bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of RB and p53 mutations in mouse lymphoma cell lines
Authors: Murakami Akira , Tanaka Harutaka
Published: February 2007

We analyzed the genomic structure and mRNA of the RB and p53 genes in four mouse lymphoid leukemia cell lines (DL‐1, DL‐5, DL‐8, and DL‐12). Although no gross structural alteration of the RB gene was observed in any cell line, abnormalities of RB mRNA were detected in at least two cell lines. RB mRNA expression was greatly reduced in DL‐12. In addition, cloning and sequencing analysis of the RB cDNA revealed that the RB mRNA in DL‐8 had a 276‐nucleotide deletion presumably consisting of exons 10, 11, and 12, suggesting that altered splicing resulted in the loss of these exons. Analysis of the p53 gene indicated that DL‐5 had a deletion in both alleles and expressed a smaller mRNA. These results suggest that mutations of the RB or p53 genes, or both, are associated with lymphoid leukemogenesis in mice.
Mutations in the p53 tumor suppressor gene in human cutaneous squamous cell carcinomas
Authors: Pierceall William E. , Mukhopadhyay Tapas , Goldberg Leonard H. , Ananthaswamy Honnavara N.
Published: February 2007

In this study, we analyzed 10 human squamous cell carcinomas (SCCs) for alterations in the p53 tumor suppressor gene in exons 4 through 9 by single‐strand conformation polymorphism (SSCP) analysis. We found that 2 of 10 SCCs displayed unusual SSC Pallelesat exon 7 of the p53 gene. Subsequent cloning and sequencing of PCR‐amplified exon 7 DNA from these two tumors revealed that one had a G→A transition at the first position of codon 244, predicting a glycine‐to‐serine amino acid change, while the other tumor exhibited a G→T base change at the second nucleotide of codon 248, predicting an arginine‐to‐leucine substitution. Because the mutations in the p53 tumor suppressor gene in both tumors were located opposite potential pyrimidine dimer sites (C‐C), it is consistent with these mutations having been induced by the ultraviolet radiation present in sunlight. These studies demonstrate that inactivation of the p53 tumor suppressor gene, as well as activation of ras oncogenes, may be involved in the pathogenesis of some human skin cancers.
v‐Ha‐ras‐induced mouse skin papillomas exhibit aberrant expression of keratin K13 as do their 7,12‐dimethylbenz[a]anthracene/12‐O‐tetradecanoylphorbol‐13‐acetate‐induced analogues
Authors: Sutter Christian , Strickland James E. , Welty David J. , Yuspa Stuart H. , Winter Hermelita , Schweizer Jürgen
Published: February 2007

Introduction of the v‐Ha‐ras gene into primary epidermal keratinocytes, followed by grafting of these cells to animals, leads to the formation of benign epidermal tumors that resemble papillomas induced chemically by a two‐stage carcinogenesis protocol. In this study, we investigated v‐Ha‐ras‐induced papillomas for aberrant expression of type I keratin K13, previously described in 7,12‐dimethylbenz[a]anthracene/12‐O‐tetradecanoylphorbol ‐13‐acetate (DMBA/TPA)‐induced mouse epidermal tumors. Papillomas produced from three independent infection series were removed 3 wk after grafting concomitant with control grafts originating from mock‐, neo‐, and v‐fos‐infected primary keratinocytes. Combined analysis of the grafts by western blotting of extracted keratins and immunofluorescence studies of frozen sections with a K13‐monospecific antibody revealed K13 expression in all v‐Ha‐ras‐induced papillomas and absence of this keratin in all control grafts. K13‐positive cells in papillomas were restricted to the suprabasal cell layers of the lesions and, at this stage of papilloma development, occurred as foci of varying extensions. Analysis of genomic DNA from v‐Ha‐ras‐induced papillomas for the methylation state of a CpG dinucleotide in the distant promoter region of the K13 gene revealed the occurrence of unmethylated DNA copies that were generated at the expense of methylated DNA copies ubiquitously present in normal epidermis. The ratio of unmethylated to methylated DNA copies correlated with the extent of suprabasal K13 protein expression. Thus, all features of aberrant K13 expression previously described in DMBA/TPA‐induced papillomas were shared by v‐Ha‐ras‐induced papillomas.
Expression of human O6‐methylguanine‐DNA methyltransferase in chinese hamster ovary cells and restoration of cellular resistance to certain N‐nitroso compounds
Authors: Wu Zining , Chan Chung‐Leung , Eastman Alan , Bresnick Edward
Published: February 2007

We have constructed a plasmid in which the expression of human O6‐methylguanine‐DNA methyltransferase (MGMT) cDNA is driven by the Rous sarcoma virus promoter sequence. Transfection of this plasmid into Chinese hamster ovary (CHO) cells results in expression of MGMT and in cellular resistance to N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) and 1‐(2‐chloroethyl)‐1‐nitrosourea (CNU), but not to N‐nitroso‐N‐ethylurea. The specific activity of MGMT in transfected CHO cells correlated well with their resistance to MNNG and CNU. Southern analysis showed that the plasmid had been integrated into the CHO cell genome. Western analysis of extracts from transfected CHO cells using an antibody against a peptide corresponding to the carboxyl‐terminal end of the human MGMT protein demonstrated a single band with a molecular size of 24–25 kDa; no such band was observed in extracts from wild‐type CHO cells. These transfected cells may therefore be used to study the role of MGMT in the repair of alkylating DNA lesions and to determine its importance in carcinogenesis as well as in chemotherapy.
Regulation of 2‐acetylaminofluorene‐and 3‐methylcholanthrene‐mediated induction of multidrug resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat liver
Authors: Gant Timothy W. , Silverman Jeffrey A. , Bisgaard Hanne Cathrine , Burt Richard K. , Marino Pamela A. , Thorgeirsson Snorri S.
Published: February 2007

Previous studies by this laboratory have indicated that expression of the multidrug resistance (mdr) gene can be increased in vivo by exposure to a variety of xenobiotics. Because of the nature of these compounds, it was proposed that mdr gene expression might, at least in part, be regulated by the arylhydrocarbon (Ah) receptor. In the present study, we used a primary hepatocyte culture model to examine the relationship between induction of cytochrome P450IA and mdr expression in vitro. Both 3‐methylcholanthrene (MC) and 2‐acetylamino‐fluorene (AAF) were efficient inducers of mdr expression in this model. Induction of mdr gene expression by both MC and AAF obeyed a log10 concentration/response relationship. In contrast, 2,3,7,8‐tetrachlorodibenzo‐P‐dioxin did not induce mdr expression at concentrations that yielded maximum induction of cytochrome P450IA expression. These data suggest that mdr induction was not mediated via the Ah receptor. Nuclear run‐off analysis indicated that both AAF and MC induced mdr expression by increasing transcription. Primer extension analysis indicated that mdr gene transcription was initiated at one major site 151 bp upstream of the ATG site in both the uninduced and induced state in vivo and in vitro. The sequence of the primer and the site of initiation of gene transcription indicate that the main gene induced was the mdr1b gene.
Ultraviolet stimulation of intermolecular homologous recombination in Chinese hamster ovary cells
Authors: Nairn Rodney S. , Adair Gerald M. , Christmann Cynthia B. , Humphrey Ronald M.
Published: February 2007

We previously showed that ultraviolet (UV) irradiation of cotransfected plasmid DNA molecules stimulated genetic transformation that depended on intermolecular homologous recombination in Chinese hamster ovary (CHO) cells. Repair‐proficient cells and an excision repair complementation class 1 (ERCC1) UV‐sensitive DNA repair‐deficient mutant responded similarly to UV stimulation in cotransfections with plasmids containing linker insertion‐disrupted copies of the herpes simplex virus thymidine kinase (HSV‐TK) gene. In this study, we cotransfected homologous DNA molecules containing nonoverlapping deletions of the hamster adenine phosphoribosyltransferase (APRT) gene into APRT‐deficient CHO ERCC1 (UVL‐10) and ERCC2 (UVL‐1) excision‐repair mutants and parental repair‐proficient CHO cells. UV damage in cotransfected circular plasmid molecules stimulated transformation in repair‐proficient cells and an ERCC1 mutant, but not in an ERCC2 mutant. Linearization of plasmids prior to cotransfection greatly enhanced transformation frequencies in all three cell lines, but UV stimulation using linear recombination substrates was no longer evident. Our results suggest (i) that the ERCC1 gene defect in CHO UVL‐10 cells does not affect UV stimulation of homology‐dependent extra‐chromosomal recombination, and (ii) that a CHO cell ERCC2 excision‐repair mutant, although recombination proficient, may exhibit altered recombination in response to UV damage.
Expression and localization of the matrix metalloproteinase pump‐1 (MMP‐7) in human gastric and colon carcinomas
Authors: McDonnell Susan , Navre Marc , Coffey Robert J. , Matrisian Lynn M.
Published: February 2007

The expression of members of the family of matrix‐degrading metalloproteinases (MMPs) is believed to contribute to the complex process of invasion and metastasis. In this study, specific cDNA probes for three members of the stromelysin subfamily of MMPs–stromelysin (MMP‐3), stromelysin‐2 (MMP‐10), and pump‐1 (MMP‐7)–were used to examine the expression of these three different MMPs in human gastric and colonic carcinomas and in adjacent normal mucosa. The expression of pump‐1 mRNA in malignant colon and stomach samples was striking. In a total of 10 gastric carcinoma samples examined, eight (80%) expressed pump‐1 transcripts; similarly, 6 of 8 (75%) colon carcinoma samples were also positive. Stromelysin and stromelysin‐2 mRNAs were not detected in any of these samples. Expression of the MMPs examined was not detected in any of the adjacent, grossly normal tissue samples. Using in situ hybridization and affinity purified anti‐pump‐1 antibodies, the expression of pump‐1 mRNA and protein was localized to tumor cells and was not detected in stromal or lymphocytic cells. This data suggests that the inappropriate expression of pump‐1 by malignant cells may contribute to the neoplastic phenotype.
Detection of extrachromosomal circular DNA sequences from tumor cells by an alkaline lysis, Alu‐polymerase chain reaction technique
Authors: Sen Subrata , Rani Sudha , Freireich Emil J. , Hewitt Roger , Stass Sanford A.
Published: February 2007

Extrachromosomal circular DNAs ranging in size from submicroscopic molecules of approximately 100 kb to cytogenetically resolvable structures of 1000+ kb called minute and double‐minute chromosomes have been shown to harbor amplified genes in primary tumor cells, tumor cell lines, and drug‐resistant cells grown in vitro. The presence of these molecules in transformed and malignant cells tends to reflect genetic instability and also suggests their role in tumor progression. Using a colon carcinoma cell line, we developed a technique to detect extrachromosomal circular DNA‐specific sequences by Alu‐polymerase chain reaction. Circular DNA was enriched by selective alkaline denaturation of genomic DNA. We have successfully performed this procedure with a minimum of 5 × 105 cells. The technique does not require any prior knowledge of the sequences located on the covalent circular DNA molecules for their detection. The procedure should be useful as a routine screen of primary tumor cells for the presence of extrachromosomal circular DNA and should permit the preparation of specific probes to aid in their detailed characterizations. © 1992 Wiley‐Liss, Inc.
Influence of poly(ADP ribose) polymerase depletion on promotion of liver carcinogenesis
Authors: Cesarone Carmelo F. , Suzuki Hisanori , Scovassi Anna I. , Scarabelli Linda , Izzo Raffaella , Giannoni Paolo , Mariani Cristina , Miwa Masanao , Orunesu Mauro , Bertazzoni Umberto
Published: February 2007

In previous studies we demonstrated that liver poly(ADP ribose) polymerase (pADPRP) activity was lost in animals exposed to N‐2‐acetylaminofluorene (2AAF) according to the Teebor and Becker experimental model (Cancer Res 31:1–3, 1971). In addition, we used the resistant hepatocyte model of Solt and Farber (Nature 263:702–703, 1976) to further investigate pADPRP activity during the multistep process of liver carcinogenesis. A marked depletion of the catalytic protein was evidenced after 2AAF exposure, confirming previous results and indicating a specific effect of 2AAF on this nuclear enzyme that controls conformational changes of chromatin and regulates several catalytic activities in the nucleus. The levels of pADPRP mRNA, measured by northern blot analysis using both experimental models, indicate that the enzyme depletion is not due to a loss of transcript. Moreover, these data indicate that pADPRP depletion, caused by 2AAF, was also maintained during liver compensatory growth, which is known to induce a rapid and marked increase in pADPRP activity and protein level. Treatment of 2AAF‐exposed animals with N‐acetyl‐L‐cysteine not only efficiently protected against DNA damage, but also prevented a rapid depletion of the catalytic protein. Interestingly, these data indicate that the marked loss of liver pADPRP occurred during the promotion step induced by 2AAF feeding and that this loss was observed using different models for experimental hepatocarcinogenesis. This phenomenon can be ascribed to a highly defective transcript that cannot be correctly translated into the specific protein or to a rapid degradation of the translated protein. © 1992 Wiley‐Liss. Inc.
Induction and progression of the transformed phenotype in cloned rat embryo fibroblast cells: Studies employing type 5 adenovirus and wild‐type and mutant Ha‐ras oncogenes
Authors: Boylan John F. , Shih Thomas Y. , Fisher Paul B. , Zimmer Stephen G.
Published: February 2007

Transformation of cloned rat embryo fibroblast (CREF) cells with the wild‐type 5 adenovirus (wtAd5) transforming genes E1A and E1B (which extend from 0 to 11.2 map units) results in morphologically transformed cells that exhibit an increased saturation density in monolayer culture and display an anchorage‐independent phenotype. WtAd5‐transformed CREF (wtAd5 CREF) cells do not, however, induce tumors when injected subcutaneously into athymic nude mice or syngeneic Fischer rats. We have analyzed the effect of the ras oncogene and sitespecific mutants in the ras oncogene that result in p21 proteins with altered biochemical properties on the oncogenic and metastatic properties of singly (ras) and doubly (ras + wtAd5) transformed CREF cells. Transformants expressing the wild‐type ras p21 protein and ras mutants producing p21 proteins that retained GTP‐binding properties grew in agar, induced tumors in nude mice and syngeneic rats, and metastasized to the lungs of rats when injected into their tail veins. In contrast, cells transformed with the ras mutant 116K (which contains a mutation at residue 116 that produces a Lys instead of an Asn and does not bind GTP or induce CREF cells to grow in agar) did not become morphologically transformed and were not oncogenic when injected subcutaneously into either nude mice or Fischer rats; further, such cells were not metastatic when injected into the tail veins of Fischer rats. When the wild‐type ras or the ras mutants, including 116K, were expressed in nontumorigenic E1A‐plus‐E1B‐expressing wtAd5 CREF cells, transformed cells induced tumors in both types of animals. The CREF cells doubly transformed with 116K + wtAd5, unlike transformants containing the wild‐type ras and the other ras mutants that still retained GTP binding, were still unable to induce lung metastases. In addition, 116K + wtAd5—transformed CREF cells also did not display any alterations in morphology distinguishable from wtAd5 CREF cells and were not able to grow in agar with increased efficiency. These results indicate that the loss of GTP‐binding ability by this mutant p21 ras protein eliminated the ability of these proteins to induce an oncogenic phenotype in an immortal but normal CREF cell line. However, the mutant ras could cooperate with wtAd5 transforming genes in transformed CREF cells to make these cells progress to an oncogenic (but not metastatic) phenotype. © 1992 Wiley‐Liss, Inc.
Testicular adenoma and seminal vesicle engorgement in polyomavirus large‐T antigen transgenic mice
Authors: Chalifour Lorraine E. , Mes‐Masson Anne‐Marie , Gomes Margaret L. , Wang Nai‐Sang
Published: February 2007

Six lines of transgenic mice harboring the cDNA for polyomavirus large‐T antigen (PVLT) linked to the mouse metallothionein‐1 promoter were isolated. The transgene was expressed in testes in all lines isolated and in testes and seminal vesicles in two lines. Three lines developed enlarged testes and seminal vesicles. Development of the phenotype was divided into three stages separable by age and pathology. In stage 1, birth to 6 mo, PVLT was expressed in testes but no pathology was noted; in stage 2, 6–10 mo, PVLT was expressed solely in testes and not in seminal vesicles, yet the seminal vesicles were enlarged; and in stage 3, 10 mo and older, both testes and seminal vesicles expressed PVLT and both were enlarged. Testes were up to sevenfold heavier and increased up to fourfold to fivefold in each dimension. Seminal vesicles were enlarged up to 20‐fold as the result of an accumulation of seminal vesicle fluid. In addition to the four major proteins of seminal vesicle fluid, extra proteins, initially found in stage 2, were increased in stage 3 seminal vesicle fluid. The Leydig cell was the dominant cell type in affected testes, there were few or no normal Sertoli cells or seminiferous tubules remaining by stage 3. The Leydig cells were physiologically active, as indicated by a 8.5‐fold higher testosterone level in sera from stage 3 affected mice compared with sera from age‐matched normal males. PVLT was present in the nuclei of the Leydig cells and was able to confer an immortal phenotype in vitro. Formation of the Leydig cell adenoma was dependent on PVLT expression, but since PVLT expression occurred much earlier than did pathology, additional secondary factors must determine the delay in phenotype development. © 1992 Wiley‐Liss, Inc.
Association of induction of a fully tumorigenic phenotype in murine radiation‐induced T‐lymphoma cells with loss of differentiation antigens, gain of CD44, and alterations in p53 protein levels
Authors: Gjerset Ruth A. , Arya Jyoti , Volkman Sarah , Haas Martin
Published: February 2007

We investigated the mechanism of radiation induction of murine thymic lymphomas by studying the characteristics of primary x‐ray–induced thymic lymphoma (PXTL) cell lines and of their oncogene‐induced, progressed progeny. It is widely thought that proto‐oncogene alterations are associated with the induction of murine lymphomas; however, few, if any primary murine radiation‐induced lymphomas possess (proto‐)oncogene alterations. Independently derived cell lines grown directly (i.e., without in vivo transplantation) from thymic lymphomas of irradiated C57BL/6 mice possess the properties of immortalized pre‐T cells and lack many of the characteristics of “tumor cells”. PXTL cells are poorly tumorigenic upon transplantation, do not clone in methylcellulose cultures, are growth factor dependent and autocrine, and lack consistent chromosome and oncogene abnormalities. However, the thymic lymphomas are malignant and cause the death of each afflicted mouse. PXTL cells expressed two immunologically distinct forms of the tumor suppressor protein p53 that have moderately increased stability (t1/2 = 1 h) when compared with p53 of normal splenic T lymphocytes. Early PXTL cells could progress in vitro to a fully tumorigenic phenotype after infection with retroviruses encoding the c‐myc and v‐ras oncogenes. Progressed T‐lymphoma cells acquired growth factor independence, a highly transplantable and tumorigenic phenotype, and the ability to quantitatively clone in methylcellulose cultures. Progressed lymphoma cells coordinately downregulated the expression of five T‐cell differentiation markers, upregulated the expression of CD44 (Pgp‐1), and harbored vastly elevated levels of two immunologically distinct forms of p53. Our results suggest that the early thymic lymphomas consist of differentiation‐inhibited, immortal pre‐T cells that are precursors to progressed, fully tumorigenic T‐lymphoma cells. © 1992 Wiley‐Liss, Inc.
Overexpression of the c‐erbB‐2/neu–encoded p185 protein in primary lung cancer
Authors: Shi Daren , He Gongping , Cao Shilong , Pan Wensheng , Zhang Hua‐Zhong , Yu Dihua , Hung Mien‐Chie
Published: February 2007

The c‐erbB‐2/neu gene encodes a transmembrane protein of 185 kDa (p185) with tyrosine kinase activity and extensive sequence homology to epidermal growth factor receptor. Amplification and overexpression of the c‐erbB‐2/neu gene has been shown in certain human tumors and is postulated to be important in human carcinogenesis. High levels of expression of the c‐erbB‐2/neu gene have been reported in non‐small‐cell lung cancer (NSCLC) cell lines and primary tumors from the United States. Since geographical and cultural factors may contribute to the development of certain types of cancer, we examined p185 expression in 120 tumors from Chinese patients with lung cancers of different cell types and used immunohistochemical staining to determine the extent and general significance of p185 expression in human primary lung cancer. Our results demonstrate that 58.8% of the NSCLCs expressed p185 and that expression of p185 was observed only in NSCLC and not in small‐cell lung cancers. Thirty‐three of 41 adenocarcinomas and 24 of 55 squamous cell carcinomas among the NSCLCs examined were found to express p185 at levels different from those of normal lung. For the squamous cell carcinomas, p185 expression was correlated with lymph node metastasis (P < 0.01), but for the adenocarcinomas, it was not (P > 0.05). In addition, expression of p185 in NSCLC was significantly more frequent in patients in advanced clinical stages. Our findings indicate that p185 expression is a frequent event and a general phenomenon in NSCLC and is correlated with poor clinical prognostic indicators, suggesting that expression of p185 may be of potential prognostic importance in NSCLC. © 1992 Wiley‐Liss, Inc.
Induction of CYP1A1 gene expression in H4‐II‐E rat hepatoma cells by benzo[e]pyrene
Authors: Houser William H. , Raha Abhijit , Vickers Michelle
Published: February 2007

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducible enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH‐induced expression of the CYP1A1 gene appears to be regulated by several trans‐acting factors, including the Ah receptor and the 4S PAH‐binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 45 PAH‐binding protein in induction of the CYP1A1 gene in H4‐II‐E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O‐deethylase (EROD) activity, in a concentration‐dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot‐blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3‐methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose‐gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 45 PAH‐binding protein, but not to the Ah receptor. These results suggest that the 45 protein may play a role in the PAH‐induced expression of the CYP1A1 gene in rat H4‐II‐E cells. © 1992 Wiley‐Liss, Inc.
Alterations in tumor angiogenesis associated with stable expression of the HIV tat gene
Authors: Huber Brian E. , Richards Cynthia A. , Martin J. Louise , Wirth Peter J.
Published: February 2007

Recent evidence suggests that the human immunodeficiency virus type 1 (HIV) trans‐activator gene (tat) has transforming properties and may be a causative factor in the development of certain types of cancers, in particular Kaposi's sarcoma (i.e., Vogel J. et al. Nature 335:606–611, 1988). To help elucidate the potential role or roles of the HIV tat gene in neoplastic transformation, cell lines were constructed that constitutively express a functional tat gene product. HeLa cells were coelectroporated with two plasmids, one containing the HIV tat gene in an expression cassette and another containing the dominant selectable marker gene xanthine guanine phosphoribosyltransferase (XGPRT). After XGPRT selection, single‐cell clones that expressed a functional tat protein were identified by measuring chloramphenicol acetyltransferase (CAT) activity after electroporating a plasmid containing the CAT gene transcriptionally controlled by HIV trans‐activation‐responsive region (tar). Phenotypic alterations resulting from the expression of tat were then determined. Control cells and tat‐expressing cells grew at similar rates in culture. However, when grown as tumors in nude mice, tat‐expressing cells produced a lower percentage of tumors, and the tumors that were produced either regressed, stopped growing, or grew at a very reduced rate compared with cells not expressing tat. These differences may have resulted from a tat‐associated reduction in neovascularization in the tumors. A comparison of total cellular proteins by two‐dimensional polyacrylamide gel electrophoresis indicated only one reproducible alteration in a polypeptide of approximately 44 kDa and p1 of approximately 6.2 associated with tat expression. These cells may be very useful in future in vitro and in vivo studies designed to examine the effects of HIV tat on endothelial and vascular smooth‐muscle cells and the role of tat in the etiology of Kaposi's sarcoma. © 1992 Wiley‐Liss, Inc.
Expression of c‐erb B‐2/neu proto‐oncogene in human prostatic cancer tissues and cell lines
Authors: Zhau Haiyen E. , Wan De Sen , Zhou Jianxin , Miller Gary J. , von Eschenbach Andrew C.
Published: February 2007

Both amplification and overexpression of c‐erb B‐2/neu have been associated with the progression and possible prognosis of a number of human cancers. In this study, we demonstrated that c‐erb B‐2/neu may also play an important role in human prostate cancer. Our conclusion is based on the following observations: (1) A monoclonal antibody raised against a peptide sequence from the C‐terminal domain of the human c‐erb B‐2/neu gene product reacted positively with 68.7% (11 of 16) of the human prostatic cancer tissue extracts analyzed by western blot procedure. These results were supported by the immunohistochemical staining of the prostatic cancer specimens; 80% (12 of 15) showed positive staining, primarily around the plasma membranes of the prostatic cancer cells, c‐erb B‐2/neu oncoprotein was not detectable in normal prostate tissues (five examined by immunohistochemical staining and three by western blotting) or in human benign prostatic hyperplasia (two examined by immunohistochemical staining and six by western blotting) and was expressed less abundantly with lower intensity in “normal” human prostate tissues adjacent to cancerous prostate tissue (5 of 12 examined by immunohistochemical staining). We observed no evidence of c‐erb B‐2/neu gene amplification in 10 fresh human prostatic cancer specimens examined by Southern blotting and in the cultured human prostatic cancer cell lines PC‐3, DU‐145, and LNCaP. (2) The c‐erb B‐2/neu protein was detected in both androgen‐receptor‐positive (LNCaP) and ‐negative (PC‐3 and DU‐145) human prostate cancer cell lines. Positive immunostaining of c‐erb B‐2/neu protein was found to be associated predominantly with the plasma membranes of PC‐3 cells, but was also found to be widespread in the cytoplasmic region of the LNCaP cells and in the perinuclear region of the DU‐145 cells. (3) Like prostate‐specific antigen (PSA) expression, c‐erb B‐2/neu mRNA expression was also positively regulated by androgen in androgen‐receptor‐positive LNCaP cells in vitro and LNCaP tumors in vivo. When LNCaP tumors were grown in castrated male hosts, levels of c‐erb B‐2/neu and PSA mRNA expression decreased initially, but rebounded at 3 wk to levels comparable to those expressed by tumors maintained in intact adult male hosts. © 1992 Wiley‐List, Inc.
Resistance of adult keratinocytes to differentiation‐induced decrease in Ha‐ras mrna levels observed in newborn keratinocytes
Authors: Betz Natalie A. , Pelling Jill C.
Published: February 2007

During two‐stage mouse skin tumorigenesis, the mouse c‐Ha‐ras oncogene undergoes activation by point mutation after initiation with polycyclic aromatic hydrocarbons. Furthermore, initiated epidermal cells containing an activated Ha‐ras oncogene have been shown to be resistant to calcium‐induced terminal differentiation. However, the relationship between Ha‐ras expression and the differentiation process is not well understood in either normal or initiated cells. Before attempting to explore the role of Ha‐ras expression in epidermal differentiation during tumorigenesis, we felt that investigation of Ha‐ras gene expression in normal primary epidermal cells undergoing differentiation was warranted, since primary cultures of normal newborn and adult keratinocytes presumably contain the stem cells from which skin tumors arise. In the present studies, northern blot analysis was used to compare Ha‐ras expression in normal newborn and adult epidermal cells undergoing differentiation. Steady‐state levels of Ha‐ras mRNA remained unchanged in primary cultures of normal adult epidermal cells during calcium‐induced differentiation, whereas steady‐state levels of Ha‐rastranscripts decreased during calcium‐induced differentiation in primary newborn epidermal cells. Differentiation was induced by switching the adult and newborn keratinocytes from medium containing 0.05 mM Ca2+ to medium containing one of three different calcium concentrations (0.15, 0.5, or 1.2 mM Ca2+). The decrease in Ha‐ras mRNA levels observed during differentiation in newborn keratinocytes occurred as an intermediate event in the differentiation process, was specific for the Ha‐ras gene, and was not due to a general decrease in transcriptional activity during differentiation. Characteristic patterns of keratin 14 gene expression and cornified envelope formation were observed, verifying that the differentiation process had been induced in both the primary adult and newborn epidermal cells. That adult keratinocytes are resistant to the differentiation‐induced reduction in Ha‐ras mRNA expression observed in newborn keratinocytes may explain the difference in in vivo tumorigenic potentials of newborn and adult skin. © 1992 Wiley‐Liss, Inc.
Relationship between the expression of differentiation‐specific keratins 1 and 10 and cell proliferation in epidermal tumors
Authors: Kartasova Tonja , Roop Dennis R. , Yuspa Stuart H.
Published: February 2007

In normal epidermis, the expression of keratins 1 and 10 is associated with the loss of proliferative capacity and the onset of terminal differentiation. Keratins 1 (K1) and 10 (K10) are commonly expressed in the differentiating layer of benign tumors, but are lost during progression from the benign to the malignant state in skin carcinogenesis. Active gene constructs of mouse K1 and K10 were introduced into papilloma and carcinoma cell lines derived from keratinocytes to analyze the consequences of the expression of these keratins on the organization of the endogenous cytoskeletal network and on the mitotic activity of the recipient cells. Exogenous K1 integrated into the preexisting keratin K5/K14 network of both SLC‐1 carcinoma and 308 papilloma cells. The formation of a recombinant cytoskeleton was more restricted for K10 than for K1 and appeared to be related to a requirement for cessation of cell division before K10 could integrate. The integration of exogenous K1 filaments into the endogenous keratin network was compatible with sustained proliferation of SLC‐1 carcinoma cells in vitro. However, the exogenous gene was not expressed in tumor grafts in vivo. In contrast, stable K1 or K10 transfectants could not be selected in 308 cells, suggesting that benign tumor cells expressing suprabasal keratins cannot sustain proliferation. Published 1992 Wiley‐liss. Inc.
Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells
Authors: Klinedinst Donna K. , Drinkwater Norman R.
Published: February 2007

We used a shuttle vector based on the Epstein‐Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40°C for various times to induce up to 20 apurinic (AP) sites per 9.7‐kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid‐encoded herpes simplex virus type 1‐thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10‐fold to 13‐fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46–55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100–150 bp) and characterized by short (1–5 bp) regions of sequence homology at the end‐points. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP‐site‐induced template dislocation during DNA synthesis. A broad spectrum of base‐substitution mutations, which accounted for 19–36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43–55%) > A°C (18–21%) > T (9–14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.© 1992 Wiley‐Liss, Inc.
Analysis of point mutations in murine c‐Ha‐ras of skin tumors initiated with dibenz[a,j]anthracene and derivatives
Authors: Gill Rosalynn D. , Beltrán Linda , Nettikumara Anusha N. , Harvey Ronald G. , Kootstra Arend , Digiovanni John
Published: February 2007

This study was designed to evaluate the point mutations in the murine c‐Ha‐ras gene of skin papillomas induced by initiation with dibenz[a,j]anthracene(DB[a,j]A), its bay‐region anti‐diol epoxide ((± anti‐DB[a,j]A‐DE), and a 7,14‐dimethyl analogue (7,14‐diMeDB[a,j]A). Recent studies (Nair RV, et al., Chem Res Toxicol 4:115–122, 1991) in our laboratory have revealed both deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from the anti‐ and syn‐diol epoxides of DB[a,j]A in cultured mouse epidermal cells after exposure to this hydrocarbon. Using PCR amplification and direct sequencing, we found specific A182±T transversion mutations (eight of 10 tumors) in codon 61 of c‐Ha‐ras in papillomas induced by initiation with DB[a,j]A. Analysis of papillomas generated by initiation with the more biologically potent analogue 7,14‐diMeDB[a,j]A revealed that five of five tumors exhibited A182±T transversions in codon 61. The nature of the changes in the two DB[a,j]A tumors not showing codon 61 mutations in Ha‐ras is currently not known since these tumor DNAs also did not possess c‐Ha‐ras mutations at codons 12,13, or 59. Interestingly, papillomas produced by initiation with (±)anti‐DB[a,j]A‐DE also possessed A182±T transversion mutations in codon 61 of c‐Ha‐ras (five of five tumors). These data suggest that dAdo adducts derived from both parent hydrocarbons may play an important role in their tumor‐initiating activity and possibly implicate a specific diol epoxide‐dAdo adduct in this process. © 1992 Wiley‐Liss, Inc.
Alternative splicing of neurofibromatosis type 1 gene transcript in malignant brain tumors: PCR analysis of frozen‐section mRNA
Authors: Mochizuki Hiroshi , Nishi Toru , Bruner Janet M. , Lee Polly S. Y. , Levin Victor A. , Saya Hideyuki
Published: February 2007

The neurofibromatosis type 1 (NF1) gene encodes a 360‐residue region showing significant homology to the catalytic domains of both mammalian GTPase‐activating protein (GAP) and yeast IRA protein. The product of the GAP‐related domain of the NF1 gene (NF1‐GRD) has been shown to stimulate ras GTPase and consequently to inactivate ras protein. We previously reported that the NF1‐GRD has two types of transcripts, type I and type II, which are generated by an alternative splicing mechanism, and that the differential splicing of the NF1‐GRD may be related to differentiation of neuroectodermal cells. Here we examined the differential expression of type I and type II transcripts of NF1‐GRD in clinical samples of supratentorial malignant brain tumors by the RNA‐polymerase chain reaction (PCR) method using frozen tissue sections. Our observations revealed that normal cerebrum predominantly expressed the type II NF1‐GRD transcript, whereas primitive neuroectodermal tumors predominantly expressed the type I transcript. Additionally, although the type I/type II ratio in astrocytomas varied widely among tissue samples, all glioblastomas showed higher type I/type II ratios than adjacent brain samples. The RNA‐PCR analysis using frozen tissue sections is a useful and sensitive method for detecting genetic markers in clinical tissue samples. © 1992 Wiley‐Liss, Inc.
Molecular cloning, analysis, and chromosomal localization of a mouse genomic sequence related to the human papillomavirus type 18 E5 region
Authors: Kahn Tomas , Friesl Holger , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Gissmann Lutz , Kramer Judith , Hausen Harald Zur
Published: February 2007

The E5 open reading frame (ORF) from bovine papillomavirus type 1 (BPV 1) as well as the E5 ORFs from human papillomaviruses (HPV) type 6 and type 16 have been reported to transform immortalized rodent cells. In an analysis of murine and human tumors for the presence of putative papillomavirus‐related sequences, we cloned amplified cellular sequences from the mouse cell line Eb that cross‐hybridized with the E5 ORF of HPV 18. A 2.1‐kb fragment termed HC1 was sequenced. In normal murine cells, it was present as a single‐copy genomic sequence located on chromosome 8. A region of 213 nucleotides corresponded to the E5 gene (HC1 E5), based on the best alignments and on the presence of direct and inverted repeats bearing a central sequence motif. These structural elements are also present in the HPV 18 E5 ORF. HC1 E5 contained an ORF that was transcribed bidirectionally. The transcription in the E5 direction was enhanced in RNA obtained from organs and tumors from carcinogen‐treated animals and C127 cells. The polypeptide deduced from the sequence was related to E5 proteins from genital papillomaviruses, to the putative product of the Q300 mouse gene, and to several viral and human growth factors. The data suggest that there may be several cellular counterparts to the viral E5 proteins. © 1992 Wiley‐Liss, Inc.
Kinds of mutations induced by aflatoxin B1 in a shuttle vector replicating in human cells transiently expressing cytochrome P450IA2 cDNA
Authors: Trottier Yvon , Waithe William I. , Anderson Alan
Published: February 2007

Transient expression of rat liver cytochrome P450IA2 cDNA was combined with the use of a shuttle vector as a mutational target to determine the frequency and types of mutation caused by the conversion of aflatoxin B, into genotoxic metabolites within human cells. Ad293 cells were first transfected with p91‐IA2, a rat liver P450lA2 cDNA expression vector, or with p91‐lA2(i) (a control vector that has the P450 cDNA in the inverted orientation) and incubated for 24 h to permit P450IA2 accumulation. Cells were then transfected with the pS189 shuttlevector plasmid, which carries the Escherichia coli supF gene as a mutational target, and incubated for a further 24 h in the presence of aflatoxin B, to permit promutagen activation and pS189 replication. In shuttle vectors replicated in p91‐lA2–transfected cells, the supF point‐mutation frequency increased with increasing concentration of aflatoxin β1. This frequency was nine to 23 times greater than the background point‐mutation frequency obtained with aflatoxin B1‐treated control (p91‐IA2(i)‐transfected) cells. The large majority of the aflatoxin B1–induced supF point mutations were base substitutions, mostly G:C ± T:A transversions. This mutagenesis system permits the molecular analysis of mutations induced by specific P450/promutagen pairs in a shuttle vector replicating in human cells and will permit the investigation of host cell mechanisms involved in the generation of these mutations. © 1992 wiley‐Liss, Inc.
Alterations in the structural gene and the expression of p53 in rat liver tumors induced by aflatoxin B1
Authors: Lilleberg Stan L. , Cabonce Marc A. , Raju Narayan R. , Wagner Lynne M. , Kier Larry D.
Published: February 2007

Rat hepatocellular carcinomas (HCCs) induced by aflatoxin B1 (AFB) treatment were examined for changes in the p53 tumor suppressor gene and in p53 suppressor gene expression. A high proportion of HCCs (nine of 11 tumors in six of eight animals) exhibited new p53 restriction fragments, indicating genomic alterations of one of the p53 alleles. Each tumor with an altered p53 restriction‐fragment pattern exhibited a new fragment in one of two size classes (3 kb or 7 kb with EcoRl digestion) that were missing portions of the 3' end of the p53 gene. These findings indicate that apparently similar genomic rearrangements or deletions occurred independently in AFB‐induced tumors. When compared with nontumor liver tissue from the same animal, the tumors with p53 gene alterations showed dramatically reduced levels of p53 mRNA and protein and greatly increased levels of histone H2B and retinoblastoma tumor suppressor (Rb) mRNA. In two HCCs showing no evidence of p53 restriction‐fragment alterations, mutant p53 protein was detected. Mutant protein was also detected in two liver samples containing an adenoma and altered foci. These data suggest that alterations of the p53 tumor suppressor gene are involved in the induction of rat HCC by AFB. © 1992 Wiley‐Liss, Inc.
Cellular pattern of multidrug‐resistance gene expression during chemical hepatocarcinogenesis in the rat
Authors: Nakatsukasa Harushige , Evarts Ritva P. , Burt Richard K. , Nagy Peter , Thorgeirsson Snorri S.
Published: February 2007

Increased expression of multidrug‐resistance (mdr) gene transcripts and of the encoded protein, P‐glycoprotein, is found in many types of tumors. The biological significance of mdr overexpression during the stepwise process of neoplastic development, however, is not well understood. To assess the possible significance of mdr overexpression in carcinogenesis, we examined the cellular distributions of both mdr gene transcripts and P‐glycoprotein during hepatocarcinogenesis induced in rats by the Solt‐Farber protocol and then compared them to the distributions of the placental form of glutathione S‐transferase (GST‐P), a known marker of preneoplastic and neoplastic lesions in the liver. In situ hybridization and immunohistochemical techniques were employed. Neither mdr transcripts nor P‐glycoprotein was expressed in oval cells that appeared early in the carcinogenic process. GST‐P was strongly expressed in the early focal lesions, whereas the levels of mdr transcripts and P‐glycoprotein expressed were low and heterogeneous. Expression of mdr transcripts and P‐glycoprotein was increased and became more uniform in hyperplastic nodules and carcinomas, although considerable heterogeneity of expression was still found, particularly at the nodular stage. These data suggest that increased expression of mdr is associated with later stages of neoplastic development in the liver. Furthermore, that no chemical treatment of the animals was employed when the expression of mdr was increasing in the preneoplastic and neoplastic lesions suggests that the enhanced mdr expression is intrinsic to the carcinogenic process. © 1992 Wiley‐Liss, Inc.
Differential susceptibility to carcinogen‐induced amplification of sv40 and dhfr sequences in sv40‐transformed human keratinocytes
Authors: Rossman Toby G. , Wolosin Dora
Published: February 2007

Gene amplification contributes to carcinogenesis by enhancing proto‐oncogene activity and causing chromosomal instability. The ease of detecting amplified tumor‐virus sequences has encouraged use of this system as a surrogate for studying the molecular events involved in endogenous gene amplification. We report here a new system for studying carcinogen‐induced amplification of both endogenous and viral sequences in the SV40‐transformed human keratinocyte line AG06. Treatment with carcinogens induced a transient dose‐dependent amplification of the integrated SV40 sequences. The amplified sequences appeared in the extra‐chromosomal fraction. Treatment of these cells with carcinogens prior to methotrexate (MTX) selection increased the frequency of MTX‐resistant colonies, 67% of which exhibited dihydrofolate reductase (dhfr) amplification. The abilities of five carcinogens with different DNA‐damaging activities (the DNA‐damaging agents N‐methyl‐N‐nitro‐N‐nitrosoguanidine, mitomycin C (MMC), ultraviolet light C, and X‐rays and the non‐DNA‐damaging agent arsenite) to induce SV40 and dhfr amplification at concentrations that result in 50% clonal survival were compared. All four DNA‐damaging carcinogens (as well as growth arrest) were able to elicit some SV40 amplification, but responses varied markedly, from 1.8‐fold for X‐rays to sevenfold to eightfold for MMC. There was no correlation between the ability to elicit the two amplification responses. Arsenite, which did not induce SV40 amplification, was the best inducer of MTX resistance. These results point to different controls involved in the induction of viral and dhfr amplification. The signal for amplification of viral genes may be triggered by DNA damage and growth arrest, whereas amplification of dhfr, and perhaps other endogenous sequences, seems to be triggered by other signals as well. © 1992 Wiley‐Liss, Inc.