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T Mitchell

Publications and source records attributed to T Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed1987

Effects of interferon-gamma on proto-oncogene expression during induction of human monocytic differentiation.

Activation of the proto-oncogenes c-fos, c-fms, and c-sis has been associated with monocytic differentiation. In the present study, we monitored the relationship of c-myc, c-fos, c-fms, and c-sis expression to interferon-gamma (IFN-gamma)-induced monocytic differentiation of human HL-60 promyelocytic leukemia cells. Treatment of HL-60 cells with 500 U/ml IFN-gamma arrested cell proliferation after 3 days. Appearance of the monocytic phenotype was manifested within 24 hr of IFN-gamma exposure by: increased nitroblue tetrazolium reduction; increased cell surface expression of the HLA-DR, Mo1, and MY4 antigens; and induction of transcripts for the second component of complement (C2) and tumor necrosis factor. In contrast, c-myc expression decreased as a later event after 72 hr of IFN-gamma exposure, and c-fos transcripts remained undetectable until 5 days of treatment. Furthermore, c-fms RNA and transcripts for the macrophage marker apolipoprotein E were induced only after 7 days. Finally, expression of the c-sis proto-oncogene at the RNA and protein levels remained undetectable after induction with IFN-gamma. These findings would thus suggest that declines in c-myc RNA, as well as induction of c-fos, c-fms, and c-sis expression, are not requisite events in the commitment of HL-60 cells to IFN-gamma-induced monocytic differentiation. Expression of c-fos and c-fms, however, is associated with acquisition of markers associated with maturation to macrophages.

Antigens, Surface↗
PubMed1987

Expression of the c-raf protooncogene in human hematopoietic cells and cell lines.

The murine sarcoma virus 3611 contains the transforming v-raf gene that has partial nucleotide homology with the src family of tyrosine kinase-encoding oncogenes. Although this virus induces fibrosarcomas in mice, a recombinant murine retrovirus carrying both the raf and myc oncogenes induces immunoblastic lymphomas and immortalizes mouse macrophages in vitro. The present study has thus monitored the expression of c-raf in human hematopoietic cells. The results demonstrate the presence of a 3.6-kb c-raf transcript in HL-60 promyelocytic leukemic cells. The induction of HL-60 cell differentiation along the monocytic or granulocytic lineages had no detectable effect on the level of c-raf transcripts. Furthermore, in contrast to c-myc and c-fms expression, inhibition of protein synthesis with cycloheximide had no detectable effect on c-raf expression. Similar levels of c-rafRNA were also found in other human cell lines derived from myeloid, B cell, and T cell tumors, as well as in normal granulocytes, monocytes, and macrophages. These findings suggest that the c-raf protooncogene is widely expressed in multiple hematopoietic lineages.

Cell Differentiation↗
PubMed1986

Roles of H1 domains in determining higher order chromatin structure and H1 location.

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.

Animals↗
PubMed1986

Incorporation of 9-beta-D-arabinofuranosyl-2-fluoroadenine into HL-60 cellular RNA and DNA.

The incorporation of 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) into HL-60 cellular nucleic acids was monitored by cesium sulfate gradient centrifugation. The results demonstrated that F-ara-A incorporated into both RNA and DNA. These findings are in contrast to those previously obtained with 1-beta-D-arabinofuranosylcytosine (ara-C) and 9-beta-arabinofuranosyladenine (ara-A) which demonstrated incorporation of these nucleosides only in DNA. F-ara-A inhibited HL-60 proliferation, and the incorporation of F-ara-A into both DNA and RNA correlated with loss of clonogenic survival. Furthermore, cytostatic concentrations of F-ara-A resulted in the appearance of a more mature phenotype, a finding consistent with the effects of other inhibitors of DNA synthesis. The incorporation of F-ara-A into RNA and DNA should provide new insights regarding the mechanism of action of this agent.

Carboxylesterase↗
PubMed1986

Secretion of vasopressin, oxytocin, and two neurophysins from rat hypothalamo-neurohypophyseal explants in organ culture.

Secretion of the neurohypophyseal principles arginine vasopressin, oxytocin, vasopressin-neurophysin, and oxytocin-neurophysin by hypothalamic-neurohypophyseal explants in organ culture was studied. The viability of explant cultures was tested on day 6 by their ability to incorporate 3H-amino acid into acid-precipitable protein, specifically vasopressin-neurophysin. The explants released all four neurosecretory products in a controlled manner during 8 successive days of culture. In addition, these substances were all present in neural lobes and hypothalami at the end of the culture period. Although the tissue retained a 1:1 molar ratio between the hormone and corresponding neurophysin, the levels of neurophysins in culture media were much higher than the levels of hormones. Through immunoprecipitation techniques we determined that both arginine vasopressin and oxytocin were unstable in culture media containing explants or in medium containing 10% heat-inactivated fetal calf serum. This suggests that neurophysins are more stable indicators of secretion by explants than are the corresponding hormones. An increased release of arginine vasopressin, oxytocin, vasopressin-neurophysin, and oxytocin-neurophysin occurred in response to elevated K+ and to increased sodium ions in the medium, and these four substances were released in different molecular proportions. The results indicate that explant cultures may be useful for comparisons of secretion of the two neurophysins and, to a more limited extent, of the two hormones in response to such stimuli.

Animals↗
PubMed1986

Effects of 1-beta-D-arabinofuranosylcytosine on proto-oncogene expression in human U-937 cells.

We have previously demonstrated that declines in c-myc expression precede the induction of c-fos and c-fms transcripts during monocytic differentiation of human leukemia (HL-60 and U-937) cell lines. The present study has monitored the effects of 1-beta-D-arabinofuranosylcytosine (ara-C) on proto-oncogene expression in U-937 cells. The results demonstrate that ara-C inhibits both U-937 proliferation and c-myc expression in a concentration- and time-dependent manner. At non-toxic concentrations of ara-C, these decreases in c-myc RNA occur in the absence of changes in the level of actin transcripts. The results also demonstrate that ara-C increases c-fos but not c-fms expression. Similar findings have been obtained with retinoic acid. Furthermore, although both agents induce a more mature U-937 phenotype, ara-C is a relatively weak inducer of these cells. These findings would suggest that the changes in proto-oncogene expression induced by ara-C may be related to induction of differentiation or the inhibitory effects of this agent on proliferation.

Actins↗
PubMed1985

Reaction of substrates with 35S-thiophosphorylated succinyl-CoA synthetase of pig heart. Similarities to the case of the Escherichia coli enzyme.

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and kcat values of 3 microM and 0.23 s-1, respectively. The corresponding values with GTP as substrate were 48 microM and 65 s-1. 35S-thiophosphorylated enzyme was prepared by incubation of pig heart succinyl-CoA synthetase with [35S]GTP gamma S. A comparison was made of thiophosphoryl group release by substrates from this alpha beta (one active site) enzyme with that of the alpha 2 beta 2 (two active sites) Escherichia coli enzyme (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119; Nishimura, J. S., and Mitchell, T. (1984) J. Biol. Chem. 259, 9642-9645). It was found, as in the case of the E. coli enzyme, that thiophosphoryl group release by GDP and by succinate plus CoA was stimulated by succinyl-CoA and GTP, respectively. The same result was observed at 1, 0.1, and 0.01 mg/ml, lending assurance that these phenomena were not exhibited by an aggregated form of the pig heart enzyme. While an alternating-sites catalytic cooperativity model is not ruled out for the E. coli enzyme, it is proposed that the NTP- and succinyl-CoA-stimulated release of thiophosphoryl groups from either enzyme involves a "same-site" mechanism, to be distinguished from an "other-site" mechanism.

Acyl Coenzyme A↗
PubMed1985

Human c-myc and N-ras expression during induction of HL-60 cellular differentiation.

The genome of the human HL-60 promyelocytic leukemia cell contains amplified c-myc sequences and the transforming N-ras oncogene. The present study has monitored c-myc and N-ras expression in HL-60 cells during induction of myeloid and monocytic differentiation with dimethyl sulfoxide, hexamethylene bisacetamide, 12-O-tetradecanoylphorbol-13-acetate and 1,25 dihydroxy-vitamin D3. The results demonstrate that induction of HL-60 differentiation is associated with decreases in c-myc RNA, while there is little if any effect on expression of the N-ras gene. Although the diminution in c-myc expression occurred as an early event in the induction of HL-60 differentiation, the rate of decrease in c-myc transcripts varied with respect to cessation of proliferation. Thus, the appearance of the mature phenotype and loss of proliferative capacity are associated with declines in c-myc RNA, while these events appear to occur in the absence of significant alterations in N-ras expression.

Acetamides↗
PubMed1985

Effects of 1-beta-D-arabinofuranosylcytosine and phorbol ester on differentiation of human K562 erythroleukemia cells.

We have previously demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces hemoglobin synthesis in human K562 erythroleukemia cells. The present study extends these findings by demonstrating that ara-C treatment of K562 cells results in both increased heme synthesis and accumulation of alpha-, gamma-, epsilon-, and zeta-globin RNA. The results also demonstrate that ara-C enhances K562 cell surface expression of glycophorin. Furthermore, we demonstrate that phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) inhibits the effects of ara-C on heme production, accumulation of globin RNA, and glycophorin expression. The inhibitory effect occurs maximally when K562 cells are treated with TPA before undergoing ara-C-induced commitment to erythroid differentiation. These findings suggest that TPA inhibits an early step in the process required for ara-C to enhance expression of genes involved in the erythroid program.

Cell Differentiation↗
PubMed1984

Adenosine 5'-O-(3-thio)triphosphate, a substrate and potent inhibitor of Escherichia coli succinyl-CoA synthetase. Additional evidence for a cooperative alternating-sites mechanism.

Adenosine 5'-O-(3-thio)triphosphate (ATP gamma S) has been shown to be a potent inhibitor of Escherichia coli succinyl-CoA synthetase. This inhibition was competitive with respect to ATP and GTP (Ki values of 0.8 and 0.7 microM, respectively) and mixed with respect to CoA and succinate. ATP gamma S previously had been shown to be a weak substrate of the enzyme, probably because of the relatively sluggish reactivity of the thiophosphoryl enzyme intermediate (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119). In our work, reaction of thiophosphoryl enzyme with ADP was greatly stimulated by succinyl-CoA, an observation that is consistent with the concept of alternating-sites cooperativity. Thiophosphoryl group release did not appear to be accompanied by "other-site" phosphorylation, in contrast to ATP stimulation of thiophosphoryl group release in the presence of succinate and CoA (Wolodko et al., see above). In addition, ADP did not appear to be required in the latter reaction.

Acyl Coenzyme A↗
PubMed1984

Escherichia coli succinyl coenzyme A synthetase. Inhibition of ATP-stimulated succinate----succinyl coenzyme A exchange at low succinyl coenzyme A concentrations by an ADP trap.

The hypothesis that Escherichia coli succinyl-CoA synthetase functions by a cooperative alternating sites mechanism is based largely on the results of [18O]phosphate exchange experiments (Bild, G. S., Janson, C. A., and Boyer, P. D. (1980) J. Biol. Chem. 255, 8109-8115). In those experiments, [18O]Pi----succinate (predominantly) exchange appeared to proceed at greater rates (relative to the apparent amount of succinyl-CoA released from the enzyme) at low ATP in incubations containing ATP, CoA, succinate, [18O]Pi, 0.48 M hydroxylamine (as a succinyl-CoA trap), and a pyruvate kinase-lactate dehydrogenase ADP trap. The conclusion arrived at was that succinyl-CoA binding at one site was inversely related to ATP binding at the second site. Thus, the residence time of succinyl-CoA binding at a site would be longer at lower ATP concentrations. Our experiments show that, under the incubation conditions described by Bild et al. (Bild, G. S., Janson, C. A., and Boyer, P. D. (1980) J. Biol. Chem. 255, 8109-8115), succinyl-CoA is not efficiently trapped. Thus, at ATP concentrations from 3.6 to 150 microM, concentrations of succinyl-CoA from 13 to 78 microM were observed. Succinate----succinyl-CoA exchange reactions carried out in this range of ATP and subsaturating succinyl-CoA concentrations were found to be markedly inhibited by the addition of the ADP trap. This inhibition was more pronounced at higher ATP levels. At a saturating succinyl-CoA concentration (1.5 mM), addition of the ADP trap actually stimulated succinate----succinyl-CoA exchange. Under these conditions, ATP----Pi exchange was greatly depressed. These results are interpreted as follows. ADP is required for optimal binding of succinyl-CoA, but only when the latter is present at subsaturating concentrations; thus, the ADP trap inhibits the reaction. ATP exerts its stimulatory action on succinate---- succinyl-CoA exchange through an "other site" effect, i.e. in binding to the noncatalytic site of succinyl-CoA synthetase, it facilitates binding and release of succinyl-CoA at the catalytic site. ATP may also exert negative effects by inhibiting other site binding of ATP or by interfering with same site succinyl-CoA binding at subsaturating concentrations of the latter. These data support the notion that a half-sites mechanism applies to succinyl-CoA synthetase, but suggest that the [18O]Pi----succinate exchange data which have been instrumental in development of the cooperative alternating sites hypothesis should be re-evaluated.

Acyl Coenzyme A↗
PubMed1984

Induction of terminal differentiation in human K562 erythroleukemia cells by arabinofuranosylcytosine.

We have previously demonstrated that continuous exposure of human HL-60 human promyelocytes to 1-beta-D-arabinofuranosylcytosine (ara-C) results in the induction of terminal differentiation to monocyte-like cells. The present study extends these findings by demonstrating that ara-C induces hemoglobin synthesis in human K562 erythroleukemia cells. This effect occurs maximally at an ara-C concentration (5 X 10(-7) M) that results in K562 cytostasis. In contrast to the reversible effects of hemin and hydroxyurea on globin synthesis in this cell line, we have found that the induction of K562 hemoglobin synthesis by ara-C is irreversible. An induction of K562 hemoglobin synthesis also occurs with aphidicolin, another inhibitor of S-phase DNA synthesis, but not with vinblastine, an inhibitor of mitosis. Finally, ara-C induction of a differentiated K562 phenotype is accompanied by the loss of self-renewal capacity, a finding consistent with terminal differentiation.

Cell Differentiation↗
PubMed1984

Involvement of spermidine in proliferation and differentiation of human promyelocytic leukemia cells.

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of cell proliferation and differentiation. Previous studies, however, have demonstrated that the polyamines are essential for proliferation, but not differentiation, of HL-60 human promyelocytic leukemia cells. We have extended these findings by demonstrating a highly significant relationship between intracellular spermidine levels and HL-60 proliferation. However, in contrast to previous studies, we have also demonstrated that induction of HL-60 differentiation with dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, or retinoic acid is inhibited by alpha-difluoromethyl ornithine (DFMO) depletion of intracellular putrescine and spermidine. Further, the addition of exogenous spermidine abrogates DFMO inhibition of HL-60 differentiation, thus confirming the involvement of this polyamine in the expression of a differentiated phenotype. The discrepancy between our results and those of previous studies probably stems from the nearly complete, rather than partial, depletion of intracellular spermidine achieved in the present work. The results of the present study thus demonstrate the involvement of spermidine in both proliferation and induction of HL-60 differentiation with certain agents.

Acetamides↗
PubMed1984

Polyamine requirements for induction of HL-60 promyelocyte differentiation by leukocyte-conditioned medium and phorbol ester.

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of both proliferation and differentiation. Spermidine is required for DNA replication, and the intracellular depletion of this polyamine can be used to distinguish cellular events related to proliferation. We have demonstrated previously that depletion of intracellular spermidine results in cytostasis of human HL-60 promyelocytes. The inhibition of HL-60 proliferation is associated with inhibition of differentiation induced by dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, and retinoic acid. The present studies extend these findings by monitoring the effects of spermidine depletion and inhibition of DNA replication on induction of HL-60 differentiation by leukocyte-conditioned medium and phorbol ester. The results demonstrate that both inducers enhance intracellular polyamine levels. However, depletion of intracellular spermidine inhibits induction of HL-60 differentiation by leukocyte-conditioned medium, but not by phorbol ester. Further, the addition of exogenous spermidine abrogates the inhibition of proliferation, and leukocyte-conditioned medium induced HL-60 differentiation, confirming the requirement of this polyamine for the expression of a differentiated phenotype. The present results and our previous findings suggest that spermidine is required for induction of HL-60 differentiation by a variety of agents, including leukocyte-conditioned medium, but that phorbol ester induces HL-60 differentiation in the absence of polyamine biosynthesis and DNA replication.

Antibodies, Monoclonal↗
PubMed1983

Affinity labeling of succinyl-CoA synthetase from Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate.

The 2'3'-dialdehyde of adenosine 5'-diphosphate, oADP, exhibited the properties of an affinity label with Escherichia coli succinyl-CoA synthetase. Inactivation of this synthetase by oADP followed pseudo-first-order kinetics and was competitively blocked by ADP. The stoichiometry of labeling of the synthetase was 1 mol/mol alpha beta or, extrapolated, 2 mol/mol inactive alpha 2 beta 2 molecule. oADP also exhibited the properties of a substrate, bringing about rapid dephosphorylation of the enzyme. Further specificity of oADP was demonstrated in partially inactivated succinyl-CoA synthetase by selective inhibition of the succinate in equilibrium succinyl-CoA exchange reaction, in comparison to the CoA in equilibrium succinyl-CoA exchange reaction. Modification of the synthetase by oADP resulted in cross-linking of the enzyme, casting uncertainty over the subunit binding site for ADP. Modification of the synthetase by ADP-2'-semialdehyde occurred at a faster rate than that by oADP but exhibited biphasic inhibitor concentration dependence and did not exhibit saturability.

Adenosine Diphosphate↗
PubMed1983

Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging.

For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. We have studied the binding of 125I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind (formula; see text). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10(8) M-1 are not likely to be useful for drug targeting or tumor imaging.

Animals↗
PubMed1983

The role of social support in the functioning of patients with unipolar depression.

The authors studied 44 outpatients with unipolar depression to determine the association among social support systems, life events, social adjustment, and depressive symptoms. Social support had a reasonably high correlation with outcome measures. Patients with high social support had significantly better scores than those with low social support on the Hamilton Rating Scale for Depression and the Social Adjustment Rating Scale Self-Report. These data corroborate the hypothesis that social support systems play a critical role in the ongoing functioning of a defined group of depressed patients.

Adult↗