A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region.

The v-akt oncogene codes for a 105-kilodalton fusion phosphoprotein containing Gag sequences at its amino terminus. Sequence analysis of v-akt and biochemical characterization of its product revealed that it codes for a protein kinase C-related serine-threonine kinase whose cellular homolog is expressed in most tissues, with the highest amount found in thymus. Although Akt is a serine-threonine kinase, part of its regulatory region is similar to the Src homology-2 domain, a structural motif characteristic of cytoplasmic tyrosine kinases that functions in protein-protein interactions. This suggests that Akt may form a functional link between tyrosine and serine-threonine phosphorylation pathways.

A survey of Epstein-Barr virus DNA in lymphoid tissue. Frequent detection in Hodgkin's disease.

A total of 151 unselected malignant and nonmalignant lymphoid tissue samples were surveyed by Southern blotting for the presence of Epstein-Barr virus (EBV) DNA. Eight of 28 Hodgkin's disease (HD) samples (29%) had detectable EBV DNA. Both nodular sclerosis and mixed cellularity histologic results were positive. The tumor type with the next highest frequency, 8%, was diffuse large cell lymphoma. The presence of EBV DNA in some HD biopsies suggests that EBV may be a factor in the pathogenesis of this disease. Alternatively, its presence may be secondary to the immune deficiency characteristic of HD. The clonal B-lymphocyte expansions reported in some cases of HD may result from EBV infection.

Thymic lymphoma induction by the AKT8 murine retrovirus.

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.

The AKT1 proto-oncogene maps to human chromosome 14, band q32.

The human AKT1 gene is the proto-oncogene of the viral oncogene v-akt. The AKT1 gene has been localized to human chromosome 14, band q32, proximal to the heavy-chain immunoglobulin locus (IGHM), by analysis of human-hamster somatic cell hybrids and by in situ hybridization. Chromosome rearrangements of this band which occur in T-lymphoid malignancies and Hodgkin's disease may affect the AKT1 gene.

Amplification of the c-myc oncogene is associated with an abnormally banded region on chromosome 8 or double minute chromosomes in two HL-60 human leukemia sublines.

Two sublines of HL-60 human promyelocytic leukemia cells were examined cytogenetically with banding techniques. The karyotype of one subline was 44,X,-X,-5,-9,-10,-15,-17,+18,8q+,14q-,16q+,16q+,+mar1,+mar2 ,+mar3. The defective chromosome #8 contained an expanded chromosomal segment at the end of the long arm at band q24. The segment appeared to be a homogeneously staining region on the basis of quinacrine fluorescence banding. Using G-banding technique, this segment showed some evidence of indistinct aberrant bands and, thus, was designated an abnormally banded region (ABR). Double minute chromosomes (DM) were not seen in these cells. The second subline showed a similar karyotype; however, these cells lacked the 8q+ marker and contained one to 37 DM in approximately 90% of the cells examined. Because HL-60 cells are known to contain multiple copies of the c-myc oncogene, in situ chromosomal hybridization of a c-myc probe to HL-60 metaphase cells was performed to localize the amplified genes. The hybridization studies revealed localization to the ABR, as well as to DM, which is consistent with amplification of c-myc within these novel interchangeable chromosomal aberrations.

Primary lymphomas of the liver. Report of six cases and review of the literature.

Six cases of diffuse large cell lymphoma (DLCL) of the liver were studied with immunohistochemistry for common leukocyte antigen (CLA), lysozyme, alpha-1-antitrypsin (AAT), and kappa and lambda light chains on paraffin-embedded tissues. All six cases were positive for CLA. Four of the six cases showed staining for lysozyme and AAT (three focal and one diffuse staining). In three cases, frozen tissue for monoclonal antibodies and glutaraldehyde-fixed tissue for electron microscopic examination were available. Two of these showed B-cell phenotypes with monoclonal antibody studies. Electron microscopic examination on these two B-cell lymphomas showed scant cytoplasm and a paucity of cytoplasmic organelles. The third case did not show definite B- or T-cell surface markers but did show strong Leu-M1 and OKM1 staining. Electron microscopic examination of the tumor cells showed a prominent Golgi apparatus, abundant cytoplasm with numerous cytoplasmic organelles and phagolysosomes. However, DNA hybridization studies on this tumor showed immunoglobulin heavy and kappa light chain gene rearrangements typical of a B-cell lymphoma. All six lymphomas were solitary liver masses without evidence of disease elsewhere. The mean age for the six patients was 56.2 years (four males, two females).

Hypermethylation of the 5' region of the calcitonin gene is a property of human lymphoid and acute myeloid malignancies.

An abnormal increase in numbers of CCGG sites methylated in the 5' region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.

Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma.

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, AKT1 and AKT2, were cloned. A survey of 225 human tumors for changes involving AKT1 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.

Richter's syndrome with two different B-cell clones.

The diagnoses of chronic lymphocytic leukemia (CLL) in prolymphocytic transformation, and diffuse large cell lymphoma (DLC), were made simultaneously in a 71-year-old man. The DLC showed mu lambda surface immunoglobulin. The CLL in a lymph node and in the peripheral blood showed mu kappa. Immunoglobulin gene DNA analysis confirmed the presence of different rearrangements in the heavy and light chain genes of the CLL and DLC. Other cases reported of Richter's syndrome are discussed, and it is concluded that there may be two types of Richter's syndrome, those arising from transformation of a single clone, and those occurring from expansion of two morphologically and immunologically distinct clones, as, it is believed, is the case in this patient.

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.

Identification of ecotropic proviral sequences in inbred mouse strains with a cloned subgenomic DNA fragment.

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 ecotropic-specific 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.

Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma.

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.

Differential effect of phenethyl alcohol on mycoplasmas and enveloped viruses.

Mycoplasmas are totally inactivated by a 20-min treatment with 1% phenethyl alcohol, whereas suspensions of enveloped viruses resist the same treatment. This treatment has proved successful in eliminating mycoplasma contamination of a virus suspension.