Paul Jolicoeur

Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV/c-neu oncogene

Transgenic mice carrying the activated c-neu oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat were produced. Epithelial hyperplasia of epididymis, seminal vesicles, and salivary glands, and dysplasia of harderian glands, were induced. Moreover, in females of our four lines, independent but multiple mammary tumors arose asynchronously, between 5 and 10 months of age, as stochastic events. Histologically, poorly differentiated adenocarcinomas, with intratumor necrosis and calcifications, arose adjacent to morphologically normal epithelium. High transgene expression was detected in all mammary tumors tested and in normal mammary glands before the appearance of the tumors. Together these results suggest that the expression of the activated c-neu oncogene was necessary but not sufficient to induce malignant transformation of the mammary epithelial cells. These tumors appear to be an adequate model for human breast cancers overexpressing c-neu.

Involvement of Notch1 in the development of mouse mammary tumors.

The MMTV/neu transgenic (Tg) mice spontaneously develop mammary tumors stochastically after a long latent period, suggesting that the c-neu/erbB2 oncogene is not sufficient for tumor formation. To identify putative collaborator(s) of the c-neu/erbB2, we used the provirus insertional mutagenesis approach with mammary tumors arising in MMTV/neu Tg mice infected with the mouse mammary tumor virus (MMTV). The Notch1 gene was identified as a novel target for MMTV provirus insertional activation. In Notch1-rearranged tumors, the Notch1 gene was interrupted by the MMTV provirus insertion upstream of the exons coding for the TM domain. These insertions led to overexpression of novel 5′ truncated ∼7 kb RNA coding for 280 kDa mutant protein harboring only the Notch1 ectodomain, N(EC)mut. These may be involved in tumor formation. Another consequence of these insertions was the expression of truncated 3′ Notch1 transcripts (3.5 – 4.5 kb) and proteins (86 – 110 kDa) deleted of most of the extracellular sequences (Notch1intra). We found that 3′ truncated Notch1intra can transform HC11 mouse mammary epithelial cells in vitro. Deletion analysis revealed that the ankyrin-repeats and the domain 1 (aa 1751 – 1821) are required, while a signal peptide, the two conserved cysteines (C1652 and C1685) and the OPA and PEST sequences are dispensable for transformation. These results indicate that the N-terminally truncated Notch1intra protein behaves as an oncogene in this system.

The Pathogenicity of Human Immunodeficiency Virus (HIV) Type 1 Nef in CD4C/HIV Transgenic Mice Is Abolished by Mutation of Its SH3-Binding Domain, and Disease Development Is Delayed in the Absence of Hck

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P72XXP75XXP78) was mutated to A72XXA75XXQ78. This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck−/− (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.

Two Distinct Notch1 Mutant Alleles Are Involved in the Induction of T-Cell Leukemia in c-myc Transgenic Mice

ABSTRACT We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTVD/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutantNotch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)Mut] which, in contrast to the processed wild-type ectodomain [N(EC)WT], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (ΔCT). The type II Notch1ΔCT protein was expressed as a normally processed receptor [N(EC)WT and N(IC)ΔCT] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)ΔCT expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)Mut and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

HIV-1 Genes vpr and nef Synergistically Damage Podocytes, Leading to Glomerulosclerosis

This study aimed to identify the causative gene for HIV-1 associated nephropathy, a paradigmatic podocytopathy. A previous study demonstrated that transgenic expression of nonstructural HIV-1 genes selectively in podocytes in mice with FVB/N genetic background resulted in podocyte injury and glomerulosclerosis. In this study, transgenic mice that expressed individual HIV-1 genes in podocytes were generated. Five of six transgenic mice that expressed vpr developed podocyte damage and glomerulosclerosis. Analysis of an established vpr transgenic line revealed that transgenic mice on FVB/N but not on C57BL/6 genetic background developed podocyte injury by 8 wk of age, with later glomerulosclerosis. Four of 11 transgenic mice that expressed nef also developed podocyte injury. One transgenic line was established from the nef founder mouse with the mildest phenotype. Transgenic mice in this line developed mesangial expansion at 3 wk of age and mild focal podocyte damage at 10 wk of age. Mating with FVB/N mice did not augment nephropathy. None of the transgenic mice that expressed vif, tat, rev, or vpu in podocytes, even with the FVB/N genetic background, developed podocyte injury. For testing effects of simultaneous expression of vpr and nef, these two lines were mated. All nef:vpr double-transgenic mice showed severe podocyte injury and glomerulosclerosis by 4 wk of age. In contrast, all vpr or nef single-transgenic mice in the same litter uniformly showed no or much milder podocyte injury. These findings indicate that vpr and nef each can induce podocyte injury with a prominent synergistic interaction.

Cell cycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected with murine leukemia virus.

Abstract We have studied Moloney murine leukemia virus (MuLV) replication in newly infected NIH/3T3 cells brought to a stationary phase by serum depletion. Progeny viruses were markedly decreased under these conditions. Studies of the early phase of the virus cycle by the Southern blot hybridization procedure revealed that levels of unintegrated linear double-stranded and supercoiled viral DNAs were decreased in quiescent NIH/3T3 cells as compared to levels detected in serum-replenished cells. When serum was added to quiescent cells up to 48 hr postinfection, we could detect an increase of viral DNA, suggesting the presence of a stable intermediate encoding viral information. In order to characterize this intermediate, stationary cells were labeled with BrdU at the time of serum addition so that substituted viral DNA molecules made under serum stimulation could be separated on CsCl gradients from those made under serum depletion. The analysis of this experiment revealed that upon serum addition, the majority of viral DNA was fully substituted (HH), indicating that it must have been synthesized from an RNA template. Also, an important part of viral DNA made after serum addition had an intermediate density (HL), suggesting that incomplete molecules made in quiescent cells were completed after serum addition. Our results clearly show that host factors are required for synthesis of viral DNA in NIH/3T3 cells newly infected with MuLV.

Ahi-1, a Novel Gene Encoding a Modular Protein with WD40-Repeat and SH3 Domains, Is Targeted by the Ahi-1 and Mis-2 Provirus Integrations

ABSTRACT The Ahi-1 locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-myb proto-oncogene. Since no significant alteration of c-myb expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1 locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3′ end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1 gene. The Ahi-1 cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1 gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1 gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1 is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1 may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abl and c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abl defective retroviruses or a c-myc transgene or in tumors exhibiting deletion of Nf1.

Notch1-induced mammary tumor development is cyclin D1-dependent and correlates with expansion of pre-malignant multipotent duct-limited progenitors

Members of the Notch family are involved in the development of breast cancer in animal models and in humans. In young transgenic mice, expressing intracellular activated Notch1 (N1IC) in mammary cells, we found that CD24+ CD29high progenitor cells had enhanced survival, and were expanded through a cyclin D1-dependent pathway. This expansion positively correlated with the later cyclin D1-dependent formation of basal-like ductal tumors. This expanded population exhibited abnormal differentiation skewed toward the basal cells, showed signs of pre-malignancy (low PTEN/p53 and high c-myc) and contained stem cells with impaired self-renewal in vivo, and more numerous multipotent, ductal-restricted progenitors. Our data suggest that N1IC can favor transformation of progenitor cells early in life through a cyclin D1-dependent pathway.

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Revertants of v-fos-transformed fibroblasts have mutations in cellular genes essential for transformation by other oncogenes

Morphologic revertants of FBJ murine sarcoma virus (v-fos)-transformed rat-1 fibroblasts were isolated using a novel selection procedure based on prolonged retention of rhodamine 123 within mitochondria of v-fos-transformed versus normal fibroblasts. Two classes of revertants were isolated: class I revertants have sustained mutations in cellular genes, and a class II revertant has a nonfunctional v-fos provirus. Somatic-cell hybridization studies suggested that the revertant phenotype was recessive to the transformed phenotype. Class I revertants were also resistant to retransformation by v-gag-fos-fox, v-Ha-ras, v-abl, and v-mos, but could be retransformed by the trk oncogene and polyoma virus middle T antigen. These results suggest that the class I revertants sustained mutations in one or more cellular genes essential for transformation by some, but not all, oncogenes. Our data suggest the existence of common biochemical pathways for transformation.