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RT-PCR analysis of WAP promoter-driven eGFP expression in mammary glands of pregnant, proestrous and estrous precWAPeGFP-transgenic mice
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The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed...
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Postnatal development of the mammary gland begins during puberty with ductal proliferation and is completed at delivery with the appearance of secretory alveolar structures. Using endogenous milk protein genes and a WAP-lacZ reporter transgene, we show that the differentiation of alveolar cells is initiated in virgin mice in estrus in a limited num...
We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the maj...
Citations
... This has been attributed to the arrangement of the casein gene cluster and that the major regulatory elements required for the coordinated high-level expression of these genes during lactation may reside upstream of the cluster (Rijnkels et al., 1995), as is the case for the human b-globin locus (Grosveld et al., 1987). Whey protein gene promoters tend to require less upstream region to direct high-level expression in transgenic mice than do the casein genes (Lipnik et al., 2005;Naruse et al., 2006). For this reason 1226 bp and 2000 bp promoter regions of the highly expressed monotreme BLG and MLP genes, respectively, were assayed. ...
Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, beta-lactoglobulin (BLG), a whey protein and Monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.
... In addition to these molecular modifications, different promoters were evaluated in the ReCon vector system to facilitate tissue-targeted expression of the delivered transgene. The promoter of the whey acidic protein (WAP) encoding gene as well as the U3 region of mouse mammary tumour virus (MMTV) have been shown previously to mediate breast cell-specific expression and to be functional in a retrovirus vector context [8][9][10][11][12]. In the present study, the WAP promoter as well as a full-length and a truncated version of the MMTV U3 region were investigated and compared with the strong and constitutively active murine cytomegalovirus virus (mCMV) promoter with respect to vector compatibility and transgene expression. ...
... Murine WAP and CMV promoter sequences were amplified by PCR from precWAPeGFP [9] and PCEWm-CMV [7], respectively. The oligonucleotide primers used (RC-WAP-1-F: 5 -GATCTGTTAACCCAGGAGAAGTCACC-CTCAGATG-3 ; RC-WAP-1-R: 5 -GATCTGTTAACCTCA-GGCAAGTGACTGATGG-3 ; mCMV-PvuI-F: 5 -AAATCG-ATCG GATATACTGAGTCATTAGGGACTTTC-3 ; mCMV-PvuI-R 5 -TTATCGATCGTGATCTG CGTTCTACGG-3 ) included restriction sites for HpaI and PvuI, respectively (underlined). ...
... For this purpose, two versions of the MMTV promoter as well as the promoter of the murine WAP encoding gene were investigated. Both the MMTV and the WAP promoters have been shown previously to be mammary cell specific and functional in a retroviral vector context [8][9][10][11][12]. With respect to the MMTV promoter, the full-length U3 region as well as a truncated version basically containing the TATA and CAAT box and the HRE [28] were tested. ...
We have previously described the generation of reconstituting retroviral (ReCon) vectors designed for cancer gene therapy using cytotoxic gene products. The unique vector structure with a promoter physically separated from the transgene allows generation of stable virus producer cells irrespective of the toxic gene. The mechanism of synthesis of DNA from retroviral RNA dictates that infection leads to the reconstitution of functional expression cassettes in the target cell.
To improve vector titres, a cytomegalovirus enhancer was inserted upstream of the 5'-long-terminal repeat (LTR); the Woodchuck hepatitis virus post-transcriptional regulatory element and an elongated attachment site upstream of the 3'-LTR were included. In addition, a bacterial origin of replication was deleted and a functional internal polyadenylation signal mutated. Transcriptional targeting was attempted by introducing mammary tissue-specific promoters such as the U3 region of mouse mammary tumour virus or the promoter of the whey acidic protein encoding gene. All modifications were analysed in detail with respect to virus production and infectivity. Finally, the vector was armed with the lambda-holin encoding gene and transduced cells were analysed for cytotoxic effects.
Distinct modifications of the vector resulted in a titre improvement of more than 560-fold. Compatibility of the optimized vector with targeted cellular promoters was demonstrated. When equipped with the cytotoxic gene, stable producer cells could be successfully established and high titre virus infection resulted in rigorous target cell killing.
The ReCon vector in its optimized form is an attractive tool for cancer gene therapy approaches.
... High expression of the transferred gene is a prerequisite in most gene therapeutic approaches and a number of modifications have been introduced into retroviral vectors to boost expression (Schambach et al., 2000;Ketteler et al., 2002;Johansen et al., 2003;Hlavaty et al., 2004b). We have evaluated the use of the WPRE that has already been shown to enhance gene expression when incorporated into various viral vectors, in combination with the MMTV promoter or a shortened version of the murine WAP promoter (Öztürk-Winder et al., 2002;Lipnik et al., 2005). Therefore, we constructed a series of EGFP expressing ProCon vectors (Fig. 1) which either carry the MMTV promoter (constructs pPCEMa, pPCEMm1) or the WAP promoter (constructs pPCEW, pPCEWm1) in the U3 region of the 3′ LTR, thereby replacing the MLV U3 region. ...
The success of gene therapy approaches relies on sufficiently high levels of expression of the therapeutic gene. However, if tissue specific or tumour specific gene expression is desired, a lower level of transgene expression usually has to be accepted due to the weakness of the majority of available tissue or tumour specific promoters. This obstacle can in part be overcome by the insertion of viral cis-acting elements that enhance gene expression in various expression vector contexts regardless of the respective promoter. We designed a series of murine leukaemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors that contain the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and evaluated its use by measuring enhanced green fluorescent protein (EGFP) levels and viral titres. In viral vector packaging cells, when the EGFP encoding gene was transcribed from the MLV promoter, incorporation of the WPRE resulted in a marked improvement of the vectors in terms of EGFP expression and virus titres. However, in infected cells after promoter conversion had taken place, the effect of the WPRE became promoter and cell line dependent. When the EGFP gene was transcribed from the heterologous mouse mammary tumour virus (MMTV) promoter the same beneficial role of the WPRE on transgene expression was observed in all eight cell lines tested. In contrast, when EGFP gene expression was driven by the murine whey acidic protein (WAP) promoter, the positive effect of the WPRE could only be observed in two cell lines whereas expression was actually reduced in the six other cell lines tested. This decrease of EGFP expression was not only demonstrated at the protein level but also manifested on the RNA level.
... After infection and reverse transcription, the MLV promoter is replaced by the heterologous tissue-specific promoter which is copied from the 3′ LTR to the 5′ LTR (Mrochen et al., 1997; Saller et al., 1998). To obtain tissue-specific expression in this study, a 470-bp fragment of the murine whey acidic protein (WAP) promoter was used, which had been shown recently in a transgenic mouse model to target the expression of transferred genes exclusively to the mammary gland (Lipnik et al., 2005). Since retroviral vectors stably integrate into the genome of infected cells, first of all, the functionality of the combined Epo/E9 enhancer element in stably transfected mammary cancer cells was tested. ...
... A major prerequisite for gene therapy with viral vectors, however, is a targeted delivery of the therapeutic gene. Therefore, in the present approach synthetic hypoxia-and radiation-inducible elements were coupled to a minimal fragment of the mammaryspecific murine WAP promoter (Lipnik et al., 2005; ÖztürkWinder et al., 2002). The hybrid WAP promoter was then introduced into a MLV-based retroviral promoter conversion vector, a vector system which allows tissue-specific expression of transferred genes in infected cells (Mrochen et al., 1997; Saller et al., 1998). ...
... The MLV-based retroviral ProCon vector pPCEW (Fig. 2B) is a modification of the vector pPCEMa published previously (Hlavaty et al., 2004). The U3 region of mouse mammary tumour virus (MMTV) located in the 3′ LTR of pPCEMa was replaced by a 470-bp fragment of the murine WAP promoter (Lipnik et al., 2005) giving rise to pPCEW. For generation of pPCE-EpoE9-W (Fig. 2C) the Epo/ E9 enhancer elements from pEpoE9GFP were amplified by PCR using the oligonucleotide primers pEpoE9-for (5′- GATACACCGCGGGATCTGGCCCTACGTGCT-3′) and pEpoE9-rev (5′-GATACA CCGCGGATCGCCCAAA- TAAGGCCAA-3′). ...
To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.
... If this heterologous promoter is preferentially expressed in a given cell type, it is expected that expression of the therapeutic gene carried by the vector will be limited to the very same cells (Figure 1). Proof of concept for ProCon vectors has been obtained using murine leukaemia virus (MLV) vectors carrying a variety of nonhomologous promoters, i.e. constitutively active promoters like the cytomegalovirus (CMV) promoter or tissue specific/restrictive promoters like that of mouse mammary tumour virus (Saller et al, 1998) or whey acidic protein (Özturk-Winder et al, 2002; Lipnik et al, 2005). Inducible promoters are also useful in this context since they allow cells transduced with genes encoding proteins that are not compatible with cell growth to be obtained in the absence of the inducer (Mrochen et al, 1997). ...
... Indeed it could be shown that improvement in expression levels can be obtained regardless of the site of introduction of the WPRE in the vector genome (Hlavaty et al, 2005). Nevertheless, it should be noted that we have recently shown the ability of the WPRE to improve gene expression can be both promoter and cell type specific (Klein et al, 2006). Recently, a high incidence of liver tumours after in utero application of a third-generation equine infectious anaemia virus vectors carrying the WPRE has been observed (Coutelle et al, 2005) and it remains to be seen if this finding is generally applicable to all vector types and configurations as well as for nonfatal gene transfer. ...
Retroviral vectors were the first virus vectors to be used in gene therapy trials and have proved to be successful for the treatment of X-linked severe combined immunodeficiency. However, there are safety concerns associated with the use of retroviral vectors or indeed delivery systems based upon viruses in general. Over the last few years, we have been developing retroviral vectors with the aim of (i) removing the retroviral promoter in transduced cells (ii) obtaining limited expression of therapeutic genes in therapeutically relevant cells by the inclusion of targeting promoters in place of the retroviral promoter (iii) being able to stably produce retroviral vectors carrying toxic genes from cells. Two of these vector systems, promoter conversion vectors and reconstituting vectors, have been described in proof of principle studies, but suffered from reduced titres that precluded their effective use in the clinic. A number of vector optimisation modifications have been made to these vectors, resulting in the successful improvement of both titre and expression levels such that these vectors are now suitable for use in clinical trials. The use of such optimised vectors for in vitro and in vivo applications using a number of different genes of interest will be described. Future successful gene therapy of solid tumours may require the use of replicating vectors. The application of many of the principles learned from the vector optimisation modifications described above to replicating MLV and MMTV based vectors will be described along with data demonstrating efficient tissue specific expression targeting.
... In spite of having a successive in vivo WAP based mouse models it still remains a challenging project to develop an in vitro WAP induced primary culture cell lines derived from the WAP animal model. WAP induced cell lines have been established by cloning WAP promoter and expressing in established cell lines like T47D a human mammary carcinoma cell line(Lipnik et al., 2005). Our primary goal was to establish a cell culture model to understand SV40 induced tumorigenesis where the expression of T antigen driven by WAP promoter could be turned on and off using lactotrophic hormones in primary mammary epithelial cells from WAPT mouse tumors. ...
INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER
Midan Ai, Ph.D.
Supervisory Professor: Zhen Fan, M.D.
Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism needs to be determined. In my studies, I found that high expression of HER2 is correlated with high expression of Brk in breast cancer cell lines. Silencing HER2 expression via RNA interference in HER2 over-expressed breast cancer cells resulted in Brk protein decrease and overexpression of HER2 in HER2 low-expressed breast cancer cells up-regulated Brk expression. The mechanism study indicated that overexpression of HER2 increased Brk protein stability. Brk was degraded through a Ca2+-dependent protease pathway involving calpain and HER2 stimulated Brk expression via inhibiting calpain activity. Calpastatin is a calpain endogenous inhibitor and the calpain-calpastatin system has been implicated in a number of cell physiological functions. HER2 restrained calpain activation via up-regulating calpastatin expression and HER2 downstream signaling, MAPK pathway, was involved in the regulation. Furthermore, silencing of Brk expression by RNA interference in HER2-overexpressing breast cancer cells decreased HER2-mediated cell proliferation, survival, invasion/metastasis potential and increased cell sensitivity to HER2 kinase inhibitor, lapatinib, treatment, indicating that Brk plays important roles in regulating and mediating the oncogenic functions of HER2. The Stat3 pathway played important roles in Brk mediated cell survival and invasion/metastasis in the context of HER2-overexpressing breast cancer cells. However, transgenic mice with inducible expression of constitutively active Brk (CA) in the mammary epithelium failed to develop malignant change in the mammary glands after Brk induction for 15 months which indicated that expression of Brk protein alone was not sufficiently to induce spontaneous breast tumor. Bitransgenic mice with co-expression of HER2/neu and inducible expression of Brk in the mammary epithelium developed multifocal mammary tumors, but there were no significant difference in the tumor occurring time, tumor size, tumor weight and tumor multiplicity between the mouse group with co-expression of Brk and HER2/neu and the mouse group with HER2/neu expression only.
Breast cancer is a heterogeneous condition with no single standard of treatment and no definitive method for determining whether a tumor will respond to therapy. The development of murine models that faithfully mimic specific human breast cancer subtypes is critical for the development of patient-specific treatments. While the artificial nature of traditional in vivo xenograft models used to characterize novel anticancer treatments has limited clinical predictive value, the development of genetically engineered mouse models (GEMMs) makes it possible to study the therapeutic responses in an intact microenvironment. GEMMs have proven to be an experimentally tractable platform for evaluating the efficacy of novel therapeutic combinations and for defining the mechanisms of acquired resistance. Described in this overview are several of the more popular breast cancer GEMMs, including details on their value in elucidating the molecular mechanisms of this disorder. © 2015 by John Wiley & Sons, Inc.
Copyright © 2015 John Wiley & Sons, Inc.
Abbreviations: cytomegalovirus, (CMV); enhanced green fluorescent protein, (eGFP); mouse mammary tumour virus, (MMTV); murine leukaemia virus, (MLV); promoter conversion, (ProCon); Rev responsive element, (RRE); woodchuck posttranscriptional regulatory element, (WPRE) Summary Retroviral vectors were the first virus vectors to be used in gene therapy trials and have proved to be successful for the treatment of X-linked severe combined immunodeficiency. However, there are safety concerns associated with the use of retroviral vectors or indeed delivery systems based upon viruses in general. Over the last few years, we have been developing retroviral vectors with the aim of (i) removing the retroviral promoter in transduced cells (ii) obtaining limited expression of therapeutic genes in therapeutically relevant cells by the inclusion of targeting promoters in place of the retroviral promoter (iii) being able to stably produce retroviral vectors carrying toxic genes from cells. Two of these vector systems, promoter conversion vectors and reconstituting vectors, have been described in proof of principle studies, but suffered from reduced titres that precluded their effective use in the clinic. A number of vector optimisation modifications have been made to these vectors, resulting in the successful improvement of both titre and expression levels such that these vectors are now suitable for use in clinical trials. The use of such optimised vectors for in vitro and in vivo applications using a number of different genes of interest will be described. Future successful gene therapy of solid tumours may require the use of replicating vectors. The application of many of the principles learned from the vector optimisation modifications described above to replicating MLV and MMTV based vectors will be described along with data demonstrating efficient tissue specific expression targeting.
Breast cancer frequently metastasizes to bone, where it takes a significant toll on quality of life. Models of bone metastasis are needed in order to better understand the process of bone metastasis and to develop better treatments. Here, we discuss the available mouse models for breast cancer bone metastasis and critical techniques for imaging bone metastasis in these models.
