bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct sequencing analysis of transmembrane region of human neu gene by polymerase chain reaction
Authors: Saya Hideyuki , Ara Seiji , Lee Polly S. Y. , Ro Jungsil , Hung Mien‐Chie
Published: February 2007

The neu gene in rat neuro/glioblastoma was found to be activated by a single point mutation in the DNA sequence encoding the transmembrane region of the neu‐encoded p185 protein. The human homologue of the rat neu gene, termed c‐erbB‐2 or HER‐2, can also be activated in vitro by a similar mutation in the corresponding region. Although the human neu gene was shown to be amplified/overexpressed in a large portion of human breast and ovarian cancer, no reports indicate that the human neu gene is activated by a point mutation in human tumor. To study the possible point mutation of neu gene in human tumors, we characterized the genomic structure in the transmebrane region of human neu gene, which in turn allowed us to determine DNA sequence in this region directly following DNA amplification by polymerase chain reaction. We analyzed 7 tumor cell lines (2 breast cancer, 1 neuroblastoma, 1 rhabdomyosarcoma, and 3 glioma) and 11 tumor tissue samples (8 breast and 3 ovarian cancers). No mutation was found in the transmembrane region of human neu gene. Our results suggest that unlike the rat neuro/glioblastoma, the single point mutation in the transmembrance region of the human neu gene is a rare event in human tumors. In this study, we developed a technique for direct DNA sequencing of the transmembrane region of the human neu gene. This technique makes it possible to screen a large number of tumor samples.
Deregulation in trans of c‐myc expression in immortalized human urothelial cells and in T24 bladder carcinoma cells
Authors: Stacey Simon N. , Nielsen Inger , Skouv Jan , Hansen Claus , Autrup Herman
Published: February 2007

The expression of a number of cellular oncogenes was investigated in human urothelial cell lines with different in vitro growth properties. Constitutively elevated levels of expression of c‐myc RNA were found in Hu609, an immortalized, nontumorigenic cell line that was derived from normal urothelium, and in the bladder carcinoma cell line T24. Potential mechanisms that might underlie deregulation of c‐myc expression in these cells were investigated. It was found that the c‐myc gene was apparently intact and not amplified in Hu609 and T24. No increased stability of c‐myc RNA was detected. A c‐myc–CAT fusion construct containing 2.5 kb of normal c‐myc5′ sequences showed levels of expression that paralleled the overexpression of the endogenous gene, indicating that the high constitutive levels of c‐myc expression were due, at least in part, to alterations in the activities of cellular trans‐acting transcriptional regulators.
Enhancement of viral and DNA mediated transformation of cloned rat embryo fibroblast cells by 3‐aminobenzamide
Authors: Su Zao‐Zhong , Zhang Peiquan , Fisher Paul B.
Published: February 2007

We have analyzed the effect of the poly(ADP‐ribose) synthesis inhibitor 3 aminobenzamide (3AB) on de novo and methyl methanesulfonate (MMS) and y irradiation enhancement of viral transformation of a cloned rat embryo fibroblast cell line, CREF, by a cold‐sensitive host‐range mutant of type 5 adenovirus, H5hr1. Additionally, we have evaluated the effect of 3AB on the transformation of CREF cells following transfection with a gene conferring resistance to hygromycin (hyg) or the neomycin analogue G418 (neo) in combination with a cloned type 5 adenovirus E1A transforming gene (Ad5 E1A) or the Ha‐ras (T24) oncogene. 3AB induced a dose‐and time‐dependent increase in the level of de novo MMS‐enhanced and y irradiation‐enhanced transformation of CREF cells by H5hr1, whereas it did not induce morphological transformation in uninfected control, MMS‐pretreated, or y irradiation‐pretreated CREF cells. Temporal kinetic studies indicated that 3AB was most effective in enhancing de novo and MMS‐enhanced and y irradiation‐enhanced viral transformation when applied early after viral and carcinogen plus viral infection and when present for extended time periods (4‐5 wk). 3AB also increased the frequency of resistant colonies following transfection with several cloned genes, including hyg, neo', and protein kinase C (which also contained a neor gene), and the frequency of morphological transformation of CREF cells following cotransfection with a hyg gene and an Ad5 E1A or an activated Ha‐ras (T24) gene. In contrast, 3AB exerted the opposite effect, i.e., an inhibitory effect, when applied to NIH 3T3 cells transfected with a hyg' or neo' gene, alone or in combination with a T24 gene. The ability of 3AB to enhance the frequency of transformation of CREF cells was not associated with a selective effect on the growth of H5hr1 transformed CREF cells in monolayer or agar culture. Similarly, 3AB did not after the percentage of MMS‐or y irradiated‐pretreated H5hr1‐infected cells retaining free Ad5 DNA or the random pattern or quantity of viral DNA integration in control or carcinogen‐treated H5hr1‐transformed cells. These results suggest that cellular processes regulated by the nuclear enzyme ADPRT, or additional processes modified by 3AB, may be important mediators of stable transformation induced by transfected DNA and both de novo and carcinogen‐enhanced viral transformation of specific target cells.
Detection of genomic alterations in carcinogen‐induced mouse liver tumors by DNA fingerprint analysis
Authors: Müller Oliver , Alonso Angel , Buchmann Albrecht , Bauer‐Hofmann Richard , Bock Karl‐Walter , Schwarz Michael
Published: February 2007

DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells. Liver tumors were induced in three strains of mice, namely C57BL/6J, C3H/He and B6C3F1, by a single injection of 20 μg/g body wt. diethylnitrosamine on day 15 after birth. DNA from liver tumors was digested with Hinfl restriction enzyme and hybridized on Southern blots with wild‐type bacteriophage M13 DNA as probe. The resulting fingerprints of tumor DNA were compared with those of DNA from normal liver tissue. Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse.
Retinoid‐enhanced gap junctional communication is achieved by increased levels of connexin 43 mRNA and protein
Authors: Rogers Michael , Berestecky John M. , Hossain Mohammad Z. , Guo Huiming , Kadle Ranjana , Nicholson Bruce J. , Bertram John S.
Published: February 2007

Natural and synthetic retinoids are potent inhibitors of experimental carcinogenesis in animals and cause reversion of premalignant lesions in humans. In the model C3H 10T1/2 cell system, retinoids enhance postconfluent growth control, reversibly inhibit carcinogen‐induced transformation, and enhance gap junctional intercellular communication. These effects are highly correlated. 10T1/2 cells were found to express low levels of connexin 43, a gap junctional protein first found in the heart. After treatment of confluent 10T1/2 cells with the synthetic retinoid tetrahydrotetramethylnapthalenylpropenylbenzoic acid (TTNPB), levels of connexin 43 mRNA and protein increased within 6 h of treatment, while elevation of junctional communication was detected within 12‐18 h. The maximally effective concentration of TTNPB (10−8 M) caused an approximate 10‐fold elevation of connexin 43 gene transcripts after 72 h. Indirect immunofluorescence microscopy using a polyclonal antibody to the synthetic C‐terminal region of connexin 43 demonstrated that TTNPB induced many fluorescent plaques in regions of cell‐cell contact. These results provide a molecular basis for the retinoid‐enhanced junctional communication in 10T1/2 cells. It is proposed that one action of retinoids is to modulate the intercellular transfer of signal molecules. These could mediate many of the physiological actions of retinoids on growth control and carcinogenesis.
Factors influencing the expression of endogenous retrovirus‐like sequences in rat 6 cells
Authors: Hsieh Ling‐Ling , Weinstein I. Bernard
Published: February 2007

Previous studies from this laboratory have demonstrated increased levels of expression of endogenous rat leukemia virus (RaLV) and 30S retrovirus‐like sequences in liver and colon tumors induced by chemical carcinogens in rats. During the process of normal liver regeneration, the levels of RaLV RNA were dramatically increased, whereas the levels of 30S RNA did not change. The present study examined several factors that might influence the expression of these sequences in the Rat 6 embryo fibroblast cell line. Rat 6 cells in either log‐phase or confluent cultures were treated with cycloheximide, the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), or the activated carcinogen benzo[a]pyrene diol epoxide (BPDE) for various periods of time up to 48 h. Northern blot analyses of total RNA indicated that cells in untreated cultures in log phase had higher levels of RaLV and 30S RNAs than did confluent cells. Within 10 h cycloheximide (2 or 10 μg/mL) markedly increased the levels of RaLV and 30S RNAs in both log‐phase and confluent cells. BPDE (100 μg/mL) induced a marked increase in the levels of RaLV RNA at 4 to 10 h, which returned to the basal level by 24 h in the log‐phase cells; but no significant change was seen in the confluent cells. The level of 30S RNA also increased moderately in the BPDE‐treated log‐phase cells and was maximal at 24 h; but no change was seen in confluent cells. Treatment with TPA (100 ng/mL) induced no significant increase in either RaLV or 30S RNA levels in the log‐phase or confluent cells. The exposure of Rat 6 cells to 5‐azacytidine (3 μM for 24 h) led to a marked increase in the levels of both RaLV and 30S RNAs, which persisted during at least 15 subsequent passages in the absence of the drug. Thus, inhibition of protein synthesis, DNA damage, or hypomethylation can increase the expression of certain endogenous retrovirus‐like sequences, but an activator of protein kinase C does not.
The v‐ras oncogene inhibits the expression of differentiation markers and facilitates expression of cytokeratins 8 and 18 in mouse keratinocytes
Authors: Cheng Christina , Kilkenny Anne E. , Roop Dennis , Yuspa Stuart H.
Published: February 2007

Cultured mouse keratinocytes can be initiated in vitro by the introduction of a v‐rasHa gene by viral transduction. Previous studies indicated that v‐rasHa‐transduced keratinocytes have a high proliferation rate in medium with 0.05 mM Ca2+ and resist terminal differentiation in medium with > 0.1 mM Ca2+, a culture condition in which normal cells mature into squames. The current studies demonstrate that v‐rasHa keratinocytes do not express transcripts or protein for epidermal early differentiation markers keratins 1 and 10 when cells are challenged with 0.12 mM Ca2+, which is a signal for expression of these genes in normal cells. Both transcript and protein for the late differentiation marker loricrin are also diminished in v‐ras keratinocytes, but filaggrin, also a late differentiation‐related gene product, is expressed in nearly normal amounts but at a different Ca2+ optimum. Modification of intracellular Ca2+ with ionomycin failed to restore the expression of any suprabasal keratinocyte markers. In contrast to the effects on normal products of keratinocyte differentiation, the introduction of the v‐rasHa gene facilitated the expression of keratins 8 (K8) and 18 (K18). These keratins are characteristic of embryonic cells and cells of simple adult epithelia but not stratified squamous epithelia such as skin. Like normal differentiation markers, the expression of K8 and K18 was dependent both on the v‐ras oncogene and the Ca2+ concentration of the culture medium, with > 0.1 mM Ca2+ being optimal. At the optimal Ca2+ level, the majority of v‐ras keratinocytes expressed K8 and K18 after 96 h, and many cells had reduced amounts of the normal keratinocyte cytokeratin K14. These studies indicate that the v‐ras gene causes substantial reprogramming of epidermal physiology, producing an unusual phenotype devoid of early suprabasal markers but at least partially permissive for late marker expression. Furthermore, the Ca2+‐dependent expression of K8 and K18 suggests that a normal signalling pathway used in keratinocyte differentiation is diverted to an abnormal endpoint.
Expression of c‐myc, c‐raf‐1, and c‐Ki‐ras in azaserine‐induced pancreatic carcinomas and growing pancreas in rats
Authors: Silverman Jeffrey A. , Kuhlmann Elna T. , Zurlo Joanne , Yager James D. , Longnecker Daniel S.
Published: February 2007

We examined the pattern of expression of several proto‐oncogenes during nonneoplastic growth and in acinar cell neoplasms in the rat pancreas. The levels of c‐myc, c‐raf‐1, and c‐Ki‐ras mRNAs were increased in regenerating pancreata following surgical partial pancreatectomy and following administration of camostat. We also investigated proto‐oncogene expression associated with the progression of pancreatic cancers in azaserine‐treated rats. Injection of a single dose (30 mg/kg) of azaserine (O‐diazoacetyl‐L‐serine) to 14‐d‐old rats leads to a variety of neoplastic lesions in the rat pancreas. Total RNA was isolated from lesions in various stages of tumor progression, including adenomas, carcinomas in situ, and invasive carcinomas. We observed increased expression of c‐myc, c‐raf‐1, and c‐Ki‐ras in azaserine‐induced adenomas and carcinomas. Actin expression was also increased in these tissues, whereas amylase expression was variable. However, when compared to the normal growing pancreas, the level of proto‐oncogene expression in the adenomas and carcinomas was disproportionate to the degree of cellular division in those tissues. Thus, the alterations induced by azaserine apparently caused a deregulated increase in expression of cellular oncogenes associated with growth regulation.
Tissue‐specific expression of p53 in transgenic mice is regulated by intron sequences
Authors: Lozano Guillermina , Levine Arnold J.
Published: February 2007

Transgenic mice were produced harboring the p53 murine cDNA clone regulated by the SV40 enhancer‐promoter region 5′ to the cDNA and the small t antigen splice sites, and poly(A) addition signals 3′ to the cDNA. This construction was not expressed in these mice. The presence of several murine p53 introns in the cDNA, however, permitted expression of the transgene mRNA in several tissues of transgenic mice. The insertion of intron 4 led to the preferential expression of the transgene mRNA in spleen cells, where the endogenous p53 gene is also expressed at high levels. While intron 4 promoted high levels of p53mRNA expression in a tissue‐preferred manner in transgenic mice, there was no evidence that intron 4 could act as an enhancer of transcription in cell culture or in transgenic animals. The presence of some p53 introns appears to be critical for the regulation of this gene in vivo.
The moloney murine leukemia virus enhancer and its flanking sequences collaborate to determine virulence in T‐cell lymphomagenesis
Authors: Yuen Pick‐Hoong , Khang Yong‐Ho , Kumar Abhay , Szurek Paul F. , Maull Elizabeth A.
Published: February 2007

A panel of recombinant virus genomes was constructed by exchanging homologous genome fragments between the potent T‐cell lymphoma inducer Moloney murine leukemia virus (MoMuLV) and its closely related but significantly less virulent relative MoMuLV‐TB. Testing of these recombinant viruses in BALB/c mice established that only nucleotide changes within the Clal(‐590)‐Kpnl(36) fragment altered virulence. Fine analysis of this fragment showed that while mutations within the enhancer of MoMuLV‐TB attenuated the latency period most, mutations within the MoMuLV‐TB fragments flanking the enhancer also helped reduce the virulence of MoMuLV. The present study also suggests that the small difference in the relative number of lymphomas that developed primarily in the spleens of MoMuLV‐ or MoMuLV‐TB‐infected mice may correlate with nucleotide differences between the Clal‐Kpnl fragments of the two viruses. However, the significantly greater proportion of premature deaths observed in MoMuLV‐TB‐ relative to MoMuLV‐infected mice could not be correlated with nucleotide differences in a specific genome fragment.
Tyrosine phosphorylation of three cellular proteins correlates with transformation of rat 1 cells by pp60src
Authors: Bouton Amy H. , Kanner Steven B. , Vines Richard R. , Parsons J. Thomas
Published: February 2007

The analysis of phosphotyrosine‐containing proteins in Rat 1 cells overexpressing either the tyrosine kinase pp60c−src or genetic variants containing alterations in functional and structural domains has led to the identification of three proteins whose tyrosine phosphorylation correlated with pp60src‐induced cellular transformation. The tyrosine phosphorylation of one of these proteins, p120, has been previously shown by us and others to coincide with the presence of kinase‐activated, membrane‐associated pp60src‐in chicken embryo cells. The second protein was identified as the ras‐associated GTPase‐activating protein (GAP). The third protein whose tyrosine phosphorylation was markedly elevated in Rat 1 cells expressing activated, membrane‐bound forms of pp60src‐had an apparent molecular mass of 64–67 kDa. The electrophoretic mobility of this protein varied in cells expressing different pp60src variants. The tyrosine‐phosphorylated form of p64–67 was present in immune complexes containing GAP, suggesting a stable interaction between these two cellular proteins.
Effect of myogenic determination on tumorigenicity of chemically transformed 10T1/2 cells
Authors: Hustad Carolyn Marziasz , Jones Peter A.
Published: February 2007

Transforming agents have been postulated to interfere with cellular differentiation programs, thus causing uncontrolled growth. Inducing transformed cells to differentiate can result in loss of the transformed phenotype since many end‐stage differentiated cells are unable to divide. We attempted to bypass or suppress the tumorigenic phenotype of 3‐methylcholanthrene (MCA)‐transformed 10T1/2 cells (MCA CI 15C1) by induction of myogenic determination. MCA CI 15C1 cells were either treated with the hypomethylating drug 5‐azacytidine (5‐aza‐CR) or were transfected with the muscle determination gene MyoD1, both of which induce a myogenic phenotype in 10T1/2 cells. Colonies containing myoblast‐like cells were isolated and examined. Muscle markers were detected both in 5‐aza‐CR‐treated and in MyoD1‐transfected myogenic clones by immunofluorescence and northern analyses. The myogenic clones did not show decreased tumorigenicities relative to that of the parental cells upon subcutaneous injection in nude mice. Some of the resulting tumors, however, were classified as rhabdomyosarcomas rather than fibrosarcomas. Although induction of myogenic determination was not sufficient to abolish the tumorigenic phenotype of MCA CI 15C1 cells, several tumors showed decreased levels of MyoD1 mRNA, suggesting that growth in vivo either selected for or caused decreased determination gene expression.
Preferential mutagenesis at G·C base pairs by the anti 3,4‐dihydrodiol 1,2‐epoxide of 7‐methylbenz[a]anthracene
Authors: Bigger C. Anita H. , Flickinger Denise J. , John Judith St. , Harvey Ronald G. , Dipple Anthony
Published: February 2007

The racemic anti‐dihydrodiol epoxide of 7‐methylbenz[a]anthracene preferentially induced mutations at G·C base pairs in the pS 189 shuttle vector. Mutations were not randomly distributed throughout the supF target gene, but were concentrated at five hotspots. The hotspots for this agent did not correspond exactly to those produced by any other dihydrodiol epoxide examined to date, indicating that dihydrodiol epoxide structure and reactivity play a major role in determining mutagenic hotspots.
DNA methylation and oncogene expression in methapyrilene‐induced rat liver tumors and in treated hepatocytes in culture
Authors: Hernandez Lidia , Petropoulos Christos J. , Hughes Stephen H. , Lijinsky William
Published: February 2007

Continued exposure of rats to carcinogenic doses of methapyrilene (MP) leads to elevated levels of 5‐methyl‐deoxycytidine (5MC) in liver DNA. Since gene expression often correlates with DNA methylation, we investigated these parameters in the MP‐induced hepatocellular carcinomas of Fischer 344 rats. DNA was hypermethylated in liver tissue surrounding the tumors relative to liver tissue of untreated controls of the same age, while tumor DNA was not; DNA methylation declined to normal levels when MP treatment ceased. Gene expression analysis showed measurable levels of mRNA for c‐Ki‐ras, erb‐B, erb‐B2, hck, src, lyn, vav, trk, raf‐1, l‐myc, c‐jun, c‐yes, c‐myc, c‐abl, and p53. No significant differences in expression for these and other oncogenes were seen between tumors and surrounding livers, although erb‐B2 and vav showed visible decreases compared with normal liver. Hypermethylation of DNA and expression of these oncogenes in MP‐treated tissues were not correlated. Levels of mRNA for the same genes in MP‐treated hepatocytes in culture were similar to in vivo levels; analysis of DNA synthesis levels showed that this gene expression pattern occurred in the absence of proliferation bursts or toxicity in these cells, thus suggesting that treatment in vivo may produce the same results.
Topological properties of bovine papillomavirus type 1 (BPV‐1) DNA in episomal nucleoprotein complexes: A model system for chromatin organization in higher eukaryotes
Authors: Rösl Frank , Waldeck Waldemar
Published: February 2007

Sedimentation analysis of isolated episomal bovine papillomavirus type 1 (BPV‐1) nucleoprotein complexes in sucrose gradients and subsequent separation of the purified DNA in chloroquine gels revealed different classes of molecules, varying in their degree of superhelicity. Since torsionally stressed DNA favors the adoption of secondary structures, we employed the single‐strand‐specific S1 nuclease to detect such structural alterations in both naked DNA and native chromatin. Direct examination of nuclease digestion products in chloroquine gels showed that neither the naked DNA nor the BPV‐1 nucleoprotein complexes in isolated nuclei were cleaved randomly by the enzyme. Instead, there was a strict dependence on nuclease susceptibility and the degree of supercoiling, strongly suggesting that the structural features detected by S1 nuclease are due to the occurrence of torsionally stressed viral chromatin. Mapping analysis using the indirect end‐labeling method demonstrated an S1‐nuclease cleavage site adjacent to 20 homopurine residues known to be hypersensitive to S1 attack. Furthermore, direct methylation experiments with viral chromatin in isolated nuclei indicated that only circular, covalently closed nucleoprotein complexes served as substrate, whereas linearized BPV‐1 chromatin was not susceptible to exogenously added Hhal methylase. This observation raises the possibility that the modulation of topology in nucleosomally organized DNA might also play a role in eukaryotic DNA methylation.
Elevation of glucose transporter, c‐myc, and transin RNA levels by Ha‐rasT24 is independent of its effect on the cell cycle
Authors: Godwin Andrew K. , Lieberman Michael W.
Published: February 2007

Elevation of the steady‐state mRNA levels of glucose transporter and c‐myc are among the earliest changes in gene expression observed after Ha‐rasT24 stimulation of Rat‐1 fibroblasts to enter the cell cycle. Since the expression of these genes may be the result of either increased cell proliferation or a specific response to rasT24, we evaluated the expression of glucose transporter and c‐myc and their induction during the cell cycle in both parental Rat‐1 cells and cell lines bearing a metallothionein rasT24 fusion gene (MTrasT24). We showed that, although levels of glucose transporter and c‐myc mRNAs in Rat‐1 cells underwent a transient increase within hours of the addition of serum, epidermal growth factor, or 12‐O‐tetradecanoylphorbol‐13‐acetate to quiescent (G0) cells, the levels of glucose transporter and c‐myc mRNA otherwise remained constant throughout the normal cell cycle. In cells carrying MTrasT24 (MR5 cells), induction of rasT24 expression by ZnSO4 led to a rapid induction of glucose transporter and c‐myc mRNA expression in both quiescent (density‐arrested) and G1/S‐synchronized (aphidicolin‐blocked) cells. These increases exceeded the constitutive levels expressed in rapidly proliferating Rat‐1 cells, indicating that the ras oncogene has an effect on these genes that is independent of growth status. In addition, the transin gene, which is not expressed in proliferating Rat‐1 cells in the continuous presence of serum growth factors, was also induced after increased expression of the mutant ras gene. These results suggest that the induction of glucose transporter, c‐myc, and transin is the direct result of rasT24‐mediated alterations in cellular gene expression and is distinct from normal cell‐cycle events.
Isolation and partial characterization of a transformation‐associated sequence from human nasopharyngeal carcinoma
Authors: Cao Ya , Sun Yi , Poirier Sylvie , Winterstein Dolores , Hegamyer Glenn , Seed John , Malin Sacha , Colburn Nancy H.
Published: February 2007

A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion‐sensitive mouse JB6(P+) C141 cells. To clone this transformation‐associated sequence, we carried out three cycles of transfection, followed by cloning of anchorage‐independent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in both soft‐agar assay and nude‐mice implantation, was used to make a genomic library in the vector λ dash. Using the human repeated sequence Blur 8 to screen the library, we obtained 10 human Alu‐positive clones. A cloned Alu‐positive insert of 16 kbp, CNE 323, was characterized in detail. CNE 323 transferred moderate transforming activity when introduced into JB6 P+ cells and showed no homology to Ha‐, Ki‐, or N‐ras genes; human promotion sensitivity genes; src, myb, jun, myc, fos, raf, or int‐2 oncogenes; or epidermal growth factor receptor. The isolated CNE 323 DNA sequence appeared to preserve the genomic structure of the original sequence found in CNE2 cells and in nude mouse tumors induced by CNE2 cells or by CNE/JB6 transfectant cells, indicating that the cloned NPC sequence was activated during NPC carcinogenesis and not during transfection or construction of the library, and that the cloned sequence or a larger sequence of which it was part played a role in tumor formation. Finally, we identified a 1.3‐kb mRNA that hybridizes to a subclone of the 16‐kb NPC sequence in CNE2 cell poly (A)+ RNA.
A monoclonal antibody to rat liver cytochrome P450 IIC11 strongly and regiospecifically inhibits constitutive benzo[a]pyrene metabolism and DNA binding
Authors: Todorovic Rosa , Devanesan Prabhaker D. , Cavalieri Ercole L. , Rogan Eleanor G. , Park Sang S. , Gelboin Harry V.
Published: February 2007

The monoclonal antibody MAb 1‐68‐11, prepared to constitutive cytochrome P450 IIC11 (2c/RLM5) from male Sprague‐Dawley rat liver, was used to study the contribution of the class of cytochrome P450s epitopically related to P450 IIC11 to the regiospecific metabolism of benzo[a]pyrene (BP) and its binding to DNA. The effect of MAb 1‐68‐11 was determined on the conversion of BP to BP‐9,10‐dihydrodiol, BP‐7,8‐dihydrodiol, BP‐4,5‐dihydrodiol, BP phenols, and BP quinones, and on the P450‐dependent DNA binding catalyzed by P450 in microsomes from uninduced male and female Wistar and Sprague‐Dawley rat livers, as well as 3‐methylcholanthrene‐ and phenobarbital (PB)‐induced male Wistar rat livers. In liver microsomes from untreated male rats, MAb 1‐68‐11 inhibited BP‐9,10‐dihydrodiol formation by 80%; in liver microsomes from untreated female rats, the inhibition was 100%. BP‐7,8‐dihydrodiol formation was inhibited from 38 to 77% in microsomes from males and 50% in those from females. In microsomes from PB‐induced rats, inhibition of the 9,10‐dihydrodiol and the 7,8‐dihydrodiol was 90% and 73%, respectively, whereas BP‐4,5‐dihydrodiol formation was enhanced 80%. In microsomes from 3‐methylcholanthrene‐treated rats, no inhibition of MAb 1‐68‐11 was observed on either the metabolism of BP or its binding to DNA. In contrast, the binding of BP to DNA was completely inhibited by MAb 1‐68‐11 in microsomes from uninduced male Wistar rats and 70% in PB‐induced microsomes. 32P‐postlabeling analysis showed that formation of the major stable adduct, BP diol epoxide bound at C‐10 to the 2‐amino of deoxyguanosine, was strongly inhibited in uninduced and PB‐induced microsomes. Formation of the major labile BP‐DNA adduct 7‐(benzo[a]pyren‐6‐yl) guanine (BP‐N7Gua) was inhibited about 60% in microsomes from untreated male Wistar rats. These results show that MAb 1‐68‐11 regiospecifically inhibits cytochrome P450 IIC11 and epitopically related P450s that metabolize BP at the 7,8 and 9,10 positions. MAb 1‐68‐11 also inhibits enzyme‐catalyzed binding of BP to DNA in the specific formation of BP‐N7Gua and adducts detected by the 32P‐postlabeling technique.
Molecular mechanisms of TPA‐mediated inhibition of Gap‐junctional intercellular communication: Evidence for action on the assembly or function but not the expression of connexin 43 in rat liver epithelial cells
Authors: Asamoto Makoto , Oyamada Masahito , Aoumari Abdelhakim El , Gros Daniel , Yamasaki Hiroshi
Published: February 2007

We found that a rat liver epithelial cell line (IAR 20) expresses connexin 43, the major cardiac gap‐junction protein, but not connexin 26 or connexin 32, major liver gap‐junction proteins. The effects of TPA on connexin 43 expression in IAR 20 were investigated using northern blot analysis, western blot analysis, and an immuno‐fluorescence technique. Gap‐junctional intercellular communication (GJIC) in this cell line decreased within 60 min of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) treatment and recovered after 24 h. The number of immu‐nofluorescence spots of connexin 43 on IAR 20 was closely related to the change in GJIC induced by TPA. However, TPA did not change the level of mRNA measured by northern blot analysis. Moreover, connexin 43 protein expression analyzed by western blotting suggests that connexin 43 proteins were still present in TPA‐treated cells at a similar level. These results suggest that GJIC of these rat liver epithelial cells was mediated by connexin 43 protein and that TPA inhibited GJIC by inhibiting posttranslational processing of connexin 43 proteins, e.g., localization or assembly.
Coordinate activation of multidrug‐resistance (P‐glycoprotein) genes mdr2 and mdr3 during mouse liver regeneration
Authors: Teeter Larry D. , Chan John Y. , Kuo M. Tien
Published: February 2007

We previously reported that only one of the three mouse multidrug‐resistance (mdr) genes, mdr3, is activated in hepatocellular carcinomas (HCCs). The present study examined the expression of mdr family members during mouse liver regeneration after partial hepatectomy to determine whether the regeneration that occurs during hepatic tumorigenesis is responsible for mdr3 elevation in HCC. We demonstrated that in both C3H/HeN and B6C3/F1 mice strains, the levels of both mdr2 and mdr3 mRNAs coordinately increased five‐ to sevenfold 24 h after partial hepatectomy, whereas the levels of mdr1 mRNA were not statistically different from those in the controls. Forty‐eight hours after partial hepatectomy, mdr mRNA levels decreased and in most cases returned to normal levels after 72 h. These results indicate that mdr3 induction during hepatocarcinogenesis is not due to liver regeneration alone.
Frequent activation of the Ki‐ras oncogene at codon 12 in N‐methyl‐N‐nitrosourea‐induced rat prostate adenocarcinomas and neurogenic sarcomas
Authors: Sukumar Saraswati , Armstrong Barbara , Bruyntjes Joost P. , Leav Irwin , Bosland Maarten C.
Published: February 2007

Rat neoplasms induced by methylating carcinogens frequently contain ras genes activated by a single point mutation. Rat prostatic tumors induced by a combination of a single injection of N‐methyl‐N‐nitrosourea (MNU) and long‐term treatment with testosterone were examined for the presence of such activating point mutations in ras genes. These tumors, which arose exclusively in the dorsolateral prostate, included both adenocarcinomas and sarcomas. Activating mutations in codon 12 of the Ki‐ras gene were found in 7 of 10 carcinomas and 4 of 5 sarcomas, using selective oligonucleotide hybridization analysis of DNA amplified by the polymerase chain reaction (PCR). However, no mutated Ha‐ras oncogenes were detected. The presence of PCR‐engineered Hphl restriction sites created by the existence of a G35→A mutation in the rat Ki‐ras oncogene identified the mutation as a GC→A transition at the second position of codon 12. Production of O6‐methylguanine adducts in the Ki‐ras codon 12 followed by base mispairing during replicative DNA synthesis is thus the likely molecular mechanism of initiation of prostatic carcinogenesis by MNU in the rat. Three of the four sarcomas positive for the Ki‐ras G35→A mutation were immunohistochemically defined as of Schwann cell origin, indicating that involvement of the ras gene family is possible in tumorigenesis of this cell lineage. Loss of the wild‐type Ki‐ras allele was also observed in all four of these sarcomas.
Transient expression of the cloned mouse c‐Ha‐ras 5′ upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element
Authors: Neades Renee , Betz Natalie A. , Sheng Xiaoyang , Pelling Jill C.
Published: February 2007

The mouse Ha‐ras oncogene is activated by point mutation and overexpressed in developing papillomas during two‐stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha‐ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5′ upstream region of the mouse Ha‐ras gene so as to characterize various cis‐regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF‐1 factor. In addition, we located a 13‐base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha‐ras 5′ upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two‐stage skin tumorigenesis. Transient gene expression assays carried out after 48‐72 h indicated that a 2.3‐kb Ha‐ras 5′ fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha‐ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha‐ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes.
Evidence that toxic injury is not always associated with induction of chemical carcinogenesis
Authors: Tennant Raymond W. , Elwell Michael R. , Spalding Judson W. , Griesemer Richard A.
Published: February 2007

Long‐term rodent bioassays with chemicals administered at maximum tolerated doses identify noncarcinogens as well as carcinogens. Thirty‐one chemicals recently evaluated for carcinogenic potential by the National Toxicology Program provide unique data on the relationships between mutagenicity, toxicity, and carcinogenicity. Twenty‐two substances were classified as carcinogens, and nine showed no evidence of carcinogenicity. Although cellular proliferation does play an intrinsic role in neoplastic processes, the responses associated with chronic toxicity in these studies were not always sufficient to induce neoplasia. Regardless of their mutagenic potential, 19 carcinogens induced toxic effects at sites that did not show neoplastic changes; similar toxic lesions were also seen among the mutagenic and nonmutagenic noncarcinogens. Although many nonmutagens induced neoplasia at sites that showed toxic effects, some of the same chemicals also exhibited toxicity at other sites that showed no neoplastic effect. These results suggest that for some chemicals, properties other than mutagenicity or toxicity may be responsible for their carcinogenic potential.
Two polychlorinated hydrocarbons cause phospholipid‐dependent protein kinase C activation in vitro in the absence of calcium
Authors: Rotenberg Susan A. , Weinstein I. Bernard
Published: February 2007

Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)‐β1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol‐bis(β‐aminoethyl ether) N,N,N′,N′,‐tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor‐promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Northern blot analysis of expression of TPA‐inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds.
Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells
Authors: Wölfel Catherine , Platt Karl‐Ludwig , Dogra Satish , Glatt Hansruedi , Wächter Felix , Doehmer Johannes
Published: February 2007

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P4501A2. Full‐length cDNA encoding rat P4501A2 was obtained by searching a cDNA library made from Aroclor 1254‐induced rat liver mRNA and by joining a small 5′‐end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full‐length cDNA was inserted into a simian virus 40 early promoter‐containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate‐coprecipitation technique. G418‐resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P4501A2‐specific enzymatic activities such as hydroxylation of 17β‐estradiol and 2‐aminofluorene.
Activating point mutation in Ki‐ras codon 63 in a chemically induced rat renal tumor
Authors: Higinbotham Kathleen G. , Rice Jerry M. , Perantoni Alan O.
Published: February 2007

Renal mesenchymal tumors induced in F344 rats with methyl(methoxymethyl)nitrosamine (DMN‐OMe) have previously been shown by our laboratory to contain transforming Ki‐ras sequences, activated most commonly by a variety of codon 12 mutations. Further sequence analysis of one DMN‐OMe‐induced tumor with transforming Ki‐ras sequences detected by NIH 3T3 transfection assay but with no mutation in codon 12 detected by selective oligonucleotide hybridization has now revealed an activating point mutation in codon 63. The observed GAG→AAG transition in codon 63, which replaces glutamic acid with lysine, was the only detectable mutation in exon 1 and 2 hotspot regions of Ki‐ras in this tumor. The same mutation was also detected in Ki‐ras sequences derived from first‐ and second‐cycle transformants in NIH 3T3 transfection assays. Although random mutagene‐sis studies of cloned Ha‐ras sequences by Fasano et al. (Proc Natl Acad Sci USA 81:4008–4012, 1984) had already indicated that GAG→AAG mutations in codon 63 of ras are transforming, this is the first demonstration of the natural occurrence of this particular activating mutation in a tumor. © 1992 wiley‐List, Inc.
Acquisition of responsiveness to chemical carcinogens by rodent embryo fibroblasts expressing high levels of the c‐myc proto‐oncogene
Authors: Hsiao W. L. Wendy , Castro Mark , Giezentanner John , Fan Hung , Hanecak Ronnie
Published: February 2007

We investigated the ability of overexpression of the c‐myc proto‐oncogene to potentiate in vitro transformation by model chemical carcinogens. A mouse c‐myc gene was introduced to C3H 10T1/2 and Rat 6 embryo fibroblast cell lines via a retroviral vector containing the gene for neomycin resistance. Our present work extends previous findings by showing that individual vectored C3H 10T1/2 clones have enhanced (twofold to sevenfold) sensitivity to benzo[a]pyrene (BP) and N‐methyl‐N‐nitro‐N′‐nitrosoguanidine (MNNG). Rat 6 clones acquiring the c‐myc gene display various degrees of altered morphology. They form orderly but densely packed cells, grow to higher saturation density, and yield microcolonies in soft agar. The degree of altered growth properties is directly correlated with the level of c‐myc expression. Transient exposure of c‐myc‐expressing clones to BP and MNNG induced the formation of distinct, large colonies in soft agar, whereas the untreated cells formed microcolonies and the parental Rat 6 cells remained single cells in soft agar. We also demonstrated that the degree of responsiveness to chemical carcinogens of the clones correlates with their ability to form microcolonies in soft agar. These cells overexpressing c‐myc may be used as a model system to study the interaction between oncogenes and chemical carcinogens in the process of multistage carcinogenesis. © 1992 Wiley‐List, Inc.
Multiple molecular alterations in mouse lung tumors
Authors: Re Fabio C. , Manenti Giacomo , Borrello Maria G. , Colombo Mario P. , Fisher James H. , Pierotti Marco A. , Porta Giuseppe Della , Dragani Tommaso A.
Published: February 2007

Twenty‐five mouse lung tumors induced by a single urethan treatment in female A/J, BALB/c, and (A/J × C3H/He)F1 (AC3) mice were analyzed for the presence of mutations at codon 61 of the Ki‐ras gene and for the expression of the surfactant protein A (SP‐A). retinoblastoma (Rb), growth arrest‐specific‐3 (gas‐3), p53, c‐myc, and thymidylate synthase (TS) genes. Ki‐ras codon 61 mutations were detected in 22 of 25 tumor samples without differences among strains. In comparison with normal lungs, all the tumors showed increased SP‐A mRNA levels, indicating their derivation from alveolar type II pneumocytes or Clara cells. Rb and gas‐3 transcripts were instead found in all tumors at about tenfold and about 20‐fold reduced levels, respectively. No apparent structural alterations or loss of heterozygosity at the Rb locus was detected in any tumors. The p53 mRNA was observed without variation in quantity or size in lung tumors and normal tissue. A threefold to fivefold c‐myc overexpression was observed, without amplification of the gene. TS expression was only slightly increased, indicating no great differences in cell proliferation between lung tumors and normal tissue. Our data suggest that the pathogenesis of urethan‐induced lung tumors in mice involves specific and recurrent molecular alterations (Ki‐ras mutations, decrease of Rb and gas‐3 expression, and increase of c‐myc expression) that could represent different steps in lung carcinogenesis. © 1992 Wiley‐List, Inc.
Characterization of the syrian hamster c‐Ha‐ras gene and intron‐d‐exon transcript
Authors: Ebert Rolf , Wiseman Roger W. , Barrett. J. Carl , Reiss Eva , Rollich Günther , Schiffmann Deitmar
Published: February 2007

The coding sequences as well as 5′‐ and 3′‐flanking sequences of the Syrian hamster c‐Ha‐ras gene were deduced from cDNA clones derived from embryo fibroblast cell lines. Sequences of introns B, C, and D were obtained from genomic DNA after amplification by the polymerase chain reaction. Sequence comparisons with rat, mouse, and human c‐Ha‐ras genes revealed a high degree of homology. One of 12 cDNA clones contained intron‐D‐exon (IDX) sequences due to alternative splicing that would encode a p19 Ha‐ras gene product. Conservation between species suggests a functional role for the IDX, possibly as a negative control of p21 Ha‐ras expression. © 1992 Wiley‐Liss, Inc.
Efficient protection against oxidative DNA damage in chromatin
Authors: Ljungman Mats , Hanawalt Philip C.
Published: February 2007

The role of histones and higher order chromatin structures in protecting against oxidative DNA damage was investigated using an in vitro system consisting of nuclear and nucleoid monolayers as model chromatin substrates. These substrates, derived from human skin fibroblasts, were challenged with hydroxyl radicals produced via a Fenton reaction involving Fe(ll)‐ethylenediaminetetraacetic acid and ascorbic acid. The resulting DNA strand breaks were measured using the alkaline unwinding technique. The sequential removal of chromosomal proteins from the DNA by pretreating nuclear monolayers with increasing concentrations of salt dramatically increased the frequency of hydroxyl radical‐induced DNA strand breaks. Furthermore, the DNA in decondensed chromatin was found to contain 14‐fold fewer DNA strand breaks than naked, supercoiled DNA, whereas the DNA of “native” chromatin and “condensed” chromatin contained 100‐fold and 300‐fold fewer breaks, respectively. We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical‐induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage. © 1992 Wiley‐Liss, Inc.
Evidence for coincident mutations in human lymphoblast clones selected for functional loss of a thymidine kinase gene
Authors: Li Chuan‐Yuan , Yandell David W. , Little John B.
Published: February 2007

A mitotic “recombination‐competent state” inducible by x‐irradiation is thought to exist in yeast. We sought evidence for such a process in mammalian cells by examining the occurrence of mutations at unlinked loci in clones derived from a human lymphoblast cell line. A total of 169 independent clones that arose spontaneously or after exposure to x‐rays or ethyl methanesulfonate were selected for new somatic mutations at the thymidine kinase gene on chromosome 17q. They were subsequently screened for coincident mutations by use of variable‐number‐of‐tandem‐repeat probes located on different chromosomes. Three coincident mutations were positively identified by Southern analysis on chromosomes 7 and 14; they included one that produced a new allele and two that caused loss of allele heterozygosity. Densitometric analysis of the latter two indicated the presence of two copies of the remaining allele. Several possible coincident genetic events were also observed on chromosome 17. These findings revealed a coincident mutant fraction of about 10−2/cell, whereas the expected mutation fraction at these loci is less than 10−4/cell. These results may thus provide the first molecular evidence that a “global” mutational process capable of inducing genetic instability exists in mammalian cells. © 1992 Wiley‐Liss, Inc.