AZU-1: a candidate breast tumor suppressor and biomarker for tumor progression.

To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.

Tissue structure, nuclear organization, and gene expression in normal and malignant breast.

Because every cell within the body has the same genetic information, a significant problem in biology is to understand how cells within a tissue express genes selectively. A sophisticated network of physical and biochemical signals converge in a highly orchestrated manner to bring about the exquisite regulation that governs gene expression in diverse tissues. Thus, the ultimate decision of a cell to proliferate, express tissue-specific genes, or apoptose must be a coordinated response to its adhesive, growth factor, and hormonal milieu. The unifying hypothesis examined in this overview is that the unit of function in higher organisms is neither the genome nor the cell alone but the complex, three-dimensional tissue. This is because there are bidirectional connections between the components of the cellular microenvironment (growth factors, hormones, and extracellular matrix) and the nucleus. These connections are made via membrane-bound receptors and transmitted to the nucleus, where the signals result in modifications to the nuclear matrix and chromatin structure and lead to selective gene expression. Thus, cells need to be studied "in context", i.e., within a proper tissue structure, if one is to understand the bidirectional pathways that connect the cellular microenvironment and the genome. In the last decades, we have used well-characterized human and mouse mammary cell lines in "designer microenvironments" to create an appropriate context to study tissue-specific gene expression. The use of a three-dimensional culture assay, developed with reconstituted basement membrane, has allowed us to distinguish normal and malignant human breast cells easily and rapidly. Whereas normal cells become growth arrested and form organized "acini," tumor cells continue to grow, pile up, and in general fail to respond to extracellular matrix and microenvironmental cues. By correcting the extracellular matrix-receptor (integrin) signaling and balance, we have been able to revert the malignant phenotype when a human breast tumor cell is cultured in, or on, a basement membrane. Most recently, we have shown that whereas beta1 integrin and epidermal growth factor receptor signal transduction pathways are integrated reciprocally in three-dimensional cultures, on tissue culture plastic (two-dimensional monolayers), these are not coordinated. Finally, we have demonstrated that, rather than passively reflecting changes in gene expression, nuclear organization itself can modulate cellular and tissue phenotype. We conclude that the structure of the tissue is dominant over the genome, and that we may need a new paradigm for how epithelial-specific genes are regulated in vivo. We also argue that unless the structure of the tissue is critically altered, malignancy will not progress, even in the presence of multiple chromosomal mutations.

LIM domains of cysteine-rich protein 1 (CRP1) are essential for its zyxin-binding function.

Previous studies have demonstrated that the adhesion-plaque protein, zyxin, interacts specifically with a 23 kDa protein, called the cysteine-rich protein 1 (CRP1), which has been implicated in myogenesis. Primary sequence analyses have revealed that both zyxin and CRP1 exhibit multiple copies of a structural motif called the LIM domain. LIM domains, which are defined by the consensus CX2CX16-23HX2CX2CX2CX16-23CX2-3(C,H,D), are found in a variety of proteins that are involved in cell growth and differentiation. Recent studies have established that LIM domains are zinc-binding structures that mediate specific protein-protein interactions. For example, in the case of the zyxin-CRP1 interaction, one of zyxin's three LIM domains is necessary and sufficient for binding to CRP1. Because the CRP1 molecule is comprised primarily of two LIM domains, we were interested in the possibility that the binding site for zyxin on CRP1 might also be contained within a single LIM domain. Consistent with the hypothesis that the LIM domains of CRP1 are critical for the protein's zyxin-binding function, zinc-depleted CRP1 displays a reduced zyxin-binding activity. However, domain mapping analyses have revealed that neither of the two individual LIM domains of CRP1 can support a wild-type interaction with zyxin. Collectively, our results suggest that the binding site for zyxin on CRP1 is not contained within a single contiguous sequence of amino acids. Instead, the interaction appears to rely on the co-ordinate action of a number of residues that are displayed in both of CRP1's LIM domains.

Structural cues from the tissue microenvironment are essential determinants of the human mammary epithelial cell phenotype.

Historically, the study of normal human breast function and breast disorders has been significantly impaired by limitations inherent to available model systems. Recent improvements in human breast epithelial cell lines and three-dimensional (3-D)3 culture systems have contributed to the development of in vitro model systems that recapitulate differentiated epithelial cell phenotypes with remarkable fidelity. Molecular characterization of these human breast cell models has demonstrated that normal breast epithelial cell behavior is determined in part by the precise interplay that exists between a cell and its surrounding microenvironment. Recent functional studies of integrins in a human model system provide evidence to support the idea that the structural stability afforded by integrin-mediated cell-extracellular matrix interactions is an important determinant of normal cellular behavior, and that alterations in tissue structure can give rise to tumorigenic progression.

Comparison of three members of the cysteine-rich protein family reveals functional conservation and divergent patterns of gene expression.

Members of the cysteine-rich protein (CRP) family are evolutionarily conserved proteins that have been implicated in the processes of cell proliferation and differentiation. In particular, one CRP family member has been shown to be an essential regulator of cardiac and skeletal muscle development. Each of the three vertebrate CRP isoforms characterized to date is composed of two copies of the zinc-binding LIM domain with associated glycine-rich repeats. In this study, we have addressed the biological significance of the CRP multigene family by comparing the subcellular distributions, biochemical properties, and expression patterns of CRP1, CRP2, and CRP3/MLP. Our data reveal that all three CRP family members, when expressed in adherent fibroblasts, associate specifically with the actin cytoskeleton. Moreover, all three CRP isoforms are capable of interacting with the cytoskeletal proteins alpha-actinin and zyxin. Together, these observations suggest that CRP family members may exhibit overlapping cellular functions. Differences between the three CRPs are evident in their protein expression patterns in chick embryos. CRP1 expression is detected in a variety of organs enriched in smooth muscle. CRP2 is restricted to arteries and fibroblasts. CRP3/MLP is dominant in organs enriched in striated muscle. CRP isoform expression is also developmentally regulated in the chick. Our findings suggest that the three CRP family members perform similar functions in different muscle derivatives. The demonstration that all members of the CRP family are associated with cytoskeletal components that have been implicated in the assembly and organization of filamentous actin suggests that CRPs contribute to muscle cell differentiation via effects on cytoarchitecture.

Molecular dissection of a LIM domain.

LIM domains are novel sequence elements that are found in more than 60 gene products, many of which function as key regulators of developmental pathways. The LIM domain, characterized by the cysteine-rich consensus CX2CX16-23HX2CX2CX2CX16-21 CX2-3(C/H/ D), is a specific mental-binding structure that consists of two distinct zinc-binding subdomains. We and others have recently demonstrated that the LIM domain mediates protein-protein interactions. However, the sequences that define the protein-binding specificity of the LIM domain had not yet been identified. Because structural studies have revealed that the C-terminal zinc-binding module of a LIM domain displays a tertiary fold compatible with nucleic acid binding, it was of interest to determine whether the specific protein-binding activity of a LIM domain could be ascribed to one of its two zinc-binding subdomains. To address this question, we have analyzed the protein-binding capacity of a model LIM peptide, called zLIM1, that is derived from the cytoskeletal protein zyxin. These studies demonstrate that the protein-binding function of zLIM1 can be mapped to sequences contained within its N-terminal zinc-binding module. The C-terminal zinc-binding module of zLIM1 may thus remain accessible to additional interactive partners. Our results raise the possibility that the two structural subdomains of a LIM domain are capable of performing distinct biochemical functions.

The LIM domain is a modular protein-binding interface.

LIM domains are zinc-binding protein sequences that are found in a growing number of proteins, including certain transcriptional regulators, proto-oncogene products, and adhesion plaque constituents. Here we define the biological activity of the LIM domain through studies of an adhesion plaque protein called zyxin that displays three C-terminal LIM domains. We have used our ability to reconstitute complexes between zyxin and its two known binding partners, alpha-actinin and the cysteine-rich protein (CRP), to examine the involvement of LIM domains in protein-protein interactions. We have determined that one of the three LIM domains of zyxin is necessary and sufficient to support the association of zyxin with CRP. Our findings demonstrate that the LIM domain functions as a specific protein-binding interface.

The LIM motif defines a specific zinc-binding protein domain.

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

Platelet-derived growth factor activates phospholipase D and chemotactic responses in vascular smooth muscle cells.

Platelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response.

Assays for investigations of signal transduction mechanisms involving phospholipase D: mass measurements of phosphatidate, phosphatidylethanol, and diacylglycerol in cultured cells.

With the intent of achieving a better understanding of agonist-induced phospholipase D activity, we have developed simple, rapid assays for quantitating the mass of phosphatidate, phosphatidylethanol, and diacylglycerol. Crude lipid extracts of cultured cells are used; preliminary sample cleanup or derivatization procedures are not necessary. The assays resolve the particular lipids by short-bed/continuous-development thin-layer chromatography. Quantitative assessments are derived from photodensitometric analysis of charred lipid spots. The assays may be employed for as little as 45 pmol of diacylglycerol and 50 pmol of phosphatidate or phosphatidylethanol. The newly developed assays are compared to other procedures for quantitating lipid mediators. The utility of the assays is illustrated in experiments that use a variety of cultured cells to demonstrate the agonist activation of the phospholipase D pathway. In addition, experiments designed to screen various agonists for signal-response coupling to phospholipase D are described.

Vasopressin stimulates phospholipase D activity against phosphatidylcholine in vascular smooth muscle cells.

It is now clear that various hormones and agonists can stimulate the production of lipid mediators from non-phosphoinositide phospholipids. We have investigated the production of diacylglycerol from nonphosphoinositide sources, and we demonstrated that vasopressin and other vasoactive agents stimulate hydrolysis of phosphatidylcholine in a variety of cultured vascular smooth muscle cells of rat and human origin. We used vasopressin to characterize this response and found that vasopressin stimulates phospholipase D activity against phosphatidylcholine in A-10 vascular smooth muscle cells. The vasopressin-stimulated phosphatidylcholine hydrolysis is both time- and concentration-dependent. The half-maximal dose of vasopressin required for phosphatidylcholine hydrolysis (ED50 approximately 1 nM) correlates well with vasopressin binding to A-10 cells (Kd approximately 2 nM). The phosphatidylcholine in A-10 cells can be preferentially radiolabeled with [3H]myristic acid; subsequent treatment with vasopressin stimulates a rapid increase in 3H-labeled phosphatidate (approximately 4 X control values at 3 min), and after a short lag, 3H-labeled diacylglycerol rises and reaches maximal levels at 10 min (approximately 2 X control values). Similar temporal elevations of phosphatidate and diacylglycerol occur in A-10 cells labeled with [3H] glycerol. In A-10 cells radiolabeled with [3H] choline, the elevation of cellular phosphatidate and diacylglycerol is concomitant with the release of [3H] choline metabolites (predominantly choline) to the culture medium. The temporal production of phosphatidate and diacylglycerol as well as the release of choline to the culture medium are consistent with vasopressin activating phospholipase D. In addition, vasopressin stimulates a transphosphatidylation reaction that is characteristic of phospholipase D. The transphosphatidylation reaction is detected by the production of phosphatidylethanol that occurs when A-10 cells are incubated with ethanol and stimulated with vasopressin. The phospholipase D is active in the absence of extracellular Ca++ whereas the vasopressin-stimulated mobilization of arachidonic acid is dependent on extracellular Ca++. The data indicate that vasopressin stimulates phospholipase D which hydrolyzes phosphatidylcholine to phosphatidate. The phosphatidate is then metabolized, presumably by a phosphatidate phosphohydrolase, to produce sustained levels of cellular diacylglycerol. These sustained levels of diacylglycerol may activate protein kinase C and thereby function in the "sustained phase" of cellular responses.