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Biomedical subjects

L R Lund

Publications and source records attributed to L R Lund.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation.

Analysis of extracellular matrix degradation systems has led to the insight that in cancer invasion there is often crucial interplay between cancer cells and several types of surrounding non-neoplastic stromal cells. Likewise, in normal tissue remodeling processes, the synthesis of proteolytic components is often distributed between several cell types, and there are strong similarities between neoplastic and non-neoplastic processes in the same tissue. Thus, tissue remodeling events are excellent models for studies of extracellular proteolysis in cancer. This has become even clearer by recent analyses of genetically modified mice.

Breast Neoplasms↗
PubMed1997

Co-ordinated expression of MMP-2 and its putative activator, MT1-MMP, in human placentation.

The spatial expression of mRNA for matrix metalloproteinase 2 (MMP-2), its putative activator, the membrane-type 1 matrix metalloproteinase (MT1-MMP), and the MMP-2 substrate type IV collagen was investigated in human placentas of both normal and tubal ectopic pregnancies and in cyclic endometrium using in-situ hybridization. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. In both normal and tubal pregnancies MT1-MMP, MMP-2 and type IV collagen mRNA were highly expressed and co-localized in the extravillous cytotrophoblasts of anchoring villi, in cytotrophoblasts that had penatrated into the placental bed and in cytotrophoblastic cell islands. In addition, the decidual cells of normal pregnancies in some areas co-expressed MT1-MMP and MMP-2 mRNA, with moderate signals for both components. Fibroblast-like stromal cells in tubal pregnancies were positive for MMP-2 mRNA but generally negative for MT1-MMP mRNA. The consistent co-localization of MT1-MMP with MMP-2 and type IV collagen in the same subset of cytotrophoblasts strongly suggests that all three components co-operate in the tightly regulated fetal invasion process. The co-expression of MT1-MMP and MMP-2 mRNA in some of the decidual cells indicates that these cells are also actively involved in the placentation process.

Chorionic Villi↗
PubMed1996

Expression of urokinase-type plasminogen activator, its receptor and type-1 plasminogen activator inhibitor is differently regulated by inhibitors of protein synthesis in human cancer cell lines.

Expression of the various components of the plasminogen activation system is under tight regulation by hormones, cytokines, and growth factors under physiologic conditions. Like early-response genes, these components are modulated by inhibitors of protein synthesis in some cell lines. To clarify the specific expression and regulation of mRNAs for urokinase (uPA), its receptor (uPAR), and type-1 plasminogen activator inhibitor (PAI-1), I analyzed RNA from four human cancer cell lines by RNA blotting after treatment with cycloheximide, anisomycin, emetine or puromycin. These inhibitors, all of which induced translational arrest, induced a very diverse response in the various transcripts, suggesting that the inhibitors mediate their effects through different molecular mechanisms. Dose-response analysis showed that, in A549 cells, anisomycin strongly induced uPAR and PAI-1 mRNA at concentrations that did not cause complete inhibition of protein synthesis, whereas cycloheximide induced these transcripts in a dose-dependent manner only at concentrations sufficient to inhibit total protein synthesis by >90%. Puromycin induced the 3.4-kb transcript of PAI-1 mRNA in A549 and RD cells, whereas it decreased the expression of both the 3.4-kb and 2.4-kb PAI-1 transcripts in HT-1080 cells. Different time patterns of induction for uPA, uPAR and PAI-1 mRNA suggest that even in the same cell type, inhibitors of protein synthesis mediate their effects on various genes through different mechanisms. Thus, induction of uPA, uPAR, and PAI-1 transcripts by inhibitors of protein synthesis was dependent on the gene, the cell line, and the type of inhibitor, and inhibition of protein synthesis per se was not sufficient for induction of these transcripts.

Anisomycin↗
PubMed1996

Impaired wound healing in mice with a disrupted plasminogen gene.

Activation of plasminogen (Plg) has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling events, including wound healing. However, there has been no definitive proof of involvement of Plg in such processes. We now report that healing of skin wounds is severely impaired in mice made deficient in Plg by targeted gene disruption. The results demonstrate that Plg is required for normal repair of skin wounds in mice and support the assumption that it also plays a central role in other disease processes involving extracellular matrix degradation, such as cancer invasion.

Animals↗
PubMed1996

Differential expression of urokinase-type plasminogen activator, its receptor, and inhibitors in mouse skin after exposure to a tumor-promoting phorbol ester.

The cellular distribution of mRNAS for urokinase-type plasminogen activator (uPA), its specific receptor (uPAR), and its inhibitors (PAI-1 and -2) in mouse skin was analyzed by in situ hybridization after topical application of the tumor promoter phorbol 12-myristate 13-acetate. In the epidermis, strong signals for uPA and PAI-1 mRNA were detected 24 h after treatment in the basal and suprabasal epidermal keratinocytes in areas with pronounced hyperproliferation and increased terminal differentiation, and in some hair follicle keratinocytes. After 48 h, both uPAR and PAI-2 mRNAs were expressed in the epidermal layers from the suprabasal keratinocytes up to the differentiating cells beneath the cornified layer and in hair follicle keratinocytes. Induction of PAI-2 mRNA was detected in epidermis as early as 3 h after treatment and remained stable for up to 7 days. In the dermis, 5 h after application of phorbol 12-myristate 13-acetate to the skin, uPA mRNA was detected in fibroblast-like cells below and around the skin muscle, and PAI-1 mRNA was detected in stromal cells located above the skin muscle. After longer exposure to phorbol 12-myristate 13-acetate, the PAI-1 mRNA-expressing stromal cells were located more superficially, apparently moving toward the epidermal layer. After 9 h, most of the PAI-1 mRNA-positive cells were identified as endothelial cells. Up to 24 h after the application of phorbol 12-myristate 13-acetate, the intensity of the signal for both uPA and PAI-1 increased, followed by a gradual decrease for up to 7 days. These results show that in mouse skin treated with a tumor-promoting phorbol ester, the various components of the plasminogen activation system are expressed by both epithelial and stromal cell types, which in dermis and subcutis are located in different places, depending on the time of exposure to the phorbol ester. Our results suggest that urokinase-mediated extracellular proteolysis has diverse functional roles during the early steps of tumor promotion.

Animals↗
PubMed1996

Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways.

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when beta-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when beta-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1 beta converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisone-treated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.

Animals↗
PubMed1996

Extracellular matrix remodeling and the regulation of epithelial-stromal interactions during differentiation and involution.

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. To examine the importance of basement membrane during development in vivo, we altered the matrix metalloproteinase and tissue inhibitor of metalloproteinases balance in mammary gland. Inhibition of matrix metalloproteinase synthesis by glucocorticoids or implants or transgenic overexpression of tissue inhibitor of metalloproteinases -1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express auto-activating isoforms of stromelysin-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express beta-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that metalloproteinases and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, cell cycle, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer.

Animals↗
PubMed1995

Transcriptional and post-transcriptional regulation of the receptor for urokinase-type plasminogen activator by cytokines and tumour promoters in the human lung carcinoma cell line A549.

The receptor for urokinase-type plasminogen activator (uPAR) is an integral membrane protein that specifically binds urokinase-type plasminogen activator (uPA) and plays a crucial role in cell surface plasmin generation. We have previously found that transforming growth factor-beta, type 1 (TGF-beta 1), increases uPAR gene transcription in the human lung carcinoma cell line A549 and now report that also epidermal growth factor (EGF) and the tumour promoter phorbol 12-myristate 13-acetate (PMA) cause increased uPAR transcription and that PMA and TGF-beta 1 in addition increase the stability of uPAR mRNA, while EGF has no effect on this parameter. All three compounds also increase the uPAR protein level, as measured by cell-binding experiments with radiolabelled ligand. The increase in uPAR protein level was however considerably lower with all three compounds than the increase in mRNA level, suggesting that they also exert a translational or post-translational control. Accompanying the increase in the number of uPAR molecules there was a proportional decrease in their ligand-binding affinity, the mechanism of which is unknown. Platelet-derived growth factor, basic fibroblast growth factor and cyclic AMP analogues did not induce any change in the uPAR mRNA level in A549 cells. Previous studies have shown that expression of uPA and its type-1 inhibitor is regulated by a variety of cytokines in a cell-specific manner. The present study indicates that cytokines in addition influence cell surface plasminogen activation by regulating uPAR expression.

Carcinogens↗
PubMed1995

Neutrophils express tumor necrosis factor-alpha during mouse skin wound healing.

The expression pattern of tumor necrosis factor-alpha (TNF-alpha) mRNA and protein was examined in vivo in experimental mouse skin wounds by in situ hybridization and immunohistochemistry. TNF-alpha mRNA and protein is detected in a distinct layer of mainly neutrophils subadjacent to the would clot. The layer of TNF-alpha-positive cells extends from the margin of the advancing epithelial outgrowth to the opposing one. By in situ hybridization the TNF-alpha mRNA is detectable 12 h after wounding; the signal peaks after 72 h and remains visible up to at least 120 h after wounding. TNF-alpha mRNA could not be detected in the normal skin or in 5-hour-old wounds. Immunohistochemical staining for TNF-alpha and macrophages on adjacent sections confirms that the main part of TNF-alpha-positive cells are polymorphonuclear neutrophils and shows that most of the cells located just beneath the layer of TNF-alpha-positive neutrophils are macrophages with weak TNF-alpha immunoreactivity. The data reported here show that neutrophils serve as an important source of TNF-alpha during healing of mouse skin wounds. We suggest that this specific expression of TNF-alpha is related to the process of re-epithelialization.

Animals↗
PubMed1995

Progesterone stimulates degradation of urokinase plasminogen activator (u-PA) in endometrial stromal cells by increasing its inhibitor and surface expression of the u-PA receptor.

Progesterone stimulates differentiation and inhibits the growth of endometrial tissue. Also, progesterone reduces plasminogen activator (PA) activity, which implies reduced turnover of extracellular matrix proteins in the secretory phase. To elucidate the mechanism responsible for reduced PA activity, primary cultures of human endometrial stromal cells were stimulated with estradiol and progesterone. Conditioned media were assayed for urokinase-type and tissue-type PA (u-PA and t-PA, respectively), PA inhibitor-1 (PAI-1), and PA activity. Binding of [125I]u-PA and [125I]u-PA:PAI-1 complex to the u-PA receptor and clearance of these ligands were studied. The PA activity of conditioned medium decreased after stimulation with progesterone, and this was secondary to a decrease in u-PA, but not t-PA, and an increase in PAI-1. Northern blot analysis showed induction of PAI-1 messenger ribonucleic acid, whereas the content of u-PA messenger ribonucleic acid was not influenced. Furthermore, the number of free u-PA receptor-binding sites was increased by estradiol and progesterone. The stromal cells degraded complexed u-PA more efficiently than free u-PA, and degradation of both ligands was inhibited by colchicine, chloroquine, and methylamine. Degradation was increased after hormone treatment, and this was apparently due to increased ligand binding, because neither ligand affinity nor the relative rate of degradation was increased. Increased expression of u-PA receptor-binding sites was not regulated on the transcriptional level, but may result from posttranslational mechanisms, such as decreased turnover of the receptor. Activation of plasminogen by receptor bound u-PA initiates a cascade of proteolytic events in the extracellular matrix that is important during tissue proliferation. Our data suggest that differentiated endometrial stroma in the secretory phase regulates extracellular proteolysis by increased elimination of u-PA through increased release of PAI-1 and increased u-PA receptor density.

Adult↗
PubMed1994

Expression of uPA and its receptor by both neoplastic and stromal cells during xenograft invasion.

Recent studies have shown that molecules involved in generation and regulation of extracellular proteolytic activity are often expressed by non-malignant stromal cells during human cancer invasion. We have studied the expression of the urokinase-type plasminogen activator and the urokinase-type plasminogen activator cell-surface receptor in xenografts of human MDA-MB-231 mammary carcinoma cells growing invasively in nude mice. Northern analysis showed the presence of both human and mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA in tumor extracts. By in situ hybridization, mRNA for human urokinase-type plasminogen activator and its receptor was detected in virtually all the cancer cells, while mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA was expressed by tumor-infiltrating fibroblast-like and macrophage-like cells. In invasive areas the cells expressing the 2 murine mRNAs were either the same or located immediately adjacent to each other. This model system has several advantages for studies of the mechanism by which cancer cells induce or recruit stromal cells to produce molecules involved in proteolysis.

Animals↗
PubMed1994

The receptor for urokinase-type plasminogen activator is expressed by keratinocytes at the leading edge during re-epithelialization of mouse skin wounds.

Expression of urokinase-type plasminogen activator receptor mRNA was examined in vivo in mouse skin wounds by in situ hybridization. Urokinase-type plasminogen activator receptor mRNA was found in keratinocytes at the front of the regenerative epithelial outgrowths at the edge of 12-, 48-, and 96-hour-old wounds. The signal was strongest in the keratinocytes just beginning to move 12 h after wounding. At later time-points the signal was weaker, but still confined to keratinocytes at the wound edges. Using in situ hybridization, no cells expressing urokinase-type plasminogen activator receptor mRNA could be detected in normal epidermis, in normal-looking epidermis adjacent to the wounds, in dermis, in subcutis, or in newly formed granulation tissue. The specificity of the results was supported by the use of antisense RNA from two different non-overlapping cDNA clones and the corresponding sense RNA probes, and by Northern analysis of tissue extracts. Together with previous findings on expression of urokinase-type plasminogen activator and its type 1 inhibitor, the localized and regulated expression of urokinase-type plasminogen activator receptor mRNA during skin wound healing indicates that focal extracellular proteolysis at the cell surface generated by receptor-bound urokinase-type plasminogen activator is implicated in the migration of re-epithelializing keratinocytes.

Animals↗
PubMed1994

The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.

All known laminin isoforms are cross-shaped heterotrimeric molecules, consisting of one heavy alpha chain and two light beta and gamma chains. Recently, a cDNA encoding a new gamma chain from laminin 5 (also known as kalinin) was sequenced. This chain, named gamma 2, showed extended homology to the classical gamma 1 chain but differed from this by lacking the terminal globular domain. Recent data, indicating an important role of the gamma 2 chain gene in establishing adhesion contacts between epithelial cells and basement membranes, prompted us to investigate whether the gamma 2 chain gene is aberrantly expressed in cancer tissue, and if so whether its localization could provide clues to its possible role in cancer dissemination. Routinely processed tissue specimens from 36 cases of human cancer were investigated, including 16 cases of colon adenocarcinoma, 7 ductal mammary carcinomas, 4 squamous cell carcinomas, 3 malignant melanomas and 6 sarcomas. In situ hybridization for the detection of mRNAs for the gamma 2 chain and for the classical laminin chains alpha 1, beta 1, and gamma 1 was performed using S-35 labeled antisense RNA probes. As positive control of the specificity of the gamma 2 chain mRNA detection, two different anti-sense probes derived from two nonoverlapping cDNA clones were used. Malignant cells were found to express the gamma 2 chain in 29 of the 30 carcinomas studied and the expression was particularly high in cancer cells located at the invasion front. In contrast, mesenchymally derived cancer cells in three different types of sarcomas did not express the gamma 2 chain. In colon cancer there was a clear histological correlation between the expression of gamma 2 chain by cancer cells and their engagement in tumor budding processes. Laminin chains alpha 1, beta 1, and gamma 1 were weakly expressed throughout cancerous areas with no apparent correlation to sites of invasion. The aberrant expression of the gamma 2 chain gene seen in invasively growing cancer cells point to a role of this molecule in establishing focal adhesions of cancer cells to the extracellular matrix during their migration through surrounding normal tissue.

Adenocarcinoma↗
PubMed1994

Tumor-promoting phorbol ester transiently down-modulates the p53 level and blocks the cell cycle.

Activation of the protein kinase C signaling pathway by tumor-promoting phorbol esters, such as 4 beta-phorbol 12-myristate 13-acetate (PMA), induced a decrease in the level of p53 mRNA in several serum-starved human cell lines. Also, the tumor-promoting phosphatase inhibitor okadaic acid induced a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 mRNA was minimal after 9 h of exposure to PMA. After approximately 120 h, the p53 mRNA level was similar to the pretreatment level. PMA induced a similar transient decrease in the level of p53 protein in the A549 cell line. The decrease in the p53 mRNA level could not be explained by changes in the transcriptional rate or the p53 mRNA stability. The protein synthesis inhibitor cycloheximide completely abolished the PMA-induced down-modulation of the p53 mRNA, suggesting that a short-lived protein was involved in the down-modulation. Flow cytometric cell cycle analysis showed that the phorbol ester treatment induced a block in the late G1 phase. The blockage was transient, and its duration correlated with the level of p53 protein in the two cell lines. We propose that the protein kinase C-catalyzed phosphorylation of p53 may be a key event in the down-modulation of p53 expression as well as in the induced blockage of the cell cycle.

Cell Cycle↗
PubMed1993

An alternatively spliced variant of mRNA for the human receptor for urokinase plasminogen activator.

Using 3' RACE (rapid amplification of cDNA ends), we have isolated a cDNA variant for the receptor for human urokinase plasminogen activator (uPAR). The deduced protein includes the amino-terminal ligand binding domain in uPAR, but lacks the carboxy-terminal membrane attachment by a glycolipid anchor. Genomic DNA analysis showed that the uPAR mRNA variant is generated by alternative splicing. The new variant mRNA is expressed in various human cell lines and tissues and both variants are up-regulated by phorbol ester in A549 cells. We propose that the alternatively spliced uPAR mRNA encodes a soluble uPA binding protein, the possible function of which is discussed.

Amino Acid Sequence↗
PubMed1992

Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain.

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored three-domain membrane protein playing a central role in pericellular plasminogen activation. We have found that urokinase (uPA) can cleave its receptor between domains 1 and 2 generating a cell-associated uPAR variant without ligand-binding properties. In extracts of U937 cells there are two uPAR variants which after complete deglycosylation have apparent molecular masses of 35,000 and 27,000. Analysis with monoclonal antibodies showed that these variants represented the intact uPAR and a two-domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its generation. Purified uPAR can be cleaved directly by uPA as well as by plasmin. The uPA-catalyzed cleavage does not require binding of the protease to the receptor through its epidermal growth factor-like receptor-binding domain, since low molecular weight uPA that lacks this domain also cleaves uPAR. This unusual reaction in which a specific binding protein is proteolytically inactivated by its own ligand may represent a regulatory step in the plasminogen activation cascade.

Antibodies, Monoclonal↗