bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of cultured human breast cancer cells (AU‐565 and MCF‐7) associated with loss of cell surface HER‐2/neu antigen
Authors: Bacus Sarah S. , Kiguchi Kaoru , Chin Dot , King C. Richter , Huberman Eliezer
Published: February 2007

The relationship between terminal cell differentiation and changes in the subcellular levels of the HER‐2/neu antigen was investigated in cultured human breast cancer cells: AU‐565 cells (which overexpress the HER‐2/neu gene) and MCF‐7 cells (which do not overexpress this gene). Differentiation was achieved by treating the cells with mycophenolic acid (MPA), phorbol 12‐myristate 13‐acetate (PMA), retinoicacid (RA), or the TA‐1 monoclonal antibody to the extracellular domain of the HER‐2/neu protein. Ten to twenty percent of the cells in untreated, sparsely growing AU‐565 cultures exhibited morphological maturation characterized by large lacy nuclei surrounded by sizable flat cytoplasms. A fraction of these cells harbored milk factors such as casein and large lipid droplets. Treatment of the AU‐565 cells for 4 d with 9 μM MPA, 1.6 nM PMA, 2.5 μM RA, or 1 μg/mL TA‐1 antibody resulted in cell growth inhibition and an increase in the percentage of cells (48‐97%) that exhibit a mature phenotype. MCF‐7 cells were also susceptible to differentiation by MPA and RA, but to a lesser degree than the AU‐565 cells. Differentiation in the AU‐565 and MCF‐7 cells was associated with reduced immunostaining for the HER‐2/neu protein at the cell surface membrane and with a transient increased diffuse immunostaining for this protein in the cytoplasm.
Activating missense mutations in Ha‐ras‐1 genes in a malignant subset of Bladder lesions induced by N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine or N‐[4‐(5‐nitro‐2‐furanyl)‐2‐thiazolyl]formamide
Authors: Jones Richard F. , Debiec‐Rychter Maria , Zukowski Kim , Wang Ching Y.
Published: February 2007

Urothelial cell cultures generated from urinary bladders from a series of N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐ or N‐[4‐(5‐nitro‐2‐furanyl)‐2‐thiazolyl]formamide (FANFT)‐treated Fischer 344 rats were examined for activating missense mutations in Ha‐ras‐1 genes. Our overall objective was to identify oncogene‐activating mutations in this system and to determine what altered biological properties correlate with such genetic changes. The urinary bladders from the treated animals showed a spectrum of histopathologies, from simple hyperplasia to transitional cell carcinoma (TCC). Using restriction analysis, oligonucleotide hybridization, and DNA sequencing, we found that approximately 20% (3/14) of the bladder cell cultures had acquired oncogenic single‐base substitutions in codon 61 of Ha‐ras‐1 genes (CAA → AAA or CGA). The donor bladder lesions for these three cultures, which also harbored the same ras‐activating mutations, were all classified as stage A or B TCCs. However, four other TCCs also arising in this series were found to have normal Ha‐ras genes. Whereas approximately half of the bladder cultures derived from the carcinogen‐treated rats were nontumorigenic in athymic mice, the three cultures containing ras oncogenes were all highly tumorigenic (forming tumors within 5 wk of injection into athymic mice). These cultures also displayed a high degree of anchorage‐independent growth and NIH 3T3‐transforming activity in gene transfer assays. The nontumorigenic cultures were derived from bladder lesions that included three hyperplasias and three stage A TCCs. We conclude that ras‐activating missense mutations were present in a malignant subset of bladder lesions induced by BBN or FANFT, but most of the lesions in this system appeared to involve genetic alterations elsewhere. Thus other oncogenes besides activated Ha‐ras may apparently be associated with the same bladder histopathologies and transformation markers.
Mutational activation of c‐HA‐ras gene in squamous cell carcinomas of rat zymbal gland induced by carcinogenic heterocyclic amines
Authors: Kudo Mami , Ogura Tsutomu , Esumi Hiroyasu , Sugimura Takashi
Published: February 2007

The activation of the c‐Ha‐ras gene and its carcinogen specificity were examined in squamous cell carcinomas (SCCs) induced by the mutagenic heterocyclic amines 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 2‐amino‐3, 8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), and 2‐amino‐3, 4‐dimethylimidazo[4,5‐f]quinoline (MeIQ) in the Zymbal gland in rats. DNA fragments of the c‐Ha‐ras gene were amplified from formalin‐fixed and paraffin‐embedded tissues by polymerase chain reaction and analyzed for activating mutations involving codons 12, 13, and 61 by oligonucleotide differential hybridization and sequencing. c‐Ha‐ras mutations were found in four of seven and two of six Zymbal gland SCCs induced by IQ and MelQx, respectively. These mutations were located in either codon 13 or 61. In the case of MelQ, point mutations at the second nucleotide of codon 13 were found in nine of the total 14 Zymbal gland SCCs and in one papilloma. Of the nine SCCs that had mutations in codon 13, two possessed mutations at the second nucleotide of codon 12 as well. Most reported mutations in c‐Ha‐ras are located at codon 12 or 61, but the heterocyclic amines in this study induced mutations not only at codons 12 and 61 but also in codon 13. Transversions were the dominant mutation induced by these heterocyclic amines.
Evidence for autocrine/paracrine growth stimulation by transforming growth factor‐α during the process of skin tumor promotion
Authors: Imamoto Akira , Beltrán Linda M. , Digiovanni John
Published: February 2007

A single topical application of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to mouse skin decreased 125I‐labeled epidermal growth factor (EGF) binding in epidermal membrane preparations within 1 h while 1,8‐dihydroxy‐3‐methyl‐9‐anthrone (chrysarobin) gradually reduced binding with maximum inhibition at 15 h. Subsequently, 125I‐EGF binding increased to ∼ 200% of control in epidermal membrane preparations from both TPA‐ and chrysarobin‐treated mice. A single application of TPA but not chrysarobin resulted in a rapid translocation of protein kinase C (PKC) to the membrane; however, treatment with both promoters ultimately led to a time‐dependent loss of PKC activity in both membrane and cytosol fractions. The initial inhibition of 125I‐EGF binding was sustained for at least 24 h after single and multiple treatments with both promoting agents. Acid washing restored EGF binding to control levels in membrane preparations obtained 24 h after a single application, whereas acid washing of membrane preparations obtained 24 h after a second application of TPA or chrysarobin increased binding (2.5‐fold and 1.5‐fold that of the control, respectively). The presence of increased amounts of ligands for the EGF receptor in tumor promoter‐treated epidermis was initially confirmed in 125I‐EGF binding competition experiments using NRK‐49F cells. A single topical application of TPA or chrysarobin induced elevated levels of transforming growth factor‐α (TGF‐α) mRNA at 6 h or 15–24 h, respectively. Elevated levels of a TGF‐α precursor (21 kDa) were subsequently observed in cytosol and membrane preparations after single and multiple applications of TPA or chrysarobin. These results suggest that repeated topical application of tumor promoters may lead to sustained loss of a negative‐feedback mechanism involving phosphorylation at Thr‐654 of the EGF receptor by PKC. The concomitant elevation of ligands, such as TGF‐α, may provide a mechanism for sustained cell proliferation essential for skin tumor promotion.
Analysis of CYP1A1 promoter function by transcription in vitro
Authors: Wen Long‐Ping , Jones Keith W. , Whitlock James P.
Published: February 2007

We have analyzed the function of the CYP1A1 promoter using in vitro transcription. Using nuclear extracts from HeLa cells, we found that transcription from the promoter in vitro was constitutive. Transcription in vitro was not increased by prior exposure of the HeLa cells to the inducer 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin nor by the inclusion of a dioxin‐responsive enhancer in the DNA template. Analyses of mutants revealed that a TATAAA sequence and two recognition motifs for CCAAT box transcription factor/nuclear factor I contributed to the constitutive activity of the promoter in vitro.
Expression of RB and p53 proteins in HPV‐positive and HPV‐negative cervical carcinoma cell lines
Authors: Wrede David , Tidy John A. , Crook Tim , Lane David , Vousden Karen H.
Published: February 2007

Mutations within the tumor suppressor genes Rb‐1 and p53 are commonly found in many human malignancies, and loss of wild‐type function of both p53 and RB appear to be important events in the development of these malignancies. Interference with normal RB and p53 function in the cell has apparently also been exploited by the oncogenic genital human papillomaviruses (HPVs), which encode transforming proteins capable of binding cellular RB and p53 proteins. We have investigated the expression of RB and p53 in a series of eight cervical carcinoma cell lines, six of which contain HPV sequences and two of which have arisen apparently independently of HPV infection. In the six HPV‐positive lines, no evidence of abnormal RB or p53 protein could be detected. However, there was evidence for abnormal RB and p53 in the two HPV–negative lines. These data are consistent with the hypothesis that loss of wild‐type RB and p53 function is necessary for tumor development and that such loss can occur either by mutation within the cellular gene or by expression of viral proteins capable of complexing wild‐type cellular proteins.
Analysis of oncogene alterations in human endometrial carcinoma: Prevalence of ras mutations
Authors: Boyd Jeff , Risinger John I.
Published: February 2007

The molecular genetics of human endometrial carcinoma have yet to be defined to any significant extent. Cell lines from 11 endometrial carcinomas were examined for alterations in proto‐oncogenes that might predictably be present, based on existing data from the better‐characterized human carcinomas of the uterine cervix, ovary, and breast. Codons 12, 13, and 61 of the Ha‐ras, Ki‐ras, and N‐ras genes were examined for possible point mutations, and the c‐erbB2/neu, c‐myc, and epidermal growth factor receptor (EGFR) genes were examined for amplification or overexpression. Ras mutations were found in seven of 11 (64%) tumors, including three in codon 61 of Ha‐ras (CAG·CAT) and four in codon 12 of Ki‐ras (GGT→GAT in two and GGT→GTT in two). No evidence was found for amplification or overexpression of the c‐enbB2 or EGFR genes in any tumor. One tumor contained amplified c‐myc sequences and exhibited relative overexpression of c‐myc. These data suggest that the amplification or overexpression of several proto‐oncogenes frequently observed in other human gynecologic and breast tumors are not prevalent in endometrial carcinoma and that ras gene mutations are relatively common in this tumor type.
Ras gene mutation and amplification in human nonmelanoma skin cancers
Authors: Pierceall William E. , Goldberg Leonard H. , Tainsky Michael A. , Mukhopadhyay Tapas , Ananthaswamy Honnavara N.
Published: February 2007

Our previous studies have shown that human skin cancers occurring on sun‐exposed body sites frequently contain activated Ha‐ras oncogenes capable of inducing morphologic and tumorigenic transformation of NIH 3T3 cells. In this study, we analyzed human primary squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) occurring on sun‐exposed body sites for mutations in codons 12, 13, and 61 of Ha‐ras, Ki‐ras, and N‐ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction, followed by dot‐blot hybridization to synthetic oligonucleotide probes designed to detect single base‐pair mutations. In addition to the primary human skin cancers, we also analyzed Ha‐ras‐positive NIH 3T3 transformants for mutations in the Ha‐ras oncogene. The results indicated that all three NIH 3T3 transformants, 11 of 24 (46%) SCCs, and 5 of 16 (31 %) BCCs contained mutations at the second position of Ha‐ras codon 12 (GGC→GTC), predicting a glycine‐to‐valine amino acid substitution, whereas only 1 of 40 skin cancers (an SCC) displayed a mutation in the first position of Ki‐ras codon 12 (GGT→AGT), predicting a glycine‐to‐serine amino acid change. In addition, three of the SCCs contained highly amplified copies of the N‐ras oncogene in their genomic DNA. Interestingly, two of the SCCs containing amplified N‐ras sequences also had G→T mutations in codon 12 of the Ha‐ras oncogene. These studies demonstrate that mutations in codon 12 of the Ha‐ras oncogene occurred at a high frequency in human skin cancers originating on sun‐exposed body sites, whereas mutation in codon 12 of Ki‐ras or amplification of N‐ras occurred at a low frequency. Since the mutations in the Ha‐ras and Ki‐ras oncogenes were located opposite potential pyrimidine dimer sites (C‐C), it is likely that these mutations were induced by ultraviolet radiation present in sunlight.
Altered growth regulation of rat kidney proximal tubule epithelial cells transformed in vitro by SV40 viral DNA: Fibroblast growth factors (heparin‐binding growth factors) are potent inducers of anchorage‐independent growth
Authors: Zhang Guohong , Stevens James L.
Published: February 2007

The majority of renal cancers are thought to arise from the proximal tubule epithelium, but little is known about their etiology. In this investigation, we have established an in vitro model to study the transformation of these target cells using rat kidney proximal tubule epithelial cells (RPTE) transformed in defined medium with SV40‐viral DNA. Selection by passaging cells onto plastic surfaces yielded a population of cells (SV‐RPTE) that expressed keratin and vimentin along with SV40 large‐T antigen. The cells were morphologically transformed and lost their differentiated character as determined by several RPTE markers. SV‐RPTE cells grew in soft agar in serum‐supplemented medium containing insulin, epidermal growth factor, and cholera toxin, but were unable to grow when serum and growth factors were not combined. Acidic and basic fibroblast growth factors (aFGF and bFGF) were unique since they were the only single factor that induced anchorage‐independent growth in the presence of serum alone. Transforming growth factor‐β1 (TGF‐β1) was a potent inhibitor of anchorage‐independent growth, but the inhibition was partially overcome by a combination of growth factors. The growth factor responses of SV‐RPTE in monolayer cultures differed from those in soft agar; the cells were more sensitive to growth stimulation by insulin and insulin‐like growth factor, neither of which stimulated anchorage‐independent growth. SV‐RPTE cells in monolayer cultures had also lost the sensitivity to growth inhibition by TGF‐β1 characteristic of normal RPTE. The RPTE transformation model described here will be very useful for investigating the molecular basis and etiology of renal cancers. Furthermore, the data suggest that maintenance of the transformed phenotype by aFGF and bFGF and loss of negative growth regulation by TGF‐β1 could play a role in renal carcinogenesis.
Racial variation in the distribution of Ha‐ras‐1 alleles
Authors: Weston Ainsley , Vineis Paolo , Caporaso Neil E. , Krontiris Theodore G. , Lonergan Joyce A. , Sugimura Haruhiko
Published: February 2007

Restriction fragment length polymorphism analyses of the Ha‐ras‐1 proto‐oncogene were undertaken in white and black populations residing in the Baltimore‐Washington metropolitan area to address whether specific rare alleles of the Ha‐ras‐1 proto‐oncogene locus vary in their distribution among different racial groups. High‐molecular‐weight genomic DNA samples from the lungs of 80 lung cancer patients and 92 accident victims were digested with appropriate restriction enzymes and subjected to Southern analysis using the 6.6‐kb BamHl human Ha‐ras‐1 recombinant fragment from the plasmid pEC. Thirty allelomorphs of different sizes were detected among the 172 study subjects. An association was observed between race and specific alleles. Rare alleles were more frequent in black cancer patients and trauma victims than in whites. Within each racial category, lung cancer patients had an excess of rare alleles. These data indicate the importance of controlling for racial variation when designing studies to determine human cancer risk factors.
The role of Ha‐ras oncogenes in growth factor independence in rat mammary carcinoma cells
Authors: Chiodino Caterina , Jones Richard F. , Ethier Stephen P.
Published: February 2007

To determine if activation of the c‐Ha‐ras‐1 gene is involved in the acquisition of growth factor independence in 7,12‐dimethylbenz[a]anthracene (DMBA)‐and N‐nitrosomethylurea (NMU)‐induced rat mammary carcinomas, three strategies were used. First, Ha‐ras DNA from growth factor‐independent DMBA‐induced rat mammary tumor cells was amplified using the polymerase chain reaction and examined for the presence of mutations in the first and second exons of Ha‐ras‐1 by restriction fragment length polymorphism analysis, allele‐specific oligonucleotide hybridization, and direct sequencing. No mutations were found in the codon 12/13 or codon 61 regions of the Ha‐ras‐1 gene. Second, a similar analysis of an NMU‐induced mammary carcinoma showed that it harbored an activating mutation in codon 12 of Ha‐ras‐1. When analyzed for growth factor requirements, these cells were found to express limited growth potential in all media tested, in contrast to growth factor‐independent cells, which proliferated extensively in the presence or absence of exogenous growth factors. Third, growth factor‐dependent rat mammary tumor cells and spontaneously immortalized rat normal mammary epithelial cells were transfected with an activated form of the Ha‐ras‐1 (T24) gene, and the growth factor requirements of the transfected cells were examined. The ras‐transfected cells retained the growth factor requirements of the normal cells. In addition, ras‐transfected cells were transplanted into syngeneic rats and nude mice, and no tumors developed after 6 mo in vivo. These results indicate that, in rat mammary tumor cells, neither growth factor independence in vitro nor transplantability are directly mediated by Ha‐ras oncogenes. The results also suggest that ras activation and growth factor independence may be associated with independent pathways to malignancy in rat mammary tumorigenesis.
The B subgroup bovine papillomaviruses lack an identifiable E6 open reading frame
Authors: Jackson Maria E. , Pennie William D. , McCaffery Roselyn E. , Smith Kenneth T. , Grindlay G. Joan , Campo M. Saveria
Published: February 2007

Analysis of the corrected DNA sequence for the bovine papillomavirus type 4 (BPV4) genome revealed that there is no open reading frame (ORF) that might encode an E6 protein. The other two B subgroup bovine papillomaviruses, BPV3 and BPV6, were found to have the same arrangement of ORFs in this region as BPV4. Thus, we conclude that E6 functions are either not required by these viruses or are performed by another viral (or host) protein. Furthermore, the position that might be expected to be occupied by E6, between the long control region and the E7 ORF, contains the E8 ORF, which has the potential to encode a 42‐residue polypeptide with considerable similarity to the E5 transforming protein of BPV1. Therefore, it appears that during the evolution of the B subgroup of BPVs, genomic rearrangements may have occurred resulting in the present layout of the early ORFs.
Formation of stable p53 homotetramers and multiples of tetramers
Authors: Stenger Judith E. , Mayr Gregory A. , Mann Kristine , Tegtmeyer Peter
Published: February 2007

We have used gradient gel electrophoresis and chemical cross‐linking to analyze the quaternary structure of purified, wild‐type, murine p53. Under nondenaturing conditions, p53 electrophoreses as tetramers and multiples of tetramers. Under denaturing conditions, fully cross‐linked p53 also behaves as tetrameric structures. We confirmed the composition of the tetramers by partially cross‐linking p53 and dissociating tetramers into monomers, dimers, and trimers. © 1992 wiley‐Liss, Inc.
Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras‐transformed NIH 3T3 cells
Authors: Chambers Ann F. , Colella Rita , Denhardt David T. , Wilson Sylvia M.
Published: February 2007

We previously found that the T24 Ha‐ras oncogene induces metastatic ability in NIH 3T3 cells and that this change depends on expression of the ras oncogene. As part of our studies on mechanisms by which ras may induce metastasis, we investigated expression and activity of two cysteine proteinases, cathepsin L (major excreted protein) and cathepsin B, as well as cysteine proteinase inhibitor activity, in ras‐transformed NIH 3T3 cells. In a series of cell lines that expressed differing amounts of ras, we found a good correlation between levels of ras expression and cathepsin L expression (r = 0.80). There was also a good correlation between secreted procathepsin L protein levels and experimental metastatic ability (r = 0.88). We found a similar but less strong association between cathepsin B levels and metastatic ability in these cells (r=0.76 and r=0.72 for 2.2‐kb and 4.1‐kb transcripts, respectively). Functional cathepsin L plus B activity (both secreted and cell‐associated) was found to be higher in ras‐transformed cells and was dependent on cell confluency in culture. Coupled with increased expression and activity of cysteine proteinases, ras‐transformed NIH 3T3 cells showed reduced cysteine proteinase inhibitor activity. We conclude that the balance between expression of cysteine proteinases and their inhibitors may be coregulated by ras expression. Our results suggest that ras‐induced increases in production of degradative enzymes such as cathepsins L and B, along with decreased activities of their inhibitors, may contribute to the increased malignant properties of ras‐transformed NIH 3T3 cells. © 1992 Wiley‐Liss, Inc.
Transcripts encoding protein kinase C‐α, ‐ϵ, ‐ζ, and ‐ζ are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells
Authors: Dlugosz Andrzej A. , Mischak Harald , Mushinski J. Frederic , Yuspa Stuart H.
Published: February 2007

The protein kinase C (PKC) family of phospholipid‐dependent serine‐threonine kinases has been implicated in keratinocyte differentiation and neoplastic transformation. To determine if Ca2+‐mediated keratinocyte differentiation is associated with changes in PKC isozyme gene expression, RNA was isolated from primary mouse keratinocytes grown in medium with 0.05, 0.12, or 1.4 mM Ca2+. Based on northern blot analysis, primary keratinocytes expressed mRNA encoding PKC‐α, ‐ϵ, ‐ζ, and ‐neta but not PKC‐p or ‐y. Relatively little change was detected in the level of these transcripts in cells induced to differentiate by exposure to elevated extracellular Ca2+. Interestingly, the PKC‐ζ transcripts detected in RNA isolated from keratinocytes were approximately 200 nucleotides longer than those from mouse brain, suggesting the existence of an alternative form of this isozyme.

An early change in benign neoplastic transformation of keratinocytes is the inability to differentiate in response to Ca2+ or the PKC activator 12‐O‐tetradecanoylphorbol‐13‐acetate, which is consistent with altered PKC function in these cells. The PKC isozyme mRNA profile was examined in two benign neoplastic keratinocyte cell lines, 308 and SP‐1, which contain an activating mutation of the c‐Ha‐ras gene. Like normal keratinocytes, 308 and SP‐1 cells expressed mRNA encoding PKC‐α, ‐ϵ, ‐ζ, and ‐η However, the abundance of PKC‐η transcripts in both cell lines was reduced by 74–89% when compared with normal keratinocytes at similar Ca2+ levels. In addition, SP‐1 but not 308 cells exhibited a sevenfold increase in PKC‐η mRNA when cultured in medium with 1.4 mM Ca2+. To address whether these changes were related to the presence of an activated ras gene, RNA was isolated from primary keratinocytes transduced to a benign neoplastic phenotype with the v‐Ha‐ras oncogene. As with normal, 308, and SP‐1 cells, v‐Ha‐ras keratinocytes expressed mRNA encoding PKC‐α, ‐ϵ, ‐ζ, and ‐neta. The level of PKC‐η transcripts was similar in normal and v‐Ha‐ras keratinocytes, indicating that reduction of this mRNA in both 308 and SP‐1 cells was not a direct result of ras activation. As in SP‐1 cells, PKC‐η in v‐Ha‐ras keratinocytes was responsive to extracellular Ca2+, with a four‐fold increase in transcript abundance in 0.12 mM Ca2+ medium relative to 0.05 mM Ca2+ medium. These findings illustrate the complexity of the PKC‐signal transduction pathway in cultured mouse keratinocytes and indicate that the level of certain PKC isozyme transcripts is modulated during the neoplastic process. © 1992 wiley‐Liss, Inc.
Homologous and heterologous Gap‐junctional intercellular communication in v‐raf‐, v‐myc‐, and v‐raf/v‐myc‐transduced rat liver epithelial cell lines
Authors: Kalimi Ghulam H. , Hampton Lori L. , Trosko James E. , Thorgeirsson Snorri S. , Huggett Anthony C.
Published: February 2007

We examined gap‐junctional intercellular communication (GJIC) in a series of normal and v‐raf‐, v‐myc‐, and v‐raf/v‐myc‐transduced rat liver epithelial (RLE) cell lines using the scrape loading‐dye transfer and fluorescence‐recovery‐after‐photobleaching (FRAP) assays. Whereas the normal RLE cell line, the control helper virus‐transduced cell line, and the v‐myc‐transduced cell line all showed excellent GJIC, the v‐raf‐transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v‐raf/v‐myc‐transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. Heterologous GJIC of these oncogenetransduced cell lines was also compared with that in the normal RLE cells. A modified FRAP assay, using fluorescent‐microbead labeling to identify the oncogene‐transduced cell from surrounding normal cells, was used to quantify the heterologous GJIC. The v‐raf/v‐myc‐transformed RLE cells had no heterologous communication with the normal RLE cells, whereas v‐raf‐ and v‐myc‐transduced cell lines maintained heterologous GJIC. Northern analysis showed that connexin 43 was the only gap‐junction protein message expressed in these cell lines; connexin 32 and connexin 26 were not expressed. The levels of connexin 43 mRNA expression were relatively unchanged in all cell lines, suggesting that the reduction in GJIC was primarily at the posttranslational level. These findings suggest that reduction of homologous GJIC in v‐raf‐ and v‐raf/v‐myc‐transformed RLE cells is linked to their tumorigenic potential. Furthermore, the loss of heterologous GJIC, which we observed only in the v‐raf/v‐myc‐transformed cells, might release such cells from the growth‐regulating effects of surrounding normal cells, possibly contributing to their enhanced tumorigenic potential. © 1992 Wiley‐Liss, Inc.
Differential induction of 12‐O‐tetradecanoylphorbol‐13‐acetate sequence gene expression in murine melanocytes and melanoma cells
Authors: Brooks Gavin , Goss Martin W. , Hart Ian R.
Published: February 2007

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel‐ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281–3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐inducible sequence (TIS) gene expression in Mel‐ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel‐ab cells to the PKC‐activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80‐fold increase in steady‐state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c‐fos); TIS 10 expression was not affected). No induction of 115 gene expression was observed either in growing Mel‐ab cells maintained in 324 nM phorbol 12,13‐dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP‐elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel‐ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways. © 1992 Wiley‐List. Inc.
Additive effects of c‐erbB‐2, c‐Ha‐ras, and transforming growth factor‐α genes on in vitro transformation of human mammary epithelial cells
Authors: Ciardiello Fortunato , Gottardis Marco , Basolo Fulvio , Pepe Stefano , Normanno Nicola , Dickson Robert B. , Raffaele Bianco A. , Salomon David S.
Published: February 2007

MCF‐10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point‐mutated c‐Ha‐ras proto‐oncogene, the rat c‐neu (c‐erbB‐2) proto‐oncogene, or the human transforming growth factor‐α (TGF‐α) gene in MCF‐10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF‐10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF‐10A Ha‐ras and MCF‐10A TGF‐α cells with a recombinant retroviral vector containing the human c‐erbB‐2 proto‐oncogene and the hygromycin‐resistance gene. Ten MCF‐10A TGF‐α/c‐ethB‐2 (MCF‐10A TE) and 10 MCF‐10A Ha‐ras/c‐erbB‐2 (MCF‐10A HE) hygromycin‐resistant clones were randomly selected and expanded into cell lines. MCF‐10A TE and MCF‐10A HE clones expressed a 10‐fold to 40‐fold increase in p185 erbB‐2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum‐free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF‐α compared with MCF‐10A cells. Moreover, both MCF‐10A TE and MCF‐10A HE clones exhibited a fivefold to 20‐fold higher cloning efficiency in soft agar than MCF‐10A Ha‐ras, MCF‐10A c‐erbB‐2, or MCF‐10A TGF‐α clones. However, neither MCF‐10A TE nor MCF‐10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c‐Ha‐ras, c‐erbB‐2, and TGF‐α genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto‐oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype. © 1992 Wiley‐Liss, Inc.
Role of mutations at codon 61 of the c‐Ha‐ras gene during diethylnitrosamine‐induced hepatocarcinogenesis in C3H/He mice
Authors: Bauer‐Hofmann Richard , Klimek Fritz , Buchmann Albrecht , Müller Oliver , Bannasch Peter , Schwarz Michael
Published: February 2007

Liver tumors of certain strains of mice frequently contain mutations at codon 61 of the c‐Ha‐ras gene. In our study, we investigated the significance of these mutations in the carcinogenic process. Male C3H/He mice received a single injection of diethylnitrosamine (DEN) on day 15 after birth, and groups of animals were killed at various time intervals between 11 and 52 wk after treatment. At the earlier time points (11–29 wk), we analyzed microdissected tissue from precancerous glucose‐6‐phosphatase‐deficient liver lesions larger than approximately 200 p.m in diameter, for the presence and pattern of c‐Ha‐ras codon 61 mutations. In parallel, the growth characteristics of these liver lesions were studied by pulse labeling with [3H]thymidine and by determining the size distribution of the lesions. At the later time points (42–52 wk after DEN treatment), liver tumors were dissected and also analyzed for the presence of c‐Ha‐ras mutations. We found mutations to be already present in some of the enzyme‐altered liver lesions at weeks 11–29, suggesting that the mutations occurred early in the carcinogenic process. Whereas about 10% of the precancerous focal liver lesions showed mutations in the c‐Ha‐ras gene, the mutation frequency was increased to about 50% in the later‐appearing hepatocellular adenomas and carcinomas, suggesting that c‐Ha‐ras codon 61 mutations may provide a selective advantage to the mutated cell clones. © 1992 Wiley‐Liss, Inc.
p53 mutations are absent from carcinogen‐induced mouse liver tumors but occur in cell lines established from these tumors
Authors: Kress Stefan , König Jorg , Schweizer Jürgen , Löhrke Heinz , Bauer‐Hofmann Richard , Schwarz Michael
Published: February 2007

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas We have examined, by single‐strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen‐induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single‐base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C→C:G or C:G→G:C transversions. Mutations at codon 61 of the c‐Ha‐ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c‐Ha‐ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro. © 1992 Wiley‐Liss, Inc.
Role of activator protein‐1 and methylation function in 12‐o‐tetradecanoylphorbol‐13‐acetate—mediated inhibition of differentiation of friend erythroleukemia cells
Authors: Bertrand Olivier , Giroldi Laurence , Nakazawa Hisayoshi , Sergeant Alain , Yamasaki Hiroshi
Published: February 2007

Friend erythroleukemia cells (FELCs) differentiate after hexamethylene‐bis‐acetamide treatment. This differentiation is characterized by an increase in β‐globin gene expression that is followed by appearance of the hemoglobin. Phorbol‐ester tumor promoters, such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), inhibit differentiation of TPA‐sensitive cells but not TPA‐resistant cells. We have shown that the increase in β‐globin expression is inhibited by TPA in a TPA‐sensitive clone but not in a TPA‐resistant clone. To study the molecular mechanisms of regulation of gene expression by TPA, we examined the possible involvement of gene methylation and the TPA‐responsive element (TRE). Both clones showed similar patterns of methylation around the β‐globin gene. Moreover, TPA‐induced TRE binding and TRE enhancer activity were similar in both variants. These results suggest that the TPA inhibition of induced differentiation may not be explained by regulation of the methylation state. The activator protein‐1 also does not play a crucial role in the sensitivity of FELCs to TPA. © 1992 Wiley‐Liss, Inc.
12‐O‐tetradecanoylphorbol‐13‐acetate—induced levels of ap‐1 proteins: a 46‐kda protein immunoprecipitated by anti‐fra‐1 and induced in promotion‐resistant but not promotion‐sensitive JB6 cells
Authors: Bernstein Lori R. , Bravo Rodrigo , Colburn Nancy H.
Published: February 2007

Neoplastic transformation and transcriptional activation by activator protein‐1 (AP‐1) complex are stimulated by tumor‐promoting agents in promotion‐sensitive (P+) but not promotion‐resistant (P‐) mouse epidermal JB6 cells in culture. This implicates AP‐1 as a specific regulator of signal transduction pathways in the promotion phase of neoplastic transformation. We therefore hypothesized that the defective P‐ responsiveness may be due to limiting levels of AP‐1 protein components in those cells. In this investigation, steady‐state levels of AP‐1 protein components were measured by immunoprecipitating proteins from 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐treated P+ and P‐ cells to discern what may limit the AP‐1 response. Whereas the AP‐1 proteins junB, junD, and fosB did not show differential basal or TPA‐inducible levels in P+ and P‐ cells, a 46‐kDa species precipitated by anti‐fra‐1 antibody was TPA‐inducible in P‐ cells but not in P+ cells, and c‐jun protein was present at higher levels in TPA‐treated and untreated P+ cells than in P‐ cells. These data raise the possibility that the 46‐kDa fra‐1‐related protein may be a negative modulator of AP‐1 activity and suggest that elevated levels of this 46‐kDa species and limiting levels of c‐jun may significantly impair AP‐1 function or transformation response in P‐ cells or both. © 1992 Wiley‐Liss, Inc.
Suppression of radiation‐induced neoplastic transformation by overexpression of mitochondrial superoxide dismutase
Authors: St. Clair Daret K. , Steven Wan X. , Oberley Terry D. , Muse Kenneth E. , St. Clair William H.
Published: February 2007

Manganese superoxide dismutase (MnSOD) scavenges toxic superoxide radicals produced in the mitochondria. Transfection of the human MnSOD gene into mouse C3H 10T1/2 cells resulted in production of active MnSOD, which was properly transported into mitochondria. Overexpression of MnSOD protected cells from radiation‐, but not chemically‐induced neoplastic transformation. This finding demonstrates that oxidative stress that occurs in the mitochondria plays an important role in the development of neoplastic transformation. © 1992 Wiley‐Liss, Inc.
Keratin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: Characterization of keratin 14 and keratin 19 as antigens for the commonly used mouse monoclonal antibody OV‐6
Authors: Bisgaard Hanne Cathrine , Parmelee David C. , Dunsford Harold A. , Sechi Salvatore , Thorgeirsson Snorri S.
Published: February 2007

We have recently reported that cell lines of nonparenchymal origin isolated from rat liver and pancreas, which have been suggested to be the progeny of a facultative stem cell compartment in vivo, express an unusual combination of keratins (K). These cell lines express K8 and K14 but not K18 and K5, their normal partners in filament formation (Bisgaard HC, Thorgeirsson SS, J Cell Physiol 147:333–343, 1991). However, upon spontaneous transformation and differentiation toward a hepatoblastlike progeny, K14 expression is abrogated and replaced by expression of K18 (Wirth et al., Electrophoresis 13:305–332, 1992). In the study presented here, we confirmed by protein sequence analysis that K14 was a major component of the intermediate filaments in a nonparenchymal cell line of hepatic origin. Immunocytochemical analysis of the cells in monolayer demonstrated that K8 as well as K14 were incorporated in the cellular cytoskeleton. Further analysis by immunoprecipitation showed that filament complexes were formed between K8 and K14 as atypical partners. Thus, we concluded that in some nonparenchymal cell lines isolated from rat liver, K8 and K14 form a major intermediate filament network. Finally, we showed that an antibody widely used in studies of the cell lineages of hepatic and pancreatic tissues and their neoplasms, the mouse monoclonal antibody OV‐6, recognizes a common epitope in K14 and K19.
Induction of epidermal hyperplasia, hyperkeratosis, and papillomas in transgenic mice by a targeted v‐Ha‐ras oncogene
Authors: Greenhalgh David A. , Rothnagel Joseph A. , Quintanilla Maria I. , Orengo Christine C. , Gagne Todd A. , Bundman Donnie S. , Longley Mary A. , Roop Dennis R.
Published: February 2007

The regulatory elements of the human keratin K1 gene have been used to target expression of the v‐Ha‐ras oncogene exclusively in the epidermis of transgenic mice. We developed 12 transgenic mouse lines that express the HK1. ras transgene, producing epidermal hyperplasia in neonates and hyperkeratosis in juveniles. Eventually this skin phenotype diminished but with time adult animals developed papillomas that could persist or regress. The rate and frequency of tumorigenesis appeared to be limited, which suggests that v‐Ha‐ras requires a second or even third event to elicit and maintain a benign phenotype in transgenic mice. Since in certain transgenic lines papillomas appeared at wound sites, it appears that the promotion stimulus from wounding may be a second event. We envision that such transgenic mice that express v‐Ha‐ras in the epidermis will become a powerful model for assessing how environmental and molecular factors affect the process of multistage skin carcinogenesis in vivo, as well as a model for evaluating novel therapeutic protocols.
Correlation between lack of bone Gla protein mRNA expression in rat transplantable osteosarcomas and expression of both c‐fos and c‐jun proto‐oncogenes
Authors: Honoki Kanya , Dohi Yoshiko , Tabata Shiro , Mii Yoshio , Miyauchi Yoshizumi , Tsutsumi Masahiro , Tsujiuchi Toshifumi , Morishita Toru , Miura Syuichi , Moriyama Tadashige , Tamai Susumu , Konishi Yoichi
Published: February 2007

Alkaline phosphatase (AP) activity and expression of bone Gla protein (BGP), c‐fos, and c‐jun were compared in two transplantable osteosarcomas with high potentials for metastasis to the lung. The original spontaneous osteosarcoma (SOS) gradually became histologically undifferentiated, losing its osteogenic activity during serial transfer, whereas the chemical (4‐hydroxyaminoquinoline 1‐oxide)—induced osteosarcoma (COS) retained osteogenesis. The two osteosarcomas showed similar doubling times and levels of lung metastasis, and strong AP activity was detected on the cell membranes of both. Northern blot analysis revealed that lack of BGP mRNA expression was associated with expression of both c‐fos and c‐jun proto‐oncogenes in SOS. In contrast, neither c‐fos nor c‐jun mRNAs were detected but BGP mRNA was expressed in the case of COS. These results suggest that the c‐fos and c‐jun genes may suppress the expression of BGP mRNA relevant to differentiation and osteoid formation in rat osteosarcomas. However, this does not appear to be directly related to proliferative or metastatic biological behavior.
Induction of superoxide by 12‐O‐tetradecanoylphorbol‐13‐acetate and thapsigargin, a non‐phorbol‐ester—type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: A permissive role for the arachidonic acid cascade in signal transduction
Authors: Yoon Hyunah L. , Marcus Craig B. , Pfeifer Richard W.
Published: February 2007

Local production of reactive oxygen intermediates, e.g., superoxide anion, by tumor promoter—stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two‐stage chemical‐induced carcinogenesis in murine skin. In the studies reported herein, peritoneal MPs elicited from phorbol‐ester—sensitive SENCAR mice demonstrated a time‐ and dose‐dependent release of superoxide anion (4–6 nmol/106 cells) when stimulated by 200 nM 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in vitro; MP superoxide response was significantly inhibited (50–70%) by preincubation with 40 μM 1‐(5‐isoquinolinyl‐sulfonyl)‐2‐methylpiperazine (H‐7), a protein‐kinase inhibitor. Alternatively, TPA‐stimulated MPs derived from relatively resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain‐dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter previously shown to act independently of protein kinase C (PKC). TG‐stimulated SENCAR MPs released a significant amount of superoxide (2–3 nmol/106 cells) that was not inhibited by H‐7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with 100 μM dibromoacetophenone, an inhibitor of phospholipase A2, completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 50 μM 1‐oleoyl‐2‐acetylglycerol, a diacylglycerol analogue and PKC activator, also induced a significant amount of superoxide from SENCAR MPs only. In parallel with the superoxide findings, TPA and TG stimulated significantly greater [3H]arachidonic acid release from prelabeled SENCAR MPs (a 32% and 48% increase, respectively, over unstimulated controls) relative to MPs from B6C3F1 mice. Two‐dimensional gel‐electrophoretic analysis indicated that TPA‐induced phosphorylation of a 47‐kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig and human polymorphonuclear leukocytes) occurred in MPs from both SENCAR and B6C3F1 mice. Therefore, arachidonic acid production may be a common biochemical pathway by which phorbol‐ester— and non‐phorbol‐ester—type tumor promoters activate MPs in SENCAR mice; such a response may be “permissive” for additive (or synergistic) interactions with PKC‐driven signal pathways.
A revised map position for the Ha‐ras gene on mouse chromosome 7: Implications for analysis of genetic alterations in rodent tumors
Authors: Kemp Christopher J. , Bremner Rod , Balmain Allan
Published: February 2007

The mouse Ha‐ras gene has previously been mapped to the central region of chromosome 7, 31 cM from the centromere, using an interspecific Mus musculus/Mus spretus backcross (Saunders AM, Seldin MF, Genomics 8:525–535, 1990). However, analysis of mitotic recombinations in mouse skin tumors from intraspecific F1 hybrid mice suggested a more distal location for the Ha‐ras gene on chromosome 7 (Bremner R, Balmain A, Cell 61:407–417, 1990). In the study reported here, we demonstrated, by analysis of Ha‐ras gene mutations in skin tumors from interspecific M. spretus/M. musculus F1 hybrids, the existence only in M. spretus of a pseudogene or other Ha‐ras—related sequence that is probably the sequence originally mapped by Saunders and Seldin. The functional Ha‐ras gene maps to the distal region of chromosome 7, and it is this sequence that acquires mutations in chemically induced tumors.
Genetic predisposition and mesenchymal‐epithelial interactions in ras + myc—induced carcinogenesis in reconstituted mouse prostate
Authors: Thompson Timothy C. , Timme Terry L. , Kadmon Dov , Park Sang H. , Egawa Shin , Yoshida Kazunari
Published: February 2007

Using a mouse prostate reconstitution (MPR) model system, strain‐specific responses to the ras and myc oncogenes were investigated. When ras + myc were introduced into both the mesenchymal and epithelial compartments of the urogenital sinus, poorly differentiated prostate cancer was produced at a high frequency (>90%) in inbred C57BL/6 mice. In contrast, under similar conditions, inbred BALB/c MPRs formed benign prostatic hyperplasia that converted to cancer at a low frequency (<10%). Restricting the oncogenes to the mesenchymal or epithelial compartments revealed that oncogene activities were more pronounced in the mesenchyme of C57BL/6 mice and resulted in elevated transforming growth factor‐β1 expression along with a severe desmoplastic reaction. Heterologous MPRs composed of BALB/c mesenchyme and C57BL/6 epithelium or vice versa demonstrated that intrinsic properties of BALB/c mesenchyme can arrest the progression of ras + myc—initiated C57BL/6 epithelium from benign hyperplasia to malignant carcinoma.