IntroductionQualitative evaluation of the cellular complexity and structural integrity of organ biopsies has been used by pathologists for decades to gain insight into human diseases. The well-accepted correlation between tissue structure and health or disease conveys important lessons for the development of experimental models for the study of normal human biology and associated disease progression. Optimally, model design should recapitulate both the 3D organization and the differentiated function of any given organ but at the same time allow experimental intervention. By doing so, cell-based models facilitate systematic analyses that address, at the molecular level, how normal organ structure and function are maintained or how the balance is lost in cancer.Because of the ethical, technical and financial constraints inherent in research on human cells and tissues, the demand for models that faithfully parallel human form and function considerably outweighs the supply. We and others asserted more than two decades ago that development of physiologically relevant models of both rodent and human origin should recognize that organs and tissues function in a 3D environment (Elsdale and Bard, 1972;Hay and Dodson, 1973;Bissell, 1981;Ingber and Folkman, 1989). Further, that in the final analysis, the organ itself is the unit of function (Bissell and Hall, 1987). We now know that exposure of cells to the spatial constraints imposed by a 3D milieu determines how cells perceive and interpret biochemical cues from the surrounding microenvironment [e.g. the extracellular matrix, growth factors and neighboring cells (for reviews, see Roskelley et al., 1995;Bissell et al., 2002;Cunha et al., 2002;Ingber, 2002;Radisky et al., 2002)]. Furthermore, it is in this biophysical and biochemical context that cells display bona fide tissue and organ specificity.Here, we describe studies of epithelial-cell-based systems that demonstrate the importance of developing and utilizing 3D human organotypic models to understand the molecular and cellular signaling events underlying human organ biology (Fig. 1). In their most simplistic form, these models comprise homogeneous epithelial cell populations that are cultured within 3D basement-membrane-like matrices. These relatively simple 'monotypic' cell models have progressively evolved into 3D co-culture models containing multiple cell types, which approximate organ structure and function in vitro and enable systematic analyses of the molecular contributions of multiple cell types. Finally, we go on to explore how human 3D culture models are being coupled to existing technologies in the mouse to generate models in vivo that could elucidate the fundamental influence of stromal-epithelial interactions in normal organ function as well as those that perturb organ homeostasis and lead to disease. As a result of these advancements, we are equipped with a hierarchy of related models that appreciate the importance of 3D environments but vary with respect to cellular complexity. By using this collectio...

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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 ␣-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.Members of the cysteine-rich protein (CRP) 1 family are evolutionarily conserved proteins that have been implicated in myogenesis. CRPs exhibit a common domain structure, being composed primarily of two tandemly arrayed LIM domains (1, 2). Each LIM domain, defined by the consensus sequence

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-ll, Isl-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are remiiniscent of a metalbinding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) The LIM motif is a cysteine-rich sequence found in a diverse collection of proteins including transcription factors (1-5), a protooncogene product (6, 7), and cytoskeletal components (8-11). Many of the LIM proteins appear to be involved in regulation of gene expression and cellular differentiation during development. The specific function of the LIM domain has not been established, although it has been postulated to serve as a DNA or protein binding interface. Because of the abundance of conserved cysteine residues in the LIM consensus sequence, the motif has been widely proposed to be a metal-binding sequence. Efforts have been made to examine the metal-binding properties of LIM-motif proteins (12,13). For example, it has been demonstrated that the cysteine-rich intestinal protein (CRIP) will bind exogenously applied Zn ions (13). In addition, expression of the LIM domains of Lin-1l in Escherichia coli resulted in the isolation of a protein from inclusion bodies that contained both Zn and an Fe-S cluster (12). The observation of an Fe-S cluster in Lin-1l prompted consideration of the intriguing idea that the LIM transcription factors might be redox-regulated (12).In this report we present a comprehensive metal analysis of a LIM-domain protein isolated from its endogenous source. Specifically, we describe the metal-binding properties of the chicken cysteine-rich protein (cCRP) isolated from avian smooth muscle (10). cCRP, the chicken homologue of the human CRP (11), exhibits two LIM domains of the sequence CX2CX17HX2CX2CX2CX17CX2C (J. Pino and M.C.B., unpublished results). Our results show cCRP to be a Zn(II) metalloprotein. The implications of these results for the LIM domain structure are discussed. MATERIALS AND METHODSPurification of cCRP. cCRP was purified from fresh chicken gizzards by a procedure to be described in detail elsewhere (A. W. Crawford, J. D. Pino, and M.C.B., unpublished work). The purity of cCRP was demonstrated by SDS/PAGE and amino acid analysis. An extinction coefficient for cCRP of 2.66 x 104 M-1'cm-' was obtained by measurement of the absorbance at 280 nm followed by quantitation of the cCRP protein by amino acid analysis. Thiol titrations were carried out as described (14, 15).Metal Exchange. cCRP was prepared for metal exchange reactions by dialysis in buffer M (40 mM Tris Cl, pH 7.5/40 mM KCl). The protein was diluted 10-fold with 0.2 M potassium phosphate (pH 7.2) and subsequently incubated with mentioned quantities of metal salts. Spectra were recorded after 10 min. For measurements of binding stoichiometry, the metal-replaced samples were incubated for 1-3 hr, dialyzed in buffer M containi...

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.

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.

The Centers for Disease Control and Prevention (CDC) continues to be aware of the need for response to public concern as well as to state and local agency concern about cancer clusters. In 1990 the CDC published the “Guidelines for Investigating Clusters of Health Events,” in which a four-stage process was presented. This document has provided a framework that most state health departments have adopted, with modifications pertaining to their specific situations, available resources, and philosophy concerning disease clusters. The purpose of this present article is not to revise the CDC guidelines; they retain their original usefulness and validity. However, in the past 15 years, multiple cluster studies as well as scientific and technologic developments have affected cluster science and response (improvements in cancer registries, a federal initiative in environmental public health tracking, refinement of biomarker technology, cluster identification using geographic information systems software, and the emergence of the Internet). Thus, we offer an addendum for use with the original document. Currently, to address both the needs of state health departments as well as public concern, the CDC now a) provides a centralized, coordinated response system for cancer cluster inquiries, b) supports an electronic cancer cluster listserver, c) maintains an informative web page, and d) provides support to states, ranging from laboratory analysis to epidemiologic assistance and expertise. Response to cancer clusters is appropriate public health action, and the CDC will continue to provide assistance, facilitate communication among states, and foster the development of new approaches in cluster science.

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.