Biomedical subjects

M J Hendrix

Publications and source records attributed to M J Hendrix.

At least 91 records · Page 5Linked to original sources

PubMed1990

Artificial matrix barriers: a diffusion study utilizing dextrans and microspheres.

Eight artificial matrices (AMs) were evaluated for the ability to restrict the passage of diffusion probes. Three AMs were composed exclusively of interstitial type I collagen (Col I) and differed from each other in thickness only. Four AMs consisted of reconstituted basement membrane (RBM) -coated polycarbonate filters (containing 10 microns diameter pores) and also only differed in thickness. One AM consisted of an uncoated 10 microns pore polycarbonate filter. The diffusion probes were uncharged fluorescein isothiocyanate-labeled dextrans, having molecular weights of 17,900, 42,000, 71,200, and 148,900 and negatively charged latex microspheres, having diameters of 0.08, 0.30, and 0.95 microns. Probes were applied to the AMs, incubated for 72 hr at 37 degrees C, and then analyzed spectrophotometrically. Dextran passage was increasingly restricted for Col I matrices as either molecular weight or collagen thickness increased (range 7% to 0.7%). Thin RBM-coated filters were more permeable to dextrans (range 100% to 30%) than Col I matrices. The diffusion rate of microspheres for Col I matrices (range 3.5% to 0) was similar to both thick and thin RBM-coated filters (range 4% to 0). The uncoated filter permitted the most diffusion for both dextrans and microspheres (range 100% to 7%). These data demonstrate that the AMs presented in this study will allow direct observation of the degradative and migratory potential of cells in vitro as they interact with various extracellular matrices.

Animals↗

PubMed1990

A comparison of levels of intrinsic single strand breaks/alkali labile sites associated with human melanoma cell invasion.

Intrinsic levels of protein-free single strand breaks/alkali-labile sites in human melanoma cell populations of varying in vitro invasive capacity have been assayed with DNA filter elution methodology. DNA from two human melanoma cell lines, A375P and C8161, and from a subpopulation selected from A375P, A375P-5, were assayed to test the hypothesis that increased levels of DNA damage may be associated with the phenotype of increased invasive and metastatic capacities. The elution profiles obtained reveal statistically significant increases in the level of single strand breaks and/or alkali-labile sites (SSB/ALS) which correlate with increasing invasive and metastatic capacities. The increased levels of SSB/ALS in A375P-5 observed in freshly selected cells decline as these cells are maintained in culture. The stability of this A375P-5 phenotype correlates with previously reported levels of double minute chromosomes, an indicator of genomic instability. Alterations in average intrinsic levels of cellular lesions are therefore an additional factor to be considered in the phenotypic characterization of invasive and metastatic tumor cells and may reflect or contribute to the genomic instability characteristic of tumor cell populations.

Alkalies↗

PubMed1990

Effect of interferon-gamma on the expression of HLA-DR by human melanoma cells of varying metastatic potential.

Three human melanoma cell lines of varying invasive and metastatic potential were analyzed for their ability to express HLA-DR antigens on the cell surface as well as transcriptionally at the mRNA level in the presence and absence of IFN-gamma treatment. Cells of low and intermediate metastatic and invasive potential showed a high percentage of HLA-DR surface expression, both before (91.2-99.9%) and after (97.8-99.9%) IFN-gamma treatment, as quantitated by flow cytometry. In contrast, cells of high metastatic and invasive potential expressed barely detectable levels of HLA-DR-positive cells before IFN-gamma treatment (0.3-0.6%) and displayed elevated levels following treatment (42.3-89.4%). Allowing the highly metastatic cells to recover for 7 or 14 days following IFN-gamma treatment resulted in barely detectable levels of HLA-DR-positive cells. Northern blot analyses of HLA-DR transcription levels showed a strong expression in cells of low and intermediate metastatic and invasive potential. HLA-DR mRNA levels were not detectable in control cells of high metastatic potential nor in those cells which had undergone 7- and 14-day recovery periods following IFN-gamma treatment. There was, however, an induction of HLA-DR expression in the cells that had been treated with IFN-gamma for 72 hr and allowed no recovery period. In addition, a punctate, receptor-like pattern of immunofluorescence staining pattern for cell surface HLA-DR was seen after a 72 hr IFN-gamma treatment in the highly metastatic cells. In contrast, cells of low and intermediate metastatic potential expressed a homogeneous ring-like pattern of antigen expression.

Blotting, Northern↗

PubMed1990

Quantitation of human melanoma, carcinoma and sarcoma tumor cell adhesion to lymphatic endothelium.

We have used an in vitro adhesion assay to study the interaction of tumor cells with lymphatic endothelium, a dynamic event that leads to tumor metastasis in vivo. 3H-thymidine-labeled human tumor cells from: one primary Ewing sarcoma, two established melanoma cell lines, two colon and two breast carcinomas (one established line and one primary culture of each) were added to 24-well culture dishes containing confluent monolayers of bovine lymphatic endothelium. Radioactivity associated with either the cells in suspension or the attached cells was assessed and compared at frequent intervals up to 360 minutes. Generally, tumor cell attachment increased as a function of time reaching a plateau between 180 and 360 minutes. the modular media system described here facilitates the primary and secondary culture (or co-culture) of a variety of normal and transformed cells. Primary cultures with a rounded morphology (one breast and one colon carcinoma) showed the lowest preferential attachment for lymphatic endothelium. All established cell lines and the primary Ewing sarcoma cell line displayed a more fibroblastic morphology and achieved the highest adhesion profiles. There was a correlation between the malignancy and attachment potential for the melanoma and breast carcinoma cell lines. Collectively, these data show that established tumor cell lines with fibroblastic-like morphology exhibit more rapid adhesion than primary tumor cell cultures with more rounded morphologies. While this property may reflect in vitro selection and/or adaptation, it does correlate with the metastatic propensity for some human tumor cells.

Adenocarcinoma↗

PubMed1990

Selection of invasive and metastatic subpopulations from a heterogeneous human melanoma cell line.

The formation and propagation of several subpopulations of human melanoma cells from a heterogeneous parental population was accomplished with the use of the Membrane Invasion Culture System (MICS) in vitro under sterile conditions. Five sequentially selected subpopulations of melanoma cells showed an increasing ability to do the following: a) invade reconstituted basement membranes in vitro; b) form experimental lung metastases in vivo; and c) express steady-state levels of human type IV collagenase, a marker for metastatic potential. In addition, the morphology and expression of 35S-methionine-labeled cell surface proteins changed with sequential selection. The adaptation of the MICS assay for studying tumor cell subpopulations allows the morphological, biochemical and molecular characterization of events associated with tumor progression in an in vitro model.

Animals↗

PubMed1989

Inhibition by retinoic acid of type IV collagenolysis and invasion through reconstituted basement membrane by metastatic rat mammary adenocarcinoma cells.

The activity of type IV collagenase, which enables tumor cells to degrade collagen type IV found in the subendothelial basement membrane, has been correlated with the metastatic potential in several tumor types, including the rat 13762NF mammary adenocarcinoma cell line and its clones. In this study, we examined whether all-trans-retinoic acid (all-trans-RA) and other retinoids, which exhibit antitumor activity in vitro and in vivo, affect the collagenolytic activity of metastatic rat 13762NF mammary adenocarcinoma cells. Cells of the highly metastatic lung-colonizing clone MTF7.T35.3, derived from the 13762NF cell line, were treated for 3 days with 0.1, 1, or 10 microM all-trans-RA, harvested, and seeded on [3H]proline-labeled extracellular matrix deposited by cultured rat lung endothelial cells or on a film of purified [3H]proline-labeled type IV collagen. The amount of radioactivity released into the medium during the subsequent 24 to 72 h was measured, and it was found that all-trans-RA treatment inhibited degradation of extracellular matrix and type IV collagen by 50 to 60%. This effect was observed whether the cells had been treated with all-trans-RA in serum-free medium or in medium supplemented with heat-inactivated or acid-treated fetal bovine serum. The growth of the cells was not inhibited under these conditions, except after treatment with 10 microM all-trans-RA in serum-free medium. The reduction in collagenolytic activity was observed in viable cells as well as in conditioned medium. A 24-h exposure of cells to all-trans-RA was sufficient to cause a 30% decrease in the collagenolytic activity, and this inhibitory effect was reversible. The direct addition of all-trans-RA to conditioned medium had no effect on secreted collagenase activity. The apparent molecular weights of the collagenolytic enzymes were determined by electrophoresis of cell extracts and concentrated conditioned medium in type IV collagen-embedded polyacrylamide gels followed by renaturation and activation of the enzymes within the gels. Two major type IV collagenolytic metalloproteinases exhibiting molecular weights of 64,000 and 88,000, respectively, were detected by this method. These two enzymes were also found to have specificity for gelatin. The Mr 64,000 enzyme could be extracted from viable cells (presumably from the cell membrane) by 2% 1-butanol. Treatment with all-trans-RA decreased the level of these enzymes in the cellular, cell membrane, and conditioned medium compartments.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗

PubMed1989

Use of the Membrane Invasion Culture System (MICS) as a screen for anti-invasive agents.

The Membrane Invasion Culture System (MICS) assay was adapted for relatively rapid screening of compounds and used to identify anti-invasive drugs that inhibit human and murine tumor cell migration through a reconstituted basement membrane in vitro. Cell lines demonstrating low and high invasive and metastatic potentials were tested with all compounds for tumoricidal effects prior to evaluation in MICS at non-cytotoxic doses. The effect on invasive potential in the MICS assay was determined in 3 categories: (1) 48 hr drug pre-treatment prior to seeding in the MICS (exceptions: 90 min pre-treatment with pertussis toxin and, for some studies, continuous exposure for 2-7 days); (2) peptide or prostaglandins 2 hr after seeding and attachment to the membranes in MICS followed by continuous exposure; and (3) cells receiving neither drug nor peptide treatment and serving as controls in each MICS chamber. Since invasion involves cellular motility and deformability, some cytoskeleton disrupting agents were selected. Of these, vincristine, colcemid and colchicine inhibited invasion but taxol did not. Pre-treatment with cAMP agonists produced conflicting results: dibutyryl cAMP and 8-(4-chloro-phenylthio) cAMP resulted in 50% and 38% reduction in invasion, respectively, whereas 8-bromo cAMP stimulated invasive potential by 30%. Forskolin and cholera toxin both significantly reduced invasiveness. Pre-treatment with 5-azacytidine and araC, to consider the role of methylation and proliferations decreased invasive ability. Anti-metastatic drugs such as gamma-interferon and razoxane inhibited invasive potential but to varying degrees. Treatment of cells with prostaglandins E2, F2 alpha, A2, and D2 were ineffectual; however, indomethacin mildly inhibits invasion (less than 30%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar.

An immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar for up to 50 days was performed. Three morphological variants of developing tumor colonies are reported: 1) large light colonies, 2) small dark colonies, and 3) smooth-edged colonies. The large light colony variant is the most frequently observed in the soft agar assay (approximately 70%), followed by the dark colony variant (approximately 27%), and the smooth-edged colony variant (approximately 3%). Major morphological characteristics are associated with each variant, as shown with light microscopy (LM) and transmission electron microscopy (TEM). Both LM and TEM analyses demonstrated that the large light colony variant was hypomelanotic and contained a microfibrillar extracellular matrix (ECM). The small dark colony variant was found to be hypermelanotic and contained a less demonstrable ECM. The smooth-edged variant has an encapsulated periphery, no demonstrable ECM, and tightly packed cells with desmosome-like junctions. In order to characterize further the ECM in the most commonly observed variant, the large light colony, specific antibodies to fibronectin (FN) and collagen types IV and V (COLs IV and V) were applied and observed with immunofluorescence microscopy and immunoperoxidase. In paraffin sections of melanoma colonies, FN was observed associated with both the cell surface and the ECM. However, no specific staining was seen for COLs IV and V. In addition, ruthenium red was used to preserve and selectively bind to glycosaminoglycans (GAGs) and proteoglycans (PGs). TEM studies reveal GAG-like granules stained with ruthenium red in the fibrillar ECM and a dotted, punctate staining of the cell surface. Understanding the biological and architectural composition of developing melanoma tumor colonies in soft agar could contribute to the development of more efficient chemotherapeutic strategies.

Agar↗

PubMed1989

Cloning efficiency of human melanoma cells is modulated after invasion through a reconstituted basement membrane.

Three human malignant melanoma cell strains (C8146A, C8146C, and C83-2CY), three established human melanoma cell lines (A375P, A375M, and C8161), and one selected human melanoma subline (A375P-5) were studied to determine if invasion through a reconstituted basement membrane-coated filter (RBMF), which selects the most aggressively invasive cells, would also modulate the cloning efficiency of these cells in soft agar. With the use of the Membrane Invasion Culture System (MICS), all cell strains tested showed a significant increase in cloning efficiency (1.05-9.3-fold) following transit through the RBMF when compared to unmanipulated populations. The established cell lines (A375P, A375M, and C8161) and the A375P-5 subline showed either a decrease or unaltered status in cloning efficiency after invasion. However, all cells demonstrated a consistent decrease in clonogenicity following transit through an uncoated filter compared with RBMFs, thus suggesting the influence of the extracellular matrix on tumor cell clonogenic properties. In general, the established cell lines were more clonogenic before invasion of the RBMF compared with the cell strains, and no correlation was found between clonogenic potential and invasive or metastatic capability. These data may provide important insight into the underlying mechanisms of tumor cell invasion and the subsequent formation and dissemination of metastases in vivo.

Animals↗

PubMed1989

Nuclear protein organization and the repair of radiation damage.

A complex network of proteins having attachment sites with DNA are known to exist in mammalian cells and have been referred to as a nuclear cage, matrix, scaffold and nucleoid. Since ionizing radiation is known to induce DNA--protein crosslinks as well as DNA single- and double-stranded breaks, an investigation of the sedimentation of the nucleoid in Chinese hamster ovary (CHO)* cells before, during and after treatments with ionizing radiation was undertaken. Using neutral sucrose gradient sedimentation, it was possible to reproducibly separate the protein and DNA components of interphase nucleoids. Under conditions of radiation damage, the DNA and protein components of the nucleoid were shifted to a coincident position in the gradients consistent with the generation of single- and double-stranded DNA scissions. During DNA damage repair, an apparent recruitment of protein to the nucleoid occurred and a rearrangement of the protein sedimentation was observed as the repair of DNA progressed. These data suggest that the protein component of the nucleoid was dynamic under conditions of DNA damage repair.

Animals↗

PubMed1989

Comparison of tumor cell invasion assays: human amnion versus reconstituted basement membrane barriers.

This study compares two well-known tumor cell invasion assays: the human amnion model versus the reconstituted basement membrane (RBM) system in the membrane invasion culture system (MICS). Our purpose was to present the quantification of tumor cell invasion using visual counts and radioactivity assessment in a side-by-side comparison and then to determine reasons for discrepancies in data collection and reporting. Basically, the data showed that: (1) fewer tumor cells invade the amnion membrane compared with the RBM, and substantially more variability exists among the data generated from the amnion assay (probably due to differences in membrane thickness); and (2) the invasive ability of the tumor cells appears to be greater using the radiolabel technique in both the amnion and RBM assay, a portion of which appears to be artifactual. Using the RBM, it was possible to sequentially select several subpopulations of highly invasive tumor cells, which was not possible with the human amnion. The invasive and metastatic potentials of these subpopulations were compared with those of established cell lines (selected in vivo). When analyzed independently, a direct relationship was shown between an increase in invasive ability and an increase in metastatic potential for the sublines selected in vitro and the established lines. However, collectively, it is more difficult to correlate the invasive and metastatic profiles of the sublines versus the established lines, which can probably be attributed to selection factors present during the establishment of the individual cell populations.

Amnion↗

PubMed1989

Impaired nurses: a cost analysis.

Although the literature reflects a continuing concern about impaired practice and its associated costs, no attempt has yet been made to examine those costs in a comprehensive manner. This analysis was undertaken to determine the specific economic impact of impaired nursing practice on the employing agency, the individual nurse, and the state regulatory body. It will provide valuable data for policy development and for planning cost-effective approaches to early recognition and intervention.

Alcoholism↗

PubMed1988

Cytogenetic evidence of gene amplification as a mechanism for tumor cell invasion.

In order to study the process by which human melanoma cells achieve invasion of basement membranes, a modification of the Membrane Invasion Culture System was developed to allow the in vitro collection of human melanoma cell populations that had invaded acellular human amniotic membranes. A significant increase in the number of double-minute chromosomes (DMs) was observed in metaphase nuclei of A375P human melanoma cells which had passed through two amniotic membranes (A375P-2) over that of control cells. Eighteen percent of the first monolayer of A375P-2 cells contained 1-89 DMs/cell, whereas 3-8.3% of the control A375P cells contained 1-10 DMs/cell. There was a rapid loss of DMs in A375P-2 cells as a function of passage number. After 25 days in tissue culture, the incidence of DMs had essentially dropped below the control range. These data indicate that an unstable gene amplification event may be part of the process by which melanoma cells execute invasion through basement membranes.

Animals↗

PubMed1988

Trichloroethylene: a cardiac teratogen in developing chick embryos.

Prior studies have evaluated the teratogenicity of TCE, a contaminant of drinking water. However, none specifically examined effects of TCE on cardiogenesis. The purpose of this study was to determine if TCE is a cardiac teratogen in early embryogenesis in an avian model. Fertile White Leghorn chicken eggs were incubated under standard conditions. At stage 6, 12, 18, or 23, TCE, in concentrations of 5 to 25 microM (2 to 28 micrograms/g body weight) was injected into the air space of the egg (vol = 0.03 ml). Mineral oil and saline served as control solutions. For this double-blinded study, solutions were coded and remained so until all observations were made and recorded. Embryonic hearts (n = 1055) were examined at stage 29, 34, or 44. Gross examination was performed, followed by microdissection. Cardiac malformations were found in 7.3% of TCE-treated hearts, compared to 2.3% of saline controls (p less than 0.01), and 1.5% of mineral oil controls (p less than 0.001). No significant difference in incidence of malformations was found when comparing saline and mineral oil controls. Cardiac defects include septal defects, cor biloculare, conotruncal abnormalities, atrioventricular canal defects, and abnormal cardiac muscle. These data demonstrate that TCE is a cardiac teratogen in an avian model.

Abnormalities, Drug-Induced↗

PubMed1987

Perceptions and attitudes toward nursing impairment.

Perceptions and attitudes toward nursing impairment held by 1,047 registered nurses were examined. Factor analysis of the 32 Likert-type items in an original survey questionnaire revealed an underlying structure of nine dimensions characterizing attitudes toward impairment. Analyses of variance and covariance gave evidence that supervisors were more likely than staff nurses to perceive a need for disciplinary action in responding to cases of nursing impairment; staff nurses were more likely to view impairment as treatable. Significant differences were found among attitudes toward drug abuse, alcohol abuse, and emotional distress as forms of impairment.

Adult↗

PubMed1987

Identification of a specialized extracellular matrix component in Drosophila imaginal discs.

We have generated a monoclonal antibody that recognizes a major component of a specialized extracellular matrix in Drosophila imaginal discs. In mature larvae, antibody binding is observed almost exclusively on imaginal discs. On the basal surface of the thoracic discs, the antigen is localized to particular regions of the epithelium, and ultrastructural studies indicate that the antigen is found in a fibrous network secreted between the cells and the basal lamina. The localized expression indicates that the matrix is not simply related to disc differentiation, as all regions of the columnar disc epithelium are determined to secrete adult cuticle. A correlation of the antigen distribution with known developmental events leads us to propose that the antigen-containing network provides an extensible matrix for the rapid elongation of the disc epithelium during evagination; consistent with this, the antigen is a component of the matrix between the dorsal and ventral surfaces of the evaginated wing pouch. The antigen is very large (greater than 5 X 10(5) Da), can be labeled metabolically with methionine and sulfate, and is digested by chondroitinase ABC; these biochemical characteristics indicate that the antigen is a proteoglycan.

Animals↗

PubMed1987

A simple quantitative assay for studying the invasive potential of high and low human metastatic variants.

This paper presents a more reliable model for studying the extent of tumor cell migration and invasion in vitro. Polycarbonate filters were uniformly coated with a reconstituted basement membrane material and allowed to dry; each filter measured 0.035 mm in thickness when hydrated with media. Subsequently, the membrane-coated filters were suspended in Membrane Invasion Culture System (MICS) chambers, and high (A375M) and low (A375P) metastatic variants of human melanoma cells were seeded onto the membranes and allowed to incubate for 3 days. At the end of this period, cells were examined morphologically, and the invasive cells of both metastatic variants were collected, stained and counted microscopically. The tumor cells could be seen attached to the reconstituted basement membrane, buried within it, and forming colony-like aggregates in the matrix. It was determined that approximately twice as many A375M cells invaded the artificial biological matrix compared with the A375P cells (P less than 0.0005). Substantially more cells from each variant invaded uncoated polycarbonate filters, thus indicating the selective barrier imposed by the Matrigel. The utilization of such a reconstituted matrix for in vitro invasion studies allows one the opportunity to examine tumor cell attachment, migration and invasion of a uniform matrix over an extended period of time.

Humans↗

PubMed1987

Invasive characteristics of neural crest cells in vitro.

An investigation of the invasiveness of avian neural crest cells and neural crest-derived melanocytes through a human amniotic basement membrane (BM) was undertaken. Avian neural tube explants or derived melanocyte populations were seeded directly onto BMs in membrane invasion culture system (MICS) chambers for periods of 24, 48, and 72 h. In 36 experimental trials for each group, neither neural crest nor neural crest-derived melanocytes were observed to have invaded the BMs. In concert with these studies, coculturing of B16F10 murine melanoma cells with avian neural crest-derived melanocytes was performed in MICS chambers. Under these experimental conditions, the neural crest-derived melanocytes were able to successfully invade the BMs and to a greater extent than the B16F10 tumor cells. These data suggest that neural crest cells and neural crest-derived melanocytes do not have the ability to invade the BM alone; however, they can be induced to be invasive when cocultured in the presence of B16F10 cells. Alternatively, the B16F10 cells may create weaknesses within the BM that facilitate migration of the pigmented crest cells.

Amnion↗