The COST Programme

The Cooperation in Science and Technology Programme (COST) is an inter-governmental framework for European Co-operation in the field of Scientific and Technical Research. COST is managed by the European Science Foundation, and in May 2005 supported almost 200 Actions involving nearly 30000 scientists from 32 European member countries and more than 50 participating institutions from 11 non-member countries. These Actions are networks of coordinated national research project in fields which are of interest to a minimum number of participants (at least 5) from different member states. COST Actions cover basic and pre-competitive research as well as activities of public utility. The members of each Action are drawn from member countries which have ratified the Action, and their activities are informed by a programme accepted by the ESF management at time of application and reviewed on an annual basis throughout the Action. Each Action comprises three principal activities: the workshops where the science or technology matters of the Action are presented and discussed; the Action's short-term scientific missions, which allow contacts made during workshop networking to be developed; and, thirdly, the dissemination of the Action's science through publication or electronic communication.

Cancer develops from overlapping events that tend to deregulate the metabolism and unbalance the homeostasis of cells. Sphingolipids, major components of biological membranes, are also mediators of intracellular signalling. Their metabolism can be influenced by diverse stimuli and the accumulation or deficiency of intermediates may trigger proliferation and/or impair the ability of damaged cells to undergo apoptosis. Many sphingolipid-regulated functions are implicated in tumour initiation, promotion, progression and responsiveness to chemotherapy. In this review, evidence of the alteration of sphingolipids metabolism and signalling will be discussed in breast cancer prevention and therapy.

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-β-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-β-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2·5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

To define better the function of Nerve Growth Factor (NGF) in breast cancer progression, we investigated whether this polypeptide was able to induce breast cancer cell invasion. NGF inhibited aggregation of tumour cells through modulation of the E-cadherin/catenin complex function. In addition, NGF induced the breast cancer cells to invade into Matrigel. We focused our attention on how NGF prevents aggregation, in order to discover the signalling pathway that leads tumour cells to acquire the invasive phenotype. Moreover, studies on the identification of signalling pathways that are responsive for NGF-induced invasion will be basically described.

Transgenic and knockout mouse models are extensively used to study the mechanisms of tumour formation. The availability of mouse models to study metastatic spread of tumours is although quite limited. S100A4(mts1), that belongs to the S100 family of Ca-binding proteins, has been shown to function as a metastasis-promoting protein. We generated strains of mice with modified expression of S100A4 in order to understand the mechanism by which S100A4 protein stimulates metastatic spread of the tumour cells. Transgenic mice over-expressing the S100A4 gene in the mammary gland were crossed with GRS/A mice, characterized by a high incidence of spontaneous non-metastatic mammary tumours. The resulting hybrid mice developed metastatic tumours. Transgenic mice with ubiquitous expression of S100A4 developed vascular tumours, hemangiomas and contained enhanced levels of the S100A4 protein in the blood. Based on these observations we demonstrated that extracellular S100A4 functions as an angiogenic factor. Study of tumour development in the S100A4 – deficient mouse model demonstrated key role of extracellular S100A4 in stimulation of tumour development and metastasis formation.

Casein genes are expressed in a tissue-specific and highly coordinated manner. The main goals of casein gene promoter studies are to unravel cis- and trans-acting factors involved in the complex signalling pathway controlling milk production, and to explore the possibility of using these promoters for tissue-specific production of heterologous proteins in the mammary gland. Here we present a comparative study of the equine β-casein and κ-casein gene proximal promoters. In order to confirm the assumption that in the horse, as in other mammalian species, casein genes are organized in a cluster located on a single chromosome, we performed in situ hybridization of pro-metaphase chromosomes with two BAC clones containing different equine casein genes. Sequence analysis of the β-casein and κ-casein gene proximal promoters revealed binding sites for activators (STAT5, GRE, NF1, MAF) and repressors (YY1, PMF), characteristic for casein genes. The alignments of casein gene promoters revealed the highest sequence identity in the proximal promoter region between the equine and human β-casein gene promoters. We directly compared the activity of equine β-casein and κ-casein gene promoters in vitro using bovine mammary gland cell line BME-UV1. In this system, the κ-casein gene proximal promoter activated the reporter gene expression more efficiently than the β-casein gene promoter of approximately the same length. The 810 bp of β-casein promoter activated the reporter gene expression more efficiently than the long fragment (1920 bp) and the 1206 bp fragment of the same promoter, which included also 396 bp of 5′ UTR.

Experimental and epidemiological studies indicate that antioxidant food polyphenols could have antimitotic activities, interfering with cancer initiation, progression or mortality. Circulating polyphenols are far lower than the nominal value in foods. In the rare studies dealing with polyphenol bioavailability, it was noted that their active concentrations in the blood are <1% of their food concentration. In the present study we investigated the effect of four polyphenols (resveratrol, and the flavonoids quercetin, catechin and epicatechin, major constituents of wine) in the hormone-sensitive human cancer cell line T47D, at concentrations compatible with their calculated plasma concentrations after ingestion of a moderate quantity of wine (nM or pM). Our results indicate that cell growth was decreased, with cells being arrested at the S phase of the cycle. In addition, we provide evidence of a bimodal modulation of the NO/NOS system, affecting its activity and transcription. We show that modulation of this system is sufficient to explain polyphenol action on this cell line. This result suggests a potential importance of wine ingestion and possibly the consumption of other polyphenol-rich dietary foods and drinks in the control of breast cancer cell growth.

Among the species investigated, the rate of spontaneous cancers of mammary glands is highest in humans and dogs (Hamilton, 1974; Owen, 1979). In addition, spontaneous mammary tumours are known to occur in strains of mice (Webster & Muller, 1994), rats (Russo et al. 1990; Sukumar, 1995) and cats (Hamilton, 1974; Kessler & von Bombard, 1997). Although both oestrogen and progesterone are known to be involved in normal mammary development as well as in the proliferative changes that occur during the oestrous or menstrual cycle, pregnancy and lactation (Going et al. 1988), only the role of oestradiol has been extensively investigated. The role of progesterone in mammary cancer is not understood and, although progesterone metabolism is known to occur in mammary tissue, the potential autocrine/paracrine role of the in situ metabolites has only recently begun to be explored in human breast tissues and cell lines (Wiebe et al. 2000).

Prolactin (PRL) plays a key role in normal growth, development and differentiation of the mammary gland. Indeed, strong evidence suggests that the development of alveolar cells requires not only oestradiol and progesterone, but also PRL. In vitro, PRL has mitogenic activity on normal mouse mammary epithelial cells (reviewed in Das & Vonderhaar, 1997). In vivo, PRL also seems to be involved in such proliferative activity, although it is more difficult to distinguish the role of PRL from the influence of the hormonal milieu (Das & Vonderhaar, 1997). This physiological role of PRL in lobular development of the mammary gland is supported by results obtained from mice deficient for PRL (Horseman et al. 1997) or for its receptor (PRLR) (Ormandy et al. 1997). Although the infertility of females homozygous for the deletion of the PRLR gene (PRLR−/−) can be partially reversed by restoring progesterone levels close to normal, their mammary gland fails to differentiate during pregnancy, leading to lactation failure (Binart et al. 2000). In addition, heterozygous mice (PRLR+/−), who have half normal receptor levels, show impaired mammary gland development and fail to lactate following their first pregnancy, clearly indicating that signals mediated by the PRL/PRLR interaction have to achieve a certain level to permit mammary gland differentiation and lactation (Kelly et al. 2002). Since the pioneering work of Topper (Topper, 1970), who observed that PRL was necessary to induce casein synthesis, our understanding of the mechanism of such induction has greatly expanded. PRL appears to be the primary hormone involved in this activity, although other hormones such as insulin and glucocorticoids are also required for lactation.

The protein fraction of milk contains several components with physiological significance for the development of the newborn. Among them, immunomodulatory peptides and lactoferrin exemplify the complexity of biologically active substances of milk. Immunomodulatory peptides have latent activity within the native protein and are generated after proteolysis during gastrointestinal transit. Once they are generated, they modulate mucosal immunity, possibly by guiding the local immune system until it develops its full functionality. Lactoferrin is another milk bioactive compound with nutritional and health promoting properties; it modulates the microbial intestinal environment, displays anti-microbial activity against various pathogens and stimulates the establishment of beneficial microflora. The following overview focuses on the importance of immunomodulatory peptides and lactoferrin for the maturation of intestine and immune system that are functionally immature in the newborn.

Ataxia-telangiectasia (AT) is a rare autosomal recessive early childhood disorder, characterized by progressive neuronal degeneration, immunological deficiency, radiosensitivity and an increased risk of cancer caused in most cases by mutations in the AT-mutated gene (ATM). Epidemiological studies on AT families have shown that AT heterozygous women have an increased risk of developing breast cancer (BC). The ATM protein plays a central role in the recognition and repair of DNA double-strand breaks and the subsequent activation of cell-cycle checkpoints. Whilst AT is a rare disease, 0·5–1% of the general population are estimated to be AT mutation carriers, thus any increases in the risks of cancer associated with ATM carrier status are of public health relevance. The main results of our published studies on the risk of BC in 34 French AT families according to heterozygote status, type of ATM mutation and exogenous factors are summarized here. The risk of BC was higher in ATM heterozygous (HetATM) women and did not differ significantly according to the type of ATM mutation (missense vs truncating) carried by the AT family members but appeared associated with the position of some truncating mutations in certain binding domains of the ATM protein. The effect of exogenous factors, such as reproductive life factors and exposure to ionizing radiation, on the risk of BC according to ATM heterozygote status was assessed. There was no evidence for interaction (except for age at first full-term pregnancy). These findings does not appear to justify a separate screening program from that already available to other women with a first-degree relative affected by BC, as their risks have similar amplitude. Chest X-rays did not appear to be a risk factor for BC in our study population. More powerful studies, using data sets pooled from international sources are being set up to confirm these observations.

The fur seal is a mammal with an unusual ability to turn its milk production on and off without significantly altering the gross morphology of the mammary gland. This atypical lactation cycle is due to the fact that maternal foraging and infant nursing are spatially and temporally separate (Bonner, 1984). Maternal care involves the suckling of offspring over a period of at least 4 months, but lactation can extend to more than 12 months. Following a perinatal fast of approximately 1 week, females depart the breeding colony to forage at sea and, for the remainder of lactation, alternate between short periods ashore suckling their young with longer periods of up to 4 weeks foraging at sea. Whilst foraging at sea, milk production in the fur seal mammary gland either ceases or is reduced (Arnould & Boyd, 1995b).

Spontaneous mammary tumours are most frequently seen (apart from rodents) in women, female dogs and cats. The mammary gland is the most commonly affected organ for tumours in women and in female dogs. The mammary gland has a similar histology in the different species whereas the number of glands differs as well as the number of interlobular ducts that reach the nipple/teat. The parenchymatous tissue is composed of alveoli that turn into interlobular ducts. The whole ductal tree is outlined by a two-layered epithelium with the luminal epithelial cells adjacent to the lumen and the more sparse myoepithelial cells peripherally located to these. Different proteins such as growth factors regulate the mammary gland, as they do for all tissues in the body. In addition, sex hormones regulate the biology of the mammary gland. Oestrogen has the most pronounced effect on duct growth whereas progesterone promotes growth of the alveoli.

Naturally occurring tumours in domestic animals have been recognized as an interesting opportunity for comparative oncology (MacEwen, 1990; Vail & MacEwen, 2000). Cancer is the second most frequent cause of death in humans and the first one in dogs and cats (Jemal et al. 2003). The age-adjusted overall cancer incidence per 100000 individuals per year is comparable in humans and domestic animals, being approximately 300 in humans, 381 in dogs and 264 in cats (Vail & MacEwen, 2000). When analysing the incidence by site, breast cancer is the most frequent (32%) in women, the first of all neoplasia (52%) occurring in bitches and the third (17%) in queens after lymphohaemopoietic and skin tumours (Hayes et al. 1981; Hayes & Mooney, 1985; MacEwen, 1990; MacEwen & Withrow, 1996; Jemal et al. 2003). Several other aspects contribute to the value of domestic animals as models for human cancers (MacEwen, 1990; Vail & MacEwen, 2000). Tumours occur spontaneously in companion animals that share a similar environment with humans and therefore might be exposed to similar risk factors. The high incidence of some tumour types offers large population samples. The shorter overall lifespan of domestic animals associated with a more rapid progression of cancer allows adequate comparison of response time with humans. Biological, anatomical, histopathological, genetic, and molecular similarities between some animal and human tumours are also well established (Hansen & Khanna, 2004). Finally, testing novel therapies is more ethically acceptable when treating spontaneous diseases in companion animals rather than experimentally induced pathologies in animal models. At the same time, there is an increasing interest of owners towards the use of the most advanced therapeutic tools for companion animals despite the higher economic costs associated with these therapies.

In ruminants, protective immunoglobulins are transferred to the newborn via colostrum to mediate maternal immunity. There is a high selectivity in the transport of immunoglobulins from the maternal plasma across the mammary barrier into the colostrum, and only IgG1 is transferred in large amounts. We have recently analysed the expression of the neonatal Fc receptor (FcRn) in sheep mammary gland around parturition. Re-analysing this issue in bovine confirmed our previous data indicating that FcRn is homogeneously localized in the mammary gland acinar cells before parturition, however a remarkable difference was observed in the pattern after calving, where only the apical side of the cells was strongly stained. The presence of the FcRn in the acinar epithelial cells of the mammary gland and the obvious change in distribution before and after parturition indicate that FcRn plays an important role in the IgG transport during colostrum formation in ruminants.

For 10 years, the regulatory regions of the mouse and rabbit whey acidic protein gene have been used to express heterologous proteins in the milk of transgenic mice, as well as to produce pharmaceutical proteins, on a large scale, in the milk of transgenic livestock. To date, a broad range of expression levels have been detected, and elucidation of the structure-function relationship in these regulatory regions might help to achieve high levels of expression, reproducibly. An extended 5′ regulatory region (17·6 kb v. 6·3 kb) of the rabbit whey acidic promoter resulted in an increased frequency of rabbit whey acidic protein expression in transgenic mice. However, the expression levels were low compared with the high expression levels achieved in both transgenic mice and rabbits using the heterologous κ-casein in the 6·3 kb rabbit whey acidic protein 5′ regulatory region. These results underline the importance of the 3’ downstream regulatory regions, which still need to be better characterized in the whey acidic protein gene.

Pathogenic microorganisms invading the mammary gland induce an inflammatory reaction which includes an increase of somatic cells in milk and activation of bacteriostatic enzymes and proteins in milk. During spontaneously occurring subclinical mastitis the somatic milk cells, mainly macrophages, secrete cytokines, eicosanoids, acute phase proteins and other immunomediators. In contrast, the bacteriostatic protein lactoferrin is mainly secreted by mammary epithelial tissue, while major milk proteins like α-lactalbumin and κ-casein are down-regulated already during subclinical infection.

Changes of the mRNA expression of various immunomediators in the mammary tissue of cows during 12 h after induction of mastitis via intramammary administration of lipopolysaccharide (LPS) in several studies are reported. Six healthy lactating cows were injected in one quarter with 100 μg Escherichia coli-LPS (O26[ratio ]B6) and the contralateral quarter with saline (9 g/l) serving as control. mRNA expression in mammary biopsy samples of various inflammatory factors and milk proteins at 0, 3, 6, 9 and 12 h after LPS administration was quantified by real-time reverse transcription-PCR.

In LPS-challenged quarters tumour necrosis factor α and cyclooxygenase-2 mRNA expression increased to their highest values (P<0·05) at 3 h after LPS-challenge. Expression of lactoferrin, lysozyme, inducible nitric oxide synthase, and of the apoptotic factors caspase-3, caspase-7 and FAS was elevated (P<0·05) and peaked at 6 h after challenge. No significant increase in mRNA expression of platelet-activating factor acethylhydrolase, 5-lipoxygenase, and insulin-like growth factor 1 was found. None of the parameters tested did change significantly in the control quarters. mRNA expression of major milk proteins did not change significantly in response to the LPS challenge (αS1-casein, αS2-CN, β-CN and β-lactoglobulin) except for α-lactalbumin which decreased (P<0·05) in LPS-treated and control quarters and for κ-CN which decreased in the LPS-treated quarters. In conclusion, mRNA expression of the majority albeit not all inflammatory factors changed within hours of LPS challenge. Decreased gene expression of α-lactalbumin and κ-CN may reduce milk yield and suitability for cheese production.

At present there is no rapid microbiological method for the detection of viable Mycobacterium paratuberculosis in milk. By combining an extensive milk sample pretreatment with solid phase cytometry as the detection technique we were able to demonstrate viable mycobacterial cells in 50 ml of artificially contaminated pasteurized milk in less than one working day.