On the Properties of the Lactic Dehydrogenase

Transcription

1 On the Properties of the Lactic Dehydrogenase Agent 1, 2 CHARLES G. CRISPENS, JR., Department 01 Anatomy, University 01 Marylana School 01 Meaicine, Baltimore, Marylana SUMMARY-Results of studies on the properties of the lactic dehydrogenase (LDH) agent are described. Evidence is presented to suggest that this virus is heat labile, acid labile, and sensitive to treatment with sodium deoxycholate and diethyl ether. Exposure to ultraviolet irradiation resulted in complete inactivation after 90 minutes. No effect on virus titer was observed after incubation with papain or trypsin; hemagglutination tests against red blood cells from several species were negative. The possibility that lymphocytic choriomeningitis, pneumonia virus of mice, mouse hepatitis virus, and the LDH agent may belong in a single group is considered on the basis of similar properties shown by these viruses. The two particles reported in plasma of mice infected with the LDH agent might be differentiated by formalin treatment.-j Nat Cancer Inst 35: , THE INITIAL report of a virus-like entity in MATERIALS AND METHODS association with a variety of transplantable murine Animals.-Male and female C57BLJFg mice, neoplasms was made by Riley et al. (7) who 3 to 6 months old, were used. found that the agent was filterable, could be LDH agent.-the isolation of the LDH agent in serially transmitted, and was detectable by its this laboratory has been described (5). Four pools induction of a fivefold to tenfold increase in plasma of infected plasma were obtained from mice 10 to lactic dehydrogenase (LDH) after inoculation into 14 days after an intraperitoneal injection of virus, normal mice. Although some characteristics of and stored in rubber-stoppered vials at - 20 C this virus, the LDH agent, have been reported until used. in subsequent papers (2-4), its biological position Proteolytic (y tic en;:ymes.- enzymes.- Samples of two plasma pools remains obscure. were incubated with papain or trypsin (0.5 and This report describes the results of investigations 5.0 mg/ml of plasma) at 37 C for 1 hour. Control in our laboratory on some physical and chemical samples were treated similarly, but without.these properties of the LDH agent. These data, to enzymes. gether with observations reported previously, are 1 Received March 10, presented to provide a basis for classification of 2 Supported by Public Health Service research grant CA- this murine virus from the National Cancer Institute cca

2 976 CRISPENS Diethyl ether.-tests for ether sensitivity were made according to Andrewes and Horstmann (6). Sodium deoxycholate (DOG).-The method of Theiler (7) was used to determine DOC sensitivity. Temperature.-Samples of two plasma pools were incubated at 50, 56, and 60 C for 30 minutes. Nonheated samples were used as controls. pr.-duplicate samples of virus were diluted 1 :10 in M/15 phosphate buffer adjusted to ph 3.0 and ph 7.2, then incubated for 2 hours at 37 C. Ultraviolet irradiation.-samples (1.0 ml) of two plasma pools were placed in Syracuse watch glasses and irradiated for 0 to 90 minutes at 8 C with a Sylvania germicidal lamp (G8T5) placed at a distance of 14 cm from the fluid. The watch glasses were agitated at 10-minute intervals. Control preparations were incubated at 8 C for 90 minutes as described, but without irradiation. Formalin.-Samples (0.4 ml) of two plasma pools were mixed with 0.1 ml of a 1 : 500 formalin solution to give a final concentration of 1 : 2500 formalin, then incubated at 4 C and ph 7.2 for o to 72 hours. For controls, 0.4 ml samples were incubated with 0.1 ml of phosphate-buffered saline (PBS), ph 7.2, for 72 hours at 4 C. Remagglutination.-Tests for hemagglutination were run in Kahn tubes by use of serial twofold dilutions of infected plasma (unheated and heated at 56 C for 30 min) in PBS. Each tube, containing 0.2 ml of the diluted plasma, received 0.2 ml of a 1 percent suspension of washed red blood cells. The tests were carried out in the cold (4 C) and at room temperature (25 C); readings were made after 2 to 3 hours by the pattern method. Virus titration.-serial tenfold dilutions of test materials were prepared in cold PBS and inoculated intraperitoneally (0.1 ml/mouse) into normal animals. Blood samples were obtained by tail bleeding 1 week after inoculation; serum LDH activities were measured as described elsewhere (8). Infective titers were calculated by the method of Reed and Muench (9). RESULTS Tests for Inactivation by Proteases As shown in table 1, infective titers in test materials treated with papain or trypsin were similar to those in control preparations. As such, our data provide no evidence for inactivation of the LDH agent by proteolytic enzymes. TABLE l,-tests for inactivation of the lactjic dehydrogenase agent by proteolytic enzymes * Treatment (mg/ml for 1 hr at 37 C) Expt. 1 Expt.2 Papain Trypsin Controlt Titers expressed as reciprocals. tincubated without pro teases at 37 C for 1 hour. Ether Sensitivity The observation that there was a complete loss of infectivity in preparations incubated with 20 percent diethyl ether at 4 C for 24 hours (table 2) suggests that the LDH agent is ether labile. TABLE 2.-8ensitivity of the lactic dehydrogenase agent to diethyl ether and sodium deoxycholate (DOC) Treatment Expt.1 Ether 20 percent for 24 hours at 4 C <1.0 Control 6. 7 DOC 1 : 1000 for 1 hour at 37 C 3. 5 Controlt 6.5 -Incubated without ether at 4 C for 24 hours. tincubated without DOe at 37 C for 1 hour. T ab for Inactivation by DOe Expt.2 < Sensitivity of the LDH agent to DOC is indicated by the finding that there was a significant loss of titer, i.e., 1 logarithm or greater, after incubation for 1 hour at 37 C with this chemical in a dilution of 1: 1000 (table 2). Heat Lability The results of this experiment, summarized in table 3, show that the LDH agent is labile to JOURNAL OF THE NATIONAL CANCER INSTITUTE

3 LACTIC DEHYDROGENASE VIRUS PROPERTIES 977 heating at 56 C for M-hour. Incubation for 30 minutes at 60 C resulted in an almost complete loss of infectivity. As reported previously (70), heat inactivation of this virus is enhanced by magnesium ions (table 3). Acid Lability The data in table 4 show that virus titers were reduced significantly (3-5 log) after treatment at ph 3.0 for 2 hours. This observation suggests that the LDH agent is acid labile. Effects of Ultraviolet Irradiation The titers of the LDH agent decreased with an increase in the time at which samples were exposed to ultraviolet irradiation (table 5). Complete inactivation of this virus was found after irradiation for 90 minutes. Formalin Inactivation The results of this experiment (table 6) indicate a continual decrease in infectivity with time up to 12 hours of incubation at 4 C with 1: 2500 formalin. Thereafter, infective titers remained essentially the same, approximately 2.5 logarithms less than the original virus titer. TABLE 3.-Tests for heat lability of the lactic dehydrogenase agent Incubation Temperature period (0 C) (min) * Controlt Incubated with M MgClt. tnot beated. Expt.l Expt < TABLE 4.-Tests for acid lability of the lactic dehydrogenase agent Plasma pool lot E G H VOL. 35, NO. 6, DECEMBER 1965 ph <2.Q 2. 5 ph TABLE 5.-Tests for inactivation of the lactic dehydrogenase agent by ultraviolet irradiation ID 50/ml (log 10) Time (min) Expt. 1 Expt <1.0 <1.0 Control* Incubated 8t 8 C for 90 minutes without ultraviolet irradiation. TABLE 6.-Tests for inactivation of the lactic dehydrogenase agent by formalin Time (hr) Expt. 1 Expt Control* Incubated with phosphate-burrered solution at 4 0 for 72 hours. Tests for Hemagglutination No evidence of agglutination of chicken, sheep, rabbit, guinea pig, mouse, or human 0 erythrocytes by the LDH agent was found in hemagglutination tests conducted at 4 and 25 C. DISCUSSION The LDH agent is an unclassified virus, indigenous to mice, which has not yet been shown to be pa thogeni c (71). Although changes in the levels of various serum and plasma enzymes have been reported in infected animals (12~ 14), diagnosis of infection depends on the demonstration of a permanent elevation in blood LDH activity (1, 3, 5). Data obtained in previous studies with the techniques of filtration (3, 15) and electron microscopy (16) indicate that this virus ranges from 40 to 60 mjj. More recently, electron microscope investigations in this laboratory have shown oval particles, approximately 15 X 45 mjj, in thin sections of pellets prepared from infected mouse plasma (17). The observations that incubation

4 978 CRISPENS of extracts, obtained by treatment of this virus with phenol-ether (78) or ether (79), with ribonuclease results in a complete loss of infectivity suggest the LDH agent is a ribonucleic acid (RNA) virus. Confirmation of two other properties, ether sensitivity (3) and heat lability (3, 4), is provided by results obtained in the studies herein described. Like the LDH agent, pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), and lymphocytic choriomeningitis (LCM) are unclassified murine viruses of medium size, RNA type, heat labile, and with an essential lipid (71). Except for PVM, which agglutinates mouse and hamster erythrocytes, no agglutination of red blood cells from different species has been observed in tests with these viruses [(17) present data]. Cultivation in mouse-embryo tissue culture has been reported for each, 3 but no cytopathic effect was produced (71, 21). The many properties shared by LCM, MHV, PVM, and the LDH agent suggest that they may belong in a single group of viral agents as yet unnamed. 4 However, additional information is needed to evaluate this hypothesis more adequately. Specifically, although evidence suggests that LCM (22) and the LDH agent (present data) are acid labile, data on the stability or lability of PVM and MHV at ph 3.0 are not yet available. Further, there is little information on the symmetry of these viruses. The observation that inactivation of the LDH agent, a virus of RNA type, is enhanced by M MgCl 2 at 50 suggests the possibility of helical symmetry (23). This suggestion must, however, be regarded as tentative. The data obtained in the study of formalin inactivation are of interest since they indicate that incubation with this chemical resulted in a continual decrease in infective titer up to 12 hours, but no significant decrease in titer thereafter up to 72 hours. Although several interpretations are possible, it is suggested that treatment with formalin may represent yet another method for differentiating between the two particles recently reported in the plasma of mice infected with the LDH agent (70, 24, 25). REFERENCES (1) RILEY, V., LILLY, F., HUERTo, E., and BARDELL, D.: Transmissible agent associated with 26 types of experimental mouse neoplaslll5. Science 132: ,1960. (2) GEORGII, A., KROTH, H., and BAYERLE, H.: Weitere Untersuchungen iiber die Aktivierung der Lactatdehydrogenase im Serum durch ein Geschwulstvirus der MallS. Klin Wschr 40: 363, (3) NOTKINS, A. L., and SHOCHAT, S. J.: Studies on the multiplication and the properties of the lactic dehydrogenase agent. J Exp Med 117: , (4) RILEY, V.: Enzymatic determination of transmissible replicating factors associated with mouse tumors. Ann NY Acad Sci 100: ,1963. (5) CRISPENS, C. G., JR.: On the epizootiology of the lactic dehydrogenase agent. J Nat Cancer Inst 32: , (6) ANDREWES, C. H., and HORSTMANN, D. M.: The susceptibility of viruses to ethyl ether. J Gen MicrobioI 3: , (7) THEILER, M.: Action of sodium desoxycholate on arthropod-borne viruses. Proc Soc Exp Bioi Med 96: , (8) CRISPENS, C. G., JR.: On the transmission of the lactic dehydrogenase agent from mother to offspring. J Nat Cancer Inst 34: , (9) REED, L. J., and MUENCH, H.: A simple method of estimating fifty per cent endpoints. Amer J Hyg 27: ,1938. (10) CRISPENS, C. G., JR.: Mouse plasma lactic dehydrogenase elevation: evidence for two particles. Virology 24: , (11) ANDREWES, C. H.: Viruses of Vertebrates. Baltimore, The Williams & Wilkins Co., 1964, 401 pp. (12) PLAGEMANN, P. G. W., WATANABE, M., and SWIM, H. E.: Plasma lactic dehydrogenase-elevating agent of mice: effect on levels of additional enzymes. Froc Soc Exp BioI Med 111: , (13) NOTKINS, A. L., GREENFIELD, R. E., MARSHALL, D., and BANE, L.: Multiple enzyme changes in the plasma of normal and tumor-bearing mice following infection with the lactic dehydrogenase agent. J Exp Med 117: ,1963. (14) MAHY, B. W. J., RowsoN, K. E. K., SALAMAN, M. H., and PARR, C. W.: Plasma enzyme levels in virusinfected mice. Virology 23: ,1964. (15) ROWSON, K. E. K., MAHY, B. W. J., and SALAMAN, M. H.: Size estimation by filtration of the enzymeelevating virus of Riley. Life Sci 2: , Results obtained in more recent studies suggest that the LDH agent can be maintained, but not propagated, in cultures of mouse embryo cells (20). Definitive experiments on the in vitro cultivation of this virus appear needed to resolve this problem. During the final preparation of this manuscript, a similar proposal was made by Dr. Raymond W. Tennant at a symposium on "Viruses of Laboratory Rodents" held in Atlanta, Ga. This symposium, which includes Dr. Tennant's paper "Taxonomy of Murine Viruses" will be published as a National Cancer Institute Monograph. JOURNAL OF THE NATIONAL CANCER INSTITUTE

5 LACTIC DEHYDROGENASE VIRUS PROPERTIES 979 (16) BLADEN, H. A., JR., and NOTIGNS, A. L.: Electron microscopic demonstration of the lactic dehydrogenase agent. Virology 21: , (17) CRISPENS, C. G., JR., and BURNS, T. A.: Electron microscope investigation of lactic dehydrogenase agent. Nature (London) 204: 1302, (18) NOTKINS, A. L., and SCHEELE, c.: An infectious nucleic acid from the lactic dehydrogenase agent. Virology 20: , (19) N OTKINS, A. L.: Recovery of an infectious ribonucleic acid from the lactic dehydrogenase agent by treatment with ether. Virology 22: , (20) GEORGII, A, and LENz, 1.: Failure to propagate a lactic dehydrogenase-elevating agent from mice tumors in mice embryo cultures. Nature (London) 202: , (21) YAFFE, D.: The distribution and in vitro propagation of an agent causing high plasma lactic dehydrogenase activity. Cancer Res 22: , (22) HAMPARIAN, V. V., HILLEMAN, M. R., and KETLER, A: Contributions to characterization and classification of animal viruses. Proc Soc Exp BioI Med 112: ,1963. (23) MELNICK, J. L.: Enteroviruses. Ann NY Acad Sci 101: ,1962. (24) RlLEY, V., CAMPBELL, H. A, LOVELESS, J. D., and FITZMAURICE, M. A: Density gradient centrifugation and molecular sieve studies of lactic dehydrogenase-elevating virus-like agents. Proc Amer Assoc Cancer Res 5: 53, (25) AnAMs, D. H., and BOWMAN, B. M.: Studies on the properties of factors elevating the activity of mouseplasma lactate dehydrogenase. Biochem J 90: ,1964. VOL. 35, NO. 6, DECEMBER 1965