Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

Disruption of retinoblastoma protein family function by human papillomavirus type 16 E7 oncoprotein inhibits lens development in part through E2F-1.

Complexes between the retinoblastoma protein (pRb) and the transcription factor E2F-1 are thought to be important for regulating cell proliferation. We have shown previously that the E7 oncoprotein from human papillomavirus type 16, dependent upon its binding to pRb proteins, induces proliferation, disrupts differentiation, and induces apoptosis when expressed in the differentiating, or fiber, cells of the ocular lenses in transgenic mice. Mice that carry a null mutation in E2F-1 do not exhibit any defects in proliferation and differentiation in the lens. By examining the lens phenotype in mice that express E7 on an E2F-1 null background, we now show genetic evidence that E7's ability to alter the fate of fiber cells is partially dependent on E2F-1. On the other hand, E2F-1 status does not affect E7-induced proliferation in the undifferentiated lens epithelium. These data provide genetic evidence that E2F-1, while dispensible for normal fiber cell differentiation, is one mediator of E7's activity in vivo and that the requirement for E2F-1 is context dependent. These data suggest that an important role for pRb-E2F-1 complex during fiber cell differentiation is to negatively regulate cell cycle progression, thereby allowing completion of the differentiation program to occur.

Multiple genetic loci modify risk for retinoblastoma in transgenic mice.

PURPOSE: Forty percent of cases of retinoblastoma, a childhood malignancy of the retina, are linked to the inheritance of a mutant allele of the retinoblastoma susceptibility gene Rb1. Tumor penetrance varies among carriers in different family pedigrees, indicating that other genetic factors may modify risk for occurrence of retinoblastoma. This study was undertaken to determine whether multiple genetic loci modify the risk for retinoblastoma in mice. METHODS: A line of alphaAcry-HPV16E6/E7 transgenic mice expressing the human papillomavirus type 16 E6 and E7 oncogenes (HPV-16 E6 and E7) ectopically in the retina was characterized. E6 and E7 proteins bind to and inactivate the cellular tumor suppressor proteins p53 and Rb, respectively. RESULTS: Retinoblastomas developed rarely when the alphaAcry-HPV16E6/E7 transgene was maintained on the FVB background, but tumors arose with high frequency on C57BL/6 X FVB and C3H x FVB F1 hybrid backgrounds. The incidence of retinoblastoma in the LHbeta-TAG transgenic mice, which express simian virus 40 large tumor antigen (SV40 T-ag), was also influenced by the FVB and C57BL/6 backgrounds. Resistance of the alphaAcry-HPV16E6/E7 FVB mice to retinoblastoma mapped in part to the retinal degeneration (rd) locus. However, multiple genetic experiments indicate that resistance to retinoblastoma depends on additional loci in FVB mice. CONCLUSIONS: Multiple cellular genes can modify risk for retinoblastoma in mice.

Disruption of lens fiber cell differentiation and survival at multiple stages by region-specific expression of truncated FGF receptors.

To determine if fibroblast growth factor signaling mechanisms are required for terminal differentiation and survival of lens fiber cells, we evaluated the effects of expressing truncated fibroblast growth factor receptors (tFGFRs) in different regions of the developing lens. Two sets of transgenic mice were generated, one expressing tFGFRs from the alphaA-crystallin promoter (alphaA-tFGFR), which expresses linked genes in fiber cells throughout their differentiation program, and the other expressing tFGFRs from the gammaF-crystallin promoter (gammaF-tFGFR), which expresses linked genes beginning later during their differentiation. Histological and TUNEL analyses of lenses from alphaA-tFGFR and gammaF-tFGFR transgenic mice suggest that FGFR signaling is required for both early and late fiber cell differentiation and/or survival of the terminally differentiated cells. Additionally, multilayering and increased levels of apoptosis were observed in the anterior epithelium after the onset of fiber cell abnormalities. In situ hybridizations suggest that tFGFR transgenes were not expressed at significant levels in the epithelium. Combined with TUNEL and X-gal analyses on the lens epithelium from gammaF-tFGFR/Rosabeta-geo26 and nontransgenic/Rosabeta-geo26 chimeras, these results suggest that the organization and survival of the epithelial cells depend on appropriate structure and/or function of the differentiated fiber cells.

Antisense Myc sequences induce differentiation of F9 cells.

Down-regulation of Myc expression is the earliest documented change in gene expression in retinoic acid-induced differentiation of murine F9 teratocarcinoma cells. F9 cells transfected with plasmids expressing antisense Myc sequences under control of the simian virus 40 (SV40) early promoter exhibit a decrease in Myc protein. The result of this decrease is the spontaneous differentiation into cells that resemble retinoic acid-treated F9 cells as judged by plasminogen activator assays. In contrast, when F9 cells are transfected with a plasmid expressing Myc under control of the SV40 early promoter, resulting cell clones are resistant to differentiation by retinoic acid as shown by the lack of induction of plasminogen activator. These results suggest that down-regulation of Myc is sufficient and necessary for F9 cell differentiation.

Temporally distinct patterns of p53-dependent and p53-independent apoptosis during mouse lens development.

Programmed cell death, or apoptosis, is a critical event in the development of multicellular organisms, and its perturbation is implicated in many diseases including cancer. The tumor suppressor protein p53 is known to mediate apoptosis induced by the DNA tumor virus oncoproteins, adenovirus E1A (AdE1A) and SV40 T antigen (SV40 Tag). We have recently demonstrated that the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) modulate apoptosis when expressed in the lens of transgenic mice. In this study we have identified the pathways that mediate E7 induction and E6 inhibition of apoptosis during different stages in the development of the lens. E7 transgenic mice made p53-null were only partially rescued in their apoptotic phenotype, indicating that both p53-dependent and -independent pathways mediate E7-induced apoptosis in the lens. The E6 transgene and p53-null genotype acted additively to reduce levels of apoptosis induced by E7 in neonatal lenses, indicating that E6 modulates apoptosis at least in part through p53-independent mechanisms. The partial reduction in E7-induced apoptosis by the p53-null genotype correlated with an increased incidence of lens tumors in adult E7 transgenic mice. Analyses of embryonic lenses at E13.5, E15.5, and E17.5 revealed a temporally distinct activation of p53-dependent and -independent apoptosis in the E7 lens. During the early stages of lens development, apoptosis was highly p53-dependent, whereas at later stages, apoptosis occurred through both p53-independent and -dependent pathways. This later time correlates temporally with the time of normal fiber cell denucleation, which can be inhibited by E6 through a p53-independent mechanism. These data suggest a similarity between the mechanism regulating E7-induced, p53-independent apoptosis and the apoptotic-like developmental process of fiber cell denucleation, and the mechanisms through which E6 suppresses both processes.

Role of papillomavirus oncogenes in human cervical cancer: transgenic animal studies.

Human papillomaviruses are believed to be etiologic agents for the majority of human cervical carcinoma, a common cancer that is a leading cause of death by cancer among women worldwide. In cervical carcinoma, a subset of papillomaviral genes, namely E6 and E7, are expressed. In vitro tissue culture studies indicate that HPV E6 and E7 are oncogenes, and that their oncogenicity is due in part to their capacity to inactivate cellular tumor suppressor genes. The behavior of E6 and E7 in vitro and the genetic evidence from analysis of human cancers suggest that the E6 and E7 genes play a significant role in the development of cervical cancer. This hypothesis is now being tested using animal models. In this review, we summarize our current knowledge of the oncogenicity of papillomavirus genes that has been generated through their study in transgenic mice.

Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development.

Tumor suppressor proteins are believed to play a role in regulating cell cycle control during mammalian development. The E6 and E7 oncoproteins from human papillomavirus type 16 are known to affect cell growth control, at least in part, through their inactivation of cellular tumor suppressor gene products, p53 and Rb, respectively. Therefore, these viral proteins can serve as trans-dominant repressors of tumor suppressor gene function. To study the potential role of p53 and Rb in murine lens morphogenesis, we generated transgenic mice in which the expression of E6 or E7 was directed to the developing lens. Transgenic mice expressing E7 exhibited microphthalmia and cataracts, whereas transgenic mice expressing E6 exhibited cataracts without noticeable microphthalmia. Microscopic analysis of the lenses from neonatal and adult E7 transgenic mice revealed inhibition of lens fiber cell differentiation, induction of cell proliferation in spatially inappropriate regions of the lens, and apoptosis. Transgenic mice expressing a mutant E7 that is defective in Rb/p107 binding exhibited normal eyes, suggesting that the activity of Rb and/or Rb-like proteins is required for the perturbation of lens development and induction of apoptosis in E7 mice. Microscopic analysis of lenses from E6 neonatal and adult transgenic mice indicated the presence of nuclei in elongated fiber cells, suggesting that E6 inhibits lens fiber cell denucleation. Furthermore, expression of E6 inhibited the apoptotic-like DNA degradation observed in the lenses of nontransgenic 15.5-day embryos. In lenses from neonatal E6 x E7 double transgenic mice, the level of apoptosis was reduced compared with that seen in lenses from neonatal E7 mice. In adults E6 x E7 double transgenic mice, lens tumors developed, whereas in E6 or E7 only transgenic mice, tumors did not. Taken together, these results point to specific roles in lens morphogenesis for Rb and p53 and to the necessity of these tumor suppressor gene products in regulating exit from the normal cell division cycle in differentiating lens fiber cells.

Differentiation versus proliferation of transgenic mouse lens cells expressing polyoma large T antigen: evidence for regulation by an endogenous growth factor.

Expression of the immortalizing oncogene polyoma large T antigen (PyLT) in the lens of transgenic mice impairs fiber cell differentiation but does not induce hyperplasia or tumor development. To examine this phenotype further, we studied the expression of the lens-specific crystallin genes in normal mice and mice of the transgenic alpha PyLT1 lineage. Immunochemical analyses showed that the spatial pattern of expression of alpha, beta, and gamma crystallins was abnormal in lenses from alpha PyLT1 mice, and that abnormally differentiated lens cells expressed beta and gamma crystallins. We also found that the levels of expression of the crystallin proteins in the transgenic mice were reduced compared to those in normal mice. In vitro, epithelial lens cells from alpha PyLT1 mice expressed not only PyLT and alpha crystallins, but also beta and gamma crystallins, which occur specifically in differentiated cells. Yet, despite their nonproliferative nature in vivo and their expression of differentiated lens cell markers, lens cells from alpha PyLT1 mice proliferated indefinitely in vitro. The growth of the alpha PyLT1 lens cells in culture was inhibited by treatment with exogenous basic fibroblast growth factor, which is thought to be involved in growth and differentiation of lens cells in vivo. These results suggest that factors in the microenvironment of the eye may be important in the process of tumor formation in vivo.

In vitro inhibition of vitamin K dependent carboxylation by tetrachloropyridinol and the imidazopyridines.

The compounds 2,3,5,6-tetrachloro-4-pyridinol (TCP) and the structurally related imidazopyridines (IP) cause hemorrhage and lower the plasma prothrombin level in animals. In vitro, TCP and the IP are more potent inhibitors of both the vitamin K dependent carboxylase which catalyzes the posttranslational gamma-carboxylation of specific glutamyl residues in proteins and the related vitamin K epoxidase activity than they are either of vitamin K epoxide reductase or of NAD-(P)H-K oxidoreductase. TCP and IP, as is the case with the coumarin and indandione anticoagulants, are competitive inhibitors of NAD(P)H-K oxidoreductae with respect to NADH. The epoxide reductase from coumarin-resistant rats is quite resistant to inhibition not only by warfarin but also by the IP, and to a lesser extent by TCP. When interpreted in light of published in vivo experiments, the data suggest that the principal site of anticoagulant action of the IP, but not TCP, is the epoxide reductase. The anticoagulant effect of TCP may be inhibition of the carboxylase itself. TCP is a significantly more potent inhibitor of the carboxylase and epoxidase than the IP; it inhibits both the enzymatic activities to the same degree with 50% inhibition observed at about 10(-5) M. Inhibition of the carboxylase by TCP is not competitive with respect to the pentapeptide substrate phenylalanyl-leucylglutamylglutamylleucine nor with respect to the following components of the in vitro carboxylase assay: imidazole, pyridoxal 5'-phosphate, dithiothreitol, KCl, sodium bicarbonate, oxygen, and vitamin K. The order of addition of components of the assay relative to the addition of inhibitor did not affect the degree of inhibition. Inhibition is readily reversed in experiments designed to dissociate an enzyme-inhibitor complex. Analysis of double-inhibitor experiments suggests that TCP and IP have the same binding site on the carboxylase.

Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells.

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.

Decreased c-myc expression is an early event in retinoic acid-induced differentiation of F9 teratocarcinoma cells.

Retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells is known to be accompanied by changes in gene expression. We examined the expression of the c-myc gene during retinoic acid-induced differentiation of F9 cells. Retinoic acid caused a 50% reduction in the level of c-myc mRNA after 3 hr of treatment and a 90% reduction after 12 hr. We have also examined several features of c-myc genomic structure in F9 cells, PYS2 (differentiated parietal yolk sac) cells, and liver--namely, methylation, amplification, and gross organization. Comparison of Hpa II and Msp I digests of DNAs from F9, PYS2, and liver showed that the c-myc gene in F9 cells is hypomethylated relative to that in PYS2 cells and in liver. The Hpa II sites that undergo methylation during differentiation were found to be in the second intron. Gross structural changes of the c-myc gene have not occurred in F9 or PYS2, and the c-myc gene is not amplified.

Novel fibroblast growth factor 2 transcripts are expressed in mouse embryos.

We have discovered two new exons in the mouse fibroblast growth factor 2 (FGF-2 or bFGF) gene that can be alternatively spliced to the second coding exon of the gene. The newly identified exons 1b and 1c are located at, respectively, approximately 19 and 32 kb downstream of the canonical exon 1a. Using RT-PCR analysis, mRNAs containing exon 1c and canonical exons 2 and 3 were identified in embryonic limb, placenta, face, carcass and ocular tissues. A 3.7-kb transcript present in placenta and embryonic limb hybridizes with an exon 1c-derived probe in Northern blot analysis. Alternative splicing of exon 1c to exon 2 creates a transcript for which the predicted alternative FGF-2 (altFGF-2) polypeptide contains a novel N-terminal domain. Our data indicate that in mouse embryos multiple novel mRNA variants are transcribed from the FGF-2 locus using alternative splicing. These data suggest that proteins arising from these alternative transcripts may play a role in mouse embryogenesis.

Overexpression of FGF-2 modulates fiber cell differentiation and survival in the mouse lens.

During mammalian embryogenesis, the ocular lens forms through a temporally and spatially regulated pattern of differentiation which is thought to be coordinated at least in part by the FGF-1 and FGF-2 members of the fibroblast growth factor (FGF) family. Previous transgenic experiments in which FGF-1 or dominant negative FGF receptors were overexpressed in the lens indicated that FGF-1 could induce differentiation while differentiated lens cells rely upon FGF signaling for their survival. In this study, we asked if the 17.5 kDa FGF-2 protein was capable of inducing differentiation of lens cells in transgenic mice. Unexpectedly, differentiation was inhibited by lens-specific expression of a transgene encoding a secreted form of the 17.5 kDa bovine FGF-2 protein under the transcriptional control of the murine alphaA-crystallin promoter (alphaAIgFGF-2 transgenic mice). To address the possibility that FGF-2 functions as a modulator of fiber cell survival, alphaAIgFGF-2 transgenic mice were crossed to transgenic mice exhibiting extensive apoptosis in the lens due to the functional inactivation of the retinoblastoma protein (alphaAE7 transgenic mice). The level of apoptosis in the lenses of double transgenic mice was substantially reduced as compared to the level in lenses from alphaAE7 only mice. These studies indicate that FGF-2 can act as a modulator of the later stages of differentiation including fiber cell survival. Additionally, they imply that control of lens development by FGFs is a complex process in which FGF-1 and FGF-2 play distinct roles.

Perturbed development of the mouse lens by polyomavirus large T antigen does not lead to tumor formation.

To study how the oncogenic process may involve effects on differentiation, we overexpressed an immortalizing oncogene in a developing tissue in transgenic mice. By use of a gene fusion of the alpha A-crystallin promoter to the viral immortalizing oncogene, polyoma large T antigen (PyLT), we created transgenic mice that express PyLT specifically in ocular lens. Expression of large T antigen during embryonic development led to a perturbation in lens development, specifically, an interference with the normal program of fiber cell differentiation. This resulted in microphthalmia, which persisted throughout the life of the animal. Histological analysis revealed impairment of cell elongation, denucleation, and mitotic senescence in both primary and secondary fiber cell differentiation. Strikingly, there was no evidence for hyperplasia or for tumor development in vivo, unlike the consequences of many immortalizing oncogenes on tissues in other transgenic mice. In vitro, however, the developmentally perturbed cells derived from the transgenic lens showed high proliferative capacity. Our results suggest that a primary effect of aberrant expression of an immortalizing gene is an interference with normal tissue development; however, this interference may not necessarily induce proliferation or lead to tumor formation.

Expression and amplification in transgenic mice of a polyoma virus mutant regulatory region.

Two hybrid gene constructs consisting of wild-type and mutant polyoma regulatory regions fused to a bacterial reporter gene were inserted in the mouse germline. Both transgenes were expressed in a large number of different organs. However, marker gene expression controlled by the polyoma wild-type regulatory region was not detectable in the early embryo and remained low throughout the life of the animal while expression controlled by the polyoma F9-1 mutation was detectable in blastocysts and was significantly higher at later stages of development. The F9-1 hybrid gene was also amplifiable when large T-antigen was supplied in trans to mice or to kidney cells derived from these transgenic mice. Amplification resulted in the appearance of several hundred copies of episomal transgenes and a marked increase of marker gene RNA and protein. Our results suggest that the F9-1 mutation does not alter the target spectrum of gene expression in vivo but does create a more efficient enhancer element in the polyoma early control region. Transgene amplification based upon use of the polyoma regulatory elements may be a means of increasing expression of genes in transgenic mice.

Vitamin K-dependent gamma-carbon-hydrogen bond cleavage and nonmandatory concurrent carboxylation of peptide-bound glutamic acid residues.

The pentapeptide Phe-Leu-Glu-Glu-Leu, tritiated at the gamma carbon of each Glu residue, has been synthesized. In a system using microsomal preparations derived from rat liver, vitamin K-dependent tritium release from the L-Glu residues of this substrate can occur without the concurrent gamma-carboxylation of Glu. This tritium release reaction, which indicates cleavage of the gamma C-H bond, although easily uncoupled from CO2-dependent gamma C carboxylation, does require the reduced (hydroquinone) form of vitamin K and oxygen. The data argue against a concerted mechanism for the cleavage of the gamma C-H bond and carboxylation and against a mechanism in which the vitamin functions solely to transfer or activate CO2. Although the tritium release is related clearly to the oxidation of vitamin KH2, it is not yet established how the subsequent carboxylation proceeds. However, two carboxylation mechanisms compatible with the results are discussed.

Human and murine urokinase cDNAs linked to the murine alpha A-crystallin promoter exhibit lens and non-lens expression in transgenic mice.

cDNAs encoding either the human or the murine urokinase-type plasminogen activator (uPA) were fused downstream from the promoter-enhancer element of the murine gene encoding alpha A-crystallin, a protein found exclusively in the ocular lens. The DNAs were microinjected into fertilized mouse eggs as linear fragments free of bacterial sequences, and for each construct one line of transgenic mice was generated. In both lines transgenic uPA activity was detected in the ocular lens, in agreement with previous results reported on transgenic mice bearing genes fused to the same regulatory region. Unexpectedly however relatively high levels of this activity were found also in the retina, and furthermore, human uPA activity was found also in different parts of the brain and in the bone marrow, and to a lesser extent in the spleen, thymus and optic nerve. Transgenic uPA transcript was found in the lens, retina, brain and thymus of mice carrying the murine cDNA. Such a pattern of expression was different from that exhibited by the endogenous murine uPA gene and, excluding the lens, it appeared to be conferred by the cDNAs. The putative regulation by uPA cDNAs is suggested to be mediated through an internal enhancer-like element functioning in combination with the alpha A-crystallin promoter in a fashion independent of the specific nature of the promoter.

Epidermal cancer associated with expression of human papillomavirus type 16 E6 and E7 oncogenes in the skin of transgenic mice.

Certain "high-risk" anogenital human papillomaviruses (HPVs) have been associated with the majority of human cervical carcinomas. In these cancers, two papillomaviral genes, E6 and E7, are commonly expressed. In this study we provide evidence that expression of the E6 and E7 genes from the high-risk HPV-16 in the skin of transgenic mice potentiated the development of preneoplastic lesions, and a high percentage of these epidermal lesions subsequently developed into locally invasive cancers. High levels of E6/E7 expression were found in these tumors relative to the preneoplastic lesions, and expression was localized to the proliferating, poorly differentiated epidermal cells. Also, the p53 and Rb genes were found to be intact, not mutationally inactivated, in representative skin tumors. These findings demonstrate that the E6 and E7 genes from a papillomavirus etiologically associated with human cervical cancer can contribute to the development of epidermal cancers in an animal model.