Suppression of tumorigenicity in clones of hamster cells transformed with avian sarcoma virus

Hamster cells transformed with avian sarcoma virus, which had been selected for resistance to 8-azaguanine, were cloned. Several single cell clones were isolated which differed significantly in their tumorigenicity in comparison to the parental highly tumorigenic cells. The nontumorigenic cell clones contained rescuable avian virus. Both parental and resistant cells possessed transformed phenotype. Comparative studies with parental highly tumorigenic cells and nontumorigenic cell clones failed to detect any difference in the expression of p60src, in this phosphorylation and in phosphokinase activity. The isolated nontumorigenic cells represent cell mutants in which the viral src gene is controlled by a suppressor gene. The cells might be useful for further characterization of a putative antioncogene.     

Long-term infection with retroviral structural gene vector provides protection against bovine leukemia virus disease in rabbits

Bovine leukemia virus (BLV) infection of rabbits is a tractable model system to evaluate vaccination strategies against lymphotropic retroviruses, which represent a global human health problem. We have previously developed genetically simplified BLV structural gene vector (SGV) that replicates BLV structural and enzymatic genes independently of BLV regulatory and accessory genes. Results of a 20-month study in a rabbit model demonstrated that BLV SGV induces an antiviral immunological response and lacks pathogenicity. Here, these chronically infected-BLV SGV rabbits are assessed in a proof-of-principle study of preventative vaccination against challenge with pathogenic BLV. This study commences 24 months after BLV SGV inoculation and proceeds for an additional 20 months. The previously characterized BLV SGV rabbits and age-matched control rabbits were challenged with 1 x 10(8) fetal lamb kidney/BLV producer cells. BLV SGV rabbits seroconverted upon BLV challenge, but did not progress to BLV infection nor clinical disease. By contrast, naive rabbits became infected and succumbed to lymphotropic disease. Our findings provide proof-of-principle that chronic infection with BLV SGV induces protection against BLV infection. The data indicate that SGV based on HTLV or HIV is a promising approach against lymphotropic disease by human retroviruses.     

Product of the src gene is not phosphorylated in avian sarcoma virus B77 infected but untransformed human cells

Human embryo cells infected with the avian sarcoma virus B77 [Hu(B77)] but untransformed, contain the whole rescuable virus genome integrated in the host cell DNA. The cellular DNA induced the transformation of the infectious B77 virus after transfection of chicken cells. In the Hu(B77) cells the src gene product was expressed as a 58 kD protein which possessed phosphokinase activity but was not phosphorylated in comparison with the src production of B77 transformed rat cells RBI in which p60src is expressed. The addition of vanadate to tissue culture fluid reversibly elicited cell transformation in both control and virus infected human cells, but did not influence phosphorylation of the src gene product. The secretion of phosphoproteins in the investigated cells was different. Whereas transformed rat cells released a 62 kD transformation related phosphoprotein, the human cells did not. In tissue culture fluid from Hu(B77) cells an elevated amount of a 22 kD phosphoprotein was found in comparison with control cells. The implications of these findings are discussed with respect to the role of the v-src gene product in malignant cell transformation.     

Isolation of the missing 5′-end of the encoding region of the bovine leukemia virus cell receptor gene

Summary The missing 5-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5) was isolated from a lambda gt11 cDNA library using the³²P-labeledEcoRI-SamI fragment corresponding to the 5-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5 cDNA revealed that the 1058 bpEcoRI fragment at its 5-end contained a new 114 amino acid long sequence, and at its 3-end contained a completely identical 88 amino acid overlapping region with the 5-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.     

Tissue damage in rat ovaries subjected to torsion and detorsion: effects of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine and <Emphasis Type="Italic">N</Emphasis>-acetyl cysteine

We aimed to evaluate histopathological changes, to detect HIF-1α staining intensities and to determine MDA levels in rat ovaries, which were subjected to torsion and detorsion and treated with l-carnitine or N-acetyl cysteine (NAC). Forty-eight prepubertal female Sprague–Dawley rats were divided into five groups (n = 8): 1, control; 2, ischemia; 3, reperfusion; 4, l-carnitine; and 5, NAC groups. In groups 3, 4 and 5, an ischemic period of 3 h was followed by reperfusion for 24 h. In groups 4 and 5, ischemia was performed and either l-carnitine or NAC was infused intraperitoneally 30 min before reperfusion. Ovarian tissues were examined histopathologically; tissue MDA levels and serum IL-6 levels were determined biochemically. HIF-1α was applied to all ovaries immunohistochemically. Total tissue damage scores, tissue MDA levels and HIF-1α scores, were significantly higher in group 2 (all P < 0.001) than group 4, and group 3 than group 4 (P < 0.001, P = 0.05 and P < 0.001, respectively). They were also significantly higher in group 2 (all P < 0.001) than group 5. When group 3 is compared to group 5, total tissue damage scores and tissue MDA levels were significantly higher in the former (P < 0.01 and P < 0.001, respectively). Serum IL-6 levels were significantly higher in group 2 when compared to groups 1, 4 and 5 (all P < 0.01). The degree of tissue damage of the torsioned ovaries decreased after a reperfusion period of 24 h in the torsioned ovaries. However, ovaries of both l-carnitine and NAC groups showed better recovery than the reperfusion group. This study was accepted for poster presentation in the 21st European Congress of Pathology, held in Istanbul, Turkey, on 8–13 September 2007.     

Avian sarcoma virus transformed hamster cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity

Summary Hamster cells transformed with the Schmidt-Ruppin strain of avian sarcoma virus were selected for resistance to 5-bromodeoxyuridine (BUDR). The resistant cell lines Ha(SR)BU-25 and Ha(SR)BU-100, proliferated in the presence of 25 and 100 g/ml BUDR, respectively. The resistant cells were deficient in thymidine kinase activity. They did not grow in HATG medium and did not incorporate labelled thymidine into DNA.Several single-cell clones were isolated in medium containing 100 g/ml BUDR from Ha(SR)BU-100 cells. These isolated cell clones differed in morphology and modal number of chromosomes from each other. None of the clones incorporated thymidine into DNA.The BUDR resistant cells were tested for their tumorigenicity in young hamsters. The number of cells needed for induction of tumor growth was higher with the BUDR resistant cells than with original clone of Ha(SR) cells.All clones of thymidine kinase deficient cells were tested for the presence of the virus genome by fusion with chicken embryo cells. All clones of Ha(SR)BU-100 cells contained the virus genome, and infectious virus could be rescued from these cells.Therefore, the genome of avian sarcoma virus can persist in virogenic hamster cells growing in the presence of 5-bromodeoxyuridine. These virogenic cells deficient in thymidine kinase activity are useful for preparation of cell hybrids.