Cancer Letters, 16 (1982) 117-120 Elsevier Scientific Publishers Ireland Ltd.

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ACTIVATION OF EPSTEIN-BARR VIRUS EXPRESSION IN HUMAN LYMPHOBLASTOID P3HR-1 AND RAJI CELLS WITH PROPIONIC ACID AND WITH CULTURE FLUIDS OF PROPIONIC ACID-PRODUCING ANAEROBES

MASAMICHI KISHISHITA, SUGIO YANASE and YOHEI IT0

Deparment of Microbiology, Faculty of Medicine, Kyoto University, Kyoto 606 (Japan)

(Received 15 April 1982) (Accepted 15 April 1982)

SUMMARY

Epstein-Barr virus (EBV)-associated early (EA) and virus capsid antigens (VCA) were efficiently induced in the viral genomecarrying human lympho- blastoid cells, PSHR-1 and Raji, by the culture fluids of Propionibacterium acnes, P. avidum, P. lymphophijum and Arachnia propionica, the anaerobes which are commonly seen among the normal flora of man. The active principle for EBV-induction in the 2 cell lines was the propionic acid pro- duced by the microbes and such activity was shown to correlate with the fatty acid content of the culture media.

INTRODUCTION

It is well-established that n-butyrate effectively activates the latently persisting EBV in human lymphoblastoid cells in vitro [ES]. During our search for such EBV-activating principles in the human environment, we found that the culture fluids of Fusobacterium nucleatum and other anae- robes which contain an abundance of butyric acid also activate EBV expres- sion [3].

In this paper, we report that the EBV expression can also be activated in EBV genome-carrying cells, P3HR-1 and Raji, with culture fluids of 4 species of propionic acid-producing microbes, Propionibacterium acnes, P. avidum, P. lymphophilum and Arachnia propionica, commonly isolated from mouth, skin and intestinal tract of man [ 7,9].

MATERIALS AND METHODS

The strain of P. lymphophilum used was isolated from clinical material and the others were all standard strains obtained from the American Type

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Culture Collection and the Virginia Polytechnic Institute. They were inocu- lated in 25 ml of broth medium (trypticase, 1.5%; heart extract, 0.5%; yeast extract, 0.3%; NaCl, 0.2%; Na,HP04, 0.2%; KHZP04, 0.2%; glucose, 0.5%; sodium pyruvate, 0.5%; L-cysteine HCl, 0.03%; hemin-vitamin K3 solution, 1.0%; Tween 80,0.025%; pH 7.0) and cultivated at 37°C for 5 days by the steel wool method anaerobically (C0,/N2, 10 : 90%). The culture fluid was then passed through a Seitz bacterial filter and was used as samples for induction trials of EBV-associated antigens. Uninoculated broth medium treated in the same way was used as the control. The fatty acids in the culture fluid were assayed by conventional procedure using a Hitachi Model 163 gas chromatograph [ 21.

For EBV antigens induction trials, the microbial culture fluids and the uninoculated control medium were diluted 5- or lo-fold with RPMI-1640 tissue culture medium supplemented with 5% fetal calf serum and the reagent propionic acid (Nakarai Chemicals, Co., Kyoto) was dissolved in ethanol as 0.5 M stock solution.

RESULTS AND DISCUSSION

The effects of 4 culture fluids on activation of EBV expression in P3HR-1 cells are summarized in Table 1. After 48 h, 20-40% of the cells were EA positive and 15-20% of the cells were VCA positive as assayed by indirect immunofluorescence technique [ 11. With regard to the results of analysis of propionic acid concentration in the culture fluid, the EBV inducing

TABLE 1

ACTIVATION OF EBV EXPRESSION IN PIHR-1 CELLS WITH CULTURE FLUIDS OF PROPIONIC ACID-PRODUCING ANAEROBES

Culturea Dilutionb

P. acmes ATCC 6919 x5 P. avidum ATCC 25577 x10 P. lymphophilum 1044 x5 A. propionica VP1 0026 x 10 Control x5

Cont. of % EBV antigen-positive celisd propionic acid (mM)’ EA VCA

11.0 25.8 18.8 7.5 23.2 15.0

12.2 38.2 19.6 6.0 31.3 20.3 0 0.2-1.0

aUninocuiated broth medium was tested as the control. bDilution of bacterial culture fluids and broth medium. ‘Concentration of propionic acid analyzed by gas chromatography. d106 cells/ml were incubated with culture fluid. After 48 h, the positive cells of EBV-

associated antigens were assayed by the method described by Henle and Henle [ 11. Staining was performed with EA (+ ) VCA (+ ) sera (titer 1 : 2560,l : 1280) from nasopharyngeal carcinoma patients or EA (-) VCA (+) serum (titer 1 : 640) from a normal person at a dilution 1 : 40.

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TABLE 2

ACTIVATION OF EBV EXPRESSION IN TWO HUMAN LYMPHOBLASTOID CELL LINES WITH THE REAGENT PROPIONIC ACID

Cell line Cont. of % EBV antigen-positive cell8 propionic acid (mM) EA VCA

P3HR-1 50 30.3 18.6 10 27 .O 15.3

5 5.3 4.3 0 0.8 0.7

Raji 56 46.5 -.

10 52.0 -.

5 4.6 -.

0 1.8 -.

a106 cells/ml of P3HR-1 were incubated with the indicated concentration of the reagent. 10’ cells/ml of Raji were incubated with the reagent in combination with the final concentration 5 pglml of croton oil. After 48 h, the positive cells of EBV antigens were assayed as same protocol mentioned in Table 1.

capacity seems to correlate with its fatty acid content. Therefore, the effects of propionic acid per se were assayed for the capacity to induce EBV- associated antigens in 2 human lymphoblastoid cell lines (Table 2). The fatty acid showed the EBV inducing activity of P3HR-1 cells at the concen- tration higher than 10 m&l, in non-producer Raji cells, the combined use with croton oil enhanced induction of EA as well as n-butyrate [ 41.

Previous reports from our laboratory have shown that not only n-butyric acid but also iso- and n-valeric acid exert EBV-induc~g activity in P3HR-2 cells [ 61. Under theconditions used, such capacity of propionic acid was little or marginal, probably due to the low concentration used. The EBV-inducing capacity of propionic acid in non-producer Raji cells when used in com- bination with the tumor promoting agent, croton oil, is interesting from the point of view of skin carcinogenesis. We have recently shown that con- siderable overlapping is seen between the agents of EBV induction and those of the tumor promoters [5], The possible role of the propionic acid-pro- ducing microbes of normal cutaneous flora, P. ~crtes in particular, in the neoplastic process, has to be reconciled by further studies.

REFERENCES

Henle, G. and Henle, W. (1966) Immunofluorescence in cells derived from Burkitt’s lymphoma. J. Bacterial., 91,1248-1256. Holdernan, L.V., Cato, E.P. and Moore, W.E.C. (19’77) Anaerobe Laboratory Manual, 4th edn. Virginia Polytechnic Institute and State University, Blacksburg, VA. Ito, Y., Kish~hita, M. and Yanase, S. (1980) Induction of Epstein-Barr virus antigens in human lymphoblsstoid PIHR-1 cells with culture fluid of Fusobucferium nucleafum. Cancer Res., 46,4329-4330.

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4 Ito, Y., Kawanishi, M.;Harayama, T. and Takabayashi, S. (1981) Combined effect of the extracts from Croton tiglium, Euphorbia lathyris, or Euphorbia tirucalli and n-butyrate on Epstein-Barr virus expression in human lymphoblastoid PSHR-1 and Raji cells, Cancer Letters, 12,175-180.

6 Ito, Y., Yanase, S., Fujita, J., Harayama, T., Takashima, M. and Imanaka, H. (1981) A short-term in vitro assay for promoter substances using human lymphob~stoid cells latently infected with Epstein-Barr virus. Cancer Letters, 13,29-37.

6 Kawanishi, M. and Ito. Y. (1980) Effect of short-chain fatty acids on Epstein-Barr virus early and viral capsid antigen induction in P3HR-1 cells. Cancer Letters, 11, 129-132.

7 Kishishita, M., Ushiiima, ‘I’,, Gzaki, Y. and Ito, Y. (1980) New medium for isolating propionibacteria and its application to assay of normal flora of human facial skin. Appl. Environ. Microbial., 40, 1100-1105.

8 Luka, J., Kallin, B. and Klein, G. (1979) Induction of the Epstein-Barr virus (EBV) cycle in latently infected cells by n-butyrate. Virology, 94,228-231.

9 Moore, W.E.C., Holdeman, L-V., Fine, L. and Georg, K. (1974) Genus I. Propioni- bacterium Orla-Jensen 1909, 337, pp. 633-641, and Genus II. Arachnia Pine and Georg 1969,269, pp. 668-69. In: Bergey’s Manual of ~terminative Bacteriology, 8th edn. Editors: R.E. Buchanan and N.E. Gibbons, The Williams & Wilkins Co., Baltiiore.