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Biomedical subjects

R J Bernacki

Publications and source records attributed to R J Bernacki.

At least 73 records · Page 4Linked to original sources
PubMed1985

Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix. An in vitro model system for studying tumor cell adhesion and invasion.

Human ovarian tumors metastasize by direct extension into the peritoneal cavity leading to tumor cell implantation onto peritoneal surfaces. Successful formation of peritoneal implants is dependent on the ability of ascitic tumor cells to infiltrate the mesothelium, and become firmly adherent to the underlying extracellular matrix (ECM). In order to investigate this process in more detail, an in vitro model system was developed employing human mesothelial cells grown on ECM-coated culture dishes. The ability of human ovarian carcinoma cells derived from ascitic fluid to attach to the mesothelial cell monolayer grown on ECM, ECM alone or plastic was quantitated with the use of 51Cr radio-labelled tumor cells. Tumor cells exhibited a more rapid and firmer attachment to ECM than to the mesothelial cells or to plastic. Using agitation to stimulate peritoneal fluid dynamics and shear forces in vivo, tumor cell arrest was found to be limited to the ECM, but it occurred at a slower rate than it did without agitation. Tumor cell attachment was also restricted to areas of exposed ECM in wounded mesothelium as assessed by phase-contrast microscopy. Morphologic alterations of the mesothelium induced by tumor cells were observed with the use of scanning electron microscopy (SEM) and immunohistochemical staining which included disruption of intercellular junctions leading to retraction of mesothelial cells, exposure of underlying ECM, subsequent attachment and proliferation on ECM. This model system would appear to be useful for elucidating mechanisms of ovarian tumor cell adhesion and proliferation, and for assessing various therapeutic modalities for their ability to block tumor cell implantation, invasion and growth on peritoneal surfaces.

Animals↗
PubMed1985

An evaluation of the effects of combination chemotherapy in vitro using DNA-reactive agents.

The combined application of DNA unwinding and strand-scission agents is a novel and potentially important approach to cancer therapy, based in part on mechanistic considerations of drug action. In order to evaluate this hypothesis a number of experiments were performed in which the cellular cytotoxicity of DNA reactive agents (ethidium bromide, adriamycin or cis-platinum) were evaluated alone and in combination with bleomycin, a strand-scission agent, using a number of different tumor cell systems in vitro. The results of these studies indicated that combinations of these agents were found to be much more effective than treatment with single drugs alone. This conclusion was warranted for the action of ethidium bromide followed by bleomycin with murine L1210 leukemia, melanoma B16-BL6 and human HeLa cells, and cis-platinum followed by bleomycin with L1210 and B16-BL6 cells. These data support previous findings in which synergistic growth inhibition of L1210 cells by ethidium bromide, followed by bleomycin was explained by a two-step mechanism; first, ethidium bromide introduces changes in DNA-conformation resulting in the facilitation of a second step in which bleomycin cleaves DNA more efficiently. Therefore, the rational use of combinations of DNA reactive agents based on mechanistic considerations should result with improved therapeutic regimens for the treatment of cancer.

Animals↗
PubMed1985

Therapeutic and diabetogenic potential of two newly synthesized nitrosoureido sugars.

Two newly synthesized nitrosoureido sugars have been evaluated for their antitumor activity and diabetogenic potential in a number of in vitro and in vivo preclinical tumor model systems. 2-Amino-2-deoxy-N'-methyl-N'-nitrosoureido-1,3,4,6-tetra-O-acetyl-alpha- D- mannopyranose (MAZ), a lipophilic mannosamine derivative, and ethyl-6-deoxy-3,5-di-O-methyl-6-(3-methyl-3-nitrosoureido)-alph a- D-glucofuranoside (EDOMEN or CGP 6'809), were both found to inhibit L1210 leukemia cell growth in vitro by 50% at approximately 5.0 X 10(-5) M. At these concentrations, little effect was noted immediately on L1210 cell radiolabeled precursor incorporation; however, at higher concentrations, EDOMEN inhibited [3H]leucine and [3H]mannose incorporation, while MAZ specifically decreased L1210 cell [3H]thymidine and [3H]leucine incorporation. Inhibition of Lewis lung carcinoma and B16 melanoma cell growth by 50% in vitro was achieved at higher concentrations of these agents (10(-4) to 10(-3) M). Since the currently available nitrosoureido sugars, streptozotocin and chlorozotocin, have been observed by us to be diabetogenic, EDOMEN and MAZ were evaluated for their specific toxicity to rat pancreatic beta-cells in vitro. Cytotoxicity in beta-cell cultures was monitored both by phase-contrast microscopy and the release of insulin into the culture medium. beta-Cells were found to be 10-fold more sensitive to the toxic effects of MAZ than were pancreatic fibroblasts. EDOMEN, on the other hand, did not damage beta-cells preferentially and therefore was not considered diabetogenic. Both MAZ and EDOMEN had moderate activity as antileukemic agents in mice. At 50 mg/kg/day i.p. for 5 days, MAZ increased the life span of female DBA/2J mice with L1210 leukemia by over 50%. Similarly, doses of EDOMEN at 125 to 250 mg/kg/day i.p. for 5 days increased L1210 leukemic life span by nearly 60%. At these doses, no effect of MAZ was observed on primary Lewis lung carcinoma growth or life span of tumor-bearing C57BL/6 mice. EDOMEN, however, increased life span in Lewis lung carcinoma mice by up to 33% and caused an apparent antimetastatic effect. These studies indicate that EDOMEN may have enhanced value as a cancer chemotherapeutic agent due to its therapeutic effectiveness, lack of diabetogenic potential, and other favorable formulation properties (water solubility) as compared with other clinically available nitrosoureas.

Animals↗
PubMed1984

Synergistic interactions of ethidium bromide and bleomycin on cellular DNA and growth inhibition.

Bleomycin is an anti-tumor agent whose cytotoxicity is related to the introduction of both single-stranded and double-stranded breaks in cellular DNA. In an assay using isolated nuclei, low levels of ethidium bromide substantially increased bleomycin induced release of nuclear chromatin. Treatment of mouse L1210 leukemia cells in vitro with low levels of ethidium bromide followed 1 hr later by bleomycin produced a synergistic effect that was 8 fold greater than that expected from the additive cytotoxicity of each drug alone. Interestingly, when the order of drug addition was reversed the drug synergism was much reduced (2 fold). The combination of DNA unwinding and strand scission agents may represent a novel and rational approach to the chemotherapy of cancer.

Animals↗
PubMed1984

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells.

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.

Animals↗
PubMed1983

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro-D-galactose, on the L1210 leukemic cell ectosialyltransferase system.

In L1210 leukemia cells, 6-deoxy-6-fluoro-D-galactose specifically inhibited the incorporation of [3H]-D-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6-3H]-6-deoxy-6-fluoro-D-galactose to a nucleotide sugar was similar to that found for [3H]-D-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro-D-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5'-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro-D-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.

Animals↗
PubMed1983

The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells.

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glycoprotein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.

Agglutination↗
PubMed1983

Biochemical effects and therapeutic potential of tunicamycin in murine L1210 leukemia.

Tunicamycin, an antibiotic which specifically inhibits the dolichol-mediated synthesis of glycoproteins, significantly decreased the incorporation of tritiated D-mannose and D-glucosamine into L1210 ascites leukemia cell glycoproteins at concentrations which affected the biosynthesis of proteins minimally. Mice receiving inoculations of L1210 cells pretreated with 10 microM tunicamycin in vitro survived nearly twice as long as did mice receiving implants of untreated tumor cells. A nonlethal dose of X-irradiation (350 rads) to mice 24 hr prior to receiving their inoculation of tunicamycin-treated L1210 cells prevented this increase in life span. Thirty-eight % of the long-term surviving mice which received 1 X 10(5) L1210 cells pretreated with 10 microM tunicamycin in vitro were then resistant to a subsequent challenge with 10(6) untreated L1210 ascites cells. Direct i.p. administration of tunicamycin to mice resulted in potent liver toxicity (50% lethal dose, 2.0 mg/kg) which obviated any therapeutic efficacy when administered to L1210 ascites tumor-bearing mice. The administration of nontoxic levels of D-mannose prior to the administration of tunicamycin decreased the toxicity of the antibiotic in vivo and, when combined with D-mannose in vitro, exhibited cytotoxic additivity in terms of the inhibition of L1210 leukemic cell growth. A therapeutic regimen incorporating a 24-hr infusion of the sugar prior to multiple administrations of tunicamycin gave evidence of a small therapeutic response in terms of the survival of tumor-bearing mice. These results suggest that tunicamycin, an inhibitor of glycoprotein biosynthesis, might be able to alter tumor cell growth and immunogenicity provided that host liver toxicity is diminished.

Animals↗
PubMed1982

Activity of tunicamycin against Trypanosoma brucei in vitro and in vivo.

Tunicamycin, a specific inhibitor of glycoprotein biosynthesis, was evaluated for activity and chemotherapeutic potential against Trypanosoma brucei in vitro and in vivo. The inhibition of parasite incorporation of two radiolabeled macromolecular precursors, [methyl-3H]thymidine and D-[2-3H]mannose, was measured in short-term (4-h) microcultures. Mannose incorporation was inhibited over the concentration range of 0.01 to 10.0 microgram/ml, reaching 60% at the latter level. Thymidine incorporation was not affected. In vivo experiments indicated that a single dose of 2.0 mg of tunicamycin per kg can cure or significantly increase the life-span of mice of three different strains, each infected with T. brucei.

Animals↗
PubMed1981

A correlation between cell surface sialyltransferase, sialic acid, and glycosidase activities and the implantability of B16 murine melanoma.

A murine melanoma variant (B16-F10ir6), resistant to lymphocytic cytolysis, has been shown previously to produce lower numbers of tumor nodules in the lung of C57BL/6J mice following i.v. inoculations. These differences found in tumor implantation and lymphocyte recognition may be due to changes in surface properties of this cell line. Therefore, membrane-bound sialic acid (released by Vibrio cholerae neuraminidase treatment), ectosialyltransferase activity, and total cellular glycosidase levels were measured in this cell line and compared with levels in its parent melanoma tumor cell line, B16-F10, which was selected for its enhanced ability to form tumor nodules. The results of these studies indicate a correlation between the degree of lung implantation and the amount of tumor cell sialic acid accessible to neuraminidase cleavage, tumor cell surface sialyltransferase activity, and several cellular glycosidase activities. These results are consistent with the idea that membrane structural changes in the glycocalyx may account for the ability of a tumor cell to implant and metastasize.

Acid Phosphatase↗
PubMed1980

Synthesis and biological activity of some 1-N-substituted 2-acetamido-2-deoxy-beta-D-glycopyranosylamine derivatives and related analogs.

Several 1-N-substituted derivative [haloacetyl-, glycyl-, (dimethyl)amino-acetyl-, azidoacetyl-, trifluoroacetyl-, and trifluoromethylsulfonyl-] of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-beta-D-glucopyranosylamine (1) were synthesized as potential metabolic inhibitors of cellular-membrane glycoconjugates. Several fully acetylated derivatives were found to inhibit growth of mouse mammary adenocarcinoma TA3, leukemia L1210, or leukemia P-288 cells at 1-0.01 mM concentration in vitro. Some of these derivatives were less active after O-deacetylation. Analogs of 1 in which NH2-1 was replaced by OH- or OAc-1 were also active on the same cell systems. The growth-inhibitory activity was correlated with inhibition of the incorporation of 2-amino-deoxy-D-glucose and L-leucine into a macromolecular fraction.

Acetylglucosamine↗
PubMed1980

Halogenated L-fucose and D-galactose analogues: synthesis and metabolic effects.

Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.

Animals↗
PubMed1980

Some short-chain N6-substituted adenosine analogues with antitumor properties.

The compounds N6-allyl-, N6-isopropyl-, N6-propargyl-, and N6-(2-methylallyl)adenosine were prepared by reacting 6-chloropurine riboside with an excess of the corresponding amines in ethanol, in the presence of two acid acceptors resulting in virtually quantitative yields. The compounds showed biological activity in a number of in vitro and in vivo tumor cell systems. Very good increases in life spans of mice bearing mammary carcinoma were obtained by treatment with the N6-allyl, N6-isopropyl, and N6-propargyl analogues, respectively. In rats, the N6-allyl analogue slowed the rate of transplantable mammary tumor growth by one-fourth. The short-chain adenosine analogues are more active in the treatment of animal carcinomas than in the leukemia or sarcoma tumor cell systems.

Adenosine↗
PubMed1980

Elevation of lysosomal enzymes in primary Lewis lung tumor correlated with the initiation of metastasis.

Lysosomal enzymes were elevated about two-fold in primary s.c. Lewis lung carcinoma as compared with metastatic nodules in the lung. In a time course experiment, a general two-fold elevation of acid phosphatase and several glycosidases was observed in the primary tumor between the 14th and 17th postimplant day following s.c. inoculation of Lewis lung carcinoma. This increase in hydrolytic enzyme activity was not due to necrosis in the primary tumor since a comparison of enzyme activities in the nonnecrotic and necrotic areas demonstrated much higher activities in the nonnecrotic areas. No increases in lysosomal enzyme activity were observed with time in Sarcoma 180, a tumor which does not metastasize. There was no change with time in primary Lewis lung tumor lactate dehydrogenase activity while a 7-fold increase in serum lactate dehydrogenase activity was observed in tumor-bearing mice. Mitochondrial succinate-2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium reductase levels fell in the primary Lewis lung tumor as the tumor size increased. A positive correlation was observed between the time of the elevations of tumor lysosomal enzymes in Lewis lung carcinoma and the appearance of micro- and macrometastatic lesions in the lungs. The mechanisms accounting for the increased intratumoral lysosomal enzymes are unknown, but they may be related to macrophage infiltration or other tumor-host interactions which may facilitate the dissemination of tumor cells.

Acid Phosphatase↗
PubMed1978

Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.

Cell Line↗