bluuebunny/crossref_metadata_2025 huggingface dataset:
Mediation of suppression of c‐fos transcription in rasT24‐transformed rat cells by a cis‐acting represser element
Authors: Osei‐Frimpong Joseph , Sepulveda Jorge , Rangdaeng Samreung , Lebovitz Russell M.
Published: February 2007

Prolonged expression of activated ras mutants resulted in both neoplastic transformation and suppression of serum‐induced c‐fos expression in Rat1 fibroblasts. Expression of other serum‐inducible genes, including c‐jun and β‐actin, was not suppressed in ras‐transformed Rat1 cells, indicating that these effects are specific for c‐fos and that growth‐factor signal transduction pathways remain essentially intact. Run‐on transcription studies indicated that c‐fos transcription was blocked at the level of initiation in these cells. Transient transfection studies using 360 bp from the wild‐type c‐fos promoter as well as a series of mutated c‐fos promoter fragments linked to the chloramphenicol acetyltransferase gene indicated that repression of c‐fos was mediated by approximately 49 bp immediately upstream of the dyad symmetry element (DSE). Deletion of this region, referred to as the upstream represser region (URR), restored serum inducibility to the c‐fos promoter in ras‐transformed cells. In contrast, suppression of c‐fos transcription was not affected by either deletion of 240 bp between the DSE and the TATA element or by base‐substitution mutations that inactivate the ternary complex factor and fos‐AP‐1‐like binding sites. In addition, in vitro competition studies indicated that ras‐transformed cells express one or more represser factors that interact with as‐yet‐unidentified elements within the c‐fos promoter (possibly the URR) and block serum induction of c‐fos. These findings suggest that prolonged expression of activated ras results in the activation of one or more as‐yet‐unidentified proteins that suppress transcription of the c‐fos gene by interacting with the URR. © 1994 Wiley‐Liss, Inc.
Increased actin cable organization after single chromosome introduction: Association with suppression of in vitro cell growth rather than tumorigenic suppression
Authors: Miyamoto Shingo , Nishida Makoto , Miwa Katsuhiro , Kato Hidenori , Imamura Toshiro , Barrett J. Carl , Shimizu Motoyuki , Oshimura Mitsuo , Wake Norio
Published: February 2007

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms. © 1994 Wiley‐Liss, Inc.
Only wild‐type c‐ki‐ras codons 12, 13, and 61 in human pancreatic acinar cell carcinomas
Authors: Terhune Pamela Guay , Heffess Clara S. , Longnecker Daniel S.
Published: February 2007

Activation of the c‐Ki‐ras proto‐oncogene is more common in carcinomas of the pancreas than in other human carcinomas. Most such carcinomas are of the ductal cell phenotype. Ductal adenocarcinomas of the hamster pancreas have similar mutations, but acinar cell carcinomas of the mouse and rat pancreas lack the common c‐Ki‐ras mutations. Therefore, we examined 11 acinar cell carcinomas of the human pancreas for evidence of mutations at codons 12, 13, and 61. DNA was isolated from tumor cells in paraffin‐embedded sections. The polymerase chain reaction was used to amplify the appropriate DNA sequence, and then allele‐specific oligonucleotide hybridization and DNA sequence analysis were used to evaluate c‐Ki‐ras structure. Only wild‐type sequences were found in codons 12, 13, and 61. Thus, in both the human and rodent species, mutations of c‐Ki‐ras appear to be more important in the genesis of ductal carcinomas than in the genesis of acinar cell carcinomas of the pancreas. © 1994 Wiley‐Liss, Inc.
Altered expression of the epidermal growth factor receptor and transforming growth factor‐α during multistage skin carcinogenesis in SENCAR mice
Authors: Rho Okkyung , Beltrán Linda M. , Gimenez‐Conti Irma B. , Digiovanni John
Published: February 2007

In the study presented here, we examined the possible role of the transforming growth factor‐α (TGFα)/epidermal growth factor receptor (EGFR) system during multistage carcinogenesis in mouse skin. In this regard, the expression (mRNA and protein) of both TGFα and EGFR was examined in primary papillomas and squamous cell carcinomas (SCCs) obtained from SENCAR mice treated with standard initiation‐promotion regimens and compared with the levels of expression in normal epidermis. The level of a 4.8‐kb TGFα transcript was elevated in 100% of the skin tumors examined (both papillomas and SCCs), including papillomas obtained 13 wk after the start of promotion, compared with normal epidermis. Immunohistochemical analyses detected elevated levels of TGFα protein in these skin tumors and in papillomas as early as 10 wk after the start of promotion. The levels of EGFR transcripts were also significantly elevated in most (90%) of the skin tumors examined, including again those harvested after 13 wk of promotion. Interestingly, multiple EGFR transcripts (10.5, 5.8, 2.8, and 1.8 kb) were detected in both papillomas and SCCs. The two smaller transcripts appeared to encode truncated versions of the EGFR, and the 1.8‐kb transcript appeared to be unique to RNA samples isolated from skin tumors, based on comparative analyses of several normal tissues. As with TGFα, immunohistochemical analyses detected elevated levels of EGFR protein in these skin tumors (both papillomas and SCCs), including papillomas harvested as early as 10 wk after the start of promotion. Southern analyses of genomic DNAs for TGFα and EGFR failed to detect any cases of gene rearrangements or amplification as a possible explanation for the elevated levels of the transcripts of these two genes. These results support the hypothesis that a key step in the development of autonomous growth in mouse skin papillomas generated in SENCAR mice by an initiation‐promotion regimen may involve alterations in the synthesis of TGFα and its cognate receptor. ©1994 Wiley‐Liss, Inc.
bcl‐2 delay of alkylating agent‐induced apoptotic death in a murine hemopoietic stem cell line
Authors: Fairbairn Leslie J. , Cowling Graham J. , Dexter T. Michael , Rafferty Joseph A. , Margison Geoffrey P. , Reipert Birgit
Published: February 2007

Many cytotoxic agents kill cells by invoking a specific death pathway termed physiological cell death, or apoptosis. Treatment of a murine hemopoietic stem cell line, FDCP‐mix, with methylmethanesulfonate (MMS) orN′‐methyl‐N′‐nitrosourea (MNU) leads to death by apoptosis. Retroviral gene transfer was used to overexpress thebcl‐2 oncogene in FDCP‐mix cells, and this was associated with a delay in apoptosis in these cells after treatment with MNU and MMS and decreased sensitivity of colony formation to the cytotoxic effects of MMS. These data suggest an explanation for the refractory nature ofbcl‐2—expressing follicular lymphoma to cytotoxic chemotherapy and furthermore suggest that DNA‐damaging antitumor therapy may contribute to the progression of disease. ©1994 Wiley‐Liss, Inc.
Induction of specific cytochrome P450s involved in aflatoxin B1 metabolism in hepatitis B virus transgenic mice
Authors: Kirby Gordon M. , Chemin Isabelle , Montesano Ruggero , Chisari Francis V. , Lang Matti A. , Wild Christopher P.
Published: February 2007

The relative roles of hepatitis B virus (HBV) and aflatoxn and their possible mechanism of interaction in the etiopathogenesis of hepatocellular carcinoma (HCC) are not understood. One hypothesis is that viral infection and associated liver injury alter expression of carcinogen‐metabolizing enzymes. We tested this hypothesis in an HBV‐transgenic mouse model in which a synergistic interaction occurs between aflatoxin B1 (AFB1) and HBV in the induction of HCC (Sell et al., Cancer Res 51:1278–1285, 1991). In this transgenic mouse lineage, overproduction of the HBV large envelope protein results in progressive liver cell injury, inflammation, and regenerative hyperplasia. Initially, two cytochrome P450s of importance in AFB1 metabolism in the mice were identified, namely Cyp2a‐5 and Cyp3a, using specific antibodies and chemical inhibitors. The expression of these P450 isoenzymes and an α‐class glutathione S‐transferase (GST) isoenzyme, YaYa, were examined. Increased expression and altered distribution of Cyp2a‐5 were demonstrated, by immunohistochemical analysis, to be associated with the development of liver injury in mice and to increase with age between 1 and 12 months. Cyp3a expression was also increased in HBV‐transgenic mice, but the increase was not as clearly related to age. GST YaYa levels were the same in HBV‐transgenic mice and their nontransgenic littermates of all ages. These results show that expression of specific cytochrome P450s is altered in association with overexpression of HBV large envelope protein and liver injury in this model. This may have general relevance to human HCC, the etiology of which is associated with a diverse range of liver‐damaging agents. ©1994 Wiley‐Liss, Inc.
Association of liver fluke (Opisthorchis viverrini) infestation with increased expression of cytochrome p450 and carcinogen metabolism in male hamster liver1,2
Authors: Kirby Gordon M. , Pelkonen Päivi , Vatanasapt Vanchai , Camus Anne‐Marie , Wild Christopher P. , Lang Matti A.
Published: February 2007

Synergy between exposure to chemical carcinogens (nitrosamines) and infestation with the liver fluke Opisthorchis viverrini has been demonstrated in a hamster model of hepatocarcinogenesis (Flavell et al., Carcinogenesis 4:927–930, 1983; Thamavit et al., Carcinogenesis 8:1351–1353, 1987). to elucidate the mechanisms of this interaction we tested the hypothesis that liver parasitism might influence the expression and activity of carcinogen metabolizing enzymes. We found that one, and perhaps more, hamster liver cytochrome P450 (CYP) isozymes immunorelated to mouse CYP2A5 contributed up to 50 or 60% of the hepatic aflatoxin B1(AFB) and N‐nitrosodiethylamine (NDEA) metabolism, respectively. As inferred from average enzyme activities and from western blot, immunoinhibition, and substrate (coumarin) inhibition analyses, O. viverrini infestation increased the expression of enzymes detectable by anti‐CYP2A5 antibody as well as NDEA metabolism in male but not in female hamsters. Immunohistochemical analysis of CYP2A expression by anti‐mouse CYP2A5 antibody demonstrated that the O. viverrini‐associated increase was not uniformly distributed throughout the lever but occurred in hepatocytes immediately adjacent to areas of inflammation. Immunohistochemical analysis of AFB‐DNA adducts in the livers of O. viverrini‐infested hamsters treated with AFB showed that the highest levels of adducts were found in the regions of liver where hepatocellular expression of enzymes detectable by anti‐CYP2A5 antibody is induced. These results suggest that a high local expression of CYP isozymes in O. viverrini‐infested livers could be a contributing risk factor in the development of liver cancers associated with paraitic hepatitis. ©1994 Wiley‐Liss, Inc.
Induction of different genetic changes by different classes of chemical carcinogens during progression of mouse skin tumors
Authors: Bremner Rod , Kemp Christopher J. , Balmain Allan
Published: February 2007

By analysis of skin tumors from F1 hybrid mice we demonstrated that the genetic events that occur during tumor progression depend on the type of chemical carcinogenesis protocol used to induce tumor growth. More than 95% of tumors induced by initiation with 7, 12‐dimethylbenz[a]anthracene (DMBA) and promotion with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) exhibited mutations in Ha‐ras and trisomy of chromosome 7. Carcinomas induced with multiple DMBA treatments had a lower frequency of alterations on chromosome 7 (50%), but only in tumors with Ha‐ras mutations, and had a much wider spectrum of alterations, including trisomy, mitotic recombination, deletion, and gene duplication. Carcinomas induced with multiple N‐methyl‐N‐nitro‐N‐nitrosoguanidine treatments only rarely exhibited alterations on chromosome 7 (8%), even if they contained mutant Ha‐ras. More frequent numerical alterations of chromosome 11 were also seen in TPA‐promoted tumors (23%) than in tumors induced by multiple carcinogen treatments (8%). These results show that postinitiation events are nonrandom and fit a model in which promoting agents induce numerical chromosomal alterations but in which mutagens cause more directed mutational events. ©1994 Wiley‐Liss, Inc.
A connexin 43 antisense vector reduces the ability of normal cells to inhibit the foci formation of transformed cells
Authors: Goldberg Gary S. , Martyn Kendra Dean , Lau Alan F.
Published: February 2007

Antisense gene constructs have been very useful in the functional analysis of genes and their products. In this report we used a connexin 43 (Cx43) antisense gene construct to study the role that heterologous gap‐junctional intracellular communication (GJIC) plays in the ability of untransformed fibroblasts to suppress the foci‐forming ability of src oncogene‐transformed cells. Untransformed Rat‐1 fibroblasts transfected with the Cx43 antisenese DNA construct showed marked decreases in Cx43 RNA and protein, which were accompanied by a corresponding decrease in GJIC. These Cx43 antisense‐transfected cells maintained normal cell morphology, growth rates, and saturation densities and did not grow in soft‐agar suspension. However, in coculture experiments, the Cx43 antisense cells were less effective than vector‐alone‐transfected, sense‐trans‐fected, and untransfected cells at in hibiting foci formation of pp60v‐src‐transformed cells. These effects of junctionally competent, normal cells were associated with the existence of heterologous GJIC with the transformed cells and did not appear to result from the elaboration of a stable, diffusible inhibitory factor. Thus, gap‐junction‐mediated transfer of putative regulatory molecules may play a role in the ability of untransformed cells to suppress the expression of certain properties of transformed cells. ©1994 Wiley‐Liss, Inc.
Analysis of transcriptional activation of a cyclic AMP response element by 2,6,10,14‐tetramethylpentadecane (pristane) in JB6 mouse epidermal cells
Authors: Banbura Marcin , Ackland‐Berglund Cathleen , Lee Soong‐Ho , Hamernik Deborah , Jones Clinton
Published: February 2007

Pristane is a naturally occurring isoprenoid that is believed to be derived from the phytyl moity of chlorophyll. Thus, it is not surprising that pristane is present in many common fruits and vegetables. Furthermore, pristane can be detected in the tissues of fish and mammals. In animal models using rodents, pristane canfunction as a potent tumour promoter. At the molecular level, pristane can induce changes in the plasma membrane, alter the conformation of chromatin, and selectively activate gene expression. Addition of pristane to a mouse epidermal cell line (JB6 P+) allows these cells to grow in an anchorage‐independent manner. In contrast, JB6 P‐cells are not transformed by pristane. Our study was undertaken to correlate transformation of P+ cells with changes induced by pristane. Transcriptional activation of a cyclic AMP response element (CRE) was in duced by pristane in P+ and P‐cells. Point mutations in the CRE abolished activation by pristane, thus indicating that an intact CRE was necessary for pristane activation. In P+ cells, pristane repressed phosphodiesterase activity. However, protein kinase A was activated by pristance in P+ and P‐cells. Taken together, these results indicated pristane induced novel changes in P+cells that in turn may facilitate neoplastic transformation.©1994 Wiley‐Liss, Inc.
Cytogenetic analysis of malignant skin tumors induced in chemically treated TG.AC transgenic mice
Authors: French John E. , Libbus Bisharah L. , Hansen Laura , Spalding Judson , Tice Raymond R. , Mahler Joel , Tennant Raymond W.
Published: February 2007

TG.AC mice (which carry a v‐Ha‐ras transgene) rapidly develp papillomas in response to 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Approximately 30% of the papilomas are associated with subsequent development of maliganancies. Early‐passage spindle‐shaped tumor cells arising from explant cultures of TPA‐induced tumors in TG‐AC mice were tumorigenic when transplanted to syngeneic recipients. The v‐Ha‐ras transgene in the transplanted tumors was expressed at a high level. To identify possible genetic changes associated with the development of malignant tumors, explanted cells were cultured in vitro and assessed for karyotypic changes between the second and third passages by analyzing G‐banded metaphase chromosomes. For comparison, skin malignancies were induced in nontransgenic FVB/N mice (parent strain) by 7, 12‐dimethylbenz[a] anthracene (DMBA) Initiation and TPA promotion, and their G‐banded metaphase chromosomes were analyzed. Trisomy (in at least 50% of about 30 metaphases) of chromosome 15 (in five of 15 tumors) and chromosome 6 (four of 15 tumors) was observed in TG.AC mice, independent of chemical treatment or tumor type. Of six tumors from DMBA/TPA‐treated FVB/N mice, three had trisomy 10 or 15 (or both), and two appeared normal. The absence of trisomy 7 is notable because c‐Ha‐ras maps to that chromosome. The absence of trisomy 7 in the six FVB/N DMBA/TPA‐induced skin malignancies contrasts with DMBA/TPA‐ induced karyotypic effects in SENCAR mice. Expression of the v‐Ha‐ras transgene may have precluded the requirement for endogenous mutant ras and allelic imbalance involving chromosome 7 in TG.AC mice, but it could not have in FVB/N mice. These results suggest the possibility that the observed trissmies are consequential, rather than causal, events in the development of TG.AC or FVB/N skin malignancies. Molecular genetic analysis will be required to understand the changes associated with tumorigenesis in this transgenic line as well as in the parent mouse line. ©1994 Wiley‐Liss, Inc.
Loss of heterozygosity at chromosome 1q loci in rat mammary tumors
Authors: Gollahon Lauren S. , Chen Aaron , Aldaz C. Marcelo
Published: February 2007

To better characterize abnormalities affecting rat chromosome 1 during mammary carcinogenesis, tumors were induced by nitrosomethylurea in F1 hybrid rats polymorphic at multiple chromosome 1 loci. By means of restriction fragment length polymorphism and microsatellite length polymorphism analyses, we observed loss of heterozygosity or allelic imbalance affecting various loci on the q arm of chromosome 1 in a high percentage of the 49 tumors analyzed. Fifty percent of the tumors showed loss or imbalance affecting the most distal (1q55) INS1 (rat insulin 1 gene) locus. The MT1PA (metallothionein‐1 pseudogene a) locus was observed to be affected in 58% of tumors induced in BUF/NCr × ACI/Vsp rats. Most of the losses appeared to have occurred by mitotic recombination. No parental bias was observed on the affected chromosome 1. Tumors were also screened for mutations in codon 12 of the Ha‐ras‐1 gene, which is located on 1q. We observed an association between the presence of mutation and allelic imbalance. These studies confirm our previous cytogenetic observations of a high level of nonrandom instability affecting rat chromosome 1 during mammary carcinogenesis. The observed loss of heterozygosity may indicate the existence of a putative tumor suppressor gene within the distal half of the 1q arm. These abnormalities, however, could also be related to the early stages of Ha‐ras amplification. © 1995 Wiley‐Liss Inc.
Expression of epidermal ornithine decarboxylase and nuclear proto‐oncogenes in phorbol ester tumor promotion‐sensitive and ‐resistant mice
Authors: Kennard Merle D. , Kang Dong‐Chul , Montgomery Raechelle L. , Butler Andrew P.
Published: February 2007

This study was undertaken to assess the effects of a single or two sequential topical applications of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on the expression of c‐fos, c‐jun, junB, c‐myc, and ornithine decarboxylase (ODC) in promotion‐sensitive SSIN mice and the relatively promotion‐resistant C57BL/6 strain. Northern blot analysis demonstrated that a single promoting dose of TPA induced ODC mRNA expression 10‐to 15‐fold in both strains. Treatment of each strain with a second dose of TPA, 48 h (in C57BL/6 mice) or 72 h (in SSIN mice) after the first, led to hyperinduction of ODC activity. Although this involved transcription of new ODC mRNA, the hyperinduction of ODC enzyme activity was primarily posttranscriptional. Induction of c‐fos mRNA or protein was maximal about 3 h after a single treatment in either strain but was sustained for at least 6 h in C57BL/6 mice. In contrast, two treatments of SSIN mice with TPA caused a rapid, strong c‐fos induction 1–2 h after treatment, whereas C57BL/6 mice responded no more strongly than after a single treatment. c‐jun mRNA and protein were induced only slightly in either strain, but junB was induced about fivefold in SSIN mice and tenfold in C57BL/6 mice. Although c‐myc was induced to comparable levels in both strains, the reponse was more prolonged in C57BL/6 mice. Compared with SSIN mice, C57BL/6 mice responded to TPA treatment, in general, with changes in proto‐oncogene mRNA to a higher level or for longer or both. Thus, although small differences in the expression of these genes were observed, they were not positively correlated with the differential sensitivity of SSIN and C57BL/6 mice toward tumor promotion by phorbol esters, with the possible exception of c‐fos. © 1995 Wiley‐Liss Inc.
Hepatocarcinogenesis in p53‐deficient mice
Authors: Kemp Christopher J.
Published: February 2007

To determine whether a constitutive p53 deficiency would enhance the rate of development of chemically induced hepatocellular carcinoma, we treated groups of wild‐type, p53‐heterozygous (+/‐), and null (‐/‐) male mice with a single dose of diethylnitrosamine at 12 d of age. Although the null mice had to be killed very early, at 15 wk of age because of the development of nonliver tumors, hemangosarcoma of the liver had already developed in two of seven mice. More detailed analysis of the wild‐type and heterozygous mice showed no difference in the number, size, or growth rate of early microscopic lesions or in the number or apparent malignancy of hepatocellular adenomas or carcinomas at later time points. Thus, germline p53 deficiency does not enhance the rate of development of diethylnitrosamine‐induced hepatocellular adenoma or carcinoma but may instead favor development of hepatic hemangiosarcoma. © 1995 Wiley‐Liss Inc.
Expression of phospholipases γ1, β1, and δ1 in primary human colon carcinomas and colon carcinoma cell lines
Authors: Nomoto Koji , Tomita Naohiro , Miyake Masami , Xhu Ding‐Bang , Logerfo Paul R. , Weinstein I. Bernard
Published: February 2007

The levels of expression of phosphoinositide‐specific phospholipase Cs (PLCs) were examined in a series of primary human colon carcinomas and in eight colon carcinoma cell lines by using monoclonal antibodies and cDNA probes for PLCγ1, PLCβ1, and PLCδ1. Western and northern blot analyses of PLCγ1 revealed elevated expression of this isozyme at both the protein and mRNA levels in most tumors when compared with paired adjacent normal mucosa samples (in 11 of 13 pairs in the western blots and 8 of 9 pairs in the northern blots). On the other hand, decreased levels of the PLCδ1 protein were seen in most colon carcinomas (12 of 13 paired samples). The levels of PLCβ1 protein were too low to detect possible differences between the carcinoma and normal mucosa samples. Relatively high expression of PLCγ1 was found in almost all of the eight human colon carcinoma cell lines at both the protein and mRNA levels. Only weak expression of PLCβ1 was detected in these cell lines, by both western and northern blot analyses, and PLCδ1 protein was not detected in any of the carcinoma cell lines. These findings provide evidence that colon carcinomas display altered expression of individual isoforms of PLCs and suggest that increased expression of PLCγ1 may play an important role in colon carcinogenesis. © 1995 Wiley‐Liss Inc.
Inhibition of ras p21 membrane localization and modulation of protein kinase C isozyme expression during regression of chemical carcinogen–induced murine skin tumors by lovastatin
Authors: Khan Sikandar G. , Saxena Ramesh , Bickers David R. , Mukhtar Hasan , Agarwal Rajesh
Published: February 2007

We investigated the ras p21 membrane localization and the expression and activation of protein kinase C (PKC) isozymes in activated ras oncogene–containing tumors and assessed whether these events were related to tumors growth. We used 7,12‐dimethylbenz[a]anthracene–initiated and 12‐O‐tetradecanoylphorbol‐13‐acetate–promoted SENCAR mouse skin tumors, which were shown to contain Ha‐ras oncogene activated by point mutation at codon 61, as an in vivo model for these studies. Compared with levels in epidermis, highly elevated levels of membrane‐bound Ha‐ras p21 were observed in growing tumors, which also showed strong expression and membrane translocation of PKC ζ and βII and weak expression of PCK α. However, when ras p21 membrane localization was blocked in vivo in growing tumors by lovastatin, opposite results were evident. Compared with saline‐treated animals, in which tumor growth continued, lovastatin‐treated animals had significantly inhibited tumor growth, which led to tumor regression with concomitant inhibition of Ha‐ras p21 membrane localization. These regressing tumors from lovastatin‐treated animals also showed a decrease in the expression and membrane translocation of PKC ζ and βII but increased expression of PKC α. Taken together, our results indicate that ras p21 membrane localization and the expression and activation of PKC ζ, βII, and α may be the critical events in the regulation of the growth of tumors that contain activated ras oncogenes. © 1995 Wiley‐Liss Inc.
Novel use of a selectable fusion gene as an “In‐Out” marker for studying genetic loss in mammalian cells
Authors: Trott Deborah A. , Cuthbert Andrew P. , Todd Christopher M. , Themis Michael , Newbold Robert F.
Published: February 2007

Recent demonstrations of loss of heterozygosity in a wide variety of human cancers suggest that large multilocus genetic deletions (presumably including tumor suppressor genes) constitute a major class of genetic alteration in human carcinogenesis. Here we show that a bifunctional fusion gene (Hytk), suitable for both positive and negative selection, is an effective marker for studying genetic loss in mammalian cells with minimal interference from point‐mutational changes. Studies with a transgenic V79 cell line in which a single functional copy of Hytk was stably inserted into the genome in a retroviral vector showed that loss of the marker (and presumably flanking cellular genetic material) could be induced efficiently by ionizing radiation (γ‐rays and fast neutrons) but only weakly by the powerful point‐mutagen benzo[a]pyrene diol epoxide. In a first application of the system, we provide evidence that radiation‐induced loss can occur through an indirect mechanism after a high‐frequency event. Collectively, our results suggest that the Hytk marker should be a valuable tool for studying genome position effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progression. © 1995 Wiley‐Liss Inc.
Microsatellite alterations in human and rat esophageal tumors at selective loci
Authors: Mironov Nikolai M. , Aguelon Anne‐Marie , Hollams Elysia , Lozano Jean‐Claude , Yamasaki Hiroshi
Published: February 2007

(CA)n simple repeats in DNA were examined at 17 loci in 18 human squamous cell carcinomas of the esophagus and compared with those in normal esophageal tissue from the same patients. Six loci were examined in 32 esophageal papillomas that had been induced by N‐nitrosomethylbenzylamine in BD VI rats. Length‐altered CA repeats were found in two human tumors and four rat papillomas. Loss of heterozygosity was observed in three human tumors; two rat papillomas had lost microsatellite bands that are common in inbred BD VI rats. Both (CA)n microsatellite length alteration and loss of heterozygosity were clustered at certain loci in the human tumor samples and in the chemically induced rat esophageal tumors. Our findings indicate that genomic instability that results in alteration of repeated sequences not only occurs in human tumors but may also be a consequence of chemical carcinogenesis in rodents. © 1995 Wiley‐Liss, Inc.
Induced terminal differentiation and tumorigenic suppression in murine keratinocyte somatic‐cell hybrids
Authors: Schneider Brandt L. , Kulesz‐Martin Molly , Bowden G. Tim
Published: February 2007

The development of malignancy has been associated with both the activation of oncogenes and the inactivation of tumor suppressor genes. Whereas recent data implicate tumor suppressor genes as cell‐cycle checkpoints, the nature and timing of tumor suppressor gene inactivation during multistage carcinogenesis is still largely uncharacterized. To address this issue, we used a syngeneic mouse epidermal model system. By creating somatic‐cell hybrids between nontumorigenic × benign (291 × 291.09RAT), nontumorigenic × malignant (291 × 291.05RAT and 291 × 291.03RAT), benign × malignant (291.09RAT × 291.03RAT) and malignant × malignant (291.03RAT × 291.05RAT) clones, multiple tumor suppressor activities were detected. Most importantly, we demonstrated the first example of the complete suppression of benign papillomas in vivo, thus implicating tumor suppressor gene activity loss as an early event in skin carcinogenesis. In addition, the carcinoma phenotype was suppressed in vivo by nontumorigenic, benign, and heterologous malignant keratinocytes. The somatic‐cell hybrids expressed the differentiation‐specific keratins, K1 and K10, in response to high extracellular calcium concentrations (1.4 mM) in vitro. All of the hybrids had fewer local metastases than did the parental lines, and when tumor formation was not suppressed, the resulting tumors were highly differentiated. Polymerase chain reaction analysis of the neomycin‐resistance gene at nontumorigenic injection sites indicated an absence of injected hybrids, and subsequent analyses failed to detect nontumorigenic 291 cells 1 wk after transplantation. These data demonstrate that distinct tumor suppressor gene activities are lost at discrete stages during multistage carcinogenesis and are consistent with the hypothesis that tumor suppression can occur through induction of terminal differentiation. © 1995 Wiley‐Liss, Inc.
Benzo[a]pyrene‐induced mutagenesis of p53 hot‐spot codons 248 and 249 in human hepatocytes
Authors: Cherpillod Pascal , Amstad Paul A.
Published: February 2007

Human tobacco‐related cancers show a high frequency of G‐to‐T transversions in several mutation hot‐spot regions of the p53 tumor suppressor gene, probably the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene. To gain insight into the mechanism of formation of these G‐to‐T transversions in tobacco‐associated carcinogenesis, we studied the mutagenesis of p53 codons 247–250 by benzo[a]pyrene in human hepatocellular carcinoma cells by restriction fragment length polymorphism‐polymerase chain reaction genotypic analysis. Benzo[a]pyrene preferentially induced G‐to‐T transversion in the second and third positions of codon 248 and C‐to‐A transversion in the first position of codon 248. However, benzo[a]pyrene did not induce base‐pair changes in codon 249, which is a mutational hot‐spot in aflatoxin‐related hepatocardno‐genesis, in which predominantly G‐to‐T transversion in the third psition of codon 249 is observed. The benzo[a]pyrene‐induced G‐to‐T transversion in the middle position of codon 248, in which arginine is changed into leucine, is frequently observed in tumors of the lung. The other two benzo[a]pyrene‐induced base‐pair changes in codon 248, namely the C‐to‐A transversion in the first position and G‐to‐T transversion in the third position, do not lead to a change in the amino‐acid composition of the p53 protein. These mutations are silent and therefore are not selected in tumors. It follows that benzo[a]pyrene‐induced mutability on the DNA level in p53 codons 247–250 correlates well with the type of mutation found in tumors of the lung. Therefore, our results support the hypothesis that benzo[a]pyrene is the etiological agent in tobacco‐related Cancers. © 1995 Wiley‐Liss, Inc.
Frequent loss of heterozygosity on chromosome 4 in diethylnitrosamine‐induced C3H/MSM mouse hepatocellular carcinomas in culture
Authors: Miyasaka Kazuyoshi , Ohtake Keiko , Nomura Kimie , Kanda Hiroaki , Kominami Ryo , Miyashita Nobumoto , Kitagawa Tomoyuki
Published: February 2007

Genetic changes, in particular the loss of heterozygosity (LOH) and the presence of c‐Ha‐ras codon 61 point mutations, were investigated in diethylnitrosamine‐induced hepatocellular carcinomas (HCCs) in C3H/MSM F1 mice. (MSM are wild mice.) LOH analysis of 48 primary tumors with microsatellite probes covering at least one proximal and one distal site of each autosome revealed no obvious positive results for LOH. Analysis of 23 cell lines established from seven of these HCCs, however, showed LOH on chromosome 4 in all (seven of seven), even in early passages (G2‐G3). With regard to other chromosomes, LOH was observed only rarely on chromosomes 16 and 19. These allelotype features were maintained in later passages (G11‐G14), with only a few additional occurrences of LOH appearing on chromosomes 1, 6, and 8. Extensive analyses with multiple micro‐satellite probes from chromosome 4 and with 52 cell lines established from 24 HCCs of 18 mice revealed LOH in 22 of the tumors (92%), with the shortest region about 10 cM distal to the a‐interferon gene. No c‐Ha‐ras oncogene activation in codon 61 was observed. These data indicate that loss of tumor suppressor genes on chromosome 4 may play an important role in mouse hepatocarcinogenesis in progression in vivo or in immortalization in vitro or both. © 1995 Wiley‐Liss, Inc.
Combined effects of ionizing radiation and cycloheximide on gene expression
Authors: Woloschak Gayle E. , Felcher Paolo , Chang‐Liu Chin‐Mei
Published: February 2007

We performed experiments to determine the effects of ionizing radiation exposure on expression of genes such as β‐actin, c‐fos, histone H4, c‐myc, c‐jun, Rb, and p53 after exposure of Syrian hamster embryo (SHE) cells to the protein synthesis inhibitor cycloheximide. The purpose of these experiments was to determine the role of a labile protein in the radiation‐induced response. The results revealed that when ionizing radiation (either fission‐spectrum neutrons or γ rays) was administered 15 min after cycloheximide treatment of SHE cells, the radiation exposure reduced cycloheximide‐mediated gene induction of c‐fos, histone H4, and c‐jun. In addition, dose‐rate differences were found when radiation exposure most significantly inhibited the cycloheximide response. Our results suggest that ionizing radiation does not act as a general protein‐synthesis inhibitor and that the presence of a labile protein is required for the maintenance of specific gene transcription and mRNA accumulation after radiation exposure, especially at high dose‐rates. © 1995 Wiley‐Liss, Inc.
Contribution of O6‐methylguanine‐DNA methyltransferase to resistance to 1,3‐(2‐chloroethyl)‐1‐nitrosourea in human brain tumor‐derived cell lines
Authors: Bobola Michael S. , Berger Mitchel S. , Silber John R.
Published: February 2007

To assess the possible role of the DNA repair protein O6‐methylguanine‐DNA methyltransferase (MGMT) in resistance of brain neoplasms to the clinically important chloroethylating agent 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU), we quantitated MGMT activity, BCNU survival, and the effect of ablating MGMT activity on the sensitivity of 14 human medulloblastoma‐ and glioma‐derived cell lines. BCNU resistance, measured as 10% survival dose (LD10), differed eightfold among the lines. Elimination of measurable MGMT activity with the substrate analogue inhibitor O6‐benzylguanine (O6‐BG) revealed a variable but limited contribution of MGMT to survival. In no case did O6‐BG reduce LD10 by more than 3.4‐fold. In contrast, 06‐BG reduced the LD10 for N‐methyl‐N′‐nitro‐N‐nitrosoguanidine up to 31‐fold in the same cell lines (Bobola MS, Blank A, Berger MS, Silber JR, Mol Carcinog 13:70–80, 1995). Variability in BCNU survival, manifested as a sevenfold range of LD10, persists after measurable MGMT was eliminated, indicating that another mechanism or mechanisms is operating to limit cytotoxicity. Cells alkylated while suspended in growth medium are more resistant to BCNU and display less dependence on MGMT than cells treated while proliferating on a plastic substratum. When alkylated in suspension, most of the lines are either unresponsive to O6‐BG or contain a subpopulation that did not respond to O6‐BG. Our results demonstrate that BCNU resistance is multifactorial and that MGMT makes a modest contribution to resistance in our lines. © 1995 Wiley‐Liss, Inc.
Characterization of a murine p53SER246 mutant equivalent to the human p53SER249 associated with hepatocellular carcinoma and aflatoxin exposure
Authors: Ghebranious Nader , Knoll Brian J. , Wu Hongyun , Lozano Guillermina , Sell Stewart
Published: February 2007

A mutation in the tumor suppressor p53 gene resulting in an Arg→Ser substitution in position 249 is found frequently in human hepatocellular carcinomas associated with hepatitis B infection and with aflatoxin exposure. To determine the significance of this mutation in an in vivo experimental model using transgenic mice, we introduced a two‐nucleotide change in the mouse p53 gene at amino‐acid position 246, which is equivalent to position 249 in human p53, by the recombinant polymerase chain reaction mismatched primer method. This p53 mutation resulted in the same change, an Arg→Ser substitution, as in the human p53 gene at position 249. We now report that the protein product of this mutant mouse p53ser246 had properties similar to those of the wild‐type protein when tested by binding to (i) monoclonal antibodies PAb246 and PAb240, (ii) simian virus 40 large T antigen, and (iii) heat‐shock protein. However, it had mutant‐type transforming properties when tested for colony formation with an osteosarcoma cell line. It was not active, as is wild‐type p53, in transcription activation of the muscle creatine kinase promoter. These properties are the same as those found in the p53trp248 product of the p53 mutation associated with the Li‐Fraumeni syndrome. Although less is known about the human p53ser249 product associated with hepatocellular carcinoma, the mutant murine p53ser246 protein shares the known properties of the human gene product. © 1995 Wiley‐Liss, Inc.
Mutational specificity of 2‐amino‐3‐methylimidazo‐[4,5‐f]quinoline in the hprt locus of CHO‐K1 cells
Authors: Lee Huei , Shih Ming‐Kuei
Published: February 2007

2‐Amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), a food carcinogen formed in cooked meats, can induce gene mutation at the hprt locus of CHO‐K1 cells in the presence of hepatic 59 mix. To elucidate the molecular nature of IQ‐induced mutation, we characterized the entire coding region of the hypoxanthine phosphoribosyl‐transferase gene of 23 independent mutants derived from IQ‐treated CHO cells by direct sequencing of polymerase chain reaction‐amplified cDNA. Ten of the 23 IQ‐induced mutants examined contained single base substitutions; one mutant had three single‐base substitutions. Among the base substitutions, G·C→CG (six of 13) and A·T→CG (three of 13) transversions predominated. Most of the base‐substitution mutations occurred preferentially at a middle G and had a dA in their 3′ ends. Of the 13 other mutations (56.5%), 12 missing one or more complete exons were splice‐site mutations, and one mutant had a partial deletion of an exon. A high frequency of complete exon deletion (11 of 12) in exons 2–5 was observed. Interestingly, 75% of the mutants (nine of 12) with splice‐site mutations were induced by IQ only at higher concentrations (300–500 μM). This was probably due to the occurrence of GC base‐substitution mutations that affected hprt mRNA splicing, especially at the intron‐exon boundaries. © 1995 Wiley‐Liss, Inc.
Effects of 12‐O‐tetradecanoylphorbol‐13‐acetate on human papillomavirus type 16‐positive keratinocytes at different stages of transformation
Authors: Sears Wendy L. , Goto‐Mandeville Ryoko , Mirapuri Mona , Braun Lundy
Published: February 2007

Normal human keratinocytes grown under serum‐free conditions can be triggered to differentiate by exposure to serum or to the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). We found that TPA treatment of human papillomavirus (HPV) type 16‐immortalized cells in culture induced formation of cornified envelopes indicative of squamous differentiation. Concurrent with differentiation, TPA inhibited the expression of HPV 16 E6 and E7 mRNA transcripts. Adaptation of the immortalized cells to growth in serum‐containing medium led to the selection of a subpopulation of HPV‐transformed cells that was resistant to TPA‐induced differentiation. In this cell line, a transient suppression of HPV transcripts was observed at 5 h, whereas in differentiation‐resistant, carcinoma‐derived lines, TPA had little effect on HPV oncogene expression, c‐myc transcripts were suppressed for the duration of exposure to TPA in only the differentiation‐competent cells; c‐fos and c‐jun were transiently induced in all cell lines. Transforming growth factor‐a mRNAs were also increased approximately eightfold as HPV 16‐immortalized cells were induced to differentiate. These results demonstrate that, in HPV 16‐immortalized keratinocytes, acquisition of resistance to inducers of squamous differentiation is accompanied by altered regulation of cell growth and gene expression. © 1995 Wiley‐Liss, Inc.
Low frequency of CDKN2 mutation in endometrial carcinomas
Authors: Peiffer Stacia L. , Bartsch Detlef , Whelan Alison J. , Mutch David G. , Herzog Thomas J. , Goodfellow Paul J.
Published: February 2007

The CDKN2 gene on chromosome 9p21 encodes the p16 inhibitor of cyclin D/cyclin‐dependent kinase 4 complexes. Mutations and deletions of CDKN2 have been frequently identified in cell lines, whereas most primary tumors have demonstrated a lower frequency of alteration. To assess the role of CDKN2 in endometrial tumorigenesis, 34 tumor samples were examined for loss of heterozygosity at 9p21 and mutation in CDKN2. To identify tumors that had lost 9p21, samples were genotyped with markers flanking the CDKN2 locus. The frequency of CDKN2 mutation in endometrial carcinomas was determined by single‐strand conformation variant analysis and direct sequencing of variants. Of the 34 tumors examined, three revealed loss of 9p21 sequences. Two samples were characterized by point mutations in CDKN2, one of which also showed loss of 9p21 sequences. © 1995 Wiley‐Liss, Inc.
p53 mutation without allelic loss and absence of mdm‐2 amplification in a transplantable hamster pancreatic ductal adenocarcinoma and derived cell lines but not primary ductal adenocarcinomas in hamsters
Authors: Okita Shunji , Tsutsumi Masahiro , Onji Morikazu , Konishi Yoichi
Published: February 2007

An investigation of p53 gene mutation by single‐stranded conformation polymorphism analysis of polymerase chain reaction products followed by direct sequencing and of murine double minute 2 (mdm‐2) gene amplification by Southern blot analysis was performed, using a series of hamster pancreatic duct adenocarcinomas: 18 primary adenocarcinomas induced by N‐nitrosobis(2‐oxopropyl)amine, a transplantable adenocarcinoma (HPD), and three cell lines derived from HPD (HPD1NR, HPD2NR, and HPD3NR). A mutation in the p53 gene was detected at codon 197, resulting in an amino acid change from leucine to phenylalanine, in both HPD and the three cell lines but in none of the 18 primary adenocarcinomas. In the three HPD cell lines, which were confirmed to contain only cancer cells, a normal p53 gene allele was retained. Immunohistochemical investigation of p53 expression using polyclonal antibody Ab‐7 revealed positive nuclear staining in the HPD and two back‐transplanted tumors derived from HPD1NR and HPD2NR but not in the 18 primary adenocarcinomas. mdm‐2 gene amplification was not detected in 18 primary adenocarcinomas or any of the tumor cell lines. The results suggest that a p53 gene mutation without allelic loss, together with overexpression of p53 protein, may be a genetic alteration involved in the progression stage of multistep pancreatic carcinogenesis in hamsters and that mdm‐2 gene amplification is not important for this process. © 1995 Wiley‐Liss, Inc.
Dietary energy restriction‐induced modulation of protein kinase C ζ isozyme in the hamster pancreas
Authors: Nair Satish C. , Toshkov Ilia A. , Yaktine Ann L. , Barnett Tracy D. , Chaney William G. , Birt Diane F.
Published: February 2007

Dietary restriction in experimental animals enhances life span, delays disease, inhibits immunological perturbations, and ameliorates cancer. Protein kinase C(a) isozymes mediate signals generated by hormones, growth factors, and neurotransmitters for cell proliferation and differentiation. The results of our study showed that a C‐terminally directed anti‐PKC ζ antibody detected an 81–kDa band in the pancreases of control and energy‐restricted hamsters. Syrian golden hamsters were fed energy‐restricted diets formulated such that the hamsters received 90%(10% energy restriction(a) ), 80%(20% ER), or 60%(40% ER) of the total energy consumed by control hamsters, with the energy reduced proportionally from fat and carbohydrate. ER decreased PKC ζ isozyme levels by 40–75% in hamsters fed 10, 20, and 40% ER diets for 8 wk. PKC ζ isozyme expression was decreased by 75–80% in hamsters fed ER diets for 15 wk. Although ER caused significant decreases in PKC ζ isozyme levels compared with those of control hamsters at both time points, the relative differences in PKC ζ levels between the dietary ER groups(10, 20, and 40%) were small and not significant. A significant decrease in the body weights of ER animals compared with those of controls was observed at both time points. No differences in tomato lectin and phytohemagglutinin reactivity were observed between control animals and animals fed 10, 20, and 40% ER diets. Furthermore, the cellular expression of PKC ζ in the hamster pancreas did not differ among hamsters fed the various ER diets. These observations may be important for understanding not only the role of dietary ER in pancreatic cancers but also PKC ζ signal transduction mechanisms in normal pancreatic physiology.© 1995 Wiley‐Liss, Inc