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Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.

Although viruses comprise the most abundant genetic material in the biosphere, to date only several thousand virus species have been formally defined. Such a limited perspective on virus diversity has in part arisen because viruses were traditionally considered only as etiologic agents of overt disease in humans or economically important species and were often difficult to identify using cell culture. This view has dramatically changed with the rise of metagenomics, which is transforming virus discovery and revealing a remarkable diversity of viruses sampled from diverse cellular organisms. These newly discovered viruses help fill major gaps in the evolutionary history of viruses, revealing a near continuum of diversity among genera, families, and even orders of RNA viruses. Herein, we review some of the recent advances in our understanding of the RNA virosphere that have stemmed from metagenomics, note future directions, and highlight some of the remaining challenges to this rapidly developing field.

Transduction of oncogenes by ALVs and generation of acute transforming viruses is common in natural viral infections. In order to understand the molecular basis for the rapid oncogenicity of Fu-J, an acutely transforming avian leukosis virus isolated from fibrosarcomas in crossbreed broilers infected with subgroup J avian leukosis virus (ALV-J) in China, complete genomic structure of Fu-J virus was determined by PCR amplification and compared with those of Fu-J1, Fu-J2, Fu-J3, Fu-J4, and Fu-J5 reported previously. The results showed that the genome of Fu-J was defective, with parts of gag gene replaced by the complete v-fps oncogene and encoded a 137 kDa Gag-fps fusion protein. Sequence analysis revealed that Fu-J and Fu-J1 to Fu-J5 were related quasi-species variants carrying different lengths of v-fps oncogenes generated from recombination between helper virus and c-fps gene. Comparison of virus carrying v-fps oncogene also gave us a glimpse of the molecular characterization and evolution process of the acutely transforming ALV.

SRC family kinases (SFKs) are known regulators of multiple cellular events, including cell movement, differentiation, proliferation, survival and apoptosis. SFKs are expressed virtually by all mammalian cells. They are non-receptor protein kinases that phosphorylate a variety of cellular proteins on tyrosine, leading to the activation of protein targets in response to environmental stimuli. Among SFKs, SRC, YES and FYN are the ubiquitously expressed and best studied members. In fact, SRC, the prototypical SFK, was the first tyrosine kinase identified in mammalian cells. Studies have shown that SFKs are regulators of cell junctions, and function in endocytosis and membrane trafficking to regulate junction restructuring events. Herein, we briefly summarize the recent findings in the field regarding the role of SFKs in the testis in regulating spermatogenesis, particularly in Sertoli–Sertoli and Sertoli–germ cell adhesion. While it is almost 50 years since the identification of the oncogene v-Src encoded by Rous sarcoma transforming virus, the understanding of SFK involvement during spermatogenesis in the testis remains far behind that in other epithelia and tissues. The goal of this review is to bridge this gap.

The link between Plasmodium falciparum malaria and endemic Burkitt’s lymphoma (eBL) has been an enigma for more than 50 years, since it was first observed that the occurrence of the two coincided [1,2]. So convincing was the association that it led to the prediction that eBL was caused by an infectious agent that was spread by malarial mosquitoes. The subsequent search turned up the first human oncogenic virus, Epstein-Barr virus (EBV) [3,4]. EBV is found in nearly all cases of eBL [5] and has been shown to be a potent transforming virus for human B cells [4]. eBL is believed to arise from germinal center (GC) B cells [6] and is characterized by a typical translocation of the c-myc oncogene into one of the immunoglobulin loci [7,8]. The subsequent deregulation of c-myc expression would normally lead to rapid apoptosis of the cell, but the presence of EBV is thought to rescue these cells and allow them to survive [9]. The enigma remained, however, because EBV is not spread by mosquitoes. eBL therefore represents an intriguing and unusual situation in which interactions between a protozoan parasite (P. falciparum) and a mammalian virus (EBV) combine to cause cancer. While the mechanisms linking EBV to lymphoma development are becoming better understood, the link between P. falciparum malaria and eBL has remained completely unexplained until now. Finally, we have a breakthrough in this longstanding issue from two separate studies: one through investigation of human tissues and one in a mouse model.

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5α ( Ref1 ), showing that XMRV is resistant to TRIM5α restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.

To study the antiviral effects of lamivudine on avian leukosis virus subgroup J (ALV-J) and its inhibitory effect on the growth of fibrosarcomas caused by acute transforming avian leukosis virus, a series of experiments were performed in chicken embryo fibroblast cultures and 1-day-old chickens inoculated with an acutely transforming viral stock Fu-J (SDAU1005). This stock was prepared from an acutely fibrosarcoma of field cases in chicken farms and contained both the replication-defective virus Fu-J carrying v-fps oncogene and its helper virus ALV-J strain SDAU1005. The results from three different assays in cell cultures demonstrated the significant inhibitory effect of lamivudine on the replication of both SDAU1005 and Fu-J viruses. Furthermore, the effect was dose dependent in the concentration range of 1–4 μg/ml. In chicken experiments, lamivudine could decrease the viral loads of SDAU1005 and Fu-J in the plasma of inoculated chickens, delay the appearance of acute sarcomas, and decrease chicken mortality in the early stage. This model may be used to directly evaluate the inhibitory effects of lamivudine on such tumors and to understand the relationship between the replication-defective virus and its helper virus while also assessing tumor processes.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a skin cancer caused by a gamma- herpesvirus.The virus is a “tumor” transforming virus and thus often leads to cancer in the infected patient. Its association with AIDS over the past two decades has brought increased attention to the roll viral infection plays in the evolution of cancer. KSHV has been shown to interfere in one major pathway that controls cell growth, the apoptotic pathway, important for the maintenance of normal growth and development. Viruses require a live cell for survival and have evolved a mechanism to interfere with apoptosis to create immortal cell hosts. The virus expresses a protein, vBCL-2 that interferes with pro-apoptotic proteins and promotes tumor growth. This viral protein is a functional homology of the human Bcl2 protein and has been shown to interact with several BH3 domain containing family members. Previously we had identified an interaction between the KSHV vBcl2 protein and the human BH3 domain containing protein, BIK...

By means of correlated observations on the roentgenographic and histological structure, in an article written over 50 yr ago, Phemister [1] demonstrated diat osteosarcomas contain tumorous and normal nontumorous bone. Normal growing bone has a trabecular pattern that is specific for each bone and part of a bone as well as a characteristic microscopic palisade of cuboidal basiphilic cells named osteoblasts. Tumorous bone has a disoriented structure widi trabeculae enveloped in a random arrangement of bizarrely shaped anaplastic cells. Tumorous bone grows by proliferation of transformed osteoblasts. The alterations in gene structure and function in the transformed state are obscure, but the involvement is comparable to a mutation. The criteria for cell transformation generally include: loss of cell contact inhibition; altered morphology; increased growdi rate; increased capacity to persist in serial subcultures; chromosomal abnormalities; increased resistance to reinfection by die transforming virus; emergence of new antigens; capacity to form neoplasms [2]. Viral-induced malignant transformation constitutes a heritable change and is accompanied by the loss of regulatory controls of cell growdi. Present knowledge of nonmalignant morphodifferentiation, as well as malignant transformation, is chiefly descriptive. Data on the biochemical controls of morphodifferentiation are lacking. The process beginning widi the fertilized ovum and ending widi the mechanics of unfolding of die structural characteristics, form, and size of functional tissues defies present understanding. The problem is the prerogative of students of dieoretical, as distinguished from experimental, biology.

Immunodeficiency dramatically increases susceptibility to cancer as a result of reduced immune surveillance and enhanced opportunities for virus-mediated oncogenesis. Although AIDS-related lymphomas (ARLs) are frequently associated with known oncogenic viruses, many cases contain no known transforming virus. To discover novel transforming viruses, we profiled a set of ARL samples using whole transcriptome sequencing. We determined that Epstein-Barr virus (EBV) was the only virus detected in the tumor samples of this cohort, suggesting that if unidentified pathogens exist in this disease, they are present in

Eight Chinese field strains of subgroup J avian leukosis viruses (ALV-J) were isolated from broilers or parent stocks during January 1999 to April 2001. One strain, SD9902, was an acute transforming virus, able to induce typical myelocytomatosis in 22-38 days after inoculation of 1-day-old meat-type chicks. The envelope protein and 3'-untranslated region (UTR) of the eight field strains were compared with the U.K. prototype HPRS-103 and several U.S. field strains isolated in 1993-97. All Chinese strains shared an almost identical deletion with the U.S. strain 4817 in the E element region of 3'-UTR when compared with the prototype HPRS-103, indicating that they have a very close phylogenic relationship. Every year, China has to import grandparent stocks of meat-type chickens, mainly from the United States. Chinese isolates should represent a part in the phylogenic tree of U.S. ALV-J evolution. Envelope protein gp85 amino acid sequence analysis demonstrated that, interestingly, all recent Chinese isolates were more closely related to HPRS-103 and the earliest U.S. isolates but not to the late U.S. isolates. The result implies that envelope gp85 may not have diverged from prototype and older strains. It is also possible that some recently imported birds could have been infected by the older viruses that were introduced in the late 1990s.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming virus associated with various cancers, the most common being the skin cancer Kaposi’s sarcoma. KSHV viral proteins are known to disrupt many normal cellular pathways in the human host. One specific target is the apoptotic pathway, or the programed cell death process. The cellular Bcl-2 family proteins, critical components of apoptotic signaling, converge at the mitochondria. This broad family of proteins is comprised of both pro- and anti-apoptotic proteins, sharing homology in four conserved trans-membrane domains, BH1-BH4. KSHV encodes a protein, vBcl2, with functional and sequential homology to the anti-apoptotic Bcl-2 protein. This gene is expressed in the early stages of the viral lytic cycle and has been shown to affect the progression of apoptotic signaling in order to allow for proper viral replication and virion production. We analyzed the interaction between KSHV’s vBcl-2 protein and a human protein, BIK, a member of the BH3 only f...

Avian leukosis virus subgroup J (ALV-J), isolated in the late 1980s, predominantly causes myelocytic myeloid leukosis in meat-type chickens. In the past 10 years, ALV-J infection has become very widespread, causing serious problems to the chicken meat industry. Previously, we have shown that turkey cells can be infected in vitro with Rous sarcoma virus pseudotypes of ALV-J. In this paper, we extend those observations to show that turkey monocyte cultures can be transformed in vitro with acutely transforming ALV-J strain 966. We also show that turkeys are experimentally susceptible to infection with ALV-J prototype strain HPRS-103. However, neoplastic lesions were not observed in these birds, probably due to the short experimental period of 10 weeks. When inoculated into 1-day-old turkey poults, acutely transforming ALV-J strain 966 induced tumours between 3 and 4 weeks after infection. Most of the birds showed tumours involving the liver, with histopathological lesions of myelocytomatosis. The demonstration of the spread of HPRS-103 by contact among turkeys, although observed only at low levels in the present study, stresses the importance of segregation of turkey and chicken breeding operations to avoid the spread of ALV-J infection.

In their classic paper on the identification of the transforming principle of Rous sarcoma virus (RSV) published 1970 in PNAS (1), Peter Duesberg at the University of California, Berkeley, and Peter Vogt, then at the University of Washington, Seattle, drew a seemingly simple yet groundbreaking conclusion. When they analyzed the genomic RNAs of transforming, acutely oncogenic RSV and of transformation-defective (td) mutant derivatives, they found that all transforming virus stocks contained two classes of RNA subunits, a larger one (a) and a smaller one (b), whereas the nontransforming yet replication-competent mutants contained the smaller b subunits only. Duesberg and Vogt concluded that the larger a subunit contained the transforming principle of RSV. Based on this and on subsequent structural comparisons of the a and b subunits of biologically cloned viruses, the transforming principle was defined by the remarkably simple equation a − b = x and was later termed src (for sarcoma). The first biochemical identification of a cancer gene was achieved, initially in a chicken virus. However, the principal proof of a physical underpinning of the cancer gene hypothesis had tremendous impact on a fundamental challenge of medicine, decoding the molecular basis of human carcinogenesis.

Recently, a novel DNA virus has been molecularly cloned from Kaposi's sarcoma (KS) tissue, a tumour common in acquired immune deficiency syndrome (AIDS). Analysis of the viral genome confirms that it is a relative of human herpesviruses and the virus has been designated HHV-8. Epidemiological evidence suggests a strong aetiological link between the presence of HHV-8 DNA and/or antibodies against the virus, and KS. Additional sequence analysis suggests that the HHV-8 genome contains sequences which encode a D type cyclin and a number of other genes potentially implicated in growth deregulation which may be relevant to its proposed role as a transforming virus.

For human B lymphocytes, Epstein-Barr virus (EBV) is a polyclonal activator, inducing both proliferation and /g secretion. It is also a transforming virus capable of generating immortalized B cell lines. These .early and late functions of EBV are not apparently connected. The receptor for EBY CD21, also serves as a receptor for some complement components and is called CR2. This molecule associates with CD19 and TAPA-l on the surface of B cells. This complex is involved in signaling B cells and participates in many responses. We have observed that simultaneous ligation of CD40 and the CD21 complex, by exposure to anti-CD40 MAbs and EBy enhances both the short-term proliferation as well as the long-term transformation rate of human B lymphocytes. B cell proliferation shows synergy between anti-CD40 MAb and EBV CD19 also appears to be involved in the synergistic activation of B cells through CD40 and CD21, since ligation of CD19 with anti­ CD19 MAbs, either prior to or concomitant with exposure to anti-CD40 and EBy markedly inhibits both proliferation and subsequent B cell transformation. These observations do not elucidate the mechanisms of B cell transformation employed by EBV but they do suggest a relationship between early proliferation and later transformation induced by the virus. Anti-CD40 enhances both these effects and anti-CD19 is capable of inhibiting both. [Hum Antibod 1997; 8: 43-47]

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.

Infection of mouse fibroblasts by wild-type polyomavirus results in increased phosphorylation of ribosomal protein S6 (D.A. Talmage, J. Blenis, and T.L. Benjamin, Mol. Cell. Biol. 8:2309-2315, 1988). Here we identify pp70 S6 kinase (pp70S6K) as a target for signal transduction events leading from polyomavirus middle tumor antigen (mT). Two partially transforming virus mutants altered in different mT signalling pathways have been studied to elucidate the pathway leading to S6 phosphorylation. An upstream role for mT-phosphatidylinositol 3-kinase (PI3K) complexes in pp70S6K activation is implicated by the failure of 315YF, a mutant unable to promote PI3K binding, to elicit a response. This conclusion is supported by studies using wortmannin, a known inhibitor of PI3K. In contrast, stable interaction of mT with Shc, a protein thought to be involved upstream of Ras, is dispensable for pp70S6K activation. 250YS, a mutant mT which retains a binding site for PI3K but lacks one for Shc, stimulates pp70S6K to wild-type levels. Mutants 315YF and 250YS induce partial transformation of rats fibroblasts with distinct phenotypes, as judged from morphological and growth criteria. Neither mutant induces growth in soft agar, indicating that an increase in S6 phosphorylation, while necessary for cell cycle progression in normal mitogenesis, is not sufficient for anchorage-independent cell growth. In the polyomavirus systems, the latter requires integration of signals from mT involving both Shc and PI3K.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.

A chimeric construct (VCD) consisting of parts from viral jun, chicken c-jun, and chicken junD was cloned into the replication-competent retroviral RCAS vector. This construct, RCAS-VCD, was found to have a higher focus forming potential in quail fibroblasts than the equivalent construct RCAS-VJ-1, expressing viral jun. DNAs from RCAS-VCD and RCAS-VJ-1 were transfected into primary quail embryo fibroblasts. Cells derived from one RCAS-VCD-induced focus survived cell crisis, which became manifest after 15 passages, and could be expanded into a long-term culture. This cell line, termed VCD, has been passaged for over 100 times so far. The cells grow to very high densities and then pile up into clumps of rounded cells. The culture releases a transforming virus with a titer of 105 FFU/ml, as tested on primary quail embryo fibroblasts. The transformed phenotype of VCD cells was verified by agar colony formation. VCD cells are capable of anchorage-independent growth with a cloning efficiency of 10%. Southern blot analysis of genomic DNA from VCD cells showed proviral integration of the RCAS construct without detectable rearrangements. Northern and Western blot analyses confirmed correct expression from integrated RCAS-VCD of predicted RNAs and of the chimeric Jun(VCD) protein. jun (VCD)-transformed cells provide a constant source of homogeneous cellular material for the investigation of the molecular mechanisms of jun -induced cell transformation and for the identification of direct and indirect targets of Jun protein function.

The oncogene revolution that developed over the past 4 decades has been remarkably powerful in explaining cancer at the molecular level. These advances in cancer biology have not developed in a vacuum but rather evolved hand in hand with technological advancements in molecular biology and molecular genetics. We are now in an era of powerful postgenomic technologies. These technologies are being used successfully not only to further understand the etiology of cancer but also to develop viable weapons against it. Retrospectively, the origin of the oncogene revolution had a much earlier history than the 1970s, dating back to over a century ago and deeply rooted in the fields of bacteriophage biology and animal virology. Arguably, the seminal finding, made by Peyton Rous in 1911 while working at The Rockefeller Institute for Medical Research in New York City, was that of a virus that caused cancer. Molecular characterization of this transforming virus led to the realization that mutations in somatic genes could lead to cell transformation, the culprit being a constitutively active tyrosine kinase called Src. In 1989, almost 20 years after the discovery of v-Src, Mayer and Hanafusa, also working at The Rockefeller University (The Rockefeller Institute became The Rockefeller University in 1965), identified a different kind of retroviral oncogene, the gene product of the CT10 virus, which had molecular features similar to v-Src but was biologically and conceptually distinct. Curiously, v-Crk retained noncatalytic regulatory sequences of homology to Src (the so-called Src homology 2 [SH2] and Src homology 3 [SH3] domains) but lacked intrinsic tyrosine kinase activity. v-Crk was demonstrated to regulate tyrosine kinases in trans (and thereby named CT10 regulator of kinase), as opposed to the cis-acting type of regulation seen for Src, where the SH2 and SH3 domains regulate Src activity intramolecularly. Such distinctions in function are seen in a variety of other oncogenes, often adding complexity to a set of general rules. In looking back, the study of the relatively simple structure of v-Crk provided important conceptual foundations. First, in the years following the discovery of Crk, tremendous effort ensued to understand the biology of SH2 and SH3 domains, leading to realizations that signaling proteins possess modular protein interaction domains to build networks. Crk was the “tip of the iceberg” of this emerging field, as now a plethora of distinct protein interaction domains have evolved in metazoans, each with unique specificities. The logic of modular protein interaction domains and intracellular communication also deserves mention as it highlights many of the current principles in the study of signal transduction. These general paradigms, derived from studying Crk, and later Grb2, elegantly showed how tyrosine phosphorylation could be exquisitely localized within subcellular compartments (such as focal adhesions, plasma membranes, and endosomes). Finally, as the 3-dimensional structures of these proteins were solved, the versatility in SH2 and SH3 domain interactions became clear, showing noncanonical roles in regulating nonreceptor tyrosine kinases such as Src and Abl. At this quarter century mark after the discovery of v-Crk, I am privileged to organize a Genes & Cancer review issue dedicated to summarizing the extant progress on Crk research and related signaling pathways in cancer. When I started this project, my plan was to help those not yet captive of the field to evaluate the impact this work has as well as its historical importance in cancer biology. I therefore chose to ask scientists whom I consider, and thoughtful, and provocative to reflect on their areas of expertise. In this respect, our contributors have risen to the challenge and have brought forth analysis and observations that enable us to take a more global view of the field while appreciating where it is moving and its possible clinical relevance and application. The reviews presented in this special issue are organized into 3 general themes to emphasize how the Crk field has progressed over the past 25 years. In the first theme, Tsuda and Tanaka address the incidence of Crk in aggressive human cancers and raise provocative questions on whether Crk may be a biomarker for cancer. This theme is picked up again by Bell and Park, who describe the development of several mouse models for Crk and how these models help validate clinical expression data. Finally, Machida, Khenkhar, and Nollau take a more global position and describe translational research on how SH2 profiling can help decipher phosphotyrosine networks in cancer, which will likely become a major tool in functional proteomics in the decades to come. The second theme explores progress on the adaptor proteins that interact with Crk and how these fields themselves have advanced into dominant areas of cancer research. Deakin, Pignatelli, and Turner discuss recent advances on the paxillin family of proteins (paxillin, Hic-5, and leupaxin) and their emerging roles in invasion and metastasis. Guerrero, Parsons, and Boutin and Wallez, Mace, Pasquale, and Riedl discuss different aspects of the p130Cas/BCAR protein family. Guerrero et al. address the overlapping and distinct roles for Cas family proteins in malignancies and the unexpected role of BCAR in the resistance to antiestrogens. Wallez et al. elaborate on novel CAS interacting proteins and how they enhance Cas function in cancer. Finally, Matsui, Harada, and Sawada and colleagues discuss the role of p130Cas tyrosine phosphorylation in mechanotransduction and how tumor cells may use these unique properties to achieve cellular transformation. In the final section, the authors turn from the theme of adaptor proteins to the nonreceptor tyrosine kinases, exemplified by Abl and Src, which show highly dynamic and versatile interactions with Crk and adaptor proteins. Several of the reviews describe the complex and dynamic interactions between adaptor proteins and Abl and Src, which appear to be highly dysregulated in cancers. Kotula and colleagues provide an elegant example for how Abi1 and Crk compete for binding Abl but do so with apparent opposing biological effects. To extend this idea, Ganguly and Plattner describe an exciting new theme in cancer biology in which Abl appears to have equally important roles in solid cancers as it does in leukemia. However, unlike the situation with leukemias, where Bcr-Abl is frequently mutated, Abl is rarely mutated in solid cancers but rather activated indirectly by growth factor receptors. Finally, Krishnan, Miller, and Goldberg describe emerging research on Src in cancers and recent studies supporting a yet unrealized role for Src in cell migration and contact normalization. A theme raised by several of the contributors reflects the important point that tyrosine kinases transmit signals through adaptor proteins, but the converse is also true, where adaptor proteins engage activation loops and activate nonreceptor tyrosine kinases. Dysregulation of Crk and adaptor proteins may be as important in activating tyrosine kinase pathways as mutations are in enhancing the kinase activity. The final series ends with 2 articles that highlight the importance of Abl research for the development of new anticancer therapeutics. The review by Hantschel reveals how the SH2 and SH3 domains of Abl provide lock and key paradigms for how it is negatively regulated, while the review by Reddy and Aggarwal elegantly shows how different tyrosine kinase inhibitors are staying one step ahead of kinase domain driver mutations. These studies provided proof of principle for the approval of kinase inhibitors for the treatment of chronic myelogenous leukemia (CML) and a clear turning point in the field of translational oncology. I would like to finish with a short reflection about the history of Crk that in many ways draws parallels to the discovery of Src. Many years ago, when I was a postdoctoral fellow at The Rockefeller University in the laboratory of the late Hidesaburo Hanafusa, I asked Saburo (as a matter of fact) whether Src had anything to do with human cancer since Tyr527 mutations were seldom observed? Saburo, always short on words, frowned and gave a profound “Probably” before briskly walking away. At the same time, in the early 1990s, we had no idea that Crk and its prototypical signaling pathways via paxillin, p130Cas, and Abl would emerge as a major sentinel in human cancers. Clearly, by looking both forward and backward, the future of Crk and adaptor protein signaling is equally exciting as Src was and likely completes a new chapter in the ongoing oncogene revolution.

SummaryB cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA 2) is an acidic transcriptional transactivator of virus and cell gene expression and is essential for growth transformation of primary B lymphocytes. EBNA 2 transactivation of response elements (E2REs) can be mediated by interaction with a GTGGGAA-specific DNA-binding factor(s). We now purify the factor by S-Sepharose and EBNA 2 affinity chromatography and identify it as a single 63-kDa protein. The protein is shown to specifically coimmunoprecipitate with EBNA 2 from lymphoblasts transfected with an EBNA 2 FLAG expression vector. Mutation of GTG to TCT in a GTGGGAA motif common to the Cp, LMP2, and LMP1 promoters results in loss of recognition by p63. EBNA 2 amino acids 310-336 are sufficient for p63 binding. The only motif in this 27 amino acid sequence which is common to the EBNA 2 genes of EBV types 1 and 2 is GPPWWPP (I/V) (C/R) DP, which is therefore likely to mediate p63 interaction. Mutation of WW to SS or FF ablates interaction with p63, indicating that both the hydrophobic and aromatic characteristics of WW are essential for its "key" interaction with p63. EBNA 2 with a WW mutated to SS is also unable to marker rescue primary B lymphocyte transforming virus from cells infected with an EBNA 2-deleted virus, while otherwise isogenic wild-type EBNA 2 readily marker rescues transforming virus in parallel experiments. EBNA 2 transactivation through the Cp E2RE is completely abolished by the WW to SS mutation while transactivation of -234 to +40 LMP1 E2RE is only partially affected. These genetic and biochemical experiments support the hypothesis that EBNA 2 WW interaction with a p63 GTGGGAA-binding protein is essential for EBV-mediated cell growth transformation because it specifically associates EBNA 2 with its response elements. This enables the EBNA 2 acidic domain to transcriptionally transactivate specific genes.

Virus from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) can induce rare foci of transformation in rat 208F fibroblasts. Characterization of three such foci revealed that one produced an acutely transforming virus at a high titer. The virus consists of a mutant Nras cDNA from the HT-1080 cells inserted into a retroviral vector (added to the HT-1080 cells as a marker for infection) in place of internal vector sequences. These results show that XMRV can generate acutely transforming viruses at a low rate, as is typical of other replication-competent retroviruses, and reveal the potential for transforming virus contamination of retroviral vectors made from transformed cell lines.

Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cellsin vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection.

To investigate the cellular mechanism of transformation by retroviruses, we established a system for isolation of cellular and viral mutants for transformation of a rat cell line. Mutagenized untransformed cells of this line were infected with recombinant murine retrovirus containing the src gene of Rous sarcoma virus and the selective marker gene, neo. After reaching confluence, cells transformed by the src gene tend to overgrow and die. Utilizing this property of src transformed rat cells and the selective marker gene, we could easily select untransformed cell clones containing the retrovirus genome. Expression of the src gene product in the flat clones selected was examined by in vitro assay of src kinase activity. To determine whether the mutations of these flat clones were viral or cellular, the susceptibilities of the clones to transformation were examined after superinfection with the wild-type virus and also characterized the retroviruses recovered from these clones. With this system, two novel clones were isolated. One had a defect in viral information affecting the transformed phenotype, but still retained src kinase activity like fully transformed cells. The other showed low src kinase activity but retained wild-type transforming virus, suggesting that a cellular gene involved in viral gene expression was mutated.

Three revenants defective in the ability to form colonies in semisolid medium were isolated from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16 (HPV16). These revenants appeared to be defective in a cellular factor(s) necessary for transformation by HPV16-E6E7 genes since they still expressed a comparable amount of HPV16-E6E7 mRNA and E7 protein to the parental cells, harbored rescuable transforming virus, and were resistant to retransformation by HPV16-E6E7 genes. All these reverted phenotypes of the three mutants were recessive on somatic cell hybridization with normal cells, because all the hybrids showed transformed phenotypes.

An Epstein-Barr virus (EBV)-producer line, designated Akata, was established from a Japanese patient with Burkitt's lymphoma. The Akata line possessed the Burkitt's-type chromosome translocation, t(8q- ; 14q+), and was derived from the tumor cell. Akata cells produced a large quantity of transforming virus upon treatment of cells with anti-immunoglobulin antibodies (Takada, 1984). Southern blot analysis of viral DNA indicated that the Akata EBV is nondefective and more representative of wild-type viruses. Akata cells should be useful as a source of EBV.

Working with knowledge gained from pioneers in the RNA tumor virus field (2), the biologic activity of this NZB virus ultimately was demonstrated in 1970 by cultivating NZB mouse embryo cells with hamster or rat tumor cells containing a nonreplicating sequence of the murine sarcoma virus (3). This procedure, using conventional MLV, leads to the rescue of the sarcoma virus genome in the envelope coat of the replication-competent MLV (2). The newly formed transforming virus pseudotype now has the host range of the “helper” MLV. Supernatants from the NZB cell coculture demonstrated the presence of a replicating virus that could induce foci of transformation in rat cells but not mouse embryo cells (3). The NZB MLV host range thus seemed limited to rat cells. Subsequent work demonstrated that the helper MLV visualized in the NZB mouse cells was infectious but was not infectious at all for mouse cells (i.e., complete block). It was infectious only for cells from heterologous species, including those of avian origin (Table 1). Similar viruses were subsequently found in all laboratory strains of Mus musculus domesticus as …

In accordance with earlier studies, we detected higher numbers of Epstein-Barr virus (EBV)-carrying lymphocytes (B-EBV) in the blood of acute infectious mononucleosis (IM) patients and higher amounts of transforming EBV particles in the saliva compared to healthy seropositive individuals. B cells grew in cultures seeded with the low and high density IM lymphocytes. The majority of B cells which grew acquired the infection in vitro (2-step outgrowth), because addition of virus neutralizing antibodies considerably reduced the emergence of lymphoblastoid cell lines (LCLs). Only the minority of the explanted B-EBV cells proliferated. The antiviral drug phosphonoformate (PFA) did not influence the frequency of 2-step LCLs in the IM cultures. This may indicate that a large proportion of EBV carrying B cells have already entered the viral productive cycle in vivo and passed the PFA-sensitive stage at the time of explantation. Earlier experiments with blood of healthy seropositive individuals showed an inhibitory effect of PFA on the generation of LCLs. One healthy individual who entered this study as a control, probably had a reactivated EBV infection as judged by the anti-EA activity in his serum and the high level of virus in his saliva. He had antibodies against EBV nuclear antigens (EBNA), and therefore he did not have a primary infection at the time of the test. Judged by the number of wells with B cell growth, the frequency of virus-carrying B cells in his blood was low. It seems that anti-EBV immunity can control the number of infected B cells in the blood, but does not influence the virus load in the epithelial cells.

Lymphoid cell lines were established which stably carry the Epstein-Barr viral (EBV) BZLF1 gene on an extrachromosomal plasmid. These lines, which spontaneously synthesize the BZLF1 gene product, ZEBRA, were examined for expression of EBV genes which were activated by ZEBRA. Cell lines which acquired oriP plasmids without BZLF1 served as controls. The extent of activation differed among derivatives of four cell lines. X50-7 cells, which harbor a standard latent EBV, could be induced by ZEBRA to produce transforming virus; a cellular subclone of this line was induced to express EBV late antigens but did not release transforming virus. In two other cell lines, Raji and ER, ZEBRA activated only a group of early antigens. Using immunofluorescence and immunoblotting with monoclonal antibodies and Northern analysis five EBV early genes were shown to be induced in cells stably transformed by oriP/BZLF1 plasmids. ZEBRA itself was activated; thus BZLF1 is autostimulatory. Four other activated genes were components of the diffuse (EA-D) and restricted (EA-R) early antigens (BMRF1, BMLF1, BHRF1, and BORF2). Stable cell lines with extrachromosomal BZLF1 expression vectors will ultimately be useful in a variety of experiments designed to study regulation of this gene, to analyze the effects of mutations on ZEBRA protein function, and to define the full spectrum of viral and cellular genes which are activated by and interact with the ZEBRA protein.

A revertant cell line was selected from Moloney sarcoma virus-transformed BALB/c cells after long-term treatment with type I interferon. Despite an actively transcribed and transfectable v-mos gene, these revertant cells were nontumorigenic in nude mice. The functionality of the mos protein was investigated, focusing on the alpha 2(1) collagen promoter regulation, which is known to be affected by mos-induced trans-acting factors. Both in transient expression assays and after stable integration into the cellular genome, the transfected alpha 2(1) collagen promoter fused to the cat reporter gene was activated in the revertant while being downregulated in the original transformed cells. In retransformation assays of the revertant by Moloney sarcoma virus strains homologous to the original transforming virus, no detectable change was noted in the in vitro phenotype or in tumorigenicity. These results reveal that the mos-directed factors were no longer effective on their specific targets. Thus, the R.MSVIF cell could be either an oncoprotein-deficient or a target-related revertant. Attempts at retransformation with unrelated sarcoma viruses bearing v-sis, v-fms, or v-fos oncogenes were also negative. In contrast, tumorigenicity was obtained with the unrelated Kirsten sarcoma virus without any change in the revertant morphology or collagen expression. These findings showed that the common pathway blocked by the reversion and shared by v-mos and v-ras was not required for ras-induced tumorigenicity.

Kaposi's sarcoma (KS) is a very rare tumor except after human immunodeficiency virus type 1 (HIV-1) infection when it becomes common. Most investigators assume that the role of HIV-1 is passive, i.e., via inducing immune deficiency, thereby enhancing cancer development, and specifically, in the case of human herpesvirus 8 (KSHV) by enhancing HHV-8 replication. We suggest that the role of HIV-1 is more active in the disease process by at least two events: 1) promoting an increase in inflammatory cytokines, which through sustained release influences early stage KS by inciting local microinflammatory responses, and 2) by the Tat protein that effects growth of the inflammatory cells. Cultures of all activated endothelial spindle cells, whether hyperplastic or neoplastic, are negative for HHV-8; transmission of HHV-8 does not induce cell growth or transformation; monkeys immune suppressed by simian immunodeficiency virus infection and infected also with HHV-8 do not develop KS; polymerase chain reaction analysis of blood cells shows HHV-8 sequences in monocytes and B cells (about 20% of normal donors in Maryland); M. Starzl showed that early KS has few cells (mostly macrophage) positive for HHV-8, increasing and present in endothelial cells only late in the disease; no increase in HHV-8 has been found in association with progressive immune deficiency; and recent studies in Gambia by others showed that HHV-8 is a very common infection, and though HHV-2 is known to be relatively common, HIV-1 is unusual and so is KS. Collectively, these findings lead me to conclude that there is little evidence that HHV-8 is a transforming virus as has been repeatedly suggested and that its role in KS is more likely to be indirect (like HIV-1), perhaps necessary but hardly likely to be sufficient for KS development.

SUMMARY Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), discovered in 1994, is a human rhadinovirus (gamma-2 herpesvirus). Unlike other human herpesviruses (herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, cytomegalovirus, HHV-6, and HHV-7), it is not widespread in the general population and has many unique proteins. HHV-8 is strongly associated with all subtypes of Kaposi's sarcoma (KS), multicentric Castleman's disease, and a rare form of B-cell lymphoma, primary effusion lymphoma. In addition, HHV-8 DNA sequences have been found in association with other diseases, but the role of the virus in these diseases is largely unconfirmed and remains controversial. The seroprevalence of HHV-8, based on detection of latent and lytic proteins, is 2 to 5% in healthy donors except in certain geographic areas where the virus is endemic, 80 to 95% in classic KS patients, and 40 to 50% in HIV-1 patients without KS. This virus can be transmitted both sexually and through body fluids (e.g., saliva and blood). HHV-8 is a transforming virus, as evidenced by its presence in human malignancies, by the in vitro transforming properties of several of its viral genes, and by its ability to transform some primary cells in culture. It is not, however, sufficient for transformation, and other cofactors such as immunosuppressive cytokines are involved in the development of HHV-8-associated malignancies. In this article, we review the biology, molecular virology, epidemiology, transmission, detection methods, pathogenesis, and antiviral therapy of this newly discovered human herpesvirus.

Avian leukosis virus subgroup J (ALV-J), the most recent member of the avian retroviruses, is predominantly associated with myeloid leukosis in meat-type chickens. We have previously demonstrated that the acutely transforming virus strain 966, isolated from an ALV-J-induced tumor, transformed peripheral blood monocyte and bone marrow cells in vitro and induced rapid-onset tumors, suggesting transduction of oncogenes (L. N. Payne, A. M. Gillespie, and K. Howes, Avian Dis. 37:438–450, 1993). In order to understand the molecular basis for the rapid transformation and tumor induction, we have determined the complete genomic structure of the provirus of the 966 strain. The sequence of the 966 provirus clone revealed that its genome is closely related to that of HPRS-103 but is defective, with the entire pol and parts of the gag and env genes replaced by a 1,491-bp sequence representing exons 2 and 3 of the c- myc gene. LSTC-IAH30, a stable cell line derived from turkey monocyte cultures transformed by the 966 strain of ALV-J, expressed a 72-kDa Gag-Myc fusion protein. The identification of the myc gene in 966 virus as well as in several other ALV-J-induced tumors suggested that the induction of myeloid tumors by this new subgroup of ALV occurs through mechanisms involving the activation of the c- myc oncogene.

Publisher Summary Identification of sequences from a new human herpesvirus in tumour tissue from AIDS-related Kaposi's sarcoma (KS) is done using the technique of Representational Difference Analysis (RDA). This technique uses cycles of polymerase chain reaction (PCR) and subtractive hybridization to amplify and enrich low-abundance DNA sequences present in only one of two otherwise identical DNA populations, and is therefore ideal for determining small differences between complex genomes. KS tumour usually appears as brownish-purple lesions, often on the extremities, but may in more aggressive forms of the disease progress to involve organs such as the lungs, lymph nodes, and gastrointestinal tract. The lesions contain many types of cell but are characterized histologically by the presence of elongated spindle cells and irregular slit-like vascular spaces. Seroepidemiology and PCR studies strongly suggest that HHV-8 is the cause of Kaposi's sarcoma and primary effusion lymphoma, and is also associated with multicentric Castleman's disease. Although HHV-8 has not yet been shown to be a transforming virus in vitro, the virus encodes several proto-oncogenes capable of the deregulation of cell cycle control, inhibition of apoptosis and control of growth differentiation, which could contribute to tumour formation.

The rat leukemia virus (RaLV) is an endogenous retrovirus that is spontaneously released by Sprague-Dawley rat embryo cells. The overall structure of the RaLV genome resembles that of other simple, replication-competent retroviruses, but the sequence of the long terminal repeats (LTR) is unique and unrelated to the known retroviruses. Phylogenetically, the RaLV genome appears to be more closely related to the feline leukemia virus group of retroviruses than to the murine leukemia virus group. A remarkable feature of RaLV is that it is capable of transducing a ras proto-oncogene from rat tumor cells in the form of an acutely transforming virus, designated the Rasheed strain of the rat sarcoma virus (RaSV). With the exception of the c- ras sequence, the genomes of both RaLV and RaSV are collinear. The RaSV-encoded oncogene v-Ra- ras expresses a fusion protein with a molecular mass of 29 kDa, and it exhibits a unique structure that has not been described previously for any known virus. The 5′ end of this gene is derived from sequences encoding RaLV matrix followed by 20 bp derived from the U5 region of the RaLV LTR (RS-U5 element) which is joined at its 3′ end to sequences derived from all six (coding and noncoding) exons of the c- ras proto-oncogene at the 3′ end. This recombinational event represents a novel mechanism among the acutely transforming viruses that have been studied.

Cytokine-mediated apoptotic destruction of viral-infected cells, downregulation of virus production and inhibition of anchorage dependent (clonal) cell growth were evaluated using virus-transfected human hepatoblastoma (HepG2) cells. The cytokines evaluated were interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha) and thymosin alpha 1 (T alpha 1), all of which have previously been implicated in control of various viral infections. The viruses evaluated were Hepatitis B (HBV) and the transforming virus, SV-40. TNF-alpha-induced apoptosis in the HBV-transfected cell line and the control HepG2 cells but not the HepG2 cells transfected with SV-40 virus. IFN-alpha and T alpha 1 had no effect on apoptosis. TNF-alpha also prevented the clonal growth of the HBV-HepG2 and control HepG2 but enhanced the growth of the SV-40-transfected HepG2 cells. IFN-alpha inhibited the clonal growth of all three cell lines in contrast to T alpha 1 which inhibited the clonal growth of only the HBV-transfected cells. Although TNF-alpha, IFN-alpha, and T alpha 1 when given alone did not significantly inhibit HBV-DNA production in the culture supernatant from HBV-HepG2 cells, the combination of T alpha 1 and IFN-alpha resulted in a statistically significant inhibition of virus production. These studies demonstrate that HepG2 cells transfected with HBV and SV-40 are useful for defining the mechanisms of cytokine activity. The HBV-transfected cells are especially useful in defining possible in vivo differences in responses to cytokines with respect to HBV production, apoptosis and clonal cell growth. Multiple mechanisms through which different cytokines can influence HBV infection and hepatoblastoma growth were identified and the importance of defining effective combinations to improve therapy in vivo demonstrated.

The tissue tropism was studied for the HPRS-103 strain of avian leukosis virus, which belongs to a new envelope subgroup, designated J. Studies were conducted in blood monocyte and bone marrow cell cultures and in chickens from six lines that had been shown previously to differ in susceptibility to induction by this virus of myeloid leukosis and other tumors. Using an immunohistochemical technique to detect expression of viral group-specific antigen (Gag) in various tissues, we detected no major differences among the six lines of chickens at 3 and 7 weeks of age following infection as embryos. Thus, Gag expression did not correlate with differences in tumor susceptibility. Of the tissues examined, greatest Gag expression was observed in cells specific to the adrenal gland, heart, kidney, and proventriculus and especially in smooth muscle cells and connective tissue. After infection of 1-day-old chicks, greater tissue expression was observed in line 21 chicks, which mostly developed a tolerant viremic infection, than in Brown Leghorn chicks, which developed virus-neutralizing antibodies. An acutely transforming virus, strain 966, derived from HPRS-103-induced myeloid leukosis, showed a tropism similar to HPRS-103. The HPRS-103 strain showed a lower propensity to replicate in the medullary region of the lymphoid follicles of the bursa of Fabricius than did the RAV-1 strain of subgroup A avian leukosis virus. This low bursal tropism may be a factor in why HPRS-103 does not induce lymphoid leukosis. The HPRS-103 and 966 virus replicated in blood monocyte cultures from chickens from the six lines, indicating a tropism for the myelomonocytic cell lineage. In comparison, as previously reported, RAV-1 did not replicate well in the monocyte cultures, whereas RAV-2, a subgroup B avian leukosis virus, did replicate. The tropism of HPRS-103 for monocytes may relate to its ability to cause myeloid leukosis. Monocyte and bone marrow cell cultures from the six lines ranked similarly in differences in susceptibility to transformation by 966 virus and showed evidence that their relative susceptibilities correlated with susceptibility of chickens from these lines to induction of myeloid leukosis by HPRS-103, suggesting common tissue-specific viral and host factors involved in oncogenesis by these two viruses.

Hepatitis B virus (HBV) DNA integrates into the host DNA and shows a series of potentially oncogenetic properties, but HBV is not an acutely transforming virus, because HCC develops decades after infection. Other factors, namely cirrhosis, inflammation, alcohol intake, and viral superinfections, could promote the oncogenetic process induced by HBV-DNA integration. We studied the impact of HDV infection in the pathogenesis of HCC in 62 consecutive patients. Their mean age was 59 years (range 25-75 years), 54 were male and eight female; 58 had cirrhosis. The findings suggest that HBsAg-positive patients with HDV superinfection developed cirrhosis and HCC at an earlier age than HBsAg carriers without HDV infection. HDV appears to represent a "promotion" factor for HCC in subjects with an oncogenic risk induced by HBV. A long-lasting necroinflammatory lesion of the liver substained by productive HBV and HDV infections may be a major pathogenetic mechanism.

Recently several groups have demonstrated cytomegalovirus (CMV) protein and DNA in a large majority of glioblastomas (GBMs), though the exact role of CMV in tumors remains controversial. Although CMV is capable of activating several oncogenic pathways, it has never been shown to be a transforming virus. Because of this, we hypothesize that CMV infection can modify the rate of gliomagenesis in the context of genetic mutations known to predispose to tumor formation. To test this, we used the Smith strain mouse CMV (MCMV) to infect Mut3 (GFAP-cre; Nf1loxP/+; Trp53-/+) mice that develop normally but eventually develop spontaneous gliomas at an adult age After intraperitoneal viral inoculation, mice exhibited multisystemic infection throughout the body, including the brain. Mut3 mice infected perinatally (103 plaque forming units) developed gliomas significantly sooner than mock-infected Mut3 mice. MCMV-infected mice developed GBMs (WHO grade IV) at a higher rate than less severe tumors, whereas, mock-infected mice developed less severe gliomas (WHO grade III). Since MCMV preferentially infected the neural stem cell (NSC) niche, an area shown to generate gliomas, we interrogated changes in signaling in this population via microarray of cultured NSCs in MCMV- versus Mock-infected mice. Ingenuity Pathway Analysis identified a PDGF-B network as the highest scoring differentially regulated network between MCMV-and Mock-infected neurospheres. In vitro infection of mouse tumorspheres and human GBM tumorspheres with CMV validated the observed upregulation of PDGF-B, a molecule shown to cause gliomas de novo. Additionally, in vitro infection of human glioma stem cells with HCMV (Towne strain) or exogenous PDGF-B activated STAT3, a key regulator of gliomas. In mice infected with MCMV, PDGF-B is upregulated in CA2/3 neurons and STAT3 is activated in the subventricular zone and dentate gyrus suggesting a paracrine effect of MCMV-induced PDGF-B. Also, infected human brain tumor stem cells demonstrated an increase in proliferation via flow cytometry. Taken together, our data suggest that CMV in gliomas may accelerate GBM progression by activating PDGF-B/STAT3 signaling in the NSC population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4815. doi:1538-7445.AM2012-4815

Bryostatin 1 is a novel anti-tumor agent currently undergoing clinical trial. We investigated the effect of this drug on B-lymphocyte cell lines that carry the Epstein-Barr virus and found that it induces these latently infected cells into the production of transforming virus particles over a wide range of concentrations. These results may have clinical implications, particularly with regard to the use of the drug in the immunocompromised patient.

The immortalization and transformation of cultured human cells has far-reaching implications for both cell and cancer biology. Human cell transformation studies will increase our understanding of the mechanisms underlying carcinogenesis and differentiation. The neoplastic process can now be studied in a model human cell culture system. The accompanying biochemical and genetic changes, once identified, will help define the relationship between malignancy and differentiation. The present studies indeed demonstrate that the neoplastic process can now be studied in a human cell model system. Primary human cells treated with various carcinogens became immortalized in culture but were not tumorigenic. Additional exposure to either retroviruses, chemical carcinogenes or X-ŗay irradiation to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary cells in culture by the combined action of either a DNA transforming virus and a retrovirus or a DNA virus and a chemical or X-ray irradiation, and support an multistep process for neoplastic conversion. It has been known that normal human cells in culture are remarkably resistant to experimentally induced tumorigenicity. However, as shown above, normal human cells could now be transformed into tumorigenic cells.

Epstein-Barr virus (EBV) isolates can be broadly classified as type 1 or type 2 on the basis of allelic polymorphism of the virus-encoded nuclear antigens EBNAs 2, 3a, 3b and 3c, and individually identified based on Mr values of their EBNA proteins (EBNA type). Here we have used this natural heterogeneity amongst isolates to re-examine the question of EBV persistence in vivo, asking in particular whether virus carriage in oropharyngeal epithelium and/or in B lymphoid tissues involves infection with a single or with multiple virus strains. Firstly, 76 healthy virus carriers were classified into serotype groups on the basis of preferential antibody reactivity to type 1 EBNAs (serotype 1) or to type 2 EBNAs (serotype 2); 60 of the 76 donors were serotype 1, four of the 76 donors were serotype 2 and 12 of the 76 donors were anti-EBNA 2, 3a, 3b, 3c antibody-negative and therefore could not be serotyped. Representative donors from each group were then selected for virus isolations from blood (by spontaneous in vitro transformation) and from throat washings (by cord blood cell transformation). All 13 serotype 1 donors tested and six of seven non-serotypeable donors gave a type 1 virus isolate, whereas all four serotype 2 donors and one of the seven non-serotypeable donors gave a type 2 isolate. Multiple transforming virus isolates from any one donor, whether from blood or throat washings, were all of the one strain characteristic of that particular donor; sequential isolations showed retention of the same strain over several years. Finally, throat washing samples from these same donors were examined for amplifiable EBV DNA in the polymerase chain reaction using EBV type-specific oligonucleotide primers and probes derived from the polymorphic EBNA 2 and EBNA 3c loci. The results were consistent with earlier virus isolation studies, each individual donor showing amplification either of type 1 or type 2 sequences. We conclude that multiple EBV infections must occur rarely, if at all, in healthy virus carriers; EBV persistence in vivo is characterized by dominance of a single transforming virus strain.

The mechanisms underlying interactions between viruses and carcinogens are only beginning to be understood. Peripheral blood lymphocytes (PBLs) separated from patients with acquired immunodeficiency syndrome (AIDS) are a relevant model to study chemical carcinogen-EBV synergism, due to the high degree of EBV reactivation and potential for EBV-associated malignant lymphomas in patients with AIDS. To better understand chemical carcinogen-EBV synergism at a molecular level, we examined the organization of the terminal region of EBV genome carried by LCLs established from patients with AIDS. For this study we chose LCLs established from PBLs treated with aflatoxin B1 (AFB1), a mycotoxin and known carcinogen. From an analysis of the EcoR1 Dhet terminal region, we found the majority of the LCLs established spontaneously to be clonal in origin, but individual lineages established from the same patient often carried EBV episomal circular forms of the genome with varying numbers of fused 500-base pair terminal repeats. Linear forms of the EBV genome with varying numbers of fused 3’ and 5’ 500-base pair terminal repeats were only detected in spontaneously derived LCLs which were established from carcinogen-treated PBLs. The presence of linear EBV DNA correlated with the presence of extracellular transforming virus. LCLs established from PBLs separated from patients with AIDS following addition of exogenous EBV (B95.8 or FF41–1) most often carried episomal circular forms of EBV genome with a terminal region characteristic of the infecting viral strain. LCLs derived from EBV-infected PBLs treated with AFB1 often carried variation of the parent episomal circular form differing in terminal region varying by number of fused terminal repeats. In addition, various forms of linear EBV DNA varying by the number of fused 3’ terminal repeats could be detected in these LCLs. These results show that the chemical carcinogen AFB1 directly interact with EBV and drive EBV-infected lymphocytes toward a semi-permissive infection. Furthermore, the permissive state of an LCL correlates with changes in the organization of the terminal region of the EBV genome and provides a model to explore how chemical carcinogens enhance EBV-induced transformation and EBV replication, and induce malignant conversion of EBV-carrying LCLs.

When human peripheral blood lymphocytes (PBLs) from Epstein-Barr virus (EBV)-seropositive donors are injected intraperitoneally into SCID mice, EBV+ B cell tumors develop within weeks. A preliminary report (Mosier, D. E., R. J. Gulizia, S. M. Baird, D. D. Richman, D. B. Wilson, R. I. Fox, and T. J. Kipps, 1989. Blood. 74(Suppl. 1):52a) has suggested that such tumors resemble the EBV-positive malignancy, Burkitt's lymphoma. The present work shows that generally the human (hu) PBL-SCID tumors are distinct from Burkitt's lymphoma and instead resemble lymphoblastoid cell lines (LCLs) generated by EBV-infection of normal B cells in vitro in terms of: (a) their cell surface phenotype, with expression of B cell activation antigens and adhesion molecules, (b) normal karyotype, and (c) viral phenotype, with expression of all the transformation-associated EBV latent proteins and, in a minority of cells, productive cycle antigens. Indeed, in vitro-transformed LCLs also grow when inoculated into SCID mice, the frequency of tumor outgrowth correlating with the in vitro growth phenotype of the LCL which is itself determined by the identity of the transforming virus (i.e., type 1 or type 2 EBV). Histologically the PBL-derived hu-SCID tumors resemble the EBV+ large cell lymphomas that develop in immuno-suppressed patients and, like the human tumors, often present at multiple sites as individual monoclonal or oligoclonal foci. The remarkable efficiency of tumor development in the hu-SCID model suggests that lymphomagenesis involves direct outgrowth of EBV-transformed B cells without requirement for secondary genetic changes, and that selection on the basis of cell growth rate alone is sufficient to explain the monoclonal/oligoclonal nature of tumor foci. EBV+ large cell lymphoma of the immunosuppressed may arise in a similar way.

Epidemiologic studies have linked infection by the human T-lymphotropic virus type I (HTLV-I) with the development of adult T-cell leukemia. The low penetrance of the virus and the long latency for disease manifestation are factors that obscure the role of HTLV-I infection in oncogenesis. We have used an in vitro transformation assay system to determine directly whether the HTLV-I tax gene has transformation potential. Transfection of the tax gene alone into early-passage rat embryo fibroblasts did not induce morphological alterations. However, cotransfection of tax with the selectable marker plasmid pRSVneo gave rise to G418-resistant colonies that could be established as immortalized cell lines. Cotransfection of tax with the ras oncogene into rat embryo fibroblasts gave rise to foci of transformed cells that were highly tumorigenic in nude mice. These data represent a direct demonstration of the oncogenic potential of the tax gene in nonlymphoid cells and establish HTLV-I as a transforming virus.

We read with interest the recent article in the Journal by Strickler et al. (1), in which the authors performed a retrospective age–period–cohort analysis to estimate the incidence of pleural mesothelioma in individuals potentially exposed to simian virus 40 (SV40) -contaminated poliovirus vaccines. The authors concluded that “Age-specific trends in U.S. pleural mesothelioma incidence rates are not consistent with an effect of exposure to SV40-contaminated poliovirus vaccine” and suggested that “monitoring of vaccine-exposed cohorts should continue.” We wish to raise two issues about this and similar studies (2–4) that address “exposure” to SV40contaminated polio vaccines and the incidence of human cancers. First, although a cohort study is recognized as the best design to identify incidence and natural history of disease (with a known exposure) and may be used to assess multiple outcomes after a single exposure (5), the prevalence of SV40 infections in the human population is not known, and the available data about prevalence are limited and inconclusive (2–4). Second, an important confounding factor in the epidemiologic assessment of malignant mesothelioma is that the actual number of people infected with SV40 through the use of contaminated polio vaccines is not known and may be less than the “exposed” population size assumed in the study by Strickler et al. (1). For example, in the United States, not all vaccine lots were contaminated with SV40, formalin inactivation was expected to reduce the titer of live SV40 in the lots that were contaminated, and successful infection rates by live SV40 are unknown (2–4). Therefore, an inability to identify the population that was actually infected with SV40 through the use of contaminated polio vaccines precludes a meaningful calculation of cancer incidence in relation to exposure to those vaccines. Furthermore, the Institute of Medicine (IOM) Immunization Safety Review Committee found that epidemiologic studies of cancer rates in people potentially “exposed” to SV40-contaminated vaccines, such as the analysis by Strickler et al. (1), are inadequate to evaluate a causal relationship between exposure and disease (2). These limitations led the IOM to “not recommend additional epidemiological studies of people potentially exposed to contaminated polio vaccine” (2). However, the IOM concluded that “the biological evidence is strong that SV40 is a transforming virus” and that “the biological evidence is of moderate strength that SV40 exposure could lead to cancer in humans under natural conditions” (2). Future studies are needed to determine the prevalence of SV40 infections in different human populations. The IOM recognized that gaps in our understanding of the pathogenesis of SV40 in humans are important and recommended “targeted biological research,” including “further study of the transmissibility of SV40 in humans” (2).