Marvin A. Rich

Incorporation of cordycepin (3′-deoxyadenosine) into ribonucleic acid and deoxyribonucleic acid of human tumor cells

Abstract Cordycepin (3′-deoxyadenosine), a nucleoside analog, is inhibitory to growing human tumor cells in culture. When these tumor cells were incubated with [3H]-cordycepin, the acid-soluble pool, the RNA and the DNA were radioactive. The RNA and DNA were hydrolyzed. The radioactive cordycepin was then isolated and crystallized to constant specific activities from the terminal and internal positions of the RNA. The cordycepin was also isolated and crystallized to constant specific activity from the DNA. In addition, the [3H]cordycepin, isolated after hydrolysis of the RNA (internal position) and the DNA, was further hydrolyzed to adenine and 3-deoxyribose. The 3-deoxyribose from the RNA (internal position) and the DNA was radioactive. No radioactivity was present in the 2-deoxyribose from the 2′-deoxyadenosine in the DNA. These results provide evidence that cordycepin is incorporated into the internal and terminal positions of RNA and the DNA and that 3-deoxyribose is not converted to 2-deoxyribose in H.Ep. No. 1 cells. The isolation of radioactive 3-deoxyribose from the cordycepin located in the internal position of the RNA indicates that a 2′,5′-phosphodiester bond is formed. The location (terminal or internal position) of the cordycepin in the DNA is not known.

INHIBITION OF HUMAN TUMOR CELLS BY CORDYCEPIN.

Abstract 1. Cordycepin inhibits the growth of human tumor cells (H.Ep. No. 1) grown in culture. The growth inhibition is cytostatic rather than cytocidal. 2. Adenosine competitively prevents inhibition but does not reverse it once it has occurred. This is in accord with the hypothesis that adenosine competes with cordycepin for phosphorylation to the active nucleotide level. 3. Exposure of human tumor cells (H.Ep. No. 1) to an inhibitory concentration of cordycepin does not markedly affect the protein, ribonucleic acid or deoxyribonucleic acid content. 4. Exposure to cordycepin results in a 2- to 4-fold depression of the incorporation of [8- 14 C]adenosine into ribonucleic acid and deoxyribonucleic acid. This is not in accord with the suggestion that the sensitive site for growth inhibition is early in the pathway of purine biosynthesis.

Human Wart Virus: In vitro Cultivation

Inoculation of cell cultures of fetal skin of human and murine origin with virus extracted from human wart tissue resulted in the appearance of intracellular wart virus specific antigen, as demonstrated by fluorescent antibody techniques. Appearance of antigen was accompanied by cytopathogenicity and the accumulation of large numbers of characteristic virus particles.

Chromosome Aberrations: Their Role in the Etiology of Murine Leukemia

If the association between chromosome aberrations and leukemia is a causal one, the aberrations should be present before the appearance of tumor. In a virus-induced murine leukemia in which the pre- and early leukemic stages were defined, aneuploidy was observed only during the later stages of the disease. This suggests that the chromosome alteration results from, rather than initiates, the neoplastic transformation.

Ultracentrifugation of a murine leukemia virus in polymer density gradients

Abstract A procedure for the purification in polymer density gradients of Rauscher leukemia virus is described. This involved zonal rate sedimentation of the virus in preformed density gradients of polyglucose or Ficoll. Stock virus preparations were resolved into three visible bands after centrifugation for 35–50 minutes (SW 39 rotor, 30,000 rpm). Leukemogenic activity was recovered in the middle band, which contained large numbers of characteristics virions as revealed by electron microscopy. Complement-fixing antigens accumulated in the two upper bands. Ficoll density gradient purified virus banded in potassium citrate in a single zone with a buoyant density of 1.16 g/ml.

RNA polymerase activity following infection with murine leukemia virus

Abstract 1. 1. Enzymes capable of incorporating [2-14C]UMP into an acid-insoluble product were extracted from spleen cells of mice infected with a murine leukemia virus. Extracts obtained from infected animals showed 8 times the RNA polymerase activity of those from uninfected controls. 2. 2. Enzyme activity was localized in the nucleus and required all 4 ribonucleoside triphosphates and Mn2+. It was sensitive to actinomycin D and deoxyribonuclease. The enzyme extracted from the infected spleen exhibited maximal activity at pH 8.3 and at a Mn2+ concentration of 2 mM. Enzyme extracted from uninfected control spleens did not show these maxima and was relatively insensitive to actinomycin D. 3. 3. The enzyme product synthesized in vitro was deoxyribonuclease resistant and sensitive to ribonuclease. Nearest neighbor analyses indicated that the product of enzyme preparation from infected spleens was a heteropolymer whereas that of extract from uninfected spleens was primarily due to a terminal addition. 4. 4. These data suggested the presence of a splenic, intranuclear, DNA-dependent RNA polymerase following infection with Friend murine leukemia virus. 5. 5. The temporal relationship between the appearance of enzyme activity and virus synthesis suggests a probable involvement of this enzyme in the process of viral induced leukemogenesis.

Studies on the Mechanism of Action of Thymic Leukemogenic Virus.

Summary Murine Leukemia virus (Rich) was inoculated into only one of the 2 thy-muses of infant mice which were sacrificed 2 months later. The animals were analyzed to compare the incidence of lymphoma evolving in the inoculated thymus with the incidence of lymphoma evolving in the uninocu-lated thymus. It was found that of 30 animals suitable for analysis, in 90% of cases the tumor arose in the inoculated thymus, supporting the view that the mechanism of viral leukemogenesis is direct.

Alkaline phosphatase activity in virus-induced murine leukemia

Abstract The levels and properties of liver alkaline phosphatase were compared in extracts from normal and Rich leukemia virus-infected mice. There was an increase in phosphatase activity in the livers from the virus-infected mice. This increase in enzyme activity was accompanied with alterations in the enzyme properties. The phosphatase activity in the extracts from infected mice was not as dependent on magnesium ions as was the enzyme from normal mice. Studies on acrylamide gel showed different electrophoretic mobilities for the alkaline phosphatase from the two sources. Heat stability studies showed that the Mg 2+ -dependent species was more stable than the Mg 2+ -independent species and that the extracts from infected mice contained a much lower concentration of this Mg 2+ -dependent phosphatase than did the extract from normal mice. The apparent Michaelis constant, with respect to p -nitrophenyl phosphate, was larger (sevenfold) for the extract from the infected mice. These data show that leukemia virus infection of mice resulted not only in changes in the levels of activity, but also in changes in the properties of this enzyme.

ARTIFICIAL RESPIRATION IN MICE DURING THORACIC SURGERY: A SIMPLE INEXPENSIVE TECHNIC.

During experiments designed to determine the effect of unilateral thymectomy on the unilateral development of thymic lymphoma in mice (1,2) an apparatus for artificial respiration with positive pressure oxygen in mice was developed. Use of the device permits wide surgical exposure of the thorax and approximates the conditions accomplished by modern anesthesia machines. Because the device decreases the hazard of pneumothorax during experimental surgery on mice and is inexpensive to construct, the apparatus is presented for possible interest to other investigators. The apparatus consists of 3 parts: 1) a flow valve, bubble meter, and high pressure limit assembly; 2) a valve to provide intermittent oxygen flow, and 3) an endotracheal catheter (Fig. 1.) 1. Flow valve, bubble meter, and high pressure limit assembly. The regulation of small amounts of oxygen is accomplished by use of a screw clamp on the oxygen supply rubber tubing which leads directly from the reducing valve of a standard oxygen tank. The rate of flow of oxygen is visually observed by bubbling the gas through water. By means of a “T” tube, the high pressure limit tube is connected to the metered gas flow. This last device consists of a length of glass tubing immersed in a column of water. The height of water above the open lower end of the tube determines the pressure at which excess oxygen will blow off. The upper end of the pressure limit tube is twice as high as the water column to prevent water from rebounding into the supply line. 2.“Finger valve.” The intermittent flow of positive pressure oxygen is controlled by a manually operated open-type valve. This device is constructed by cutting a 2 cm round hole in a 4 inch length of 3/4′ polyethylene drying tube.

Significance of Increased Alkaline Phosphatase Activity in Viral-Induced Thymic Lymphoma.

Summary Mice inoculated with a leukemogenic virus (Rich) were studied to determine when alkaline phosphatase appears in thymic cells relative to the developing thymic lymphoma. The findings indicated that the enzyme increase occurs only after the tumor is definitely established, hence this change cannot be causally related to the neoplasm.