Article

A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line

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  • University of Vermont Health Network, UVM Medical Center, Burlington, VT
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Abstract

In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of int-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of int-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by int-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that int-1 plays a functional role in MMTV-induced mammary tumorigenesis.

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... Ectopic expression of the normally silent Wnt-1 locus results from the introduction of transcriptional enhancers contained in the mouse mammary tumor virus long terminal repeats (30,31). Formal proof of a causative role for Wnt-1 in mammary oncogenesis has come from experiments on gene transfer into mammary epithelial cell lines (5,41) and transgenic mice (50). ...
... In C57MG cells, an epithelial cell line derived from normal mouse mammary tissue (51), Wnt-1 expression causes a morphological transformation (5,18). Normal C57MG cells grow in a monolayer with a regular, cuboidal appearance at confluence; Wnt-1 expression causes the cells to become refractile and elongated, growing over other cells in a disorganized pattern. ...
... WNT-I was first identified as a mammary oncogene activated by MMTV (Nusse et al., 1984). It contributes to mammary oncogenesis when overexpressed (Nusse et al., 1984; Brown et al., 1986; Riisewilk et al., 1987a,b; Tsukamoto et al., 1988; Blasband et aL, 1992; Donehower et al., 1995). Experimental studies showed that WNT-l induces morphological transformation of mammary epithelial cells (lulius et al., 1999). ...
... The stabilization of catenins is post-transcriptional and results from a decrease in the rate of beta-catenin molecule degradation (Hinck et al., 1994). Wildtype APC is able to down-regulate beta-catenin levels (Munemitsu et al., 1995), while the loss of APC function reduces beta-catenin turnover and generates a signal equivalent to that of the WNT-mammary oncogene (Brown et al., 1986; Tsukamoto et al., 1988; Edwards et al., 1992; Jue et al., 1992). The stabilization of catenins by WNT-I is primarily the result of a selective increase in the amount of uncomplexed, monomeric beta-and gamma-catenin (Papkoff et al., 1996). ...
Article
Reductions incell-cell adhesion andstromal andvascular invasion areessential steps intheprogression fromlocal- ized malignancy tometastatic disease forall cancers. Proteins involved inintercellular adhesion, suchasE-cadherin andcatenin, probably play animportant role inmetastatic processes andcellular differentiation. While E-cadherin andbeta-catenin expression hasbeenextensively studied inmanyforms ofhumancancers, less isknownabout therole oftheWingless-Type- 1(WNT- 1)path- wayinhumantumors. Alarge bodyofgenetic andbiochemical evidence hasidentified beta-catenin asakey downstream compo- nent oftheWNTsignaling pathway, andrecent studies ofcolorectal tumors haveshownafunctional link amongbeta-catenin, ade- nomatous polyposis coli geneproduct (APC), andother components oftheWNT-Ipathway. WNT-Ipathway signaling isthought to bemediated viainteractions between beta-catenin andmembers oftheLEF-l/TCF family oftranscription factors. TheWNT signal stabilizes beta-catenin protein andpromotes itsaccumulation inthecytoplasm andnucleus. Inthenucleus, beta-catenin associ- ates with TCFtoformafunctional transcription factor which mediates thetransactivation oftarget genes involved inthepromotion oftumorprogression, invasion, andmetastasis, suchasC-Myc, cyclin Dl,c-jun, fra-l, andu-PAR. There isastrong correlation between theability oftheWNT-1genetoinduce beta-catenin accumulation anditstransforming potential invivo, suggesting that theWNT-lgeneactivates anintracellular signaling pathway that caninduce themorphological transformation ofcells. Forthese reasons, data obtained fromthestudy oftheWNT-1pathway could beimportant inourunderstanding ofthemechanisms ofepithe- lial tumors, ingeneral, andprobably also oforal squamous cell carcinoma, inparticular.
... Ectopic expression of Wnt-1 under the control of a mouse mammary tumor virus promoter leads to extensive mammary hyperplasia and subsequent generation of adenocarcinomas in mice (4). Cell culture experiments demonstrate that multiple Wnt gene family members including Wnt-1 can cause partial cellular transformation of some epithelial and fibroblastic cell lines (5)(6)(7)(8)(9)(10)(11)(12). Collectively, these data implicate Wnt-1 as an oncogene when inappropriately expressed. ...
... To examine the effect of Wnt-1 on Cox-2 expression, we generated fresh cell populations expressing Wnt-1 by infection of the mouse mammary epithelial cell lines C57MG and RAC311 with retrovirus encoding Wnt-1 (MVWnt-1) or control retrovirus (MV7). As observed previously (5,6), both C57/Wnt-1 and RAC/Wnt-1 cells appeared morphologically transformed and grew to higher cell densities than control cells (C57/MV7 and RAC/MV7, respectively). An additional clonal subline, RAC/Wnt-1 #9, was generated from RAC/ Wnt-1 by limiting dilution and selected because of its high degree of morphological transformation. ...
Article
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Wnt-1 acts as a mammary oncogene when ectopically expressed in the mouse mammary gland. APC is a tumor suppressor gene, mutations in which cause intestinal tumorigenesis in humans and rodents. Both Wnt-1 expression and APC mutation activate a common signaling pathway in- volving transcriptional activation mediated by b-catenin/Tcf complexes, but few targets relevant to carcinogenesis have yet been identified. Ex- pression of the inducible prostaglandin synthase cyclooxygenase-2 ap- pears critical for intestinal tumorigenesis resulting from APC mutation, suggesting that cyclooxygenase-2 might be a transcriptional target for b-catenin/Tcf complexes. Here, we have investigated the effect of Wnt-1 on cyclooxygenase-2 expression. Wnt-1 expression in the mouse mammary epithelial cell lines RAC311 and C57MG induces stabilization of cytosolic b-catenin and morphological transformation. Expression of Wnt-1 in these cells caused transcriptional up-regulation of the cyclooxygenase-2 gene, resulting in increased levels of cyclooxygenase-2 mRNA and protein. Prostaglandin E2 production was increased as a consequence of the ele- vated cyclooxygenase-2 activity and could be decreased by treatment with a selective cyclooxygenase-2 inhibitor. Cyclooxygenase-2 thus appears to be a common downstream target for APC mutation and Wnt-1 expression. In view of the critical role of cyclooxygenase-2 in intestinal tumorigenesis, cyclooxygenase-2 up-regulation in response to Wnt signaling may contrib- ute to Wnt-induced mammary carcinogenesis.
... Since we had previously observed Cox-2 up-regulation in Wnt1-expressing cell lines (13), we were interested to determine whether PEA3 factors could play a role in this up-regulation. In particular, we had observed significantly increased Cox-2 transcript in response to Wnt1 in C57MG cells, a mouse mammary epithelial cell line which undergoes Wnt1-induced morphological transformation (48). Therefore PEA3 transcripts were measured in C57MG-derived cell lines, and also in mammary tumors from Wnt1 transgenic mice. ...
Article
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The inducible prostaglandin synthase cyclooxygenase-2 (COX-2) is aberrantly expressed in intestinal tumors resulting from APC mutation, and is also transcriptionally up-regulated in mouse mammary epithelial cells in response toWnt1 expression. β-Catenin stabilization is a consequence of both APC mutation and Wnt signaling. We have previously observed coordinate regulation of the matrilysin promoter by β-catenin and Ets family transcription factors of the PEA3 subfamily. Here we show that while β-catenin only weakly activates theCOX-2 promoter, PEA3 family transcription factors are potent activators of COX-2 transcription. Consistent with this, PEA3 is up-regulated in Wnt1-expressing mouse mammary epithelial cells, and PEA3 factors are highly expressed in tumors from Wnt1 transgenic mice, in whichCox-2 is also up-regulated. Promoter mapping experiments suggest that the NF-IL6 site in the COX-2 promoter is important for mediating PEA3 responsiveness. The NF-IL6 site is also important for COX-2 transcription in some colorectal cancer lines (Shao, J., Sheng, H., Inoue, H., Morrow, J. D., and DuBois, R. N. (2000) J. Biol. Chem. 275, 33951–33956), and PEA3 factors are highly expressed in colorectal cancer cell lines. Therefore, we speculate that PEA3 factors may contribute to the up-regulation of COX-2 expression resulting from bothAPC mutation and Wnt1 expression.
... Wnt-1 was the first Wnt ligand demonstrated to cause mammary tumors. Early on, Wnt-1 expression was shown to cause striking changes in morphological and growth properties in mammary epithelial cell lines [127]. Wnt-1 was then shown to induce numerous mammary tumor formation in vivo [128] and was shown to transform primary human mammary epithelial cells alone without a cooperating oncogene [129]. ...
... Other mechanisms that are known to affect the copy number of transcriptional regulatory genes (e.g. partial replication, extrachromosomal duplications, aneuploidy, viral infection) could also result in changes in transcriptional activity, and in some cases have been linked to oncogenesis (Brown et al., 1986;Schwab et al., 1983;Varmus, 1984). 14 Genomic rearrangements. ...
Article
Historically, knowledge of gene-specific transcription has been accumulated by the study of the individual genetic and physical interactions between transcriptional regulators and the genes they regulate, often requiring considerable time and effort. Microarray technology now enables investigation of gene expression at the level of the entire genome, allowing researchers access to rich datasets and promising new levels of depth in the understanding of transcriptional regulation. Our lab has made use of these technologies both to measure the levels of all mRNA transcripts within a population of cells, as well as to locate the regions within the genome that are bound by transcriptional regulators. Such studies not only allow for the functional annotation of both genes and regulators, but can also provide clues about the identity of the regulatory regions within DNA, the structure of global regulatory networks and the regulation of DNA-binding proteins. These and other insights are presented here based on our genome-wide studies of transcriptional regulation in the yeast Saccharomyces cerevisiae.
... Additionally, group A cell lines exhibited lower expression of their target proteins FZD5, TMEM92 and HNF1B. The expression of Wnt-related genes leads to cell shape changes toward elongation (Brown et al, 1986), and the canonical Wnt/bcatenin signalling cascade has been revealed to be deregulated in PDAC cells (Zeng et al, 2006;Bu et al, 2008). b-Catenin, the central molecule of the Wnt/b-catenin pathway, connects it to the main intercellular adhesion mechanism, the cadherin/catenin complex. ...
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Background: Invasion of the surrounding tissue is part of the metastatic cascade. Here, we examined the invasion of pancreatic ductal adenocarcinoma (PDAC) cells into the mesothelial barrier and identified the related microRNA (miRNA) expression profiles. Methods: The interactions between PDAC cells and mesothelial monolayers were characterised and quantified using a specific time-lapse videomicroscopy assay. Pancreatic ductal adenocarcinoma cells were further evaluated using the adhesion assay, and miRNA, mRNA and protein expressions were determined using microarray, q-RT-PCR and western blots, respectively. These data were correlated with in vivo dissemination scores. Results: Two groups of PDAC cell lines were distinguished by their integration capacity into the mesothelial monolayer using mean elongation factors (MEFs). Adhesion assays showed a concordant relation between adhesive properties and integration capacity. The distant metastases scores were reverse correlated with MEFs. Microarray analysis of these groups revealed that miR-23a and/or miR-24 target for FZD5, HNF1B and/or TMEM92, respectively, and that they are significantly deregulated. Conclusions: MiR-23a and/or miR-24 overexpression leads to gene silencing of FZD5, TMEM92 and/or HNF1B. Their downregulation induces deregulated expression and degradation of E-cadherin and β-catenin causing destabilisation of the cadherin/catenin complex, and altered the expression of Wnt-related genes. We propose a molecular (epi)genetic mechanism by which increased EMT-like cell shape transformation and integration into mesothelial monolayers of PDAC cells can be observed.
... In 1991, Nusse R et al proposed a new nomenclature to call Int-1 and related genes Wnt, which is an amalgam of Wingless and Int (Nusse R. 1991). Wnt1 was shown to induce morphological transformation in cultured cell lines, including C57MG mammary epithelial cells (Brown AM et al. 1986;Jue SF et al. 1992), RAC311 mammary cells (Rijsewijk F et al. 1987b), PC12 pheochromocytoma cells (Bradley RS et al. 1993), and C3H 10T1/2 fibroblasts (Bradbury JM et al. 1994). Then people extended to all the other Wnts, and found that different Wnts have distinct ability in transforming activity; hence the Wnt family members were divided into two groups based on this biological activity: ...
... The importance of the model placing APC function between GSK-3 and APC was that it suggested a mechanism for APC's role in colon tumorigenesis. The loss of APC function was proposed to reduce β-catenin turnover, thereby generating a signal equivalent to that of the Wnt-1 mammary oncogene178179180181. Support for this model was provided by the finding that β-catenin was mutated in some melanoma and colon-cancer cell lines [121,122,168,176]. ...
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The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in diverse developmental processes. The loss or inappropriate activation of Wnt expression has been shown to alter cell fate, morphogenesis and mitogenesis. Recent progress has identified Wnt receptors and components of an intracellular signalling pathway that mediate Wnt-dependent transcription. This review will highlight this 'core' Wnt signal-transduction pathway, but also aims to reveal the potential diversity of Wnt signalling targets. Particular attention will be paid to the overlap between developmental biology and oncogenesis, since recent progress shows Wnt signalling forms a paradigm for an interdisciplinary approach.
... 1.1.26. epha4-Expression im kranialen Abschnitt des präsomitischen Mesoderms sowie in den zuletzt gebildeten Somiten wachsende Axone zu ihren Zielorten (Kennedy et al. 1994, Serafini et al. 1996 (Brown et al. 1986, Peters et al. 1986, McMahon u. Moon 1989, Nusse 1990. ...
... Forced expression of Wnt-1 causes mammary epithelial hyperplasia and tumor formation (Brown et al., 1986;Tsukamoto et al., 1988). In addition, ectopic expression of Wnt-1 enhances proliferation in the developing mammalian central nervous system (Dickinson et al., 1994). ...
Article
β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell–cell adhesion through its association with cadherins. To explore the in vivo effects of β-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH2-terminal truncation mutant (ΔN89β-catenin) was expressed in the 129/Sv embryonic stem cell–derived component of the small intestine of adult C57Bl/6–ROSA26↔ 129/Sv chimeric mice. ΔN89β-Catenin was chosen because mutants of this type are more stable than the wild-type protein, and phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila. ΔN89β-Catenin had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative compartment of the intestine (crypts of Lieberkuhn). The proliferative response was not associated with any discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin at the adherens junctions and basolateral surfaces of 129/Sv (ΔN89β-catenin) intestinal epithelial cells and an accompanying slowing of cellular migration along crypt-villus units. 1–2% of 129/Sv (ΔN89β-catenin) villi exhibited an abnormal branched architecture. Forced expression of ΔN89β-catenin expression did not perturb the level or intracellular distribution of the tumor suppressor adenomatous polyposis coli (APC). The ability of ΔN89β-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during the 10-mo period that animals were studied. Together, these in vivo studies emphasize the importance of β-catenin in regulating normal adhesive and signaling functions within this epithelium.
... For preparation of Wnt3a conditioned medium [16], L-Wnt3a cells (ATCC CRL-2647) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS for 4 days, and conditioned medium (CM) [16] was collected and sterilely filtered. Wnt activity in CM was measured using the Wnt-responsive mammary epithelial reporter cell line (C57MG) [17] infected with WntRgreen transcriptional reporter virus (C57MG/WGR) [18]. In these cells, the GFP reporter is activated by Wnt3a-stimulated β-catenin/TCF signaling and the percentage of activated cells relates linearly to Wnt activity. ...
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Populations: Lgr5+ and Lgr5- basal cells and c-Kit+ and c-Kit- luminal cells that spontaneously organize into a ductal structure with basal cells around the periphery and luminal cells lining an interior cavity, reminiscent of normal mammary duct structure. Lgr5+ cell-derived organoids were sustainable during prolonged passaging. In contrast, although Lgr5- cells expand into primary colonies, colony-forming efficiency immediately dissipated upon passaging. Furthermore, reproductive hormones induce epithelial cell proliferation resulting in marked increases in lumen diameter accompanied by squamous transdifferentiation. We propose this estrogen-responsive, self-organizing duct-like structure derived from single murine Lgr5+ mammary cells represents a "mini-breast" organoid.
... We thus asked whether the modulation of PLEKHA4 levels would affect Wnt signaling strength. For these assays, we used a mouse fibroblast cell line (C57MG) responsive to Wnt stimulation that contained a Wnt-inducible GFP transcriptional reporter called WntRGreen (Brown et al., 1986;Santiago et al., 2012), as well as human cell lines such as HeLa cells. ...
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... One approach towards investigating such responses is to characterize the phenotypic changes elicited by expression of the gene in cultured cell lines. Wnt-1 causes morphological transformation and deregulated growth of certain mammary epithelial cells (4,65) and also induces distinct morphological changes in PC12 pheochromocytoma cells, a neural crest-derived tumor cell line with both neuronal and epithelial characteristics (19,23,68). ...
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subscriptions, please fill the attached reprint order form. 6. Should you require information about your article (publication date, volume, page numbers, etc) please call: +30-22950-52945 or send an e-mail to journals@iiar-anticancer.org. 7. Please provide your complete address (not P.O.B.), telephone and fax numbers for the delivery of reprints and issues. 8. Please feel free to contact us with any queries that you may have (Tel./Fax: +30-22950-53389 or +30-22950-52945, e-mail: journals@iiar-anticancer.org). Abstract. Oral cancer is a common neoplasm worldwide. The incidence and mortality have also increased in recent decades. It is characterized by poor prognosis and a low survival rate despite sophisticated surgical and radiotherapeutic modalities. The WNTs comprise a large family of highly conserved growth factors associated with a number of functions. In this review, we focus largely on the canonical pathway, revealing the recent findings, in oral cancer research, thereby raising our understanding of the mechanisms of this crucial signaling in several cellular activities.
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The rat interleukin-5 (IL-5) gene was isolated from a genomic lambda phage library and a fragment containing all four exons was inserted into the retroviral vector pXT1, resulting in pXTRIL5. Upon retroviral gene transfer into two IL-5-dependent mouse cell lines, B13 and T88M, autonomously growing cells were established and B-cell growth factor activity was detected in the supernatants of the infected cells. “cDNA” versions of the rat IL-5 gene were rescued by the polymerase chain reaction (PCR) with primers specific for the flanking regions of the cloning site in pXT1. Restriction or DNA sequence analysis of five different clones revealed precise splicing in two cases, while three of the clones had retained the first intron. In addition, in two of these about 400 bp of rat IL-5 5′ flanking regions were deleted. The sequence comparison of rat, mouse, and human IL-5 genes revealed a high degree of conservation (e.g., mouse and rat were 92% homologous at the amino acid level). The combination of retroviral gene transfer and PCR may offer an alternative, efficient method for the cloning of cDNAs.
Article
The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity and subsequent work has now demonstrated its importance in critical and diverse aspects of biology. Since those early discoveries, Dishevelled has been shown to coordinate a plethora of developmental and cellular processes that range from controlling cell polarity during gastrulation to partnering with chromatin modifying enzymes to regulate histone methylation at genomic loci. While the role of DVL in development is well-respected and the cytosolic function of DVL has been studied more extensively, its nuclear role continues to remain murky. In this review we highlight some of the seminal discoveries that have contributed to the field, but the primary focus is to discuss recent advances with respect to the nuclear role of Dishevelled. This nuclear function of Dishevelled is a dimension which is proving to be increasingly important yet remains enigmatic.
Article
The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.
Article
The mouse Wnt family comprises at least 10 members sharing substantial amino acid identity with the secreted glycoprotein Wnt-1/int-1. Two of these, Wnt-1 and Wnt-3, are implicated in mouse mammary tumor virus-associated adenocarcinomas, although neither member is normally expressed in the mammary gland. These results suggest the presence of active cellular pathways which mediate the action of Wnt-1 and Wnt-3 signals. An understanding of the normal role of these signalling pathways is clearly necessary to comprehend the involvement of Wnt-1 and Wnt-3 in mammary tumorigenesis. We demonstrate here that five Wnt family members are expressed and differentially regulated in the normal mouse mammary gland. In addition, some of these genes are also expressed in both Wnt-1-responsive and nonresponsive mammary epithelial cell lines. We propose that Wnt-mediated signalling is involved in normal regulation of mammary development and that inappropriate expression of Wnt-1, Wnt-3, and possibly other family members can interfere with these signalling pathways.
Article
The mouse Wnt-1 gene plays an essential role in fetal brain development and can contribute to tumorigenesis when activated aberrantly in the mammary gland. The gene encodes secretory glycoproteins associated with the extracellular or pericellular matrix, and it has been proposed that Wnt-1, as well as its Drosophila homolog wingless, may function in intercellular signalling. We show here that fibroblasts expressing Wnt-1 protein, although not transformed themselves, are able to elicit morphological transformation of neighboring C57MG mammary epithelial cells in coculture experiments. Heparin inhibits this effect, possibly by displacing Wnt-1 protein from its normal site of action. Our results indicate that the Wnt-1 gene can act via a paracrine mechanism in cell culture and strongly support the notion that in vivo the gene may function in cell-to-cell communication.
Article
The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.
Article
The Wnt-1 and int-2 proto-oncogenes are transcriptionally activated by mouse mammary tumor virus insertion mutations in virus-induced tumors and encode secretory glycoproteins. To determine whether these two genes can cooperate during carcinogenesis, we have crossed two previously characterized lines of transgenic mice to obtain bitransgenic animals carrying both Wnt-1 and int-2 transgenes under the control of the mouse mammary tumor virus long terminal repeat. Mammary carcinomas appear earlier and with higher frequency in the bitransgenic animals, especially the males, than in either parental line. Nearly all bitransgenic males develop mammary neoplasms within 8 months of birth, whereas only 15% of Wnt-1 transgenic males and none of the int-2 transgenic males have tumors. In virgin bitransgenic females, tumors occur approximately 2 months earlier than in their Wnt-1 transgenic siblings; int-2 transgenic females rarely exhibit tumors. Preneoplastic glands from the bitransgenic animals of either sex demonstrate pronounced epithelial hyperplasia similar to that seen in Wnt-1 transgenic virgin females and males, and both transgenes are expressed in the hyperplastic glands and mammary tumors. RNA from the int-2 transgene is more abundant in mammary glands from bitransgenic animals than from int-2 transgenic animals; the increase is associated with high levels of RNA specific for keratin genes 14 and 18, suggesting that Wnt-1-induced epithelial hyperplasia is responsible for the observed increase in expression of the int-2 transgene.
Article
The mouse Wnt family includes at least 10 genes that encode structurally related secreted glycoproteins. Wnt-1 and Wnt-3 were originally identified as oncogenes activated by the insertion of mouse mammary tumor virus in virus-induced mammary adenocarcinomas, although they are not expressed in the normal mammary gland. However, five other Wnt genes are differentially expressed during development of adult mammary tissue, suggesting that they may play distinct roles in various phases of mammary gland growth and development. Induction of transformation by Wnt-1 and Wnt-3 may be due to interference with these normal regulatory events; however, there is no direct evidence for this hypothesis. We have tested Wnt family members for the ability to induce transformation of cultured mammary cells. The results demonstrate that the Wnt gene family can be divided into three groups depending on their ability to induce morphological transformation and altered growth characteristics of the C57MG mammary epithelial cell line. Wnt-1, Wnt-3A, and Wnt-7A were highly transforming and induced colonies which formed and shed balls of cells. Wnt-2, Wnt-5B, and Wnt-7B also induced transformation but with a lower frequency and an apparent decrease in saturation density. In contrast, Wnt-6 and two other family members which are normally expressed in C57MG cells, Wnt-4 and Wnt-5A, failed to induce transformation. These data demonstrate that the Wnt genes have distinct effects on cell growth and should not be regarded as functionally equivalent.
Article
Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters.
Article
The murine int-1 proto-oncogene has been implicated in neural development and, when transcriptionally activated by mouse mammary tumor virus, contributes to the genesis of mammary tumors. To understand the function of the int-1 gene product in these processes, we have characterized the biochemical properties of int-1 protein expressed in a neuroendocrine cell line transfected with int-1 cDNA. Here we provide evidence that int-1 protein is secreted and associates with the cell surface. int-1 protein was very efficiently processed and secreted through the constitutive secretory pathway, although no int-1 protein could be immunoprecipitated from the culture medium. Treatment with suramin effectively released mature int-1 proteins into the culture fluid, which suggests that secreted int-1 protein associates with the cell surface or extracellular matrix. We have also shown directly, by radioiodination of intact cells and by surface antibody adsorption, that secreted int-1 proteins can be detected on the cell surface. These data support a model in which int-1 protein is secreted and functions locally in cell-to-cell signaling.
Article
The int-1 proto-oncogene is a target for insertional activation of transcription by mouse mammary tumor virus in many murine mammary tumors. Whereas no expression of int-1 is seen in normal mammary tissue, int-1 RNA can be detected in normal mice in the neural tubes of midgestation embryos and in postmeiotic spermatocytes from adult testes. I report here the results of a study in which several different antibodies against synthetic peptides were produced and used to characterize the processing and secretion of int-1 protein. CHO cells were transfected with an inducible int-1 expression vector that was subsequently amplified to generate cell lines expressing very high levels of int-1 protein. Immunoprecipitation of [35S]cysteine-labeled cell lysates from these CHO cells yielded large amounts of four immature forms of int-1 glycoprotein (molecular weights of 36,000, 38,000, 40,000, and 42,000). A significant fraction of these int-1 species formed disulfide-linked multimers. Pulse-chase and glycosidase digestion studies demonstrated that some of the immature species of int-1 protein move through the secretory pathway and are processed to a mature heterogeneous glycoprotein with a molecular weight of about 44,000. Suramin treatment of the CHO cells during pulse-chase experiments increased the amount of 44,000-molecular-weight int-1 protein in the culture medium.
Article
Wnts are glycosylated proteins secreted from various cell types including mesenchymal, hematopoietic and epithelial cells. Directional secretion of Wnts in polarized epithelial cells is unique; Wnt11 is secreted apically, whereas Wnt5a and Wnt3a are secreted basolaterally. Here, we found that Wnt1 is equivalently secreted both apically and basolaterally in MDCK cells. Wnt1 was modified with a complex- or hybrid-type glycan at Asn29 and Asn359 and the high-mannose- or hybrid-type glycan at Asn316. Although glycosylation of Wnt11 at the N-terminal site was shown to be essential for its apical secretion, glycosylation of Asn29 of Wnt1 was not required. Instead, the apical secretion of Wnt1 was inhibited by knockdown of Sec6 and Sec8, suggesting that Wnt1 is secreted apically via exocyst-mediated transport. Basolateral secretion of Wnt1 was mediated by clathrin and AP-1, in mechanism similar to that used by Wnt5a and Wnt3a. Although Wingless was reported to be transcytosed to the basolateral region in the Drosophila wing disc, transcytosis was not involved in the basolateral secretion of Wnt1. Thus, the polarized secretion of Wnt1 is regulated by different mechanisms than other Wnts.
Article
In recent years we have witnessed a revolution in the understanding of the mechanisms which regulate mammalian cell growth. This applies both to normal cells, in which growth is tightly controlled, as well as to cancer cells, which divide in an uncontrolled fashion. With the advent of molecular biology a number of genes have been identified whose products are involved in regulating normal cell growth. In parallel research 40–60 genes that are capable of inducing a transformed phenotype and that have been termed “on-cogenes” have been identified. It is now clear that growth control genes on the one hand and oncogenes on the other are largely one and the same. Although unequivocal evidence for this notion was obtained only recently, the concept itself emerged gradually beginning with the discovery of viral oncogenes (v-onc genes) and the fact that these genes represent cell-derived sequences (termed protooncogenes, cellular oncogenes, or c-onc genes).
Chapter
Oncogenes were first characterised as distinct genes in acutely transforming RNA tumour viruses. A combination of genetic and molecular techniques confirmed that a single gene carried by the virus, the viral oncogene, was solely responsible for the malignant growth of cells in the infected host (for review, see Bishop and Varmus 1984). There is no evidence to suggest that acutely transforming retroviruses play any role in the development of human malignancies, and a broader definition of an oncogene has evolved to encompass cellular sequences that are affected by genetic changes and thereby contribute to the initiation or maintenance of the malignant phenotype. The clearest example of this occurs when rearrangement, amplification or mutation of a gene is present in a tumour cell. In this case the gene is considered to be a proto-oncogene, the implication being that the genetic changes observed represent the conversion of this normal gene into a dominantly acting oncogene. In such cases, stronger justification for the term oncogene can be obtained by reintroducing the cloned gene into non-transformed cells and demonstrating a biological effect.
Chapter
Since its discovery over 50 years ago, interest in mouse mammary tumor virus (MMTV) has been fuelled by the conviction that virally induced mammary carcinomas in the mouse will serve as an informative model system for the corresponding disease in humans, despite the absence of unequivocal indicators of viral etiology in humans. Perhaps more realistic, given the experience with other retrovirus-related malignancies (1), was the hope that the mouse model might unearth potential oncogenes that can contribute to the disease, and allow a direct evaluation of their relevance to human breast cancer and its treatment. Within the past four or five years, these hopes have been at least partially fulfilled in the identification of a series of cellular genes, the so-called int genes, that appear to have been the targets for transcriptional activation in MMTV- induced tumors. The earliest identified and best understood examples are the int-1 and int-2 genes, located on mouse chromosomes 15 and 7 respectively, although it seems certain from recent findings that other int -like genes exist and that others await discovery (2–9).
Chapter
One approach to understanding the control of growth and three-dimensional organisation of mammary epithelium is to identify genes that may be involved and then perturb their expression. We have developed a method for expressing genes specifically in mouse mammary epithelium by reconstituting mammary epithelium from genetically manipulated cells (Edwards et al., 1988; Edwards 1993). We summarise here recent results we have obtained with genes of the erbB and Wnt families.
Chapter
The Wnt gene family is a large group of related genes encoding secreted proteins that function as extracellular signaling factors. At least 18 distinct Wnt genes have so far been described in mammals, and homologs have been isolated from a broad range of metazoan species from nematodes to humans. Both genetic analysis and experimental studies have shown that Wnt genes function in a variety of critical developmental processes and these data imply that Wnt signals have the potential to regulate several aspects of cell behavior, including cell proliferation, cell fate determination, and cell-cell adhesion (for reviews, see refs. 1 and 2). While there has been enormous interest in Wnt genes because of their multiple roles in embryonic development, the original Wnt gene, mouse Wnt1, was discovered as an oncogene activated in mouse mammary carcinomas, and other members of the Wnt gene family are also strongly implicated in the genesis of such tumors (3–5). There is now compelling evidence that Wnt proteins serve to regulate mammary development in the mouse, and it is likely that they fulfill a similar role in humans. Ever since the initial identification of Wnt genes as mammary oncogenes in the mouse, it has been a reasonable possibility that aberrant expression of WNT genes could contribute to human breast cancer. This idea has been strengthened by evidence that several human WNT genes are over-expressed in breast cancers, and by recent revelations that downstream components of the Wnt signaling pathway are activated in other forms of human cancer (6,7). It is therefore timely to review the evidence linking Wnt genes to breast cancer, both in mouse model systems and in the human disease.
Chapter
Historically, retroviruses were first isolated and studied because they efficiently cause tumors in experimental and domestic animals (Rous, 1911; Ellerman and Bang, 1908). Work was particularly intense during the 1970s, under the “War on Cancer” and the Special Virus Cancer Program. Important discoveries from re-trovirology such as oncogenes and proto-oncogenes (Bishop, 1987) have had profound implications for understanding tumorigenesis, including for spontaneous human tumors. Retroviruses remain some of the best model systems for studying carcinogenesis in the whole organism, since they induce tumors with extremely high efficiency. The focus of this chapter will be retroviral carcinogenesis in small animal models. Major attention will be paid to nonacute retroviruses, since they induce disease through multiple steps, with analogies to non-virus-induced cancer as well.
Chapter
Nearly all of the known retroviral oncogenes have proto-oncogene counterparts detectable in human DNA. Many of these proto-oncogenes, activated through a variety of mechanisms, have been associated with human cancers. Perturbations in the structure or expression of certain proto-oncogenes appear to be the general means by which this activation occurs. Examples of structural changes are seen in single amino acid substitutions in the ras family of genes isolated from human leukemias, colon carcinomas, and bladder carcinomas. In chronic myelogenous leukemia, the translocation between chromosomes 9q and 22q results in the formation of a novel fusion gene termed bcr-abl whose gene product differs from that of the normal c-abl protein in size and in the ability to autophosphorylate. Perturbations in the expression of a proto-oncogene can be due to the presence of abnormal regulatory elements as in the case of translocations involving c-myc seen in Burkitt’s lymphomas, or due to amplification which augments expression by increasing the gene copy number. Amplifications of N-myc in neuroblastoma, and erB-2/neu in human breast cancer have been correlated with a more advanced stage and a poorer prognosis. Recently, specific genetic elements which suppress tumorigenicity have been described. Several such elements have been localized to human chromosomes 11 and 13q leading to the conceptualization of cancer suppressor genes. In this paradigm, the loss of both alleles of such a suppressor gene, rather than the “activation” of a proto-oncogene, is responsible for tumorigenesis. In human retinoblastomas, the inactivation of a specific gene on chromosome 13q, called rb-1, is critical for neoplastic transformation. In each case described, perturbations in any one gene may be involved in different phases (early or late) of oncogenesis depending on the disease context. Also, quite frequently, many genetic abnormalities are associated with a tumor that obscures the significance of any single genetic change. Furthermore, reliance on animal model systems to explain human malignancy must be taken with some caution since evidence exists implicating different oncogenes in the genesis of similar tumors depending on the species studied (man vs. mouse). To this point, we have suggested some associations between oncogenes and the neoplastic state. What evidence do we have that endogenous transforming genes are involved in human cancer? All of the retroviral oncogenes discovered thus far have cellular homologues (proto-oncogenes) that are thought to be involved in normal cellular function (see Chapter 5). If these normal genes are to cause cancer, their structure or their expression must be perturbed. Examples of such changes include the following: 1. Point mutations within the gene 2. Genetic rearrangements within the coding sequence of the gene 3. Genetic rearrangements outside the coding region 4. Amplification and/or overexpression of the gene Each of these mechanisms results in the “activation” of one or another of the cellular proto-oncogenes that has been associated experimentally with human cancer. An additional mechanism for which there is preliminary experimental evidence is: 5. Deletion of possible “anti-oncogenes”.
Article
The WNT/β-catenin signaling pathway plays important roles in development and homeostasis of the gastrointestinal tract. The molecular mechanisms and key factors in this evolutionary ancient signaling pathway are described in this chapter.
Article
The proto-oncogene int-1 is implicated in mouse mammary tumour formation and encodes a protein of unknown function that is probably secreted. int-1 has a highly restricted pattern of normal expression, limited to the mature testis and a transient expression during mouse development. To gain clues as to the normal function of int-1 we have examined the embryonic sites of int-1 RNA accumulation by in situ hybridization. int-1 expression is restricted to a subset of cells in the developing central nervous system. In the spinal cord, these cells comprise the non-neuronal roof plate. Possible functions of int-1 in the organization of the mouse neural tube are presented in light of the recent finding that the Drosophila homologue of int-1 is wingless, a segment polarity gene.
Article
Full-text available
A major proportion of carcinomas induced by mouse mammary tumour virus (MMTV) show evidence for proviral activation of a cellular gene, int-2, on chromosome 7. The sequence of 7869 bp of DNA spanning the transcription unit of int-2 was determined and compared with that of a series of int-2-specific cDNA clones derived from mammary tumour RNA. The predicted positions of intron-exon boundaries, established by alignment of cDNA and chromosomal DNA sequences, indicate that the gene comprises at least three exons. An open reading frame capable of encoding a protein of 245 amino acids with an estimated mol. wt of 27 kd, is flanked by substantial non-coding segments at both 5' and 3' ends. Comparison of the chromosomal DNA sequence and the predicted amino acid sequence with available data-bases has revealed no homology to other known genes. These results are discussed in relation to the status of int-2 as a candidate proto-oncogene.
Article
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The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41-kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase.
Article
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Two loci independently implicated in T-and B-lymphocyte neoplasia are shown to be equivalent. The Mis-1 locus is a common proviral integration site in retrovirally induced rat T lymphomas, while the pvt-1 locus on murine chromosome 15 frequently translocates to the kappa locus in plasmacytomas bearing 6;15 translocations. By comparing cloned sequences, we show that pvt-1 is the murine homolog of Mis-1.
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A new technique for assaying infectivity of adenovirus 5 DNA has been developed. Viral DNA was diluted in isotonic saline containing phosphate at a low concentration, and calcium chloride was added, resulting in the formation of a calcium phosphate precipitate. DNA coprecipitated with the calcium phosphate and, when the resulting suspension was added to human KB cell monolayers, became adsorbed to the cells. Following adsorption, uptake of DNA into the cells occurred during an incubation in liquid medium at 37 ° in the continued presence of extra calcium chloride.For adenovirus 5 DNA the assay resulted in up to 100-fold more plaques than could be obtained using DEAE-dextran. Furthermore a reproducible relationship between amounts of DNA inoculated per culture and numbers of plaques produced was demonstrated. The assay was most efficient at high DNA concentrations (10–30 μg/ml); below this range the addition of carrier DNA was necessary for optimum results.In addition to adenovirus 5 DNA, the technique has been used successfully to assay infectivity of DNA from adenovirus 1 and simian virus 40.
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The endogenous, vertically transmitted proviral DNAs of the ecotropic murine leukemia virus in AKR embryo fibroblasts were found to be hypermethylated relative to exogenous AKR murine leukemia virus proviral DNAs acquired by infection of the same cells. The hypermethylated state of the endogenous AKR murine leukemia virus proviruses in these cells correlated with the failure to express AKR murine leukemia virus and the lack of infectivity of cellular DNA. Induction of the endogenous AKR murine leukemia virus proviruses with the methylation antagonist 5-azacytidine suggested a causal connection between DNA methylation and provirus expression. Also found to be relatively hypermethylated and noninfectious were three of six Moloney murine leukemia virus proviral DNAs in an unusual clone of infected rat cells. Recombinant DNA clones which derived from a methylated, noninfectious Moloney provirus of this cell line were found to be highly active upon transfection, suggesting that a potentially active proviral genome can be rendered inactive by cellular DNA methylation. In contrast, in vitro methylation with the bacterial methylases MHpaII and MHhaI only slightly reduced the infectivity of the biologically active cloned proviral DNA. Recombinant DNA clones which derived from a second Moloney provirus of this cell line were noninfectious. An in vitro recombination method was utilized in mapping studies to show that this lack of infectivity was governed by mechanisms other than methylation.
Article
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Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
Article
This chapter summarizes briefly the past literature on the studies of spontaneous mouse mammary cancer and discusses in detail the discoveries that have been made only recently. Mammary tumor studies on mice began with the first description of such tumors in a wild mouse in the year 1854. This is an area of tumor biology that was studied first by pathologists, was nurtured by geneticists and endocrinologists, and is only lately being pursued vigorously by investigators from other disciplines such as virology, immunology, biochemistry, molecular biology. Mouse mammary tumors offer many advantages for tumor biological studies. The chapter also talks about mammary tumor virus (MTV), their hormonal influences on mouse mammary neoplasia, and the two categories of inbred strains of mice employed in mammary cancer research—susceptible strains and resistant strains. The mouse mammary tumor system still remains an area of interest to researchers from different disciplines. One of the great advantages of this system remains the amenability of the host animals to laboratory experimentation.
Article
We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins.
Article
This chapter focuses on the genetics of susceptibility to mammary tumorigenesis under the influence of (1) exogenous virus infections and (2) the genetic control of the release of endogenous viruses and subsequent tumorigenesis. Essentially, it describes the transmission of mammary tumor virus (MTV), genetics of susceptibility to exogenous MTV, and endogenous viruses. It also presents some instances in which mammary tumors occur without clear demonstration of virus expression. MTV has a relatively wide organ distribution; however, the mammary gland is the only tissue that can be transformed by MTV. An important aspect of endogenous mammary tumor viruses in mice is their oncogenicity for the natural host. Endogenous mammary tumor viruses are considered good tools for understanding neoplastic transformation. In contrast to the somatic provirus, the germinal provirus is present in all cells of the organism and is transmitted via the germ cells to the offspring. The somatic provirus can be present only in those cell species that have a receptor for the exogenous virus.
Article
An epithelioid cell line derived from the mammary glands of a C57BL/6 mouse and designated C57MG cell line was found to be susceptible to infection by routine mammary tumor virus (MuMTV). Although uninfected C57MG cells contain endogenous MuMTV-related DNA sequences, no RNA sequences homologous to MuMTV were detectable, even after treatment with the glucocorticoid, dexamethasone. However, after infection with MuMTV, these cells acquire additional MuMTV DNA, and viral RNA and proteins were readily detectable. Most, if not all, of the additional MuMTV DNA in infected C57MG cells appeared to be integrated. Synthesis of viral RNA and protein, and the release of virions into culture fluid by infected C57MG cells, was stimulated by incorporation of dexamethasone in the growth medium. The efficiency of infection by MuMTV in C57MG cells was similar to that in nonmurine cells. There was a direct relationship between multiplicity of infection (m.o.i.) and the average number of MuMTV RNA molecules detected per cell 5 weeks after infection. However, even at the highest m.o.i. used (4 × 105 virions/cell), a plateau in the average number of viral RNA molecules per cell was not achieved. The origin of MuMTV synthesized by infected C57MG cells, whether it is the progeny of infecting MuMTV or of the endogenous C57BL/6 MuMTV or of both, remains undetermined. We have been unable to detect any morphological or growth pattern changes in C57MG cells following infection with MuMTV.
Article
The int-1 and int-2 genes were first isolated as targets for transcriptional activation by proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus (MMTV). Since these proto-oncogenes are not expressed at detectable levels in previously tested normal tissues from adult mice, we sought to determine whether these genes might be active during embryogenesis by examining mouse embryos and cultured teratocarcinoma cells for RNA encoded by int-1 and int-2. A single size class of int-1 RNA is present only at mid-gestation stages of development (days 8 through 13) and is also detected in testes from postpuberal mice. Four species of int-2 RNA are found in peri-implantation embryos and teratocarcinoma cells and are particularly abundant in derivatives of the primitive endodermal lineage, but int-2 RNA is not detected during mid- or late gestation or in any normal adult tissues tested. Thus, these two proto-oncogenes, activated during mammary carcinogenesis by the same mechanisms, are normally expressed at different times and places in embryonic and adult mice.
Article
We constructed several retroviruses which transduced a mutant dihydrofolate reductase gene that was resistant to methotrexate inhibition and functioned as a dominant selectable marker. The titer of dihydrofolate reductase-transducing virus produced by virus-producing cells could be increased to very high levels by selection of the cells in increasing concentrations of methotrexate. Helper virus-free dihydrofolate reductase-transducing virus was also generated by using a broad-host-range amphotropic retroviral packaging system. Cell lines producing helper-free dihydrofolate reductase-transducing virus with a titer of 4 X 10(6) per ml were generated. These retroviral vectors should have general utility for high-efficiency transduction of genes in cultured cells and in animals.
Article
The mouse mammary tumor virus can induce mammary tumors in mice by proviral activation of an evolutionarily conserved cellular oncogene called int-1. Here we present the nucleotide sequence of the human homologue of int-1, and compare it with the mouse gene. Like the mouse gene, the human homologue contains a reading frame of 370 amino acids, with only four substitutions. The amino acid changes are all in the hydrophobic leader domain of the int-1 encoded protein, and do not significantly alter its hydropathic index. The conservation between the mouse and the human int-1 genes is not restricted to exons; extensive parts of the introns are also homologous. Thus, int-1 ranks among the most conserved genes known, a property shared with other oncogenes.
Article
The int-1 mammary oncogene is activated by proviruses of the Mouse Mammary Tumor Virus in many different mammary tumors. We have inserted a genomic fragment containing the protein-encoding domain of the gene into the retroviral shuttle vector pZIPneoSV(X)1. After one round of virus replication we recovered recombinant proviral DNA containing a correctly spliced copy of int-1.In vitro transcription of this cDNA version of int-1 using SP6 polymeraae and translation in a reticulocyte lysate yielded a protein of approximately 37,000 daltons. High expression of int-1 in NIH-3T3 cells infected with recombinant virus did not lead to morphological transformation.
Article
The induction of tumours by retroviruses lacking transduced oncogenes can involve the transcriptional or functional activation of cellular proto-oncogenes by an integrated provirus. Thus, the two cellular genes int-1 and int-2, identified as common targets for activation by mouse mammary tumour virus (MMTV), may constitute previously unrecognized oncogenes. In tumours, proviral insertion at these loci leads to expression of messenger RNAs which are undetectable in normal mammary glands. Here we report that in a survey of the two transcriptional activity and structural integrity of the two int loci in 30 BR6 mouse mammary tumours, around 50% of the tumours expressed both of these genes, in ostensibly monoclonal cell populations. Our data suggest that int-1 and int-2 may act cooperatively in the genesis of mammary carcinomas. However, because three tumours (10%) involved neither gene, and because in five cases activation occurred in the apparent absence of an adjacent provirus, it is clear that other loci and mechanisms contribute to tumorigenesis.
Article
Retrovirus vectors allow efficient transfer of genetic material into cells. We describe an improved method for making cell lines which secrete broad host range retrovirus vectors in the absence of helper virus. This method was used to make virus-producing cell lines from several retrovirus vector constructions that encode dominant selectable markers. Virus titers from such lines exceeded 10(6) colony-forming units per milliliter of medium exposed to the cells. Cell lines that secreted certain vectors remained free of helper virus, while cell lines made using other vectors always secreted helper virus. Secretion of helper virus apparently depended on recombination between vector and the retrovirus packaging system, and factors involved in this event were investigated.
Article
Nine epithelial cell strains were established from normal mouse liver, mammary gland, ovary, and ear skin. After overnight digestion by collagenase in growth medium, intact glandular fragments produced many islands of epithelial outgrowth when plated in an enriched medium. Contaminating fibroblasts were removed by selective trypsinization. These strains continue to show morphologic and ultrastructural features typical of epithelial cells after 10-50 subcultures. In addition, several strains continue to form secretory vesicles, or hemicysts, on confluent cell sheets. All but 1 strain maintained a majority population of diploid cells, but all strains show increasing numbers of near triploid cells. Five strains produced tumors (benign cystadenomas, adenocarcinomas, and sarcomas) when inoculated into isogeneic mice. Type C oncornaviruses were seen in all but 2 strains.
Article
Many mammary tumors induced by mouse mammary tumor virus (MMTV) contain a provirus in the same region of the host-cell genome, leading to expression of a putative cellular oncogene called int-1. Here we present the structure and nucleotide sequence of int-1. We have established several proviral insertion sites exactly by nuclease S1 analysis or by molecular cloning and DNA sequencing. The protein-encoding domain of int-1 is distributed over four exons. At the 5' end of the gene two overlapping exons were detected, one of which is preceded by a TATA box. The deduced int-1-encoded protein has 370 amino acids, with a preponderance of hydrophobic residues at the NH2 terminus. Proviruses are found at both sides of the gene, usually oriented away from the gene. Downstream integrations occur frequently in the long 3' untranslated region of the last exon. One upstream provirus is inserted in the 5' untranslated region and, unlike the other upstream insertions, in the same orientation as the int-1 gene. Proviral integrations always leave the protein-encoding domain intact, providing further evidence that the int-1 protein contributes an essential step in mammary tumorigenesis.
Article
We have asked whether oncogenesis by the mouse mammary tumor virus (MMTV), a slowly oncogenic retrovirus, involves integration of viral DNA within a certain region of the host genome. We first identified a C3H mouse mammary tumor bearing a single new MMTV provirus and cloned a 19 kilobase (kb) DNA restriction fragment containing a junction of viral and host sequences. Host sequences from this clone were used to retrieve 25 kb of the uninterrupted locus (termed MMTV int1) from a bacteriophage library of normal mouse DNA. Hybridization with subcloned DNA fragments of MMTV int1 detected abnormal restriction fragments in digests of DNA from 18 of 26 C3H mammary tumors. The rearrangements all appeared to be due to the insertion of an MMTV provirus, and the integration sites were located in at least five clusters over a total distance of 19 kb. A polyadenylated 2.6 kb RNA species transcribed from int1 was found in the few tumors tested, but not in lactating mammary glands from C3H mice. Of 12 tested viral oncogenes, none exhibited homology with cloned DNA from this locus. We propose that tumorigenesis by MMTV is strongly favored by proviral insertion within the int1 locus, perhaps as a consequence of enhanced expression of a novel cellular oncogene.
Article
We have prepared specific probes for unique-sequence cellular DNA adjacent to each of the newly integrated proviruses in tumors induced by mouse mammary tumor virus (MMTV). The use of such probes to screen a large number of independent mammary tumors in the BR6 strain of mouse has indicated that in at least 17 out of the 40 tumors examined so far, an MMTV provirus has integrated into a common chromosomal domain. A 10 kb Eco RI fragment of single copy DNA from this region has been isolated and partially characterized by restriction enzyme mapping. Of the proviruses located within this fragment in different tumors, all but one are complete, in the same orientation, and clustered within about 3 kb of cellular DNA. These findings are consistent with an insertional mutagenesis model for tumorigenesis by MMTV, in which the integration of a provirus in a particular region of cellular DNA may activate a neighboring oncogene. The region we describe here appears to be different from that reported for mammary tumors in the C3H strain of mouse.
Article
Most mammary carcinomas induced in C3H mice by the mouse mammary tumour virus (MMTV) bear a new proviral insertion within a highly conserved locus on chromosome 15 called int-1. A transcriptional unit within this locus is inactive in all tested normal tissues but expressed at low levels in mammary tumours with proviral insertions positioned on either the 5' and 3' sides of the gene. Transcription of the proviruses proceeds away from int-1; thus an indirect mechanism appears to activate expression of this putative oncogene.
Article
Approximately 50% of tumors induced by mouse mammary tumor virus (MMTV) contain an acquired provirus within a limited region of chromosomal DNA, termed int-2. We have extended our previous characterization of this locus and have mapped provirus integration sites in 21 independent tumors. Although integration occurs at multiple sites, proviruses within int-2 are distributed into two oppositely oriented groups whose transcription is directed away from a central domain. Provirus insertion in int-2 is accompanied by expression of RNA derived, at least in part, from this central domain. Since the RNA is not detected in normal mammary tissue, we conclude that MMTV integration activates the expression of a cellular gene within int-2 and that this event may contribute to tumorigenesis.
Article
A number of mink cell focus-forming (MCF) proviruses was molecularly cloned from mouse lymphoma DNA. From each clone, flanking probes were prepared to detect common integration regions in other MuLV-induced lymphomas. One clone frequently revealed variations in the molecular structure of the corresponding region (Pim-1) in other lymphomas. The results show the following. Changes in the Pim region are seen in 24 out of 93 lymphomas tested. Over 50% of the early T-cell lymphomas show integration in the Pim-1 region. The alterations are seen in different mouse strains and with various MuLVs. The observed variations are caused by the integration of predominantly MCF genomes. All integrations occur in a region spanning less than 20 kb and are associated with the transcriptional activation of a distinct region within the Pim-1 domain. The activated region does not show any homology with 13 known and three putative oncogenes.
Article
We develop a murine retrovirus shuttle vector system for the efficient introduction of selectable and nonselectable DNA sequences into mammalian cells and recovery of the inserted sequences as molecular clones. Three protocols allow rapid recovery of vector DNA sequences from mammalian cells. Two of the methods rely on SV40 T-antigen-mediated replication of the vector sequences and yield thousands of bacterial transformants per 5 X 10(6) mammalian cells. The majority of plasmids recovered by all three protocols exhibited the proper structure and were as active as the parental vector in the generation of transmissible retrovirus genomes upon transfection of mammalian cells. One of the rescue methods, which relies on "onion skin" replication and excision of an integrated provirus from the host chromosome, enables facile recovery of the chromosomal site of proviral integration. The system was also used to generate, and then efficiently recover, a cDNA version of a genomic insert from the adenovirus E1A region.
Article
The chromosomal breakpoint of chronic lymphocytic leukemia (CLL) cells of the B-cell type carrying the translocated long arms of chromosomes 11 and 14 [t(11;14) (q13;q32)] was cloned. The breakpoint was found to be within the joining segment of the human heavy chain locus on the translocated long arm of chromosome 14. A probe that is specific for chromosome 11 and that maps immediately 5' to the breakpoint on the 14q+ chromosome was isolated. The probe detected a rearrangement of the homologous genomic DNA segment in the parental CLL cells and also in DNA from a diffuse large cell lymphoma with the t(11;14) translocation. This rearranged DNA segment was not present in Burkitt lymphoma cells with the t(8;14) translocation or in nonneoplastic human lymphoblastoid cells. The probe can thus be used to identify and characterize a gene located on band q13 of chromosome 11 that appears to be involved in the malignant transformation of human B cells carrying the t(11;14) translocation. This gene, named bcl -1, appears to be unrelated to any of the known retrovirus oncogenes described to date.
Article
A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.
Article
This chapter discusses the molecular basis of nonviral tumorigenesis. Indirect experiments have suggested that DNA damage is a central step in transformation, largely because DNA-damaging agents tend to be carcinogens. However, this realization of the importance of DNA damage does not facilitate an understanding of which DNA sequences must be damaged in order to induce transformation. The chapter illustrates how the process of gene transfer makes possible the resolution of some of the above issues. It discusses a model that invokes a cellular oncogene as being centrally important in the process of oncogenic transformation. According to this model, such an oncogene derives from a normally innocuous cellular DNA sequence whose inappropriate activation during oncogenesis confers a novel transforming potency on the altered gene. This study concludes two points. First, the oncogenic sequence is of endogenous cellular origin. Second, the DNA sequences encoding transformation comprise a discrete unit of function and may represent an allelic variant of a normal cellular gene. This, in turn, implies that the activation of critical cellular genes, not the global activation of large portions of the genome, is important for oncogenic transformation.
A new technique for the assay of the infectivity of adenovirus 5 DNA.
  • Graham F.L.
  • van der Eb A.J.
Mammary neoplasia in mice.
  • Nandi S.
  • McGrath C.M.
Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15.
  • Nusse R.
  • van Ooyen A.
  • Cos D.
  • Fung Y.K.
  • Varmus H.E.
Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosomal translocation.
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  • Coce C.M.
Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15
  • Nusse