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Human lymphocyte response to d -valine media
635 Biochimica et Biophysica Acta, 451 (1976) 635--637 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 21436
HUMAN LYMPHOCYTE RESPONSE TO D-VALINE MEDIA
PAUL J. BENKE and DAVID DITTMAR Mailman Center and University of Miami School of Medicine, Miami, Fla. 33152 (U.S.A.) (Received September 2nd, 1976)
Summary Failure of human lymphoid cell lines to grow in D-valine-substituted media is associated with the lack of D-amino acid oxidase activity in these cells.
Circulating human lymphocytes are like cultured fibroblasts and unlike peripheral leukocytes and cultured epithelial cells because they lack enzymic D-amino acid oxidase activity (EC 1.4.3.3) and are unable to convert D-amino acid isomers to their biologically active L-forms [1, 2]. Gilbert and Midgeon [2] have used this to advantage in selecting for epithelial cells in culture against fibroblasts in explants from kidney slices. We report that this system also selects against stimulation of peripheral human lymphocytes and growth of cell lines of human lymphocytes in vitro. Human lymphocytes cell lines were obtained by adding EB virus to isolated peripheral lymphocytes [3]. They were grown in 15% heat-inactivated fetal calf serum and RPMI 1640 media (Grand Island Biological Co., New York, N.Y.). Fetal calf serum was dialyzed for 24 h against phosphate-buffered saline and filtered through a Millipore 22 pm filter before experiments. Thymidine incorporation was used as a measure of the response to phytohemagglutination in Hypaque-Ficoll-isolated lymphocytes [4]. D-Amino acid oxidase activity was assayed by a modification of a spectrophotometric technique which employs oxyhemaglobin as an O2 donor and absorbance indicator [ 5]. The assay mixture contained 9.2 mM pyrophosphate buffer, pH 8.3, 18 mM FAD, 92.0 mM D-alanine and 0.03 mM purified oxyhemaglobin prepared according to the method of Hultborn [6]. The response of isolated peripheral lymphocytes to phytohemagglutination. in D-valine-substituted media depended on the source of the D-valine as shown in Table I. [a H]Thymidine incorporation in D-valine media was 6% of that found in L-valine media utilizing D-valine with an optical rotation o f - 2 6 . 5 ° (ideal -26.7°). Thymidine incorporation was three-fold higher in D-valine from a second source with an optical rotation of -25.6 °. Presumably this source has
636
TABLE I E F F E C T O F M E D I A W I T H D - V A L I N E S U B S T I T U T E D F O R L - V A L I N E ON [ 3 H ] T H Y M I D I N E I N C O R P O R A T I O N IN P H Y T O H E M A G G L U T I N I N - S T I M U L A T E D L Y M P H O C Y T E S
Theoretical [ a ] D vaiine is - 2 6 . 7 °. Experiment
N u m b e r of experiments
Percent thymidine i n c o r p o r a t i o n c o m p a r e d t o control
[ ~ ] D vaiine
x 100 L-vaiLne
( M e a n +- S.D.) 1 2
6 3
6 +- 2 3 9 +- 1 4
-26.5 ° -25.6 °
more L-valine contamination. It can be seen in Fig. 1 that lymphocytes do not grow in tissue culture media lacking L-valine or with D-valine substituted for L-valine. Using a more sensitive fluorimetric assay, we have confirmed the results of Cline and Lehrer [1] which suggest that isolated peripheral lymphocytes do not demonstrate D-amino acid oxidase activity. We find that lymphocyte cell lines in tissue culture also do not demonstrate D-amino acid oxidase activity. The failure of humm~ lymphocytes to respond to phytohemagglutination in D-valine-substituted media and the failure of human l y m p h o c y t e cell lines to grow in D-valine-substituted media is related to their lack of D-amino acid oxidase activity. These findings have two practical applications. First, it may be possible to select against the lymphocyte donor strain in somatic cell hybridization experiments. This might be useful when one parent cell is a l y m p h o c y t e cell line, lacking D-amino acid oxidase, and the second parent type is an epithelial or hepatic cell strain with the enzyme. If the second cell strain lacks hypoxanthine-guanine phosphoriboryltransferase activity, double selection with D-valine and 8-azaguanine could be employed. Second,
20t-
!:i J// 2~ii:~i:i~i
,
........................................:.:.. ...........: ...............
/ DAYS Fig. 1. G r o w t h of l y m p h o c y t e cell lines in v a r i o u s m e d i a . L y m p h o c y t e s w e r e divided into flasks c o n t a i n ing RPM1 1 6 4 0 m e d i a l (e----4) and R P M I 1 6 4 0 l a c k i n g L-vaiine ( s - - -m) or w i t h D-valine s u b s t i t u t e d for L-vallne (A. . . . a). Cells w e r e c o u n t e d daily in a C o u l t e r C o u n t e r .
637 strenuous limitation of one or more L-amino acids and replacement with the D-isomer in the diet may select against cancer or leukemia cells which lack D-amino acid oxidase. This is analogous to the asparaginase selection [7] where leukemia cells lack the enzymic capacity to synthesize an amino acid essential for leukemia cell growth. This research was supported b y an American Cancer Society Institutional Grant (IN-SIP) and a Basil O'Connor grant from the National Foundation March of Dimes. References 1 2 3 4 5 6 7
Cline, M.J. and Lehrer, R.I. (1969) Proc. Natl. Acad. Sci. U.S. 62, 756--763 Gilbert, S.F. and Midgeon, B.R. (1975) Cell 5, 11--17 Steel, C.M. (1972) J. Natl. Cancer Inst. 48, 6 2 3 - - 6 2 8 Bain, B. and Pshyk, P. (1972) Transplant. Proc. 4, 163--164 Barzu, O. and Borza, V. (1967) Anal. Biochem. 2 1 , 3 4 4 - - 3 5 7 Hultborn, R. (1972) Anal. Biochem. 47, 442--450 Tall~l, I~., Tan, C. and Oettger, H. (1970) Cancer 25, 306--320
BBA Report BBA 21436
HUMAN LYMPHOCYTE RESPONSE TO D-VALINE MEDIA
PAUL J. BENKE and DAVID DITTMAR Mailman Center and University of Miami School of Medicine, Miami, Fla. 33152 (U.S.A.) (Received September 2nd, 1976)
Summary Failure of human lymphoid cell lines to grow in D-valine-substituted media is associated with the lack of D-amino acid oxidase activity in these cells.
Circulating human lymphocytes are like cultured fibroblasts and unlike peripheral leukocytes and cultured epithelial cells because they lack enzymic D-amino acid oxidase activity (EC 1.4.3.3) and are unable to convert D-amino acid isomers to their biologically active L-forms [1, 2]. Gilbert and Midgeon [2] have used this to advantage in selecting for epithelial cells in culture against fibroblasts in explants from kidney slices. We report that this system also selects against stimulation of peripheral human lymphocytes and growth of cell lines of human lymphocytes in vitro. Human lymphocytes cell lines were obtained by adding EB virus to isolated peripheral lymphocytes [3]. They were grown in 15% heat-inactivated fetal calf serum and RPMI 1640 media (Grand Island Biological Co., New York, N.Y.). Fetal calf serum was dialyzed for 24 h against phosphate-buffered saline and filtered through a Millipore 22 pm filter before experiments. Thymidine incorporation was used as a measure of the response to phytohemagglutination in Hypaque-Ficoll-isolated lymphocytes [4]. D-Amino acid oxidase activity was assayed by a modification of a spectrophotometric technique which employs oxyhemaglobin as an O2 donor and absorbance indicator [ 5]. The assay mixture contained 9.2 mM pyrophosphate buffer, pH 8.3, 18 mM FAD, 92.0 mM D-alanine and 0.03 mM purified oxyhemaglobin prepared according to the method of Hultborn [6]. The response of isolated peripheral lymphocytes to phytohemagglutination. in D-valine-substituted media depended on the source of the D-valine as shown in Table I. [a H]Thymidine incorporation in D-valine media was 6% of that found in L-valine media utilizing D-valine with an optical rotation o f - 2 6 . 5 ° (ideal -26.7°). Thymidine incorporation was three-fold higher in D-valine from a second source with an optical rotation of -25.6 °. Presumably this source has
636
TABLE I E F F E C T O F M E D I A W I T H D - V A L I N E S U B S T I T U T E D F O R L - V A L I N E ON [ 3 H ] T H Y M I D I N E I N C O R P O R A T I O N IN P H Y T O H E M A G G L U T I N I N - S T I M U L A T E D L Y M P H O C Y T E S
Theoretical [ a ] D vaiine is - 2 6 . 7 °. Experiment
N u m b e r of experiments
Percent thymidine i n c o r p o r a t i o n c o m p a r e d t o control
[ ~ ] D vaiine
x 100 L-vaiLne
( M e a n +- S.D.) 1 2
6 3
6 +- 2 3 9 +- 1 4
-26.5 ° -25.6 °
more L-valine contamination. It can be seen in Fig. 1 that lymphocytes do not grow in tissue culture media lacking L-valine or with D-valine substituted for L-valine. Using a more sensitive fluorimetric assay, we have confirmed the results of Cline and Lehrer [1] which suggest that isolated peripheral lymphocytes do not demonstrate D-amino acid oxidase activity. We find that lymphocyte cell lines in tissue culture also do not demonstrate D-amino acid oxidase activity. The failure of humm~ lymphocytes to respond to phytohemagglutination in D-valine-substituted media and the failure of human l y m p h o c y t e cell lines to grow in D-valine-substituted media is related to their lack of D-amino acid oxidase activity. These findings have two practical applications. First, it may be possible to select against the lymphocyte donor strain in somatic cell hybridization experiments. This might be useful when one parent cell is a l y m p h o c y t e cell line, lacking D-amino acid oxidase, and the second parent type is an epithelial or hepatic cell strain with the enzyme. If the second cell strain lacks hypoxanthine-guanine phosphoriboryltransferase activity, double selection with D-valine and 8-azaguanine could be employed. Second,
20t-
!:i J// 2~ii:~i:i~i
,
........................................:.:.. ...........: ...............
/ DAYS Fig. 1. G r o w t h of l y m p h o c y t e cell lines in v a r i o u s m e d i a . L y m p h o c y t e s w e r e divided into flasks c o n t a i n ing RPM1 1 6 4 0 m e d i a l (e----4) and R P M I 1 6 4 0 l a c k i n g L-vaiine ( s - - -m) or w i t h D-valine s u b s t i t u t e d for L-vallne (A. . . . a). Cells w e r e c o u n t e d daily in a C o u l t e r C o u n t e r .
637 strenuous limitation of one or more L-amino acids and replacement with the D-isomer in the diet may select against cancer or leukemia cells which lack D-amino acid oxidase. This is analogous to the asparaginase selection [7] where leukemia cells lack the enzymic capacity to synthesize an amino acid essential for leukemia cell growth. This research was supported b y an American Cancer Society Institutional Grant (IN-SIP) and a Basil O'Connor grant from the National Foundation March of Dimes. References 1 2 3 4 5 6 7
Cline, M.J. and Lehrer, R.I. (1969) Proc. Natl. Acad. Sci. U.S. 62, 756--763 Gilbert, S.F. and Midgeon, B.R. (1975) Cell 5, 11--17 Steel, C.M. (1972) J. Natl. Cancer Inst. 48, 6 2 3 - - 6 2 8 Bain, B. and Pshyk, P. (1972) Transplant. Proc. 4, 163--164 Barzu, O. and Borza, V. (1967) Anal. Biochem. 2 1 , 3 4 4 - - 3 5 7 Hultborn, R. (1972) Anal. Biochem. 47, 442--450 Tall~l, I~., Tan, C. and Oettger, H. (1970) Cancer 25, 306--320