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J Botling

Publications and source records attributed to J Botling.

11 recordsLinked to original sources
PubMed2000

The role of RAR and RXR activation in retinoid-induced tissue factor suppression.

Excessive expression of tissue factor (TF) is a common finding in leukaemic cells and may contribute to thrombotic complications in patients. Retinoic acid has been shown to induce differentiation and reduce TF expression in acute promyelocytic leukaemia (APL) cells in vitro, and to induce remission in APL patients. Treatment of the APL cell line NB4 with the specific retinoic acid receptor-alpha (RARalpha) agonists Ro4-6055 or TTNPB resulted in down-regulation of TF expression and in induction of differentiation. The activation of RARbeta, RARgamma or retinoid X receptor (RXR) did not suppress the constitutive TF expression in NB4 cells. Moreover, the RARalpha antagonist Ro41-5253 blocked the retinoid-induced down-regulation of TF. In contrast, in the monoblastic U-937 cell line only a partial suppression of TF antigen expression and activity was observed by treatment with the RAR agonist TTNPB or the RXR agonist SR11237 alone. However, the combination of TTNPB and SR11237 resulted in a pronounced down-regulation of TF expression and induction of differentiation in U-937 cells. We show for the first time that the activation of both subunits of the RARalpha-RXR transcriptional complex is needed for TF suppression in U-937 cells, whereas in NB4 cells RARalpha activation alone is sufficient. Thus, distinct molecular mechanisms for TF suppression seem to be operating in leukaemic cell lines of different origin.

Base Sequence↗
PubMed1997

Tissue factor expression in human monocytic cell lines.

Tissue factor (TF) is a main initiator of the coagulation protease cascade. Control of the expression of this protein in monocytes is essential, since these cells are the only circulating blood cells responsible for TF expression. In this report we have used two human cell lines, arrested at different stages of monocytic differentiation, to study TF expression. The monoblastic cell line U-937 had a constitutive expression of TF surface protein and low TF mRNA levels detected by immunofluorescence or quantitative reverse transcriptase polymerase chain reaction respectively. The phorbol-12-myristate-13-acetate (PMA) was a potent enhancer of TF expression in U-937. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF) had no effect on TF expression in U-937. The Mono Mac 6 cell line, with phenotypic features similar to that of mature monocytes, expressed lower basal levels of TF mRNA and surface TF antigen. However, in Mono Mac 6 cells TF expression was induced in response to LPS and TNF. These results indicate differences in basal and induced TF expression between U-937 and Mono Mac 6 cell lines.

Carcinogens↗
PubMed1997

Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation.

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. In this report we show that efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. Also, we demonstrate that the allosteric inhibition of RXR is not obligatory and that RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differ between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. Finally, the AF2 activation domain of both subunits contribute to activation even if only one of the receptors is associated with ligand. Our data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner.

Cell Differentiation↗
PubMed1997

Tissue and differentiation specific expression on the endogenous retrovirus ERV3 (HERV-R) in normal human tissues and during induced monocytic differentiation in the U-937 cell line.

ERV3 (HERV-R) is a complete, single copy human endogenous retrovirus located on the long arm of chromosome 7. The open reading frame in its envelope gene has been conserved during evolution but the gag and pol genes contain in-frame termination codons. To find a suitable experimental model system for analysis of the functions of the ERV3 genome, an extensive screening study of different normal and neoplastic human tissues was performed. Most tissues express low levels of the ERV3 env mRNA although high expression levels are observed in placenta, sebaceous glands, adrenals, testis, bronchial, epithelium and the monocytic cell line U-937. In U-937 cells the ERV3 env expression varied in a manner related to the differentiation status of the cells; being highest in the terminally differentiated non proliferating cells. U-937 cells can be induced to differentiate from the monoblastic to the mature monocyte/macrophage stage upon stimulation by several substances such as phorbolesters (TPA), Vitamin D3, Retinoic Acid (RA) and combinations of some cytokines. We conclude that the ERV3 locus is expressed in a tissue and differentiation specific way and that the U-937 cell line is a suitable model system to further analyze the proposed functions of ERVs such as immunomodulation, cell fusion and protection against exogenous retroviral infections.

Cell Differentiation↗
PubMed1996

Expression of the endogenous retrovirus ERV3 (HERV-R) during induced monocytic differentiation in the U-937 cell line.

ERV3 (HERV-R) is a complete human endogenous retrovirus located on the long arm of chromosome 7. LTR-env-gene-spliced mRNA of 9 and 3.5 Kb is widely expressed in human tissues and cells, but gag-pol mRNA has not been found. Further, the env gp70 gene contains an open reading frame throughout its length and its expression has recently been detected as a full-length protein. The highest expression of ERV3 detected so far is in placenta and the lowest in cytotrophoblasts and choriocarcinoma cell lines. In this report we have studied ERV3 mRNA and protein expression in the human monoblastic cell line U-937 during differentiation into monocytes/macrophages. Differentiation of U-937 cells was induced by 1,25a-dihydroxyvitamin D3 (vitD3), retinoic acid (RA), gamma interferon (IFN-gamma) and phorbol-myristate-acetate (PMA-TPA). The expression of ERV3 env mRNA was found to be differentiation-associated, with high expression detected in the late stages of monocytic development. Using TPA, the expression of ERV3 env was detected as 9- and 3.5-kb transcripts by Northern blotting, as mRNA by in situ hybridization and as a cytoplasmic 65-kDa protein by immunofluorescence and Western blots. Low levels of basal expression were found, with up-regulation of both message and protein at 24 to 48 hr after addition of TPA. Induction with vitD3, IFN-gamma and RA produced higher levels of mRNA at earlier time points. It is concluded that the U-937 cell line represents an excellent model system for further studies to study the relationship between ERV3 expression and cellular differentiation.

Calcitriol↗
PubMed1996

Anti-cancer drug characterisation using a human cell line panel representing defined types of drug resistance.

Differential drug response in a human cell line panel representing defined types of cytotoxic drug resistance was measured using the non-clonogenic fluorometric microculture cytotoxicity assay (FMCA). In total 37 drugs were analysed; eight topoisomerase II inhibitors, eight anti-metabolites, eight alkylating agents, eight tubulin-active agents and five compounds with other or unknown mechanisms of action, including one topoisomerase I inhibitor. Correlation analysis of log IC50 values obtained from the panel showed a high degree of similarity among the drugs with a similar mechanism of action. The mean percentage of mechanistically similar drugs included among the ten highest correlations, when each drug was compared with the remaining data set, was 100%, 92%, 88% and 52% for the topoisomerase II inhibitors, alkylators, tubulinactive agents and anti-metabolites respectively. Classification of drugs into the four categories representing different mechanisms of action using a probabilistic neural network (PNN) analysis resulted in 29 (91%) correct predictions. The results indicate the feasibility of using a limited number of cell lines for prediction of mechanism of action of anti-cancer drugs. The present approach may be well suited for initial classification and evaluation of novel anti-cancer drugs and as a potential tool to guide lead compound optimisation.

Antineoplastic Agents↗
PubMed1996

Vitamin D3- and retinoic acid-induced monocytic differentiation: interactions between the endogenous vitamin D3 receptor, retinoic acid receptors, and retinoid X receptors in U-937 cells.

Retinoic acid (RA) and 1,25 alpha-dihydroxycholecalciferol (VitD3) are potent regulators of hematopoletic differentiation. Yet, little is known as to how the RA and VitD3 receptor network operates in hematopoietic cells, and whether receptor interactions can explain the interplay between the RA- and VitD3-signaling pathways during differentiation. Therefore, we analyzed the expression, DNA binding, and transcriptional activity of the endogenous RA and VitD3 receptors [retinoic acid receptors (RARs), retinoid X receptors (RXRs), and VitD3 receptor (VDR)] in the U-937 cell line, in which RA and VitD3 induce distinct monocytic differentiation pathways. VitD3 induction resulted in the formation of VDR/RXR DNA-binding complexes on both VitD3 response elements and RA response elements (RAREs). However, transcriptional activation was only observed from a VitD3 response element-driven reporter construct. Several DNA-binding complexes were detected on RAREs in undifferentiated cells. Stimulation by RA resulted in increased RAR beta/RXR DNA binding, activated RARE-dependent transcription, and increased expression of RAR-beta. Concomitant stimulation by VitD3 inhibited the RA-stimulated formation of RAR beta/RXR heterodimers, favoring VDR/RXR binding to the RARE. Also, VitD3 inhibited the expression of CD23 and CD49f, characteristic markers of retinoid-induced U-937 cell differentiation. In contrast, neither the RA-stimulated, RARE-mediated transcription nor the induced RAR-beta expression was suppressed by VitD3, suggesting that VitD3 selectively inhibited the retinoid-induced differentiation program but not the RARE-mediated signal. These results demonstrate a complex role for VitD3 in modifying the retinoid differentiation pathway and may have implications for differentiation-inducing therapy of hematopoietic tumors.

Cell Differentiation↗
PubMed1995

CD49f (alpha 6 integrin) and CD66a (BGP) are specifically induced by retinoids during human monocytic differentiation.

Retinoic acid (RA) and 1,25(OH)2-cholecalciferol (VitD3) are potent regulators of normal and malignant myeloid cells. In the human monoblast cell line U-937 they induce terminal differentiation, and the resulting phenotypes display both common and distinct, inducer-specific, properties. This paper shows that in U-937 cells the two retinoids, all-trans and 9-cis RA, induced the expression of CD49f (alpha 6 integrin subunit) and CD66a (biliary glycoprotein, BGP) mRNA and protein. In contrast, expression of CD49f and CD66a was not found in untreated or VitD3-induced cells. Cytokine-induced modulation of CD49f and CD66a expression was restricted to the retinoid-induced U-937 cells. The retinoid specific induction of CD49f and CD66a was confirmed in the related monoblastic cell line THP-1. Human blood monocytes and the monocytic cell line Mono Mac 6 responded poorly to RA, with respect to the regulation of CD49f and CD66a expression, indicating that early monocytic precursors were targets for the retinoid-specific regulation. Thus, the expression of CD49f and CD66a is developmentally regulated and specifically induced by all-trans and 9-cis Ra in human monocytic cells.

Antigens, CD↗
PubMed1994

Development of vincristine resistance and increased sensitivity to cyclosporin A and verapamil in the human U-937 lymphoma cell line without overexpression of the 170-kDa P-glycoprotein.

A vincristine (Vcr)-resistant subline of the human histiocytic lymphoma cell line U-937 (U-937-vcr) has been established and characterized with respect to its phenotypic features, including growth rate, surface marker expression and ability to respond to differentiation-inducing agents. The sensitivity of U-937-vcr cells to the direct cytotoxicity of cyclosporin A (CsA) and verapamil (Ver), and the capacity of these drugs to modify Vcr resistance, were also examined. The U-937-vcr cells exhibited a more than 200-fold resistance to Vcr, and cross-resistance to vinorelbin and taxol. Also, there was a slight cross-resistance to colchicine, doxorubicin and VP16. However, the response of U-937-vcr to CsA or Ver alone was substantially altered, with a marked decrease in their respective IC50s. The U-937-vcr cells did not show increased levels of pgp 170. We conclude that the development of Vcr resistance was not associated with a change in the major phenotypic properties of the U-937 cell line, and that resistance modifier hypersensitivity was not associated with increase in pgp 170 expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Functional antagonism between vitamin D3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiation of U-937 cells.

1,25 alpha-Dihydroxicholecalciferol (VitD3) and retinoic acid (RA) are important regulators of the proliferation and differentiation of several cell types. This paper describes how the expression of the monocyte-macrophage Ag, CD14, and the low affinity Fc receptor for IgE, CD23, were inversely regulated during VitD3- and RA-induced monocytic differentiation of human U-937 monoblasts. PMA induced the expression of both CD14 and CD23 mRNA and protein. Exposure to VitD3 rapidly induced the de novo expression of CD14 mRNA and protein. The addition of cycloheximide completely blocked the VitD3 induction of CD14 mRNA expression, indicating that the induction was dependent on ongoing protein synthesis. While inducing CD14 expression, VitD3 concomitantly suppressed the basal, PMA-, and RA-inducible CD23 expression in a dose-dependent manner. In contrast, U-937 cells induced by RA strongly increased their expression of CD23 mRNA and protein, whereas they completely lacked detectable CD14 cell surface or mRNA expression. Furthermore, the VitD3- and the PMA-induced CD14 expression was inhibited as a temporal consequence of the RA-induced differentiation. The results suggest that there exists a functional antagonism between VitD3 and RA that may have important implications for the regulation of certain immune and inflammatory responses through their inverse effects on CD14 and CD23 gene expression.

Antigens, CD↗