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R Radinsky

Publications and source records attributed to R Radinsky.

At least 19 recordsLinked to original sources
PubMed1997

Influence of the host microenvironment on the clonal selection of human colon carcinoma cells during primary tumor growth and metastasis.

The purpose of this study was to determine the subpopulation dynamics of human colon carcinoma (HCC) cells growing at orthotopic (cecum, liver) or ectopic (subcutis, kidney, spleen) sites in nude mice and to correlate any outgrowth of distinct clones with the differential expression of metastasis-related genes. Low metastatic KM12C HCC cells were genetically tagged with a retrovirus harboring the neomycin-resistance (Neo(R)) gene. Southern blot analyses demonstrated only minor resolution of the Neo(R) hybridization pattern in DNA isolated from primary tumors growing orthotopically or ectopically, suggesting a polyclonal outgrowth. In contrast, a major resolution of the Neo(R) hybridization pattern was observed in liver-specific metastases, demonstrating the outgrowth of single dominant clones. Expression of epidermal growth factor receptor (EGR-R) increased 20-60% in the liver metastases vs spleen tumors and the KM12C Neo(R) cells. Transforming growth factor alpha (TGF-alpha), amphiregulin (AR), and c-met showed only modest differences in mRNA expression. A 20-80% increase in type IV collagenase mRNA levels was also observed in all tumor specimens. Furthermore, expression of the multi-drug resistance gene PGY-1 and the carcinoembryonic antigen (CEA) gene were elevated in the liver metastases compared with the spleen tumors and cultured cells. Transcript levels of the angiogenic factors interleukin-8 and basic fibroblast growth factor did not correlate with clonal outgrowth. These data demonstrate a correlation between EGF-R, type IV collagenase, CEA, and PGY-1 gene expression and the production of liver metastases. Our results suggest that distinct HCC clones differentially expressing specific mRNA transcripts for metastasis-related genes are the forerunners of the experimental liver metastatic lesions.

Animals↗
PubMed1996

Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by cell density.

To determine the effect of cell density on vascular endothelial growth factor (VEGF) expression and the mechanism of this effect, four human colon cancer cell lines were grown as sparse or confluent monolayers or as spheroids. VEGF mRNA increased > 2-fold in cells grown as confluent monolayers or spheroids compared with cells grown as sparse monolayers. Semiquantitative reverse transcription-PCR demonstrated a 2-fold increase in the larger VEGF mRNA isoform (189 bp) in confluent cells. Sparse cells grown in conditioned medium from confluent cells demonstrated a > 2-fold increase in VEGF mRNA. These data suggest that VEGF expression may be regulated by an unidentified soluble factor.

Base Sequence↗
PubMed1996

Regulation of carcinoembryonic antigen expression in human colon carcinoma cells by the organ microenvironment.

The expression of carcinoembryonic antigen (CEA) is thought to be involved in homotypic adhesion and has been associated with the progression of human colon carcinomas (HCC) to the metastatic state. Three cell lines established from surgical specimens of Dukes' stage D (KM20) or Dukes' stage B (KM12C, KM12SM) with high and low preoperative CEA serum levels, respectively, were studied subsequent to growth in culture, in the subcutis (ectopic) or cecal wall (orthotopic) of nude mice. In all cell lines, CEA expression was higher in cecal tumors than in subcutaneous lesions. The degree of differentiation and CEA expression by HCC growing in the cecal wall of nude mice correlated with the pathological diagnosis and preoperative CEA level of the original patients. To better understand the regulation of CEA expression, the HCC cells were grown in culture as sparse and confluent monolayers or as multicell spheroids. The CEA expression level increased in all three cell lines growing as confluent monolayers and was highest in multicell spheroids. Treatment of sparse monolayer cultures of KM12SM cells with mitomycin-C inhibited cell division and was associated with higher production of CEA protein. Moreover, conditioned media from confluent monolayer cultures or from spheroids up-regulated CEA production in sparse monolayer cells. These data show that CEA expression in HCC cells may be regulated by cell density and by factors from the organ environment.

Animals↗
PubMed1996

Molecular determinants in the biology of liver metastasis.

A primary goal of cancer research is an increased understanding of the molecular mechanisms mediating the process of cancer metastasis. Analyses of colon cancer cells (the seeds) and the microenvironment (the soil) has increased our understanding of the biologic mechanisms mediating liver-specific metastasis. Insight into the molecular mechanisms regulating the pathobiology of cancer metastasis as well as a better understanding of the interaction between the metastatic cell and the host environment should produce a foundation for new therapeutic approaches. In this article we summarize experimental observations demonstrating the importance of specific factors that regulate various steps in the metastatic cascade. Furthermore, this article emphasizes the importance of the host organ's microenvironment and its role in liver metastasis formation. The production of metastases depends, in part, on the interaction of particular tumor cells with specific organ environments. Therefore, the successful metastatic cell must be viewed currently as a cell receptive to its environment. The analyses presented herein add important evidence to support the concept that cancer metastasis is not a random process; it is a highly regulated process that can be analyzed on the molecular level. To the clinician, it is readily apparent that by the time metastasis forms, most steps in the metastatic cascade have been completed. Therefore, therapy targeted to downregulate or interrupt the last stages of metastasis, proliferation and angiogenesis, should be the areas of greatest investigation in regards to treating established metastasis, whether they are microscopic or macroscopic.

Animals↗
PubMed1995

Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis?

Fas/APO-1, a member of the NGF/TNF receptor superfamily expressed on the cell-surface of normal and malignant cells, is known to induce cell death by apoptosis. In the present study, we have investigated Fas/APO-1 gene defects in a human osteosarcoma cell line resistant to the apoptosis-inducing effects of anti-Fas. cDNA cloning and sequencing revealed that these cells contained both 'authentic' and mutant Fas/APO-1 containing a 63 base pair in-frame deletion spanning the transmembrane domain, designated DFas/APO-1. Direct evidence for the existence of a soluble Fas/APO-1 protein was obtained by immunoprecipitation and Western blotting. Taken together with prior studies demonstrating a role for Fas/APO-1 and Fas ligand, respectively, in tumor target cell killing by cytotoxic T-lymphocytes, production of soluble Fas/APO-1 might have significant implications in malignant disease pathogenesis.

Antigens, Surface↗
PubMed1995

Transcriptional induction of the melanocyte-stimulating hormone receptor in brain metastases of murine K-1735 melanoma.

Metastatic K-1735 murine melanoma cells are amelanotic in culture or in the subcutis of syngeneic mice. When injected into the internal carotid artery, these cells produce melanotic brain metastases. The production of melanin in tumor cells growing in the brain was directly correlated with induction of melanocyte-stimulating hormone receptor (MSH-R) steady-state mRNA transcripts. K-1735 cells isolated from brain lesions and implanted into the subcutis or grown in culture lose MSH-R transcripts and become amelanotic. In contrast to K-1735 cells, B16-BL6 melanoma cells constitutively produce melanin and express high levels of MSH-R mRNA regardless of the site of growth. Somatic cell hybrids between K-1735 and B16 cells produced melanin and expressed high levels of MSH-R mRNA transcripts, regardless of the site of growth, suggesting the dominance of the B16 phenotype. Treatment with alpha-MSH failed to upregulate MSH-R expression in cultured K-1735 cells or to maintain MSH-R expression in K-1735 cells isolated from brain metastases to be grown in culture. Responsiveness to alpha-MSH as determined by cell proliferation, melanin production, and intracellular accumulation of cyclic AMP directly correlated with MSH-R expression. These data demonstrate that a specific organ environment influences the phenotype of metastatic cells by regulation of specific genes that encode for cell surface receptors.

Animals↗
PubMed1995

Modulation of tumor cell gene expression and phenotype by the organ-specific metastatic environment.

The mechanistic basis of a metastatic cell's ability to proliferate in the parenchyma of certain organs and develop organ-specific metastases is under intense investigation. Signals from paracrine or autocrine pathways, alone or in combination, may regulate tumor cell proliferation with the eventual outcome dependent on the net balance of stimulatory and inhibitory factors. This article summarizes recent reports from our laboratory and others demonstrating that the organ microenvironment can profoundly influence the pattern of gene expression and the biological phenotype of metastatic tumor cells, including induction of melanocyte stimulating hormone receptor and production of melanin, regulation of terminal differentiation and apoptosis, resistance to chemotherapy, and regulation of growth at the organ-specific metastatic site. These recent data from both murine and human tumor models support the concept that the microenvironment of different organs can influence the pattern of gene expression and hence the phenotype of tumor cells at different steps of the metastatic process. These findings have obvious implications for the therapy of neoplasms in general and metastases in particular.

Animals↗
PubMed1995

Heterogeneity of cytokine and growth factor gene expression in human melanoma cells with different metastatic potentials.

The purpose of this study was to determine the mRNA expression level of multiple cytokine and growth factor genes in human malignant melanoma. Melanoma cells were isolated from several surgical specimens, adapted to growth in culture, characterized for their ability to produce experimental metastases in nude mice, and assessed for cytokine and growth factor steady-state gene expression. Highly metastatic in vivo- and in vitro-derived variants isolated from a single melanoma, A375, were also analyzed. Northern blot analyses revealed that all melanomas analyzed constitutively expressed steady-state mRNA transcripts for the growth and angiogenic factors, basic fibroblast growth factor (bFGF), and transforming growth factor alpha (TGF-alpha), which correlated with metastatic propensity. Only one highly metastatic melanoma, TXM-1, originally isolated from a lymph node metastasis, expressed mRNA transcripts specific for monocyte chemotactic and activating factor (MCAF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Similarly, of the nine melanomas examined, only TXM-1 expressed interleukin (IL)-1 alpha, IL-1 beta, and IL-6, important immunomodulatory cytokines. These data demonstrate the differential and heterogeneous expression of cytokine and growth factor genes in human malignant melanoma.

Animals↗
PubMed1994

Human recombinant interferons-beta and -gamma decrease gelatinase production and invasion by human KG-2 renal-carcinoma cells.

We investigated the effects of human recombinant interferons (r-IFNs) on gelatinase production and invasion by human renal-cell carcinoma (HRCC). Incubation of KG-2 HRCC with human r-IFN-beta or -gamma (but not -alpha) suppressed transcription of the 72-kDa gelatinase gene and, hence, production of gelatinase activity. These inhibitory effects of interferons (IFNs) were independent of their antiproliferative effects. Treatment of KG-2 cell with r-IFN-beta or -gamma significantly inhibited cell invasion through reconstituted basement membrane toward chemoattractants produced by kidney fibroblasts. The inhibitory activity of r-IFNs was specific to the KG-2 cells since gelatinase activity by various fibroblasts was unaffected. These findings suggest that r-IFN-beta or IFN-gamma may be used to inhibit the invasive potential of HRCC.

Carcinoma, Renal Cell↗
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↗