Notch-1 signaling promotes the cyclinD1-dependent generation of mammary tumor-initiating cells that can revert to bi-potential progenitors from which they arise.

In a previous work, we reported that young transgenic (Tg) mice expressing the intracellular domain of Notch1 (N1(IC)) showed expansion of lin(-) CD24(+) CD29(high) mammary cells enriched for stem cells and later developed mammary tumors. Mammary tumor formation was abolished or greatly reduced in cyclin D1(-/-) or cyclin D1(+/-) N1(IC) Tg mice, respectively. Here, we studied the epithelial cell subsets present in N1(IC)-induced tumors. CD24(-) CD29(int) and CD24(+) CD29(high) cells were found to be present at low numbers in tumors. The latter had the same properties as those expanded in young Tg females, and neither cell population showed tumor-initiating potential nor were they required for maintenance of tumors after transplantation. CD24(int) CD29(int) cells were identified as tumor-initiating and mammosphere-forming cells and represent a large percentage tumor cells in this model. Their number was significantly lower in tumors from cyclin D1(+/-) N1(IC) Tg mice. Using cyclin D1 shRNA knockdown, we also show that N1(IC)-induced tumor cells remain addicted to cyclin D1 for growth and survival. Interestingly, at lower levels of cyclin D1 or after transplantation in the presence of normal mammary cells, these N1(IC)-expressing tumor cells reverted to a state of low malignancy and differentiate into duct-like structures. They seem to adopt the fate of bi-potential stem/progenitor cells similar to that of the expanded CD24(+) CD29(high) stem/progenitor cells from which they are likely to be derived. Our data indicate that decreasing cyclin D1 levels would be an efficient treatment for tumors induced by N1 signaling.

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 (N1(IC)) in mammary cells, we found that CD24(+) CD29(high) 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 N1(IC) can favor transformation of progenitor cells early in life through a cyclin D1-dependent pathway.

Dysregulated expression of mitotic regulators is associated with B-cell lymphomagenesis in HOX11-transgenic mice.

Dysregulated expression of the homeobox gene, HOX11 is a frequent etiologic event in T-cell acute lymphoblastic leukemias. HOX11-transgenic mice (IgHmu-HOX11Tg)-expressing HOX11 in the B-cell compartment develop B-cell lymphomas with extended latency. The latency suggests that additional genetic events are required prior to the onset of malignant lymphoma. We report the identification of 17 HOX11 collaborating genes, revealed through their propensity to be targeted in a proviral insertional mutagenesis screen. Seven integrations disrupted genes in mitotic spindle checkpoint control, suggesting that cells with elevated HOX11 expression are especially sensitive to dysregulation of chromosome segregation during mitosis. IgHmu-HOX11Tg primary B-lymphocyte cultures exposed to the aneugenic agents, colchicine and colcemid, exhibited increased incidences of chromosome missegregation as assessed by cytokinesis-block micronucleus assays. Additionally, IgHmu-HOX11Tg cultures were shown to exhibit aberrant bypass of spindle checkpoint arrest, as assessed by the increased presence of cycling cells determined by assessment of DNA content and by BrdU immunolabelling. Western immunoblotting revealed elevated expression of the mitotic effector molecules, cyclin A, cyclin B1 and cdc20 in IgHmu-HOX11Tg cultures. Moreover, spontaneously arising lymphoid neoplasms in IgHmu-HOX11Tg mice frequently exhibit aberrant expression of mitotic regulators, concomitant with increased development of micronuclei, abnormal mitotic checkpoint control and increased incidences of abnormal karyotypes when expanded in culture. Collectively, these findings indicate that abnormal regulation of spindle checkpoint control as a result of HOX11 overexpression leads to a heightened predisposition for development of aneuploidy, contributing to oncogenesis.

Expression and analysis of the Epstein-Barr virus BARF1-encoded protein from a tetracycline-regulatable adenovirus system.

Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin. Potential functions of the BARF1 early gene in EBV oncogenesis emerged from our observations showing expression of BARF1-encoded protein in nasopharyngeal carcinoma biopsies, and induction of either malignant transformation (in rodent fibroblast and human B cell lines) or immortalization (in monkey primary epithelial cells) following BARF1 transfection. We previously reported expression of the BARF1 product as a cytoplasm/membrane-associated protein from 293-tTA cells infected with a BARF1-recombinant adenovirus. Since constitutive expression of BARF1 from this heterologous system became inefficient, we developed a tetracycline-regulatable recombinant vector expressing BARF1 and green fluorescent protein from a dicistronic message. As here reported, stable and efficient expression of BARF1 from this vector in either permissive or non-permissive cell lines, allowed the first sequencing identification and further molecular characterization of BARF1-encoded protein.

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.

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 (P(72)XXP(75)XXP(78)) was mutated to A(72)XXA(75)XXQ(78). 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.

Distinct regulatory elements are required for faithful expression of human CD4 in T cells, macrophages, and dendritic cells of transgenic mice.

To identify the regulatory elements controlling expression of the human CD4 (hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3' end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5' end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8alpha(+) dendritic cells (DCs) from lymph node and thymus were found to be different from those required for macrophage expression. The results indicate that expression of hCD4 in T cells, macrophages, and DCs is controlled by distinct regulatory elements.

The AIDS disease of CD4C/HIV transgenic mice shows impaired germinal centers and autoantibodies and develops in the absence of IFN-gamma and IL-6.

The mechanisms responsible for degeneration of germinal centers (GC) and follicular dendritic cell (FDC) networks during progression to AIDS remain elusive. Here, we show that CD4(+) T cells from CD4C/HIV-1 Tg mice, which develop a severe AIDS-like disease, express low levels of CD40 ligand. Accordingly, GC formation, FDC networks, and immunoglobulin isotype switching are impaired in these animals. However, Tg B cells respond to in vitro CD40 stimulation. Total serum IgG levels are reduced in Tg mice, whereas total IgM levels are increased with a significant amount showing DNA specificity. IFN-gamma- and IL-6-deficient CD4C/HIV Tg mice also develop the AIDS-like disease and produce auto-Ab. Thus, CD4C/HIV Tg mice have immune dysfunction accompanied by autoimmune responses.

Pathogenesis of defective retroviruses.

Concerns about the presence of contaminating defective retroviruses in cells used for vaccine preparations have been raised. These concerns mainly reflect the fact that (i) most species harbour several copies of endogenous defective and/or non-defective retroviral sequences in their genome; (ii) these sequences have frequently been found to participate in recombination events giving rise to novel viral entities; and (iii) several strains of defective retroviruses have been shown to be pathogenic. Here, a brief description of the main features of defective retroviruses is provided. In addition, the characteristics and plasticity of defective retroviruses are illustrated with a defective retrovirus inducing a mouse disease, murine AIDS (MAIDS).

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice.

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/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 mutant Notch1 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 (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] 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)(DeltaCT) 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.

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 approximately 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.

Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene.

It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.

Elevated frequency of loss of heterozygosity in mammary tumors arising in mouse mammary tumor virus/neu transgenic mice.

Loss of heterozygosity (LOH) analysis was performed on 62 mammary tumors that were induced in (BALB/c x C57BL/6)F1 mouse mammary tumor virus/neu transgenic mice. Eighty-six simple sequence length polymorphism markers were used to cover all of the somatic chromosomes. Frequency of LOH was observed to be significant for chromosomes 4 (50%), 19 (32%), and 8 (21%). On chromosome 4, at least three distinct regions of allelic deletions could be identified: one proximal to 22 cM; the second close to the p16INK4a/p15INK4b locus, which is commonly deleted in various tumors; and the third one in the proximity of Mom1. The frequency of LOH on chromosome 19 was the same for the four markers used. Our data suggested the presence of two distinct LOH loci, one proximal to 47 cM and the other at the distal region. On chromosome 8, possibly two distinct LOH loci could be recognized, one around 52 cM and the other one at 67 cM or distal to it. These regions map close to E-cadherin (Cdh1) and M-cadherin (Cdh15) loci, respectively. Because LOH sites are thought to harbor tumor suppressor genes, this allelotype screening has allowed the mapping of putative tumor suppressor genes that may be implicated, in collaboration with the erbB-2/neu oncogene, in the development of mammary tumors in these transgenic mice.

Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells.

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34(+) hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony-stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit-dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL- or dn-SCL- expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro-B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

Establishment of MAIDS-defective virus-infected B cell lines and their characterization.

Mice inoculated with the murine AIDS (MAIDS)-defective virus develop severe B and T cell dysfunctions. The primary event in the development of this disease is the infection and polyclonal expansion of the target cells of this defective virus, which have been reported to belong to the B cell lineage. To further study the central role that these cells play in the development of MAIDS, we attempted to establish MAIDS-defective virus-infected B cell lines in vitro. We succeeded in establishing two cell lines, SD1 and CSTB5, from the enlarged organs of C57BL/6 mice inoculated with helper-free stocks of the MAIDS-defective virus. Both cell lines are not transplantable in syngeneic C57BL/6 mice or in nude or CD8-/- mice and are apparently not malignant. They both belong to the B lineage, as their immunoglobulin (Ig) genes, but not the T cell receptor (TcR) beta locus, are rearranged, suggesting that they are relatively mature B cells. However, analysis of cell surface marker expression by FACS revealed a surface phenotype similar to that of pre-B cells (MHC I+, MHC II+, B7.2+, sIgM-, sIgG-, kappa-, B220-, CD5-, Thy1.2-, TcR-, CD3-, CD4-, CD8-, Mac-1-, 33D1-). Additionally, the CSTB5 cells express CD40 and the SD1 cells express CD43. Both cell lines contain the MAIDS-defective provirus and express the expected 4.2-kb viral RNA and the corresponding Pr60gag protein. The CSTB5 cells are nonproducer, while the SD1 cell line produces what appears to be an endogenous MuLV. The phenotype of these cell lines is very similar to what is known about the target B cells of this virus in vivo. These new established cell lines are likely to be useful in elucidating the mechanism(s) by which the MAIDS-defective virus causes its target B cells to proliferate and induce T cell anergy in infected animals.

A full-length Notch1 allele is dispensable for transformation associated with a provirally activated truncated Notch1 allele in Moloney MuLV-infected MMTV(D)/myc transgenic mice.

The Notch1 gene was previously found to be targetted by provirus insertion in a high proportion of T-cell lymphomas arising in Moloney MuLV-inoculated MMTV(D)/myc transgenic mice. Proviral activation of Notch1 was associated with overexpression of truncated Notch1, deleted of the sequences coding for the extracellular domain. The high levels of truncated Notch1 RNA and proteins in these tumors are thought to be involved in the oncogenic transformation. However, in addition to these truncated RNA and proteins, high level expression of full-length Notch1 RNA and proteins was also observed in several tumors, suggesting that they could also contribute to the transformation process. To test this hypothesis, we used a genetic approach and studied MMTV(D)/myc transgenic mice in which one of the Notch1 alleles was mutated by targeted mutagenesis (Notch1+/- mice). Heterozygote (Notch1+/-) and wild-type (Notch1+/+) transgenic mice were inoculated with Moloney MuLV and the frequency of Notch1 rearrangements was compared between both groups. Notch1 was rearranged at similar frequencies in both groups, indicating that the full-length Notch1 allele is dispensable in tumors harboring an activated Notch1 allele.

Inducible overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette.

We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(delta2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce deltaR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5 deltaR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy.

Use of mouse mammary tumour virus (MMTV)/neu transgenic mice to identify genes collaborating with the c-erbB-2 oncogene in mammary tumour development.

Mouse mammary tumour virus (MMTV)/neu transgenic mice develop clonal or oligoclonal mammary tumours stochastically. The pathology of these tumours is very similar to that of human breast tumours. Moreover, these mouse tumours metastasize in the lungs. We present evidence that this mouse model of human breast tumours can be instrumental in identifying novel genes of two distinct classes (activated oncogenes or tumour suppressor genes) which may collaborate with the c-erbB-2/neu transgenic oncogene.

New adenovirus vectors for protein production and gene transfer.

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10-15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression.