The promoter of the CHO APRT gene contains three regulatory regions.
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Adenine Phosphoribosyltransferase↗
Biomedical subjects
M W Taylor
Publications and source records attributed to M W Taylor.
Reversal of interferon-gamma-resistant phenotype by poly(I:C): possible involvement of ISGF2 (IRF1) in interferon-gamma-mediated induction of the IDO gene.
Indoleamine 2,3-dioxygenase (IDO) is induced in many cell lines by interferon-gamma (IFN-gamma) treatment. IDO mRNA increases rapidly from 4 h after IFN-gamma treatment to at least 24 h after treatment in ME180 cells. The IFN-gamma-resistant mutant of ME180, IR3B6B, expresses only one-sixth the amount of IDO message after IFN-gamma treatment and very low levels of IDO. However, pretreatment of these mutants with poly(I:C) restores normal levels of IDO mRNAs and IDO activity. Since IRF1 mRNA induction is also low in IR3B6B cells after IFN-gamma treatment, we examined whether there was any relationship between IRF1 induction and IDO induction by IFN-gamma. The steady-state level of IRF1 mRNA was elevated by treating IR3B6B cells with poly(I:C) and IFN-gamma. Poly(I:C)-mediated reversal of IFN-gamma-resistant phenotype and induction of IDO and IRF1 messages are inhibited by 2-aminopurine. Transient transfection of IRF1 cDNA in ME180 cells resulted in activation of IDO transcription. Nuclear extracts prepared from IFN-gamma-treated ME180 and IR3B6B cells affected differently the mobility of a 80-bp DNA fragment of the 5' regulatory region of IDO gene. Pretreatment of IR3B6B cells with poly(I:C) and addition of IFN-gamma resulted in increased DNA binding of nuclear proteins to the DNA. Pre- and post-treatment of nuclear extract of IFN-gamma-treated ME180 cells with anti-IRF1 antibody resulted in a super shift in mobility of the probe with the abolishment of normal gel-shift pattern.(ABSTRACT TRUNCATED AT 250 WORDS)
2-Aminopurine↗
IDO mutants cross resistant to type I interferon retain p91-dependent gene induction.
Genetic analyses of mutants have yielded valuable information about p91-associated interferon signal transduction. It was thus discovered that p91 is an essential protein for the induction of both type I and type II interferons. We previously reported the development of ME180 mutants resistant to interferon-gamma because of a signaling defect resulting in the loss of IDO induction. IDO does not respond to type I interferon despite an ISRE-like sequence upstream of the coding region. However, the IDO mutants were found to be cross-resistant to the growth-inhibitory effects of type I interferon. We therefore examined the effects of both types of interferon on interferon-stimulated gene mRNA accumulation and examined alterations in cellular protein introduced by the mutation. The induction of the p91-responsive gene 6-16 was not altered in either of the mutants, and the early-induced gene IRF1 exhibited differences only in the kinetics of mRNA accumulation. The later induced gene, p68, also exhibited different kinetics, possibly reflecting the changes in IRF1. Immunoprecipitated p91 exhibited normal, interferon-induced phosphorylation in both mutants. Two-dimensional gel electrophoresis revealed that the mutant cells contained 20 peptides with altered biochemistry. These results suggest that IDO induction is controlled by a distinct set of proteins not directly correlated with p91 activation.
Cell Division↗
Interleukin 2 protects hairy leukemic cells from lymphokine-activated killer cell-mediated cytotoxicity.
Interleukin 2 (IL-2) activates natural killer cells and generates lymphokine-activated killer (LAK) cell-mediated cytotoxicity. In "adoptive immunotherapy," a combination of LAK administration and IL-2 infusion was found to be effective therapy for some tumors and ineffective for others. Here we report a novel function for IL-2, its ability to protect tumor cells (cell lines obtained from hairy cell leukemia patients) against LAK activity. The protective effect induced by IL-2 is similar to that induced by interferon (IFN). Protection by both cytokines requires new mRNA/protein synthesis; both IL-2 and IFN reduce the ability of tumor target cells to trigger LAK effector cells following binding between these two types of cells. However, endogenous IFN is not the mediator of the IL-2 protective effect against LAK activity since monoclonal antibodies against IFN-alpha and IFN-gamma did not abolish the protective effect of IL-2. In addition, IL-2 does not induce the expression of class I major histocompatibility complex antigens on the target cell surface, believed to be the signal for the IFN-induced protection against natural killer and LAK activities. Finally, leukemic cells resistant to IFN-alpha did respond to IL-2 treatment and became less sensitive to LAK cytotoxicity. Thus the ability of IL-2 to protect tumor cells from LAK activity may explain the lack of response to adoptive immunotherapy in tumors that express the IL-2 receptor.
Cytotoxicity, Immunologic↗
IFN-gamma-mediated antimicrobial response. Indoleamine 2,3-dioxygenase-deficient mutant host cells no longer inhibit intracellular Chlamydia spp. or Toxoplasma growth.
The role of indoleamine 2,3-dioxygenase (IDO) in IFN-gamma-mediated inhibition of intracellular parasite growth has been examined previously, although earlier work has been largely correlative. In this study, we defined more completely the role of IDO in the IFN-antimicrobial response. Two mutant cell lines, derived from ME180 cells and exhibiting reduced IDO activity (IR3B6A, IR3B6B) were characterized to determine if they retained the capacity to inhibit intracellular Chlamydia and Toxoplasma growth. Mutant cells treated with IFN-gamma exhibited reduced capacity to suppress pathogen growth. The expression of several IFN-regulated genes also was measured to confirm that the inability to inhibit pathogen growth was because of the lack of IDO. The expression of class II MHC, intracellular adhesion molecule-1, MxA, and P68 kinase genes was induced in the IFN-gamma-treated wild type ME180 cells, but was variable in the mutant cell lines, supporting the hypothesis that IFN-gamma-induced production of IDO is a key IFN-gamma-mediated antimicrobial mechanism.
Animals↗
Combination treatment of 2-chlorodeoxyadenosine and type I interferon on hairy cell leukemia-like cells: cytotoxic effect and MHC-unrestricted killer cell regulation.
Hairy cell leukemia (HCL) is a lymphoproliferative disorder of B lymphocytes. Interferons (IFNs), especially of the alpha (alpha) subtype, have shown a significant antitumor effect in HCL patients. However, the therapeutic effect of IFN-alpha is still rather limited. The purine analogue 2-chlorodeoxy-adenosine (2-CdA) was reported recently to be an effective agent in the treatment of HCL. In the present study, we find that the HCL cell lines HS-1 and HS-2 as well as Eskol and its IFN-resistant clone (IREs-4) are sensitive to the cytotoxic activity of 2-CdA. Combination treatment of IFN-Con1 and 2-CdA results in a synergistic effect at low doses but an additive inhibitory effect at higher concentrations. IREs-4 cells responded only to 2-CdA treatment. All the HCL cell lines are resistant to natural killer (NK) cell-mediated cytotoxicity (CMC) but are relatively sensitive to IFN-Con1-primed or interleukin-2 (IL-2)-primed NK-CMC activities. No inhibition in killing ability was measured when only the effector cells (NK) were treated with 2-CdA. Pretreatment of the HCL target cells with 2-CdA increases their susceptibility to NK-CMC. Pretreatment with IFN-Con1 can reduce the susceptibility of target cells to NK-CMC in HS-1, HS-2, and Eskol cells but not in the IFN-resistant clone IREs-4. 2-CdA abolished this IFN-induced protection against NK-CMC. Normal fibroblasts only responded to treatment with relatively high doses of 2-CdA, and only a moderate additive cell growth inhibitory effect was seen in combination of 2-CdA with IFN-Con1. Only high doses of 2-CdA increased the susceptibility of fibroblast culture to NK-CMC. Thus, combination of IFN-Con1 and 2-CdA results in an in vitro enhancement of the direct antiproliferative/cytotoxic activity of each treatment alone and increases the efficacy of the NK activity against the HCL cell lines.
Cell Division↗
Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8.
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Animals↗
An interferon resistant variant of the hairy-cell leukemic cell line, Eskol: biochemical and immunological characterization.
The B-lymphoblastoid cell line Eskol, which is composed of differentiated cells resembling hairy-cell leukemia, has been used to study the effects of type I interferon in vitro. In order to study the mechanism of delayed interferon therapy resistance, a hairy-cell leukemia-like clonal cell line (IREs-4) was isolated from Eskol after 4 months of exposure to r-metIFN-con1. When compared to Eskol cells, the IREs-4 cells were resistant to the antiproliferative effect of type I interferons as well as interferon induced protection against LAK cells. Treatment of IREs-4 with type I interferon did not induce MHC antigens, although both MHC class I and II antigens were induced in Eskol. Binding studies indicated the presence of equal numbers of high affinity binding sites with similar affinities on both cell lines. The resistant phenotype appears to result from an intracellular event which is essential to interferon signal transduction. It is hypothesized that this variant may reflect heterogeneity in the normal population of hairy-cell leukemia cells, and may explain the partial resistance of HCL patients to IFN therapy.
Blotting, Northern↗
Reversal of an interferon-gamma-resistant phenotype by poly(I:C): possible role of double-stranded RNA-activated kinase in interferon-gamma signaling.
Indoleamine 2,3-dioxygenase (IDO) is induced in neoplastic cell lines by interferon-gamma (IFN-gamma) treatment. In ME180 cervical carcinoma cells, there is a rapid increase in IDO mRNA accumulation beginning at 4 h after IFN-gamma treatment and continuing for at least 24 h. The IFN-gamma-resistant mutant of ME180, IR3B6B, expresses very low levels of IDO message after IFN-gamma treatment. However, pretreatment of this mutant with poly(I:C) restores normal levels of IDO mRNAs and IDO enzyme activity. Poly(I:C) mediated reversal of the IFN-gamma-resistant phenotype and induction of IDO mRNA are inhibited by 2-aminopurine. In vitro phosphorylation of calf thymus histone using the immunoprecipitated p68 kinase prepared from IFN-gamma-treated ME180 and IR3B6B cells revealed the deficiency of activation of this kinase in IR3B6B cells after IFN-gamma treatment, and treatment of this mutant cells with poly(I:C) restores p68 kinase activity. From these results, we conclude that a double-stranded RNA-dependent kinase is activated by IFN-gamma treatment and its activation correlates with IFN-gamma-mediated induction of the IDO gene.
Drug Resistance↗
Consensus interferon induces peak mRNA accumulation at lower concentrations than interferon-alpha 2a.
The biological activity of a novel recombinant interferon, r-metIFN-con1, which represents a consensus sequence of the most commonly appearing amino acids at each locus of 14 naturally occurring IFN-alpha s, was assessed and compared to that of IFN-alpha 2a. The increase in cellular mRNA levels for three IFN-inducible genes served as a quantitative measure of the effectiveness of the stimulation by each of the IFNs. Three cell lines were treated with equimolar amounts of two IFNs encompassing a 5 log range and mRNA was extracted at five different times after treatment. In all cases, r-metIFN-con1 produced mRNA increases at lower concentrations than IFN-alpha 2a. HLA-DR alpha mRNA, which is not affected by IFN-alpha in ME180 or Daudi cells, was also not affected by r-metIFN-con1. However, in Eskol cells, both IFNs effected an increase in HLA-DR alpha mRNA to similar levels. The r-metIFN-con1 was effective at approximately 10-fold lower molar concentrations. At effective concentrations (10-fold lower molar dose of r-metIFN-con1), both IFNs produced similar kinetics of accumulation of all three mRNAs tested. r-metIFN-con1 is therefore more effective than IFN-alpha 2a at the level of mRNA regulation as well as the antiviral and antiproliferative activities that have been reported previously.
Amino Acid Sequence↗
Correction of a deletion mutant by gene targeting with an adenovirus vector.
The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.
Adenine Phosphoribosyltransferase↗
Memory of butyrate induction by the Moloney murine sarcoma virus enhancer-promoter element.
We reported previously that a housekeeping gene could be converted to a butyrate-inducible gene by replacing its cognate promoter with the Moloney murine sarcoma virus enhancer-promoter element. In this study we report that the activated transcriptional state could be propagated from mother to daughter cells after the withdrawal of the inducer.
Adenine Phosphoribosyltransferase↗
Sodium butyrate selectively induces transcription of promoters adjacent to the MoMSV viral enhancer.
The long terminal repeat region of the Moloney murine sarcoma virus (MoMSV) was cloned upstream from the Chinese hamster ovary adenine phosphoribosyltransferase (APRT)-encoding gene (APRT) in order to enhance synthesis of the APRT protein. The replacement of the native promoter with the viral enhancer-promoter increased the enzymatic activity of APRT two- to threefold. Addition of sodium butyrate (NaBu) to the cell growth medium induced APRT activity ten- to 20-fold above wild-type levels in both transient and stable transfectants. The introduction of the APRT native promoter between the MoMSV enhancer-promoter and structural gene reduced the magnitude of the NaBu response. The bacterial cat gene was also stimulated by NaBu when linked to the viral enhancer-promoter. No NaBu response was found in constructs lacking the MoMSV enhancer region. Northern analysis and nuclear run-on experiments indicated that NaBu enhanced transcription of APRT mRNA in both transiently and stably transfected cells, but not in cells inhibited by cycloheximide. Thus, a butyrate-response element (BRE) is associated with the MoMSV enhancer and the action of the MoMSV BRE is promoter-dependent.
Adenine Phosphoribosyltransferase↗
A dual anti-tumor effect of a combination of interferon-alpha or interleukin-2 and 5-fluorouracil on natural killer (NK) cell-mediated cytotoxicity.
Previous in vitro and in vivo studies have shown a synergism between interferon (IFN) and 5-fluorouracil (5-FU) against different tumor cell lines. In the present study we report that the combination of IFN-alpha and 5-FU has a significant effect not only on the inhibition of tumor cell growth but also on the regulation of natural killer cell-mediated cytotoxicity (NK-CMC). The addition of 5-FU to effector cell population neither affects NK cell activity nor activation of NK cells by IFN or by interleukin (IL)-2. However, pretreatment of target cells with 5-FU increased their susceptibility to NK activity and abolished the protective effect induced by IFN against NK-CMC. This dual effect of IFN-alpha and 5-FU was found to be applicable to target cells of different origins including a cervical carcinoma cell line (ME-180), a hairy cell leukemia-like cell line (Eskol), a CML cell line (K-562) and a primary culture of AIDS-related Kaposi's sarcoma cells. Similar results were found with IL-2 treatment of Eskol cells but not other cells. Combination of IL-2 with 5-FU resulted in enhancement of the sensitivity of the cells to NK activity and abolished the protection against NK-CMC. Based on these results we propose that the combination of IFN-alpha and 5-FU not only has a direct growth inhibitory effect on tumor cells but also has a regulatory role on the immunological arm of the NK-CMC. Moreover, since the combination gave the same pattern of response in different tumor cells, both NK-sensitive and NK-resistant, this combination treatment may be a candidate for clinical trials in various types of tumors.
Cell Division↗
A comparison of interferon-Con1 with natural recombinant interferons-alpha: antiviral, antiproliferative, and natural killer-inducing activities.
The antiviral, antiproliferative, and natural killer (NK) cell activation by recombinant human interferon-consensus (IFN-Con1) has been compared with that of two other type I IFNs: IFN-alpha 2a (Roferon) and IFN-alpha 2b (Intron A). The specific activity (antiviral units/mg) of IFN-Con1 was 10-fold higher than that of the other two IFNs in the vesicular stomatitis virus (VSV)-HeLa antiviral assay. The antiproliferative activity on a molar basis of IFN-Con1 on Daudi cells and Eskol (a human leukemic hairy cell-like cell line) was significantly greater than that of IFN-alpha 2a and IFN-alpha 2b. IFN-Con1 also enhanced or induced NK cell killing of target cells to a greater extent than that of IFN-alpha 2a and IFN-alpha 2b. However, on antiviral unit basis, the activities were similar. These results would suggest that IFN-Con1 may be more effective at lower protein concentrations in clinical applications than other available IFNs.
Amino Acid Sequence↗
Cytokine and natural killing regulation of growth of a hairy cell leukemia-like cell line: the role of interferon-alpha and interleukin-2.
Hairy cell leukemia (HCL) is a lymphoproliferative disorder of B-lymphocytes, with pathological manifestations usually including splenomegaly and pancytopenia. Naturally occurring and recombinant interferons (IFNs), specifically of the alpha subtype, have shown a significant anti-tumor effect in HCL patients, with improvement of hematologic parameters within the first few months of treatment. The mechanisms responsible for the beneficial action of IFN-alpha in HCL patients are unclear, but several hypotheses have been suggested. Recently, a continuous line of cells (Eskol) from a patient diagnosed with hairy cell leukemia was established and shown to have several properties of a leukemic hairy cell. In the present study, we investigated the direct effect of IFN-alpha and interleukin (IL-2) on the Eskol cell line, and lymphokine regulation of natural killing (NK) activity against these cells. It was found that IFN-alpha has a direct antiproliferative effect on Eskol cells. Furthermore, Eskol cells were found to be completely resistant to NK-cell mediated cytotoxicity (CMC) but were somewhat sensitive to either IFN-alpha-primed NK or lymphokine-activated killer (LAK) cells-CMC. The resistance of Eskol cells to NK-CMC is due to a low binding ability to effector cells. Moreover, it was found that like IFN, IL-2 can protect Eskol cells from activated NK-CMC. Both cytokines reduced the ability of Eskol cells to induce NK-cytotoxic factor (NKCF) release from NK cells following conjugate formation between Eskol cells and effector cells. Moreover, cycloheximide treatment abolished the protective effect against NK-CMC induced by IFN-alpha or by IL-2. Therefore, it seems that the protective effect against NK-CMC induced by both cytokines is mediated via the same mechanism.
Cell Division↗
A possible role for interferon-alpha and activated natural killer cells in remission of AIDS-related Kaposi's sarcoma: in vitro studies.
The effect of consensus interferon-alpha on the growth of AIDS-related Kaposi's sarcoma-derived cells was studied. Interferon caused a low but significant in vitro inhibition of cell growth. Whereas Kaposi's sarcoma cells were resistant to the cytotoxic effect of natural killer (NK) cells, treatment of NK cells with either interferon or interleukin-2 activated the cell-mediated cytotoxic response. Pretreatment of the Kaposi's sarcoma cells with interferon reduced their sensitivity to interferon-primed natural killer cell or lymphokine (interleukin-2)-activated killer (LAK) cell cytotoxicity. The resistance of Kaposi's sarcoma cells to NK cell-mediated cytotoxicity did not reside in conjugate formation but was due to an inability of Kaposi's sarcoma cells to induce NK cytotoxic factor. Although interferon reduces the sensitivity of Kaposi's sarcoma cells to interferon-primed NK cell and LAK cell activities, the level of natural killing susceptibility remained significantly higher than the poor sensitivity of Kaposi's sarcoma cells to unprimed NK cell activity. Thus, the potential antitumor effect of interferon against Kaposi's sarcoma cells could be mediated both directly (antiproliferative) and/or indirectly by activation of NK cells.
Acquired Immunodeficiency Syndrome↗
Expression of the APRT gene in an adenovirus vector system as a model for studying gene therapy.
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Adenine Phosphoribosyltransferase↗