A Novel Interferon-Inducible Gene Expressed during Myeloid Differentiation

T. Niikura, et al.

Blood Cells, Molecules, and Diseases (1997) 23(17) Sept 15: 337–349 Article No. MD970151

A Novel Interferon-Inducible Gene Expressed during Myeloid Differentiation Submitted 08/13/97 (communicated by Ernest Beutler, M.D., 08/21/97)

Takako Niikura, Roli Hirata, Susan C. Weil ABSTRACT: The acute promyelocytic leukemia cell line, NB4, can be induced to differentiate to mature granulocytes by retinoic acid treatment. A novel retinoic acid-inducible cDNA clone, designated RI58, was isolated from a cDNA library constructed from retinoic acid-treated NB4 cells by differential hybridization. RI58 cDNA encodes a protein of 58kDa which has a similarity in its amino acids sequence to interferon (IFN)-inducible proteins. In addition, RI58 was induced by recombinant human IFN-a (rhIFN-a) in NB4 cells. RI58 was detectable within 4 hours post-stimulation with rhIFN-a, while it took as long as 1day after retinoic acid stimulation. Culture supernatant from retinoic acid-treated NB4 cells also induced RI58 expression similarly as rhIFN-a. This activity in culture supernatant was inhibited by anti-leukocyte IFN antiserum which showed specific reactivity to rhIFN-a. These results indicate that RI58 is induced by retinoic acid stimulation through autocrinally secreted IFN-a from NB4 cells. In the retinoic acid-treated NB4 cells, the expression of RI58 was increased along the process of differentiation. On the other hand, it was expressed constitutively in untreated non-hematopoietic cell lines and mature hematopoietic cell lines. Keywords: acute promyelocytic leukemia, retinoic acid, interferon, interferon-inducible gene, TPR motif

INTRODUCTION

bind to specific cis-elements located in the promoter region of retinoic acid-target genes. In acute promyelocytic leukemia cells, PML/ Retinoic acid receptor a fusion protein changes this signal pathway and causes the block of differentiation (1). Retinoic acid has been used as one of the most effective agents for the therapy of acute promyelocytic leukemia patients. The treatment of acute promyelocytic leukemia cells with retinoic acid induces terminal differentiation of acute promyelocytic leukemia blasts into granulocytes in vivo and in vitro (6-8), though molecular events of this effect are not yet fully understood. Retinoic acid has been shown to activate the expression of interferon regulatory factor (IRF)-1 gene directly in myeloid cells,

Acute promyelocytic leukemia is a unique form of acute myeloid leukemia, in which myeloid differentiation is arrested at the promyelocyte stage (1,2). Acute promyelocytic leukemia is characterized by a specific translocation t(15;17) that fuses the genes for two proteins, promyelocytic leukemia (PML) and retinoic acid receptor a (3). Retinoic acid receptor a is a member of the family of nuclear hormone receptors that include retinoic acid receptor a, b and g. Retinoic acid is a vitamin A metabolite that has effects on development, proliferation and differentiation in a variety of systems (4,5). Retinoic acid receptors when stimulated by retinoic acid, directly

Department of Pathology, Cell Biology, and Medicine, Thomas Jefferson University, Philadelphia, PA 19107-5099. Reprint requests to: Susan C. Weil, M.D., HHMI, CRB Room 328, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6148. phone (215)898-0398, fax (215)573-2000.

1079-9796/97 $25.00 Copyright r 1997 by The Blood Cells Foundation, La Jolla, California, USA All rights of reproduction in any form reserved.

Published by Academic Press Established by Springer-Verlag, Inc. in 1975

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of the IFN-inducible protein family including IFI-56K. We report the isolation and characterization of this novel human protein and demonstrate that RI58 is induced by retinoic acid and IFN treatment of NB4 cells. Further, retinoic acid appeared to induce this protein via stimulation of endogenous IFN-a in NB4 cells.

including acute promyelocytic leukemia cell line NB4 cells (9). IRF-1 is a transcriptional activator which binds to the promoter region of interferon (IFN) and IFN-inducible genes. Retinoic acid also induces the expression of signal transducers and activators of transcription (STAT)-1 protein, which is the common mediator for IFN signaling pathways (10). These indicate that retinoic acid has a potential to modulate the expression of IFN and IFN-inducible genes. IFNs have been known to play important roles in viral and parasitic infections and in immune responses (11,12). IFNs also function as differentiation factors, cell growth inhibitors and modulators of gene expression, depending on the target cells (11). IFN-a, therefore, has been used therapeutically in hairy cell leukemia (13), chronic myeloid leukemia and other malignant diseases (14). A combination of retinoic acid and IFNs have been demonstrated to exhibit additional or syngenical effects to the growth inhibition of NB4 cells (15) and non-promyelocytic leukemia cells (16,17), respectively. In NB4 cells, IFNs augment the expression of retinoic acid receptor a, PML and PML/ retinoic acid receptor a (10,15,18). These findings suggest that IFN may play a key role during myeloid differentiation IFNs induce a set of proteins and alter the levels of some constitutively-expressing proteins in treated cells (19,20). More than 30 IFNinducible proteins have been identified, including b2-microglobulin and major histocompatibility antigens. Among IFN-inducible proteins, P1 kinase and Mx proteins have antiviral properties, and 17kDa protein causes the arrest of cell growth. 28,58-oligoadenylate synthetase has been reported to contribute to both the resistance to virus infection and the control of cell growth (19,20). However, the function of most IFN-inducible proteins is unknown. During a study of neutrophil differentiation using acute promyelocytic leukemia cell line NB4 (7), we isolated a cDNA clone, termed pHRI58. The corresponding mRNA and protein are induced in NB4 cells by retinoic acid. The protein product, RI58, is a new member

MATERIALS AND METHODS Cell Culture Acute promyelocytic leukemia cell lines NB4 (7) and acute myeloid leukemia (AML) cell line HL-60 (21), histiocyte lymphoma cell line U937 (22), T-lymphoblastoid cell lines CEM-SS (23) and HUT78 (24) were maintained in RPMI1640 medium (GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin (GIBCO BRL). Epidermoid carcinoma cell line HeLa (25) and lung carcinoma cell line TKB-2 (26) were maintained in DMEM (GIBCO BRL) supplemented with 10% FBS and penicillinstreptomycin. Cells at a density of 1-2x105/ml were treated with 1 µM all-trans retinoic acid (SIGMA, St Louis, MO) or 100 international units(IU)/ml recombinant human interferon-alpha-2a (rhIFN-a, Roferon-A, Hoffmann-La Roche Inc., Nutley, NJ). Isolation of cDNAs Upregulated by Retinoic acid Treatment NB4 cells were treated with retinoic acid for 6 days and poly(A)1RNA was isolated. A cDNA library was constructed in Lambda ZAP II vector (Stratagene, La Jolla, CA). A total of 106 recombinants were screened by differential hybridization31 with 32P-labeled cDNA probes synthesized from poly(A)1RNA of retinoic acid-treated NB4 cells or untreated cells. Rapid amplification of cDNA ends (RACE) was performed using the 58RACE System (GIBCO BRL). Primer1 (58-GTCTGAGTGTTCT TGCTGG-38) was

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Monoclonal Antibody Production

used for mRNA reverse transcription and primer2 (58-CAUCAUCAUC AUGTTCCAAGCACTCAAGGGCGTC-38) was used for polymerase chain reaction (PCR) amplification. A composite transcript was determined from the overlapping clones.

The 38-terminus of RI58 cDNA (nucleotides 435-1626, 376 amino acids) was amplified by PCR using primers 58-CTGGGTGTATTATCAT ATGGAC-38 and 58-ACAAACAGGAGGATCC AAAATGA-38. The underlined regions designate the position of NdeI and BamHI restriction sites, respectively. The amplified product was digested with NdeI and BamHI, and cloned into NdeIBamHI site of the pET-15b vector (Novagen, Madison, WI). The production and nickel column purification of the histidine-tagged protein were performed as described by the manufacturer under denaturing conditions. Purified protein was used for immunization of mice. A portion of the protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electroeluted in 4mM TrisHCl pH8.4 and 2mM EDTA and used for the last boost. Screening of hybridomas with enzymelinked immunoassay was performed with electroeluted protein. Isotype of monoclonal antibody (MAb) 3-16 was determined as IgG1 by using Mouse Monoclonal Antibody Isotyping Kit (ISO strip, Boehringer Mannheim Co., Indianapolis, IN).

RNA Isolation and Northern Hybridization RNA was isolated using the acid guanidium isothiocyanate/phenol/chloroform method (28). Total cellular RNA (5 µg) was electrophoresed in 1% agarose-formaldehyde gels and transferred to nylon membranes (Hybond-N, Amersham, Airlington Heights, IL). A part of cDNA (nucleotides 412-1625) was isolated and labeled with [32P]dCTP (NEN, Boston, MA) by random hexamer primer extention using an Oligolabelling Kit (Pharmacia LKB Biothchnology, Piscataway, NJ) to a specific activity of at least 1x108 cpm/µg. The membranes were hybridized with [32P]dCTP-labeled cDNA (106 cpm/ml) in 1% sodium dodecyl sulfate (SDS), 1M sodium chloride, 10% dextran sulfate and 100mg/ml denatured salmon sperm DNA at 65 C overnight and washed in 2xSSC at room temperature for 5 min twice, 2xSSC 1%SDS at 65 C for 30 min twice, then 0.1xSSC at room temperature for 15 min twice. The blots were exposed to films (Kodak X-OMAT AR, Kodak, Rochester, NY) at 270 C using intensifying screens.

Western Blot Analysis Cells were harvested, washed with PBS and lysed in Laemmli SDS sample buffer (29). Samples were separated on a 10% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by using semi-dry blotter. Primary antibody (MAb 3-16 hybridoma supernatant) was diluted 1:4 and secondary antiserum (peroxidase-conjugated goat antimouse IgG1IgM, Jackson ImmunoResearch, West Grove, PA) was diluted 1:10,000. Detection of RI58 was performed by using ECL Western blotting system (Amersham).

DNA Sequencing DNA sequencing was performed either using an automatic DNA sequencer (Applied Biosystems Model 373A DNA Sequencing System) by the Nucleic Acid Facility of Thomas Jefferson University or manually using the Sequenase Version 2.0 DNA Sequencing Kit (United States Biochemical Co., Cleveland, OH) and 35S-labeled dATP (NEN) according to manufacturer’s instruction. The deduced amino acid sequence was compared to the protein database using the blastp algorithm provided by the National Center for Biotechnology Information.

Conditioned Medium Preparation and Inhibition Experiments Culture medium of NB4 cells was changed to fresh medium on the 3rd day of retinoic acid

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treatment and further cultured for 2 days. Then culture supernatant was harvested and used as a conditioned medium (CM) after filtration. For stimulation by CM, NB4 cells were cultured in medium containing 50% (volume) of CM and harvested at the indicated times. For the inhibition test, the CM was incubated with anti-human leukocyte interferon antiserum (Sigma) for 6 hours at 4 C prior to the treatment of NB4 cells.

The IFI family proteins contain tetratricopeptide repeat (TPR) motif (35). The TPR motif is composed of 34 amino acid repeats. The most conserved residues in the TPR motif are 8 hydrophobic amino acids which contribute to the formation of a distinct secondary structure, snap helix (36). According to the criteria used by Honore´ B et al. (37), allowing the presence of up to 5 mismatches out of these 8 residues, 14 putative repeats were present in RI58 (Figure 3). When overlapping repeats were excluded, 9 such repeats were present in the amino acid sequence of RI58.

RESULTS Isolation of pHRI58 cDNA Clone

Induction of RI58 by Retinoic Acid

The pHRI58, 3851 bp, was isolated from a cDNA library synthesized from mRNA of retinoic acid-treated NB4 cells by differential screening (GENBANK; U34605). To complete 58 region of this cDNA, the RACE technique was performed and an extra 73 bp of 58 region was obtained. The total sequence (3924 bp) consists of 117 nucleotides of 58 untranslated region (UTR), a single open reading frame (ORF) of 1449 nucleotides beginning with an ATG putative initiation codon and ending with a TAA stop codon, and 2339 nucleotides of 38 noncoding region with an AATAAA polyadenylation signal 14 nucleotides upstream of poly(A) tail (19 nucleotides) (Figure 1). The ORF encodes a protein of 482 amino acids with calculated molecular weight of 55, 841.

Total RNA from retinoic acid-treated and untreated NB4 cells was analyzed by Northern hybridization with a probe corresponding to the RI58 ORF (Figure 4A). In NB4 cells 4.1, 2.8 and 1.6 kb bands with almost the same intensity were induced by retinoic acid treatment. These signals were observed as early as 1 day after stimulation. The intensity of the signals increased significantly by day 2 and remained at a high level up to day 6. Allowing for a poly(A) tail of 100 to 200 nucleotides, the cDNA clone we obtained (Figure 1) likely corresponds to the 4.1kb mRNA. Accordingly, a shorter cDNA clone which starts at position 1114 and ends at position 2830 followed by poly(A) was also isolated, even though we could not identify the apparent polyadenylation signal responsible for this termination. However, a partial cDNA clone (237bp, Embl Z40106) (38) identical to the RI58 38UTR region was found in database with a termination at 7 nucleotides upstream of the pHRI58 short clone’s 38end (data not shown). As both of our short clone and this partial cDNA were isolated from human cDNA libraries constructed from oligo-(dT)-primed mRNAs, both are likely to be natural products. Thus, we believe this short clone corresponds to the 2.8 kb mRNA, according to its termination position. For protein expression analyses MAbs against the recombinant RI58 expressed in bacteria were produced. The specificity one of these MAb, 3-16,

Amino Acid Sequence Homology A homology search of the RI58 amino acid sequence against the protein database revealed significant homology to a family of IFN-inducible proteins, IFI-56K, IFI-54K, ISG-54K and IRG-2 (30-33) (Figure 2). RI58 showed highest similarity to IFI-56K (55% identity). This similarity is higher than that found between IFI-56K and IFI-54K (46%) (34). Overall, amino-terminal amino acids are more conserved than carboxyterminal sequence among those five proteins. The amino acid sequence of RI58 also showed the high similarity with GARG-16 and GARG-39 (35), mouse homologues of IFI-56K and IFI-54K respectively (data not shown).

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retinoic acid-treated NB4 cells was examined to see if it could induce RI58 in NB4 cells. RI58 expression was induced by CM from retinoic acid-treated cells within 4 hours and continued to increase for 1 day after treatment. This pattern was identical to that of cells treated with 100 IU/ml of rhIFN-a. Culture medium from untreated cells did not induce RI58 expression (Figure 6A). To confirm that the effector in CM is IFN-a, an inhibition test using anti-leukocyte IFN antiserum was performed. The antiserum inhibited RI58 induction by CM in a dose-dependent manner (Figure 6B). Further, this antiserum inhibited the induction of RI58 by rhIFN-a, but not by rhIFN-b and rhIFN-g (data not shown). These results indicate that RI58 was induced in NB4 cells by retinoic acid through the stimulation of autocrinal IFN-a. In the case of human mature neutrophils, it has been reported that granulocyte-colony-stimulation factor (G-CSF) induces IFN-a production from these cells (39). However, involvement of G-CSF in the induction of IFN-a by retinoic acid in NB4 cells was unlikely, since 10,000 IU/ml of G-CSF could not induce RI58 expression in these cells (data not shown).

was confirmed by Western blot analysis (data not shown). In retinoic acid-treated NB4 cells, Western blots probed with anti-RI58 MAb 3-16 showed a single protein band of 58 kDa (Figure 5A). A significant upregulation was observed on day 1 and expression increased markedly through day 6 of retinoic acid treatment. The expression level of protein paralleled that of the mRNAs, suggesting that RI58 protein expression induced by retinoic acid is regulated at the transcriptional level, although mRNA stability may also play a role. Induction of RI58 by IFN Since RI58 showed a high similarity with a class of IFN inducible proteins (Figure 2), in particular IFI-56K, we asked if RI58 could also be induced by IFN. Figure 4B demonstrates that rhIFN-a induced all three species of RI58 mRNAs to the levels similar to retinoic acid-treated cells. Detectable signals were observed as early as 2 hours post-stimulation and higher expression of mRNAs were observed at 4 and 8 hours poststimulation. The level of expression slightly dropped by day 1, increased again at day 2 and remained high up to day 6. Protein expression level increased at 4 hours, reaching a maximum on day 1 and remained constant to day 6 (Figure 5B). The RI58 was also induced by rhIFN-b and -g in NB4 cells. In rhIFN-b-treated cells, RI58 expression was similar to that seen in rhIFN-a-treated cells, whereas the response to rhIFN-g was weaker (data not shown).

Expression of RI58 in Various Cell Lines We examined the cell type distribution of RI58 using hematopoietic and non-hematopoietic cell lines (Figure 7). An AML cell line HL-60 expressed RI58 after both rhIFN-a and retinoicacid treatment. The level of expression induced by retinoic acid was lower in HL-60 cells compared to that in NB4 cells. Histiocytic cell line U937 and T cell line CEM-SS expressed RI58 after rhIFN-a treatment like NB4 cells. On the other hand, another T cell line HUT78 and epidermoid carcinoma cell line HeLa expressed RI58 apparently constitutively without stimula-tion, although slightly higher expression was seen after rhIFN-a treatment. A lung carcinoma cell line TKB-2 expressed RI58 constitutively and the expression level increased only slightly by rhIFN-a treat-

Induction of RI58 by Retinoic Acid via Autocrine IFN Stimulation Time course analyses showed that the induction of RI58 mRNA and protein was significantly more rapid in cells treated with IFN as compared to these treated with retinoic acid. This suggests that RI58 induction by retinoic acid may require the synthesis of an intermediate effector, possibly IFN. Therefore, conditioned medium (CM) from

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ment. These results suggest that the regulation of RI58 expression varies in different cell lines.

retinoic acid-treated NB4 cells (acute promyelocytic leukemia cell line) by differential hybridization. Although RI58 cDNA was isolated from retinoic acid-treated cells, conditioned medium and induction inhibition experiments indicated that RI58 was induced by retinoic acid through autocrinal production of IFN-a in NB4 cells.

DISCUSSION A novel human cDNA clone, pHRI58, was isolated from a cDNA library constructed from

Figure 1. Nucleotide sequence and predicted amino acid sequence of RI58. Numbers on the left refer to the nucleotide number. Numbers on right refer to the amino acid number starting with the initiating methionine.

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Figure 2. Amino acid sequence alignment of RI58 with four IFN-inducible proteins, IFI-56K, IFI-54K, ISG-54K and IRG-2. Numbers on the left refer to the amino acid number. The identical amino acids among the proteins are highlighted, and gaps are introduced for optimal alignment.

Figure 3. TPR motif of RI58. 34 amino acid repeats in RI58 were aligned to the TPR consensus sequence. Numbers on the left refer to the first amino acid position (Figure 1) in each repeat. The amino acids identical to the conserved residues of the TPR consensus sequence are highlighted.

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Figure 4. RI58 mRNA induction by retinoic acid (A) and IFN-a (B). Total RNA (5 µg) in each lane was electrophoresed on a 1% formaldehyde-agarose gel, transferred to nylon membrane and hybridized with RI58 ORF region probe. Total cellular RNA was extracted from NB4 cells treated with 1 µM retinoic acid (A) or 100 IU/ml rhIFN-a (B) for indicated times. Molecular mass markers are indicated on the left. Lower panels show 18S and 28S rRNAs stained with ethidium bromide.

Figure 5. RI58 expression induced by retinoic acid (A) and IFN-a (B) in NB4 cells. NB4 cells were treated with 1 µM retinoic acid (A) or 100 IU/ml rhIFN-a (B) for indicated times. Total cellular proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-RI58 Mab 3-16. Arrows indicate the position of specific band. Molecular mass markers are indicated on the left.

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Figure 6. RI58 induction in NB4 cells by conditioned medium treatment (A) and its inhibition by antisera (B). NB4 cells were treated with 1 µM retinoic acid for 3 days, the medium was replaced and the cells were further incubated for 2 days. Culture medium was filtered and applied as conditioned medium. (A) Cells were treated with 1 µM retinoic acid, 100 IU/ml rhIFN-a (IFN), conditioned medium from retinoic acid treated NB4 cells (CM) or conditioned medium from untreated NB4 cells (control) for the indicated times. (B) Cells were treated for 1 day with conditioned medium from retinoic acid (RA) treated NB4 cells (CM) or conditioned medium from untreated NB4 cells (control) after incubation with anti-IFN antisera for 6 hours at 4 C. Numbers indicate the amount of antisera as neutralization units per ml. Total cellular proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-RI58 Mab 3-16. Arrows indicate the position of specific band. Molecular mass markers are indicated on the left.

Figure 7. RI58 expression in various cell lines before and after induction by IFN-a. Cells were untreated (-) or treated with 100 IU/ml rhIFN-a for 1 day (1) or with 1 µM retinoic acid for 6 days (RA).Total cellular proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-RI58 Mab 3-16. The arrow indicates the position of specific band. Molecular mass markers are indicated on the left.

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lines (43,44). Their genes are located on chromosome 10 at bands q23-q24 (45), and are under the regulation of the IFN-stimulated-response-element (ISRE), which is a binding site of STAT proteins (46,47). The gene for ISG-54K is also under the regulation of an ISRE (32). These proteins are induced through the Janus kinase (Jak)-STAT pathway by IFN stimulation (32,46,47). On the other hand, mouse homologues, GARG-16, GARG-39 and GARG-49/ IRG2, are induced by both IFN and lipopolysaccharide via a protein kinase C pathway (33,35). RI58 was not induced by a protein kinase C activator, phorbol 12-myristate 13-acetate in NB4 cells (data not shown). Therefore, it is likely that RI58 is induced via Jak-STAT pathway by IFN in NB4 cells. In addition to the direct stimulation of Jak-STAT pathway by IFN, the indirect modulation of this pathway by retinoic acid is reported. The promoter region of STAT1, the common mediator of IFN signaling cascade, contains the element which is activated by the retinoic acid receptor a. It suggests that retinoic acid can modulate the expression of the IFN-responding genes at the level of STAT1 (10,40). Retinoic acid also directly activate the expression of IRF-1 (9). IRF-1 and TFIIB, a component of the basic transcriptional machinery, cooperatively enhance the ISRE promoter activity (48). Regarding these findings, the possibility that the expression of RI58 in NB4 cells is modulated by retinoic acid, through these cascades without processing the autocrinal IFN production, can not be rule out. On the other hand, RI58 was expressed constitutively in mature hematopoietic cell line (HUT78) and non-hematopoietic cell lines (HeLa and TKB-2) (Figure 7). Wang et al. reported that cooperative enhancement of ISRE promoter by IRF-1 and TFIIB occurred TATA-independently in P19 embryonal carcinoma cells, but TATA-dependently in NIH3T3 cells, indicating the presence of a cell type-specific factor (48). The expression of RI58 may be also modulated by other factor(s) in the cell type-specific manner. All members of IFI protein family including RI58 have TPR motif which is a 34-amino acid

Recently, Gianni et al. reported the secretion of IFN-a from retinoic acid-treated NB4 cells by specific ELISA (40). Retinoic acid directly activates the expression of IFN regulatory factor (IRF)-1, the activator for both IFN and IFNinducible genes (9). In COS cells, overexpression of IRF-1 cDNA resulted in the induction of the endogenous IFN-a and IFN-b genes (41). Similary, the induction of IRF-1 by retinoic acid may cause the production of IFN in NB4 cells. In addition to NB4, retinoic acid induces IRF-1 gene expression in other myeloid leukemia cell lines, HL-60 and U937 (9). After retinoic acid treatment, HL-60 cells and U937 cells differentiate into granulocytes and monocytes, respectively (8,42). IFN markedly induced the expression of RI58 in both line cells. However, the expression level of RI58 in retinoic acid-treated HL-60 cells was very low compared to that in NB4 cells (Figure 7). Further, conditioned medium from retinoic acid-treated HL-60 cells could not induce the expression of RI58 in NB4 cells (data not shown). In U937 cells, the expression of RI58 was not observd by retinoic acid treatment (data not shown). Since RI58 expression in NB4 cells seemed to be induced by autocrinal IFN, these results may indicate that unlike NB4 cells, these line cells produce no or very low amount of IFN during retinoic acid-induced differentiation process. Therefore, high-level production of autocrinal IFN as a response to the retinoic acid stimulation may be unique to the acute promyelocytic leukemia cells. RI58 could be induced by exogenous IFN alone (Figures 4 and 5), and RI58 amino acid sequence shows a high similarity to the IFI protein family. Therefore, this protein should be considered to belong to IFI protein family. This small group of INF-inducible proteins with unknown function consists of human proteins IFI-56K (also called 56K-Da protein) and IFI-54K (also called ISG-54K), hamster protein ISG-54K and their mouse homologues, GARG-16, GARG-39 and GARG-49/IRG-2 (30-33,35). Among them, IFI56K and IFI-54K are induced by IFNs, doublestranded RNA and virus infection in many cell

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repeated motif and frequently tandemly arrayed. TPR proteins are widely present from bacteria to human, and they are involved in cell cycle control, transcription repression, protein transport, stress response, protein kinase inhibition and neurogenesis (36). TPRs can mediate protein-protein interactions which are composed of both TPR-TPR proteins and TPR-nonTPR proteins. For example, yeast TPR proteins, Cdc16p, Cdc23p and Cdc27p, form a complex and this interaction is essential for cell cycle progression (49). In eukaryotic cells, chaperone cofactors, Hip and Hop proteins which are TPR proteins, regulate the activity of molecular chaperone, Hsc70 which is non-TPR protein, during the protein biogenesis (50). Although the function of IFI protein family is still unknown, the presence of TPR motif suggests that they may interact among them or with other proteins. Further studies investigating the function of RI58 will be necessary to fully understand the exact function of IFI protein family. Understanding of the function of RI58 will be extremery important. This should also contribute to a better understanding fo the molecular effects of retinoic acid and IFN on acute promyelocytic leukemia cells.

5. 6.

7.

8.

9.

10.

11.

12.

13. 14.

ACKNOWLEDGMENT We thank Dr. Gideon Dreyfuss and the members in his lab for helpful discussions. We also thank Hoffmann-La Roche Inc. for providing the rhIFN-a.

15.

REFERENCES 1.

Grignani F, Fagioli M, Acalay M, et al. Acute promyelocytic leukemia: from genetics to treatment. Blood 83: 10-25, 1994 2. Trayner ID, Farzaneh F. Retinoid receptors and acute promyelocytic leukaemia. Eur J Cancer 29A: 20462054, 1993 3. The´ H, Chomienne C, Lanotte M, Degos, L Dejean A. The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor a gene to a novel transcribed locus. Nature 347: 558-561, 1990 4. Gudas LJ. Retinoids and vertebrate development. J Biol Chem 269: 15399-15402, 1994

16.

17.

347

Love JM, Gudas LJ. Vitamin A, differentiation and cancer. Curr Opin Cell Biol 6: 825-831, 1994 Huang M, Ye Y, Chen S, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72: 567-572, 1988 Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 77: 1080-1086, 1991 Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci USA 77: 2936-2940, 1980 Matikainen S, Ronni T, Hurme M, Pine R, Julkunen I. Retinoic acid activated interferon regulatory factor-1 gene expression in myeloid cells. Blood 88: 114-123, 1996 Weihua X, Kolla V, Kalvakolanu DV. Modulation of interferon action by retinoids. J Biol Chem 272: 9742-9758, 1997 Hauser H. Interferons, in Habenicht A (ed): Growth factors, differentiation factors, and cytokines. Berlin, Springer-Verlag, 1990, pp.243-253. Arai K-I, Lee F, Miyajima A, Miyatake S, Arai N, Yokota T. Cytokines: coordinators of immune and inflammatory responses. Annu Rev Biochem 59: 783836, 1990. Thompson JA. Fefer A, Interferon in the treatment of hairy cell leukemia. Cancer 59: 605-609, 1987. Gutterman JU, Blumenschein GR, Alexanian R, et al. Leukocyte interferon-induced tumor regression in human metastatic breast cancer, multiple myeloma, and malignant lymphoma. Ann Intern Med 93: 399-406, 1980. Gianni M, Zanotta S, Terao M, Rambaldi A, Garattini E. Interferons induce normal and aberrant retinoic-acid receptors type a in acute promyelocytic leukemia cells: Potentiation of the induction of retinoiddependent differentiation markers. Int J Cancer 68: 75-83, 1996. Zheng A, Savolainen E-R, Koistinen P. All-trans retinoic acid combined with interferon-alpha effectively inhibits granulocyte-macrophage colony formation in chronic myeloid leukemia. Leukemia Res 20: 243-248, 1996. Ferrero D, Carlesso N, Bresso P, et al. Suppression of in vitro maintenance of non-promyelocytic myeloid leukemia clonogenic cells by all-trans retinoic acid: Modulating effects of dihydroxylated vitamin D3, ainterferon and ‘stem cell factor’. Leukemia Res 21: 51-58, 1997.

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18.

19. 20. 21.

22.

23. 24.

25.

26.

27. 28.

29.

30.

31.

32.

33.

T. Niikura, et al.

Nason-Burchenal K, Gandini D, Botto M, et al. Interferon augments PML and PML/RARa expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line. Blood 88: 3926-3936, 1996. Sen GC, Lengyel P. The interferon system. J Biol Chem 267: 5017-5020, 1992. Staeheli P. Interferon-induced proteins and the antiviral state. Adv Virus Res 38: 147-200, 1990. Collins SJ, Gallo RC, Gallagher RE. Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270: 347-349, 1977. Sundstro¨m C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer 17: 565-577, 1976. Nara PL, Fischinger PJ, Quantitative infectivity assay for HIV-1 and -2. Nature 332: 469-470, 1988. Gootenberg JE, Ruscetti FW, Mier JW, Gazdar A, Gallo RC. Human cutaneous T cell lymphoma and leukemia cell lines produce and respond to T cell growth factor. J Exp Med 154: 1403-1418, 1981. Scherer WF, Syverton JT, Gey GO. Studies on the propagation in vitro of poliomyelitis viruses. J Exp Med 97: 695-710, 1953. Endo K, Kamma H, Ogata T. Two murine monoclonal antibodies against human lung cancer-associated antigens. Cancer Res 46: 6369-6373, 1986. Sargent TD. Isolation of differentially expressed genes. Methods Enzymol 152: 423-432, 1987. Chomozynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extracton. Anal Biochem 162: 156-159, 1987. Laemmli UK. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227: 680-685, 1970. Wathelet M, Moutschen S, Defilippi P, et al. Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons. Eur J Biochem 155: 11-17, 1986. Levy D, Larner A, Chaudhuri A, Babiss LE, Darnell JE. Interferon-stimulated transcription: isolation of an inducible gene and identification of its regulatory region. Proc Natl Acad Sci USA 83: 8929-8933, 1986. Bluyssen HAR, Vlietstra RJ, van der Made A, Trapman J. The interferon-stimulated gene 54K promoter contains two adjacent functional interferon-stimulated response elements of different strength, which act synergistically for maximal interferon-a inducibility. Eur J Biochem 220: 395-402, 1994. Lee CGL, Demarquoy J, Jackson MJ, O’Brien WE. Molecular cloning and characterization of a murine

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

348

LPS-inducible cDNA. J Immunol 152: 5758-5767, 1994. Wathelet MG, Clauss IM, Content J, Huez GA. The IFI-56k and IFI-54k interferon-inducible human genes belong to the same gene family. FEBS Lett 231: 164-171, 1988. Smith JB, Herschman HR. The glucocorticoid attenuated response genes GARG-16, GARG-39, and GARG49/IRG2 encode inducible proteins containing multiple tetratricopeptide repeat domains. Arch Biochem Biophys 330: 290-300, 1996. Lamb JR, Tugendreich S, Hieter P. Tetratrico peptide repeat interactions: to TPR or not to TPR? Trends Biochem Sci 20: 257-259, 1995. Honore´ B, Leffers H, Madsen P, Rasmussen HH, Vandekerckhove J, Celis JE. Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to the stress-inducible yeast protein STI1. J Biol Chem 267: 8485-8491, 1992. Auffray C, Behar G, Bois F, et al. Image: Integated molecular analysis of the human genome and its expression. C R Acad Sci, III, Sci Vie 318: 263-272, 1995. Shirafuji N, Matsuda S, Ogura H, et al. Granulocyte colony-stimulating factor stimulates human mature neutrophilic granulocytes to produce interferon-a. Blood 75: 17-19, 1990. Gianni M, Terao M, Fortino I, et al. Stat1 is induced and activated by all-trans retinoic acid in acute promyelocytic leukemia cells. Blood 89: 1001-1012, 1997. Fujita T, Kimura Y, Miyamoto M, Barsoumian EL, Taniguchi T. Induction of endogenous IFN-a and IFN-b genes by a regulatory transcription factor, IRF-1. Nature 337: 270-272, 1989. Nilsson K, Ivhed I, Forsba¨ck K. Induced differentiation in human hematopoietic cell lines, in Anderson LC, Gahmberg CG, Ekblom P (eds): Gene expression during normal and malignant differentiation, London, Academic Press, 1985, pp 57-72. Wathelet MG, Clauss IM, Content J, Huez GA. Regulation of two interferon-inducible human genes by interferon, poly(rI).poly(rC) and viruses. Eur J Biochem 174: 323-329, 1988. Wathelet MG, Clauss IM, Nols CB, Content J, Huez GA. New inducers revealed by the promoter sequence analysis of two interferon-activated human genes. Eur J Biochem 169: 313-321, 1987. Lafage M, Clauss I, Couez D, Simonetti J, Wathelet MG, Huez G. The interferon- and virus-inducible IFI-56k and IFI-54k genes are located on human chromosome 10 at bands q23-q24. Genomics 13: 458-560,1992.

T. Niikura, et al.

Blood Cells, Molecules, and Diseases (1997) 23(17) Sept 15: 337–349 Article No. MD970151

46.

Pellegrini S, Schindler C. Early events in signalling by interferons. Trends Biochem Sci 18: 338-342, 1993. 47. Darnell JE, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415-1421, 1994. 48. Wang I-M, Blanco JCG, Tsai SY, Ozato K. Interferon regulatory factors and TFIIB cooperatively regulate

interferon-responsive promoter activity in vivo and in vitro. Mol Cell Biol 16: 6313-6324, 1996. 49. Lamb JR, Michaud WA, Sikorski RS Hieter PA. Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J 13: 4321-4328, 1994. 50. Frydman J, Hohfeld J. Chaperones get in touch: the Hip-Hop connection. Trends Biochem Sci 22: 87-92, 1997.

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