ALK (anaplastic lymphoma kinase) is a tyrosine kinase receptor, expressed as part of the chimeric NPM-ALK protein, in anaplastic large cell lymphomas (ALCLs) exhibiting the t(2;5)(p23;q35) translocation. As a result of this translocation, the NPM (nucleophosmin) gene is fused to the portion of the ALK gene encoding its intracytoplasmic segment. In normal mouse tissues, mRNA encoding the Alk receptor has been found only in neural cells, suggesting involvement of this receptor in the development of the nervous system. The purpose of the present study was to examine the presence of ALK transcripts and protein in normal human tissues and a variety of cell lines and human tumors. Emphasis was placed on neuroblastomas because other tyrosine kinase receptors are expressed in human neuroblastomas. Fifty-six cell lines, including 29 lines of neural origin, and lymphoid and nonlymphoid tissue specimens, including 24 neuroblastomas, were investigated for ALK expression, using reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. The results confirmed that mRNA encoding ALK protein was not detectable in any normal or neoplastic hematopoietic tissue tested, except for t(2;5)-positive ALCL. The salient finding was that 13 of the 29 cell lines of neural origin and 22 of 24 neuroblastomas were found to express ALK transcripts and ALK protein. However, no correlation was evident between any known prognostic factors and the level of ALK expression.

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Mutagen treatment of mouse P815 tumor cells produces tum-variants that are rejected by syngeneic mice because these variants express new surface antigens. These "tum antigens" are recognized by cytolytic T lymphocytes but induce no detectable antibody response. Transfection of P815 cell line P1.HTR with DNA of tum-variant P91 yielded transfectants expressing tum antigen P91A. They were detected by their ability to stimulate proliferation of cytolytic T lymphocytes [Wolfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26,[178][179][180][181][182][183][184][185][186][187]. A cosmid library of a cell line expressing antigen P91A was transfected into P1.HTR. Transfectants expressing the antigen were obtained. By packaging directly the DNA of a transfectant with A phage extracts, we obtained a small cosmid population containing as major component a cosmid that transferred the expression of P91A. The assay of various restriction fragments of this cosmid led to the isolation of an 800-base-pair fragment containing the P91A sequence required for transfection. Comparison with a homologous cDNA showed that this fragment contained only one of the several exons of the P91A gene. The normal and the tumforms of the gene differ by one nucleotide located in this 137-base-pair exon. The essential role of this mutation, which produces an amino acid change, was confirmed by site-directed mutagenesis. No significant sequence similarity was found between the 800-base-pair fragment and any recorded gene.In vitro mutagen treatment of mouse tumor cell lines generates at high frequency immunogenic variants that are rejected by syngeneic mice (1). Since they fail to form tumors, these stable variants have been named "tum-" as opposed to the original "tumr+" cell, which forms progressive tumors. Most tum-variants express transplantation antigens not found on the tum+ cell. The existence of these "tumantigens" can be demonstrated in vivo by cross-immunization experiments or in vitro with cytolytic T lymphocytes (CTLs) (2).We have studied a series of tum-variants derived from mastocytoma P815, a tumor induced iq a DBA/2 mouse with methylcholanthrene (3). For most P815 tum-variants, stable CTL clones have been obtained that show a strict specificity for the immunizing variant and thus define one or several tum-antigens (4). Despite numerous attempts, we failed to obtain antibodies against these variaqt-specific antigens.The diversity of tum-antigens is considerable: the analysis of 15 P815 tum-variants demonstrated the existence of a different antigen on each of them (5). To understand the underlying genetic mechanism, we undertook the cloning of the "tum-genes" that determine the expression of tumantigens. As a first step, we attempted DNA-mediated transfer of antigen P91A, one of the two tum-antigens of P815 variant P91. Other workers have described the production of transfectants for surface antigens (6, 7), but in these instances, the antigen-expressing transfectants were detected ...

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta-and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta-and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses. IMPORTANCEControl of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC). Herpesviruses are enveloped viruses containing relatively large, double-stranded DNA genomes. They are divided into three subfamilies (alpha-, beta-, and gammaherpesviruses) according to sequence homology, cellular tropism, and productive cycle behavior under laboratory culture conditions. Nine herpesviruses have been identified in humans. Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV) are alphaherpesviruses that due to neurotropism cause recurrent skin lesions, meningitis, and rare but very serious encephalitis in the case of HSV-1; human cytomegalovirus (HCMV), human herpesviruses 6A and 6B (HHV-6A and HHV-6B), and human he...

That the expression of late genes is coupled to viral genome replication is well established for all herpesviruses, but the exact mechanisms of their regulation, especially by viral proteins, are poorly understood. Here, we report the identification of the Epstein-Barr virus (EBV) early protein BcRF1 as a viral factor crucial for the activation of late gene transcription following viral DNA replication during the productive cycle. In order to study the function of the BcRF1 protein, we constructed a recombinant EBV lacking this gene. In HEK293 cells, this recombinant virus underwent normal DNA replication during the productive cycle but failed to express high levels of late gene transcripts or proteins, resulting in a nonproductive infection. Interestingly, a TATT motif is present in the promoter of most EBV late genes, at the position of the TATA box. We show here that BcRF1 forms a complex with the TATT motif and that this interaction is required for activation of late viral gene expression. Moreover, our results suggest that BcRF1 acts via interaction with other viral proteins.

Seven cases of large B-cell lymphoma which define a previously unrecognized subgroup are reported. Morphologically they are comprised of monomorphic large immunoblast-like cells, containing large central nucleoli, which tend to invade lymphatic sinuses. Superficially they resemble anaplastic large cell lymphoma (ALCL) but they lack CD30. These lymphomas express epithelial membrane antigen (as do ALCL), but also contain intracytoplasmic IgA of a single light chain type (five cases) and an endoplasmic reticulum–associated marker detected by antibody VS38. They lack lineage-associated leukocyte antigens with the exception of CD4 (5 of 5 cases) and CD57 (5 of 7 cases). They are labeled by antibodies detecting both the intracytoplasmic and extracellular regions of the ALK receptor kinase, suggesting that they express the full-length form of this molecule. This was confirmed by Western blotting (in the one case tested) which showed a band of 200 kD in tumor cell lysates, and by polymerase chain reaction (PCR) amplification of mRNA encoding intracellular and extracellular ALK sequences (in the two cases tested). There was no evidence by cytogenetics (one case analyzed) or reverse transcriptase-PCR (three cases tested) of the 2; 5 translocation or the resultant NPM-ALK gene, as is commonly found in ALCL. All but one of the patients were male and all but one were adults, and in all but the latter case the disease followed an aggressive course.

Anaplastic large cell lymphomas (ALCL) are frequently associated with the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene at 5q35, which encodes a nucleolar protein involved in shuttling ribonucleoproteins from the cytoplasm to the nucleus, to the anaplastic lymphoma kinase (ALK) gene at 2p23, encoding a tyrosine kinase receptor. In this report, we describe a typical case of ALCL whose malignant cells exhibited a novel (1;2)(q25;p23) translocation. These cells expressed ALK protein, but, in contrast to t(2;5)-positive ALCL (which show cytoplasmic, nuclear, and nucleolar staining), labeling was restricted to the malignant cell cytoplasm. Using a polymerase chain reaction (PCR)-based technique to walk on chromosome 2 from the known ALK gene across the breakpoint, we showed that the gene involved at 1q25 is TPM3, encoding a nonmuscular tropomyosin. We subsequently identified, using reverse transcription-PCR analysis of cases showing similar ALK cytoplasm-restricted staining, fusion of the ALK andTPM3 genes in 2 other cases of ALCL. The TPM3 gene has been previously found in papillary thyroid carcinomas as a fusion partner with the TRK kinase gene. We showed that TPM3 is constitutively expressed in lymphoid cell lines, suggesting that, in these t(1;2)-bearing ALCL cases, the TPM3 gene contributes an active promoter for ALK expression. Activation of the ALK catalytic domain probably results from homodimerization of the hybrid protein TPM3-ALK, through the TPM3 protein-protein interaction domain. The present cases of ALCL associated with a novel t(1;2)(q25;p23) demonstrate that at least one fusion partner other than NPM can activate the intracytoplasmic domain of the ALK kinase.

Seven cases of large B-cell lymphoma which define a previously unrecognized subgroup are reported. Morphologically they are comprised of monomorphic large immunoblast-like cells, containing large central nucleoli, which tend to invade lymphatic sinuses. Superficially they resemble anaplastic large cell lymphoma (ALCL) but they lack CD30. These lymphomas express epithelial membrane antigen (as do ALCL), but also contain intracytoplasmic IgA of a single light chain type (five cases) and an endoplasmic reticulum–associated marker detected by antibody VS38. They lack lineage-associated leukocyte antigens with the exception of CD4 (5 of 5 cases) and CD57 (5 of 7 cases). They are labeled by antibodies detecting both the intracytoplasmic and extracellular regions of the ALK receptor kinase, suggesting that they express the full-length form of this molecule. This was confirmed by Western blotting (in the one case tested) which showed a band of 200 kD in tumor cell lysates, and by polymerase chain reaction (PCR) amplification of mRNA encoding intracellular and extracellular ALK sequences (in the two cases tested). There was no evidence by cytogenetics (one case analyzed) or reverse transcriptase-PCR (three cases tested) of the 2; 5 translocation or the resultant NPM-ALK gene, as is commonly found in ALCL. All but one of the patients were male and all but one were adults, and in all but the latter case the disease followed an aggressive course.