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C H Graham

Publications and source records attributed to C H Graham.

At least 55 records · Page 3Linked to original sources
PubMed1994

Extent of vascularization as a prognostic indicator in thin (< 0.76 mm) malignant melanomas.

Angiogenesis in malignant melanoma (MM) was evaluated by comparing mean vessel number (MVN) in Spitz's nevi (SN), thick and thin MMs that metastasized, and thick and thin MMs with > or = 10-year survival. Vessels were identified with antibodies against factor VIII-related antigen (FVIII) and CD34 in 37 MMs (17 < or = 1.9 mm and 20 > or = 4.0 mm) with > or = 10-year follow-up and 10 SN from children (< or = 9 years old). Fields (x250) with the highest vessel density were counted by independent observers blinded to clinical outcome. There were no differences in MVN between SN versus MMs (P = 1.0), but the distribution of vessels was much more uniform in SN. Seven MM pairs (> or = 5.5 mm) and five pairs (< or = 0.75 mm) were matched by sex, age, site, stage, and primary treatment (paired t-test). In the pairs > or = 5.5 mm, there was no correlation with MVN with either metastasis or death (FVIII P = 0.98; CD34 P = 0.85). Among the thin paired lesions, high MVN (FVIII = 46, CD34 = 39) was significantly related not only to metastasis (FVIII P = 0.04, CD34 P = 0.03) but also to death (FVIII P = 0.04, CD34 P = 0.05). MVN does not separate SN versus MM nor predict outcome in thick (> or = 4.0 mm) MMs; however, high MVN (> or = 42 average) is predictive of metastasis and death in MMs < or = 0.75 mm. Larger matched studies are indicated to confirm this observation.

Adult↗
PubMed1994

Variant sublines of early-stage human melanomas selected for tumorigenicity in nude mice express a multicytokine-resistant phenotype.

Surgical removal of early-stage radial growth phase or vertical growth phase primary cutaneous human melanomas usually results in cure of the disease. Hence there are few examples of genetically-related paired human melanoma cell lines for study in which one member of the pair is from a curable early-stage lesion and the partner is a more aggressive malignant variant. A rapid method of obtaining such variants is described. It consists of injecting cells from established early-stage radial growth phase or vertical growth phase melanoma cell lines--which are normally non- or poorly tumorigenic in nude mice--into such hosts, where the cell inoculum is co-mixed with Matrigel, a reconstituted basement membrane extract. This resulted in the rapid formation of progressively growing solid tumors from which permanent cell lines were established. Subsequently, the sublines were found to be frankly tumorigenic upon retransplantation into new nude mouse hosts in the absence of Matrigel co-injection. This process was repeated a second time, resulting in the isolation of secondary sublines, manifesting a stepwise increase in tumorigenic properties. The tumorigenic variant sublines were examined for their relative sensitivity to a panel of different cytokines that are normally growth inhibitory for melanoma cells from early-stage primary lesions. All the sublines were found to express an increased resistance to the cytokines transforming growth factor-beta, interleukin-6, interleukin-1 and tumor necrosis factor-alpha, and did so in a stable manner. Thus the results support the hypothesis that a progressive multicytokine resistance accompanies the progression of human melanoma. The availability of such related sublines should provide a valuable resource to help study the changes associated with, and perhaps causative of, disease progression in human malignant melanomas.

Animals↗
PubMed1993

Acquired multicellular-mediated resistance to alkylating agents in cancer.

EMT-6 murine mammary tumor sublines highly resistant to cyclophosphamide, cis-diamminedichloro-platinum(II), or N,N',N"-triethylenethiophosphoramide were generated in vivo by sequential treatment of tumor-bearing mice with the respective drugs. Previous studies demonstrated the drug-resistant phenotypes of the sublines were not expressed in vitro when the cells were grown as monolayer cultures. We now show that expression of drug resistance--including patterns of cross-drug resistance observed in vivo--can be fully recapitulated in vitro when the cells are grown under in vivo-like, three-dimensional conditions--namely, as multicellular tumor spheroids. Moreover, the spheroids generated from all of the drug-resistant sublines manifested a much more compact structure. Immediate drug-sensitivity testing of single cells released by trypsin treatment from compact drug-resistant spheroids revealed that such cells lost much of their drug-resistant properties. The results suggest a possible mechanism of acquired drug resistance in tumors based on the response of a cell population (i.e., multicellular or tissue resistance) as opposed to classic (uni)cellular resistance mechanisms.

Alkylating Agents↗
PubMed1993

Establishment and characterization of first trimester human trophoblast cells with extended lifespan.

We established trophoblast cell cultures with extended lifespans by introducing into first trimester human trophoblasts the gene encoding simian virus 40 large T antigen. The transfected trophoblasts were characterized according to their expression of various morphological and functional markers. Both parental (HTR-8) and transfected (HTR-8/SVneo) lines were morphologically similar and positive for cytokeratin, confirming their epithelial (trophoblastic) identity. Whereas the parental cells senesced after 12-14 passages, the transfectants have been in culture for over 32 passages. Human chorionic gonadotrophin was detected only in the HTR-8/SVneo cells and not in the parental cells. Both lines required at least 5% serum in order to sustain growth in vitro and responded to transforming growth factor-beta (TGF-beta) with reduced [3H]-thymidine incorporation in a dose-dependent manner. Treatment with TGF-beta also resulted in decreased secretion of plasminogen activators (PAs) and reduced PA activity by both lines. Both cell lines secreted mostly 72-kDa type IV collagenase as determined by substrate gel zymography, but the level of secretion of this enzyme was not significantly affected by TGF-beta in either line. Even though both lines exhibited similar in vitro invasive abilities, only the invasiveness of the parental cells was reduced by TGF-beta. Neither parental or transfected cells were capable of growth in soft agar and no sign of tumor formation was evident more than 5 months after subcutaneous inoculation of the transfected cells into nude mice. These results indicate that apart from their ability to sustain prolonged growth in culture, the transfected HTR-8/SVneo cells share a number of phenotypic properties with the parental trophoblast cells. For this reason, these transfected trophoblasts may prove to be an important tool for the study of placental function and/or tumor progression.

Animals↗
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↗
PubMed1982

Liver adenoma with granulomas. The appearance of granulomas in oral contraceptive-related hepatocellular adenoma and in the surrounding nontumorous liver.

A 31-year-old woman who had taken oral contraceptives regularly for seven years was found to have a hepatocellular adenoma in the left lobe of her liver. An unusual microscopic feature of the resected specimen was the presence of numerous noncaseating, epithelioid granulomas in both the adenoma and the surrounding nontumorous liver. The literature contains brief mentions of granulomas occurring within an occasional hepatocellular adenoma, but a search uncovered no reports of granulomas occurring in both the tumor and the surrounding nontumorous liver. This further evidence of the occurrence of epithelioid granulomas with hepatocellular adenomas suggests a possible relationship between tumor antigens and the granulomatous response.

Adenoma↗
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↗