A-type Particles Research Articles

Lupus erythematosus‐like features in patients with cutaneous T‐cell lymphoma

The development of lupus erythematosus-like (LE-like) features in patients with cutaneous T-cell lymphoma (CTCL) has not been reported previously in the literature. Both diseases, however, have been etiologically linked to retroviruses. Our purpose was to report four cases of patients with CTCL who developed LE-like features during the course of their disease, and to evaluate for evidence of antibodies to retroviruses in the sera of these patients. Four patients with biopsy-proven CTCL with clinical or histologic features of systemic lupus erythematosus (SLE) were evaluated for clinical and laboratory criteria for SLE. Only one patient demonstrated four American Rheumatism Association (ARA) criteria sufficient for the diagnosis of SLE. The remaining three patients demonstrated one or two criteria for SLE. In addition, the sera of these patients were examined by Western blot analysis for evidence of human immunodeficiency virus type I (HIV-I), human T-cell lymphotrophic virus type I (HTLV-I), or human intracisternal A-type particle type I (HIAP-I) retroviral proteins. Each patient demonstrated antibodies to some of the retroviral proteins examined. The sera of two patients reacted to proteins for HIAP-I, and the sera of two patients reacted to p24 gag proteins of HIV-I. No patient reacted to HTLV-I proteins. Our report identifies four patients with CTCL who developed LE-like features during the course of their disease. Although the etiology of CTCL and SLE has not been well established, each has been linked to retroviruses. Evidence of antibodies to retroviral proteins was identified in each of our patients by Western blot analysis. Although the clinical and laboratory findings in these cases do not resolve the etiologic role of retroviruses in CTCL or SLE, they suggest that retroviruses may have a role in the pathogenesis of the clinical phenomenon reported in these four patients.

Detection of retroviral antibodies in primary biliary cirrhosis and other idiopathic biliary disorders

Retroviruses have been implicated in the aetiology of various autoimmune diseases. We used immunoblots as a surrogate test to find out whether retroviruses play a part in the development of primary biliary cirrhosis. We did western blot tests for HIV-1 and the human intracisternal A-type particle (HIAP), on serum samples from 77 patients with primary biliary cirrhosis, 126 patients with chronic liver disease, 48 patients with systemic lupus erythematosus, and 25 healthy volunteers. HIV-1 p24 gag seroreactivity was found in 27 (35%) of 77 patients with primary biliary cirrhosis, 14 (29%) of 48 patients with systemic lupus erythematosus, 14 (50%) of 28 patients with chronic viral hepatitis, and nine (39%) of 23 patients with either primary sclerosing cholangitis or biliary atresia, compared with only one (4%) of 24 patients with alcohol-related liver disease or alpha1-antitrypsin-deficiency liver disease, and only one (4%) of 25 healthy volunteers (p=0.003). Western blot reactivity to more than two HIAP proteins was found in 37 (51%) of patients with primary biliary cirrhosis, in 28 (58%) of patients with systemic lupus erythematosus, in 15 (20%) of patients with chronic viral hepatitis, and in four (17%) of those with other biliary diseases. None of the 23 patients with either alcohol-related liver disease or alpha1-antitrypsin deficiency, and only one of the healthy controls showed the same reactivity to HIAP proteins (p<0.0001). Our results showed a strong association between HIAP seroreactivity and the detection of autoantibodies to double-stranded DNA. HIAP seroreactivity was also strongly associated with the detection of mitochondrial, nuclear, and extractable nuclear antigens. The HIV-1 and HIAP antibody reactivity found in patients with primary biliary cirrhosis and other biliary disorders may be attributable either to an autoimmune response to antigenically related cellular proteins or to an immune response to uncharacterised viral proteins that share antigenic determinants with these retroviruses.

The role of intracisternal A-type particles in the evolution of factor-independent murine hematopoietic cell lines.

Cocultivation of a clonal factor-dependent hematopoietic cell line (FDC-P1JL26) with an irradiated bone marrow stromal cell line (D2XRII) significantly increased the frequency of isolation of factor-independent subclones. Eight out of nine factor-independent subclonal lines showed expression of IL-3, GM-CSF or both cytokine mRNAs by reverse-transcription polymerase chain reaction (RT-PCR) and seven of these expressed biologically active GM-CSF or IL-3. In three cell lines that synthesized biologically active IL-3 (FIJ1, FIJ4D and FIJ10D) insertion of an IAP sequence into the IL-3 gene was detected by PCR analysis and the insertions were confirmed by DNA sequence analysis of PCR or RT-PCR fragments. In the four cell lines in which no IL-3 expression was detected no IAP insertions were detected. Rearrangements of the GM-CSF gene were detected in three factor-independent cell lines and an insertion of an IAP into the GM-CSF gene was confirmed by DNA sequence analysis of PCR fragments. In contrast to results with IL-3, insertion of an IAP into the GM-CSF gene did not correlate with GM-CSF expression. In one cell line that contained an IAP insertion into the GM-CSF gene, no GM-CSF was detected by biological assay nor by RT-PCR. Retrotransposition of IAPs may be responsible for the emergence of factor-independent cells in our cocultivation system and other IAP insertions may prove to be responsible for the factor-independent phenotype seen in the non-autocrine factor-independent cell line, FI7CL2.

Intracisternal A-Type Particles Express Their Proteinase in a Separate Reading Frame by Translational Frameshifting, Similar to D-Type Retroviruses

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum of rodent cells. IAP genomes share extensive sequence homologies with D-type retroviruses, but were presumed to express the viral proteinase (PR) as part of thegagopen reading frame (ORF) while D-type retroviruses express PR in a separate ORF. Here we show that expression of the murine IAP element MIA14 yields three major translation products, corresponding to the Gag, Gag-PR, and Gag-PR-Pol polyproteins. Sequence analysis revealed that MIA14 PR is encoded in its own reading frame, separate fromgagandpol.Frameshifting occurred with an efficiency of approximately 25% between thegagandproORFs and 35% betweenproandpol.The region containing the putativegag-proframeshift signal consists of a heptanucleotide slippery sequence (A6C) and a stem–loop structure probably forming a pseudoknot. Deletion of this structure element almost completely abolished frameshifting. Insertion of an additional base next to the frameshift signal placedgagandproin the same ORF and resulted in predominant formation of Gag-PR and Gag-PR-Pol polyproteins which were not processed followingin vitrotranslation. Expression of a similar construct in tissue culture cells, on the other hand, led to efficient intracellular processing of the mutant polyproteins.

Plasma membrane targeting of chimeric intracisternal A-type particle polyproteins leads to particle release and specific activation of the viral proteinase.

Retrovirus morphogenesis involves assembly of structural Gag polyproteins with subsequent budding from the plasma membrane, followed by proteolytic cleavage by the viral proteinase (PR) and extracellular maturation to the infectious virion. Intracisternal A-type particles (IAPs) are defective retroviruses that assemble and bud at the membranes of the endoplasmic reticulum (ER), where they remain as immature particles consisting exclusively of uncleaved polyproteins. To analyze requirements for intracellular polyprotein transport and PR activation, we constructed deletion and substitution mutations in the IAP gag gene, including the putative ER-targeting signal. Mutant polyproteins were transported to various intracellular locations, including the nucleus, the cytoplasm, the ER, and the plasma membrane. Interestingly, assembly of capsid-like particle structures occurred at almost all sites. However, only those polyproteins transported to the plasma membrane were efficiently and specifically cleaved by viral PR, with cleavage occurring predominantly within the virus particle. Thus, at least in the experimental system presented here, retroviral particle assembly can occur at almost any location within the cell, while polyprotein processing and, consequently, virion maturation are confined to a specific cellular site. These results suggest that a factor restricted to the plasma membrane is required to trigger PR activation and maturation of infectious retroviruses.

Loss of intracisternal A-type retroviral particles in BL6 melanoma cells transfected with MHC class I genes.

Electron microscopy of B16 melanoma and its sublines revealed that these cells produce numerous intracisternal A-type retroviral particles (IAPs). To identify and sequence the melanoma-associated IAPs of C57BL/6 mice, a cDNA library was constructed from IAP-producing BL6.8 cell RNA and screened using MIA14 IAP DNA as a probe. A 6-8 kb mRNA was identified that represents the full-length message for a new subfamily of IAP, termed MeIAP. The melanoma-derived IAP cDNA showed high similarity to MIA14 with major differences in the LTR. A nine base motif of the R region showed that this IAP differs from other previously sequenced IAPs. Analysis of the individual clones from BL6 melanoma revealed that IAPs were produced only in the clones that failed to express H-2Kb molecules. No IAPs were found in the melanoma clones that expressed endogenous H-2Kb. To analyse further the association between MHC class I genes and IAP production, the H-2Kb-negative clones of BL6 melanoma were transfected with various H-2 genes. Transfection of the H-2Kb or H-2Kd, but not H-2Dd or H-2Ld genes resulted in the elimination of IAPs. Northern blot analysis revealed that loss of IAPs in the H-2K gene-transfected BL6 melanoma cells was due to lack of IAP transcripts. Elimination of IAPs in the H-2Kb-positive BL6 melanoma cells was also accompanied by alterations in expression of various cellular genes and changes of their phenotypic properties.

Biochemical characterization of the reverse transcriptase of a human intracisternal A-type particle (HIAP).

The discovery of novel human intracisternal A-type particle (HIAP) that may be associated with the autoimmune disease Sjŏgren's syndrome has been previously reported. Although the HIAP retrovirus has been shown to be antigenically related to HIV-1, the viruses were distinguishable by different hydrodynamic mobilities through a sucrose gradient by morphology and intracellular location, and by differing divalent cation requirements for their in vitro reverse transcriptase (RT) reactions. In this report, additional biochemical characteristics are provide that further differentiate the HIAP RT from HIV-1 RT. Data are also presented that distinguish the HIAP RT from the known cellular DNA polymerases.

Antibodies against Retroviral Proteins and Nuclear Antigens in a Subset of Idiopathic CD4+T Lymphocytopenia Patients

Idiopathic CD4+ T lymphocytopenia (ICL) is an immunodeficiency syndrome characterized by severe depletion of CD4+ T lymphocytes, but in which human immunodeficiency virus cannot be detected. Peripheral blood mononuclear cells (BPMCs) from an ICL patient were cocultured with HUT78 T-lymphoblastoid cells, and an acute cytopathic effect and formation of multinucleated cells were observed. A human intracisternal A-type retroviral particle designated HIAP-II was detected in cells surviving the acute cytopathic effect. Eight of 13 ICL patients in a blinded screen of a serological panel provided by the National Centers for Disease Control and Prevention (CDC) had serum antibodies that specifically reacted with HIAP-II associated proteins by Western immunoblotting. None of 19 control sera in the panel that were unreactive with HIV Gag proteins produced a positive result on HIAP-II immunoblots. Comparable results were obtained in a blinded screen of a second CDC serological panel. Sera from 8 of 14 ICL patients in the second serological panel were positive for antinuclear autoantibodies (ANAs) commonly observed in patients with systemic autoimmune diseases. These results suggest the possible involvement of an A-type retrovirus or autoimmunity in development of ICL in a subset of patients.

Plasmacytoma-Associated Neuronal Glycoprotein,Pang,Maps to Mouse Chromosome 6 and Human Chromosome 3

A new member of the immunoglobulin/fibronectin superfamily of adhesion molecules,Pang(plasmacytoma-associated neuronal glycoprotein), was recently isolated from a plasmacytoma. In previous studies,Pangwas found to be normally expressed in the brain and ectopically activated by intracisternal A-type particle long terminal repeats in plasmacytomas. In this study,Pangwas initially mapped to mouse Chr 6 by somatic cell hybrid analysis and further positioned on the chromosome betweenWnt7aandPcp1.Southern blot analysis of human–rodent somatic cell hybrids together with predictions from the mouse map location indicate that human PANG is located at 3p26.

Construction of a mouse blastocyst cDNA library by PCR amplification from total RNA.

Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT-PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains beta-actin, intracisternal A-type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones.

Evidence for a retroviral trigger in Graves' disease.

An apparently high frequency of Graves' disease encountered in New Orleans, Louisiana, prompted an investigation for a possible infectious agent that might be triggering the disease in genetically susceptible individuals. We studied 40 patients with Graves' disease, and compared them to the following groups of controls: age and gender matched healthy subjects; patients with multinodular goiter (non-autoimmune thyroid controls); patients with chronic lymphocytic thyroiditis (autoimmune thyroid disease controls) and additional organ or tissue specific autoimmune controls exclusive of thyroid autoimmunity, including patients with Type I diabetes and other endocrine autoimmune complex disorders. Serum antibodies against a prototypic strain of a human intracisternal A-type retroviral particle type 1 (HIAP-1) were detected by a sensitive and specific immunoblotting assay. In 87.5% (35/40) of the Graves' disease patients there was a positive reaction against several HIAP-1-associated proteins, predominantly 97 Kd and 80 Kd, with only 5 showing no reactivity to any. In contrast, 2% (2/105) of sera from normal controls showed positive reactivity. Furthermore, only 10% (1/10) of sera from multinodular goiter control patients and 10% (1/10) of Hashimoto's patients showed reactivity (p < 0.0005). Sera from 3 of 20 (15%) of Type I diabetic patients none of whom had Graves' disease, showed reactivity but there was no reactivity in 9 other patients with one or more of the endocrine autoimmune complex disorders, including Addison's disease, vitiligo, myasthenia gravis and pernicious anemia. In addition we studied two individuals with Graves' disease from each of two families residing outside Louisiana, all of whom were positive for these antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracisternal A-type particle elements as genetic markers: detection by repeat element viral element amplified locus-PCR

We describe a novel, PCR-based technique termed REVEAL-PCR for examining the inheritance of intracisternal A-type particles (IAP). Amplifications use an unlabeled primer to SINE repeats and a radiolabeled primer to the IAP long terminal repeat; labeled products, which can be resolved on sequencing gels, are formed when IAPs lie in proximity to SINEs. With this technique we have identified a total of 124 polymorphisms in the BXH and CXS recombinant inbred strains. We suggest that this method will be equally applicable for examining other gene families present at around a thousand copies per genome.

PANG, a gene encoding a neuronal glycoprotein, isectopically activated by intracisternal A-type particle long terminal repeats inmurine plasmacytomas.

Plasmacytomagenesis provides a murine model to decipher progressive genetic events culminating in a B-cell neoplasia. Activation of the c-myc protooncogene by chromosomal translocation is considered an initiating event. Intracisternal A-type particles (IAPs) are defective retroviral-like structures present in the endoplasmic reticulum of plasmacytomas (PCTs). IAP proviral insertions have been documented to engender negative or positive effects on the expression of nearby cellular genes. We have isolated a gene, PANG (plasmacytoma-associated neuronal glycoprotein), that is ectopically transcribed in a number of PCTs due to IAP long terminal repeat (LTR) activation. A full-length PANG cDNA was isolated from an MPC-11 plasma cell tumor cDNA library and encodes a polypeptide of about 113 kDa with six immunoglobulin C2-like and four type III fibronectin-like domains. PANG bears a striking resemblance to axonal glycoproteins TAG-1 and F11 known to function in neuronal outgrowth. An extensive survey revealed a predominant 3.6-kb PANG transcript in 60% (30 of 50) of PCTs as well as unique smaller and larger species. All other normal and transformed lymphoid and nonlymphoid cell lines and normal tissues were negative for PANG expression except for the brain, wherein unique 4.0- and 6.1-kb transcripts were detected. Reverse transcriptase PCR analysis revealed IAP LTR fusion to PANG mRNAs in five PCTs and in a neuroblastoma line. The 5' end of a mouse brain PANG cDNA was identical to the MPC-11 PANG transcript except for the precise replacement of its 5' LTR sequence.

RNA synthesis and ultrastructure of equine zygotes and embryos

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.

A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum.

Morphogenesis of retroviruses involves assembly of the structural Gag and Gag-Pol polyproteins with subsequent budding of the virus particle from the plasma membrane and proteolytic cleavage by the viral proteinase. The matrix (MA) domain, representing the N-terminal segment of Gag, plays a critical role in this process. We constructed an in-frame deletion in the MA coding region (lacking codons 16 to 99) of the human immunodeficiency virus (HIV) type 1 gag gene. Following transient transfection of the complete proviral DNA carrying the deletion, the mutant polyprotein was synthesized and proteolytically processed like the wild-type polyprotein. However, release of virus particles was reduced approximately 10-fold. The extracellular particles that were released did not contain viral glycoproteins and were noninfectious. Electron micrographs revealed budding of virus particles into the endoplasmic reticulum (ER) of transfected cells and large numbers of particles within the ER. These particles were all immature and morphologically indistinguishable from intracisternal A-type particles, a class of murine endogenous retrovirus elements. Budding structures at the plasma membrane were rarely seen and only a few extracellular particles were observed, but in contrast to those in the ER, these particles had the morphology of mature particles, similar to that of wild-type HIV, except for the lack of surface projections.

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.

Are endogenous retroviruses involved in humanautoimmune disease?

A role for viruses in the etiopathogenesis of human autoimmune diseases has long been suspected but has not yet been proven. In Sjögren's syndrome (SS), there is continuing experimental support for the possible involvement of Epstein-Barr virus. Since the advent of AIDS, there is also great interest in retroviruses and autoimmune disease. We previously reported that 30% of SS patients and 36% of systemic lupus erythematosus (SLE) patients have serum antibodies to the p24 gag protein of HIV-1. We now report that two mechanisms classic for retroviruses (molecular mimicry and immunosuppression) may be operative in SS and SLE. The p24 gag protein shares a proline-rich epitope with the Sm nucleoprotein to which many SLE patients have antibodies. The impaired lymphocyte activation seen in peripheral blood T cells in SS patients is also seen in a human T cell line infected with an A-type retroviral particle linked to SS. Many studies suggest that endogenous retroviral sequences are important in immunoregulation. We now suggest that endogenous retroviral sequences may also be important in the etiology and pathogenesis of SS and SLE.

Establishment and characterization of cell lines from human adenovirus type 12-induced murine tumors producing endogenous virus particles.

Two cell lines designated IC-KMS and D-KMS were established from human adenovirus type 12-induced tumors of C3Hf/OK mouse. The cell lines retained the characteristics of the original tumor i.e., production of numerous C-type and intracisternal A-type particles, integration of Ad12 E1 region DNA and amplification of the myc gene family. Chromosomal analysis revealed chromosome aberrations in both IC-KMS and D-KMS cells. The modal chromosome number of IC-KMS cells was 54 and that of D-KMS cells was 48. Metacentric chromosomes and minichromosomes were found. Trisomy of chromosome 3, 7 and 12 was seen frequently in D-KMS cells. Although DNA aneuploidy was revealed by flow cytometry, the DNA indices of these cells showed no relation to the copy number of integrated Ad12 DNA. These cells have been propagated by serial culture during the past 17 months. Production of endogenous virus particles is a unique characteristic of IC-KMS and D-KMS cells. These cell lines would be useful materials for examining the contribution of Ad12 carcinogenesis to activation of endogenous virus particles, and also the correlation between Ad12 carcinogenesis and cancer-related genes.

Aldolase-DNA interactions in a SEWA cell system

In this report we demonstrate the novel finding that aldolase A interacts with DNA sequences in mouse SEWA sarcoma cells. This interaction was initially observed through the identification of a 40 kDa protein which was eluted from a DNA affinity chromatography column consisting of the long terminal repeat (LTR) of the endogenous intracisternal A-type particle (IAP). Microsequencing analysis identified this 40 kDa protein as the glycolytic enzyme, aldolase A. The use of specific anti-aldolase antibodies enabled the identification and subsequent purification of aldolase from the nuclear protein fraction of two SEWA sublines, one that is adherent and one that grows in suspension. In order to confirm our initial finding that aldolase is capable of interacting with DNA, proteins from each subline were immunopurified with anti-aldolase antibodies, eluted and then tested for their ability to interact with IAP-LTR DNA sequences. Interestingly, only aldolase derived from the anchorage dependent SEWA cells was capable of interacting with the IAP-LTR, however, several cell lines derived from human tumors also exhibited this activity. Subsequent studies revealed the ability of aldolase to interact with some but not every DNA sequence tested, implying that there may be a minimal DNA conformation and/or sequence requirement for this activity. The presence of aldolase A in the nuclei and its ability to interact with certain DNA sequences suggest a novel role for this metabolic enzyme.

Interaction of SEWA sarcoma cell proteins with the intracisternal A-type particle long terminal repeat DNA sequence.

Intracisternal A-type particle (IAP) transcripts are endogenous retrovirus-like sequences expressed during specific stages of normal development and in a variety of murine tumors. In this study, we have analyzed two cell lines derived originally from the SEWA murine osteosarcoma and grown either as ascites or as solid tumors, for proteins that might regulate IAP expression. We found that subline AA7-NA, originally derived from the ascites tumor, expressed about five times more IAP RNA than the AS12-AD subline, which was derived from a solid tumor. In view of this finding, we examined the binding of cellular proteins from the two cell lines to the 5' end of an IAP long terminal repeat sequence. Gel retardation assays of DNA-protein complexes and DNase I footprinting assays identified several DNA sequences within the long terminal repeat fragment that were protected by protein extracts from both SEWA sublines. Gel retardation assays using specific synthetic oligonucleotide sequences that correspond to two of these protected regions revealed different patterns of DNA-protein complexes with extracts from the two SEWA sublines. These data suggest that expression of IAP sequences is regulated by complex mechanisms involving several proteins that appear to differ between the two sublines.