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C H Graham

Publications and source records attributed to C H Graham.

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

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR.

Biological Transport↗
PubMed1998

Stimulation of plasminogen activator inhibitor-1 expression in immortalized human trophoblast cells cultured under low levels of oxygen.

Low levels of oxygen characterize certain biological settings such as the first trimester human placenta and rapidly growing tumors. Hypoxia in tumors and expression of high levels of tumor-associated plasminogen activator inhibitor-1 (PAI-1) indicate a poor prognosis for some cancer patients. Furthermore, PAI-1 may promote tumor invasion and metastasis by modulating cell adhesion and detachment from the extracellular matrix. In this study, we used immortalized human trophoblasts (HTR-8/SVneo cells) derived from first trimester placenta to study the effect of exposure to low levels of oxygen on PAI-1 expression. Cell viability following 24-h exposure to 1% oxygen was similar to that of cells cultured under 20% oxygen. Exposure to hypoxia resulted in time-dependent increases in PAI-1 mRNA and protein levels, as determined by Northern blot analysis and enzyme-linked immunosorbent assay. Culture with cobalt chloride or Tiron also resulted in increased PAI-1 mRNA levels, while carbon monoxide inhibited the hypoxia-mediated increase, thereby indicating that a heme protein is involved in the stimulation of PAI-1 expression by hypoxia. Incubation with transforming growth factor-beta1 (TGF-beta1) also resulted in increased levels of PAI-1 mRNA. However, addition of a neutralizing anti-TGF-beta antibody to hypoxic cultures did not abrogate the increase in PAI-1 mRNA levels, suggesting that hypoxia stimulates PAI-1 expression via a pathway that does not require TGF-beta production. These results indicate that, through their effect on PAI-1 expression, oxygen levels may play an important role in modulating cellular migration and invasion.

Cell Survival↗
PubMed1998

Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway.

Hypoxia underlies a number of biologic processes in which cellular migration and invasion occur. Because earlier studies have shown that the receptor for urokinase-type plasminogen activator (uPAR) may facilitate such events, we studied the effect of hypoxia on the expression of uPAR by first trimester human trophoblasts (HTR-8/SVneo) and human umbilical vein endothelial cells (HUVEC). Compared with control cells cultured under standard conditions (20% O2), HTR-8/SVneo cells and HUVEC cultured in 1% O2 expressed more uPAR, as determined by flow cytometric and [125I]-prourokinase ligand binding analyses. Increased uPAR expression paralleled increases in uPAR mRNA. The involvement of a heme protein in the hypoxia-induced expression of uPAR was suggested by the observations that culture of cells with cobalt chloride, or sodium 4, 5-dihydroxybenzene-1,3-disulfonate (Tiron), an iron-chelating agent, also stimulated uPAR expression, and that the hypoxia-induced uPAR expression was inhibited by adding carbon monoxide to the hypoxic atmosphere. Culture of HTR-8/SVneo cells with vascular endothelial growth factor (VEGF) did not increase uPAR mRNA levels, suggesting that the hypoxia-mediated effect on uPAR expression by these cells did not occur through a VEGF-dependent mechanism. The functional importance of these findings is suggested by the fact that HTR-8/SVneo cells cultured under hypoxia displayed higher levels of cell surface plasminogen activator activity and greater invasion through a reconstituted basement membrane. These results suggest that hypoxia may promote cellular invasion by stimulating the expression of uPAR through a heme protein-dependent pathway.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1998

Differential display analysis of oxygen-mediated changes in gene expression in first trimester human trophoblast cells.

Physiologic or pathologically induced periods of exposure to relatively low levels of oxygen during pregnancy affect the expression and function of certain genes in the placenta. In this study, the differential display technique was utilized to identify genes that are regulated in cultured cytotrophoblast cells by exposure to low levels of oxygen. Using this approach, four genes, which have been designated HRF-1, HRF-2, HRF-6, and HRF-8, were cloned and partially characterized. Northern blot analysis showed that clones HRF-1 and HRF-2 were downregulated in response to exposure to low levels of oxygen, whereas expression of HRF-6 and HRF-8 was increased. DNA sequencing and sequence analysis revealed that HRF-1 may represent an alternatively spliced or tissue-specific form of the Kruppel family zinc finger protein znfp104 gene. Clone HRF-2 showed a high degree of identity with exons 9, 10 and 11 of N33, a gene that is located within a homozygously deleted region of metastatic prostate cancer. Clones HRF-6 and HRF-8 did not exhibit significant sequence identity with known sequences in GenBank and may represent novel genes. None of these genes have previously been shown to be present in trophoblast cells, nor have their expressions been shown to be regulated by oxygen. This study demonstrates that the differential display technique is a novel and effective method to analyse oxygen-mediated changes in gene expression in trophoblast cells.

Adult↗
PubMed1998

Heme oxygenase and nitric oxide synthase in the placenta of the guinea-pig during gestation.

Nitric oxide (NO) and carbon monoxide (CO) are novel gaseous chemical messengers that play key roles in cell function and cell-cell communication in many organ systems, including the cardiovascular system. Although the presence of NO synthase (NOS) in the placenta and its role in the regulation of fetoplacental and uteroplacental blood flow are well established, little is known about placental expression and activity of heme oxygenase (HO), the enzyme that catalyses the oxidation of heme to CO, biliverdin and iron, during gestation. The objectives of this study were to elucidate the localization of HO-1 and HO-2 isoforms relative to NOS III protein, and to determine the enzymatic activity of HO in the placenta of the guinea-pig during gestation. Placentae were obtained from pregnant guinea-pigs at gestational day (GD) 34, 50, 62 and full term (term, about GD 68). Immunohistochemical localization of HO-1, HO-2 and NOS III protein was conducted using selective polyclonal antibodies. HO activity was determined by using a gas chromatographic method to measure the rate of formation of CO from heme. Faint staining for HO-1 was observed in the adventitial layer of larger fetal blood vessels of the placenta at GD 34. The intensity of this staining was higher at GD 50 and GD 62, and decreased at full term. Similar areas in serial sections of placentae obtained at these selected times during gestation exhibited lower staining intensity when incubated with anti-HO-2 antiserum. Placental HO activity was significantly increased (P<0.05) at GD 62 compared with GD 34, GD 50 and full term. NOS III (endothelial constitutive NOS) staining was highest at GD 34, decreasing thereafter, and was localized mostly to trophoblast lining maternal channels. The data demonstrate that, in the guinea-pig, placental HO and NOS differ in tissue localization during the second half of gestation, with expression of HO protein and its catalytic activity being higher during near-term pregnancy. In a preliminary immunohistochemical investigation of the full-term human placenta, HO-1 protein was localized primarily in the adventitial region of fetal blood vessels of stem chorionic villi. In view of the vasodilator action of CO and NO, the HO and NOS systems may play key roles in the regulation of placental haemodynamics.

Adult↗
PubMed1997

Cellular localization of gp46 at the human fetal-maternal interface.

gp46 is a collagen-binding heat-shock glycoprotein with a possible role in the biosynthesis of collagen as well as in cell differentiation and fusion. In this study, the relative levels of gp46 protein and its mRNA transcript were examined, as well as the mRNA levels of collagen types I and IV in first trimester and term human placental tissues. Western blot analysis revealed substantially higher levels of gp46 in first trimester placentae than in term placentae. Similarly, elevated levels of type IV collagen transcript were detected in first trimester relative to term issues. Interestingly, the levels of gp46 and type I collagen mRNA remained unchanged. Immunohistochemical analysis of first trimester tissues demonstrated intense gp46 staining in mononucleated villous and extravillous cytotrophoblasts, decidual cells and in the villous connective tissue stroma. Syncytiotrophoblast in the same tissues also exhibited gp46 staining but at a reduced intensity. In chronic villi of term placentae, faint gp46 staining was only observed in the syncytiotrophoblast layer. However, as in the first trimester placentae, intense labelling was evident in the extravillous cytotrophoblasts and decidual cells of these tissues. These results suggest a developmental regulation of gp46 expression at the fetal-maternal interface during pregnancy and suggest a possible functional link between gp46 and collagen type IV. during gestation.

Antibodies, Monoclonal↗
PubMed1996

Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells.

BACKGROUND: De novo or acquired resistance to chemotherapeutic drugs continues to be one of the most important obstacles hindering the successful treatment of cancer patients. Consequently, enhancing the efficacy of conventional chemotherapeutic drugs has become an important research goal. Our previous studies using the mouse EMT-6 mammary carcinoma selected for resistance to various alkylating agents in vivo demonstrated that such acquired drug resistance may be manifested in vitro only in cells growing in a three-dimensional configuration but not in conventional monolayer culture. We also found that this phenomenon, which we refer to as "acquired multicellular resistance," is associated with an increase in intercellular adhesion or compaction of the alkylating agent-resistant cell lines grown as aggregates in three-dimensional culture. PURPOSE: The present study further investigates the impact of three-dimensional architecture on acquired multicellular drug resistance and its influence on cell cycle kinetics, cell cycle arrest, and cell survival. METHODS: To test the hypothesis that an increase in three-dimensional compaction is related to the drug resistance properties of the cells, we did the following: 1) selected clones of the EMT-6 cell line that spontaneously formed tightly or loosely adherent aggregates and assessed their respective drug resistance properties in vitro; 2) assayed tumorigenic potential of the tight and loose clones after exposure to defined concentrations of the activated form of cyclophosphamide, 4-hydroperoxycyclophosphamide (4-HC) in vitro; and 3) treated the tight clones with hyaluronidase, an agent capable of disrupting EMT-6 spheroids, and assayed what effect this treatment had on chemosensitivity. We used fluorescence-activated cell sorter analysis to monitor any potential alterations in cell cycle kinetics. RESULTS: The increase in compaction in three-dimensional culture was sufficient to confer resistance to 4-HC. This increase in intercellular adhesion was also associated with a lower proliferating fraction of tumor cells and with an almost completely diminished ability of the cells to arrest in the G2/M phase of the cell cycle after drug exposure. Furthermore, these changes were detectable only in three-dimensional culture, not in conventional monolayer culture. In conventional monolayer culture, all cell types consistently showed a high level of proliferation and arrested in G2/M after exposure to 4-HC. Moreover, hyaluronidase was able to disrupt intercellular adhesion and chemosensitize tumor cells both in vitro and in vivo in an ascites model. CONCLUSION: Earlier studies have demonstrated that hyaluronidase is able to sensitize tumor cells to various anticancer agents. Our studies now demonstrate that this sensitization can occur by a mechanism independent of increased drug penetration. This mechanism is likely to be related to the "anti-adhesive" effect of hyaluronidase, which overrides cell contact-dependent growth inhibition, recruits cells into the cycling pool, and renders tumor cells more sensitive to cytotoxic agents that preferentially kill rapidly dividing cells. IMPLICATIONS: Other tumor-specific "anti-adhesives" should be explored that can be effective chemosensitizers when used in combination with cell cycle-specific drugs for the treatment of small, solid tumors.

Animals↗
PubMed1996

Altered expression of gelatinase and surface-associated plasminogen activator activity by trophoblast cells isolated from placentas of preeclamptic patients.

OBJECTIVE: This study compared in vitro degradative properties of trophoblasts isolated from placentas of preeclamptic patients with those of trophoblasts isolated from placentas of patients with uncomplicated pregnancies. Specifically, the expression of gelatinases, plasminogen activators, and plasminogen activator inhibitor type 1 by these cells was examined. STUDY DESIGN: Gelatinase and plasminogen activator secretion were determined by zymography, whereas antigenic levels of urinary-type plasminogen activator and plasminogen activator inhibitor type 1 in culture-conditioned medium were determined by enzyme-linked immunosorbent assay. We also evaluated expression of cell-surface plasminogen activator activity with a sensitive assay that uses the fluorescent plasmin substrate H-D-Val-Leu-Lys-7-amino-4-methylcoumarin. RESULTS: Cells from both normal and preeclamptic placentas secreted variable levels of gelatinase, of which the most predominant was matrix metalloproteinase-9 (gelatinase B). In 60% of the media conditioned by preeclamptic cells the matrix metalloproteinase-9 present was exclusively of a higher molecular weight (> 92 kd) than the predominant enzyme secreted by trophoblasts isolated from normal placentas. Incubation of the preeclamptic cell culture-conditioned media with p-aminophenylmercuric acetate, an activator of metalloproteinases, resulted in a decrease in the apparent molecular weight of the enzyme, indicating that most of the matrix metalloproteinase-9 contained in these samples was in the inactive form. Although trophoblasts from normal and preeclamptic placentas secreted similar amounts of urinary-type plasminogen activator and plasminogen activator inhibitor type 1, the latter cells expressed significantly less (p < 0.001) cell surface plasminogen activator activity. CONCLUSION: These results suggest that abnormal uteroplacental blood flow in preeclampsia (as a result of either shallow invasiveness of the uterine arteries or excessive fibrin deposition within the intervillous spaces) might result from altered expression of active proteinases by trophoblasts.

Adult↗
PubMed1996

Expression of atrial natriuretic peptide by third-trimester placental cytotrophoblasts in women.

Atrial natriuretic peptide (ANP) is a vasoactive molecule with a potential role in modulating uteroplacental blood flow. We examined the expression of ANP in the third-trimester fetomaternal interface by immunohistochemistry performed on sections of placental tissue as well as by reverse transcriptase-polymerase chain reaction (RT-PCR) and RIA performed on isolated, purified term trophoblasts. Results from immunohistochemical studies on ten normal term placental samples revealed labeling of extravillous interstitial and cytotrophoblastic shell trophoblasts. The trophoblastic identity of the cells was determined by labeling of adjacent serial sections with anti-cytokeratin antibody. Labeling of decidual tissue was also observed in some samples (3 of 10 samples), whereas villous trophoblastic tissue showed no labeling. RT-PCR analysis of RNA extracted from isolated third-trimester trophoblasts revealed the presence of a 0.45-kb and band corresponding to ANP mRNA. Sequencing of the amplified cDNA confirmed its identity. RIA on lysates of isolated trophoblasts revealed the presence of ANP at a level of 41 pg +/- 8 pg/10(7) cells (n = 4; range = 16.8-50.4 pg/10(7) cells). These results indicate that placental cytotrophoblastic tissue is a source of ANP. This molecule might play a role in the regulation of uteroplacental and/or fetoplacental blood flow.

Atrial Natriuretic Factor↗
PubMed1996

Human trophoblast cell adhesion to extracellular matrix protein, entactin.

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion. METHODS: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta. RESULTS: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both beta 1 and beta 3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, beta 2 and beta 4 integrin subunits were not detected. In addition, we found that alpha v beta 3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The beta 3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections. CONCLUSION: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by beta 1 and/or beta 3 class integrins.

Antigens, CD↗
PubMed1996

Altered expression of the tumor suppressor/oncoprotein p53 in SV40 Tag-transformed human placental trophoblast and malignant trophoblast cell lines.

The expression of the tumor suppressor/oncoprotein p53 has been investigated in normal human placental villous trophoblast, in vitro propagated invasive extravillous trophoblast, SV40 tumor antigen (Tag)-immortalized extravillous trophoblast, human cytomegalovirus (hCMV)-infected syncytiotrophoblast and malignant trophoblast (choriocarcinoma) cell lines (JAR, JEG-3 and BeWo) using quantitative enzyme-linked immunosorbent assay (ELISA) and Western immunoblot methods using monoclonal antibodies specific for wild-type and mutant p53. The normal villous and extravillous trophoblast cells expressed low levels of the wild-type p53 protein, whereas normal terminally differentiated multinucleated syncytiotrophoblast cells, as well as hCMV-infected syncytiotrophoblast, showed a higher expression of the wild-type p53 protein. SV40 Tag-immortalized invasive trophoblast cells also showed a high expression of the wild-type p53 protein which remained complexed with the Tag protein. All the choriocarcinoma cell lines over expressed the mutant form of the p53 protein. The increased expression of p53 protein in the SV40 Tag-immortalized invasive trophoblast and choriocarcinoma cells paralleled with increased expression of the mouse double minute 2 (mdm2) oncogenic protein. Transforming growth factor (TGF)-beta inhibited proliferation of normal extravillous trophoblast cells. The antiproliferative effects of TGF-beta were reduced in SV40 Tag-immortalized cells and non-detectable in choriocarcinoma cell lines JAR, BeWo and JEG-3. The inactivation of p53 owing to complexing with Tag in the immortalized premalignant trophoblast and p53 mutation in the malignant trophoblast may be responsible for their aberrant proliferation and refractoriness to antiproliferative effects of TGF-beta observed in these cells as compared to the normal trophoblast. These results may suggest the role of p53 protein in trophoblast differentiation, transformation and tumorigenesis.

Animals↗
PubMed1996

Analysis and significance of the malignant 'eclipse' during the progression of primary cutaneous human melanomas.

Why is it that primary melanomas which are less than 0.76 mm in thickness are almost always curable by surgery whereas thicker lesions are associated with a worse prognosis? Put in another way, why is it that such small increases in tumor thickness beyond 0.76 mm are often associated with the eventual formation of distant metastases and death? Part of the answer lies in the dramatic qualitative changes which can accompany small increases in the size of primary human melanomas. Thus, primary melanomas less than 0.76 mm in thickness usually contain very low proportions of metastatically competent tumor cells, whereas slightly thicker lesions can contain very high proportions of such cells, resulting from a selective growth advantage of the latter in the dermal mesenchyme. This overgrowth process is akin to a 'malignant eclipse' phenomenon (by analogy with a solar eclipse). We have been studying the causes of the malignant eclipse in melanoma, for which there are at least four possibilities: 1) an increase in autocrine, mitogenic growth factors by melanoma cells; 2) a decreased rate of apoptosis in the same population; 3) an acquired resistance to paracrine growth inhibitory factors; and 4) an increased ability to induce an angiogenic response. Evidence exists for all four possibilities. Our experimental approach to studying this problem has relied heavily on the use of cell lines obtained from early stage radial growth phase or vertical growth phase lesions which have a clinical-like inability to grow progressively in nude mice, and variants obtained from such lines which are aggressively tumorigenic. Using such paired lines, and other experimental systems, we have obtained evidence that shows early stage melanoma cell lines may be deficient in inducing angiogenesis, are highly sensitive to the growth inhibitory effects of a plethora of cytokines, including transforming growth factor beta, interleukin-6, and oncostatin M, and are more sensitive to undergoing spontaneous apoptosis in several conditions including when growth in anchor-age-independent, 3-dimensional tissue culture. How this information may impact tumor prognosis and the design and effects of new strategies to treat melanoma, especially antiangiogenesis strategies, is discussed.

Animals↗
PubMed1995

Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture.

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.

Cell Movement↗
PubMed1994

Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents.

BACKGROUND: Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating agents in vivo after serial passage into syngeneic mice manifest their resistance in vitro only when cultured as three-dimensional multicellular aggregates or spheroids. PURPOSE: We examined whether a single exposure of mouse EMT-6 or human MDA-MB-231 breast cancer cells to alkylating agents in vitro is sufficient for the induction of a resistance phenotype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. METHODS: Mouse EMT-6 and human MDA-MB-231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed after a subsequent alkylating-agent exposure of cells cultured as monolayers or three-dimensional aggregates. RESULTS: A single in vitro exposure to 12.5-microM cisplatin (CDDP) for 5 days or 25 microM 4-hydroperoxycyclophosphamide (4-O2H CTX) for 1 or 3 days without changing the medium was sufficient to induce transient but substantial resistance in EMT-6 cells as determined by clonogenic assays. Such resistance was not detected when monolayer cell cultures were used. The concentration of 4-O2H-CTX and the length of time the cells remained in three-dimensional culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimensional cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architecture of the cell aggregates, which now formed much more compact multicellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enough to bring about resistance in MDA-MB-231 cells detectable only in three-dimensional cultures, as well as the change in three-dimensional architecture. CONCLUSIONS: Rapid acquisition of resistance likely represents a physiologic mechanism of adaptation operative at the multicellular level rather than a stable genetic change and may be one of the reasons for the rapid development of drug resistance acquired by tumors in vivo. IMPLICATIONS: In vivo drug exposure may result in transient and low levels of drug resistance that may nevertheless be clinically relevant.

Animals↗
PubMed1994

Intrinsic or acquired drug resistance and metastasis: are they linked phenotypes?

Evidence is reviewed which suggests a linkage may exist between certain forms of de novo or acquired drug resistance and metastasis. This includes the finding that expression of certain dominantly acting mutant oncogenes or tumor suppressor genes, i.e., genes which normally act to "drive" tumor progression and metastasis, can also affect the expression of drug resistance. Moreover, this can be accompanied by altered expression of certain cellular genes thought to be involved in expression of drug resistance. A direct linkage between acquired drug resistance and metastasis would suggest that tumor sublines selected for drug resistance should manifest more aggressive malignant properties than their drug-sensitive counterparts. While this does not appear to be true for drug resistant sublines selected in vitro, indeed such cell lines frequently manifest diminished in vivo tumorigenic and/or metastatic competence, there is some evidence to support such a correlation exists for tumor cell lines that are selected in vivo for drug resistance. Attention is also drawn to the fact that new linkages between metastasis and drug resistance may be uncovered by analyzing the ability of tumor subpopulations to acquire drug resistance after one or several previous exposures to chemotherapeutic drugs, as opposed to examining intrinsic drug resistance only. Furthermore, ability to detect induced or acquired drug resistance in vitro may be strongly influenced by the types of assay used to detect and monitor drug resistance. In particular, three-dimensional cell culture systems may reveal acquired or induced "multicellular" drug resistance in situations where conventional two-dimensional culture systems do not. Use of three-dimensional culture systems may therefore reveal as yet undiscovered associations between the phenotypes of metastasis and drug resistance.

Animals↗
PubMed1994

Resistance of malignant trophoblast cells to both the anti-proliferative and anti-invasive effects of transforming growth factor-beta.

Human placental trophoblast invasion of the uterus is a highly controlled event. We had shown that transforming growth factor-beta (TGF-beta) produced in the pregnant uterus controls invasiveness and reduces proliferation of first trimester placental trophoblasts in vitro. The anti-invasive effect of TGF-beta was due, at least in part, to induction of tissue inhibitor of metalloproteinases (TIMP)-1. In the present study we compared the effects of TGF-beta on proliferation ([3H]-TdR incorporation) and invasiveness (3-day Matrigel invasion assay) of JAR and JEG-3 choriocarcinoma cells vs normal first trimester human trophoblast cells. Transcripts of type IV collagenases (72- and 92-kDa enzymes, i.e., gelatinases A and B) and their inhibitors (TIMP-1 and TIMP-2) in these cells were measured by Northern analysis, and secretion of gelatinases and plasminogen activators (PAs) was evaluated by gel zymography. The results revealed that: (a) TGF-beta inhibited invasiveness and proliferation of normal trophoblast but not JAR and JEG-3 choriocarcinoma cells; (b) gelatinase A mRNA, expressed by the normal trophoblast and JAR cells, was upregulated in the presence of TGF-beta; (c) gelatinase B mRNA was not detected in the total RNA preparations of treated or untreated normal trophoblast or choriocarcinoma cells; (d) TGF-beta significantly upregulated the levels of TIMP-1 mRNA in the normal trophoblasts, but this transcript was very low in treated as well as untreated choriocarcinoma cells; TGF-beta also upregulated the 3.5-kb TIMP-2 message in the normal trophoblast; (e) gelatin zymography revealed a distinct band of approximately 68-kDa (gelatinase A) in the conditioned media of normal trophoblast and JAR cells; however, TGF-beta did not change the level of secretion of this gelatinase; and (f) the normal trophoblast also exhibited significant PA secretion (casein zymography) which was reduced in the presence of TGF-beta. PA secretion by the malignant trophoblast cells was low and unaffected by TGF-beta. These findings suggest that choriocarcinoma cells may become refractory to the mechanisms which control normal trophoblast proliferation and invasiveness. Concurrent resistance to antiproliferative and anti-invasive molecules such as TGF-beta may be highly relevant to tumor progression.

Cell Division↗