bluuebunny/crossref_metadata_2025 huggingface dataset:
Malignant transformation of NIH 3T3 fibroblasts by human c‐sis is dependent upon the level of oncogene expression
Authors: Macarthur Linda H. , Clarke Michael F. , Westin Eric H.
Published: February 2007

High‐level expression of the c‐sis oncogene, which encodes the β chain of platelet‐derived growth factor, transforms immortalized rodent fibroblasts in vitro to a malignant phenotype. c‐sis gene expression has been demonstrated in a variety of human tumors, although generally at levels much lower than those shown to transform cells in vitro. We examined the effect of lower levels of c‐sis expression on the phenotype of NIH 3T3 fibroblasts. Clones with various levels of c‐sis expression were generated by transfecting NIH 3T3 cells with a plasmid that expressed the human c‐sis cDNA and the TN5 neomycin‐resistance gene. G418‐resistant clones, which expressed the c‐sis cDNA, were selected and characterized. Alterations in the phenotype of the clones that expressed c‐sis ranged from increased growth in soft agar to malignant tumor formation in nude and syngeneic mice. Increased levels of c‐sis cDNA expression correlated with the acquisition of features of transformation in a dose‐dependent manner and altered the cellular phenotype in a manner consistent with the progression of cells towards malignancy. These data support a model in which low levels of sis gene expression in tumors contribute to the acquisition of some features of transformation but require complementation by other genes or factors to produce a fully malignant phenotype. © 1992 Wiley‐Liss, Inc.
Expression of the transin, c‐fos, and c‐jun genes in rat transplantable osteosarcomas and malignant fibrous histiocytomas
Authors: Honoki Kanya , Tsutsumi Masahiro , Tsujiuchi Toshifumi , Kondoh Satoshi , Shiraiwa Kazumi , Miyauchi Yoshizumi , Mii Yoshio , Tamai Susumu , Konishi Yoichi , Tim Bowden G.
Published: February 2007

The expression of the transin, c‐fos, and c‐jun genes was assessed in transplantable osteosarcomas and malignant fibrous histiocytomas, as well as in pancreatic duct adenocarcinomas and hepatocellular carcinomas of rats and hamsters. Northern blot analysis revealed that both an undifferentiated osteosarcoma of spontaneous origin (SOS) and 4‐hydroxyaminoquinoline 1‐oxide (4‐HAQO)‐induced malignant fibrous histiocytomas with metastatic potential to the lung showed remarkably increased expression of transin mRNA transcripts. This was not the case for the other tumors. Interestingly, levels of transin mRNA were lower in lung metastatic lesions than in primary subcutaneous SOS tumors. The primary SOS and MFH expressed both c‐fos and c‐jun genes in conjunction with the transin gene, whereas the non‐transin expressers, a 4‐HAQO‐induced osteosarcoma (COS) and the pancreatic duct adenocarcinomas, demonstrated one or the other, but not both. These results suggest a possible involvement of transin expression in the progression of spontaneous osteosarcomas and 4‐HAQO‐induced malignant fibrous histiocytomas in rats. Expression of the c‐fos and c‐jun genes may play a regulatory role in this process. © 1992 wiley‐Liss, Inc.
ras Oncogene activation in mammary carcinomas induced by n‐methyl‐n‐nitrosourea in copenhagen rats
Authors: Lu Shi‐Jiang , Archer Michael C.
Published: February 2007

The occurrence of Ha‐ras and Ki‐ras oncogenes was investigated in mammary tumors produced by treating genetically resistant Copenhagen (Cop) rats with N‐methyl‐N‐nitrosourea. G35A codon 12 mutations in both Ha‐ras and Ki‐ras genes were analyzed by a polymerase chain reaction/liquid hybridization and gel retardation assay. More than half of the adenocarcinomas analyzed contained an activated Ha‐ras gene. This was also the predominant mutation in similar tumors from susceptible Buf/N rats, suggesting a common mechanism of initiation. In contrast, only two of 15 mammary adenosquamous carcinomas from the Cop rats contained an activated Ha‐ras gene, suggesting a different initiation mechanism for most of these tumors. Ki‐ras activation was found in none of five and one of five adenocarcinomas from Buf/N and Cop rats, respectively, and in none of 13 adenosquamous carcinomas from Cop rats. These results suggest that Ki‐ras activation does not play a major role in the initiation of the mammary tumors. © 1992 Wiley‐Liss, Inc.
Transcriptional inhibition by carcinogenic chromate: Relationship to DNA damage
Authors: Manning Francis C. R. , Xu Jian , Patierno Steven R.
Published: February 2007

Hexavalent chromium compounds are carcinogenic to humans, are potent inducers of tumors in experimental animals, and can neoplastically transform cells in culture. In this study, the effects of sodium chromate on the expression of the 78‐kDa glucose‐regulated protein (GRP78) gene and on general transcription were investigated with respect to the DNA damage induced by this agent. DNA single‐strand breaks, DNA‐protein cross‐links, and chromium‐DNA adducts were present in CHO cells immediately after 2 h of treatment with sodium chromate. Subsequently, these types of damage were repaired at different rates. Single‐strand breaks were essentially repaired after 8 h. By 24 h posttreatment, no cross‐links remained in cells exposed to 30 or 150 μM chromate, although cells treated with the 300‐μM concentration still contained cross‐links at that time. DNA‐chromium adducts remained unrepaired for at least 32 h. The moderate constitutive level of GRP78 mRNA was not affected by chromate. Chromate did, however, suppress induction of this gene by tunicamycin in a concentration‐ and time‐dependent manner. Thirty micromolar sodium chromate (96% survival), which caused the least DNA damage, had no effect on GRP78 induction, general RNA synthesis, or mRNA synthesis. Induction of GRP78 was suppressed immediately and 12 h after treatment with 150 μM chromate (54% survival), although there was a partial recovery of induction at 24 h after treatment, which correlated with the repair of DNA‐protein cross‐links. In contrast, both total cytoplasmic RNA and mRNA synthesis were suppressed by approximately 60–75% for at least 32 h by 150 μM chromate. At the 300‐μM concentration (8% survival), where DNA‐protein cross‐links persisted beyond 24 h, GRP78 induction was totally suppressed for at least 24 h, while total RNA and mRNA synthesis were suppressed by 80–90% for at least 32 h. Overall, the effects of chromate on GRP78 induction correlated most closely with the presence of DNA‐protein cross‐links, but suppression of total RNA and mRNA synthesis correlated with the presence of DNA‐chromium adducts. These results indicate that chromate exerts differential effects on the induction of the GRP78 gene and on general transcription. © 1992 Wiley‐Liss, Inc.
Overexpression of an elongation factor‐1 γ‐hybridizing rna in colorectal adenomas
Authors: Ender Bulent , Lynch Patrick , Kim Yeul Hong , Inamdar Nikhil V. , Cleary Karen R. , Frazier Marsha L.
Published: February 2007

While it is apparent that colorectal carcinogenesis results from a series of genetic alterations manifested phenotypically by the adenoma‐to‐carcinoma sequence, the early events that occur in the process of tumorigenesis have not been elucidated. We previously demonstrated that human elongation factor‐1 (EF‐1)γ‐hybridizing RNA was overexpressed in 25 of 29 colorectal carcinomas. To determine if the overexpression of this mRNA occurs early in tumor development, we examined 25 adenomas and corresponding normal‐appearing distant mucosae from 20 patients without familial adenomatous polyposis (FAP). We observed overexpression at a level of twofold or more in 14 (56%) of the 25 adenomas, indicating that overexpression of EF‐1γ RNA is often a relatively early event in the development of non‐FAP colorectal cancer.
Different levels of v‐Ha‐ras p21 expression in primary keratinocytes transformed with harvey sarcoma virus correlate with benign versus malignant behavior
Authors: Brissette Janice L. , Missero Caterina , Yuspa Staurt H. , Dotto G. Paolo
Published: February 2007

Primary mouse keratinocytes transformed with an activated ras oncogene transduced by helper‐free Harvey sarcoma virus (HaSV) form predominantly benign tumors. In contrast, keratinocytes transformed with helper‐associated HaSV form malignant tumors. We report here that this different tumorigenic behavior correlated with a much higher level of v‐Ha‐ras p21 protein in cells transformed with the helper‐associated virus. The high level of v‐ras expression in these cells was due to viral spread beyond the initial infection. The low level of v‐ras p21 expression that resulted from single‐hit infection with helper‐free virus, together with the intrinsic heterogeneity of primary keratinocytes, explains, at least in part, the different tumorigenic behavior of keratinocytes transformed by the two types of viruses.
Expression pattern of proteins that bind to the ultraviolet‐responsive element (TGACAACA) in human keratinocytes
Authors: Yang Yang‐Ming , Rutberg Susan E. , Foiles Peter G. , Ronai Zeev
Published: February 2007

We previously identified an ultraviolet (UV)‐responsive element (URE; TGACAACA) that plays a role in both transcription and replication of polyoma sequences. Mouse polyclonal antibodies were raised against affinitypurified URE‐bound proteins to characterize their expression patterns. These antibodies specifically recognized two of four URE‐bound proteins, of 40 and 68 kDa. The 68‐kDa protein was constitutively expressed in human keratinocytes, while the expression of the 40‐kDa protein was induced by UV irradiation. Of the two, the 68‐kDa protein bound to the URE with greater affinity than the 40‐kDa protein, as determined by southwestern analysis. The expression of the 40‐kDa protein increased as early as 1 h after UV irradiation of both rat fibroblasts and human keratinocytes and correlated with increased binding to the URE in an electrophoretic mobility shift assay. Other types of damage, as well as heat shock and serum stimulation, also induced the expression of this protein, suggesting that it may play a role in cellular response to stress or damage. The 40‐kDa protein was expressed at the highest levels in the S phase of the cell cycle and was induced by aphidicolin, suggesting that it has a role in DNA replication. All together, these results suggest that exposure of human keratinocytes to damage‐ and stress‐inducing agents modulates the expression of proteins that may play a role in regulating cellular response to DNA damage.
Differential activity of the RVF enhancer element in the decreased expression of the neu oncogene in NR‐6 cells versus parental Swiss Webster 3T3 cells
Authors: Yan Duen‐Hwa , Hung Mien‐Chie
Published: February 2007

The rat neu oncogene encodes a growth factor receptor—like glycoprotein, termed p185, that shares structural similarity with epidermal growth factor receptor (EGFR), particularly in the tyrosine‐kinase domain. The Swiss Webster 3T3 murine embryo fibroblast (SW3T3) variant NR‐6, in contrast to the parental cell line, does not express EGFR mRNA. After transfection of an activated rat neu cosmid clone, we demonstrated in this report that whereas SW3T3 cells readily expressed the exogenous rat p 185 protein, NR‐6 cells did not express detectable levels of this protein product. By transfection of plasmids containing the chloramphenicol acetyltransferase (CAT) gene driven by the neu promoter and subsequent CAT assays, we also showed that neu gene promoter activity was significantly less in NR‐6 cells than in SW3T3 cells and that the neu promoter sequence responsible for this decreased transcription was a previously identified RVF enhancer element (Yan D‐H, Hung M‐C, Mol Cell Biol 11:1875–1882, 1991). That is to say, the RVF enhancer element of the neu promoter did not function as an enhancer in NR‐6 cells. To investigate the mechanism responsible for the inactivation of RVF in NR‐6 cells, we used southwestern blot analyses and demonstrated that the 60‐kDa RVF polypeptide was present in both NR‐6 and SW3T3 cell nuclear extracts. This result indicates that the DNA‐binding activity of RVF was similar in these two cell lines; therefore, loss of RVF enhancer activity in NR‐6 cells is probably due to inactivation of the trans‐activating function and not DNA binding activity of RVF.
Reversal of ras‐induced inhibition of gap‐junctional intercellular communication, transformation, and tumorigenesis by lovastatin
Authors: Ruch Randall J. , Madhukar Burra V. , Trosko James E. , Klaunig James E.
Published: February 2007

The plasma‐membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farneslyl synthesis and p21 ras farnesylation are inhibited by hydroxmethylglutaryl‐CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v‐Ha‐ras‐transformed rat liver epithelial cell line (WB‐ras cells) and if changes were associated with the enhancement of gap‐junctional intercellular communication (GJIC). WB‐ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1–0.5 μM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB‐ras tumors were induced in male rats by intraportal‐vein injection of WB‐ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB‐ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin‐treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.
Creation of an active estrogen‐responsive element by a single base change in the flanking sequence of a cellular oncogene: A possible mechanism for hormonal carcinogenesis?
Authors: Nawaz Zafar , Stancel George M. , McDonnell Donald P. , Hyder Salman M.
Published: February 2007

Estrogens are considered to act as promoters in a multistep process of hormonal carcinogenesis, although the molecular mechanisms by which these hormones act in tumorigenesis are unclear at present. Estradiol is known to induce expression of certain proto‐oncogenes, and this led us to examine potential regulatory regions of the cellular c‐fos oncogene. The 5′‐flanking region of the murine c‐fos contains a 13‐bp palindromic sequence (GGTCTnnnAGACC) with striking homology to the consensus estrogen‐responsive element (ERE) GGTCAnnnTGACC. However, the c‐fos sequence did not bind the human estrogen receptor or confer hormonal responsiveness in a yeast‐based transcriptional test system. Importantly, a single base change in the fifth position of the c‐fos sequence (GGTCTnnnAGACC to GGTCA/GnnnAGACC) produced an element that bound the estrogen receptor and conferred estrogen‐dependent transcriptional activation of a reporter gene. This suggests a specific hypothesis by which estrogens could act as tumor promoters. In this paradigm, the regulatory region of the cellular oncogenes, tumor suppressor genes, and growth‐factor genes contain inactive sequences with close homologies to hormone‐responsive elements. Initiation occurs when some agent (e.g., a chemical carcinogen) causes a mutation in such a sequence to create a functional hormone‐responsive element. Estrogens, acting through their receptors and the mutated element, can then activate the target gene to stimulate cell proliferation and increase the population of initiated cells.
Direct growth stimulation of normal human epithelial cells by mutant p53
Authors: Wyllie Fiona S. , Lemoine Nick R. , Barton Claire M. , Dawson Tim , Bond Jane , Wynford‐Thomas David
Published: February 2007

We developed a high‐titer amphotropic retroviral vector that expresses mutant (Ala 143) human p53 to test directly the response of genetically normal human epithelial cells to p53 mutation. Contrary to our prediction, we found that in pancreatic epithelium (whose tumors display a high frequency of p53 mutation) but not in thyroid (whose tumors show an exceptionally low mutation frequency), expression of mutant p53 induced a dramatic, though self‐limiting, proliferative response. This result questions the assumption that p53 mutation is relevant only to the later stages of tumorigenesis.
Quantitation of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline and 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline DNA adducts in specific sequences using alkali or uvrABC excinuclease
Authors: Nouso Kazuhiro , Bohr Vilhelm A. , Schut Herman A. J. , Snyderwine Elizabeth G.
Published: February 2007

2‐Amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) and 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MelQx) are carcinogens found in cooked meats that form DNA adducts upon metabolic activation. Purified DNA from Chinese hamster ovary (CHO) cells was reacted in vitro with the active metabolites N‐acetoxy‐lQ or N‐acetoxy‐MelQx, and the adduct levels in the 5′ dihydrofolate reductase (DHFR) gene and downstream region were quantitated by Southern hybridization. Adducted and restricted DNA was treated with Escherichia coli uvrABC excinuclease or alkali (0.1 N NaOH, 37°C, 60 min) to incise DNA at IQ and MeIQx adduct sites. The DNA was then denatured with formamide, electrophoresed on a neutral agarose gel, transferred to a support membrane, and hybridized with sequence‐specific DNA probes. Both uvrABC and alkali reduced the intensity of Southern hybridization in proportion to the number of IQ or MeIQx adducts in DNA, indicating that these adducts are substrates for uvrABC and that they form alkali‐labile lesions in DNA. IQ and MeIQx adduct levels were the same in the 5′ DHFR gene and in the downstream region. Southern hybridization analysis of pBR322 containing known levels of IQ or MeIQx adducts showed that the efficiency of cutting IQ or MeIQx adducts by uvrABC excinuclease and alkali was approximately 30% and 15%, respectively. 32P‐postlabeling studies examining adduct level in bulk DNA further showed that the adduct profiles were identical in pBR322, CHO DNA, and cultured CHO cells exposed to the reactive metabolites of IQ or MeIQx. The results indicate that IQ and MeIOx adducts can be quantitated in specific genomic sequences and that this method should be directly applicable to studies of gene‐specific repair of these adducts in cultured cells.
Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell‐mediated chromosome transfer
Authors: Satoh Hitoshi , Lamb Patricia W. , Dong Jin‐Tang , Everitt Jeff , Boreiko Craig , Oshimura Mitsuo , Barrett J. Carl
Published: February 2007

To map tumor suppressor genes for lung adenocarcinomas, we introduced normal human chromosomes 3, 7, and 11 into the A549 tumor cell line by microcell‐mediated chromosome transfer to test which chromosomes had the ability to suppress tumorigenicity. These human chromosomes, which contain the neomycin gene as a selectable marker, were transferred into A549 lung adenocarcinoma cells at frequencies of 0.3–1.8 × 10−6. Two microcell hybrid clones with an introduced chromosome 3, two with an introduced chromosome 7, and six with an introduced chromosome 11 were isolated and examined for their growth properties and tumorigenicity in nude mice. Whereas parental A549 cells formed tumors with an average latency of 68 d, both microcell hybrids with an introduced chromosome 3 failed to form tumors for over 360 d. Similar tumorigenicity results were obtained when the clones were implanted into denuded tracheas, a more orthotopic transplantation site. The two clones with an introduced chromosome 7 were still tumorigenic; they formed tumors within 100–123 d after injection and grew progressively, although the tumors grew slightly slower than the parental cells did. Among the six clones with an introduced chromosome 11, one clone was still highly tumorigenic but did not contain an extra copy of an intact introduced chromosome 11. Three clones with a single intact copy of introduced chromosome 11 formed tumors with latency periods significantly longer than those of the parental cells. Two clones had two copies of the introduced chromosome 11, and both failed to form tumors within 1 yr of injection. These results indicate that chromosomes 3 and 11 can suppress the tumorigenicity of A549 lung adenocarcinoma cells.
High variability of nitrosamine metabolism among individuals: Role of cytochromes P450 2A6 and 2E1 in the dealkylation of N‐nitrosodimethylamine and N‐nitrosodiethylamine in mice and humans
Authors: Camus Anne‐Marie , Geneste Olivier , Honkakoski Paavo , Béréziat Jean‐Claude , Henderson Colin J. , Wolf Roland C. , Bartsch Helmut , Lang Matti A.
Published: February 2007

We undertook this study to answer several questions regarding nitrosamine metabolism. Kinetics of nitrosamine metabolism showed the involvement of at least two enzymes in the dealkylation of N‐nitrosodiethylamine (NDEA) and N‐nitrosodimethylamine (NDMA) in mouse liver microsomes. Coumarin inhibited both reactions competitively. On the other hand, microsomal coumarin 7‐hydroxylase was inhibited by NDMA (Ki 2.7 mM) and NDEA (Ki 0.013 mM). The big difference in the Ki values suggests a higher affinity of NDEA than NDMA to Cyp2a‐5 (mouse cytochrome P450coh). A specific antibody against Cyp2a‐5 inhibited more of the microsomal NDEA (up to 90%) than NDMA (up to 40%) dealkylation. The converse was true with anti‐Cyp2e‐1 antibody. These results suggest that the primary substrate for Cyp2a‐5 is NDEA and for Cyp2e‐1, NDMA. Western blot analysis of human liver microsomes showed a great interindividual variation in the amounts of CYP2A6 (human cytochrome P450coh) and CYP2E1. Also, courmarin 7‐hydroxylation and nitrosamine dealkylation varied greatly among individuals. A high correlation (r = 0.93, P < 0.001) was found between NDEA and coumarin metabolism. Both activities were associated with CYP2A6. On the other hand, little or no correlation was found between microsomal CYP2A6 and CYP2E1 or between CYP2E1 and NDEA dealkylation. Immunoinhibition of human microsomal NDEA metabolism by CYP2a‐5 antibody varied greatly among individuals (10–90%), suggesting, as in the case of mice, that NDEA is metabolized primarily by CYP2A6, at least in some individuals. Taken together the data suggest that (1) the metabolic activation of nitrosamines in humans varies greatly among individuals; (2) different nitrosamines may partially be metabolized by different cytochrome P450 isozymes; and (3) because of similarities between nitrosamine metabolism in mice and humans, inbred strains of mice would be relevant experimental models for studying nitrosamine activation.
Nucleotide sequence analysis of c‐raf‐1 cDNA and promoter from a radiation‐resistant human squamous carcinoma cell line: Deletion within exon 17
Authors: Patel Bharvin K. R. , Kasid Usha
Published: February 2007

The c‐raf‐1 proto‐oncogene is the cellular homologue of v‐raf, the oncogene of the acutely transforming retrovirus 3611‐MSV. The product of c‐raf‐1 (raf‐1) is a 74‐kDa cytoplasmic serine/threonine protein kinase. We previously reported that antisense human c‐raf‐1 cDNA transfection results in reduction of the endogenous c‐raf‐1 transcript, decreased tumor growth rate, and enhanced radiation sensitivity of SQ‐20B tumor cells established from a radiation‐resistant laryngeal squamous cell carcinoma. In the study reported here, we used cDNA‐linked polymerase chain reaction amplification and nucleotide sequencing to examine the structure of the 3233‐bp SQ‐20B c‐raf‐1 cDNA. The 812‐bp c‐raf‐1 promoter region was analyzed by genomic DNA amplification followed by cloning and sequencing. Sequence comparison with a previously published c‐raf‐1 sequence indicated no structural changes within the coding region of SQ‐20B c‐raf‐1. However, a 4‐bp deletion was observed in the 3′ untranslated region within exon 17. This deletion was also present in a c‐raf‐1 cDNA clone isolated from a SQ‐20B cDNA library. While the possibility of a 3′ transcriptional control mechanism cannot be ruled out, it appears that the raf‐1 protein kinase may regulate the development of radioresistant malignancies via interaction with other molecules in the damage and repair—related signal transduction pathways. © 1993 Wiley‐Liss, Inc.
Timing of replication of differentially transcribed genes in syrian hamster embryo fibroblasts
Authors: Sorscher David H. , Barrett J. Carl , Cordeiro‐Stone Marila
Published: February 2007

Syrian hamster embryo cell lines have been used as models of neoplastic progression in vitro. Changes in phenotype and biological properties have been observed in these cell lines in association with modulation of gene transcription. We explored this natural evolution of cells in culture to investigate the reported relationship between transcriptional activity and replication in early S phase. In this study we used two nontumorigenic cell lines that had either retained (supB+) or lost (supB‐) the ability to suppress tumorigenicity of malignantly transformed hamster fibroblasts (BP6T). In association with the loss of suppressor gene function, supB‐ cells have downregulated the expression of the H19 and tropomyosin‐I (TM‐I) genes, which are actively transcribed in supB+ cells. Synchronous populations of supB+ and supB‐ cells were pulse‐labeled with [3H]thymidine and bromodeoxyuridine at 1‐h intervals during S phase; the replicating DNA was isolated by centrifugation in cesium chloride gradients and hybridized to 32P‐labeled gene probes. No correlation was found between the timing of gene replication and the status of expression of these two genes. TM‐I replicated during the first hour and H19 replicated between the second and third hours of the S phase in the expressing and nonexpressing cell lines. Immunoglobulin gene sequences, known to be late‐replicating in fibroblasts, replicated at the end of the S phase. These results suggest that downregulation of transcription is not always accompanied by a concomitant change in time of gene replication from early to late S phase. © 1993 Wiley‐Liss, Inc.
Preferential incorporation of 3′‐azido‐2′,3′‐dideoxythymidine into telomeric DNA and Z‐DNA—containing regions of chinese hamster ovary cells
Authors: Olivero Ofelia A. , Poirier Miriam C.
Published: February 2007

3′‐Azido‐2′,3′‐dideoxythymidine (azidothymidine; AZT) induces bone marrow toxicity in patients chronically given therapeutic doses of drug and is tumorigenic in rodents, inducing squamous cell tumors in vaginal tissues of mice and rats. In the study reported here, we explored the incorporation of AZT into specific regions of mammalian chromosomal DNA. CHO cells were exposed to AZT for 4 h, allowed to complete at least one cell cycle, and then arrested in metaphase with colchicine. Regions of concentrated AZT incorporation were identified in individual metaphase chromosomes by immunohistochemistry using antiserum specific for AZT and a secondary antiserum with a streptavidin—Texas red end point. These studies demonstrated that most of the intensely staining regions were chromosomal ends or telomeres. When 18 metaphases were examined, all telomeres but one (39 of 40) were positive at least once. Using an anti—Z‐DNA antibody, chromosomal regions containing DNA in Z conformation were also localized by immunohistochemistry using a rhodamine‐conjugated secondary antibody. When metaphase chromosome spreads were stained for either AZT or Z‐DNA, ideograms showing localization of AZT (18 metaphases) and DNA in Z configuration (26 metaphases) were drawn for every chromosome of each metaphase examined. These ideograms demonstrated that 60% of the regions that stained positive for AZT were also positive for Z‐DNA. Furthermore, slides incubated with both antibodies, using streptavidin—Texas red to identify AZT and fluorescein to identify Z‐DNA, confirmed colocalization of the two markers. Additional experiments exploring the induction of chromatin bridges in AZT‐treated cells suggest that the analogue may be able to bind to and disrupt the normal functioning of telomeric DNA.
Effect of retroviral integration on control of expression of a tumor marker in hela cells
Authors: Gross Monika M. , Janiaud Paul , Stanbridge Eric J.
Published: February 2007

Fusion between HeLa cells and normal human fibroblasts results in the suppression of tumorigenicity. Under prolonged culture conditions, rare tumorigenic segregants arise and have been shown to reexpress a cell surface antigen, p75, which is also present in the HeLa parent but not in the fibroblast parent or in the nontumorigenic HeLa x fibroblast hybrid. Expression of p75 strictly correlates with tumorigenicity in HeLa and human somatic cell hybrids, as has been shown by chromosomal segregation and after γ‐irradiation. Using insertional mutagenesis, we induced expression of p75 in the nontumorigenic hybrid. Three cell clones were isolated that expressed p75 at different levels. Two of these clones exhibited a high level of p75 expression and displayed an altered morphology similar to that of the previously characterized tumorigenic segregants and consistent with the appearance of tumorigenicity. When injected into athymic nude mice, two clones were found to be tumorigenic, one from the onset of subculturing and the second only after further propagation for approximately 50 population doublings. The third clone showed very low p75 expression, had no altered morphology, and was nontumorigenic.
Common target for 12‐O‐tetradecanoylphorbol‐13‐acetate and ras in the transcriptional enhancer of the growth factor—inducible JE gene
Authors: Koike Manabu , Kuroki Toshio , Nose Kiyoshi
Published: February 2007

JE gene expression in the mouse osteoblast cell line MC3T3‐E1 is activated transiently by the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). In ras‐transformed MC3T3‐E1 cells the JE gene is constitutively expressed at a high level, whereas in their raf‐transformed counterparts it is not constitutively expressed or inducible by TPA. Using these cells, we investigated a specific sequence recognized by nuclear factors in the 5′‐upstream region of the rat JE gene. By gel‐mobility shift assays, we determined that the amount of nuclear factors that bind to the region ‐ 130 to ‐ 96 bp upstream from the cap site of the rat JE gene (JE‐1 probe) increased after TPA treatment of MC3T3‐E1 cells. However, in the ras transformants it was elevated constitutively, and in the raf transformants it was not detectable. Both unlabeled JE‐1 probe and a probe containing a TPA‐responsive element (TGACTCA) competed with the binding of these nuclear factors. Preincubation of the nuclear extracts with fos‐ or jun‐specific antibodies interfered with the binding of the factors to the JE‐1 probe. The essential sequence in the JE‐1 element for the binding of nuclear factors was found to be TTACTCA. c‐fos and c‐jun proteins synthesized in vitro could bind to the DNA fragment containing this sequence, but the binding was weaker than to the TPA‐responsive element (TGACTCA). These results suggest that the sequence TTACTCA in the JE‐1 element is a common target of TPA and ras and that protein complexes containing fos‐ and jun‐related proteins recognize the sequence.
Retinoic acid receptor and retinoid X receptor expression in retinoic acid—resistant human tumor cell lines
Authors: van der Leede Bas‐Jan M. , van den Brink Christina E. , Van der Saag Paul T.
Published: February 2007

Retinoic acid (RA) has profound effects on cell proliferation and differentiation both in vitro and in vivo. Many human cell lines are known to be sensitive to the growth‐inhibitory action of RA. We analyzed established human solid tumor—derived cell lines for their RA sensitivity. Growth inhibition by RA in monolayer was examined by [3H]thymidine incorporation and cell proliferation. Here we report that 11 widely used human cell lines were RA resistant. The majority are carcinoma derived (A‐431, BT‐20, C‐41, ACHN, HCT116, 293, A549, and PA‐1); two are sarcoma derived (Saos‐2 and A673); and one is a melanoma cell line (A‐375). Since nuclear retinoid receptors are implicated in the biological effects of RA, we examined the expression of retinoic acid receptors (RARs) RARα, RARβ, RARγ, and the retinoid X receptors (RXRs) RXRα, RXRβ, and RXRγ in the RA‐resistant cell lines by northern blotting and by RNase protection analysis for RARβ. RARα transcripts were constitutively expressed in all cell lines. By contrast, RARβ was expressed in only seven RA‐resistant cell lines (Saos‐2, ACHN, 293, A549, A‐375, A673, and PA‐1), and its level was enhanced by RA in some cases. In most cell lines, RARγ expression was low and was not affected by RA. The RXR genes showed a very distinct expression pattern in the group of selected cell lines. In general, RXRα was the most abundantly expressed subtype, RXRβ was expressed at low levels, and RXRγ could not be detected. In none of the RA‐resistant cell lines was RXR expression modulated by RA. The results presented here indicate that the resistance of these human tumor cell lines to RA cannot be simply correlated with expression of RAR or RXR or both.
Toxicity of phorbol esters for human epithelial cells expressing a mutant ras Oncogene
Authors: Dawson Tim , Bond Jane , Eccles Neil , Wynford‐Thomas David
Published: February 2007

Phorbol esters and related compounds provide a promising source of potential anticancer agents. The mechanism of their toxicity, however, is unclear, and interpretation has been complicated by the conflicting responses exhibited by different transformed cell lines. Previously we showed that in primary thyroid follicular cells, expression of mutant p21ras conferred a striking sensitivity to the toxic effects of phorbol esters. We have now extended this work using a thyroid cell line with an inducible mutant ras gene to exclude the possibility that this result was a trivial consequence of the marked growth stimulation induced in these cells by mutant p21ras. Furthermore, by assessing the action of a panel of phorbol esters and a potential chemotherapeutic agent, bryostatin, we demonstrated that this phenomenon was only a function of biologically active phorbol esters. These results provide a molecular rationale for the development of phorbol ester analogues as chemotherapeutic agents.
Ras protein p21 processing enzyme farnesyltransferase in chemical carcinogen—induced murine skin tumors
Authors: Agarwal Rajesh , Khan Sikandar G. , Athar Mohammad , Zaidi Syed I. A. , Bickers David R. , Mukhtar Hasan
Published: February 2007

Farnesylation of ras protein p21 is crucial for the protein's membrane localization, which is essential for its cell‐transforming activity, which in turn is thought to be critical for the ultimate induction of cancer. The cytosolic enzyme farnesyltransferase plays a major role in posttranslational modification of p21, but the level of farnesyltransferase activity in mammalian tumors and its relationship to the processing of cytosolic p21 that leads to tumorigenesis are unknown. We report here that farnesyltransferase activity was significantly higher in chemical carcinogen—induced benign skin papillomas in SENCAR mice than in the epidermises of control animals. The enzyme is primarily epidermal in origin, and kinetic studies with cytosol from epidermis and papillomas showed that the reaction was linear with respect to time, substrate concentration, and protein content. Skin papillomas showed significantly elevated levels of both cytosolic and membrane‐bound Ha‐ras p21, whereas far lesser cytosolic and almost negligible amounts of membrane‐bound p21 were present in the epidermis of control mice. There was a positive correlation between increased enzyme activity in papilloma cytosol and the processing of overexpressed cytosolic Ha‐ras p21 for its localization to membrane.
Correlation of p53 mutations with epidermal growth factor receptor overexpression and absence of mdm2 amplification in human esophageal carcinomas
Authors: Esteve Asuncion , Montesano Ruggero , Hollstein Monica , Lehman Theresa , Harris Curtis C. , Jiang Wei , Weinstein I. Bernard , Ruol Alberto , Peracchia Alberto
Published: February 2007

Esophageal carcinomas from 24 patients, most of whom were smokers and consumed alcoholic beverages daily, were analyzed for mutations in exons 5–8 of the p53 tumor suppressor gene. Mutations were identified by polymerase chain reaction amplification and direct sequencing in 12 of 24 (50%) of the samples; almost half of the mutations were at A:T base pairs. Nuclear accumulation of p53 protein, determined by immunohisto‐chemistry with the CM‐1 polyclonal antibody, was observed in all cases in which a missense mutation in the p53 gene was detected. None of the 24 carcinomas had amplification of the mdm2 gene, an alternate pathway to p53 loss of function. Alterations involving three other cancer‐related genes associated with human esophageal carcinogenesis, c‐erbB‐1/epidermal growth factor receptor (EGFR), c‐myc, and retinoblastoma (Rb), were examined by Southern blot or immunohistochemical analysis in the same sample set to explore the possibility of a link between oncogene activation and loss of tumor suppressor function. While no associations were observed between amplification of the c‐myc or EGFR genes and p53 abnormalities, a significant correlation (P < 0.01) was seen between the presence of p53 mutation and EGFR overexpression. Absence of Rb protein, measured immunohistochemically, was observed in four tumors, none of which had aberrations of the p53 gene.
Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2‐acetylaminofluorene–induced hepatocarcinogenesis
Authors: Ringer David P. , Howell Boyd A. , Norton Tom R. , Woulfe Gary W. , Duffel Michael W. , Kosanke Stanley D.
Published: February 2007

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2‐ acetylaminofluorene (2AAF)–induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N‐hydroxy‐2AAF sulfotransferase activity was found to decrease 80–90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF‐mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF‐induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action. © 1994 Wiley‐Liss, Inc.
Increased expression of stromelysin‐3 in basal cell carcinomas
Authors: Majmudar Gopa , Nelson Bruce R. , Jensen Timothy C. , Voorhees John J. , Johnson Timothy M.
Published: February 2007

We examined the expression of two groups of matrix metalloproteinases (MMPs), stromelysin and interstitial collagenase, in human skin cancer by northern blot analysis and in situ hybridization. Stromelyins‐3 (ST‐3) mRNA was overexpressed more than tenfold in 17 of 19 (89%) specimens of basal cell carcinoma (BCC) but in only three of 13 (23%) cutaneous squamous cell carcinomas (SCCs). Stromelysin‐1 and ‐2 (ST‐1/2) mRNA was overexpressed in three of 19 (16%) BCC and three of 13 (23%) SCC. Collagenase mRNA was overexpressed in nine of 19 (47%) BCC and three of 13 (23%) SCC. No mRNA for ST‐3, ST‐1/2, or collagenase was detected by northern analysis in 21 specimens of adjacent normal skin. Because of these findings, we examined the specific location of the ST‐3 mRNA in BCC specimens by in situ hybridization. ST‐3 mRNA was particular abundant in the characteristic stroma adjacent to the invasive basaloid tumor islands of the BCC and absent in the malignant cells. Moreover, ST‐3 mRNA was expressed and induced by phorbol ester treatment in adult dermal fibroblasts but not in keratinocytes. In vitro studies have shown that MMPs are involved in the degradation of extracellular matrix molecules. Our finding of ST‐3 mRNA overexpression in 17 of 19 (89%) BCC specimens is consistent with a role for this molecule in local invasion of stroma by BCC. Our in situ hybridization data suggested that while ST‐3 is not expressed by malignant basal cells themselves, these tumor cells may induce the expression of ST‐3 in adjacent nonmalignant stromal elements such as fibroblasts. © 1994 Wiley‐Liss, Inc.
G→A mutations in p53 and Ha‐ras genes in esophageal papillomas induced by N‐nitrosomethylbenzylamine in two strains of rats
Authors: Lozano Jean‐Claude , Nakazawa Hisayoshi , Cros Marie‐Pierre , Carbral Ricardo , Yamasaki Hiroshi
Published: February 2007

In human esophageal cancers, no ras gene mutations but a relatively high prevalence of p53 gene mutations have been reported. We found a high prevalence of point mutations in Ha‐ras and p53 genes in N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal tumors in two strains of rats (BD VI and F344). Our analysis showed the point mutation GGA←GAA (expected from the known mechanisms of action of NMBA) at Ha‐ras codon 12 in 22 of 46 (48%) and 22 of 38 (58%) papillomas from BD VI and F344 rats, respectively. There was no significant difference in the prevalence of ras mutations in tumors induced by high doses (5.0 mg/kg) and low doses (2.5 mg/kg) of NMBA. Eleven papillomas from each strain were analyzed for p53 mutations. The prevalent mutations found were G←A and C←T transitions. The frequency of p53 mutation was 36% (four of 11) for each strain. No apparent hot‐spot codon or exon was found in the p53 gene, and two papillomas contained double mutations in this gene. The high prevalence of G←A mutations in the rat Ha‐ras gene contrasts with that in the human gene, in which no ras mutations have been found in primary tumors, and suggests either that the biology of esophageal carcinogenesis differs in humans and rats or that nitrosamines are not the major etiological risk factor for human esophageal cancers. © 1994 Wiley‐Liss, Inc.
Mutation and altered expression of p53 genes in experimental rat bladder tumor cells
Authors: Jones Richard F. , Matuszyk Janusz , Debiec‐Rychter Maria , Wang Ching Y.
Published: February 2007

p53 genes were analyzed for mutations and expression in a series of 24 tumors or hyperplastic lesions of the urinary bladder induced in F344 rats by carcinogen treatment. Of these, 18 were analyzed as short‐term urothelial cultures. Polymerase chain reaction–single‐strand conformation polymorphism analysis and DNA sequencing were used to detect alterations in p53 genes or cDNAs, and the relative amounts of p53 protein per cell were estimated by immunohistochemical staining. Missense substitutions were found in the exon 5–9 region of two of five cell cultures analyzed from lesions induced by the bladder carcinogen N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine. One of these was a papillary nodular hyperplasia, indicating that p53 mutations can be present in low‐ as well as high‐stage/grade bladder lesions. p53 mutations were not found in the exon 5–9 region in cells of any of eight bladder lesions induced by N‐[4‐(5‐nitro‐2‐furyl)‐2‐thiazolyl]formamide (FANFT), including five transitional cell carcinomas (TCCs), or either of two TCCs induced by N‐methylnitrosourea. Two of nine TCCs induced by the N‐glucuronide of N‐hydroxy‐2‐aminofluorene were found to have p53 mutations. One of these was evidently altered by three genetic events: a missense substitution in exon 8, a nonsense mutation in exon 6, and silencing of the “nonsense” allele (i.e., only the p53 missense mutation was detected). Immunohistochemical analysis with monoclonal antibody PAb240 (which preferentially binds to mutant p53 protein) detected p53 antigen only in those samples in which missense p53 mutations were found. With monoclonal antibody PAb421 (which detects mutant and wild‐type p53), p53 antigen was also detected in cells from F542, a bladder tumor induced by FANFT in which no p53 mutations were found. Northern blot hybridization analysis showed that p53 transcripts were elevated twofold to threefold in several cases, including F542, suggesting that constitutive overexpression of wild‐type p53 may occur in some bladder neoplasias. These data support the view that p53 may be involved in multiple rate‐limiting steps in neoplastic transformation and may be a continuing target during bladder carcinogenesis. The data also contribute to evidence that certain chemical carcinogens may directly alter p53 genes during tumorigenesis. © 1994 Wiley‐Liss, Inc.
Expression in lung tumors and genetic mapping of the novel murine protein kinase Cη
Authors: Canzian Federico , Gariboldi Manuela , Manenti Giacomo , De Gregorio Laura , Osada Shin‐Ichi , Ohno Shigeo , Dragani Tommaso A. , Pierotti Marco A.
Published: February 2007

Protein kinase Cη (nPKCη) is a member of the protein kinase C family with a unique tissue distribution in skin and lung. We analyzed nPKCη expression in normal murine lung and in 36 lung tumors induced by urethane in AC3 F1 and F2 mice. The nPKCη‐related transcript was present at fivefold to tenfold lower levels in tumors than in normal lung controls. We mapped two distinct loci for nPKCη on murine chromosome 12, using linkage analysis in an interspecific test cross. The results indicate that the mouse Pkcn‐rs1 and Pkcn‐rs2 loci were about 30 cM from the centromere and about 2 cM from each other. © 1994 Wiley‐Liss, Inc.