In vitro invasion assay using matrigel™: a reconstituted basement membrane preparation.

Basement membranes, specialized extracellular matrices composed of collagens, laminins, and proteoglycans, form thin, continuous sheetlike structures that separate epithelial tissues from adjacent connective tissues. The crossing of basement membranes by cancer cells is a crucial aspect of metastasis-it must occur in order that cancer cells can invade lymphatic or blood vessels during dissemination and also when they penetrate into the target organ tissue where they will eventually colonize to form secondary tumors. The assay system described in this chapter utilizes the solubilized basement membrane preparation Matrigel™ and measures the ability of cells to attach to the matrix, invade into and through the matrix, and migrate towards a chemoattractant. It is technically straightforward and requires no specialist equipment and provides a useful tool for assessing the invasive ability of cancer cells, exploring the functional role of specific cell surface molecules/receptors in this process and screening for inhibitors of invasive ability, thus contributing to current knowledge of the molecular events occurring during the invasive process.

Lectin histochemistry to detect altered glycosylation in cells and tissues.

Lectins are naturally occurring carbohydrate-binding molecules. A very wide range of purified lectins are commercially available which exhibit a diversity of carbohydrate-binding preferences. They can be used in the laboratory to detect carbohydrate structures on, or in, cells and tissues in much the same way that purified antibodies can be employed to detect cell- or tissue-bound antigens using immunocytochemistry. As lectins can distinguish subtle alterations in cellular glycosylation, they are helpful in exploring the glycosylation changes that attend both transformation to malignancy and tumour progression. In this chapter, methodologies are given for appropriate preparation of many types of cell and tissue preparations, including cells cultured on coverslips (which can be used for live-cell imaging), cell smears, and frozen (cryostat) and fixed, paraffin wax-embedded tissue sections. Heat- and enzyme-based carbohydrate retrieval methods are covered. Basic detection methods, which can be readily adapted to the researcher's needs, are given for direct (labelled lectin), simple indirect (labelled secondary antibody directed against the lectin), and avidin-biotin (biotinylated lectin) and avidin-biotin complex. The use of both the enzyme label, horseradish peroxidase, and fluorescent labels is considered.

PLU-1/JARID1B/KDM5B is required for embryonic survival and contributes to cell proliferation in the mammary gland and in ER+ breast cancer cells.

The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER+ breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER+ breast cancer.

Molecular interactions in cancer cell metastasis.

Metastasis, the process by which cancer cells leave the primary tumour, disseminate and form secondary tumours at anatomically distant sites, is a serious clinical problem as it is disseminated disease, which is often impossible to eradicate successfully, that causes the death of most cancer patients. Metastasis results from a complex molecular cascade comprising many steps, all of which are interconnected through a series of adhesive interactions and invasive processes as well as responses to chemotactic stimuli. In spite of its clinical significance, it remains incompletely understood. This review provides an overview of some of the molecular interactions that are critical to metastasis. It summarises the principle molecular players in the major steps of the metastatic cascade. These are: (1) tumour angiogenesis, (2) disaggregation of tumour cells from the primary tumour mass, mediated by cadherins and catenins, (3) invasion of, and migration through, the basement membrane (BM) and extracellular matrix (ECM) surrounding the tumour epithelium, and subsequent invasion of the BM of the endothelium of local blood vessels. This is mediated through integrins and proteases, including urokinase form of plasminogen activator (uPA), matrix metalloproteinases (MMPs) and cathepsins, (4) intravasation of the tumour cells into the blood vessels prior to hematogeneous dissemination to distant sites, (5) adhesion of the circulating tumour cells to the endothelial cell lining at the capillary bed of the target organ site. This occurs through adhesive interactions between cancer cells and endothelial cells involving selectins, integrins and members of the immunoglobulin superfamily (IgSF), (6) invasion of the tumour cells through the endothelial cell layer and surrounding BM (extravasation) and target organ tissue and (7) the development of secondary tumour foci at the target organ site.

Functional analysis of the transcription repressor PLU-1/JARID1B.

The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G(2)/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B.

Breast cancer associated transcriptional repressor PLU-1/JARID1B interacts directly with histone deacetylases.

The PLU-1/JARID1B nuclear protein, which is expressed in a high proportion of breast cancers, but shows restricted expression elsewhere, belongs to the ARID family of proteins, known to play important roles in development, differentiation, transcriptional regulation and chromatin remodeling. PLU-1/JARID1B is a strong transcriptional repressor, and here we show that the protein localizes in MAD bodies when cotransfected with class IIa histone deacetylases (HDACs) or N-CoR. Direct binding to class I and class IIa HDACs is demonstrated, while the interaction with N-CoR appears to be indirect. The domains involved in the HDAC4-PLU-1/JARID1B interaction were investigated in detail, and the data show that 2 PHD domains in PLU-1/JARID1B, which are involved in transcriptional repression, are also crucial for binding to a domain in the 5' region of HDAC4, overlapping the MEF-2 binding region. Physiological relevance of this interaction in the mammary gland is suggested from the observation that HDAC4 and PLU-1/JARID1B are coexpressed in the pregnant and involuting mouse mammary gland and are both silenced at lactation. Significantly, the expression of both proteins is seen in breast cancers.

HPA binding and metastasis formation of human breast cancer cell lines transplanted into severe combined immunodeficient (scid) mice.

Six human breast cancer cell lines were injected subcutaneously into scid mice and their in vivo growth behaviour and HPA binding pattern were analysed. Furthermore, the role of HPA binding glycoconjugates concerning the adhesion to endothelial cells in vitro was investigated. Four of the tested cell lines engrafted in the scid mouse model but they showed considerable variations concerning their growth behaviour, their metastatic potential and their HPA binding pattern. HPA inhibited adhesive interactions between cell lines derived from metatstatic sources and tumour necrosis factor (TNF)alpha stimulated endothelial cells. The transplantation of HPA defined breast cancer cell lines into scid mice is a useful animal model for the research of breast cancer and its metastasis. The HPA binding glycoconjugates appear to be associated with adhesive interactions between metastasising tumour cells and endothelial cells.

PLU-1, a transcriptional repressor and putative testis-cancer antigen, has a specific expression and localisation pattern during meiosis.

PLU-1, a large multi-domain nuclear protein with strong transcriptional repression activity, is a member of the ARID family of DNA binding proteins. In previous studies, high levels of expression of Plu-1 mRNA and PLU-1 protein were detected in breast cancers, while expression in normal adult tissues was detected only in the testis, ovary and transiently in the mammary gland of the pregnant female. Due to its high levels of expression in the testis and to its specific relationship to cancer, PLU-1 has been proposed to belong to the family of testis-cancer antigens. In this study we attempted to determine putative functions for PLU-1 during spermatogenesis. To address this, we analysed the pattern of expression and localisation of this protein in mouse testicular cells during postnatal development and adulthood. Using in situ hybridisation and immunostaining of testis sections we show that Plu-1 mRNA and PLU-1 protein are both highly expressed in the mitotic spermatogonia. Expression is reduced dramatically in the early prophase I stages (zygotene, leptotene), but reappears at pachytene and is still detectable in diplotene cells. We also found that PLU-1 localises diffusely over the nucleus, which indicates a potential chromatin binding ability of this protein. Consistent with this notion, we found that PLU-1 is present in the chromatin fraction in biochemical cell fractionation experiments using both somatic and meiotic cells. Our data point to a role for PLU-1 in meiotic transcription, which may be restricted to certain meiotic stages and may be mediated by the ability of this protein to associate with the chromatin.

Investigations into the potential role of aberrant N-acetylgalactosamine glycans in tumour cell interactions with basement membrane components.

The expression of the aberrant N-acetylgalactosamine (GalNAc) glycoconjugates, detected by binding of the lectin from Helix pomatia (HPA) is reported to be associated with metastatic competence and poor prognosis in a range of human adenocarcinomas, but the functional significance of the glycoconjugates in metastatic mechanisms is unknown. We have employed seven cell lines derived from normal breast epithelium, primary breast cancer and breast cancer metastases which stably express varying levels of HPA-binding glycoconjugates consistent with their derivation and phenotype. These cell lines have been thoroughly characterised and express identical profiles of HPA-binding glycoconjugates as tumour cells derived from clinical samples. Their ability to adhere to, and invade through, basement membrane components was investigated in a matrigel assay system, and the functional role of the aberrant GalNAc glycans assessed by competitive inhibition experiments using HPA. The behaviour of the cell lines in these assay systems was entirely consistent with their derivation and phenotype, but there was no evidence that the glycoconjugates of interest were functionally involved in adhesion or invasion mechanisms. Research in our laboratory is ongoing to seek a functional role for the HPA-binding glycoconjugates in other aspects of the metastatic cascade.

Characterisation and developmental expression of mouse Plu-1, a homologue of a human nuclear protein (PLU-1) which is specifically up-regulated in breast cancer.

PLU-1 is a novel breast cancer associated nuclear protein containing highly conserved domains including the PLU domain, putative DNA/chromatin binding motifs, and PHD/LAP domains. Here we report the cloning of the mouse homologue (Plu-1), and document its expression in adult tissues, mammary tumours and the embryo. The overall homology with human PLU-1 is 94% at the protein level, with almost 100% identity in the conserved domains, suggesting functional conservation. As with human PLU-1 the expression of Plu-1 in adult tissues is restricted, with high expression being seen only in testis, while expression in mammary tumours from c-neu transgenic mice is high. Plu-1 is also differentially expressed in the adult mammary gland. In the developing embryo Plu-1 is expressed in a temporally restricted fashion with tissue specific expression being limited to parts of the developing brain, whisker follicle, mammary bud, thymus, limbs, intervertebral disc, olfactory epithelium, teeth, eye, and stomach. The temporal and spatial expression patterns of the transcription factors Bf-1 and Pax9, recently found to bind to PLU-1 through the PLU domain overlap with Plu-1 expression during development. Thus Plu-1 appears to play an important role in mouse embryonic development which may involve interaction with Pax9 and Bf-1.

Characterisation and developmental expression of mouse Plu-1, a homologue of a human nuclear protein (PLU-1) which is specifically up-regulated in breast cancer.

PLU-1 is a novel breast cancer associated nuclear protein containing highly conserved domains including the PLU domain, putative DNA/chromatin binding motifs, and PHD/LAP domains. Here we report the cloning of the mouse homologue (Plu-1), and document its expression in adult tissues, mammary tumours and the embryo. The overall homology with human PLU-1 is 94% at the protein level, with almost 100% identity in the conserved domains, suggesting functional conservation. As with human PLU-1 the expression of Plu-1 in adult tissues is restricted, with high expression being seen only in testis, while expression in mammary tumours from c-neu transgenic mice is high. Plu-1 is also differentially expressed in the adult mammary gland. In the developing embryo Plu-1 is expressed in a temporally restricted fashion with tissue specific expression being limited to parts of the developing brain, whisker follicle, mammary bud, thymus, limbs, intervertebral disc, olfactory epithelium, teeth, eye, and stomach. The temporal and spatial expression patterns of the transcription factors Bf-1 and Pax9, recently found to bind to PLU-1 through the PLU domain overlap with Plu-1 expression during development. Thus Plu-1 appears to play an important role in mouse embryonic development which may involve interaction with Pax9 and Bf-1.