Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.

We have previously isolated from human seminal plasma a CD4 ligand, the gp17 glycoprotein, which shares sequence identity with three previously identified proteins : secretory actin-binding protein (SABP) from seminal plasma, gross-cystic-disease fluid protein-1 5 (GCDFP-15) and prolactin-inducible protein (PIP) from breast tumor cells. Functions of these glycoproteins are unknown. To further characterize the physical interaction between gpl7 and CD4 we used surface plasmon resonance and demonstrated that gp17-CD4 binding affinity is high. Competition experiments indicated that gp17 interferes with human immunodeficiency virus (HIV) envelope proteidCD4 binding, although it binds to a site distinct from but close to the gpl20-binding site. We observed moreover that gp17 inhibits syncytium formation between transfected cells expressing the wild-type HIV-1 envelope glycoprotein and CD4, respectively. Our results suggest that gp17, which may function as an immunomodulatory CDCbinding factor playing a role at insemination, may also play a role in controlling HIV spread in the sexual tract.Keywords: human seminal-plasma glycoprotein 17 ; prolactin-inducible protein ; secretory actin-binding protein ; gross-cystic-disease fluid protein 15 ; human immunodeficiency virus glycoprotein 120.CD4 is a transmembrane glycoprotein that acts as a coreceptor in antigen recognition of peptides associated with class I1 major histocompatibility complex proteins (MHC 11) [1, 21. CD4 appears to contact non-polymorphic regions of class I1 molecules [3, 41 leading to the formation of a ternary complex with the T-cell receptor (TCR) [5]. Human CD4 also serves as the primary cellular receptor for human immunodeficiency virus type-I (HIV-1) retroviruses [6, 71. Binding of the viral envelope glycoprotein gp120 to CD4 mediates attachment and penetration of HIV particles [8]. In this regard other non-CD4 cellular receptors have been recently identified [9-121 that mediate some other aspects of HIV infection. It has been demonstrated that gp120 requires only the first immunologlobulin-like domain of the CD4 molecule for high-affinity binding [13, 141. Extensive site-directed mutagenesis studies have mapped the gp120 highaffinity binding site to a single amino acid stretch (residues 39-59) within the first domain of CD4 [15-221. Strategies, aimed at blocking the binding of gp120 to CD4 by recombinant soluble CD4 molecules, have been developed with some success in vitm using laboratory virus strains, but not with primary isolates or in vivo [23].In this context, we have previously reported on an assay system which allows the easy identification of natural and synthetic

BackgroundThe PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes.Principal FindingsMicroarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP−] cells were identified. Functional and regulatory network analyses based on a knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The network identified appears associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines, and contains many genes with a STAT5 regulatory motif in their promoters.ConclusionsOur global exploratory approach identified biological pathways modulated along with PIP expression, providing further support for its good prognostic value of disease-free survival in breast cancer. Moreover, our data pointed to the importance of a regulatory subnetwork associated with PIP expression in which STAT5 appears as a potential transcriptional regulator.