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C H Graham

Publications and source records attributed to C H Graham.

13 recordsLinked to original sources
PubMed1992

Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells.

BACKGROUND: We have previously shown that down-modulation (i.e., by antisense expression vector) of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a noninvasive, nontumorigenic cell line led to an acquisition of an invasive, tumorigenic, and metastatic ability in these cells. PURPOSE: Our purpose was to examine whether increased levels of murine TIMP-1 can directly suppress the invasive ability of malignant cells. METHODS: Murine B16-F10 melanoma cells were transfected with an expression vector to overproduce TIMP-1. Among these transfectants, we isolated five clonal cell lines (2-5, 2-8, 2-10, 6-5, and 6-9) that showed upregulation (i.e., overexpression) of TIMP-1. RESULTS: These cell lines had an increased basal level of TIMP-1 messenger RNA. TIMP-1 expression was under the control of the mouse metallothionein-I promoter, and four of these five clones (2-5, 2-8, 6-5, and 6-9) showed a threefold to 10-fold induction of TIMP-1 message when they were treated with 20 microM cadmium for 4 hours. An increase in TIMP-1 message led to an increase in TIMP-1 protein activity measured in the conditioned medium of clones 2-10 and 6-5. The invasive ability of the TIMP-1-upregulated cells was tested in a matrigel transwell invasion assay. All of the upregulated clones showed a significant reduction in their invasive ability, relative to the invasive ability of parental B16-F10 and the control 1-2 cell line; this reduction correlated with the level of TIMP-1 overexpression. Cadmium induction of TIMP-1 messenger RNA resulted in a further suppression of the invasive ability of the two inducible cell lines tested (i.e., 2-8 and 6-5). CONCLUSIONS: Our data demonstrate that a specific upregulation of murine TIMP-1 expression in B16-F10 melanoma cells directly suppresses their invasive ability.

Animals↗
PubMed1992

Characterization of urokinase receptor expression by human placental trophoblasts.

The processes of implantation and placentation are both dependent on the invasion and remodeling of the uterine endometrium and vasculature by trophoblasts. Because the secretion and autocrine binding of urokinase (uPA) appears to be a common mechanism used by cells to facilitate plasmin-dependent tissue invasion, we measured the production of uPA and expression of uPA receptors by trophoblasts. Prourokinase bound specifically, reversibly, and with high affinity to cultured trophoblasts, via the uPA epidermal growth factor-like domain. Trophoblasts derived from two first-trimester placentae bound more prourokinase than cells isolated from term placentae. Furthermore, in vitro differentiation of cultured cytotrophoblasts into syncytiotrophoblasts was associated with diminished expression of urokinase receptors and a parallel decrease in the cellular content of uPA receptor mRNA. Trophoblasts also secreted prourokinase and plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2). Although prourokinase was secreted in amounts sufficient to endogenously saturate trophoblast uPA receptors, trophoblasts secreted greater amounts of PAI-1 and PAI-2 than uPA, and no net plasminogen activator activity was detected in trophoblast conditioned medium. In contrast, plasminogen added directly to cultured trophoblasts was readily converted to plasmin. Although the invasion and remodeling of uterine tissues by trophoblasts is a complex process dependent on several proteases of varying specificity, our findings suggest that the expression and modulation of urokinase receptors on the trophoblast cell surface may play an important role in this process.

Cells, Cultured↗
PubMed1992

Localization of transforming growth factor-beta at the human fetal-maternal interface: role in trophoblast growth and differentiation.

We examined the localization of transforming growth factor (TGF)-beta in first-trimester and term human decidua and chorionic villi and explored the role of this factor on the proliferation and differentiation of cultured trophoblast cells. Two antibodies, 1D11.16.8, a mouse monoclonal neutralizing antibody capable of recognizing both TGF-beta 1 and TGF-beta 2 and CL-B1/29, a rabbit polyclonal antibody capable of recognizing TGF-beta 2, were used to immunolocalize TGF-beta in fixed, paraffin-embedded, or fixed, frozen sections of placenta and decidua, providing similar results. Intense labeling was observed in the extracellular matrix (ECM) of the first-trimester decidua and cytoplasm of term decidual cells. Syncytiotrophoblast cell cytoplasm as well as the ECM in the core of the chorionic villi of both first-trimester and term placentas exhibited a moderate degree of labeling. Strong cytoplasmic labeling was observed in the cytotrophoblastic shell of the term placenta. To examine the role of TGF-beta on trophoblast proliferation and differentiation, early passage cultures of first-trimester and primary cultures of term trophoblast cells were established and characterized on the basis of numerous immunocytochemical and functional markers. These cells expressed cytokeratin, placental alkaline phosphatase, urokinase-type plasminogen activator, and pregnancy-specific beta glycoprotein, but not factor VIII or 63D3; they also produced hCG and collagenase type IV. Exposure of first-trimester trophoblast cultures to TGF-beta 1 significantly inhibited proliferation in a dose-dependent manner. An antiproliferative effect was also noted in the presence of TGF-beta 2. These effects were abrogated in the presence of the neutralizing anti-TGF-beta antibody (1D11.16.8) in a concentration-dependent manner. In a 3-day culture, exogenous TGF-beta 1 stimulated formation of multinucleated cells by the first trimester as well as term trophoblast cells. Addition of neutralizing anti-TGF-beta antibody to first-trimester trophoblast cells stimulated proliferation beyond control levels in a 24-h culture and reduced formation of multinucleated cells in a 3-day culture, indicating the presence of endogenous TGF-beta activity. These results indicate that TGF-beta produced at the human fetal-maternal interface plays a major regulatory role in the proliferation and differentiation of the trophoblast.

Alkaline Phosphatase↗
PubMed1991

Mechanism of control of trophoblast invasion in situ.

We have previously shown that first trimester human trophoblast cells share in vitro invasive properties with malignant cells. In this study we show that the in situ control of trophoblast invasion is provided by the uterine microenvironment. Trophoblast cells were labeled with 125I-deoxyuridine and examined for their ability to invade an epithelium-free human amniotic membrane in vitro under various conditions. The degree of invasion was determined as the percentage of the radioactivity retained within the membrane. Conditioned media from first trimester human decidual cells (DCM) suppressed invasion of trophoblast cells in the amnion invasion assay. This suppression was prevented by addition of neutralizing anti-TGF beta antibody or neutralizing antibody to tissue inhibitor of metalloproteinases (TIMP-1) to the DCM, and mimicked by TGF beta 1. These antibodies also augmented invasion beyond control levels, suggesting that trophoblast cells may also produce these factors. A bioassay for TGF beta activity, measured by antiproliferative effect on the mink lung epithelial cell line Mv 1 Lu, revealed that decidual cells produced this factor only in the latent form, whereas the active form was produced by the trophoblast. A decrease in collagenase type IV activity in the conditioned media of trophoblast cultures was observed when TGF beta 1 was added to these cultures. Removal of endogenous TGF beta in trophoblast cultures by addition of anti-TGF beta antibody resulted in down-regulation of TIMP message as determined by Northern analysis. These results indicate that a) decidua-derived (and to a minor extent trophoblast-derived) TGF beta is the prime mediator in the control of invasion by first trimester trophoblast, the latent form of TGF beta likely being activated by trophoblast-derived proteinases; b) induction of TIMP by TGF beta in both trophoblast and decidua is the final pathway in this control.

Antibodies↗
PubMed1990

Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors.

Implantation and subsequent placental development in many species including the human are dependent on trophoblast invasion of the uterine epithelium, the underlying basement membrane, connective tissue and blood vessels. However, trophoblast invasion in situ is strictly controlled by the microenvironment provided by the pregnant uterus. Key mechanisms underlying various steps in trophoblast invasion of basement membrane and stroma are similar to those identified in the case of invasive tumor cells: (a) attachment to basement membrane by binding to laminin and possibly other basement membrane components; (b) detachment from the basement membrane matrix prior to its penetration, a process that requires the presence of complex-type oligosaccharides on the cell surface; (c) breakdown of basement membrane components by trophoblast-derived metalloproteases (type IV and interstitial collagenase) and serine proteases (plasminogen activator). Type IV collagenase activity is stimulated by binding to laminin, a molecule also secreted by the trophoblast. Activation of trophoblast-derived metalloproteases appears to be plasmin-dependent. Plasmin results from the cleavage of plasminogen by trophoblast-derived plasminogen activator. Control of trophoblast invasion in situ is mediated by decidua-derived transforming growth factor beta (TGF beta) which in turn induces tissue inhibitor of metalloproteases (TIMP) both in the decidua and the trophoblast. We suggest that this control of trophoblast invasiveness is regulated both spatially as well as temporally during gestation. A preprogrammed decline in trophoblast invasiveness with increasing gestational age remains an additional possibility. The nature of the loss of control of trophoblast invasiveness in choriocarcinoma remains to be identified. Refractoriness to TGF beta action remains to strong possibility.

Basement Membrane↗
PubMed1990

Activation of maternal killer cells in the pregnant uterus with chronic indomethacin therapy, IL-2 therapy, or a combination therapy is associated with embryonic demise.

We have previously shown that NK lineage cells migrate to the murine decidua of pregnancy; but with advancing gestation, they are progressively inactivated in situ by prostaglandins of the E series (PGE2) secreted by decidual cells and decidual macrophages. We have also shown that the same mechanism inactivates all killer lineage cells in the human decidua, and that this inactivation is at least in part due to a down-regulation of IL-2 receptors and an inhibition of IL-2 production in situ. We examined whether chronic indomethacin therapy (to block prostaglandin synthesis), or a systemic administration of a high dose of IL-2, or a combination of both agents administered to pregnant mice could activate killer cells in situ and interfere with the progress of pregnancy; and if so, whether there was a causal relationship between the two events. Pregnant CD1 mice (Day 5 of gestation) were subjected to chronic indomethacin therapy (14 micrograms/ml in drinking water up to Day 15, or 50 micrograms twice daily sc or ip up to Day 10), high dose IL-2 therapy (25,000 Cetus U of human recombinant IL-2, ip every 8 or 12 hr for 3-5 days), or a combination of the two. These treatments led to pregnancy loss in 89-100% of mice, in contrast to 1% loss in control, vehicle-treated mice. Uterine mononuclear cells isolated from the embryo resorption sites exhibited high killer activity against YAC-1 lymphoma as well as murine trophoblast targets, with NK-like phenotype (Asialo GM-1+, Thy-1-) after indomethacin therapy and LAK-like phenotype (AGM-1+, Thy-1+) after IL-2 or indomethacin + IL-2 therapy. That AGM-1+ killer cells resulted in the pregnancy loss was suggested by the findings that in two of three separate experiments, iv injections of AGM-1 ab into pregnant indomethacin + IL-2-treated mice nearly completely prevented the fetoplacental demise (reducing it to 7.7% from 100%). These results reveal that PGE2-mediated inactivation of killer lineage cells in the decidua in situ is conducive to the survival of the conceptus.

Abortion, Induced↗
PubMed1989

Hemopoietic origin of certain decidual cell precursors in murine pregnancy.

The possible hemopoietic origin of certain precursors of uterine decidual cells appearing during normal murine pregnancy was investigated in semiallogeneic hemopoietic chimeras with retained or regained fertility. Chimeras were produced by three different methods in two donor-host combinations: F1 [BALB/c female x C3H/HeJ male] cells introduced into the parental strain BALB/c female hosts or F1 [CBA/J female x C57BL/6 male] cells introduced into CBA/J female hosts. Prenatal chimeras (PN) were made by reconstituting mouse fetuses (day 13-17) with 10(6)-10(7) adult bone marrow or fetal liver cells through the yolk sac and they were allowed to be delivered naturally. Neonatal chimeras (NN) were made by injecting 1-2 x 10(7) adult bone marrow cells into the anterior facial vein of neonatal mice (less than 24 hr old). In both cases, experimental animals were raised to maturity. Ovary-transplanted chimeras (OT) were made by injecting 10(7) bone marrow cells into lethally irradiated (9.5 Gy) young adult female mice, followed 6 weeks later with bilateral orthotopic transplants of syngeneic ovary grafts to restore fertility. All female chimeras produced by the three different methods were mated with syngeneic male partners to produce normal pregnancy. The extent of chimerism at the cellular level was determined in all cases by a radioautographic identification of the H-2 phenotype of splenic lymphocytes and decidual cells and macrophages in the collagenase-dispersed decidua at day 11-16 of normal pregnancy, following a sandwich labelling with monospecific anti-H-2 antibodies and 125I-protein A. Morphological discrimination of typical decidual cells from macrophages in the collagenase-dispersed decidua was carried out on the basis of several distinctive markers: presence of surface Dec-1 and Thy-1 and absence of surface F4/80 or latex phagocytosis for decidual cells, in contrast to macrophages which were phagocytic and expressed F4/80 but not Dec-1 or Thy-1. While the degree of hemopoietic chimerism (judged by the incidence of donor-derived lymphocytes in the spleen) varied from animal to animal, in all three groups (PN, NN, and OT) comprising a total of 26 chimeras, the percentage of typical decidual cells expressing donor H-2 phenotype showed an excellent correlation with that for small lymphocytes in the spleen. These results reveal that at least a subpopulation of typical decidual cells of the pregnant uterus has a hemopoietic genealogy. A possible familial relationship of these cells to granulated metrial gland cells remains unclear.

Animals↗
PubMed1976

Effects of occlusal instability.

Changes that may occur in an occlusion are basically either horizontal or vertical. Horizontal changes need not be damaging and may be readily accommodated. Vertical changes can be more damaging and require a more extensive treatment procedure.

Adult↗
PubMedDate not supplied

Mechanisms of placental invasion of the uterus and their control.

Trophoblast cells of the placenta in many species have acquired mechanisms to invade the uterus, inclusive of its blood vessels, to establish efficient fetomaternal exchange of molecules. This invasion is strictly controlled both spatially and temporally and, in humans, usually continues until midgestation. Key mechanisms underlying various steps in trophoblast invasion are: (i) the attachment to the basement membrane, most likely by binding to laminin; (ii) the detachment from the basement membrane matrix, a process requiring the presence of complex-type oligosaccharides on the cell surface; and (iii) the breakdown of basement membrane components, mediated by secretion of metalloproteases (such as type IV collagenases) and serine proteases (plasminogen activator). Activation of trophoblast-derived metalloproteases appears to be plasmin dependent. Trophoblast invasiveness in situ is controlled by the microenvironment, owing to local production of anti-invasive factors by the decidual tissue of the uterus. One of these factors is TIMP (tissue inhibitor of metalloproteases), which neutralizes metalloproteases in an equimolar ratio. Another is TGF-beta (transforming growth factor-beta), which has a dual effect: it induces TIMP-1 secretion by the trophoblast and decidual cells and promotes differentiation of invasive trophoblast cells into multinucleated giant cells, which are presumably noninvasive. Thus, TGF-beta provides the key control of trophoblast invasiveness in situ. This control is lost in certain choriocarcinomas. In contrast to the response shown by the normal trophoblast, JAR and JEG-3 choriocarcinoma cell invasiveness does not seem to be inhibited by TGF-beta. In fact, in preliminary studies, JAR cells responded to TGF-beta by increased invasiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗