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Biomedical subjects

M S Rieber

Publications and source records attributed to M S Rieber.

At least 19 recordsLinked to original sources
PubMed2000

Lower cyclin H and cyclin-dependent kinase-activating kinase activity in cell cycle arrest induced by lack of adhesion to substratum.

Knowledge about adhesion checkpoints is important to counteract dissemination of cells from solid tumors. Lack of anchorage in adherent cells is associated with growth arrest and inhibition of cyclin-dependent kinases (cdks) required to drive cell cycle progression. Because cyclin-cdk complex activation requires CDK-activating kinase comprising cdk7 and cyclin H, we now investigated their relationship to decreased proliferation by lack of cell spreading. This report shows that either UV irradiation on an adhesive substrate or culture on a nonadhesive substrate produced K1735 melanoma growth arrest. Inhibition of proliferation by UV primarily induced the cdk inhibitor p21WAF1 without a significant effect on cyclin H and cdk7. In contrast, lack of adhesion to substratum decreased cyclin H but not cdk7 with accumulation of a slower migrating, presumably unphosphorylated cdk4 isoform. These results were paralleled by decreased cdk7-mediated phosphorylation of GST-cdk2 and lower activation of a baculovirus-derived cdc2-cyclin B kinase complex. This is the first report showing that cyclin H-mediated down-regulation of cdk-activating kinase activity is involved in growth arrest induced by lack of anchorage.

Cell Adhesion↗
PubMed2000

Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells.

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).

3T3 Cells↗
PubMed1999

Tumor suppression without differentiation or apoptosis by antisense cyclin D1 gene transfer in K1735 melanoma involves induction of p53, p21WAF1 and superoxide dismutases.

In mammalian cells, terminal differentiation is mutually exclusive with proliferation. However, resistance to differentiation-inducing therapy requires alternative strategies to control poorly responsive tumors. We now show that retroviral transfer of the antisense cyclin D1 gene to differentiation-refractory K1735 melanoma leads to loss of in vivo tumorigenicity, shortened replicative ability, induction of the tumor suppressor p53 protein and of the cdk-inhibitor p21WAF1, increased beta-galactosidase pH 6.0 activity, and elevation in the ratio of superoxide dismutases to peroxidases, all properties associated with replicative senescence. However, pigmentation and tyrosinase expression, characteristic of differentiated melanocytic cells or apoptosis-associated PARP cleavage, were not increased by antisense cyclin D1 transduction. Our data suggests that targetting cyclin D1 inhibition suppresses melanoma tumorigenicity by promoting a cytostatic differentiation-independent pathway, mediated by activation of p53 and anti-oxidant functions.

Animals↗
PubMed1997

Sensitization to DNA damage by okadaic acid or bromodeoxyuridine involves unequal effects on melanoma cell adhesion and differentiation.

Because melanoma tumors originate partly from excessive UV exposure but become relatively resistant to radiation, we have now compared the effects of okadaic acid, a phosphatase inhibitor, with that of the thymidine analog bromodeoxyuridine as sensitizers of DNA damage in B16 melanoma. We now show that 25 nM okadaic acid promotes DNA fragmentation in B16 melanoma, increasing cell detachment as well as pigmentation, a characteristic of melanocytic cell differentiation. At lower levels, okadaic acid synergizes with UV exposure to increase DNA fragmentation. Although bromodeoxyuridine also caused DNA damage, it did not increase pigmentation and it suppressed cell detachment. Okadaic acid was also more effective in promoting DNA laddering in growing versus quiescent melanocytes. Because DNA damaging effects of okadaic acid are mediated by different pathways from those used by nucleoside analogs, like bromodeoxyuridine, we propose their concurrent effect with radiation as sensitizers to DNA damage.

Animals↗
PubMed1994

UV radiation induces DNA fragmentation and cell death in B16 melanoma sensitized by bromodeoxyuridine: impaired c-jun induction and defective tyrosine phosphorylation signalling.

The relevance of tyrosine phosphorylation and c-jun protooncogene expression to radiation sensitization was investigated in B16 melanoma. These cells are sensitized by bromodeoxyuridine (BrdU), a thymidine analog, showing extensive DNA fragmentation reminiscent of apoptosis, after UV radiation. UV-irradiated unsensitized cells did not reveal DNA fragmentation but showed increased expression of c-jun and greater protein tyrosine phosphorylation in response to sodium vanadate, an inhibitor of tyrosine phosphatases. However, these responses were inhibited in UV-irradiated BrdU-treated cells. Our data suggest that the bromodeoxyuridine-induced sensitization to radiation can lead to DNA fragmentation and cell death, partly because of a defective tyrosine kinase signalling and an impaired c-jun expression, both of which appear important for cell survival in response to UV radiation.

Animals↗
PubMed1994

Cyclin-dependent kinase 2 and cyclin A interaction with E2F are targets for tyrosine induction of B16 melanoma terminal differentiation.

L-Tyrosine promotes a dramatic increase in melanogenesis and an apparent replicative senescence in B16 melanoma (M. Strasberg-Rieber and M. Rieber, Cancer Res., 53:2469-2471, 1993). Since cyclins are implicated in controlling cell proliferation and differentiation, we have now investigated their relationship to melanocytic growth arrest and pigmentation. In B16 melanoma cells enriched in G1 by serum starvation or synchronized in late G1/early S phase by exposure to hydroxyurea, L-tyrosine overrides mitogenic signals and induces terminal differentiation without cytotoxicity. This correlates with a decrease in cyclin A and cyclin E-dependent kinase 2 activity and with an altered interaction of cyclin A with the transcription factor E2F. This activity involves a lower level of the catalytic cdK2 kinase protein without a concomitant decrease in cyclin A or cyclin E. Upon addition of serum or removal of hydroxyurea, cells resume cell cycle progression and the ability to form tumors in vivo, but these properties are irreversibly inhibited in tyrosine-treated cells. Our data suggest that targeted inactivation of cdK2 with specific inducers of differentiation favors reacquisition of tumor growth control.

Animals↗
PubMed1993

Specific tyrosinases associated with melanoma replicative senescence and melanogenesis.

Replicative senescence occurs in normal cells, in contrast to their malignant counterparts which are generally immortal in vitro. We now show that induction of melanogenesis in subconfluent B16 melanoma cells deprived of growth factors can lead to irreversible growth arrest but continued cell viability, concurrent with the expression of specific glycosylated high molecular weight tyrosinases. These tyrosinase activities identify withdrawal from the cell cycle since they were not detected in reversibly arrested quiescent melanocytes, serum-deprived melanoma, or apoptotic melanoma. Our data suggest that different tyrosinases can distinguish cycling and noncycling cells of melanocytic origin and also imply that replicative senescence can be restored in some tumor cells when induced to terminal differentiation in the absence of growth-promoting agents.

Animals↗
PubMed1993

Early inhibition of protein phosphatases preferentially blocks phorbol ester-stimulated mitogenic signalling in melanocytes: increase in specific tyrosine phosphoproteins.

Inhibition of protein phosphatases has been suggested as an alternative mechanism of tumor promotion (H. Fujiki, Mol. Carcinog. 5:91, 1992). We have now used early melanocyte passages dependent on phorbol esters and serum for growth and later passages with partial phorbol ester independence, to investigate the role of protein phosphatases on melanocyte DNA synthesis. Neither okadaic acid, an inhibitor of ser/thr protein phosphatases, nor vanadate, an inhibitor of tyrosine phosphatases, can stimulate basal or serum-stimulated mitogenesis in contrast to phorbol esters. Moreover, both phosphatase inhibitors are able to suppress serum and phorbol ester-stimulated mitogenesis, if added within 4 hours of growth activation. Inhibition of mitogenesis by either inhibitor correlated with an early increase in a common set of tyrosine phosphoproteins, which included a major 33 Kd species. Our data suggest that protein phosphatase inhibitors are growth suppressors and antagonize phorbol ester effects in cells of melanocytic origin, implying an early requirement for protein phosphatase activity during mitogenic signalling in these cells.

Animals↗
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↗