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Biomedical subjects

R E Gallagher

Publications and source records attributed to R E Gallagher.

At least 55 records · Page 3Linked to original sources
PubMed1989

Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides.

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.

Cell Differentiation↗
PubMed1989

Variable regulation of sensitivity to retinoic acid-induced differentiation in wild-type and retinoic acid-resistant HL-60 cells.

The initial cell association and metabolic conversion of retinoic acid (RA) by HL-60 cells in serum-free, transferrin/insulin-supplemented, RPMI 1640 medium was greater than or equal to 10-fold greater than in RPMI 1640 medium containing 10% fetal bovine serum (FBS). This was paralleled under the serum-free conditions by 10-fold greater sensitivity to RA-induced differentiation, which was partially reversed by the addition of purified bovine serum albumin to the same concentration present in 10% FBS. In serum-free HL-1 medium, HL-60 cell sensitivity to RA-induced differentiation was approximately 250-fold less than in serum-free RPMI 1640 medium but, in this comparison, there was little difference in RA cell association or metabolism. A greater than 200-fold RA-resistant HL-60 subline had RA cell-association and metabolism rates similar to those of wild-type cells under all culture conditions. No significant qualitative differences in the high performance liquid chromatography elution patterns of polar metabolites were observed under any circumstances. These results indicate that inherent cellular properties, not associated with gross differences in RA uptake or metabolism, primarily determined the relative sensitivity or insensitivity of HL-60 cells to RA-induced differentiation but that RA responsiveness was markedly regulated by extracellular factors, one of which, serum albumin, appeared to act by decreasing the initial cell association and metabolism of RA, whereas other, as yet unidentified exogenous factors, may have acted independently of these functions.

Cell Differentiation↗
PubMed1989

Stability of multiple antigen receptor gene rearrangements and immunophenotype in Hodgkin's disease-derived cell line L428 and variant subline L428KSA.

The Hodgkin's disease (HD) derived cell line L428 and a phorbol ester-selected subline L428KSA, which have been independently passaged in tissue culture for several years, were studied for possible antigen receptor gene and immunophenotypic differences. Multiple but identical alterations of these genes were found, including: the deletion of one and rearrangement of the other immunoglobulin (Ig) heavy chain allele; the rearrangement of one kappa and one lambda light chain allele; and the rearrangement of one T cell receptor (TCR) beta allele. Restriction mapping of the Ig heavy chain locus indicated that rearrangement of the retained allele produced a JH-C gamma 4 fusion product by an isotype switch mechanism. The 14q+ chromosome [t(14q32;?)] present in both cell cultures derived either from translocation 5' (telomeric) to the rearranged JH allele or 3' (centromeric) to the deleted Ig heavy chain allele and did not involve detectable rearrangement of the c-myc, bcl 1, or bcl 2 oncogenes. No differences in the immunophenotype were found between the L428 and L428KSA cells: both expressed leukocyte activation antigens and some determinants associated with myelomonocytic cells but no lymphoid markers. It is postulated that these phenotypic characteristics derived from secondary genetic events/differentiative reprogramming which produced extinction of primary lymphoid characters, including terminal deoxynucleotidyl transferase (TdT) essential to generation of the Ig and TCR gene rearrangements, and expression of an incomplete set of myelomonocytic markers.

Blotting, Southern↗
PubMed1989

Expression of retinoic acid receptor-alpha mRNA in human leukemia cells with variable responsiveness to retinoic acid.

Retinoic acid receptor (RAR)-alpha mRNA expression was studied in a variety of myeloid leukemia cells with variable responsiveness to the induction of terminal differentiation by retinoic acid (RA). Cells from both the wild-type (wt), RA-responsive HL-60 promyelocytic leukemia cell line and a selected greater than or equal to 300-fold RA-resistant subline expressed approximately equal amounts of two RAR-alpha transcripts, 4.0 and 3.1 kb in size. In wt cells, the RAR-alpha did not change during induction of granulocyte differentiation by RA or macrophagic differentiation by 12-0-tetradecanoylphorbol-13-acetate (TPA). Relative to HL-60 cells, other cultured and fresh myeloid leukemia cells expressed 2.5-fold less to equal amounts of the RAR-alpha transcripts. The relative expression in six cases of acute promyelocytic leukemia (APL; two RA-responsive; one, previously treated with 13-cis-RA in vivo, equivocally RA-responsive) and one case of acute myelogenous leukemia (AML) with promyelocytosis (RA unresponsive) was 0.91 +/- 0.14 versus 0.53 +/- 0.14 for eight cases of nonpromyelocytic AML (p congruent to 0.001). Lymphoid leukemia cells expressed 2- to 5-fold less RAR-alpha mRNA. No qualitative variations in the mRNA transcripts were observed, although the 3.1 kb transcript was relatively decreased in three cases. The RAR-alpha gene was not amplified or detectably rearranged in any DNA source, although an apparent EcoRI restriction fragment length polymorphism was observed. It is concluded (a) that the steady-state level of RAR-alpha mRNA is not tightly correlated with natural responsiveness/unresponsiveness or, in some instances, acquired resistance to RA-induced differentiation and (b) that further studies are needed to determine if the mean 1.7-fold higher RAR-alpha mRNA level in APL cells could be an essential factor in the RA-responsiveness of APL cells, as primarily regulated at a different molecular level.

Adult↗
PubMed1988

Cell cycle-dependent reactivity with the monoclonal antibody Ki-67 during myeloid cell differentiation.

The specificity and sensitivity of the monoclonal antibody Ki-67 in identifying proliferating cell compartments was tested with the human promyelocytic leukemia cell line HL-60 using multi-parameter flow cytometry. While correlated measurements of DNA content and Ki-67 immunofluorescence indicated that the antigen was present in all phases of the cell cycle, reactivity with the antibody was highest in proliferating S and G2+M cells. The analysis of the BrdU content of cells sorted on the basis of reactivity with Ki-67 showed a correlation between Ki-67 reactivity and BrdU uptake. In HL-60 cells induced to differentiate with dimethyl sulfoxide (DMSO), the loss of reactivity with Ki-67 paralleled the exit of cells from the cell cycle. This was not observed in DMSO-resistant HL-60 cells. These results validate the usefulness of the Ki-67 antibody for determining the proliferative stage of mammalian cells in culture.

Antibodies, Monoclonal↗
PubMed1987

Effects of interferon and retinoic acid on the growth and differentiation of clonogenic leukemic cells from acute myelogenous leukemia patients treated with recombinant leukocyte-alpha A interferon.

Studies with human myeloid leukemia cell lines indicate that combined interferon (INF) and retinoic acid (RA) have greater effects in inhibiting cell growth and in inducing terminal differentiation than either agent alone. Consequently, we studied the effects of these agents, singly and in combination, on fresh leukemic blast cells obtained from 13 acute myelogenous leukemia (AML) patients, most of whom were subsequently treated with recombinant leukocyte-alpha A interferon (rINF-alpha A). The in-vitro response to rINF-alpha A and RA was assessed in an established myeloid leukemic blast cell clonogenic assay containing conditioned medium from phytohemagglutinin-stimulated lymphocytes. Strong inhibition of colony cell growth (greater than or equal to 50%) was observed in 4/10 cases treated with rINF-alpha A alone, but only at high concentration (greater than or equal to 2500 U/ml) and in 4/10 cases treated with RA alone (5 X 10(-8) M or 5 X 10(-7) M). Combined rINF-alpha A and RA augmented the inhibition of primary or secondary colony cell growth in 5/8 evaluable cases. Stimulation of leukemic cell differentiation was observed in 1/8 cases by rINF-alpha A alone and in 4/7 cases by RA alone. Combined rINF-alpha A and RA enhanced cell differentiation in 4/7 cases. In addition, increased inhibition of clonal cell growth and/or differentiation by RA alone was observed in 2/5 cases following in-vivo rINF-alpha A treatment. These results suggest that treatment with combined rINF-alpha A and RA may be rewarding in some cases of AML.

Cell Differentiation↗
PubMed1986

Evaluating the cancer education program: examining a range of approaches.

The objectives of this article are to provide people who are responsible for cancer education programs with a broadened perspective of the rich array of evaluation strategies available and responsive to the evaluation needs of cancer education programs, and to summarize selected literature resources useful for the development of an evaluation plan. This article reviews limitations found in previous evaluations of medical education programs and provides a descriptive survey of diverse strategies for the evaluation of cancer education programs. The characteristics, requirements, utility, audience, constraints, and resources are presented. Two cancer related case studies are discussed in detail as illustrations of the diversity of strategies available to evaluate cancer education programs.

Evaluation Studies as Topic↗
PubMed1986

Hyposialylation of differentiation-inducer-resistant HL-60 cells.

The total sialic acid concent of retinoic acid (RA)-resistant or 6-thioguanine (6TG)-resistant HL-60 cells was more than tenfold lower and of dimethylsulfoxide (DMSO)-resistant HL-60 cells was approximately twofold lower than that of parental, wild-type (wt) HL-60 cells. Neuraminidase-inaccessible, ie residual cell-associated sialic acid after neuraminidase treatment, was four- to twelvefold lower in the three differentiation-inducer-resistant sublines than in the parent line. Neuraminidase treatment of 125I-labeled surface membrane glycoproteins (SMGs) from wt HL-60 cells converted the two-dimensional gel electrophoretic pattern to one having features in common with RA- and 6TG-resistant cells. However, neuraminidase treatment did not alter the sensitivity of wt HL-60 cells to differentiation induction by RA, hypoxanthine (purine base), or DMSO. These results indicate that differences in peripheral, neuraminidase-accessible sialic acids are important determinants of the gel electrophoretic mobility of the SMGs of the HL-60 line and sublines but are not likely related to the differentiation-resistance mechanism. Further studies are required to determine if hyposialylation of cryptic, neuraminidase-inaccessible sites has functional significance.

Cell Differentiation↗
PubMed1985

Interhemispheric transfer and the completion of reversible operations in non-conserving children.

Evidence that concerns interhemispheric transfer deficits in young children is reviewed, and it is suggested that the late maturation of the corpus callosum may prevent the transfer and thus verbal expression of knowledge concerning equivalence relationships in non-conservers - operations normally performed by the right hemisphere. Because previous findings with "split-brain" patients indicate that interhemispheric transfer can be achieved when questions are phrased in the context of a reward (e.g., "preference"), questions concerning the conservation of a non-equivalent volume were posed to 65 non-conservers in terms of an imagined reward (preference condition). Questions also were phrased so as to require comparative-relational determinations as well as expectation of an empirical reversal of the stimulus conditions so as to determine differences/similarities in the reasoning process. Significantly more non-conservers demonstrated an awareness of empirical reversal or performed the preference relational operation correctly as compared to conditions that required comparative-relational judgments. The evidence also suggests the presence of two fundamentally distinct cognitive approaches to the problem, which appear to resist integration in children of this age group.

Child Development↗
PubMed1985

Characterization of differentiation-inducer-resistant HL-60 cells.

Sub-lines of the cultured human promyelocytic leukemia cell line HL-60 were individually selected for their ability to sustain exponential growth in the presence of 3 structurally-unrelated inducers of granulocytic differentiation - retinoic acid (RA), dimethylsulfoxide (DMSO), and 6-thioguanine (6TG). Selections were made by step-wise augmentation to final drug concentrations of 10(-3)mM RA, 169mM (1.2%) DMSO and 0.12mM (20 micrograms ml-1) 6TG. In addition to growth resistance, cells in each sub-line displayed variable cytodifferentiation resistance to each of the 3 selective agents, which was quantitated as the ratio of the concentration of drug required to induce differentiation in 50% of the cells in each resistant sub-line versus comparably-passaged wild-type HL-60 cells. The levels of resistance/cross-resistance were as follows: RA-resistant (res) sub-line greater than 2700-fold to RA, 1.3-fold to DMSO and greater than 1.5-fold to hypoxanthine (HXN; the noncytotoxic purine base inducer analogue of 6TG); DMSO-res sub-line 2.5-fold to DMSO, 137-fold to RA and greater than 1.5-fold to HXN; and 6TG-res sub-line greater than 1.5-fold to HXN, 9-fold to RA and 1.6-fold to DMSO. These sub-lines were not cross-resistant to sodium butyrate (NaBut), a monocyte inducer, or to 12-0-tetradecanoylphorbol 13-acetate (TPA), a macrophage inducer. HL-60 sub-lines selected by exposure to a single high concentration of 5-bromo-2'-deoxyuridine (BUdR; 3.3 X 10(-2)mM) or oubain (Ou; 5 X 10(-3)mM) were not or were slightly cross-resistant to either granulocyte or monocyte inducers. Although some variations in line/sub-line phenotype were observed, this was minor compared to the quantitative variations in response to individual inducing agents. The RA-res and 6TG-res sub-lines contained numerous double minute chromosomes (indicators of amplified genes) which were either absent or present in much smaller numbers in the parental wild-type cells or in the other drug-resistant sub-lines. There was little change or a decrease in the amplification level of the known amplified oncogene c-myc in the various drug-resistant sub-lines compared to wild-type HL-60 cells. These results (a) confirm that the neutrophilic granulocytic and monocytic/macrophagic differentiation programs in HL-60 cells are mechanistically different and separable; (b) suggest that both agent-specific and common quantitative alterations contribute to the mechanism(s) for resistance to granulocyte differentiation; and (c) suggest that the latter quantitative defects could be related to amplification of genes other than c-myc.

Bromodeoxyuridine↗
PubMed1985

Surface membrane glycoproteins of wild-type and differentiation-inducer-resistant HL-60 cells.

Surface membrane glycoproteins (SMGs) of cells from the parental wild-type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two-dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritide) and peptide (1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pl], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55- to 60-kD neutrophil-associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross-resistance mechanism(s) for RA/purine bases but not for DMSO.

Cell Differentiation↗
PubMed1984

Two brains, one child: interhemispheric information transfer deficits and confabulatory responding in children aged 4, 7, 10.

In an investigation of developmental trends in the ability to transfer information between the cerebral hemispheres, 138 children, ages 4, 7, and 10, viewed pictures presented to either the right or left hemisphere and were asked to describe what was viewed. Responses were socored for the number of accurate pictorial features reported (inclusion score), confabulation (erroneous embellishment), perceptual, semantic, and syntactic errors. Misses (failure to provide a response) were also noted. A preponderance of errors were found, such that 4-year olds engaged in a significant degree of confabulatory responding following right vs. left presentation. A significant inverse relationship was found between inclusion and confabulation such that the larger the gap in information reported the greater was the tendency to insert (confabulate) erroneous material. In addition, all children were given a tactile-shape recognition task employing both transfer and non-transfer conditions and requiring tactile exploration and visual recognition. Significantly more errors were found on the transfer vs. non transfer task among the 4-year olds only. In that deficits in information transfer were not complete but only partial, and given that significant intra-hemispheric (non-transfer) errors were also discovered, it was concluded that the hemispheres of children age 4 are not completely but only partially disconnected, and that intra-hemispheric (cortical) immaturity plays a significant role in the production of processing deficits.

Cerebral Cortex↗
PubMed1984

Cytotoxic and cytodifferentiative components of 6-thioguanine resistance in HL-60 cells containing acquired double minute chromosomes.

As an experimental strategy for potentially dissociating and studying the cytotoxic and cytodifferentiative antileukemic effects of 6-thioguanine (6-TG), cultured human promyelocytic leukemia cells (HL-60) were serially selected for growth in increasing concentrations of 6-TG (0.5 to 50 micrograms/ml). Three acquired characteristics, cytotoxic resistance, cytodifferentiative resistance, and double minute chromosomes (DM), were monitored at successive 6-TG selection levels. Approximately 200-fold resistance to the cytotoxic effect of 6-TG was acquired at the first selection step, and it neither increased at higher 6-TG selection levels nor reverted to greater sensitivity in cells subcultured off of drug. This was due to the irreversible loss of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. In contrast, a lesser, not completely quantifiable, degree of resistance developed to the cytodifferentiative effects of the purine nucleobases hypoxanthine and 6-TG which varied as a function of 6-TG selection pressure. Numerous DM, not observed in the parental wild-type HL-60 cells, appeared at 6-TG (0.5 micrograms/ml) selection which varied substantially in parallel with 6-TG selection pressure up to 6-TG (20 micrograms/ml). At higher selection levels (50 micrograms/ml or prolonged culture on 20 micrograms/ml), a marked decrease in DM occurred which was associated with the acquisition of new marker chromosomes. The most consistent marker was a chromosome 6 with additional material in the short arm (6p+); this was noted as a single copy in the basal 6-TG/20 subline but as two copies (trisomy 6; 2p+) in independently selected higher 6-TG-resistant subcultures. These cytogenetic findings suggest the presence of amplified genes which increased in number and shifted from a predominance in extra-chromosomal DM to intrachromosomal sites as a function of 6-TG selection. Among the 6-TG-resistant sublines, there was no change or a decrease in the amplification level of the known amplified oncogene c-myc from that demonstrated in parental HL-60 cells. Although proof requires detailed analyses with specific gene probes, the overall results imply that: (a) the cytotoxic component of the resistance is due to an invariant loss of HPRT which, therefore, is not likely to be related to amplified genes; (b) the cytodifferentiative component of the resistance is due to a positively selectable mechanism which could be directly or indirectly related to 6-TG-selected amplified genes; and (c) variations in the cytogenetic indicators of amplified genes and the resistance to 6-TG cannot be simply ascribed to quantitative variations in c-myc amplification.

Animals↗
PubMed1983

Cell surface membrane protein changes during the differentiation of cultured human promyelocytic leukemia HL-60 cells.

The human promyelocytic leukemia cell line HL-60 was induced to differentiate in vitro by treatment with dimethyl sulfoxide or retinoic acid. Morphological maturation was accompanied by a total loss of transferrin binding and a 7-fold increase in the percentage of cells reducing nitro blue tetrazolium. Cell surface membrane proteins and glycoproteins were labeled with 125I by the lactoperoxidase-H2O2 or 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodo-Gen) methods and analyzed by two-dimensional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A minimum of 12 cell surface proteins were unchanged, 3 proteins (Mr 95,000, 87,000, and 77,000) were lost, and up to 7 new proteins (Mr 270,000, 240,000, 150,000, 135,000, 58,000, 56,000, and 50,000) appeared during HL-60 cell differentiation. The kinetics of disappearance of one major labeled cell surface protein (Mr 95,000) within two days during treatment with retinoic acid correlated with the loss of cellular transferrin binding. This protein was identified as the transferrin receptor by affinity absorption of extracts of 125I-surface protein-labeled cells to transferrin-Sepharose beads. The affinity-purified component had molecular weights of 190,000 and 95,000 under nonreducing and reducing conditions, respectively, confirming its dimeric structure. Two-dimensional electrophoresis of cell surface membrane-labeled proteins of normal human granulocytes confirmed the absence of the transferrin receptor and identified cell surface proteins with molecular weight and pI values corresponding to three of the new cell surface proteins which appeared during HL-60 maturation. The most intensely labeled of these had a molecular weight of about 55,000, and was confirmed as being identical to the corresponding Mr 58,000 HL-60 cell surface membrane protein by one-dimensional peptide-mapping analysis. This prominent new Mr 55,000 to 58,000 protein increased continuously throughout retinoic acid-induced maturation and was identified as a major terminal myeloid differentiation cell surface membrane protein.

Cell Differentiation↗