Effect of Antimitotic Agents on Intracellular Reovirus Antigen* REX S. SPENDLOVE, EDWIN H. LENNETTE, JEAN N. CHIN, AND CHARLES 0. KNIGHT (Viral and Rickeitsial Di8ease Laboratory, California State Department of Public Health, Berkeley, California) SUMMARY Intracellular reovirus antigen localizes in the area occupied by the achromatic figure of mitotic cells. In untreated interphase cells, viral antigen forms a filarnen thus, intracytoplasmic reticulum, but in the presence of certain spindle poisons it consolidates into clumps. The specificity of this latter phenomenon was investigated in this study. The results obtained are as follows. 1. Six of eight spindle poisons tested were found to have a disruptive effect on the intracellular organization of reovirus antigen; antimitotic agents that have not been reported to disrupt the mitotic spindle had no effect. 2. Antigen of all three reovirus types formed filaments in interphase cells, localized with the mitotic apparatus, and responded similarly to the same concentrations of spindle poisons. The concentration of spindle poison required to clump type 1 reovirus antigen (the only type tested) varied in different cells. 3. The effectof those spindle poisonstested was found to be reversible. 4. Colchicine had no effect on the development of infectious virus and did not re duce the infectivity of infectious culture fluids. These results indicate that spindle poisons affect intracellular reovirus antigen in directly by their action on the spindle or on a closely related organelle of similar physi cochemical structure. Recent communications (12, 13) from this laboratory 1\IATERIALS AND METHODS have presented the findings of studies in which the de Most materials and methods used in this investigation velopment of viral antigen, stainable by fluorescent anti have been described in detail previously (11—13). body, was followed in HeLa and FL cells infected with Viruses.—Type 1 reovirus (Lang strain) was obtained reovirus. These findings indicated that viral antigen is from Dr. Herbert Wenner as infectious HeLa cell culture associated with the mitotic apparatus of the host cell. fluid. Type 2 reovirus (D5 Jones strain) was obtained Antigen seen in rnitotic cells was located in the area from Dr. Albert B. Sabin and type 3 reovirus (Abney occupied by the achromatic figure (spindles, asters, and strain) from Dr. Leon Rosen; both strains were supplied centnioles); antigen produced in cells (interphase and in the form of infectious fluid from monkey kidney cell mitotic) cultivated in the presence of certain spindle cultures. poisons formed intracytoplasmic clumps. In interphase Ce118.—TheFLline of human amnion cellswas obtained cells infected in the absence of these spindle poisons, the from the Naval Biological Laboratory, Berkeley, Call antigen occurred as filaments. Antigen clumping ap fornia, and was carried for more than 276 passages in a peared to be related to C-rnitosis; i.e., it occurred when lactalbumin hydrolysate-yeast extract medium plus 10 cells were treated with agents which block rnitosis in per cent human serum before use in these experiments. metaphase by disrupting spindle organization. Miss Hope Hopps supplied the grivet monkey kidney cells; Additional evidence supporting the conclusion that the tb@iscell line (BS-C-1) was at the fiftieth passage when re spindle poisons act on some subcellular organelle of reo ceived and had been passaged 45 times by us in a lactal virus-infected cells and not on the virus per se was sought bumin hydrolysate-yeast extract medium plus 20 per to determine whether this phenomenon could be used as cent calf serum when used in this work. A clone of ham a tool to study the mitotic apparatus and the mechanism ster kidney cells (BHK21-C13) was obtained from Dr. of action of spindle poisons. The results of such studies Ian MacPherson of the Wistar Institute, Philadelphia. are reported in this communication. These cells were cultured in a medium composed of 10 per cent unheated calf serum and 10 per cent autoclaved * The work on which this paper is based was supported by tryptose phosphate broth (Difco) in Eagle's basal medium Grant AI-01475-07 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States modified to contain twice the normal concentration of Public Health Service. amino acids and vitamins (7). The human diploid cell Received for publication June 23, 1964. strains (embryonic kidney and lung) were provided by 1826 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. SPENDLOVE et al.—Antimitotic Agents and Reovirus 1827 Dr. Jack H. Schieble of this laboratory. They were quinoline (Merck and Company Inc., Rahway, New Jer cultivated in Eagle's medium containing Earle's salts sey) ; 5-bromo-2'-deoxyuridine (California Corporation plus 10 per cent fetal bovine serum. for Biochemical Research, Los Angeles) ; 5-fluorouradil Fluorescent antibody technw.—The fluorescent antibody (La Roche Laboratories, Nutley, New Jersey) ; amethop conjugates used in this and previous studies (11) were pre term (K and K Laboratories, Jamaica, New York); pared by adding fluorescein isothiocyanate to a 5 per cent DL-p-fluOrOphenylalafllne, adenosine triphosphate, and protein solution at a ratio of 0.25 mg. of dye per 1.0 ml. creatine phosphate (Nutritional Biochemicals Corpora of protein solution (a 1:200 fluorescein :protein ratio). tion, Cleveland) ; 2 ,4-dinitrophenol, sodium azide, and The free fluorescein was removed by passage of the conju iodoacetate (Matheson, Coleman, and Bell, East Ruther gates through a column of Sephadex G-50. ford, New Jersey) ; and sodium cyanide (Baker and Adam Cover slip cultures to be stained were treated as fol son, Albany, New York). The Myleran, mitomycin C, lows : media were removed, the cultures were dried in air, 6-mercaptopurine, and chlorambucil were generously fixed in acetone for 5 mm., dried, and stained with fluores supplied by Dr. Howard Bond of the National Cancer cent antibody for 30 min. (A number of other fixatives Institute, Bethesda, Maryland. were tested, and it was found that most lipid solvents The vinblastine sulfate was diluted in phosphate could be used successfully if the preparations had been buffered saline. Coumarin was dissolved in lactalbumin dried in air prior to fixation. Those tested and found to hydrolysate-yeast extract medium, and all other anti be satisfactory were acetone, benzene, chloroform, ether, mitotic agents and inhibitory compounds were dissolved and xylene. Some intensity of fluorescence was lost with in distilled water. All reagents were sterilized by filtra 10 per cent formalin, and absolute ethanol was found to tion through a Seitz pad. be a poor fixative. The time of fixation could be reduced Evaluation of effect of test chemwala.—The various cell to less than 1 mm., but the results were not consistently cultures used were grown on 15-mm. circular cover slips good with such short fixation times.) The results with all in 60-mm. Petri dishes. The cultures were washed twice methods of fixation were the same. The viral antigen in with lactalbumin hydrolysate-yeast extract medium and interphase cells was in the form of filaments, and the were infected with reovirus types 1, 2, or 3. The corn antigen in mitotic cells was localized in the area of the pounds to be tested were added in a total volume of 4 ml. spindles. of lactalbumin hydrolysate-yeast extract medium plus Infected cell assay method.—One-day-old cultures of FL 10 per cent human serum prior to or after infection, and cells grown to confluency on circular cover slips were were left on the cultures for the time periods indicated inoculated with 0.02 ml. of the appropriate virus dilutions for each experiment. After an appropriate period of (11). The cultures were incubated for 2 hr., after which incubation, the medium was removed and the cultures were a lactalbumin hydrolysate-yeast extract medium with 10 stained with fluorescent antibody as described. per cent human serum was added to the cultures. After The effect of the antimitotic agents on the formation approximately 20 hr. of incubation, the medium was re of intracellular reovirus antigen was determined by differ moved; the cells were dried, fixed in acetone, and stained ential counts of the number of cells in which all of the with fluorescent antibody. The cultures were scanned antigen was present in the form of clumps and those which and the infected cells were counted under a Zeiss fluores contained filaments of antigen. In the early phases of cence microscope, with the use of the lox objective and this study, two end points were determined : the lowest the 12.5x eyepieces. To obtain complete illumination of concentration causing complete clumping, and the lowest the field, it was necessary to adjust the condenser to concentration having no effect. Untreated control cul bring it closer to the bottom of the microscope slide than tures were found to contain an occasional cell in which was possible with the original factory adjustment. Also, all of the antigen was clumped; therefore, 95 per cent was excess mounting medium was removed by placing the arbitrarily selected for both end points. microscope slide momentarily on a Celluwipe with the Enzyme treatment.—a-Chymotrypsin (three times crys side containing the mounted cover slip down. tallized), deoxyribonuclease I (beef pancreas, once crys The proportionality found between the concentration of tallized), and ribonuclease (beef pancreas, three times virus added to the cultures and the number of infected crystallized) were obtained from Worthington Biochemical cells observed proved the reliability of the method. Corporation, Freehold, New Jersey. Chymotrypsin and Anti-mitotic and other inhilitory compounds.—The anti ribonuclease were dissolved in distilled water, and deoxy mitotic agents that affected the localization of intracellular ribonuclease was dissolved in 0.2 M MgSO4 .