that papillomatogenesis in HK1.TGFα mice proceeds frequently via a pathway independent of Ha‐ras activation or overexpression, and, thus, that other events are required for autonomous growth and malignant conversion. © 1994 Wiley‐Liss, Inc.
Infrequent mutation of ha‐ras and p53 in rat mammary carcinomas induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine
Authors: Ushijima Toshikazu , Kakiuchi Hideki , Makino Hiroshi , Hasegawa Ryohei , Ishizaka Yukihito , Hirai Hisamaru , Yazaki Yoshio , Ito Nobuyuki , Sugimura Takashi , Nagao Minako
Published: February 2007

2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine(PhlP) is the most abundant of the heterocyclic amines, a group of potent carcinogens contained in cooked meat and fish. Female F344 rats fed a diet containing 100 or 400 ppm PhIP developed mammary carcinomas within 104 or 52 wk, respectively, at the rate of 47% for each group; these carcinomas were examined for mutations in three members of the ras gene family and in the p53 gene. Single‐strand conformation polymorphism (SSCP) analysis and direct sequencing demonstrated a G←A transition at the second position of Ha‐ras codon 12, with the resultant substitution of glutamic acid for glycine, in two of 10 carcinomas induced by 100 ppm PhIP and in one of seven induced by the 400 ppm dose. No mutations in Ki‐ras or N‐ras were detected. cDNA polymerase chain reaction‐SSCP analysis and direct sequencing demonstrated a G←Ttransversion at the third position of p53 codon 130, with the resultant substitution of asparagine for lysine, in one of the 10 carcinomas induced by 100 ppm of PhIP for which freshly frozen samples were available. PhlP‐induced rat mammary carcinogenesis can be regarded as a unique system in that rat mammary carcinomas are negative for ras and p53 mutations. © 1994 Wiley‐Liss, Inc.
Transforming growth factor‐α expression in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: Responses to lipopolysaccharide and 12‐O‐tetradecanoylphorbol‐13‐acetate
Authors: Pfeifer Richard W. , Adams Lorrie A.
Published: February 2007

Recent findings in our laboratory indicated that peritoneal macrophages (MPs) elicited from phorbol ester‐sensitive SENCAR mice generated significant amounts of superoxide when stimulated by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in vitro; negligible responses were observed for MPs derived from relatively resistant B6C3F1 mice. We hypothesized a similar strain‐dependent secretion of transforming growth factor‐α (TGF‐α) by TPA‐stimulated MPs. TGF‐α secreted by MPs was quantitated by competitive enzyme‐linked immunosorbent assay. After 72 h (maximal secretion), for MPs derived from B6C3F1 mice, in vitro exposure to 10 μg/mL lipopolysaccharide (LPS; non‐lipid‐A‐detoxified) resulted in maximal induction (708 pg/mL), in vivo exposure to intraperitoneally (i.p.) administered TPA (2 μg/mouse) alone resulted in a minimal response (32 pg/mL), and prior in vivo exposure to TPA significantly inhibited (more than 90% suppression) the LPS‐stimulated MP response in culture (i.e., to 62 pg/mL). Although significant amounts of TGF‐α could be detected in both SENCAR‐and B6C3F1‐derived MPs (i.e., approximately 2–3 ng/5x106 cells), SENCAR MPs did not secrete TGF‐α in response to either TPA or LPS. In addition, the use of the semiquantitative reverse transcription‐polymerase chain reaction to detect TGF‐a‐specific mRNA did not support the strain dependency observed for LPS‐stimulated TGF‐α secretion, i.e., detectable transcripts were observed in MP RNA derived from both strains. In conclusion, although TPA itself demonstrated negligible effects on TGF‐α expression in murine MPs, prior in vivo exposure inhibited LPS‐stimulated transcriptional activation and intracellular TGF‐α production. The negligible TGF‐α secretion determined for LPS‐stimulated SENCAR‐derived MPs suggested the possibility of a strain‐specific defect in the posttranslational processing of the proTGF‐α molecule. © 1994 Wiley‐Liss, Inc.
Expression and localization of matrix‐degrading metalloproteinases during colorectal tumorigenesis
Authors: Newell Ken J. , Witty Jean P. , Rodgers William H. , Matrisian Lynn M.
Published: February 2007

The metalloproteinase matrilysin is widely expressed in the epithelial tumor cells of malignant colorectal adenocarcinomas. Approximately 50% of benign adenomas also express low levels of matrilysin that is focally localized. The expression of stromelysin‐1, stromelysin‐3, and gelatinase A was observed in the stromal component of several carcinomas and was not present in adenomatous tissue. The expression of interstitial collage‐nase and gelatinase B was observed in occasional adenomas and carcinomas. Stromelysin‐2 transcripts were not detectable in any of the samples examined. Tissue inhibitor of metalloproteinase‐1 gene expression was widespread and was observed in both epithelial and stromal cells of adenomas and carcinomas. These results indicate that matrilysin gene expression is an early event in colorectal tumorigenesis and that the expression of stromelysin‐1, stromelysin‐3, and gelatinase A is primarily a late event. The observed gene expression patterns suggest that matrilysin may participate in early events in tumor progression and that multiple members of the metalloproteinase family may work in concert to facilitate late‐stage tumor invasion and metastasis. © 1994 Wiley‐Liss, Inc.
A switch from stromal to tumor cell expression of stromelysin‐1 mRNA associated with the conversion of squamous to spindle carcinomas during mouse skin tumor progression
Authors: Wright Jane H. , McDonnell Susan , Portella Giuseppe , Bowden G. Tim , Balmain Allan , Matrisian Lynn M.
Published: February 2007

We previously reported that the expression of stromelysin‐1 (ST‐1), a matrix‐degrading metalloproteinase, correlates with tumor progression in the mouse skin model of carcinogenesis. Using in situ hybridization techniques, we confirmed in this study the expression of ST‐1 mRNA in mouse skin keratinocytes treated with the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate and also observed dramatic expression of ST‐1 message in underlying fibroblastic cells. Benign tumors formed by an initiation/promotion protocol expressed low levels of ST‐1 mRNA, which was localized exclusively to stromal tissue surrounding the tumor cells. Squamous cell carcinomas, produced either by chemical carcinogenesis or by injection of cultured cells derived from chemically initiated squamous cell tumors, expressed high levels of ST‐1 mRNA, which was also localized to adjacent stromal tissues. In contrast, aggressive, highly metastatic spindle cell tumors expressed ST‐1 mRNA in the tumor cells as well as in normal, adjacent stroma. These results suggest that the change from ST‐1 expression in surrounding stromal cells to its expression in the tumor cells themselves is associated with the conversion of squamous to spindle carcinomas and may play a causal role in the ability of these cells to invade and metastasize. © 1994 Wiley‐Liss, Inc.
Increased expression of matrix metalloproteinase‐3 (stromelysin‐1) in cultured fibroblasts and basal cell carcinomas of nevoid basal cell carcinoma syndrome
Authors: Majmudar Gopa , Nelson Bruce R. , Jensen Timothy C. , Johnson Timothy M.
Published: February 2007

Nevoid basal cell carcinoma syndrome (NBCCS), an autosomal dominant disorder, is characterized by the development of numerous cutaneous basal cell carcinomas (BCCs). To better characterize this disorder, we examined the expression of matrix metalloproteinase‐3 (MMP‐3; also known as stromelysin‐1) in BCC tumor specimens, adjacent normal skin, and fibroblasts isolated from the normal‐appearing skin of NBCCS patients. Three of three BCC tumors obtained from NBCCS patients overexpressed MMP‐3 mRNA. In contrast, only 25% of BCC specimens in patients without NBCCS demonstrated overexpression of MMP‐3 mRNA. Moreover, fibroblasts isolated and cultured from all nine uninvolved skin specimens of NBCCS patients overexpressed MMP‐3 mRNA. MMP‐3 mRNA was not detected or was detected at very low levels in normal skin and fibroblast cultures isolated from normal skin in nonsyndrome patients. ©1994 Wiley‐Liss, Inc.
Ki‐ras mutations in 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone—initiated and butylated hydroxytoluene—promoted lung tumors in A/J mice
Authors: Matzinger Steve A. , Gunning William T. , You Ming , Castonguay Andre
Published: February 2007

The promotional effects of butylated hydroxytoluene (BHT) on lung tumorigenesis induced by 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) was evaluated in a two‐stage model of lung tumorigenesis in the A/J mouse. Mice were treated in two separate experiments with 1.54 mmol/kg (9.1 mg/mouse) NNK, which induced an average of 8.4 and 9.1 tumors/mouse in the experiments. Animals fed a diet that contained 1 g/kg BHT after administration of the carcinogen in these two experiments demonstrated an increase of the tumor multiplicity by 63% and 43%. Multiplicity of forestomach tumors was not effected by BHT in the diet. No differences in lung tumor morphology were seen as a result of the promoting effect of BHT. Mutations in the Ki‐ras oncogene from lung tumors induced by NNK (19 tumors) or by NNK plus a diet containing BHT (34 tumors) were characterized by polymerase chain reaction, single‐strand conformational polymorphism, and direct sequencing. All 19 NNK‐induced tumors not promoted with BHT contained activated Ki‐ras genes with GC→AT transitions at the second base of codon 12. Only 11 of 34 NNK‐induced and BHT‐promoted tumors (32%) had this characteristic Ki‐ras alteration. These data suggest that the NNK‐initiated mouse lung tumorigenesis pathway, which involves the specific mutation of the Ki‐ras oncogene, is altered to a predominantly non‐ras mechanism when these tumors are promoted by BHT in the diet. ©1994 Wiley‐Liss, Inc.
Evidence for a p53‐independent pathway for upregulation of SDI1/CIP1/WAF1/P21 RNA in human cells
Authors: Johnson Mac , Dimitrov Diana , Vojta Patrick J. , Barrett J. Carl , Noda Asao , Pereira‐Smith Olivia M. , Smith James R.
Published: February 2007

SDI1 is an inhibitor of DNA synthesis that we isolated by expression screening cDNAs prepared from senescent, terminally nondividing human cells. Other groups then cloned this gene as a cyclin‐dependent kinase (cdk)‐interacting protein (CIP1, p21) that inhibits cdks; the gene was also isolated by screening for genes transactivated by p53 (WAF1). p53 levels are low in senescent and quiescent contact‐inhibited or serum‐deprived normal human cells, which we have found express high levels of SDI1 mRNA. This indicates that alternate pathways for upregulation of message level of this gene may exist. We therefore proceeded with the study presented here, treating human cells with a variety of growth‐arrest‐inducing agents, including some that damaged DNA, and found that RNA levels of SDI1 were increased in all cases that resulted in growth inhibition. More important, with the exception of γ‐radiation, most of these agents were able to elevate SDI1 message levels in cells lacking wild‐type p53. At least two distinct kinetic profiles for RNA induction were observed, one that implicated p53 transactivation and occurred early enough to cause arrest, and another that clearly was p53 independent and suggested a role for the SDI1 gene product in the maintenance rather than in the cause of inhibition of DNA synthesis. ©1994 Wiley‐Liss, Inc.
Articles activation of promutagens in a human bronchial epithelial cell line stably expressing human cytochrome P450 1A2
Authors: Macé Katherine , Gonzalez Frank J. , McConnell Ian R. , Garner R. Colin , Avanti Ornella , Harris Curtis C. , Pfeifer Andrea M. A.
Published: February 2007

Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T‐antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T‐antigen‐positive cells (BEAS‐2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S‐transferrase π, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS‐2B cells either by infection with a high‐titer recombinant retrovirus (pXT‐1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B‐1A2 and B‐CMV1A2, and B‐CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50‐ to 400‐fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B1) (AFB1) than the corresponding control cell lines. The cytotoxic effects of AFB1 were paralleled by increased metabolism of AFB1 and enhanced formation of the AFB1‐N7 guanine adduct in B‐CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter‐driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase l enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens. ©1994 Wiley‐Liss, Inc.