Biomedical subjects

M J Morin

Publications and source records attributed to M J Morin.

32 records · Page 2Linked to original sources

PubMed1990

Increased cell surface EGF receptor expression during the butyrate-induced differentiation of human HCT-116 colon tumor cell clones.

Several clonal sublines of HCT-116 human colon adenocarcinoma cells were isolated and characterized on the basis of their growth characteristics, intrinsic enterocyte-like differentiation (as assessed by alkaline phosphatase and lactase activities), and responses to butyrate, an inducer of colon tumor cell maturation. The HCT-116 sublines were found to be heterogeneous and several phenotypically distinct clones were identified. Further characterization of these clones indicated that the effects of butyrate on cell growth, alkaline phosphatase activity, and lactase activity were distinct and separable. The growth of all of the clones were inhibited by butyrate (IC50 values varied from 0.44 to 1.5 mM), but the effects of this agent on alkaline phosphatase and lactase activities varied widely. In several sublines butyrate had no effect on either enzyme while in others one or both activities were induced. Additionally, the binding of 125I-epidermal growth factor (EGF) to cell surface receptors was found to be proportional to the expression of lactase activity in the cell. The D3 clone and other sublines with intrinsic lactase activities greater than 100 nmol/mg/min expressed a class of high-affinity EGF receptors (e.g., D3 cells had 3.48 X 10(4) EGF receptors/cell with a kd of 0.61 nM). Other clones with less lactase activity had undetectable levels of 125I-EGF binding. In clones which exhibited greater than twofold increases in lactase activity in response to butyrate, the expression of a large number of low-affinity EGF receptors was also induced. In one such clone, the P1 subline, lactase activity was increased from 70 nmol/mg/min to 230 nmol/mg/min after 96 h in 2 mM butyrate, and the expression of EGF receptors was increased from undetectable levels to 1.18 X 10(5) EGF receptors/cell (kd of 3.2 nM). Northern blot analysis indicated that the increased 125I-EGF binding after butyrate treatment may have been due, in part, to a greater than twofold accumulation of EGF receptor mRNA. In addition, the expression of the messages for transforming growth factor alpha (TGF-alpha) and transforming growth factor beta (TGF-beta) was examined in butyrate-treated cells. While TGF-alpha mRNA levels were found to correlate with EGF receptor message levels in the HCT-116 clones, TGF-beta mRNA expression was not found to correlate with the butyrate-induced growth inhibition or with increases in EGF receptor expression, alkaline phosphatase activity, or lactase activity in these cells.

Alkaline Phosphatase↗

PubMed1989

Human multidrug resistant KB cells overexpress protein kinase C: involvement in drug resistance.

Among the many phenotypic characteristics of multidrug resistance (MDR), the presence of P-glycoprotein is nearly always observed, and it appears that the plasma membrane of the multidrug resistant cell is integrally involved in controlling drug resistance. Another membrane-associated protein kinase, protein kinase C (PKC), has been shown to regulate the flow of information to the cell interior and to control the efflux of a number of different compounds. We therefore initiated a study of PKC and MDR. We found that multidrug resistant sublines from both mouse sarcoma 180 and human KB lines exhibited 80-90% increases in basal PKC activity. The mechanism of the increase appears to be quite different in the two cell lines. The human KB cells overexpress the alpha isozyme of PKC, commensurate with the increase in alpha-PKC protein, whereas the mouse cells do not overexpress alpha-mRNA but increase alpha-PKC protein. Furthermore, it appears that PKC activity plays a functional role in drug resistance, since inhibition of endogenous PKC activity by staurosporine resulted in decreased resistance to Adriamycin. We also found that phosphorylation of MDR cell membrane vesicles by purified PKC, followed by immunoprecipitation of P-glycoprotein with monoclonal antibody C219, resulted in a level of phosphorylation of P-glycoprotein that was greater than the endogenous phosphorylation level. The data presented indicate that MDR cells of diverse species exhibited enhanced PKC activity but that the mechanisms were different. The increased kinase activity may have biological relevance to MDR since PKC appears to be coupled to P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Rapid increase in the activity of DNA topoisomerase I, but not topoisomerase II, in HL-60 promyelocytic leukemia cells treated with a phorbol diester.

There is significant evidence to suggest that protein kinase C and DNA topoisomerases are functionally linked in signal transduction pathways. Much of this is based on the observation that phosphorylation of topoisomerase II by protein kinase C may lead to its activation in vitro and that inhibitors of topoisomerase II block phorbol diester-induced differentiation in HL-60 cells. In the present study, the activities of the DNA topoisomerases I and II have been quantitated to examine their regulation in phorbol diester-treated HL-60 cells undergoing differentiation. The activity of topoisomerase I increased rapidly after treatment with phorbol myristate acetate (PMA); it increased maximally (150% of control activity) at 3 hr post-treatment and remained elevated for at least 24 hr. Conversely, from the onset of exposure to PMA through 12 hr, there was no measurable alteration in topoisomerase II activity in PMA-treated cells. Moreover, there was a measurable decrease in topoisomerase II activity at the later time points, a result that occurred concomitantly with the loss of proliferative potential in differentiating HL-60 cells. Similar results were obtained when the activities of both enzymes were measured in nuclear extracts. The apparent increase in topoisomerase I activity was not due to an increase in the mass of the enzyme after PMA treatment, as measured by both western blotting and by the formation of camptothecin-dependent, topoisomerase I-DNA complexes. Taken together, these data suggest that the activities of the topoisomerases I and II may have been regulated independently in PMA-treated HL-60 cells, that the activity of topoisomerase II was not increased under conditions in which protein kinase C was activated in vivo, and that an increase in the activity of topoisomerase I may have had a role in the mechanism through which HL-60 cells underwent monocytic maturation in response to phorbol diesters.

Camptothecin↗

PubMed1987

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation.

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.

Calcimycin↗

PubMed1987

Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site.

A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for von Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span approximately 175 kilobases of human DNA sequence, and hybridization analysis suggests that the vWf coding unit is approximately 150 kilobases in length. Within one of these clones, the vWf transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical "TATA box," a downstream "CCAAT box," and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWf translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species.

Amino Acid Sequence↗

PubMed1987

Function of the anthracycline amino group in cellular transport and cytotoxicity.

Using a number of derivatives of doxorubicin (Adriamycin) and daunomycin, we have examined how substitution of the anthracycline amine affected net cellular accumulation and cytotoxic potency in HL-60 leukemia cells. Octanol/buffer partitioning demonstrated that each of the derivatives had an amino group titratable between pH 5 and 8, with the exception of derivatives containing a cyanomorpholino-substituted amine, which had a significantly lower pKa value. The steady state cellular drug levels for the Adriamycin and daunomycin series decreased in the following order: N,N-dimethyl-greater than morpholino-greater than parent greater than cyanomorpholino-. Thus, the net cellular accumulation of an anthracycline was found to be influenced by the basicity of the amino group; drugs with a non-basic amino group exhibited reduced uptake. Soft agar clonogenic assays showed the following order of cytotoxicity for both series: cyanomorpholino-much greater than parent greater than morpholino-approximately equal to N,N-dimethyl-. The data demonstrate an inverse correlation between uptake and potency; thus, differences in net cellular accumulation do not account for the order of anthracycline potency.

Amines↗

PubMed1986

Mechanisms involved in the induction of malignant cell differentiation.

Cancer appears to be a disease of altered maturation, with changes in genetic expression leading to a situation in which the physiological regulation of cellular proliferation and maturation are altered. Environmental factors as well as defined chemical agents have been demonstrated to have the capacity to convert neoplastic cells to end-stage forms with a finite life span through a process characteristic of cellular maturation. The correction of genetic defects by these inducers of differentiation does not appear to be required; the critical feature is that the differentiated cells assume a state in which they no longer possess the capability for continued cellular replication. The extrapolation of these advances, accomplished in experimental systems, to clinical practice should yield significant decreases in the neoplastic cell burden without the degree of morbidity produced by aggressive therapy with cytodestructive agents, especially when employed in multidrug combinations. The ultimate introduction of differentiation as a therapeutic approach to cancer treatment if attained, however, will require a variety of principles to be established, so that optimum efficacy may be obtained from each agent, the fabrication of new agents with major changes in the ratio of the concentrations required to produce cytotoxicity relative to those necessary to initiate maturation is attained, and the elucidation of non-antagonistic combinations of differentiation inducing agents with or without cytotoxic drugs is achieved to combat the problem of tumor cell heterogeneity.

Animals↗

PubMed1985

Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells.

The role of C-kinase in the induction of maturation of HL-60 promyelocytic leukemia cells was examined using two activators of this kinase, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG). At 10(-8) M, a concentration that induced maturation, TPA effectively stimulated C-kinase activity in cell-free preparations by increasing the affinity of the enzyme for Ca2+. Similar activation was observed with 20 micrograms/ml of OAG. At these concentrations, addition of either compound to intact cells stimulated the phosphorylation of cellular proteins. Treatment with TPA resulted in an increased phosphorylation of 14 proteins, 9 of which also changed in response to OAG. In addition to the effects on protein phosphorylation, TPA and OAG both affected choline lipid metabolism. TPA at 10(-8) M stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. OAG at 20 micrograms/ml had quantitatively similar effects on the labeling of the former two lipids, but did not affect incorporation of choline into lysophosphatidylcholine. Despite the similar biochemical effects of TPA and OAG, the diglyceride was unable to induce HL-60 cell maturation as measured by inhibition of cell growth, development of nonspecific esterase activity, phagocytosis, adherence of cells to plastic, and loss of transferrin receptor activity. The lack of effect is not due to metabolism of OAG; maturation could not be induced by treating cells with fresh OAG every 2 h for a period of 12 h. These results suggest a dissociation of the activation of C-kinase and the induction of HL-60 cell maturation by TPA.

Cell Differentiation↗

PubMed1985

Molecular cloning of cDNA for human von Willebrand factor: authentication by a new method.

We have identified a 2.4 kb partial cDNA clone (pDL34) for human von Willebrand factor (vWf) mRNA. pDL34 was selected by screening an endothelial cell cDNA library with a radiolabeled reverse transcript of mRNA obtained by specific immunoisolation of vWf polysomes from endothelial cells. pDL34 selectively hybridized to an endothelial cell-associated 9.5 kb mRNA. To confirm its identity, SP6 RNA polymerase was used to generate in vitro transcripts of the cDNA. This synthetic RNA, truncated at its 5' end, directed the synthesis of several unique polypeptides in rabbit reticulocyte lysates. These polypeptides were immunoprecipitated by polyclonal and monoclonal anti-vWf antibodies. These results indicate that pDL34 contains an authentic partial copy of vWf mRNA. In vitro transcription of partial cDNA clones and translation of the resulting RNAs may be a useful general method of verifying the identity of various cDNA clones in circumstances where antibodies are available and protein sequence is not.

Antibodies, Monoclonal↗

PubMed1984

Inhibition of glycoprotein biosynthesis by the inducers of HL-60 cell differentiation, aclacinomycin A and marcellomycin.

The trisaccharide-containing anthracyclines aclacinomycin A (ACM) and marcellomycin (MCM) have been shown by our laboratory to be potent inducers of HL-60 leukemia cell maturation, while the monosaccharide-containing anthracyclines Adriamycin and pyrromycin were inactive and significantly less potent, respectively, as initiators of the differentiation of these malignant cells. We have now observed that ACM and MCM are potent inhibitors of both total glycoprotein synthesis and the formation of lipid-linked oligosaccharide intermediates in intact HL-60 cells. This inhibitory activity was both concentration and time dependent and was maximal after 12 hr exposure to 30 nM ACM or MCM, conditions under which both cell growth and total protein synthesis were maintained at levels equal to those of untreated cells. In contrast, exposure of HL-60 cells to pyrromycin or Adriamycin, even at cytotoxic concentrations, did not result in specific decreases in the synthesis of glycoproteins containing asparagine-linked oligosaccharides. Maximum inhibition of the formation of lipid-linked intermediates by ACM or MCM preceded the loss of cell surface transferrin-binding activity (maximal at 24 hr), which in turn preceded the minimal exposure time necessary for significant induction of differentiation by either of the active anthracyclines (36 hr). These findings demonstrate a new biochemical site of action for ACM and MCM and suggest that this activity is involved in the induction of terminal differentiation of HL-60 leukemia cells by these antitumor agents.

Aclarubicin↗

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↗

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↗