bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractProtein 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

AbstractConsumption 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

AbstractTo 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

AbstractThe 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

AbstractO6‐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

AbstractAberrant 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

Abstract1‐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

AbstractThe 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

AbstractWe 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

AbstractIn 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

AbstractEvidence 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

AbstractMany 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

Abstract2‐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

AbstractThe 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.