bluuebunny/crossref_metadata_2025 huggingface dataset:
Similar patterns of hypomethylation in the β‐hydroxy‐β‐methylglutaryl coenzyme a reductase gene in hepatic nodules induced by different carcinogens
Authors: Rossiello Maria Rosaria , Rao Prema M. , Rajalakshmi Srinivasan , Sarma Dittakavi S. R.
Published: February 2007

Our earlier studies demonstrated that the β‐hydroxy‐β‐methylglutaryl coenzyme A (HMG CoA) reductase gene is hypomethylated and overexpressed in hepatic nodules initiated by 1,2‐dimethylhydrazine (1,2‐DMH). The study presented here examined whether the pattern of DNA methylation of the HMG CoA reductase gene in hepatic nodules reflected carcinogen‐DNA interaction during initiation. Accordingly, hepatic nodules were generated in male Fischer 344 rats with either 1,2‐DMH or aristolochic acid (AA), which interact predominantly with the guanine and adenine bases in DNA, respectively. DNA from individual nodules was restricted with Hpall, Mspl, and Hhal, and the fragments obtained were hybridized to a cDNA probe for HMG CoA reductase. The results indicated that the hypomethylation pattern in the reductase gene in the nodules initiated with these two carcinogens was similar, although they interacted with different bases in the DNA. The question still remained whether the DNA fragments obtained by digesting the two sets of nodules with the restriction endonucleases were from the same domains in the genome of HMG CoA reductase. To examine this, probes for the different domains of the HMG CoA reductase gene were generated from the cDNA using the restriction enzyme Accl. Three probes were obtained: (i) a 5′‐end fragment corresponding to the membrane‐spanning region, (ii) a second fragment corresponding to the 3′‐end of the protein, and (iii) a third fragment spanning the region between (i) and (ii). Each of these probes was radiolabeled and hybridized to the Hpall‐and Hhal‐generated fragments from the DNA of hepatic nodules initiated with 1,2‐DMH and AA. Irrespective of the carcinogen used for initiation, hypomethylation occurred in all three domains of the gene. More important, the pattern of hypomethylation was similar in the nodules initiated by the two carcinogens. Furthermore, an overall similarity was seen in the hypomethylation patterns in the c‐myc and c‐Ha‐ras genes in the DNA of nodules initiated with either 1,2‐DMH or AA. These results raise the possibility that the pattern of hypomethylation established in the hepatic nodules may not directly reflect the interaction between the initiating carcinogen and DNA but may represent a unique phenotype of hepatic nodules. © 1994 Wiley‐Liss, Inc.
Clonal variation of tumorigenic potential in v‐Ha‐ras‐transformed human bronchial epithelial cells: Relationship to ras oncogene expression and CAD gene amplification
Authors: Forrester Kathleen , Kispert Jennifer , Sanchez Janet H. , Gerwin Brenda I. , Tlsty Thea D. , Harris Curtis C.
Published: February 2007

Infection of an SV40 large‐T antigen‐“immortalized” human bronchial epithelial cell line with a Zip‐v‐Ha‐ras retroviral vector resulted in a mass culture that was tumorigenic in athymic nude mice. A tumor cell line derived from passage of the mass culture in vivo, however, exhibited increased tumorigenicity and v‐Ha‐ras expression. To examine and compare the molecular events involving the ras oncogene during cell transformation in vitro and subsequent tumor formation in vivo, clonal cell populations were isolated from the v‐Ha‐ras‐transformed mass culture. While the clonal cell lines exhibited diverse tumorigenic profiles, these differences did not correlate with v‐Ha‐ras expression. However, the expression of the activated ras gene, while not necessary for growth in vitro, did appear to be associated with a selective growth advantage in vivo. In addition, the modulation of gene amplification ability in these cells was not associated with the induction of tumorigenicity or v‐Ha‐ras expression. ©1994 Wiley‐Liss, Inc.
Far less frequent mutations in ras genes than in the p53 gene in skin tumors of xeroderma pigmentosum patients
Authors: Sato Mayumi , Nishigori Chikako , Lu Yanjun , Zghal Mohamed , Yagi Takashi , Takebe Hiraku
Published: February 2007

Mutations in Ha‐ras, and n‐rasNras genes in squamous and basal cell carcinomas in patients with xeroderma Pigmentosum (XP) were examined by the polymerase chain reaction followed by single‐strand conformation polymorphism analysis and direct base sequencing. No mutation was detected in codons 12, 13, and 61 of the ras genes in XP skin tumors. This was in contrast with previous findings of a high frequency of mutation in the p53 gene in skin tumors in XP patients. A novel mutation in codon 6 of the ki‐ras gene was detected in a squamous cell carcinoma. The mutation was a C→T transtion at a dipyrimidine (5′‐CT) sequence and could have been produced by solar ultraviolet light. The mutated ras gene did not have the ability to transform NIH/3T3 cells. In three tumors, multiple base substitutions were detected in exon 1 of the Ki‐ras and N‐ras genes. These results and our previous work on p53 gene mutations suggest that mutations in ras genes are far less frequent than in the p53 gene in the skin tumors in XP patients and that ras genes are less important in skin tumorigenesis in XP patients that is the p53 gene.
Differential cytotoxicity in mouse epidermal JB6 cells: A potential mechanism for oxidant tumor promotion
Authors: Jain Pramod T. , Chang Seung H. , Berezesky Irene K. , Amstad Paul , Cerutti Peter A. , Trump Benjamin F.
Published: February 2007

It has been suggested that superior antioxidant defense systems protect promotion‐sensitive (p + t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion‐resistant (p—) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p—cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p— cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p— cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p— cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters. ©1994 Wiley‐Liss, Inc.
Cloning of the syrian hamster p53 gene: Structural and functional characterization of the upstream promoter region
Authors: Albor Amador , Laborda Jorge , Notario Vicente
Published: February 2007

We isolated the p53 gene from Syrian hamster embryo cells by cosmid cloning procedures. The organization of the hamster p53 gene was similar to that of other mammalian p53 genes; it had 11 exons, a noncoding exon 1, and a long intron 1 (about 6.5 kb). The upstream p53 promoter was isolated, and the nucleotide sequence of a region encompassing 694 bp upstream from the exon/intron 1 boundary plus the first 6 nt of intron 1 was determined. This genomic region was highly homologous to those of mice and humans but contained a repetitive element not present in either species. Sequence comparisons with the murine and human promoters revealed the presence of similar transcription‐factor binding motifs mapping within a 431‐bp Sacl‐Pstl fragment. Transient transfection assays of primary Syrian hamster embryo cells and neoplastic cell lines with recombinant constructs in which the Sacl‐Pstl fragment was placed upstream of a bacterial chloramphenicol acetyltransferase (CAT) gene revealed the efficient expression of CAT activity. Primer extension analyses identified several putative transcription initiation sites within the p53 upstream promoter, the strongest of which was located 315 bp upstream from the exon/intron 1 junction and about 30 bp upstream from the region encompassing the regulatory motifs. ©1994 Wiley‐Liss, Inc.
Expression of multiple isoforms of protein kinase C in normal human colon mucosa and colon tumors and decreased levels of protein kinase C β and η mRNAs in the tumors
Authors: Doi Sadyuki , Goldstein Cebra , Hug Hubert , Weinstein I. Bernard
Published: February 2007

Previous studies have suggested that protein kinase C (PKC) may play an important role in colon carcinogenesis and that human colon tumors have less total PKC enzyme activity than normal tissue. Because PKC is a multigene family that enocodes for at least 11 distinct isofroms, in the study reported here we analyzed the expression of six of these isoforms at the mRNA level by northern blot hybridization in 22 pairs of primary colon tumors (of various stages), and adjacent normal mucosa samples. We found that the normal mucosa samples expressed the mRNA of the following isoforms of PKC, in decreasing order of abundance: PKCδ>PKC>PKCα>PKCβ>PKCϵ. There was no consistent difference in the levels of PKCα, PKCδ, and PKCη. mRNAs between the normal mucosa and the tumor samples. PKCγ was expressed at a very low level in two of the colon tumors but could not be detected in the remaining tumors of any of the normal mucosa samples. The levels of both PKCβ and PKCη mRNAs were singificantly lower in the tumor samples than in the normal mucosa samples, and this was true of adenomas as well as Dukes' stage A, B, and Cadenocarcinomas. Furthermore, the devrease in PKCη mRNA appeared to be greater in the more poorly differentiated carcinomas. This finding in of interest becouse PKCη is normally expresed in the more differentiated cells of epithelial tissues. The devreased levels of both PKCβ and PKCη mRNAs ocurred early in the multistage process of colon carcinogenesis, as it was also seen in adenomas. The functional significane of these changes remains to be determined.
A p53 mutation in exon 5 associated with adenovirus transformation
Authors: Eyler Yvonne L. , Siwarski David F. , Huppi Konrad E. , Lewis Andrew M.
Published: February 2007

The adenovirus type 5 (Ad5) 55‐kDa E1B oncoprotein has been shown to form complexes with the p53 tumor suppressor protein. These complexes are thought to interfere with normal p53 activity and may be responsible for the paucity of p53 mutations in cells transformed by these viruses. This report describes an example of a p53 mutation in exon 5 in an Ad5‐transformed cell line that exhibited less expression of E1B 55‐kDa protein and a longer tumor‐latency phenotype than another Ad5‐transformed cell line expressing wild‐type p53. The finding of a p53 mutation in an Ad5‐transformed cell line is unusual, especially considering the current theory that p53‐E1B interactions play an important role in adenovirus transformation. This mutation could represent an alternative method of inactivating p53 function in the absence of sufficient levels of E1B 55‐kDa oncoprotein. © 1995 Wiley‐Liss Inc.
Mutation, loss of heterozygosity, and recombination of the p53 gene in mouse forestomach tumors induced by 2‐amino‐3,4‐dimethylimidazo[4,5‐f]quinoline
Authors: Ushijima Toshikazu , Makino Hiroshi , Okonogi Hideo , Hosoya Yoko , Sugimura Takashi , Nagao Minako
Published: February 2007

2‐Amino‐3,4‐dimethylimidazo[4,5‐f]quinoline (MelQ), a food mutagen, induces forestomach tumors in CDF1 mice. We established a polymerase chain reaction (PCR)–single‐strand conformation polymorphism (SSCP) analysis system to detect mutations in the mouse p53 gene exons 2–10, which encompass all five regions conserved among species, and a system to examine loss of heterozygosity (LOH) that uses newly identified polymorphisms between BALB/c and DBA mice, the parental strains of CDF1 mice. Four original forestomach tumors (one papilloma, two carcinomas, and one lymph‐node metastasis) and four cell lines derived from four independent forestomach tumors were examined with the PCR‐SSCP system and by polymorphism analysis. Of the four original tumors, the papilloma had a G→A transition at the second position of codon 171, and one carcinoma had a G→T transversion at the second position of condon 113 with loss of the wild‐type allele, whereas the other two carcinomas had no detectable mutations. Of the four cell lines, two had a base substitution and LOH, and the other two had double mutations (a base substitution and a deletion). By amplification of the double mutations in a fragment, the two cell lines were shown to have four kinds of alleles, indicating induction of recombination within the p53 gene. Our results show that our PCR‐SSCP analysis system is efficient for detecting p53 mutations in mouse genomic DNA and that alteration of the p53 gene plays a significant role in MelQ‐induced mouse forestomach carcinogenesis. © 1995 Wiley‐Liss Inc.
Deletion analysis of protein kinase c inactivation by calphostin C
Authors: Rotenberg Susan A. , Huang Michael H. , Zhu Jianwei , Su Lihe , Riedel Heimo
Published: February 2007

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC‐α (a Ca2+‐dependent conventional isoform) and PKC‐α (a Ca2+‐independent novel isoform) are similarly inactivated by calphostin C (75–100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC‐α required for inactivation by calphostin C. A series of N‐terminal–truncation mutants of bovine PKC‐α expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild‐type PKC‐α by 80–90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND 140) was inactivated by only 20%. These findings imply that the aa sequence 92–140 is a structural determinant of PKC‐α inactivation by calphostin C. This sequence contains one of the phorbol ester‐binding sites (aa 102–144), which is highly conserved among most PKC isoforms including PKC‐α. In addition to aa 92–140, PKC‐stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect of calphostin C action is discussed. © 1995 Wiley‐Liss Inc.
Upregulation of gap junctional communication and connexin43 gene expression by carotenoids in human dermal fibroblasts but not in human keratinocytes
Authors: Zhang Li‐Xin , Acevedo Patrick , Guo Huiming , Bertram John S.
Published: February 2007

Consumption of dietary carotenoids has been statistically associated with decreased risk of cancer at several anatomic sites. In a model murine system of carcinogenesis (the 10T1/2 assay), we have previously shown that carotenoids can inhibit chemically and physically induced neoplastic transformation. This action is strongly correlated with the ability of carotenoids to increase gap‐junctional communication (GJC) by induction of connexin43 (Cx43) gene expression. Here we extend these studies to human foreskin‐derived dermal fibroblasts and keratinocytes. In fibroblasts, β‐carotene and canthaxanthin at concentrations between 10−5 and 3 × 10−6 M were found to strongly enhance GJC in a dose‐ and time‐dependent manner. This was accompanied by an increase in the number of immunofluorescent junctional plaques recognized by an anti‐Cx43 antibody and by an increase in Cx43 protein level as determined by western blot analysis. No decrease in proliferation rates was detected by [H3]thymidine labeling. Human keratinocytes grown in monolayer culture did not respond to carotenoids in terms of GJC as measured by dye transfer, immunofluorescent analysis of Cx43 distribution, or Cx43 levels as measured by western blotting. Both cell types accumulated high levels of carotenoids. Because canthaxanthin, which has no known provitamin A activity in mammals, is as active in fibroblasts as is β‐carotene, the carotenoid with the highest provitamin A activity, the induction of GJC and Cx43 expression by carotenoids in human dermal fibroblasts seems unrelated to their provitamin A status. The lack of response of keratinocytes suggests differences in regulation of Cx43 expression or in carotenoid processing. © 1995 Wiley‐Liss Inc.
Phenotypic variability in induction of p‐glycoprotein mrna by aromatic hydrocarbons in primary human hepatocytes
Authors: Schuetz Erin G. , Schuetz John D. , Thompson Melissa T. , Fisher Robert A. , Madariage Juan R. , Strom Stephen C.
Published: February 2007

To determine whether human liver responds to treatment with aromatic hydrocarbons (AHs) with induction of the multidrug resistance (mdr) gene product P‐glycoprotein and whether AH induction of mdr involves the Ah receptor, we compared induction of mdr mRNA with induction of cytochrome P450 (CYP)1A1 mRNA in AH‐treated cultures of primary human hepatocytes. Hepatocytes from all 15 individuals tested responded to treatment with 3‐methylcholanthrene (MC) or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) with induction of CYP1A1 mRNA. However, only 62% and 55% of the preparations responded to treatment with MC and TCDD, respectively, with induction of mdr mRNA. Indeed, in some individuals mdr mRNA was suppressed by MC and TCDD despite robust CYP1A1 induction. These studies provide the first evidence that not only does individual variation in mdr induction by AH exist but that AHs regulate mdr in humans by a novel mechanism distinguishable from the classical Ah receptor pathway. The dramatic variability in AH induction of mdr may be a predictive risk factor that will help to identify an individual's risk of AH‐associated toxicities. © 1995 Wiley‐Liss Inc.
Differential protein kinase C ligand regulation detected in vivo by a phenotypic yeast assay
Authors: Shieh Hui‐Ling , Hansen Hans , Zhu Jianwei , Riedel Heimo
Published: February 2007

The molecular dissection of protein kinase C (PKC) action has been based in part on time‐consuming functional assays such as the mouse skin model for testing the tumor promoter activity of phorbol esters and related PKC activators. To help overcome the limitations imposed by the complexity of such assays, we developed the yeast Saccharomyces cerevisiae as an alternative, rapid, and simple experimental system. This model has a specific phenotype, an increase in the cell doubling time, that is proportional to the level of enzymatic activity of expressed mammalian PKC isoforms. We used this phenotype to assay and compare the regulation of native bovine PKCα and mutants in the conserved regulatory region C1 in vivo by various activators: two diterpenes, the phorbol ester phorbol‐12‐myristate‐13‐acetate (PMA) and mezerein, and the indole alkaloid indolactam V. We found that PMA activated PKC mutants lacking either Cys‐rich, zinc finger‐like repeat of the conserved region C1 to comparably reduced levels, whereas indolactam V activated native PKCα but none of the mutants at normal doses. In contrast, mezerein activated native PKCα and a mutant lacking the second Cys repeat equally well but mutants lacking the first Cys repeat of C1 at a greatly reduced level. These differential rsponses were supported by the observed in vitro PKC catalytic activities. Therefore, PMA regulates PKCα activity comparably well via either Cys repeat, whereas mezerein regulation predominantly occurs via the first Cys repeat of C1. Indolactam V activation was less potent, it was greatly reduced in the absence of either Cys repeat, and displayed no preference. We introduce this phenotypic assay as a rapid and general screen for the PKC‐activating or possibly inhibitory potential of drug candidates and to identify the PKC regulatory sites involved in these interactions. © 1995 Wiley‐Liss Inc.
Differential expression of the o6‐alkylguanine‐DNA alkyltransferase gene in normal human breast and skin tissue: In situ mapping of cell type‐specific expression
Authors: Wani Gulzar , D'Ambrosio Steven M.
Published: February 2007

O6‐Alkylguanine is a major toxic, mutagenic, and carcinogenic lesion in cellular DNA that is repaired by O6‐alkylguanine‐DNA alkyltransferase (ATase). The expression of this gene is directly related to the cellular sensitivity of alkylating agents, with levels of this protein varying widely among human organs, tumors, and cell types. To better understand specific cell‐type responses to repairing O6‐alkylguanine lesions in DNA, we used colorimetric in situ hybridization, with an ATase‐specific antisense oligomer probe, to map the cellular distribution of ATase mRNA in tissue sections of normal adult human breast and neonatal foreskin tissues. This is the first report of mapping ATase gene expression directly in normal human breast and skin tissues. Paraffinembedded tissue sections were hybridized with a digoxigenin‐labeled, 39‐base antisense ATase oligomer. Hybridization of the probe to cells expressing the ATase gene was visualized after immunodetection with an alkaline phosphatase‐conjugated anti‐digoxigenin antibody. After color development, we simultaneously identified tissue architecture and cell types and measured the expression of the ATase gene. There was no hybridization‐specific color when sections were mock hybridized, hybridized with a sense probe, or treated with RNase. In the breast tissue, 93% of the cells in the loosely connective tissue and 84% of the myoepithelial cells expressed high levels of ATase mRNA. Most of the luminal ductal epithelial cells (61%) were devoid of stain, indicating undetectable levels of ATase mRNA. In skin dermis, 93% of the fibroblasts appeared to express high levels of ATase mRNA. Within the epidermis, approximately 64% of the basal and 65% of the granular epithelial cells expressed ATase mRNA. Expression was undetectable in the epithelial cells of the suprabasal layer of the epidermis. There was very little interindividual variation (<17%) in the distribution of expression of ATase within the same cell types of different individuals. These data illustrate the differential potential of individual cell types within the organ matrix to repair O6‐alkylguanine damage in cellular DNA. This data may provide insights into the understanding of cell type‐specific responses to carcinogens. © 1995 Wiley‐Liss Inc.
Evidence for ras gene mutation in 2‐amino‐3‐methylimidazo[4,5‐f]quinoline–induced colonic aberrant crypts in the rat
Authors: Tachino Nicholas , Hayashi Rick , Liew Christina , Bailey George , Dashwood Roderick
Published: February 2007

Aberrant crypt foci (ACF) are putative peneoplastic lesions that develop after treatment of animals with colon carcinogens, including cooked‐meat heterocyclic amines such as 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ). Male F344 rats given IQ by gavage on alternating days for 2 wk (130 mg/kg body weight) and killed 12 wk after the final carcinogen dose had an average of 4.4 ACF/colon and an average of 3.2 crypts/focus. The DNA from these ACF was amplified by the polymerase chain reaction and analyzed by 3′‐primer mismatch and direct sequencing methods for mutations in the Ki‐ras proto‐oncogene. Of the 37 IQ‐induced ACF screened, three contained a GGT→GAT mutation in codon 12 and one contained a GGC→GCC mutation in codon 13. The approximately 11% frequency of mutation in IQ‐induced ACF is within the range of previous ACF studies of azoxymethane, which reported a 7–37% incidence of Ki‐ras mutaion. These findings suggest that for both compounds, ras mutations occur during early stages of colorectal tumorigenesis. However, while ras mutations can be detected with increasing frequency in azoxymethane‐induced adenomas and carcinomas, they are reportedly absent in IQ‐induced colon tumors. Thus, for IQ and related compounds additional factors (possibly increased cell proliferation) may be important in the later stages of colorectal tumorigenesis. © 1995 Wiley‐Liss Inc.
No involvement of Ki‐ras or p53 gene mutations in colitis‐associated rat colon tumors induced by 1‐hydroxyanthraquinone and methylazoxymethanol acetate
Authors: Suzui Masumi , Yoshimi Naoki , Ushijima Toshikazu , Hirose Yoshinobu , Makita Hiroki , Wang Aijin , Kawamori Tshihiko , Tanaka Takuji , Mori Hideki , Nagao Minako
Published: February 2007

1‐Hydroxyanthraquinone (1‐HA), which is present in some herbs, and methylazoxymethanol (MAM) acetate, a metabolite of azoxymethane, show synergistic carcinogenicity in rat colon, and 1‐HA induces ulcerative changes with simultaneous severe inflammation of the entire colon. In this study, mutations in Ki‐ras (exons 1 and 2) and p53 (exons 4–7) were studied by polymerase chain reaction (PCR)–single‐strand conformation polymorphism (SSCP) analysis. Of 18 adenomas and 38 adenocarcinomas induced in male F344 rats (52 tumors induced by 1‐HA plus MAM acetate, three by 1‐HA alone, and one by MAM acetate alone), no mutations in Ki‐ras of p53 were detected under two conditions of PCR‐SSCP analysis. Because human colon carcinomas from patients with ulcerative colitis have a very low incidence of Ki‐ras mutation, this experimental system would be a good animal model of human colon carcinomas with ulcerative colitis and of human colon carcinomas without Ki‐ras of p53 mutations. © 1995 Wiley‐Liss Inc.
Elevation of transforming growth factor‐α mRNA and protein expression by diverse tumor promoters in sencar mouse epidermis
Authors: Kiguchi Kaoru , Beltrán Linda M. , You Jinsong , Rho Okkyung , Digiovanni John
Published: February 2007

The study presented here was designed to further investigate the role of transforming growth factor‐α (TGFα) in skin tumor promotion by examining the ability of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and several non–phorbol ester promoters to alterTGFαmRNA and protein levels in mouse epidermis. Total RNA was isolated from SENCAR mouse epidermis at various times after single topical treatments with TPA (3.4 nmol), chrysarobin (220 nmol), okadaic acid (2.5 nmol), and thapsigargin (8.5 nmol). Northern analyses of these isolated RNA samples revealed that all four tumor promoters transiently elevatedTGFαmRNA levels. Whereas TPA, okadaic acid, and thapsigarin elevatedTGFαmRNA levels over similar time courses (peak at 4–8 h), chrysarobin elevatedTGFαmRNA levels with a markedly delayed time course (peak at 24–48 h). More detailed studies with TPA also revealed that multiple treatments (four over a 2‐wk period) transiently elevatedTGFαmRNA in both the epidermis and the dermis. The time courses for changes inTGFαmRNA after multiple TPA treatments were similar for both tissues. To facilitate studies of alteredTGFαmRNA expression in mouse epidermis and possibly other mouse tissues, a semiquantitative reverse transcriptase–polymerase chain reaction method was developed. This method faithfully revealed changes inTGFαmRNA levels with all four tumorpromoting agents similar to those determined by northern blot analyses. Immunofluorescence analysis of frozen sections from promoter‐treated skin revealed elevated TGFα protein levels in both epidermis and dermis, although staining was most intense in the epidermal layer. Immunofluorescence analysis of epidermal hyperplasia adjacent to a full‐thickness wound also demonstrated significant epidermal TGFα staining. Collectively, these results indicate that mechanistically diverse tumor promoter stimuli elevateTGFαmRNA and protein in SENCAR mouse epidermis. Elevated levels of TGFα may play an essential role in mitogenic stimulation during tumor promotion by diverse promoting stimuli. © 1995 Wiley‐Liss Inc.
Ki‐ras mutations with frequent normal allele loss versus absence of p53 mutations in rat prostate and seminal vesicle carcinomas induced with 3,2′‐dimethyl‐4‐aminobiphenyl
Authors: Masui Tsuneo , Shirai Tomoyuki , Imaida Katsumi , Hirose Masao , Futakuchi Mitsuru , Tatematsu Masae , Ito Nobuyuki
Published: February 2007

We have developed a prostate carcinogenesis model in Fischer 344 rats using 3,2′‐dimethyl‐4‐aminobiphenyl (DMAB) as a carcinogen to examine various potential modifying factors. In this study, mutational changes in the ras and p53 genes were assessed in DMAB‐induced rat prostate and seminal vesicle carcinomas by single‐strand conformation polymorphism analysis and subsequent direct DNA sequencing. Eight of 22 prostate adenocarcinomas (three of nine (33.3%) from the ventral lobe and five of 13 (38.5%) from the dorsolateral lobe, including three transplantable tumors) and one of 11 seminal vesicle adenocarcinomas (9.1 %) demonstrated point mutations in the Ki‐ras gene. One prostate malignant fibrohistiocytoma examined was negative. Among the positive cases, five (three ventral prostate carcinomas and two transplantable tumors) also showed loss of the normal allele. In contrast, other than one mutation in the p53 gene in the malignant fibrohistiocytoma, there were no mutations in the Ha‐ras or p53 genes. These results indicate that mutational activation of the Ki‐ras gene, but not of the Ha‐ras or p53 genes may play a mechanistic role in prostate and seminal vesicle carcinogenesis by DMAB and that a loss of the normal allele of the Ki‐ras gene may also be involved in the process. © 1995 Wiley‐Liss, Inc.
Characterization of the basal and carcinogen regulatory elements of the rat mdr1b promoter
Authors: Silverman Jeffrey A. , Hill Barbara A.
Published: February 2007

In this report we characterized the transcriptional regulation of the rat mdr1b gene by xenobiotics. The expression of this gene was increased in primary rat hepatocytes and in the H4‐II‐E hepatoma cell line by exposure to carcinogens such as aflatoxin B1, N‐acetoxy‐2‐acetylaminofluorene, and methyl methanesulfonate. Nuclear run‐on experiments indicated that the higher steady‐state levels of mdr1b mRNA were due to an increase in transcription. The 5′‐flanking region of the mdr1b gene was isolated, sequenced, and functionally characterized in transient and stable transfection assays. A single transcription start site was identified for this gene; no alternate start sites were used after induction with aflatoxin B1. Deletion analysis of this promoter demonstrated that the sequence between nt −214 and −178 was critical for basal promoter activity. This region did not contain any consensus‐binding sites for previously identified transcription factors. A negative regulatory region was also identified between nt −940 and −250. No specific carcinogen‐responsive element was identified; the xenobiotic response required a large part of the promoter. These data suggest that the carcinogen induction of mdr1b expression is mediated through sequences that overlap or that are identical to the basal promoter element. © 1995 Wiley‐Liss, Inc.
Abnormalities of the RB1 and DCC tumor suppressor genes: Uncommon in human pancreatic adenocarcinoma
Authors: Barton Claire M. , McKie Arthur B. , Hogg Annette , Bia Brita , Elia George , Phillips Stewart M. A. , Ding Shi‐Fa , Lemoine Nicholas R.
Published: February 2007

Evidence of RB1 allele loss was found in only 6% of pancreatic cancers, and we found no significant sequence abnormalities nor loss of RB protein expression in a panel of tumors and cell lines. Using reverse transcription‐polymerase chain reaction and Southern blot analysis, we found no evidence for loss of DCC expression in pancreatic cancer cell lines, and allele loss only rarely in tumor biopsies. These findings suggest that abnormalities of RB1 and DCC are unlikely to play a major role in pancreatic carcinogenesis. © 1995 Wiley‐Liss, Inc.
Effect of increased glucocorticoid responsiveness in transformed mouse lung cells
Authors: Li Jia , Beer David G.
Published: February 2007

Many transformed mouse lung cells, including LM2 cells, contain activating mutations in the Ki‐ras gene and show reduced responsiveness to growth inhibition by glucocorticoids. LM2GR cells, which are LM2 cells stably transfected with a rat glucocorticoid receptor (GR) gene, were used to determine whether increasing glucocorticoid responsiveness can influence aspects of the transformed phenotype. LM2GR cells grew slower and had a lower final saturation density than the parental LM2 cells. Expression of growth‐related genes was examined by northern blot analysis. The cells were serum‐deprived and treated with fetal bovine serum (FBS), steroid‐stripped FBS (ssFBS), dexamethasone, or 12‐O‐tetradecanoylphorbol‐13‐acetate. The level and pattern of Ki‐ras mRNA expression was similar in both LM2 and LM2GR cells, but histone H4 mRNA was expressed in a more regulated fashion in LM2GR cells. The induction of c‐jun and c‐fos mRNA expression lasted longer in the LM2GR cells treated with ssFBS; however, the maximal induction was greater in the LM2 cells treated with FBS. LM2GR cells demonstrated similar activator protein‐1 (AP‐1) activity but higher GR activity than LM2 cells as determined by using AP‐1‐chloramphenicol acetyltransferase (CAT) and mouse mammary tumor virus‐CAT transient transfection assays, consistent with the higher level of GR mRNA in LM2GR cells. Both cell lines exhibited the ability to grow in soft agar and to form tumors in nude mice. These results indicate that introduction of a functional GR transgene into LM2 cells can increase glucocorticoid responsiveness and alter the expression of genes involved in growth regulation but cannot overcome anchorage‐independent cell growth or tumorigenicity, apparently because of the presence of an activated Ki‐ras gene. © 1995 Wiley‐Liss, Inc.
A human factor that recognizes DNA substituted with 2‐chloroadenine, an antileukemic purine analog
Authors: Hentosh Patricia , Tibudan Martin , Grippo Paul
Published: February 2007

2‐Chloro‐2′‐deoxyadenosine (cladribine), an analog of deoxyadenosine, is an important new drug for the treatment of hairy cell leukemia and other forms of adult and pediatric leukemia. By a gel‐shift binding assay, we identified an activity in HeLa nuclear extracts that recognizes and binds to oligonucleotides substituted with 2‐chloroadenine (ClAde). The activity was specific for ClAde residues because control oligomers did not readily compete out the complex. The binding factor was a monomeric protein that was resistant to inactivation by heating at 45°C but sensitive to heating at 65°C, proteinase K treatment, and 5 mM ZnCl2. This protein, designated ClAde recognition protein (CARP), appeared to be related to a protein that recognized other forms of DNA damage. Gel‐shift binding reactions with ultraviolet (UV)‐irradiated oligomers revealed a UV‐specific protein/DNA complex that had an electrophoretic mobility similar to that of the CARP/DNA complex, and CARP binding to ClAde‐containing oligomers was readily competed out by UV‐irradiated DNA. Moreover, CARP activity was present in extracts prepared from UV‐sensitive xeroderma pigmentosum group A cells but not in a subset of cells from group E, suggesting that CARP was similar to a previously described repair associated factor, xeroderma pigmentosum‐E binding factor. Our findings support a possible repair process for ClAde residues incorporated into cellular DNA. © 1995 Wiley‐Liss, Inc.
Ultraviolet light‐responsive element (TGACAACA,) ‐binding proteins in cells of xeroderma pigmentosum patients
Authors: Yang Yang Ming , Ronai Zeev
Published: February 2007

The ultraviolet light (a) ‐responsive element (a) is an octamer sequence, TGACAACA, that shares homology with cyclic AMP‐responsive element and activator protein 1 target sequences. Because URE‐binding proteins have been shown to play a role in cellular response to DNA damage, we determined their expression and DNA‐binding activities in repair‐deficient cells. Of the complementation groups tested, only xeroderma pig‐mentosum (a) ‐C cells induced expression of c‐jun after UV irradiation; this correlated with XP‐C binding to the URE and resembled the pattern observed with normal human fibroblasts. In other cases either a decrease (XP‐A) or no change (XP‐D) in URE‐binding activities was noticed, which may be associated with decreased c‐fos and poor c‐jun expression after UV irradiation. That XP‐C cells were the only complementation group exhibiting URE‐binding activities similar to those of repair‐proficient cells points to the possible correlation between proper repair of transcriptionally active genes and the expression and activities of proteins implicated in the cellular response to UV irradiation. ©1995 Wiley‐Liss, Inc.
Alterations of multiple tumor suppressor genes (p53 (17p13), p16INK4 (9p21), and DBM (13q14)) in B‐CELL chronic lymphocytic leukemia
Authors: Newcomb Elizabeth W. , Rao Laxmi S. , Giknavorian Sonia S. , Lee Seung Y.
Published: February 2007

B‐cell chronic lymphocytic leukemia (B‐CLL) samples were screened for alterations in multiple tumor suppressor genes (p53 (17p13), p16 INK4 (9p21), and disrupted in B‐cell malignancy (DBM) (13q14)) by using poly‐merase chain reaction‐based assays. Eleven percent (11 of 96) of the B‐CLL cases analyzed in this study and a previous study had mutations in the p53 gene. In contrast, analysis of the p 16 gene showed none of 80 B‐CLL cases had mutations and five cases (6%) had homozygous deletions. Deletions of 13q14 (DBM) occurred in 18% (17 of 96) of patients surveyed. Thus, 28 of 96 cases showed an alteration in one or more of the three tumor suppressor loci examined. However, cases with p53 mutations rarely showed simultaneous loss of DBM. Our results suggest that inactivation of the tumor suppressor genes p53 and DBM may be mutually exclusive, thus providing alternate pathways for tumor development in B‐CLL patients. © 1995 Wiley‐ Liss, Inc.
Activating mutations in human C‐Ha‐ras‐1 protooncogene induced by stereoisomeric fjord‐region benzo[c]chrysene diol‐epoxides
Authors: Du Ming‐Qing , Seidel Albrecht , Phillips David H.
Published: February 2007

The mutagenicity of fjord‐region benzo[c]chrysene diol‐epoxide (a) stereoisomers ((+) anti‐BcCDE, (‐)anti‐BcCDE, (+)syn‐BcCDE, and (‐)syn‐BcCDE) was studied in a forward‐mutation system. pEC plasmid containing the human c‐Ha‐ras‐1 proto‐oncogene was reacted in vitro with each optically active isomer separately and transfected into NIH/3T3 cells. Morphologically transformed foci were cloned, and DNA obtained from these foci was tested for the presence of Ha‐ras‐1 sequence by Southern blot analysis. A total of 50 transformed foci (11‐14 for each diastereomer) were generated. To determine the nature of mutations responsible for activating the proto‐oncogene, regions of the gene likely to contain the activating mutations were amplified by polymerase chain reaction and then subjected to hybridization with specific oligonucleotides. Gene mutations in 42 of 50 transformed foci were characterized by these methods, and most were found at codon 61 (27), followed by codons 12 (13) and 13 (a). All mutations observed were either G → T or A → T transversions. Thirty‐six were G → T transversion mutations occurring at codons 61, 12, and 13. The remaining six were A → T transversions at codon 61. BcCDE stereoisomers may specifically attack guanine and adenine and result in the mutations observed. Some differences in codon preference but not in the types of mutations were found among these optically active isomers. © 1995 Wiley‐ Liss, Inc.
Analysis of oncogenes, tumor suppressor genes, autocrine growth‐factor production, and differentiation state of human osteosarcoma cell lines
Authors: Isfort Robert J. , Cody David B. , Lovell Glenda , Doersen Claus‐Jens
Published: February 2007

Human osteosarcoma and fibrosarcoma cell lines were investigated for alterations in oncogenes, tumor suppressor genes, and growth factors, all of which have been implicated in tumor formation. Characterization of oncogenes that are involved in osteosarcoma formation, including the c‐fos and c‐myc oncogenes, indicated that all six osteosarcoma cell lines examined had 5‐ to 20‐fold amplification of the c‐myc oncogene, whereas neither of two fibrosarcoma cell lines had c‐myc amplification. Interestingly, only three of six osteosarcoma cell lines displayed altered c‐myc immediate‐early gene function. c‐fos was found to be normal, both at the gene and functional levels, in all six osteosarcoma and both fibrosarcoma cell lines tested. Characterization of two tumor suppressor genes, p53 and RB1, that have been implicated in osteosarcoma formation indicated that p53 was altered in five of six osteosarcoma cell lines, whereas RB1 was altered in only two of six of these cell lines. Neither RB1 nor p53 was found to be altered in the fibrosarcoma cell lines tested. An additional transformation marker, autocrine growth‐factor production, was observed in all six osteosarcoma cell lines and both fibrosarcoma cell lines examined. Finally, the differentiation state of the osteosarcoma cell lines was investigated via the bone differentiation markers alkaline phosphatase and osteocalcin. Alkaline phosphatase activity was observed in four of six osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined. The alkaline phosphatase activity was a result of the expression of the bone/liver/kidney alkaline phosphatase isoform. High‐level osteocalcin expression was observed in one of the osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined, although all cell lines demonstrated low‐level osteocalcin expression. Together, these data demonstrate that relatively undifferentiated osteosarcomas commonly display c‐myc amplification, p53 and RBI mutation, and autocrine growth‐factor production, all of which may play a role in osteosarcomagenesis. © 1995 Wiley‐ Liss, Inc.
Hepatocarcinogenesis in BXH recombinant inbred strains of mice: Analysis of diverse phenotypic effects of the hepatocarcinogen sensitivity loci
Authors: Lee Gang‐Hong , Drinkwater Norman R.
Published: February 2007

The hepatocarcinogen sensitivity (Hcs) loci were originally identified as determinants of the approximately 50‐fold higher susceptibility of male C3H/HeJ (C3H) mice to perinatally induced hepatocarcinogenesis relative to male C57BL/6J (B6) mice. These two inbred strains also differ in other phenotypes related to hepatocarcinogenesis, including their incidences of spontaneous liver tumors and the properties of neoplastic hepatic lesions. To test the hypothesis that the Hcs loci also influence these phenotypes, we characterized male mice from B6, C3H, and nine BXH recombinant inbred (a) strains for spontaneous liver tumor development, the frequency of activating mutations in tumors, and the presence of cytoplasmic inclusions in preneoplastic lesions. By comparing these results to the relative susceptibilities of the parental and Rl strains to N, N‐diethylnitrosamine (a) ‐and N‐ethyl‐N‐nitrosourea‐induced hepatocarcinogenesis in preweanling male mice, we concluded that the C3H alleles of the Hcs loci also positively influence the spontaneous development of liver tumors in male animals. While strain‐dependent differences in the frequency of Ha‐ras‐1 activation in DEN‐initiated liver tumors were observed, this phenotype was not correlated with susceptibility to liver tumor induction. The formation of eosinophilic intracytoplasmic inclusion bodies observed specifically in B6 liver tumors, which has been suggested to be associated with the resistance of this strain to hepatocarcinogenesis, also segregated independently of the Hcs loci. © 1995 Wiley‐ Liss, Inc.
Characterization of novel phorbol ester‐ and serum‐responsive sequences of the rat ornithine decarboxylase gene promoter
Authors: Mar Penny K. , Kumar Addanki P. , Kang Dong‐Chul , Zhao Biwei , Martinez Luis A. , Montgomery Raechelle L. , Anderson Larry , Butler Andrew P.
Published: February 2007

Ornithine decarboxylase (a), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promoters. We used transient expression assays and DNA‐protein binding studies to examine the regulation of ODC promoter activity by phorbol esters and serum growth factors. A fragment of the ODC 5′ flanking region (nt ‐1156 to + 13) was sufficient to confer 12‐O‐tetradecanoylphorbol‐13‐acetate (a) ‐responsive expression to a luciferase reporter gene when transfected into H35 cells. However, induction by TPA was not observed in Rat2 fibroblasts, although refeeding of serum‐starved Rat2 cells with fresh serum‐containing medium rapidly induced a fivefold to sixfold increase in ODC promoter activity, maximal about 8 h after refeeding. Deletion analysis demonstrated that several sequences contributed to basal ODC promoter activity but that nt ‐92 to +13 was sufficient for induction by TPA or by serum. This sequence lacked canonical TPA‐responsive elements, and an activator protein‐1 (AP‐1) consensus oligonucleotide failed to compete effectively for proteins binding to this region. Two of four protein complexes observed by gel‐shift analysis of nt ‐92 to +13 were competitively inhibited by wild‐type but not mutant oligonucleotides encompassing a variant cyclic AMP‐response element (a) (ODC nt ‐50 to ‐42); however, a consensus CRE did not compete. Mutagenesis of this site demonstrated that it contributes to basal expression of the ODC promoter but not to TPA or serum responsiveness. Thus, we conclude that the proximal ODC promoter (nt ‐92 to +13) responds to TPA and serum stimulation in a cell‐type‐specific manner that is not mediated by canonical AP‐1 elements. © 1995 Wiley‐ Liss, Inc.
Performance evaluation of a production/inventory system with periodic review and endogenous lead times
Authors: Boute Robert N. , Lambrecht Marc R. , Van Houdt Benny
Published: February 2007

This paper considers a discrete time, single item production/inventory system with random period demands. Inventory levels are reviewed periodically and managed using a base‐stock policy. Replenishment orders are placed with the production system which is capacitated in the sense that there is a single server that sequentially processes the items one at a time with stochastic unit processing times. In this setting the variability in demand determines the arrival pattern of production orders at the queue, influencing supply lead times. In addition, the inventory behavior is impacted by the correlation between demand and lead times: a large demand size corresponds to a long lead time, depleting the inventory longer. The contribution of this paper is threefold. First, we present an exact procedure based on matrix‐analytic techniques for computing the replenishment lead time distribution given an arbitrary discrete demand distribution. Second, we numerically characterize the distribution of inventory levels, and various other performance measures such as fill rate, base‐stock levels and optimal safety stocks, taking the correlation between demand and lead times into account. Third, we develop an algorithm to fit the first two moments of the demand and service time distribution to a discrete phase‐type distribution with a minimal number of phases. This provides a practical tool to analyze the effect of demand variability, as measured by its coefficient of variation, on system performance. We also show that our model is more appropriate than some existing models of capacitated systems in discrete time. © 2007 Wiley Periodicals, Inc. Naval Research Logistics, 2007