A previous report described the isolation of a directly transforming retrovirus, AKT8, from a spontaneous thymoma of an AKR mouse. The AKT8 provirus has now been molecularly cloned from a transformed, nonproducer cell line. The virus genome contains both viral and nonviral, cell-related sequences; the nonviral sequence has been designated v-akt, the presumed viral oncogene of the AKT8 virus. This gene lacks homology to the 16 other oncogenes tested. The cloned provirus has undergone a partial deletion, during cell passage in vitro, that prevents direct demonstration of the transforming ability of this molecular clone. Two human homologues of the v-akt oncogene, AKTI and AKT2, were cloned. A survey of 225 human tumors for changes involving AKTI led to the discovery of a 20-fold amplification of this gene in one of the five gastric adenocarcinomas tested. The results demonstrate that AKT8 has the characteristic structure of a directly transforming retrovirus and that it contains a gene derived from highly conserved cellular sequences that may be involved in the pathogenesis of some human malignancies.

Murine leukemia viruses capable of malignant transformation of mink tissue culture cells have been isolated from an AKR thymoma cell line and from a spontaneous reticulum cell sarcoma in an NIH Swiss mouse partially congenic for the AKR ecotropic virus-inducing locus Akv-2. In contrast to the recently described mink cell focus-inducing strains of murine leukemia virus, at least one of the two transforming strains is replication defective. Nonproducer mink cells carrying the genome 'of the transforming virus of AKR origin have been isolated, and pseudotype transforming viruses generated.We have recently reported the detection of a unique category of murine leukemia virus (MuLV) (2) supplemented with 10% heat-inactivated fetal calf serum, penicillin/streptomycin/glutamine, and 5 X 10-5 M mercaptoethanol. Established cultures were split 1 to 2 every 2-3 days.The mink lung cell line (ATCC no. CCL-64) (3) was used in assays for transformation and fluorescent antibody (FA) focus induction. The cells were maintained on 10% heated fetal calf serum and penicillin/streptomycin/glutamine in Dulbecco's modified basal medium. Inoculations were as described (4), except that polybrene (Abbott; 16 ,g/ml for the first day of infection) rather than DEAE-dextran was used for transformation assays with cell-free virus. Infectious center assays with mitomycin C-treated cells, and FA focus induction tests were done as described (4).Agar Assay. Assay for growth of cells in semisolid medium was based on the method of Macpherson (5). Cells were trypsinized and suspended at the desired concentration (usually 1 to 2 X 106 cells per ml) in 1 ml of tissue culture medium. Two milliliters of agar medium containing 10% heated fetal calf serum, penicillin/streptomycin/glutamine, and 0.5% agar or Abbreviations: MCF, mink cell focus-inducing; MuLV, murine leukemia virus; FA, fluorescent antibody. agarose in Eagle's minimal essential medium was mixed with the cells and layered over 7 ml of hardened 0.5% agar mediumin a 60-mm petri dish. The cells were fed with 3 ml of 0.3% agar medium at 5-7 days.Transformed clones were established by picking colonies from agar with a pasteur pipette under microscopic visualization. The colony-containing agar plug was placed in a 35-mm petri dish containing 1 ml of tissue culture medium. The colony was teased free from the agar with sterile needles, and a further 1 ml of medium was added. Visible clusters of adherent cells appeared in 5-7 days. The clonal lines were carried in Eagle's minimal essential medium with 10% heated fetal calf serum and penicillin/streptomycin/glutamine. RESULTS Isolation of transforming virusesT-8 Strain. The initial isolate was from an in vitro thymoma cell line, AKT-8, from a spontaneously lymphomatous AKR/J mouse. The massively enlarged thymus was minced and a portion suspended in EHAA medium. Large tissue fragments were allowed to settle for 10 min, and the supernatant was transferred to a 75-cm2 tissue culture flask. After the lymphocytes were well established in culture...

EBV DNA has been detected by Southern blot hybridization in 20-25% of Hodgkin's disease tumor specimens and localized to the Reed-Sternberg cells by in situ hybridization. In the present investigation we used a 3H-labelled EBER I anti-sense RNA for in situ hybridization of archival formalin-fixed paraffin-embedded Hodgkin's disease specimens previously shown by Southern Blot hybridization to be EBV-positive. In 6 of 8 specimens neoplastic cells showed an intense signal in virtually all of the tumor cells. The background lymphocytes, eosinophils, plasma cells and histiocytes did not demonstrate significant hybridization. In each case hybridization tended to spare the nucleoli. Hybridization was detected in specimens with histologies of mixed cellularity and nodular sclerosis. The intensity of signal relative to background is better than that in previous studies utilizing 35S-labelled large internal repeat probes for EBV in Reed-Sternberg cells. The exposure time is one week in contrast to the 4-5 weeks reported by others for detection of EBV in Hodgkin's disease. Both increased relative intensity and shorter exposure requirements may be attributed to the very high number of EBER transcripts in the target cells. The demonstration that EBER I is expressed is consistent with a role for EBV in growth regulation of Reed-Sternberg cells and suggests that the virus is not merely a silent passenger in Hodgkin's disease.

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropicsecific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size. The chromosomal DNA of inbred and feral mice contains multiple copies of sequences reactive with murine leukemia virus (MuLV) nucleic acid probes (1-7). Among these sequences are complete, potentially infectious genomes of ecotropic and xenotropic viruses (8), the complete genetic information of amphotropic and additional xenotropic viruses that apparently cannot be expressed as infectious viruses in inbred mice (ref. 9; unpublished data), proviral DNA that is expressed only in the form of viral antigens (10-12), and probably many subgenomic viral DNA segments that are not expressed.Because of their abundance in mouse chromosomal DNA and the extensive polynucleotide sequence homology between large portions of the genomes of the various classes of MuLV, little is known about the molecular organization of the endogenous proviral DNA sequences of the particular MuLV types. Although absorption of labeled MuLV cDNA with purified viral RNA from a heterologous MuLV decreases the crossreaction with endogenous sequences (6, 7), such probes still hybridize to a significant extent to related proviruses present in mouse DNA. Clearly, the preparation of nucleic probes that recognize type-specific regions of proviral DNA would offer great advantages.The molecular cloning of AKR ecotropic MuLV (AKV) DNA (13) has made it possible to generate such a probe. We have subcloned, in Escherichia colt K-12 with a pBR322 vector, a portion of the AKV env gene region that is specific for ecotropic viruses in the operational sense that it does not react either with cellular DNA prepared from the prototype ecotropic-negative mouse strain NFS/N or with genomic DNA of a xenotropic MuLV.We describe here the construction and characterization of the recombinant plasmid, illustrate the strategy developed for using the cloned DNA to identify endogenous ecotropic proviruses, and report the presence of a unique set of nucleotide sequences reactive with this probe in the DNA of several mouse strains that, like NFS/N, are biologically negative for...

The directly transforming murine retrovirus, AKT8, was isolated from a spontaneous AKR thymoma and carries the cell-derived viral oncogene, akt. We have now shown that this virus produces thymic lymphomas after inoculation of susceptible mouse strains. The presence of the AKT8 genome in the DNA of the virus-induced tumors was demonstrated by Southern blotting using an akt-specific probe. These results establish the in vivo pathogenicity of the AKT8 virus and its akt oncogene, and imply a potential role for the cellular akt proto-oncogene in tumor development.