PubMed Health⌕ Search

Biomedical subjects

M S Rieber

Publications and source records attributed to M S Rieber.

At least 19 recordsLinked to original sources
PubMed1992

Accessibility to DNA in carcinoma chromatin is promoted by nanomolar okadaic acid: effect on AT-rich DNA binding proteins.

Differential accessibility to DNA in tumor cell chromatin is important to growth, differentiation apoptosis, and the targeting of DNA modifying drugs. We now show that endonuclease accessibility to DNA in the nuclei of A431 human carcinoma cells is increased within 90 min by nontoxic nanomolar levels of okadaic acid, known to inhibit protein phosphatase 2A. This genomic hypersensitivity was partly enhanced by joint treatment with epidermal growth factor and okadaic acid but did not appear without the latter. Nuclei with greater DNA susceptibility showed a decrease in M(r) 80,000 DNA binding protein doublet specific for dAT-rich sequences concurrent with the "apparent" hyperphosphorylation of a M(r) 70,000 nuclear matrix protein. We propose that some of the tumor-promoting effects of okadaic acid may be partly associated with its ability to promote genomic susceptibility.

Adenine↗
PubMed1992

Extracellular RGD-binding proteins modulate cell adhesion.

Cell/fibronectin adhesion in extracellular matrices is partly mediated by integrin receptor recognition of RGD domains in fibronectin. Since blood contains significant levels of soluble fibronectin we have now investigated the occurrence of extracellular RGD-binding proteins. Attachment assays indicate that extracellular RGD-binding proteins prevent cell adhesion, suggesting their potential as novel secreted modulators of blood-borne cell adhesive interactions. These extracellular RGD-binding proteins also showed electrophoretic changes with reducing agents, suggestive of intrachain disulphide bonds, like those found in RGD-binding integrins. However, they differed from the latter in their electrophoretic profile, which was greatly dependent on the presence of protease inhibitors. Plasma from tumor-bearing mice showed a greater proportion of fast-migrating RGD-binding species under reducing condition compared to similarly treated normal plasma, suggesting that tumor development is associated with a partial degradation of extracellular RGD-binding proteins.

Animals↗
PubMed1991

Differential genomic susceptibility in malignancy correlates with changes in ATATAT DNA-binding proteins.

Accesibility to DNA in the nucleus is important for the regulation of gene expression and for the effect of DNA-modifying drugs. We have now studied differential genome susceptibility in normal melanocytes and the corresponding malignant melanoma. DNA hypersensitivity assays revealed a markedly lesser degradation in melanoma nuclei compared to that in melanocytes. Cross-linking of DNA to nuclear proteins by ultraviolet light showed a cell-type dependent inverse correlation of genomic susceptibility with binding of (dA.dT) (dA.dT) sequences, compared to that shown with (dG.dC) (dG.dC), regardless of methylation in cytosines. Exposure to cholera toxin partly reversed genomic susceptibility and increased DNA/protein cross-linking in melanocytes. In contrast, melanoma cells showed decreased DNA/protein interactions and greater genome susceptibility after exposure to cholera toxin or okadaic acid. Our data suggest that a molecular mechanism for differential genome exposure in cancer cells involves a modified expression of sequence-specific DNA-binding proteins.

Animals↗
PubMed1990

Cell adhesion regulates melanoma specific differentiation and interactions with the 3' region of the tyrosinase gene.

Decreased attachment to substratum has now been found to increase melanosome formation and cell-cell interaction in B16 melanoma. Since melanosome formation involves tyrosinase gene expression, we assayed for differential RNA expression by hybridization with probes from the distal ends of this gene, detecting unequal reactivity only with the 3' end probe. The same DNA showed binding of 2 nuclear proteins of 50 and 60 kd in unanchored cells, in contrast with a decreased binding of the 60 kd species, in nuclear extracts from attached cells. No comparable differences were detected with a gamma-actin DNA of identical length, suggesting that the changes observed are sequence-specific. Our studies suggest that the adhesion-mediated modulation of pigmentation in B16 melanoma correlates with differential macromolecular interactions with the 3' end of the tyrosinase gene.

Animals↗
PubMed1990

Tumor hypersensitive DNA is enriched in c-myc sequences and reacts differentially with normal and malignant genomic DNA.

We now show that exposure of B16 melanoma cells to bromodeoxyuridine increases cell-substratum interactions concurrent with an increase in genome susceptibility to nucleases. Hypersensitive DNA was isolated after mild nicking of nuclei with DNase I followed by repair with DNA polymerase I in the presence of biotin-19-SS-dUTP and affinity chromatography on streptavidin-agarose. Dot blot studies showed that the hypersensitive DNA is enriched in c-myc sequences compared to total tumor genomic DNA, and hybridizes preferentially to the latter, compared to normal genomic DNA, particularly when prepared from BrdU-treated cells. Since hypersensitive DNA can hybridize with multiple Alu sequences in the genome, we postulate that one of the mechanisms for its differential reactivity may be by recognition of an unequal number of Alu repeats in normal and tumor genomic DNA.

Animals↗
PubMed1989

Differential response of adherent and unanchored melanoma cells to bromodeoxyuridine evidenced by specific lectin-binding protein changes.

The possible differential response of adherent and nonadherent cells of the same tumor type to pyrimidine analogues has been investigated. We show that bromodeoxyuridine (BUdR) increases interactions of attached cells with their substrate without markedly affecting the cell adhesion properties of the same cells when these are not anchored. However, evidence for an adhesion-independent response of both cell types to BUdR has been obtained with lectin binding assays using 125I-labelled Lens culinaris agglutinin (LCA). This revealed a greatly increased binding of LCA to a large glycoconjugate in all cultures exposed to the halogenated pyrimidine. Attachment-dependent effects of BUdR were manifested in flattened cells by a greater LCA-binding to a 240-kDa protein and by increased interaction of 125I-labelled wheat-germ agglutinin (WGA) with a 200-kDa protein and a large glycoconjugate sharply defined in electrophoresis. Although both tumor cell aggregates and anchored cells exhibit detectable responses to pyrimidine analogues such as BUdR, the corresponding effects are thus manifested unequally in cells with different adhesion properties.

Animals↗
PubMed1989

DNA on membrane receptors: a target for monoclonal anti-DNA antibody induced by a nucleoprotein shed in systemic lupus erythematosus.

Antibodies to double stranded (ds) DNA correlate with clinical evolution in systemic lupus erythematosus (SLE) although little is known about the immunogen and target for these antibodies, since ds DNA is poorly immunogenic. We now show that monoclonal anti DNA antibodies similar to those detected in human SLE can be produced by immunization of genetically non-autoimmune mice with a human circulating DNA-protein complex increased in the circulation of SLE patients. One such monoclonal antibody showed antinuclear reactivity, interacting with a 74 kd DNA-binding membrane protein, in reactions prevented by absorption with ds DNA cellulose. Our data suggests that anti ds DNA antibody reactions in SLE may be triggered by circulating nucleoproteins and directed toward membrane receptors capable of interacting with extracellular DNA.

Antibodies, Antinuclear↗
PubMed1989

Changes in cytoskeleton-associated molecules in cells with different degrees of proliferation and metastatic ability.

An antiserum prepared against the Triton-insoluble cytoskeleton of in vivo grown B16 melanoma tumor has been used to analyze the differential expression of cytoskeleton-associated molecules in cells with different degrees of proliferation and metastatic ability. This antiserum identified a major 97 kd molecule associated with the cytoskeletal fraction in B16 melanoma tumors, mouse embryo and in proliferating lymphocytes, with no reactivity with the 97 kd species in non proliferating lymphocytes. The antiserum revealed immune reactivity with a 180 kd Triton-insoluble species in normal adult mouse liver and kidney. A comparison of tumor cells with differing metastatic ability also showed a minor 180 kd component in poorly metastatic cells which appeared decreased and partly degraded in its more invasive counterpart. The differential recognition of a 97 kd species in resting and proliferating lymphocytes, as well as the different cleavage of a 180 kd species in tumor cells of differing metastatic ability, implies a role for these molecules in cell proliferation. The fact that these differences can be detected with an antiserum to tumor cell cytoskeleton suggests that this Triton-insoluble fraction may be a good source of molecules involved in growth control.

Animals↗
PubMed1988

Decrease in tumor-cell attachment and in a 140-kDa fibronectin receptor correlate with greater expression of multiple 34-kDa surface proteins and cytoplasmic 54-kDa components.

B16 melanoma cells attach to matrix-bound fibronectin but fail to adhere to albumin-coated surfaces supplemented with soluble fibronectin. Attachment to substratum is also decreased in the presence of an adhesion-disrupting antibody, or when cells are seeded on substrates poorly adhesive for these cells, such as collagen gels. We have now investigated some of the more general adhesion-related alterations that occur between flattened and poorly attached cells. Immune blots of octylglucoside extracts with the adhesion-disrupting IgG revealed a 140-kDa component in flattened cells, in contrast to the increased detection of a 54-kDa species in a comparable assay with rounded cells. Surface iodination also showed a decreased external exposure of a 140-kDa fibronectin binding species and an increased labelling in multiple 34-kDa protein species, in cells with decreased attachment to substratum. Analysis of 35S-methionine-labelled cell aggregates cultured on collagen gels also revealed a decrease in the 140-kDa region and a greater labelling of multiple 54-kDa components, compared to the same cells flattened on fibronectin. A change in 54- and 34-kDa species was also seen in matrix-associated components of rounded cells that failed to attach with soluble fibronectin. Since the 34-kDa species increase in poorly adherent cells is mainly detected by iodination, and the 54-kDa species increase in the same cells is partly associated with the corresponding detergent-insoluble matrices, we propose that these 2 novel proteins may relate to cell rounding, through a transmembrane modulation involving both surface membrane and cytoskeletal structures.

Animals↗
PubMed1988

Human repetitive and unique sequences coexist in a large circulating DNA species found in cryoprecipitates from SLE patients.

Cryoprecipitates from systemic lupus erythematosus (SLE) patients with high levels of anti DNA antibodies show a sharply migrating large circulating DNA species of about 17-20 kb (M. Rieber et al., Clin. exp. Immunol. (1986) 66, 61). We have now used Southern blot analysis of circulating DNA from different individuals to analyse the relative cross-hybridization of circulating DNA from different individuals, as well as their homology with genomic DNA from different species. Molecular hybridization showed significant homology of the various circulating DNA examined, only with human genomic DNA, but limited cross-reactivity among circulating DNA from different individuals. This suggests that the circulating DNA is composed of sequences repeated in human genomic DNA and by specific sequences unique to circulating DNA from some individuals. Our data suggests the possibility of using probes derived from the specific sequences now reported in the circulating DNA, in gene typing and in the analysis of susceptibility to disease.

Base Sequence↗
PubMed1987

Relationship of a Mr 140 fibronectin receptor and other adhesion-related glycoproteins to tumor cell-cell interaction.

Primary melanocytes attach poorly to collagen type IV and laminin, in contrast to their firm attachment to collagen type I/III and fibronectin [Gilchrest et al., In vitro (Rockville), 21: 114-120, 1985]. We have now found that metastatic B16 melanoma cells attach well to collagen type IV, laminin, vitronectin, and fibronectin but show a selective defect in attachment and cell aggregation on native collagen type I. Both flattened and aggregated melanoma cells revealed the presence of a Mr 120,000 surface-iodinated species with affinity for a matrix containing the hexapeptide (glycylarginylglycylaspartylserylproline) which includes the fibronectin cell attachment sequence, but only flattened cells showed significant exposure of a Mr 140,000 iodinated component with affinity for a large cell attachment-promoting fibronectin polypeptide. Decrease of the Mr 140,000 fibronectin-binding external protein in the collagen-cultured melanoma cells was also associated with an inability to respond to the cell attachment activity of fibronectin, laminin, or vitronectin added to the collagen gels. Metabolic labeling with [3H]glucosamine and electrophoretic analysis showed that lack of attachment and cell aggregation was associated with an increase in high molecular weight wheat germ agglutinin-binding glycoconjugates and an increase in Mr 55,000 concanavalin A-binding glycoprotein species. Our data suggest that: (a) melanoma cell attachment requires the expression of the Mr 140,000 fibronectin receptor which appears to be down regulated in cells exposed to poorly adhesive substrates; (b) expression of the Mr 120,000 iodinated species with affinity for the fibronectin attachment sequence (arginylglycylaspartic acid) may be necessary but not sufficient for firm cell-substratum interactions; (c) increased tumor cell-cell interaction may involve a decreased attachment to substrate and the expression of different glycoproteins which may modulate cell-cell association.

Amino Acid Sequence↗
PubMed1986

120 and 80 kd detergent-insoluble glycoproteins as markers of differentiation and adhesion in B16 melanoma.

Surface glycoproteins correlating with cell adhesion and differentiation have now been studied in pigmented and amelanotic B16 melanoma cell monolayers and aggregates, labelled with 3H-glucosamine. Solubilization with the non-ionic detergent octyl glucoside revealed mostly adhesion-related changes, which involved a decrease in 140 and 110 kd glycoproteins in both pigmented and amelanotic cell aggregates. Nevertheless, differentiation-regulated changes were preferentially evident in the remaining detergent-insoluble glycoproteins. Pigmented cell monolayers showed a preferential increase in glycoprotein species of about 120 kd and 80 kd, which were not detected in amelanotic monolayers or in pigmented or amelanotic cell aggregates. Our findings suggest the relevance of these cytoskeleton associated glycoproteins as markers of differentiation and adhesion in melanoma cells.

Cell Adhesion↗
PubMed1986

Substrate-dependent effect of epidermal growth factor on intercellular adhesion and synthesis of triton-insoluble proteins in human carcinoma A431 cells.

Human epidermoid carcinoma A431 cells attach more rapidly to collagen type-I and -IV substrates than to surfaces coated with laminin or fibronectin. The diminished intercellular interaction and rounding up manifested when these cells are exposed to epidermal growth factor (EGF) in tissue culture plastic or collagen films is not shown when the assay is performed on 3-dimensional collagen. In the latter substrate, cells exposed to EGF reveal greater cell cohesion with interdigitations and desmosomal junctions, compared to the limited intercellular interaction detected in similarly treated cells assayed in tissue culture substrate. Although the protein synthesis inhibitor, cycloheximide, did not prevent these EGF-mediated changes, metabolic labelling indicated a substrate-dependent effect of EGF on the synthesis of proteins associated with the Triton-insoluble cytoskeletal matrix. The involvement of cytoskeleton components in the EGF effects on intercellular adhesion was shown by its susceptibility to cytochalasin B, known to disorganize actin-containing microfilaments. Some of the mechanisms of epithelial morphogenesis and the influence of extracellular matrix components on cell-receptor/growth-factor interactions may now be suitably analyzed by examining the EGF effects on A431 cells grown on different substrata.

Carcinoma, Squamous Cell↗
PubMed1986

Novel DNA-protein complex and a large DNA in SLE cryoprecipitates.

Agarose gel electrophoresis of cryoprecipitates from systemic lupus erythematosus (SLE) patients revealed the presence of a slowly migrating DNAse I-sensitive DNA species at the top of the gel. Upon deproteinization, electrophoretic migration was modified favouring the migration of a 17.5 kb DNA fragment. Mixing experiments adding human serum or plasma to a lambda phage DNA digest revealed a DNA-protein interaction shown by an accumulation of high mol. wt polynucleotide at the top of the gels, and a slowed migration of the DNA bands. No comparable effect was observed when serum albumin was added to the lambda DNA digest. Dot hybridization analysis showed preferential reactivity of the 17.5 kb DNA to human DNA, implying its human origin. Our data suggests that most of this high molecular weight DNA exists as a DNA-2 protein complex. Our mixing experiments also suggest the occurrence of an excess of free DNA antibodies. We propose that the DNA-protein association may play a role in the stabilization and immunogenicity of the nucleoprotein complex.

Antigen-Antibody Complex↗
PubMed1985

Improved detection of labile cell-surface components with zinc chloride-aprotinin: demonstration of glycoprotein differences in K-1735 metastatic melanoma variants.

Metabolic labelling of K-1735 melanoma variants with 3H-glucosamine and cell harvesting with the commonly used protease inhibitor phenylmethylsulfonylfluoride revealed a Triton-insoluble fibronection-like 230 kd component in poorly metastatic cells. This component was not evident in highly metastatic cells. Significantly improved surface labelling and detection of the 230 kd glycoprotein in the highly metastatic variant was achieved by zinc chloride-aprotinin treatment of cells prior to harvesting. This procedure also revealed an increase in a trypsin-sensitive glycoprotein of higher molecular weight in the Triton-insoluble fraction of the highly metastatic cell variant. Glycoprotein labelling in this fraction showed an electrophoretic pattern strongly resembling that reported by others for the high-molecular-weight human melanoma-associated glycoprotein complex. The differential detection of the high-molecular-weight glycoprotein species in melanoma variants with differing metastatic abilities in an animal model provides a means of studying their possible relevance to metastatic melanoma. Our data also suggest that zinc chloride-aprotinin can be used to improve the detection of labile cell-surface components.

Animals↗
PubMed1984

Relationship of a novel extracellular matrix glycoprotein to cell detachment in highly metastatic B16 melanoma: modulating effect of bromodeoxyuridine.

Growth of highly invasive B16 melanoma BL6 cells with bromodeoxyuridine (BUdR) decreases in vitro cell detachment and modulates extrapulmonary growth in vivo. We now show: (1) The presence of an 80 kd glycoprotein in the Triton-insoluble matrix of control BL6 cells but not in the corresponding fractions from BUdR-treated BL6 cells and poorly metastatic F1r cells. (2) The matrix fractions from the two last mentioned cells reveal Triton-insoluble glycoproteins of about 55-58 kd. (3) Mild trypsin treatment of intact cells before matrix preparation leads to the preferential disappearance of the 80 kd component from control BL6 matrix, suggesting its extracellular localization. (4) Prevention of Triton-mediated BL6 matrix detachment by zinc chloride pretreatment, and analysis of different BL6 clones with significant metastatic behavior, also revealed the presence of 80-90 kd matrix-associated glycoproteins in control but not in corresponding BUdR-grown cultures. Since BUdR decreases cell detachment, extrapulmonary metastasis and the levels of the 80-90 kd Triton-insoluble glycoprotein species in metastatic B16 melanoma, and this matrix component is also decreased in poorly metastatic F1r cells, we propose an involvement of this glycoconjugate in tumor cell detachment and metastatic behavior.

Animals↗
PubMed1983

Epidermal growth factor promoted changes in the triton-insoluble cytoskeletal matrix from human epidermal carcinoma cells: effect of bromodeoxyuridine.

We have used monolayers of bromodeoxyuridine (BrdU)-grown and control human epidermoid carcinoma (A431) cells to investigate the polypeptide changes resulting when cells are rounded by epidermal growth factor (EGF). Whereas no significant change was detected in Triton-soluble components, the urea-solubilized matrix fraction revealed greater levels of a 20 kd species in cells exposed to EGF, compared with the same cells not exposed to the growth factor. The corresponding urea fraction from BrdU-grown cells showed decreased levels of the 20 kd species as a result of exposure to EGF. Further evidence for a differential effect of EGF resulting from growth of the cells with the pyrimidine analog was observed in the matrix fraction soluble in SDS, which revealed a decrease in a 20 kd species resulting from exposure of control cells to EGF, with no comparable effect in BrdU-grown cells. Our results suggest that EGF induces a change in the properties of matrix-associated components of low molecular weight in an effect which appears to be modified by prior growth of cells with BrdU.

Bromodeoxyuridine↗