Induction of antioxidant enzyme activity by hyperoxia (60 % O2) in the developing chick embryo.

1. At premature birth, man and animals are exposed to relatively high oxygen levels, compared with intra-uterine conditions, at a time when their antioxidant enzyme (AOE) system is still immature. Using the chick embryo as a study model, we investigated changes in the AOE system in response to hyperoxia applied at different time points during the incubation period. Relations between hyperoxia and AOE activity were studied in selected organs (brain, heart, liver, intestine and lungs) of developing chick embryos (during the second half of the incubation period). 2. Incubated White Leghorn eggs were divided into four groups: control (n = 100) and three test groups exposed for 48 h to 60 % O2 on day 10 (test group 1, n = 80), day 14 (test group 2, n = 60) and day 18 (test group 3, n = 30). Superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) enzyme activities were measured in homogenates of the brain, heart, liver, intestine and lungs. 3. Exposure to hyperoxia at different time points during incubation resulted in a 2- to 10-fold increase in SOD activity in all organs except the brain. Catalase and GPx enzyme activities were only induced in test group 1, 48 h after initiation of hyperoxia. 4. In the developing chick embryo, hyperoxia can produce a temporary induction of AOE activity, which is dependent on the AOE, organ, incubation time and time point of exposure.

Reducing effects of garlic constituents on DNA adduct formation in human lymphocytes in vitro.

A water extract of raw garlic (RGE) and two organosulfur compounds, diallyl sulfide and S-allylcysteine (SAC), were evaluated for their relative effectiveness in reducing benzo[a]pyrene (BaP)-DNA adduct formation in stimulated human peripheral blood lymphocytes in vitro. In replicate experiments, RGE significantly inhibited BaP-DNA adduct formation at concentrations of 0.001, 0.01, and 0.1 mg/ml. SAC also significantly decreased BaP-DNA adduct formation at concentrations of 0.01 and 0.1 mg/ml. For diallyl sulfide, no significant reduction in BaP-DNA adduct formation was found. BaP-DNA adduct formation was not associated with cell viability or proliferation of peripheral blood lymphocytes after the various treatments. No clear scavenging activity was detected for the garlic constituents. Aryl hydrocarbon hydroxylase activity was not decreased, nor was formation of sulfate and glucuronide conjugates of 3-hydroxy-BaP increased in the presence of RGE and SAC, indicating that increased glutathione S-transferase activity or a more efficient repair of BaP-DNA adducts may explain the observed effects. In addition, reactive oxygen species-induced 8-oxodeoxyguanosine in DNA was reduced in the presence of SAC. It is concluded that raw garlic and SAC may be useful in the prevention of BaP-associated tumorigenesis and that further evaluation of their preventive potential in humans at risk appears feasible.

Molecular cloning of the Rauscher murine leukemia virus. Disease spectrum and integration pattern.

After transfection of NIH 3T3 cells with DNA from molecularly cloned Rauscher MuLV, virus was isolated which showed a disease spectrum comparable to that of R-MuLV cloned biologically by endpoint dilution. In both cases sites of proviral integration vary from 2-5 per leukemic tissue and occur apparently at random.

Oncogene expression in Rauscher murine leukemia virus induced erythroid, myeloid and lymphoid cell lines.

A comparative study on the expression of nuclear and cytoplasmic oncogenes was carried out using the Northern blotting technique, in Rauscher virus induced primary leukemias and the more malignant transformed cell lines derived from them. The latter grow permanently in vitro. Hyperplastic spleens obtained from mice recovering from anemia were analysed as controls. In addition to the detection of mRNAs, Southern blotting was carried out to observe whether rearrangement or amplification of oncogenes had occurred. The results show that the nuclear oncogenes c-myc, c-myb and p53 are strongly expressed in leukemic tissue, whereas c-fos transcripts show a much weaker hybridization. The expression of two of these oncogenes, c-myc and c-myb was followed during differentiation in myeloid leukemic cells and showed a gradual decrease when compared with the actin gene, which is constitutively transcribed. A large number of cytoplasmic oncogenes is expressed in the leukemic cells lines, i.e. c-abl, c-fms, c-fes, c-src, c-ros, c-H-ras, c-K-ras and N-ras. Of these, transcripts coding for c-abl and c-src were absent in blast cells of acute erythroid leukemias. Transcripts coding for c-erb, c-mos and c-sis could also not be detected. A number of putative oncogenes which are reported to play a role in Moloney and Friend virus induced leukemias for instance pim-1, fis-1, fim-1 and fim-2 were also used for screening. Only expression of pim-1 in Rauscher virus induced myeloid leukemic cells and in primary acute erythroid leukemias could be observed. At the DNA level no rearrangement or amplification of any of the oncogenes investigated could be detected. The results show that a number of oncogenes are expressed simultaneously in the same leukemic tissue or cell lines. It therefore seems likely that the presence of transcripts of different oncogenes is associated with the progression of leukemia, but is not the primary cause of leukemogenesis or of the transformation of these cells into established cell lines.

Syngeneic monoclonal antibodies directed against Rauscher virus-induced myeloid leukemic cells: isolation and characterization.

Hybridomas producing syngeneic monoclonal antibodies (MAbs) were prepared by fusion of spleen cells of BALB/c mice, which were immunized with sublethal doses of RMB-I cells. This cell line originates from a Rauscher virus (R-MuLV)-induced myeloid leukemia and forms tumors when re-inoculated into mice. MAbs were characterized as regards their reactivity against virally and non-virally induced cell lines. Two selected MAbs, IC5F5 and 4D2B4, were analyzed further. Their binding to subcellular structures was determined, and so were the properties of the antigens to which they are directed. MAb IC5F5 is of the IgG2A and 4D2B4 of the IgG2b subclass. Both bind to R-MuLV-infected or -transformed cell lines and are not mutually competitive. The antibodies do not react with other murine and human myeloid leukemic cells. As shown by immuno-electron microscopy, these MAbs have affinity to the cell membrane of non-virus producing RMB-I cells. When lysates of purified virus were analyzed, the MAbs were found to be directed to the gag precursor protein Pr65, and one of them (IC5F5) also to be directed to the core protein p12. In RMB-I cells, binding occurs to a 50-kDa glycoprotein and 2 proteins of 26 and 29 kDa. Since RMB-I cells do not produce virus, but express aberrant viral proteins, these MAbs are tumor-specific and useful for immunotherapy.

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies.

The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes 125I and 131I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with 131I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with 125I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.

Pyruvate kinase inhibition in the diagnosis of gliomas with an intermediate degree of malignancy.

The aim of the investigation was to see if the histological diagnosis of brain tumors showing an intermediate degree of malignancy can be improved by the measurement of L-alpha-alanine inhibition of pyruvate kinase isoenzymes. The inhibition of pyruvate kinase activity was measured in 51 gliomas with different grades of malignancy. It was confirmed that benign tumors have a low level of inhibition (less than 50%) and that the more malignant the tumor, the higher the level of inhibition became, reaching more than 75%. However, when grade II and III astrocytomas and grade II and III oligodendrogliomas were analyzed, their level of inhibition was found to be variable. Grade II showed low and moderate levels of inhibition and grade III moderate and high levels. In turn, inhibition levels ranging from 50 to 75% were not only found in brain tumors with an intermediate grade of malignancy, but also in a number of benign and malignant tumors. When the survival times of patients with brain tumors were compared with both the histological diagnosis and pyruvate kinase inhibition, the prediction of the survival time on the basis of low and high levels of inhibition correlated well with the histological diagnosis. In contrast, when moderate levels of inhibition were measured, the prediction of the patients' survival remained uncertain and no improvement was found in the prediction for tumors showing an intermediate degree of malignancy on the basis of histology.

A Rauscher-virus-induced T-lymphocyte cell line. Induction of differentiation under influence of dimethylsulfoxide and phorbolesters.

DBA/2 mice which are neonatally infected with Rauscher helper virus (R-MuLV) develop predominantly lymphatic leukemias. From one of these lymphatic leukemias we established a permanent cell line which we named RLD (Rauscher Lymphoid DBA/2). Phenotyping of this cell line with a panel of monoclonal antibodies directed to cell-surface determinants shows that RLD cells have T-cell characteristics: they bind monoclonal antibodies directed to the antigens Thy-1, T-200 and Lyt-1; they do not react with anti-Lyt-2 antibodies, nor do they react with antibodies directed to determinants on B cells or myelomonocytic cells. RLD cells show a high activity of the nuclear enzyme terminal deoxyribonucleotidyl transferase (TdT). RLD cells are able to differentiate after in vitro stimulation with 1% DMSO or with 30 nM tetradecanoylphorbol-1.3-acetate (TPA). This differentiation process is reflected by (1) changes in the 2D gel electrophoresis pattern of metabolically labelled proteins, (2) a decrease in TdT activity and (3) changes in the expression of cell-surface markers. Flow cytometric analysis of stimulated RLD cells shows a strong increase in the Lyt-1 expression. Together these data indicate that RLD cells are immature T lymphocytes which upon appropriate stimulation differentiate along the line of T helper cells.