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D T Denhardt

Publications and source records attributed to D T Denhardt.

At least 73 records · Page 4Linked to original sources
PubMed1993

Temporal expression of tissue inhibitors of metalloproteinases in mouse reproductive tissues during gestation.

Tissue inhibitors of metalloproteinases (TIMPs) appear to play an important regulatory role in tissue remodelling and invasion by malignant cells. Since pregnancy involves morphological changes in existing maternal tissues, as well as a strictly controlled invasion by fetal trophoblasts, we have examined the temporal expression of TIMP-1, TIMP-2, and specific metalloproteinases in the mouse uterus, decidua, placenta, amnion, and ovaries throughout gestation by examining mRNA levels on northern and slot blots. Maximal levels of TIMP-1 mRNA were observed from day 6 to day 10 in the uterus, decidua, and placenta. In clear contrast to the early burst of TIMP-1 mRNA accumulation, the level of TIMP-2 mRNA increased steadily throughout gestation in the uterus, decidua, and amnion, while in the placenta it showed a sevenfold increase after day 14. In amnion, TIMP-1 was induced specifically on day 18. Interestingly, the normally high level of TIMP-1 mRNA seen in the ovaries of virgin mice was low during gestation, until day 18 and postpartum, when a sixfold increase over the levels in virgin ovary was observed. In contrast, ovarian TIMP-2 mRNA showed a marginal increase during gestation. The temporal pattern of 72 kDa gelatinase type A followed that of TIMP-2 in the decidua and ovary. Stromelysin-2 mRNA was detected at term only in ovary and decidua. Our data show that the temporal accumulation of TIMP-1 and TIMP-2 mRNA is precisely coordinated in each of the tissue compartments and is independently regulated during the in vivo remodeling of reproductive tissues in gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amnion↗
PubMed1993

Tissue inhibitor of metalloproteinases (TIMP, aka EPA): structure, control of expression and biological functions.

The TIMPs play an important role in regulating the activity of the secreted metalloproteinases (collagenases, stromelysins, gelatinases). Two different TIMPS have been well characterized, each capable of inhibiting all tested eukaryotic metalloproteinases but showing specific binding to a particular gelatinase at a site distinct from the active site. They influence the activation of the prometalloproteinase and act to modulate proteolysis of extracellular matrix, notably during tissue remodeling and inflammatory processes. On certain cell types, they can exhibit growth factor-like activity, and they can inhibit the tumorigenic and metastatic phenotype of cancer cells.

Amino Acid Sequence↗
PubMed1993

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.

Animals↗
PubMed1993

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

Amino Acid Sequence↗
PubMed1993

Osteopontin: a protein with diverse functions.

In this review most of the various known, suspected, or postulated functions of osteopontin, a secreted highly acidic phosphoprotein, are discussed in terms of what we currently know about the protein. These include 1) binding of OPN both to cells via a GRGDS cell adhesion sequence that recognizes the alpha v beta 3 integrin and to extracellular matrix components via poorly characterized motifs, 2) regulation of the formation and remodeling of mineralized tissue, 3) recruiting and stimulating macrophages and lymphocytes as part of a nonspecific response to microbial infections, 4) multiple interactions with Ca2+ that likely influence OPN protein conformation and may be important in Ca(2+)-mediated or Ca(2+)-dependent processes, 5) inhibiting the growth of calcium oxalate crystals by disruption of the growing crystal lattice, 6) effects on gene expression, Ca2+ regulation, and nitric oxide production, and 7) involvement in cell migration. OPN production is frequently augmented when cell signaling pathways are activated by any of a variety of stimuli, for example in cancer cells.

Amino Acid Sequence↗
PubMed1993

Heterogeneity of osteopontin expression among nephrons in mouse kidneys and enhanced expression in sclerotic glomeruli.

BACKGROUND: Osteopontin (OPN) is a secreted Ca(2+)-binding phosphoprotein able to mediate cell attachment to bone via an RGD sequence and the alpha v beta 3 integrin. OPN mRNA is found at high levels in the kidney, and the protein is found in the urine. Because published reports of where the protein is produced conflict, we undertook a comprehensive study to localize OPN expression. EXPERIMENTAL DESIGN: In situ hybridization with a mouse cDNA probe and immunohistochemical staining with three different antisera to mouse OPN were used to identify those cells that contained significant levels of mRNA and protein, respectively. RESULTS: Both methods of analysis revealed that OPN expression in the normal mouse kidney was primarily restricted to the thick ascending limbs of the loop of Henle and to the distal convoluted tubules. Protein was detected predominantly at the apical surface of cells lining the lumen of a subset of tubules. The alpha v beta 3 integrin, which is a receptor for vitronectin and osteopontin, was uniformly localized by immunostaining not on the apical surface but rather to the baso-lateral surface of cells in the distal part of the tubule. OPN expression was not detected in healthy glomeruli, proximal tubules, thin limbs of the loop of Henle, collecting ducts, or interstitial fibroblasts. In contrast to the localization of Tamm-Horsfall protein expression, in all distal nephrons, expression of OPN was detected by both methods of analysis in only some nephrons. OPN expression (relative to male mice) was somewhat increased in female, pregnant and lactating mice and markedly increased in the parietal epithelium of glomeruli undergoing sclerosis in aging mice. OPN was also detected in the macula densa. CONCLUSIONS: OPN is synthesized and secreted into the tubule fluid by the luminal epithelia of the distal portions of a subset of kidney nephrons. As animals age expression is found in more proximal portions of the tubule. OPN may contribute to, or be a consequence of, glomerular sclerosis, and may be an indicator of subclinical injury or infection.

Aging↗
PubMed1992

Mechanism of action of antisense RNA. Sometime inhibition of transcription, processing, transport, or translation.

Anyone considering the use of AS RNA, generated endogenously, to inhibit gene expression should plan to generate several independent transfectants with nonoverlapping sequences; strategies that maximize both the transcription rate and the stability of the AS RNA are obviously desirable. Reasons why different results are obtained in different systems or with different constructs likely include the specific nucleotide sequence under investigation, the location of the AS gene in the nucleus relative to the endogenous gene, and the rate-limiting step in the expression of the target gene. Splicing may not be necessary, but an efficient polyadenylation signal likely is. Employment of a ribozyme-mediated strategy, discussed by various investigators in this volume, may be beneficial. There is no reason at present to conclude that any gene, however abundant its transcript might be, is inherently recalcitrant to AS-mediated down-regulation of expression.

Biological Transport↗
PubMed1992

Expression and purification of mouse TIMP-1 from E. coli.

Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of secreted glycoproteins involved in regulating extracellular matrix degradation in both normal and malignant tissues. We have expressed a cDNA clone of mouse TIMP-1 as a 22-kDa protein with 12 cysteine residues in E. coli and purified protein that shows inhibitory activity against collagenase following renaturation by chemical means. The low specific activity and circular dichroism measurements suggest, however, that the renaturation of the mouse recombinant (non-glycosylated) protein is not efficient under the conditions we have used, indicative of either thermodynamic instability or the transition to kinetic intermediates which have very low in vitro refolding rates.

Animals↗
PubMed1992

Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells.

We previously found that the T24 Ha-ras oncogene induces metastatic ability in NIH 3T3 cells and that this change depends on expression of the ras oncogene. As part of our studies on mechanisms by which ras may induce metastasis, we investigated expression and activity of two cysteine proteinases, cathepsin L (major excreted protein) and cathepsin B, as well as cysteine proteinase inhibitor activity, in ras-transformed NIH 3T3 cells. In a series of cel lines that expressed differing amounts of ras, we found a good correlation between levels of ras expression and cathepsin L expression (r = 0.80). There was also a good correlation between secreted procathepsin L protein levels and experimental metastatic ability (r = 0.88). We found a similar but less strong association between cathepsin B levels and metastatic ability in these cells (r = 0.76 and r = 0.72 for 2.2-kb and 4.1-kb transcripts, respectively). Functional cathepsin L plus B activity (both secreted and cell-associated) was found to be higher in ras-transformed cells and was dependent on cell confluency in culture. Coupled with increased expression and activity of cysteine proteinases, ras-transformed NIH 3T3 cells showed reduced cysteine proteinase inhibitor activity. We conclude that the balance between expression of cysteine proteinases and their inhibitors may be coregulated by ras expression. Our results suggest that ras-induced increases in production of degradative enzymes such as cathepsins L and B, along with decreased activities of their inhibitors, may contribute to the increased malignant properties of ras-transformed NIH 3T3 cells.

3T3 Cells↗
PubMed1992

Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.

Animals↗
PubMed1992

p53 mutations in spontaneously immortalized 3T12 but not 3T3 mouse embryo cells.

We have asked whether p53 mutations are involved in the process of spontaneous immortalization of mouse embryo cells. Cells from Swiss mouse embryos were used to prepare 3T3 and 3T12 lines according to the protocol of Todaro & Green [(1963). J. Cell Biol., 17, 299-313]. After the cells emerged from crisis, p53 sequences were amplified by polymerase chain reaction (PCR) from both RNA and DNA. The sequence of the aggregated cDNA from each of six 3T3 lines showed no evidence of mutation. PCR-amplified p53 cDNA from two 3T3 lines was cloned, and individual clones in M13mp19 were partially sequenced. One cell ine showed a single, non-coding nucleotide change in 2/8 independent clones. Nine cDNA clones from the second 3T3 lines were sequenced, and no single nucleotide changes appeared more than once. The mutations which appeared only once were not detected in clones of genomic DNA. Since these apparent mutations are probably reverse transcriptase or Taq polymerase errors, we conclude that both the 3T3 lines contained only wild-type p53. In two out of three independent 3T12 lines however, missense mutations were readily observed in the aggregate cDNA sequence. Restriction fragment length polymorphism and Southern blot analyses of the genomic DNA indicated that these cells were homozygous for the mutations. The p53 protein molecules in four cell lines were analysed by immunoprecipitation: one 3T12 line showed the pattern of antibody reactivity characteristic of some p53 mutants, while the others displayed the wild-type pattern. We conclude that p53 mutations arise and are strongly selected for during immortalization according to the 3T12 but not the 3T3 protocol.

Amino Acid Sequence↗
PubMed1992

Oncogenic consequences of down-modulating TIMP expression in 3T3 cells with antisense RNA.

We have used recombinant DNA technology to engineer a set of murine 3T3 cell lines that vary in the extent to which they express TIMP, tissue inhibitor of metalloproteinases, and we have found that both invasiveness and tumorigenic potential are conferred when TIMP production is impaired. These cell clones were produced by transfecting immortal Swiss 3T3 cells with plasmid constructs capable of expressing antisense TIMP RNA under the control of the mouse metallothionein promoter (Khokha & Denhardt, AntiCancer Res. 7: 653-660, 1987). The ability of these cells to invade the amnion membrane in vitro was dependent upon metalloproteinase expression (Yagel et al., JNCI 81: 768-775, 1989). Cells expressing TIMP at a reduced level acquired the ability to form tumors in nude mice (Khokha et al. SCIENCE 243: 947-950, 1989). These results suggest not only that TIMP controls the invasive character of the immortal 3T3 cell, but also that it determines cellular tumorigenic potential in the mouse. We presume that these phenotypes are conferred as a consequence of a net increase in extracellular matrix metalloproteinase activity resulting from the reduced quantities of TIMP secreted. The lag in formation of tumors by the cells down-modulated for TIMP production suggests that further changes in gene expression may be necessary. In support of this hypothesis, recent experiments indicate that the expression of genes encoding one or more matrix metalloproteinases is increased in cell lines derived from tumors that have developed from the engineered cells. Thus, in the immortal 3T3 cell line, TIMP has the properties of a tumor-suppressor gene, or anti-oncogene.

3T3 Cells↗
PubMed1991

Developmental expression of cathepsin L and c-rasHa in the mouse placenta.

An investigation is described of the expression of the cysteine proteinase cathepsin L during placental development. In addition, whether cathepsin L expression is linked to c-rasHa expression in development, as it is in metastatic cells, is examined. Large amounts of cathepsin L and its transcript are present in the mouse placenta, more than six times more than in adult kidney and liver. Throughout gestation, cathepsin L and its transcript are located in the giant cells and spongiotrophoblasts of the placenta. Several forms of different mobility on denaturing gels are found in the placenta. Their apparent molecular weights, as determined from the gels, are 43,000, 39,000, 29,000, and 20,000. The 39-kDa form is procathepsin L. The 29-kDa and 20-kDa forms are lysosomal cathepsin Ls. The 39-kDa procathepsin L and the 20-kDa mature cathepsin L are the most abundant species in the placenta and are present in about equal amounts throughout gestation. At any time during gestation, placental minces synthesize and secrete only procathepsin L. The amniotic fluid of the fetus contains the 43-kDa form of cathepsin L and procathepsin L, but no detectable amounts of mature cathepsin L. By contrast, serum from nonpregnant or pregnant mice contains three forms of cathepsin L (i.e., the 43-kDa form, procathepsin L, and mature cathepsin L). Cathepsin L and the rasHa oncogene are expressed in two coincident waves corresponding to periods during which the placenta is invasive and just before parturition. The presence of large amounts of cathepsin L in the placenta suggests that the proteinase has a significant function there. Expression of cathepsin L in the placenta is potentially under the control of the ras gene product p21; both are under developmental control.

Animals↗
PubMed1991

Increased proteinase expression during tumor progression of cell lines down-modulated for TIMP levels: a new transformation paradigm? [corrected].

We have reported that down-modulation of tissue inhibitor of metalloproteinases (TIMP) by means of antisense RNA converts non-tumorigenic Swiss 3T3 cells into malignant cells capable of forming metastasizing tumors in nude mice [Science 243:947 (1989)]. We now describe changes in the expression of specific genes associated with tumor progression of two lines down-modulated with TIMP, LA1 and LA7. Six independent variant cell lines, generated from different primary tumors produced by LA1 and LA7, lacked (like LA1 and LA7) many characteristics of typical transformed cells. However, their tumorigenicity in nude mice was enhanced; tumors appeared with a shorter lag (1-3 weeks versus 8-10 weeks for the parental clones, LA1 and LA7) and grew very rapidly. Increases, substantial in some cases, in the expression of a cysteine proteinase, cathepsin L, and metalloproteinases homologous to rat transin (stromelysin) and transin-2 were characteristic of these variant clones. The mRNA levels encoding the transformation-associated secreted phosphoprotein (osteopontin) and the calcium-binding protein calcyclin were also augmented. No evidence for gene amplification was found, and we did not detect any change in the mRNA levels of the proto-oncogenes that were examined. These novel cell lines represent a new paradigm for the transformed cell. Our data suggest that a reduction in TIMP secretion enhances the cell's oncogenic capacity by altering the extracellular environment in a way conducive to further changes in gene expression necessary for tumor progression.

Animals↗
PubMed1991

Spontaneous immortalization of mouse embryo cells: strain differences and changes in gene expression with particular reference to retroviral gag-pol genes.

We have studied the kinetics with which cultures of primary mouse embryo cells pass through the crisis period, escape their terminal differentiation (cellular senescence), and give rise to an immortal cell line. The process is strain-dependent, with cells from the outbred Swiss CD-1 mouse being considerably more adept at forming an immortal 3T3 line than cells from the inbred SWR line; Balb/c cells appeared intermediate in their behavior. The continued presence of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the poly(ADPribose)polymerase inhibitor 3-aminobenzamide affected the kinetics but did not seem to alter the outcome. Changes in expression of various genes, including those encoding mitogen-regulated protein (proliferin), endogenous gag-pol retrovirus sequences, insulin-like growth factor II, and a variety of protooncogenes, were monitored during the process of immortalization, and although certain changes were reproducibly characteristic of cells from a given mouse strain passed according to a specific regimen, none of the observed changes were reproducibly characteristic under all conditions of immortalization. In particular, our data indicate the absence of a strict correlation between cellular immortalization and the activation of endogenous gag-pol expression. We conclude from our observations that the establishment of permanent lines from primary mouse embryo cells in serum-containing medium reflects the selection of a variant subpopulation of cells that did not preexist but rather arose in response to the specific culture conditions by a process resembling differentiation. Multiple and complex changes in gene expression occur that are affected by the culture conditions and the strain (genotype) of the mouse.

Animals↗
PubMed1991

Regulation of the expression of mitogen-regulated protein (MRP; proliferin) and cathepsin L in cultured cells and in the murine placenta.

The genes encoding mitogen-regulated protein (MRP; also called proliferin; PLF) and procathepsin L (CL; also called major excreted protein; MEP) are expressed to high levels in the mouse placenta. Although they are both regulated by epidermal growth factor (EGF) and fibroblast growth factor (FGF) in 3T3 cells, expression of these genes is differently regulated with growth state. The expression patterns of MRP and CL as a function of murine development are also different. Basal and growth factor-stimulated levels of MRP expression are much higher in growing than in quiescent 3T3 cells, whereas CL levels are similar. These changes in gene expression in cultured quiescent cells parallel the changes in MRP and CL expression observed in the late-gestational quiescent placenta. These results suggest growth factors may regulate the expression of these genes, but other influences also regulate the expression of MRP and CL in vivo.

3T3 Cells↗