Showing papers in "Journal of Cellular Biochemistry in 1996"

Cadherin-catenin complex: protein interactions and their implications for cadherin function.

Abstract: Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. In epithelial cells, E-cadherin represents a key molecule in the establishment and stabilization of cellular junctions. On the cellular level, E-cadherin is concentrated at the adherens junction and interacts homophilically with E-cadherin molecules of adjacent cells. Significant progress has been made in understanding the extra- and intracellular interactions of E-cadherin. Recent success in solving the three-dimensional structure of an extracellular cadherin domain provides a structural basis for understanding the homophilic interaction mechanism and the calcium requirement of cadherins. According to the crystal structure, individual cadherin molecules cooperate to form a linear cell adhesion zipper. The intracellular anchorage of cadherins is regulated by the dynamic association with cytoplasmic proteins, termed catenins. The cytoplasmic domain of E-cadherin is complexed with either β-catenin or plakoglobin (γ-catenin). β-catenin and plakoglobin bind directly to α-catenin, giving rise to two distinct cadherin-catenin complexes (CCC). α-catenin is thought to link both CCC's to actin filaments. The anchorage of cadherins to the cytoskeleton appears to be regulated by tyrosine phosphorylation. Phosphorylation-induced junctional disassembly targets the catenins, indicating that catenins are components of signal transduction pathways. The unexpected association of catenins with the product of the tumor suppressor gene APC has led to the discovery of a second, cadherin-independent catenin complex. Two separate catenin complexes are therefore involved in the cross-talk between cell adhesion and signal transduction. In this review we focus on protein interactions regulating the molecular architecture and function of the CCC. In the light of a fundamental role of the CCC during mammalian development and tissue morphogenesis, we also discuss the phenotypes of embryos lacking E-cadherin or β-catenin. © 1996 Wiley-Liss, Inc.

BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy.

Abstract: The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases.

HA receptors: regulators of signalling to the cytoskeleton.

Abstract: Hyaluronan (HA) is a ubiquitous component of the extracellular matrix (ECM) and occurs transiently in both the cell nucleus and cytoplasm. It has been shown to promote cell motility, adhesion, and proliferation and thus it has an important role in such processes as morphogenesis, wound repair, inflammation, and metastasis. These processes require massive cell movement and tissue reorganization and are always accompanied by elevated levels of HA. Many of the effects of HA are mediated through cell surface receptors, three of which have been molecularly characterized, namely CD44, RHAMM, and ICAM-1. Binding of the HA ligand to its receptors triggers signal transduction events which, in concert with other ECM and cytoskeletal components, can direct cell trafficking during physiological and pathological events. The HA mediated signals are transmitted, at least in part, by the activation of protein phosphorylation cascades, cytokine release, and the stimulation of cell cycle proteins. A variety of extracellular signals regulate the expression of both HA and the receptors necessitating that HA-receptor signalling is a tightly controlled process. Regulated production of soluble forms of the receptors, alternately spliced cell surface isoforms, and glycosylation variants of these receptors can dramatically modulate HA binding, ligand specificity, and stimulation of the signalling pathway. When these processes are deregulated cell behaviour becomes uncontrolled leading to developmental abnormalities, abnormal physiological responses, and tumorigenesis. The elucidation of the molecular mechanisms regulating HA-mediated events will not only contribute greatly to our understanding of a variety of disease processes but will also offer many new avenues of therapeutic intervention.

NCI-Navy Medical Oncology Branch cell line data base

Abstract: The cell line data base described in this paper includes both clinical information about the patients from whom the cell lines were derived and information about the in vitro analyses performed of the cell lines. The cell line data base has evolved as a part of a systematic effort by a research group at the NCI since 1976 to generate human cell lines as biological tools to study cancer and other diseases. The cell lines were generated from clinical specimens obtained as part of a series of Institutional Review Board-approved clinical protocols. The preponderance of the data is on lung cancer cell lines, though a broad range of other cancers are represented. A bank of over 300 human cell lines including cancer cell and in some instances autologous B-lymphoblastoid cells from the NCI-VA and NCI-Navy Medical Oncology Branch are reposited at the American Type Culture Collection. The cell lines are available for the research community. The entire data base is available on the American Type Culture Collection Web Site (WWW:http:@www.atcc.org/).

Regulation of leukocyte integrin function: Affinity vs. avidity

Abstract: Leukocytes circulate freely in the bloodstream until receiving signals which activate adhesive mechanisms essential for immune responsiveness. Key mediators of these adhesion events are heterodimeric cell surface receptors called integrins. It is now apparent that several components may contribute to successful integrin-mediated adhesion: alterations in individual receptors lead to enhanced affinity for ligand; integrin clustering causes an increase in avidity; by spreading, the adhering cell is less susceptible to shear force. Model systems have allowed us to examine the contribution of each of these factors in generating adhesion. In more physiologically relevant situations, it can now be questioned whether integrin-mediated adhesion is regulated via alterations in receptor affinity or avidity, or whether both these mechanisms are involved.

Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells.

Abstract: Bone formation involves several tightly regulated gene expression patterns of bone-related proteins. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone-related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4-10), bone matrix formation/maturation (days 10-16), and mineralization stages (days 16-30). During the proliferation period (days 4-10), the expression of cell-cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10-16), type I collagen expression and biosynthesis, fibronectin, TGF-beta 1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF-beta 1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16-30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone-related proteins are temporally regulated during the MC3T3-E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone-related proteins in relation to the different stages during the osteoblast differentiation.

Expression of bone‐specific genes by hypertrophic chondrocytes: Implications of the complex functions of the hypertrophic chondrocyte during endochondral bone development

Abstract: Endochondral bone formation is one of the most extensively examined developmental sequences within vertebrates. This process involves the coordinated temporal/spatial differentiation of three separate tissues (cartilage, bone, and the vasculature) into a variety of complex structures. The differentiation of chondrocytes during this process is characterized by a progressive morphological change associated with the eventual hypertrophy of these cells. These cellular morphological changes are coordinated with proliferation, a columnar orientation of the cells, and the expression of unique phenotypic properties including type X collagen, high levels of bone, liver, and kidney alkaline phosphatase, and mineralization of the cartilage matrix. Several studies indicate that hypertrophic chondrocytes also express osteocalcin, osteopontin, and bone sialoprotein, three proteins which until very recently were widely believed to be restricted in their expression to osteoblasts. Recent studies suggest that the hypertrophic chondrocytes are regulated by the calcitropic hormones, morphogenic steroids, and local tissue factors. These considerations are based on the regulation by 1,25 (OH)2D3 and retinoids of the cartilage specific genes as well as osteopontin and osteocalcin expression in hypertrophic chondrocytes. They are also based on the effects on growth plate development caused by 1) transgenic ablation of autocrine/paracrine regulators such as PTHrP and of the transcriptional regulator c-fos and 2) naturally occurring genetic mutations of the FGF receptor. These studies further suggest that specific transcriptional factors mediate exogenous regulatory signals in a coordinated manner with the development of bone. While it has been widely demonstrated that the majority of hypertrophic chondrocytes undergo apoptosis during terminal stages of the developmental sequence, their response to specific exogenous regulatory signals and their expression of bone-specific proteins give rise to questions about whether all growth chondrocytes have the same developmental fates and have identical functions. Furthermore, specific questions arise as to whether there are similar mechanisms of regulation for commonly expressed genes found in both cartilage and bone or whether these genes have unique regulatory mechanisms in these different tissues. These recent findings suggest that hypertrophic chondrocytes are functionally coupled during endochondral bone formation to the recruitment of osteoblasts, vascular cells, and osteoclasts.

Retinoblastoma protein family in cell cycle and cancer: A review

Abstract: Two genes, p107 and Rb2/p130, are strictly related to RB, the most investigated tumor suppressor gene, responsible for susceptibility to retinoblastoma. The products of these three genes, namely pRb, p107, and pRb2/p130 are characterized by a peculiar steric conformation, called "pocket," responsible for most of the functional interactions characterizing the activity of these proteins in the homeostasis of the cell cycle. The interest in these genes and proteins springs from their ability to regulate cell cycle processes negatively, being able, for example, to dramatically slow down neoplastic growth. So far, among these genes, only RB is firmly established to act as a tumor suppressor, because its lack-of-function is clearly involved in tumor onset and progression. It has been found deleted or mutated in most retinoblastomas and sarcomas, but its inactivation is likely to play a crucial role in other types of human cancers. The two other members of the family have been discovered more recently and are currently under extensive investigation. We review analogies and differences among the pocket protein family members, in an attempt to understand their functions in normal and cancer cells.

Apoptosis mediated by the TNF-related cytokine and receptor families

Abstract: T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin beta-receptor (LT beta R), among others. The MHC-encoded cytokines, TNF and LT alpha, form homomeric trimers, whereas LT beta assembles into heterotrimers with LT alpha, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LT beta R are involved in induction NF kappa B and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the "death domain" homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways.

Inverted repeats, stem‐loops, and cruciforms: Significance for initiation of DNA replication

Abstract: Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.

Variable effects of tyrosine kinase inhibitors on avian osteoclastic activity and reduction of bone loss in ovariectomized rats

Abstract: We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID50s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of 3H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID50s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.

Domains of laminin

Abstract: Extracellular matrix molecules are often very large and made up of several independent domains, frequently with autonomous activities. Laminin is no exception. A number of globular and rod-like domains can be identified in laminin and its isoforms by sequence analysis as well as by electron microscopy. Here we present the structure-function relations in laminins by examination of their individual domains. This approach to viewing laminin is based on recent results from several laboratories. First, some mutations in laminin genes that cause disease have affected single laminin domains, and some laminin isoforms lack particular domains. These mutants and isoforms are informative with regard to the activities of the mutated and missing domains. Second, laminin-like domains have now been found in a number of other proteins, and data on these proteins may be informative in terms of structure-function relationships in laminin. Finally, a large body of data has accumulated on the structure and activities of proteolytic fragments, recombinant fragments, and synthetic peptides from laminin. The proposed activities of these domains can now be confirmed and extended by in vivo experiments. © 1996 Wiley-Liss, Inc.

Risk biomarkers and current strategies for cancer chemoprevention

Abstract: Quantifiable, well-characterized cancer risk factors demonstrate the need for chemoprevention and define cohorts for chemopreventive intervention. For chemoprevention, the important cancer risk factors are those that can be measured quantitatively in the subject at risk. These factors, called risk biomarkers, can be used to identify cohorts for chemoprevention. Those modulated by chemopreventive agents may also be used as endpoints in chemoprevention studies. Generally, the risk biomarkers fit into categories based on those previously defined by Hulka: 1) carcinogen exposure, 2) carcinogen exposure/effect, 3) genetic predisposition, 4) intermediate biomarkers of cancer, and 5) previous cancers. Besides their use in characterizing cohorts for chemoprevention trials, some risk biomarkers can be modulated by chemopreventive agents. These biomarkers may be suitable surrogate endpoints for cancer incidence in chemoprevention intervention trials. The criteria for risk biomarkers defining cohorts and serving as endpoints are the same, except that those defining cohorts are not necessarily modulated by chemopreventive agents. A primary criterion is that the biomarkers fit expected biological mechanisms of early carcinogenesis—i.e., differential expression in normal and high-risk tissue, on or closely linked to the causal pathway for the cancer, and short latency compared with cancer. They must occur in sufficient number to allow their biological and statistical evaluation. Further, the biomarkers should be assayed reliably and quantitatively, measured easily, and correlated to cancer incidence. Particularly important for cancer risk screening in normal subjects is the ability to use noninvasive techniques that are highly specific, sensitive, and quantitative. Since carcinogenesis is a multipath process, single biomarkers are difficult to correlate to cancer, as they may appear on only one or a few of the many possible causal pathways. As shown in colorectal carcinogenesis, the risks associated with the presence of biomarkers may be additive or synergistic. That is, the accumulation of genetic lesions is the more important determinant of colorectal cancer compared with the presence of any single lesion. Thus, batteries of biomarker abnormalities, particularly those representing the range of carcinogenesis pathways, may prove more useful than single biomarkers both in characterizing cohorts at risk and defining modulatable risks. Risk biomarkers are already being integrated into many chemoprevention intervention trials. One example is the phase II trial of oltipraz inhibition of carcinogen-DNA adducts in a Chinese population exposed to aflatoxin B1. Also, urine samples from subjects in this trial will be screened for the effect of oltipraz on urinary mutagens. A second example is a chemoprevention protocol developed for patients at high risk for breast cancer; the cohort is defined both by hereditary risk and the presence of biomarker abnormalities. Modulation of the biomarker abnormalities is a proposed endpoint. Also, dysplastic lesions, such as prostatic intraepithelial neoplasia, oral leukoplakia and colorectal adenomas, have been used to define high-risk cohorts and as potential modulatable surrogate endpoints in chemoprevention trials. J. Cell. Biochem. 25S:1–14. © 1997 Wiley-Liss, Inc.11

New agents for cancer chemoprevention.

Abstract: Clinical chemoprevention trials of more than 30 agents and agent combinations are now in progress or being planned. The most advanced agents are well known and are in large Phase III chemoprevention intervention trials or epidemiological studies. These drugs include several retinoids [e.g., retinol, retinyl palmitate, all-trans-retinoic acid, and 13-cis-retinoic acid], calcium, Beta carotene, vitamin E, tamoxifen, and finasteride. Other newer agents are currently being evaluated in or being considered for Phase II and early Phase III chemoprevention trials. Prominent in this group are all-trans-N-(4-hydroxy phenyl)retinamide (4-HPR) (alone and in combination with tamoxifen), 2-difluoromethylornithine (DFMO), nonsteroidal antiinflammatory drugs (aspirin, piroxicam, sulindac), oltipraz, and dehydroepiandrostenedione (DHEA). A third group is new agents showing chemopreventive activity in animal models, epidemiological studies, or in pilot clinical intervention studies. They are now in preclinical toxicology testing or Phase I safety and pharmacokinetics trials preparatory to chemoprevention efficacy trials. These agents include S-allyl-l-cysteine, curcumin, DHEA analog 8354 (fluasterone), genistein, ibuprofen, indole-3-carbinol, perillyl alcohol, phenethyl isothiocyanate, 9-cis-retinoic acid, sulindac sulfone, tea extracts, ursodiol, vitamin D analogs, and p-xylyl selenocyanate. A new generation of agents and agent combinations will soon enter clinical chemoprevention studies based primarily on promising chemopreventive activity in animal models and in mechanistic studies. Among these agents are more efficacious analogs of known chemopreventive drugs including novel carotenoids (e.g., alpha-carotene and lutein). Also included are safer analogs which retain the chemopreventive efficacy of the parent drug such as vitamin D3 analogs. Other agents of high interest are aromatase inhibitors (e.g., (+)-vorozole), and protease inhibitors (e.g., Bowman-Birk soybean trypsin inhibitor). Combinations are also being considered, such as vitamin E with l-selenomethionine. Analysis of signal transduction pathways is beginning to yield classes of potentially active and selective chemopreventive drugs. Examples are ras isoprenylation and epidermal growth factor receptor inhibitors.

Integrin‐dependent signal transduction

Abstract: Integrins are receptor molecules for extracellular matrix molecules (e.g., the beta(1) family), serum components (alpha(v) family) and immunoglobulin family adhesion molecules (beta(2) family). Integrin-dependent adhesion has also been shown to have metabolic consequences. Adhesion to a variety of extracellular matrix proteins, such as fibronectin, collagen, and laminin, is a potent regulator of cell growth, differentiation, and gene expression. Ligand binding or aggregation of integrin receptors initiates a number of metabolic changes including activation of serine/threonine and tyrosine kinases, increased Ca2+ influx, increased cytoplasmic alkalinization, and altered inositol lipid metabolism. In some instances activation of transcription factors and induction of gene expression have also been demonstrated. Components of key signaling pathways involving integrins are beginning to be identified. Some studies have shown that integrins form multi-component complexes with signal transduction molecules. Elucidating the interactions of the signal transduction molecules with each other and with the integrin cytoplasmic domains will be key to understanding the initial events of signal transduction through the integrins.

Structural and functional diversity of cadherin superfamily: Are new members of cadherin superfamily involved in signal transduction pathway?

Abstract: A large number of cadherins and cadherin-related proteins are expressed in different tissues of a variety of multicellular organisms. These proteins share one property: their extracellular domains consist of multiple repeats of a cadherin-specific motif. A recent structure study has shown that the cadherin repeats roughly corresponding to the folding unit of the extracellular domains. The members of the cadherin superfamily are roughly classified into two groups, classical type cadherins proteins and protocadherin type according to their structural properties. These proteins appear to be derived from a common ancestor that might have cadherin repeats similar to those of the current protocadherins, and to have common functional properties. Among various cadherins, E-cadherin was the first to be identified as a Ca(2+)-dependent homophilic adhesion protein. Recent knockout mice experiments have proven its biological role, but there are still several puzzling unsolved properties of the cell adhesion activity. Other members of cadherin superfamily show divergent properties and many lack some of the expected properties of cell adhesion protein. Since recent studies of various adhesion proteins reveal that they are involved in different signal transduction pathways, the idea that the new members of cadherin superfamily may participate in more general cell-cell interaction processes including signal transduction is an intriguing hypothesis. The cadherin superfamily is structurally divergent and possibly functionally divergent as well.

Temporal study of the activity of matrix metalloproteinases and their endogenous inhibitors during wound healing.

Abstract: The restoration of functional connective tissue is a major goal of the wound healing process. This regenerative event requires the deposition and accumulation of collagenous and noncollagenous matrix molecules as well as the remodelling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). In this study, we have utilized substrate gel electrophoresis, radiometric enzyme assays, and Western blot analyses to determine the temporal pattern of appearance and activity of active and latent MMPs and their inhibitors during the entire healing process in a partial thickness wound model. Through the use of substrate gel electrophoresis, we studied the appearance of proteolytic bands whose molecular weight was consistent with their being members of the MMP family of enzymes. Proteolytic bands whose molecular weight is consistent with both the active and latent forms of MMP-2 (72 kDa, Type IV gelatinase) were detected in wound fluid of days 1-7 after wounding. The number of active MMP-2 species detectable in wound fluid was greatest during days 4-6 after wounding. The most prominent proteolytic band detected each day migrated with a molecular weight consistent with it being the latent form of MMP-9 (92 kDa, Type V pro-collagenase). In contrast to MMP-2, the active form of this enzyme was never detected. The presence of MMP-1 (interstitial collagenase) was detected by immunoblot in the wound fluid from days 1-6 post-injury. Using a radiometric enzyme assay for collagenase inhibitory activity we have also determined the time course of activity of endogenous matrix metalloproteinase inhibitors. We have correlated these data to the known cellular events occurring in the wound during this time period as well. This study establishes a prototypical pattern of MMP appearance in normal wound healing. It may also provide potential intervention sites for the therapeutic use of inhibitors of aberrant MMP activities which characterize chronic wounds.

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

Abstract: The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identi- fied the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.

Tamoxifen induces TGF-beta 1 activity and apoptosis of human MCF-7 breast cancer cells in vitro.

Abstract: We report here that the antiestrogen tamoxifen (TAM) induces cell death in human breast cancer cell line MCF-7. We assessed the type of cell death induced by TAM in this breast cancer cell line on the basis of morphological and biochemical characteristics. Dying cells showed morphological characteristics of apoptosis, such as chromatin condensation and nuclear disintegration. DNA isolated from these cells revealed a pattern of distinctive DNA bands on agarose gel. The DNA fragmentation in MCF-7 cells induced by TAM could also be detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Northern blot hybridization revealed a substantial increase in the amounts of TRPM-2 and TGF-beta 1 mRNAs in MCF-7 cells after treatment with TAM. In contrast, the mRNA level of the estrogen-induced pS2 gene was strongly suppressed. The biological activity of TGF-beta was increased at least fourfold in the media from MCF-7 cells treated with TAM. The results presented in this study suggest that TAM induces apoptosis of MCF-7 cells and it may be mediated by the secretion of active TGF-beta.

PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components

Abstract: The PML protein is a human growth suppressor concentrated in 10 to 20 nuclear bodies per nucleus (PML bodies). Disruption of the PML gene has been shown to be related to acute promyelocytic leukaemia (APL). To obtain information about the function of PML bodies we have investigated the 3D-distribution of PML bodies in the nucleus of T24 cells and compared it with the spatial distribution of a variety of other nuclear components, using fluorescence dual-labeling immunocytochemistry and confocal microscopy. Results show that PML bodies are not enriched in nascent RNA, the splicing component U2-snRNP, or transcription factors (glucocorticoid receptor, 'TFIIH, and E2F). These results show that PML bodies are not prominent sites of RNA synthesis or RNA splicing. We found that a large fraction of PML bodies (50 to 80%) is closely associated with DNA replication domains during exclusively middle-late S-phase. Furthermore, in most cells that we analysed we found at least one PML body was tightly associated with a coiled body. In the APL cell line NB4, the PML gene is fused with the RARa gene due to a chromosomal rearrangement. PML bodies have disappeared and the PML antigen, i.e., PML and the PML-RAR fusion protcin, is dispersed in a punctated pattern throughout the nucleoplasm. We showed that in NB4 cells the sites that are rich in PML antigen significantly colocalize with sites at which nascent RNA accumulates. This suggests that, in contrast to non-APL cells, in NB4 cells the PML antigen is associated with sites of transcription. The implications of these findings for the function of PML bodies are consistent with the idea that PML bodies are associated with specific genomic loci. L! 1996 Wiley-Liss, Inc.

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Abstract: Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C2C12) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products.

Oncogene alterations in primary, recurrent, and metastatic human bone tumors

Abstract: We investigated the structure and the expression of various oncogenes in three of the most common human bone tumors-osteosarcoma (36 samples from 34 patients), giant cell tumor (10 patients), and chondrosarcoma (18 patients)-in an attempt to identify the genetic alterations associated with these malignancies. Alterations of RB and p53 were detected only in osteosarcomas. Alterations of c-myc, N-myc, and c-fos were detected in osteosarcomas and giant cell tumors. Ras alterations (H-ras, Ki-ras, N-ras) were rare. Chondrosarcomas did not contain any detectable genetic alterations. Our results suggest that alterations of c-myc, N-myc, and c-fos oncogenes occur in osteosarcomas, in addition to those previously described for the tumor suppressor genes RB and p53. Moreover, statistical analyses indicate that c-fos alterations occur more frequently in osteosarcoma patients with recurrent or metastatic disease.

Effect of acidic phospholipids on sphingosine kinase.

Abstract: Sphingosine-1-phosphate (SPP) is a unique sphingolipid metabolite involved in cell growth regulation and signal transduction. SPP is formed from sphingosine in cells by the action of sphingosine kinase, an enzyme whose activity can be stimulated by growth factors. Little is known of the mechanisms by which sphingosine kinase is regulated. We found that acidic phospholipids, particularly phosphatidylserine, induced a dose-dependent increase in sphingosine kinase activity due to an increase in the apparent Vmax of the enzyme. Other acidic phospholipids, such as phosphatidylinositol, phosphatidic acid, phosphatidylinositol bisphosphate, and cardiolipin stimulated sphingosine kinase activity to a lesser extent than phosphatidylserine, whereas neutral phospholipids had no effect. Diacylglycerol, a structurally similar molecule which differs from phosphatidic acid in the absence of the phosphate group, failed to induce any changes in sphingosine kinase activity. Our results suggest that the presence of negative charges on the lipid molecules is important for the potentiation of sphingosine kinase activity, but the effect does not directly correlate with the number of negative charges. These results also support the notion that the polar group confers specificity in the stimulation of sphingosine kinase by acidic glycerophospholipids. The presence of a fatty acid chain in position 2 of the glycerol backbone was not critical since lysophosphatidylserine also stimulated sphingosine kinase, although it was somewhat less potent. Dioleoylphosphatidylserine was the most potent species, including a fourfold stimulation, whereas distearoyl phosphatidylserine was completely inactive. Thus, the degree of saturation of the fatty acid chain of the phospholipids may also play a role in the activation of sphingosine kinase. © 1996 Wiley-Liss, Inc.

Differential gene expression of extracellular matrix components in dilated cardiomyopathy

Abstract: Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13-21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP-1). Northern blot analysis using MMP-1 cDNA probe indicated that MMP-1 was induced three- to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP-1 and MMP-1 levels were 37 ± 8 ng/mg and 9 ± 2 ng/mg in normal tissue specimens (P < 0.01) and 2 ± 1 ng/mg and 45 ± 11 ng/mg in DCM tissue (P < 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP-1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP-1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic-fibroblast cell ECM-proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy. © 1996 Wiley-Liss, Inc.

Expression of bone matrix proteins during dexamethasone‐induced mineralization of human bone marrow stromal cells

Abstract: Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10(-10) M with maximum values achieved at 10(-8)M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1 ,25(OH)(2)D(3) during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells.

Evolutionary conservation of a genetic pathway of programmed cell death

Abstract: Genetic analysis of programmed cell death in Caenorhabditis elegans has led to the identification of 13 genes that constitute a developmental pathway of programmed cell death. Two of the three key genes in this pathway, ced-9, a cell death suppressor, and ced-3, a cell death inducer, were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1β converting enzyme, respectively. These results suggest that the genetic pathway of programmed cell death may be evolutionarily conserved from worms to mammals. © 1996 Wiley-Liss, Inc.

Secreted MUC1 mucins lacking their cytoplasmic part and carrying sialyl-Lewis a and x epitopes from a tumor cell line and sera of colon carcinoma patients can inhibit HL-60 leukocyte adhesion to E-selectin-expressing endothelial cells.

Abstract: A secreted MUC1 mucin from the spent medium of the colon carcinoma cell line COLO 205 carrying sialyl-Lewis a and x epitopes (H-CanAg) was purified by trichloroacetic acid precipitation and Superose 6 gel filtration. The purified H-CanAg inhibited adhesion of the leukocyte cell line HL-60 to E-selectin transfected COS-1 cells or interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells. Sera from two patients with advanced colon carcinoma containing high concentrations of sialyl-Lewis a and x activity inhibited HL-60 cell adhesion to E-selectin-expressing COS-1 cells and IL-1 beta-activated endothelial cells. After affinity column absorption of the sialyl-Lewis a activity, the sera also lost most of their sialyl-Lewis x activity and at the same time their adhesion inhibitory effect. A large part of the sialyl-Lewis a/x activity in the two patients was found in fractions containing mucins having a MUC1 apoprotein, as shown by its size, and reactivity with the two anti-MUC1 apoprotein monoclonal antibodies, Ma552 and HMFG-2. The cell-adhesion inhibitory effect of the purified sialyl-Lewis a-carrying MUC1 mucin fraction from the sera of the two patients was stronger than that of smaller sized sialyl-Lewis a-carrying mucin-type glycoproteins also found in the patient sera. The MUC1 mucin fraction secreted by the COLO 205 cells and from the two sera were all shown to lack their C-terminal portion, in contrast to the MUC1 mucin from cells. It is hypothesized that sialyl-Lewis a- and/or x-containing mucins, especially MUC1, secreted by tumors can interact with E-selectin on endothelial cells and thus inhibit leukocyte adhesion.

Syndecans, signaling, and cell adhesion

Abstract: Syndecans are transmembrane proteoglycans which can participate in diverse cell surface interactions, involving extracellular matrix macromolecules, growth factors, protease inhibitors, and even viral entry. Currently, all extracellular interactions are believed to be mediated by distinct structures within the heparan sulfate chains, leaving the roles of chondroitin sulfate chains and extracellular portion of the core proteins to be elucidated. Evidence that syndecans are a class of receptor involved in cell adhesion is mounting, and their small cytoplasmic domains may link with the microfilament cytoskeleton, thereby mediating signaling events. The molecular details are unknown, but the conservation of regions of syndecan cytoplasmic domains, and a strong tendency for homotypic association, support the idea that the ligand-induced clustering may be a discrete source of specific transmembrane signaling from matrix to cytoskeleton, as proposed for other classes of adhesion receptors.

PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis

Abstract: The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-El preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and p-glycerophosphate, and samples were collected from 0-26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-EI and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-El and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using 1251-PTHrP. The numbers of receptors per pg DNA were greatest at days 3 and 5 for MC3T3-El and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-EI cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-EI cells had twice the PTH- stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was adminis- tered to MC3T3-EI cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/ PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro.

Cell culture methods for the establishment of the NCI series of lung cancer cell lines

Abstract: More than 200 human small cell lung cancer and non-small cell lung cancer cell lines were established over 15 years mainly by utilizing the serum-free, hormone and growth factor supplemented, defined media HITES and ACL4. Use of modified, established cell culture techniques such as the mechanical spillout method for the releasing of cell aggregates from tumor tissue, ficoll gradient centrifugation for the separation of tumor cells from erythrocytes and tissue debris, and an apparatue consisting of a platinum tubing attached to a suction flask for removal of spent medium have greatly contributed to the success in culturing tumor cells. Characterization of these lung cancer cell lines have extended our knowledge of lung cell biology. Studies elucidating the nutritional requirements of lung cancer cell growth may be helpful for the manipulation of these tumors in patients.