Biomedical subjects

R Radinsky

Publications and source records attributed to R Radinsky.

At least 37 records · Page 2Linked to original sources

PubMed1994

Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells.

BACKGROUND: The major cause of death from cancer is metastases that are resistant to conventional therapies. The resistance of metastatic tumor cells to chemotherapy can be caused by their intrinsic properties, such as increased expression of the mdr genes. PURPOSE: The purpose of our present study was to determine some of the mechanisms by which the organ microenvironment influences the response of tumor cells to chemotherapy. METHODS: Murine CT-26 colon cancer cells growing in continuous culture (parental cells) were harvested and injected subcutaneously into the lateral flank (to produce subcutaneous tumors) or the lateral tail vein (to produce experimental lung metastases) of 10 8-week-old syngeneic male BALB/c mice. Seven days after tumor-cell injection, the mice were given intravenous injections of either doxorubicin (10 mg/kg) or 0.9% NaCl (controls). This in vivo injection was repeated 7 days later. Mice with subcutaneous tumors and lung metastases were killed by cervical dislocation on day 21, and tumor samples from control mice were harvested and adapted to culture. The sensitivity of the cultured cells to doxorubicin and fluorouracil (5-FU) was determined at multiple time points. Levels of mdr-1 DNA were measured by slot-blot and Southern-blot analyses. mdr mRNA expression levels were measured by Northern-blot analysis using mdr-1- and mdr-3-specific hybridization probes, and P-glycoprotein level was determined by fluorescence-activated cell sorting using different monoclonal antibodies. RESULTS: Treatment with doxorubicin produced 80% growth inhibition of CT-26 subcutaneous tumors but had little effect on the number (and size) of experimental lung metastases. Collectively, the results suggest that the multidrug-resistant phenotype developed in CT-26 cells growing in the lung environment. Cultures established from lung metastases were initially resistant to doxorubicin (but not to 5-FU) and showed elevated expression of mdr-1 mRNA transcripts and P-glycoprotein. This resistance could be overcome by verapamil and disappeared after 21 days in culture. No mdr gene amplification was detected. The expression level of mdr-specific mRNA (predominance of mdr-1) and P-glycoprotein was directly associated with resistance to doxorubicin. CONCLUSIONS: Results of this study have demonstrated that the in vivo sensitivity of murine CT-26 colon carcinoma cells to doxorubicin depends on the organ environment. The organ environment can influence the P-glycoprotein-mediated multidrug-resistant phenotype in tumor cells, and the increased expression of P-glycoprotein is transient; once removed from the environment (lung), the cell's resistance reverts to that of the sensitive parent cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice.

We correlated the steady state transcription and protein secretion of interleukin 8 (IL-8) in 13 different human melanoma cell lines with their ability to grow and produce metastasis in nude mice. Highly metastatic cells expressed higher steady state levels of IL-8 mRNA transcripts than did low metastatic cells. In situ mRNA hybridization analyses confirmed the pattern of mRNA expression on a cellular level. Increased mRNA expression directly correlated with secretion of IL-8 protein as determined by enzyme-linked immunosorbent assay. Recombinant IL-8 stimulated the proliferation of low metastatic A375P cells in a dose-dependent manner, a stimulation that was abrogated by the use of a polyclonal antibody against IL-8. The data suggest that IL-8 can be an autocrine growth factor for human melanoma cells and that IL-8 is involved in melanoma metastasis.

Animals↗

PubMed1994

Anti-Fas on nonhematopoietic tumors: levels of Fas/APO-1 and bcl-2 are not predictive of biological responsiveness.

Fas/APO-1 is a cell surface protein known to trigger apoptosis in a variety of cell types upon specific antibody binding. Although extensively studied on normal and malignant hematopoietic cells, little is known about Fas/APO-1 on nonhematopoietic cells. In the study presented here, we have examined Fas/APO-1 expression and function on 11 human tumors of nonhematopoietic origin. By flow cytometric analysis, Fas/APO-1 was expressed on 10 of the 11 tumors at levels comparable to those previously reported for lymphoid cells sensitive to the cytolytic effects of anti-Fas. Despite abundant cell surface expression, only 4 of the 10 Fas-positive tumors were sensitive to the cell-killing effects of anti-Fas. Moreover, anti-Fas enhanced the growth of 2 of 10 Fas-positive tumors. Additional studies using cycloheximide demonstrated that de novo protein synthesis was required for anti-Fas-triggered growth stimulation and, at least in one case, was responsible for the resistance to antibody-induced apoptosis. The biological effects initiated by anti-Fas engagement, however, were not correlated with endogenous bcl-2 expression. This report documents that: (a) Fas/APO-1 is widely expressed on cultured nonhematopoietic tumors; (b) the inherent susceptibility to anti-Fas-induced apoptosis is not correlated with expression of the Fas/APO-1 protein; (c) Fas/APO-1 engagement can result in growth enhancement; and (d) protective/growth-promoting proteins other than bcl-2 may contribute to the diverse spectrum of biological effects induced by anti-Fas engagement of the Fas/APO-1 protein.

Antigens, Neoplasm↗

PubMed1994

Modulation of tumor cell response to chemotherapy by the organ environment.

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression of mdr1 mRNA. However, the organ-specific mechanism for upregulating mdr1 and P-glycoprotein has yet to be elucidated.

Animals↗

PubMed1994

Terminal differentiation and apoptosis in experimental lung metastases of human osteogenic sarcoma cells by wild type p53.

Human SAOS-2 osteogenic sarcoma cells are not metastatic in nude mice and do not express p53. We have selected a variant line (SAOS-LM2) that is tumorigenic and metastatic in nude mice. These cells were transfected with the p53 wild-type (p53wt) or mutated (p53mut 143A) gene, whose expression was verified by reverse transcriptase PCR, cDNA sequencing, and protein immunoprecipitation. Cells were injected i.v. into nude mice, and 4 months later, the mice were necropsied. All cell lines produced a similar number of visible lung metastases, albeit of different sizes. Microscopic examination revealed that most lung metastases in mice injected with p53wt cells (but not p53mut 143A or control cells) consisted of osteoid matrix and apoptotic cells. Expression of either p53wt or p53mut 143A verified the origin of the metastases. These data suggest that transfection of SAOS-LM2 cells with p53wt is associated with in vivo induction of terminal differentiation and apoptosis that inhibit progressive growth of metastases.

Animals↗

PubMed1993

A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas.

We have developed a rapid colorimetric in situ mRNA hybridization procedure to analyze epidermal growth factor receptor (EGF-R) transcripts in paraffin-embedded surgical specimens of human colon carcinomas. This technique is based on the use of 24-base oligonucleotide probes labeled with 6 biotin molecules at the 3' end. mRNA integrity was verified using a hyperbiotinylated 30-residue-long deoxythymidylate oligonucleotide probe, and the specificity of the reaction was confirmed by using labeled EGF-R-specific sense and antisense probes. Avidin alkaline phosphatase detection and the capillary technology used in the Microprobe System allowed for completion of the procedure in under 5 h. The human A431 epidermoid carcinoma cells growing in culture and fixed with formalin as well as paraffin-embedded sections of this tumor growing s.c. in nude mice served as positive controls. In situ hybridization with antisense EGF-R oligonucleotide probes directly correlated with EGF-R mRNA and protein levels observed by Northern blot and immunohistochemistry, respectively. In situ hybridization of paraffin-embedded sections of primary human colon carcinoma and metastases from liver and lymph node revealed cell-specific staining with EGF-R antisense oligonucleotide probes that correlated directly with Northern blot and immunohistochemistry analyses. Since this rapid and sensitive in situ mRNA hybridization technique can be used in properly preserved paraffin-embedded tissue, it allows for retrospective analyses of human tumor specimens using archival material.

Animals↗

PubMed1993

Paracrine growth regulation of human colon carcinoma organ-specific metastasis.

The process of cancer metastasis consists of a series of steps resulting in the spread of malignant cells beyond the site of origin and formation of metastases in distant organs. The outcome of this nonrandom process depends, in part, on the interaction of unique tumor cells with a compatible organ microenvironment. The molecular basis of the intrinsic capacity of distinct malignant cells to colonize specific organs and the degree to which host factors influence this process is under intense investigation. Biological analyses of human colon carcinoma tumors obtained from surgical specimens and implanted orthotopically into athymic nude mice revealed that these tumors are heterogeneous for metastatic properties. Moreover, recent evidence using this model suggest that whereas nonmetastatic and highly metastatic cells can grow at local sites, growth in the secondary liver-specific site was associated only with highly metastatic HCC cells. These cells also respond to mitogenic signals produced by damaged normal tissues, suggesting that physiological signals can be utilized by neoplastic cells. Molecular characterization of highly metastatic HCC cells selected in the nude mouse model as well as in situ mRNA hybridization of archival HCC surgical specimens for specific growth factor receptors correlated with the malignant cell's ability to respond to organ-specific growth factors. This article will focus on biological and molecular evidence supporting the hypothesis that organ-derived, paracrine growth factors regulate the site-specific growth of receptive malignant cells that possess the appropriate receptors.

Animals↗

PubMed1993

The immunogenic properties of drug-resistant murine tumor cells do not correlate with expression of the MDR phenotype.

Alterations in the immunogenic properties of tumor cells frequently accompany selection for multiple-drug-resistant (MDR) variants. Therefore, studies were performed to examine the hypothesis that overexpression of membrane P-glycoprotein, commonly observed in MDR tumor cells, is associated with enhanced immunogenic properties. Immunogenicity was determined by (a) the ability of drug-sensitive parental UV2237M fibrosarcoma cells and drug-resistant UV2237M variant cells to immunize normal mice against rechallenge with parental tumor cells and (b) the ability of normal syngeneic mice to reject cell inocula that caused progressive tumor growth in immunocompromised mice. Variant UV2237M cell lines included subpopulations selected for a six- to ten-fold increase in mRNA for P-glycoprotein and expression of the MDR phenotype (resistance to doxorubicin) and cells sensitive to doxorubicin (and no expression of MDR properties) but resistant to ouabain. All UV2237M drug-resistant cells were highly immunogenic in immunocompetent mice, regardless of their MDR phenotype. Additional studies showed that CT-26 murine adenocarcinoma cells, sensitive or resistant to doxorubicin (expressing high levels of P-glycoprotein), injected into normal syngeneic Balb/c mice produced rapidly growing tumors. The data do not demonstrate a correlation between the immunogenic properties of drug-resistant tumor cells and the expression of P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

A rapid colorimetric in situ mRNA hybridization technique using hyperbiotinylated oligonucleotide probes for analysis of mdr1 in mouse colon carcinoma cells.

We describe the development of a rapid colorimetric in situ hybridization technique utilizing oligonucleotide probes labeled with six biotin molecules at the 3' end to detect mdr1 in mouse colon cancer cells growing in culture and in vivo. mRNA integrity was verified by the use of a multibiotinylated poly d(T) oligonucleotide, and the specificity of the reaction was confirmed by use of labeled sense and anti-sense probes in serial cryostat sections and cultured cells. The multiple biotin label produced a strong signal after a short hybridization time. Avidin-alkaline phosphatase detection and the capillary technology used in the Microprobe Accelerated System allowed completion of the procedure in less than 5 hr. Excellent correlations with the MDR phenotype of the cells, Northern blot analysis, and immunohistochemistry recommend this procedure for identifying cells that express the MDR phenotype in culture and in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Expression level of the nm23 gene in clonal populations of metastatic murine and human neoplasms.

The purpose of this study was to determine whether nm23 steady-state mRNA expression levels correlate with metastatic potential of mouse K-1735 melanoma cells, human KM12 colon cancer cells, and human SN12 renal cancer cells. Since neoplasms are heterogeneous and contain subpopulations of cells with different metastatic potentials, we analyzed multiple sets of nonmetastatic and metastatic clones isolated from each neoplasm. In addition, we also examined nine somatic cell hybrids produced by the fusion of nonmetastatic and metastatic K-1735 clones. In the mouse melanoma, we found heterogeneity in nm23-1 steady-state expression levels among the clones and hybrids that did not correlate with their metastatic phenotype. Clones isolated from human colon or renal carcinomas expressed similar levels of nm23-HI regardless of metastatic potential in nude mice. All of the human tumor cells were heterozygous for the nm23-HI-specific allelic DNA fragments, with no allelic deletions or gross alterations detected. Since the failure of tumor cells to produce metastasis can be due to multiple deficiencies, these data stress the importance of using independent clones with different metastatic potentials for the analysis of gene regulation of this process.

Alleles↗

PubMed1992

The use of molecular genetic markers to demonstrate the effect of organ environment on clonal dominance in a human renal-cell carcinoma grown in nude mice.

Transduction of the SN12C human renal-cell carcinoma line with the neoR gene produces a genetically "tagged" cell population within which individual clones can be identified if they dominate the tumor during its growth in vivo. We used this technique to determine whether the clones that dominate the primary local tumor and its metastases are the same or different when the tumor is growing in different organ sites in nude mice. The results show that clonal dominance is influenced by the organ environment in which the primary tumor grows, i.e., distinct clones dominated in the kidney, colon and subcutaneous sites. In addition, tumors grown in the orthotopic site (kidney) were all populated by the same dominant clones, and each distant visceral metastasis retained the same clonality. SN12C neoR-cells grown in an epithelial, ectopic site (colon) produced tumors with uniquely different dominant clones, and their visceral metastases retained the dominant pattern expressed by the parent tumor from which they were derived. In contrast, SN12C tumors growing subcutaneously showed a random pattern of clonal dominance in both their primary and metastatic sites. Parallel cytogenetic analyses could not demonstrate these patterns. We conclude that the organ environment significantly influences clonal dominance of human renal carcinoma and that tumor injection into orthotopic sites may produce a more reproducible selection of dominant clones.

Animals↗

PubMed1991

Clonal dominance of select subsets of viral Kirsten ras(+)-transformed 3T3 cells during tumor progression.

Long-term culturing of viral Kirsten-ras-oncogene (v-Ki-ras)-transformed BALB/c 3T3 cells (KiMSV) selectively enriches for cells which have deleted the viral oncogene. In contrast, v-Ki-ras in vivo is amplified and expression increased in all late-stage tumors and lung metastases relative to late-passage KiMSV cells being injected. The nature and significance of these selection processes, in terms of the v-Ki-ras gene, have been explored using genetically-tagged cells, as have the properties of v-Ki-ras- revertant subclones. Inoculation of KiMSV late-passage cells (containing less than 5% v-Ki-ras+ cells) into nude mice, generated primary and lung metastatic tumors with the v-Ki-ras gene at increased dosage in all tumors and their single-cell clones, isolated at both early and late stages of tumor development; this demonstrates early and specific in vivo selection for v-Ki-ras+ cells in both induction and progression of tumors in this system. v-Ki-ras- revertant subclones, isolated from late-passage KiMSV cells and inoculated individually into athymic nude mice, yielded tumors for only 1 of the 4 revertants, with no evidence for v-Ki-ras sequences in these tumor cells, thereby revealing a v-Ki-ras-independent mechanism for tumor formation in a small subset of revertant cells. Mixtures of the 4 subclones yielded tumors in all animals, although at a much longer latent period than observed with v-Ki-ras+ cells. Experiments with mixtures of v-Ki-ras- revertant cells and pSV2neo0tagged/v-Ki-ras+ cells (both complex NeoR cell mixtures and individual NeoR clones tested) at various cell ratios revealed clonal variability among v-Ki-ras+ cells for dominance during tumor formation. Moreover, the complex NeoR cell mixtures yielded both primary and metastatic tumors with simplified patterns of pSV2neo integration sites, suggesting that secondary genetic or epigenetic events, in addition to v-Ki-ras, contribute to the tumor-progressing phenotype. These experiments taken together demonstrate (a) clone-specific early selection of distinct v-Ki-ras+ cells amongst themselves and over v-Ki-ras- cells in both induction and progression of tumors, (b) reduced tumorigenic competence of v-Ki-ras- revertant cells, with a small subset displaying a v-Ki-ras-independent mechanism for tumor formation in this BALB/c 3T3 system, and (c) the significance of additional genetic or epigenetic events for tumor-progressing competence in unique subsets of v-Ki-ras+ cells.

Animals↗

PubMed1991

Growth factors and their receptors in metastasis.

The process of metastasis is not random. Rather, the outcome depends on the interaction of unique metastic cells with different organ microenvironments. Although the ability of some tumor cells to reach and proliferate in the parenchyma of some organs is responsible for the development of site-specific metastasis, the molecular basis of organ-specific metastasis is unclear. This article will address the molecular mechanisms involved in the interaction between favored tumor cells and a compatible organ environment in terms of growth factors and those receptors participating in paracrine and autocrine signal transduction pathways.

Animals↗

PubMed1990

Adhesion of Kirsten-ras+ tumor-progressing and Kirsten-ras- revertant 3T3 cells on fibronectin proteolytic fragments.

Adhesion has been evaluated for tumor cell populations derived from Kirsten murine sarcoma virus (KiMSV)-transformed BALB/c 3T3 cells responding to substrata coated with intact plasma fibronectin (pFN), a family of related proteolytic fragments from pFN or cellular fibronectins (FNs), and the heparan sulfate-binding platelet factor-4 (PF4). Both early-passage KiMSV cells, harboring the viral Kirsten ras oncogene (v-Ki-ras+), and late-passage KiMSV cells, in which most cells have lost the oncogene (v-Ki-ras-), are compared with primary tumor and lung metastatic tumor cells after three routes of injection into nude mice; nontumorigenic v-Ki-ras- revertant cells have been cloned from the late-passage KiMSV population. Attachment of early-passage KiMSV, primary tumor, and lung metastatic tumor cells was optimal and resistant to soluble RGDS peptide in the medium on intact pFN, on fragment F-155 from pFN containing the RGDS cell-binding domain and the heparinII domain, and on PF4 but decreased for metastatic cells on F110 containing only the RGDS domain (and sensitive to RGDS peptide). Cytoplasmic spreading of early-passage KiMSV and all tumor cells was good to excellent in polygonal patterns on pFN and on F155, while most cells remained round on F110. Responses for KiMSV and tumor cells varied on different heparin-binding proteins; cells remained rounded or detached on F38 derived from pFN or on PF4 but spread effectively with long linear process extension on cellular FN-derived fragments F44 + 47 harboring the extra domaina sequence. That F44 + 47 may contain a new cell-binding site for v-Ki-ras+ cells was also indicated by resistance to bacterial heparitanase in cell responses on F44 + 47 but not on PF4 and extensive catabolism of proteoglycans in the substratum-attached material of these cells. v-Ki-ras- revertant cells, nontumorigenic in nude mice, have reacquired 3T3-like responses to proteolytic fragments, including much more effective spreading on PF4 or on F38 substrata, and have reverted in generating microfilament stress fibers on pFN, a competence lacking in all v-Ki-ras+ cells. These results indicate that (a) v-Ki-ras+ primary and metastatic tumor cells respond similarly to most proteolytic fragments of FNs harboring known binding domains, with a few exceptions; (b) v-Ki-ras gene expression correlates with a new cell surface receptor activity recognized by extra domaina-containing fragments from cellular FNs; and (c) loss of the viral oncogene to generate v-Ki-ras- revertant cells reverts their FN-mediated adhesion responses.

Actins↗

PubMed1990

Genetic control of cancer metastasis.

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Animals↗

PubMed1988

Clonal diversity of the Kirsten-ras oncogene during tumor progression in athymic nude mice: mechanisms of amplification and rearrangement.

Single-cell clones from primary and lung metastatic tumors have been evaluated for the state of the viral-Kirsten-ras oncogene (v-Ki-ras) by Southern blot analysis after injection of Kirsten sarcoma virus-transformed BALB/c 3T3 cells (KiMSV, with a replication-defective provirus) into athymic nude mice by four different injection routes. While all clones of early-passage KiMSV cells contained an EcoRI-generated 5.3-kilobase DNA fragment at high dosage level, most clones of late-passage cells had lost this v-Ki-ras fragment or had greatly diminished levels. However, all clones of all tumors (greater than 90 tested) obtained after injection of these late-passage cells contained a dosage of the 5.3-kilobase v-Ki-ras band similar to that of the early-passage KiMSV cells, suggesting either a very strong selection for v-Ki-ras-bearing cells of the early-passage type in tumor formation and/or the ability of a subset of late-passage cells to amplify this gene to some minimal dosage level. Both flow cytometric analyses for DNA content and quantitation of chromosomes showed that all primary and lung metastatic tumors had more than twice the number of chromosomes as the late-passage KiMSV cells; however, four of 80 late-passage cells had a chromosome count in the range of tumors, consistent with their importance in tumor generation and possibly amplification of the v-Ki-ras-bearing chromosome. Clonal analyses of lung micrometastatic tumors revealed a v-Ki-ras blot pattern identical to that of the s.c. primary tumors. However, two of five lung metastases from the footpad (as large rapidly growing nodules) and i.v. routes had multiple copies of v-Ki-ras in new sites; a second injection round led to even greater complexity in v-Ki-ras patterns in clones of lung tumors. Two assays were used to demonstrate that these new v-Ki-ras integrations were generated by superinfection with a "helper" retrovirus, not sarcomagenic by itself in the nude mice, that led to rescue/reinfection of tumor cells with the defective Kirsten sarcoma proviral genome--cellular transformation of 3T3 or C3H10T1/2 cells and RNA dot blot analyses for medium-secreted retrovirus specific for LTR or v-Ki-ras sequences. This "helper" retrovirus could not be detected in early- or late-passage KiMSV cells used for inoculation but could be detected in certain tissues of normal nude mice, demonstrating its in vivo origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Amplification and rearrangement of the Kirsten ras oncogene in virus-transformed BALB/c 3T3 cells during malignant tumor progression.

Analyses of the cellular and viral Kirsten ras genes (c-Ki-ras and v-Ki-ras, respectively) during malignant tumor progression were performed by using Kirsten murine sarcoma virus-transformed BALB/c 3T3 cells that harbor a replication-defective provirus. After injection into athymic nude mice by four different routes, primary tumors and secondary lung metastases were isolated, adapted to in vitro growth, and analyzed for DNA levels and mRNA expression of both genes for comparison with the originally injected transformed cells and untransformed 3T3 cells. For all tumors (primary or secondary), the v-Ki-ras gene was amplified and v-Ki-ras mRNA expression was highly elevated above that observed in the original transformed cell population. In two of five lung metastases from the i.v. and footpad injection routes, rearranged Ki-ras DNA sequences were observed. Micrometastases from the s.c. route of injection did not display these alterations. Injection of footpad lung tumor cells with rearrangements into a second group of animals led to multiple lung metastases with even further rearrangements correlating with more effective lung colonization/growth ability (overt lung tumors in five of eight animals less than 20 days after injection). However, reinjection of an i.v. lung tumor with rearranged Ki-ras led to no further rearrangements in the lung microfoci tumors isolated greater than 40 days after injection. These data suggest (i) the significance of amplification and elevated expression of v-Ki-ras in tumor formation, (ii) correlation of this amplification with more effective tumor progression, and (iii) the selective advantage that cells with Ki-ras DNA sequence additions have in the formation of overt lung tumors.

Animals↗

PubMed1986

Fibronectin binding to gangliosides and rat liver plasma membranes.

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.

Animals↗