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Epstein-Barr virus (EBV) receptors were implanted into the membranes of receptor-negative cells, using Sendai virus envelopes as vehicles. The presence of the receptors in the target cell membrane was demonstrated by monitoring the fate of radioiodinated donor membranes. Receptors could be detected for at least 36 hr after implantation. [3H]Thymidine-labeled EBV bound efficiently to receptor-implanted cells but not to control cells. Binding was inhibited by an excess of nonlabeled virus. Of the [3H]thymidine-labeled EBV DNA, 50-75% was found inside the receptor-implanted, EBV-exposed cells 24 hr after the infection. The viral genome was functionally active in B lymphocyte-derived cell lines of human, murine, and baboon origin; in T lymphocyte-derived lines of human and murine origin; in mouse fibroblasts; and in freshly explanted mouse lymphocytes, as shown by the expression of EBV-determined nuclear, early, and viral capsid antigens.

T lymphocytes recognize their antigenic targets as peptides associated with major histocompatibility complex molecules. The HLA-A11 allele, a preferred restriction element for Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocyte responses, presents an immunodominant epitope derived from the EBV nuclear antigen 4. Subpicomolar concentrations of a synthetic nonamer peptide, IVTDFSVIK, corresponding to amino acids 416-424 of the EBV nuclear antigen 4 sequence, can sensitize phytohemagglutinin-stimulated blasts to lysis by EBV-specific HLA-A11-restricted cytotoxic T-lymphocytes. We show that micromolar concentrations of this peptide induce assembly and surface expression of HLA-A11 in an A11-transfected subline of the peptide transporter mutant cell line T2. Using the IVTDFSVIK peptide and a series of synthetic nonamer peptides, differing from the original sequence by single amino acid substitutions, we have defined a motif for HLA-A11-binding peptides. This predicts the presence of a hydrophobic amino acid in position 2, amino acids with small side chains in positions 3 and 6, and a lysine in position 9. Using this motif, we have identified a peptide in the carboxyl-terminal end of wild-type p53, ELNEALELK, which is able to induce HLA-A11 assembly as efficiently as the IVTDFSVIK viral peptide.

We have isolated phage clones from Drosophila melanogaster genomic and cDNA libraries containing a sequence homologous to the murine Int-1 protooncogene. The Drosophila gene is represented by a single locus at position 28A1-2 on chromosome 2. The gene is expressed as a 2.9-kilobase-long polyadenylylated mRNA in embryo, larval, and pupal stages. It is hardly detectable in adult flies. The longest open reading frame of the cDNA clone corresponds to a protein 469 amino acids long. Alignment of the predicted amino acid sequences shows that the Drosophila protein is 86 amino acids longer than its murine counterpart. In spite of the difference in length, the two proteins are highly conserved with an overall sequence homology of 54%. Both Drosophila and murine Int-1 proteins begin with a hydrophobic leader sequence and contain cysteine residues and sites for glycosylation (four in the murine protein and one in the Drosophila protein) in conserved positions, suggesting that they play important functional roles.

Presented is a detailed molecular analysis of the rearranged c-myc oncogene from ABPC45, an unusual plasmacytoma that was originally classified as translocation-negative. Previous data obtained by high-resolution chromosome banding suggested that this tumor was a member of a small group distinguished by the absence of rcpt (12;15) or (6;15) and further characterized by a band deletion near the c-myc locus on chromosome 15. However, genomic Southern blotting and analysis of the cloned oncogene in the present study reveal that (i) chromosome 12 sequences lie 365 base pairs 5' of the rearranged c-myc; (ii) this DNA consists of immunoglobulin alpha switch region and 5' immunoglobulin mu switch region sequences that are rearranged in an aberrant fashion; and (iii) the immunoglobulin heavy-chain gene enhancer element now resides approximately 2.5 kilobase pairs 5' of c-myc. We infer from these and other data that the rearrangement of c-myc in ABPC45 occurred via a multistep switch region-mediated process and that a reciprocal translocation has indeed taken place. Unlike many other plasmacytomas, this event did not interrupt the normal c-myc transcription unit. Rather, disruption of gene regulation is manifested in part by a change in relative usage of the two promoters normally used by the unrearranged gene. In contrast to several of its counterparts in Burkitt lymphomas, DNA sequence analysis of the translocated c-myc gene of ABPC45 reveals that it has not acquired point mutations in the noncoding first exon. These results strongly imply that a cis-acting regulatory element normally located 5' of exon 1 is lost and that heavy-chain constant region or enhancer sequences exert similar cis effects on translocated c-myc loci.

We have detected an endoribonucleolytic activity in human cell extracts that processes the Escherichia coli 9S RNA and outer membrane protein A (ompA) mRNA with the same specificity as RNase E from E. coli. The human enzyme was partially purified by ion-exchange chromatography, and the active fractions contained a protein that was detected with antibodies shown to recognize E. coli RNase E. RNA containing four repeats of the destabilizing motif AUUUA and RNA from the 3' untranslated region of human c-myc mRNA were also found to be cleaved by E. coli RNase E and its human counterpart in a fashion that may suggest a role of this activity in mammalian mRNA decay. It was also found that RNA containing more than one AUUUA motif was cleaved more efficiently than RNA with only one or a mutated motif. This finding of a eukaryotic endoribonucleolytic activity corresponding to RNase E indicates an evolutionary conservation of the components of mRNA degradation systems.

A soluble complement-fixing antigen carried by Epstein-Barr virus (EBV)-transformed human cells has been previously extracted from cell nuclei and purified by DNA-cellulose chromatography [Luka, J., Siegert, W. & Klein, G. (1977) J. Virol., in press]. On addition of this antigen to methanol/acetic acid-fixed metaphase chrmosomes, followed by exposure to human sera containing antibodies against the EBV-determined nuclear antigen (EBNA), brilliant positive staining was obtained by anti-complement immunofluorescence. There was no staining after exposure to EBV-negative sera. Moreover, a nuclear protein fraction, prepared from an EBV-negative cell line in an analogous fashion, failed to induce the staining reaction. These data identify the soluble purified antigen as the EBV-determined nuclear antigen. The purified antigen has a molecular weight of 174,000 +/- 15,000, as determined by sucrose gradient centrifugation and gel filtration experiments. In neutral buffers containing 0.5-1.0 M NaCl, the antigen dissociates into a form of approximately one-half the original molecular weight with retained complement-fixing activity. This "monomer" has a molecular weight of 98,000 +/- 8,000.

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment of RB and p53 function.

The p53 tumor-suppressor protein has previously been shown to bind double-stranded and single-stranded DNA. We report that the p53 protein can bind single-stranded DNA ends and catalyze DNA renaturation and DNA strand transfer. Both a bacterially expressed wild-type p53 protein and a glutathione S-transferase-wild-type p53 fusion protein catalyzed renaturation of different short (25- to 76-nt) complementary single-stranded DNA fragments and promoted strand transfer between short (36-bp) duplex DNA and complementary single-stranded DNA. Mutant p53 fusion proteins carrying amino acid substitutions Glu-213, Ile-237, or Tyr-238, derived from mutant p53 genes of Burkitt lymphomas, failed to catalyze these reactions. Wild-type p53 had significantly higher binding affinity for short (36- to 76-nt) than for longer (> or = 462-nt) single-stranded DNA fragments in an electrophoretic mobility-shift assay. Moreover, electron microscopy showed that p53 preferentially binds single-stranded DNA ends. Binding of DNA ends to p53 oligomers may allow alignment of complementary strands. These findings suggest that p53 may play a direct role in the repair of DNA breaks, including the joining of complementary single-stranded DNA ends.

Epstein-Barr virus nuclear antigens (EBNAs) are expressed in a cell-phenotype-dependent manner. EBNA 1 is regularly expressed in all Epstein-Barr virus-carrying cells, whereas EBNAs 2-6 are only expressed in Epstein-Barr virus-carrying cells with a lymphoblastoid phenotype including group III Burkitt lymphoma (BL) lines positive for B-cell activation markers. Transcripts are initiated at the BCR2 or exceptionally at one BWR1 promoter in lymphoblastoid cell lines and group III BL lines. In group I BL lines, nasopharyngeal carcinoma, and the somatic cell hybrids, where EBNAs 2-6 are downregulated, the BCR2/BWR1 promoter complex is inactive or switched off. Upregulation of EBNAs 2-6 in group III BL cells and in 5-azacytidine-treated group I BL cells accompanies the activation of the silent BCR2/BWR1 promoters. Activation of BCR2 parallels demethylation of at least one CpG pair in the same promoter region. The activity of BCR2/BWR1 promoter complex depends on a particular B-cell phenotype. EBNA 1 transcription must be initiated at another promoter in cells that express only EBNA 1.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.

Experiments have been carried out to establish the relative position of the mouse Ig heavy chain locus and the c-myc oncogene. In mouse plasmacytoma with the typical rcpt(12;15) chromosome translocation the c-myc oncogene is juxtaposed to one of the heavy chain genes in a head-to-head orientation. Since the relative orientations of the c-myc locus and the Ig heavy chain gene cluster on the corresponding mouse chromosomes had not been settled, it was not known whether the rearranged c-myc gene is transposed to chromosome 12 or remains on chromosome 15. To decide which of the two alternatives is correct, we separated the translocation chromosomes by fluorescence-activated chromosome sorting. The separated chromosomal fractions were hybridized with myc-specific DNA probes corresponding to the first or second/third exons in a chromosome spot assay. The results presented here indicate that the c-myc gene in mouse plasmacytoma carrying the typical translocation, as in the human Burkitt lymphoma analogous translocation, transposes to the chromosome carrying the Ig heavy chain locus. These results also establish the orientations of the Ig heavy chain locus and the c-myc locus on their normal chromosomes.

Recombinant vectors carrying DNA fragments from the BamHI E region of the B95-8 Epstein-Barr virus (EBV) genome were transfected into COS-1 cells, and the transient expression of EBV-encoded nuclear antigens (EBNAs) was analyzed by using polyvalent human antisera and rabbit antibodies to synthetic peptides. Vector DNA containing two rightward open reading frames in the BamHI E fragment, BERF2a and BERF2b, induced the expression of a nuclear antigen identical serologically and with respect to size to the larger of the two polypeptides previously designated as EBNA4 in B95-8 cells. An antigen corresponding to the smaller polypeptide was induced in cells transfected with constructs that contained two neighboring reading frames, BERF3 and BERF4. This antigen also reacted with a rabbit antiserum to the synthetic peptide 203, deduced from BERF4. Thus, the findings show that the two components of the EBNA4 doublet in B95-8 cells are encoded by separate genes. The antigen encoded by BERF2a and/or BERF2b has been designated as EBNA4 and the antigen encoded by BERF3 and/or BERF4 has been designated as EBNA6. Polyvalent human antisera detected EBNA4 and EBNA6 in 9 of 11 lymphoid cell lines carrying independent EBV isolates. In the remaining two lines, either EBNA4 or EBNA6 was not detectable.

The patterns of long-chain neutral glycolipids and gangliosides of established, L-cell-derived murine fibroblast lines and their low- and high-tumorigenic hybrids were studied.

Human B cell lymphoma and murine T cell leukemia can be initiated by several agents. The present paper formulates some thoughts on the role of cytogenetic changes in the subsequent neoplastic process. Initiation creates long-lived preneoplastic cells. In some respects, they are comparable to in vitro-transformed ("immortalized") cell lines that maintain a diploid karyotype and are not tumorigenic in vivo. The development of a tumorigenic ("autonomous") clone is dependent on additional changes at the genetic level. In human B and murine T cell lymphoma, there are characteristic nonrandom chromosomal changes. The 14q+ marker appears to play a key role in human B cell lymphomas. The reciprocal 8;14 translocation in Burkitt lymphoma is a specialized subclass within this category. In murine T cell leukemia, trisomy 15 is the predominant change. The clustering of these nonrandom changes to tumors derived from a certain cell type rather than to tumors induced by a given etiological agent has important implications for the understanding of the genetic control of cellular responsiveness to growth-regulating forces in vivo.

In vitro binding of a Herpesvirus ateles (HVA)-associated soluble antigen to amphibian erythrocyte nuclei was demonstrated by the acid-fixed nuclear binding technique in combination with anticomplement immunofluorescence. Incubation of concentrated salt-extracted soluble antigens derived from HVA-carrying marmoset lines with methanol/acetic acid-fixed erythrocytes of frogs and salamanders resulted in a brilliant nuclear fluorescence after exposure to a live virus-boostered, anti-HVS-positive squirrel monkey serum. Anti-HVS-negative sera did not stain. The activity of the positive serum could be abosrbed completely with extracts of HVA-carrying cells but not with Epstein-Barr virus-carrying or Herpesvirus papio-carrying cells. The HVA-associated antigen was also present in lytically HVA-infected marmoset kidney cells.

The expression of Epstein-Barr virus (EBV)-determined antigens associated with growth-transformation of B cells was studied by immunoblotting with human sera from healthy donors. Four antigens were detected in EBV-carrying cell lines and in B lymphocytes early after infection with the transforming B95-8 substrain of virus. They were not found in uninfected cells, nor could they be demonstrated with sera lacking antibodies to EBV antigens. All four antigens were nuclear. Each of them varied in size in the different cell lines. The two antigens with the lowest molecular weight were identified as EBV-determined nuclear antigens (EBNAs) 1 and 2. The two high molecular weight antigens (140-160 kDa and 150-180 kDa, respectively) were detected with 6 of 16 EBV antibody-positive sera. These proteins appeared to be antigenically unrelated to each other and to EBNAs 1 and 2 and were designated EBNAs 3 and 4. Like EBNAs 1 and 2, they bound to double- and single-stranded DNA in vitro.

Four peptides were synthesized on the basis of amino acid sequences deduced from a highly spliced transcript encoded by the Bam W, Y, and H fragments of the Epstein-Barr virus (EBV) genome [Bodescot, M., Chambraud, J. B., Farrell, P. J. & Perricaudet, M. (1984) EMBO J. 3, 1913-1917]. Rabbit antisera against three of the four peptides identified a nuclear polypeptide that varied between 22 and 70 kDa in molecular size. Four of 20 EBV-positive human sera contained antibodies against this polypeptide. Since this is the fifth EBV-determined nuclear antigen (EBNA) discovered in growth-transformed cells, it is designated EBNA5. The antigen was detected in virus nonproducer lines (less than 0.01% EBV early antigen expression) and is thus not dependent on the viral cycle. It was differentially expressed depending on the origin of the lines. All 10 lymphoblastoid cell lines tested expressed EBNA5, but it could not be detected in 10 of 11 EBV-carrying Burkitt lymphoma lines. Infection of tonsillar lymphocytes with the B95-8 strain of EBV induced six EBNA5-specific polypeptides that varied between 41 and 70 kDa in molecular size with regular increments of 6 kDa. This may be due to the fact that the EBNA5 coding sequence includes the Bam W internal repeat. Parallel infection of the EBV-negative Burkitt lymphoma line Ramos with the same viral substrain did not induce detectable levels of EBNA5, nor was this antigen present in permanently EBV-converted Ramos sublines. These findings imply that the expression of the viral genome varies among B cells having different phenotypes.

A heat-stable DNA-binding protein with subunits of about 53 kilodaltons (kDal) was purified from two virally transformed human cell lines (Epstein-Barr virus-positive Raji and Namalwa) and two mouse tumor cell lines (methylcholanthrene-induced Meth A sarcoma and TA3 mammary carcinoma). All four 53kDal proteins showed closely related total amino acid compositions, similar peptide maps, and identical NH2-terminal amino acid sequences for 20 residues. These 53-kDal proteins are therefore evolutionarily highly conserved, independent of whether they originate from virally or chemically transformed cells. The NH2-terminal sequence and the protein chain as a whole are not hydrophobic; however, some unexpected residue distributions were observed. Comparisons with other proteins reveal no clear sequence similarity with known tumor antigen structures, homologous immunoglobulins, or some other proteins of known sequence. Epstein-Barr virus-determined nuclear antigen also appears to have a different NH2-terminal sequence. Thus, the results show that the 53-kDal proteins represent a unique protein type with little species variation; this finding suggests that these proteins must perform an important common function in different transformation systems.

Spontaneously arising immunocytomas in Lou/Wsl rats contain a consistent translocation between chromosomes 6 and 7. The c-myc gene has been localized to chromosome 7 and has been shown to be rearranged in the majority of the rat immunocytomas. We now report the cloning of the rearranged 11-kilobase EcoRI c-myc fragment from the IgE-secreting IR75 tumor. Sequence analysis revealed that the cytogenetically visible t(6;7) translocation must have involved several events in this tumor. One event has led to the juxtaposition of c-myc and the switch mu region, in a head-to-head orientation. The breakpoint is approximately 850 base pairs upstream from the proximal c-myc promoter on chromosome 7. This area is distinct from the more common mouse plasmacytoma- and Burkitt lymphoma-associated translocation breakpoints and also differs from the known murine retroviral insertion sites. A second rearrangement has led to the transposition of sequences upstream from the switch gamma 1 region to the c-myc-distant end of the switch mu region, tail-to-tail. This requires at least two events, including one inversion. In addition to showing that identical loci (c-myc, immunoglobulin) are juxtaposed via chromosomal translocations in three different tumors (Burkitt lymphoma, mouse plasmacytoma, and rat immunocytoma) in different species (human, mouse, and rat), the multiple rearrangements in IR75 and some other tumors emphasize the selective value of c-myc activation by an immunoglobulin locus in the tumorigenic process.

Two synthetic peptides corresponding to residues 1-20 and 10-20, respectively, of one type of a cellular protein called "p53" have been linked to a carrier protein and injected into rabbits to raise antibodies. The antibodies obtained were capable of reacting with the native protein, as judged by an enzyme-linked immunosorbent assay, protein A-linked staining of immunoblots after NaDodSO4 gel electrophoresis, and immunoprecipitation. The immunoassay titers against the protein were lower for these antibodies than for antisera derived from immunization with purified p53. However, staining with the immunoblot method showed that the antipeptide antibodies against p53 were uniquely specific. The data suggest that at least two different types of p53 molecules occur. The cellular protein previously isolated from human cells transformed by Epstein-Barr virus and from murine tumors induced by methylcholanthrene appears to be larger than the p53 reported in relation to simian virus 40- or adenovirus-transformed cells and to some other tumors. Some interrelationships have not been excluded, but it is clear that the two protein molecules do not behave identically. The reactions of the antipeptide antibodies with the intact protein have implications in regard to protein conformations. The strict specificities of such antibodies allow the generation of distinct sets of reagents useful for quantitation, purification, and cloning.

Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) assay, in line with the original definition of the EBV-determined nuclear antigen (EBNA) [Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499-520]. Eight EBNA and EBV DNA-carrying lines showed nuclear staining with the antipeptide antibody, whereas five EBV DNA negative lines failed to stain. The staining pattern was more discretely punctate than the finely dispersed diffuse EBNA staining obtained with human antisera. Human EBV antibody-positive but not EBV-negative sera reacted with the synthetic peptide in an ELISA test. The peptide-specific antibodies were purified from the sera of healthy EBV-seropositive persons by affinity chromatography with the peptide. They gave an EBV-specific, brilliant punctate nuclear ACIF staining similar to that of the rabbit antipeptide antibodies. It was concluded that the glycine-alanine structure encoded by the IR3 region contains a native determinant of EBNA, detected by the ACIF test. Immunoblotting with the rabbit and human peptide-specific antibodies identified poly-peptides that varied between 70 and 92 kilodaltons in size in different EBV-positive cell lines, corresponding closely to a previously identified variation pattern in the size of EBNA. In addition, rabbit antipeptide antibodies identified two cellular polypeptides, 44 and 49 kilodaltons in size.

Renal cell carcinoma (RCC) and normal kidney tissues have been examined from 34 patients with sporadic, nonhereditary RCC. Eighteen of the 21 cytogenetically examined tumors (86%) had a detectable anomaly of chromosome arm 3p distal to band 3p11.2-p13, manifested as a deletion, combined with the nonreciprocal translocation of a segment from another chromosome or monosomy 3. Restriction-fragment-length polymorphism analysis showed loss of D1S1 heterozygosity in 16 of the 21 cases (76%). D3S2 heterozygosity was lost in 2 of 11 cases (18%). The variability of the breakpoint between 3p11.2 and 3p13 and the absence of a consistently translocated segment from another chromosome suggests a genetic-loss mechanism, while the activation of a dominant oncogene appears less likely. Together with the previously demonstrated involvement of the 3p14.2 region in a familial case, these findings suggest that RCCs may arise by the deletion of a "recessive cancer gene," as do retinoblastoma and Wilms tumor. The relevant locus must be located on the telomeric side of the D1S1 locus on the short arm of chromosome 3.

Six independently maintained sublines of FLEB 14, a fetal-liver-derived Epstein-Barr virus-transformed pro-B cell line that has not yet rearranged its immunoglobulin genes, were examined after in vitro propagation during 19-36 months. Two lines showed no immunoglobulin heavy chain gene rearrangement, whereas one allele was rearranged with breakpoints inside the switch region of the mu chain gene in the remaining four. These rearrangements had been generated by the translocation of different chromosome fragments to the immunoglobulin heavy chain gene cluster-carrying 14q32 band in each of the four lines. Previously, a similar rearrangement was found in a fifth subline concurrently with a reciprocal 6;14 translocation. The transposed pieces have been derived from chromosomes 16 and 18 in two of the more recently rearranged lines. Their origins could not be determined in the remaining two lines, but they were different from each other and the other three 14q+ markers. The 14q+ marker-carrying variant has replaced its diploid progenitor suggesting that the translocation has conveyed some in vitro growth advantage on its carrier. This was also supported by the duplication of the 14q+ marker and the loss of its normal chromosome 14 homologue in one subline during serial culturing. The vulnerability of the switch region of the mu chain gene to illegitimate recombination at the pro-B stage and the possible relevance of this finding for the origin of the Burkitt lymphoma-associated 8;14 (immunoglobulin heavy chain gene cluster/MYC) translocation is discussed.

Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation.

Spontaneous tumours are defined as tumors that develop in the absence of all experimental interference. In contrast to the widely documented, strong rejection reactions against most virus-induced tumors, spontaneous tumors evoke little or no detectable rejection reaction in intact or preimmunized syngeneic hosts. The difference can be viewed in relation to the contrasting natural history of the two conditions. Spontaneous tumors evolve in several steps, as a fule. "Tumor progression" is a microevolutionary process at the level of the somatic tissue where successive clonal variants replace each other. Each new variant gains the upper hand due to its greater independence of some restricting host mechanism. Independence of immune restrictions must be part of this process. Host selection for immune resistance apparently plays no major role here, presumably because most of the naturally occurring tumors arise after the host has passed the peak of its reproductive period. Protection against the oncogenic effects of ubiquitous tumor viruses is, on the other hand, the result of host selection for immune mechanisms favoring prompt rejection of virus-transformed cells. This is neither synonymous with nor related to protection against the viral infection per se, which is frequently successful and usually quite harmless. A certain relationship can be perceived between the degree of viral ubiquity and the strength of immune protection against the corresponding tumor cells. Natural selection for host recognition of commonly occurring, virally induced changes in neoplastic cell membranes can be surmised to occur, at least in part, by the fixation of appropriate immune responsiveness (Ir) genes. The role of Ir genes for tumor recognition can be approached by the genetic analysis of the F1 hybrid resistance effect. Unresponsiveness to spontaneous tumors may be overcome by target-cell modification, e.g., by chemical coupling, somatic cell hybridization, or viral "xenogenization".

Soluble membrane fractions derived from Raji cells trigger lymphocytes of Epstein-Barr virus (EBV)-seropositive, but not EBV-seronegative, individuals to release a lymphokine that inhibits leukocyte migration. The reaction can be blocked by the sera of patients with EBV-DNA-carrying tumors, Burkitt lymphoma, or nasopharyngeal carcinoma. Absorption of these sera with EBV-positive, but not EBV-negative, cells abrogates their blocking activity. These findings suggest that the antigen responsible for the leukocyte migration inhibition reaction is an EBV-encoded or an EBV-induced membrane component. The antigen is not identical with EBV-associated nuclear antigen or any other known antibody-detected EBV antigen.

Five distinct Epstein-Barr virus (EBV)-determined nuclear antigens (EBNA-1 to EBNA-5) were recently identified. Antibody responses to these antigens could conceivably differ, and thus prove of serodiagnostic value, in EBV-associated disease processes. As a first step, murine or human cell lines transfected with appropriate EBV DNA fragments and stably expressing either EBNA-1 or EBNA-2 were used to determine the frequency and time of emergence of antibodies to these two antigens in the course of acute and chronic infectious mononucleosis (IM) and to assess their titers in so-called chronic active EBV infections. Following IM, antibodies to EBNA-2 arose first and, after reaching peak titers, declined again in time to lower persistent or even nondetectable levels. Antibodies to EBNA-1 emerged several weeks or months after anti-EBNA-2 and gradually attained the titers at which they persisted indefinitely. The ratios between the anti-EBNA-1 and anti-EBNA-2 titers therefore were generally well below 1.0 during the first 6-12 months after IM and turned to well above 1.0 during the second year. In clear cases of chronic IM, the inversion of this ratio was delayed or prevented. In the less well-defined chronic EBV infections, low ratios were observed in only some of the patients. Because many of these illnesses were not ushered in by a proven IM and often showed EBV-specific antibody profiles within the normally expected range, a causal role of the virus in these cases remains doubtful.

Treatment of H-2-deficient nonmetastatic B16 melanoma cells with physiological doses of interferon gamma (IFN-gamma) reduced cellular growth in vitro but induced a shift to the lung-colonizing phenotype as assessed after intravenous injection of the treated cells. As little as 1 antiviral unit of recombinant IFN-gamma per ml induced B16 cells to form 3-40 pulmonary metastases in each injected mouse, whereas a 1000-fold higher concentration of IFN-beta was required to see similar effects. IFN-gamma may induce cell-surface molecules that contribute to the metastatic ability of the tumor cells. The efficient enhancement of metastatic ability after IFN-gamma treatment of the B16 cells was paralleled by an increased H-2 antigen expression and decreased sensitivity to natural killer cells. The experiments support the idea that metastasis may not depend exclusively on stable genetic changes or heterogeneity within a tumor population but may be also influenced through the modulation of the phenotype by physiological or pharmacological agents. The results are also discussed with regard to the role of different effector cells in tumor cell clearance and in relation to lymphokine-based strategies for therapy.

The GC-BJAB cell line, which carries the Epstein--Barr virus (EBV), was derived from an EBV-genome-negative lymphoma line (BJAB) by EBV infection in vitro [G. B. Clements, G. Klein, and S. Povey (1975) Int. J. Cancer, in press]. Both lines grow at a similar rate at 37 degrees but they differ at other temperatures. BJAB grows well at 34 degrees, 37 degrees, and 39 degrees. GC-BJAB grows at 37 degrees and 39 degrees, but grows poorly at 34 degrees. At 37 degrees, GC-BJAB cultures can be maintained at the viable state for a long time after having reached saturation density (approximately 10(6) cells per ml). In contrast, BJAB cultures die very soon after having attained similar maximum density. Since the identity of the two cell lines has been critically established [Clements et al.; E. Svedmyr and M. Jondal (1975) Proc. Nat. Acad. Sci USA 72, 1622--1626; G. Klein, to be published; J. Zeuthen, personal communication] the remarkable differences in their growth properties must be attributed to the EBV genome.

Trypsin-Giemsa banding studies on T cell leukemias induced in Robertsonian translocation mice by dimethylbenz[a]anthracene and Moloney leukemia virus show a trisomy of chromosome 15 even in cases in which chromosome 15 has undergone centromeric fusion with chromosomes 1, 5, or 6. These results suggest that the duplication of gene(s) located on chromosome 15 is of critical importance for murine T cell leukemia development.