The genetic changes that occur in cancer, whether these be mutations or alterations in levels of gene expression, become evident as changes in the phenotype of a specific cell type. In characterizing these phenotypic changes in malignancy, it is therefore important to work with the appropriate cells or cell lines. Breast cancers show the phenotype of the luminal epithelial cell (1), which can be cultured from milk, and cell lines have been developed from these milk cells which retain the luminal phenotype (2). One of these cell lines (MTSV1-7) has been used to look at the effect of overexpression of various oncogenes and proto-oncogenes on the behavioral properties of this cell type (3, 4).Overexpression of the c-ErbB2 receptor has been observed in a proportion of breast cancers and found to correlate with a poor prognosis (5), making signaling from this receptor an important parameter for investigation. To study the function of c-ErbB2 in human mammary epithelial cells, the receptor was overexpressed in MTSV1-7 cells to produce the ce-1 cell line (3). Unlike other receptors in the ErbB family, the c-ErbB2 homodimer has no known ligand, although c-ErbB2 can function as a heterodimeric receptor for the heregulin family of ligands with c-ErbB3 or c-ErbB4 or for EGF with c-ErbB1 (6 -9). Signaling from c-ErbB2 in overexpressing cells is, however, thought to be constitutive, operating through autophosphorylation of the homodimer, which forms because of overexpression (10).To look for genes whose expression is reversibly regulated by c-ErbB2 signaling as a homodimer, we have down-regulated c-ErbB2 phosphorylation using an antibody that has been shown to inhibit signaling from the receptor in breast cancer cell lines (11). In the humanized form (12), the antibody is under investigation in the clinic for the treatment of breast cancer (13). cDNAs prepared from ce-1 cells, treated or untreated with the antibody 4D5, were used to differentially screen filters from a fetal brain library using a computerized analysis (14), and a partial clone was isolated representing a novel sequence. Clones covering the full-length sequence (6.4 kb) 1 of the novel PLU-1 gene 2 were subsequently isolated by screening a breast cancer cDNA library.

The PLU-1 gene is expressed at the level of message in breast cancers and breast cancer cell lines and shows restricted expression in normal adult tissues with the exception of testis. The predicted protein sequence contains several domains, including the PLU domain, which is shared by other proteins involved in transcription and/or development. We have developed a polyclonal antiserum to a C-terminal fragment of the PLU-1 protein, which shows little homology to other family members. Immunohistochemic analysis with the antiserum ␣-PLU-1C confirmed the nuclear localisation of PLU-1. ␣-PLU-1C also reacted with the mouse homologue of PLU-1 (mPlu-1) but not with the closest family member, RBP2. Using Western blot analysis, PLU-1 was shown to be well expressed in breast cancers and breast cancer cell lines, while it was not detected in a range of normal adult tissues. Our results suggest that the PLU-1 protein may belong to the class of testis/cancer antigens. © 2002 Wiley-Liss, Inc. Key words: breast cancer: PLU-1 protein; testis/cancer antigenThe PLU-1 gene was identified as being downregulated in a human mammary epithelial cell line (MTSV1-7) transfected with HER2/c-erbB2, 1 after treatment with the 4D5/HERCEPTIN antibody, which inhibits c-erbB2 signaling. 2 However, in situ hybridisation and Northern blot analyses showed that PLU-1 mRNA is expressed in most breast cancers and breast cancer cell lines, regardless of the level of c-erbB2. 3 However, Northern blot analysis of total mRNA from normal human adult tissues showed high levels of expression in testis and detectable levels in placenta, but levels were undetectable in the other tissues examined.Following our own report on the identification and characterisation of PLU-1, other investigators reported on the cloning of cDNAs of splice and transcriptional variants of the PLU-1 gene. These were referred to as RBP2-H1 4 and RBBP2H1A, 5 respectively. The nomenclature of the splice and transcriptional variants of PLU-1 reflects the high homology between the predicted protein sequence of PLU-1 and the RBP2 6 in conserved domains, particularly in the novel PLU domain. 3,7 PLU-1 binds to a conserved consensus sequence in 2 transcriptions factors through the novel PLU domain. 8 Additionally, PLU-1 was a potent transcriptional repressor and may be involved in gene regulation in breast cancer. We also defined the sequence of mPlu-1 cDNA, which shows at the amino acid level an overall homology with human PLU-1 of 94% and almost 100% identity within the conserved domains. 9 The very conserved sequence of PLU-1 from human to mouse in combination with an almost identical expression pattern in adult tissues and breast cancers in both species suggests a conserved function in breast cancer.The later studies by Vogt et al. 4 and Kashuba et al. 5 on the RNA variants of PLU-1 examined mRNA expression using polyAϩ RNA and probes that would pick up all 3 transcripts. Expression was reported to be less restricted in normal tissues than we observed using total RNA. However, as we repo...

We have earlier shown that antisense morpholino oligomers are able to restore dystrophin expression by systemic delivery in body-wide skeletal muscles of dystrophic mdx mice. However, the levels of dystrophin expression vary considerably and, more importantly, no dystrophin expression has been achieved in cardiac muscle. In this study, we investigate the efficiency of morpholino-induced exon skipping in cardiomyoblasts and myocytes in vitro, and in cardiac muscle in vivo by dose escalation. We showed that morpholino induces targeted exon skipping equally effectively in both skeletal muscle myoblasts and cardiomyoblasts. Effective exon skipping was achieved in cardiomyocytes in culture. In the mdx mice, morpholino rescues dystrophin expression dose dependently in both skeletal and cardiac muscles. Therapeutic levels of dystrophin were achieved in cardiac muscle albeit at higher doses than in skeletal muscles. Up to 50 and 30% normal levels of dystrophin were induced by single systemic delivery of 3 g kg -1 of morpholino in skeletal and cardiac muscles, respectively. High doses of morpholino treatment reduced the serum levels of creatine kinase without clear toxicity. These findings suggest that effective rescue of dystrophin in cardiac muscles can be achieved by morpholino for the treatment of Duchenne muscular dystrophy.

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Abstract. Overexpression of the B cell leukemia/lymphoma-2 (bcl-2) gene has been shown to confer a survival advantage on cells by inhibiting apoptosis. In epithelia, the bclo2 gene is also related to development and differentiation, and the protein is strongly expressed in the embryo in the epithelial cells of the developing mammary gland. To investigate directly the effect of bcl-2 on human epithelial cells, we used an amphotropic recombinant retrovirus to introduce the gene into nontumorigenic cell lines developed from luminal epithelial cells cultured from milk. Here we demonstrate that while bcl-2 overexpression does not directly induce the tumorigenic phenotype, it provides a survival advantage to the mammary epithelial cells by inhibiting cell death at confluence or under conditions of serum starvation, bcl-2 can also affect the phenotype of the original epithelial cells, and promote epithelial-mesenchymal conversion, accompanied by loss of the cell adhesion molecules E-cadherin and 0t2~l integrin. The extent of the epithelial-mesenchymal conversion varies with small differences in the phenotype of the parental line and with the level of expression of Bcl-2 and in some cases cell lines emerge with a mixed phenotype. The increased survival of Bcl-2--expressing cells at confluence results in multilayering, and the development of three-dimensional structures. Where a mixed phenotype is observed these structures consist of an outer layer of polarized epithelial cells separated by a basement membrane-like layer from an inner mass of fibroblastoid cells. Branching morphogenesis of bcl-2 transfectants is also observed in collagen gels (in the absence of fibroblast growth factors). The results strongly indicate that by increasing their survival under restrictive growth conditions, and by modifying the epithelial phenotype, bcl-2 can influence the specific morphogenetic behavior of mammary epithelial cells.

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In this report, we have analyzed the function of two Sp1 sites present in the epithelium-specific MUC1 promoter. Using promoter-reporter gene (CAT) constructs, we found that mutagenesis of either of the Sp1 binding motifs at ؊576/؊568 and ؊99/؊90, reduced transcription in MUC1-expressing epithelial cell lines. However, abolition of the binding site at ؊99/؊91 by mutagenesis also resulted in increased transcriptional activity in non-epithelial cell lines, suggesting involvement of the site in tissue-specific expression. In vitro binding assays revealed a novel binding motif at ؊101/؊89 (AGGGGGCGGGGTT), which overlaps but differs from the Sp1 consensus motif by having an adenine residue in the 5-flanking sequence. The 5-flanking sequence appeared to be important for binding of an Sp1-unrelated factor (SpA) but not for binding of Sp1. Site-directed mutagenesis of the motif into a site able to bind Sp1, but unable to bind SpA, resulted in an increased level of transcription of the CAT reporter gene in all cell lines tested, suggesting a repressive effect of the novel factor on transcription. The ratio between the Sp1 and SpA binding activity in nuclear extracts correlated with both promoter activity and the levels of endogenous transcription in different breast cancer cell lines. Our results are consistent with the idea that the relative activities of the two factors may be involved in the upregulation of expression of the MUC1 gene seen in breast and other carcinomas.

Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker-Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with a proline to leucine missense mutation (P448L). Our results showed that intraperitoneal administration of AAV9-FKRP resulted in systemic FKRP expression in all striated muscles examined with the highest levels in cardiac muscle. Consistent with our previous observations, FKRP protein is localized in the Golgi apparatus in myofibers. Expression of FKRP consequently restored functional glycosylation of α-DG in the skeletal and cardiac muscles. Significant improvement in dystrophic pathology, serum creatine kinase levels and muscle function was observed. Only limited FKRP transgene expression was detected in kidney and liver with no detectable toxicity. Our results provided evidence for the utility of AAV-mediated gene replacement therapy for FKRP-related muscular dystrophies.