Journal of Ncurochernisrr,v. 1977. Vol. 29. pp. 83-K6 Pcrgamon Press. Printed in Great Britain.
ELEVATED INTRACELLULAR GLYCINE ASSOCIATED WITH HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE DEFICIENCY IN GLIOMA CELLS'
s. D. SKAPER' and J. E. SEEGMILLER3 Department of Medicine, University of California, San Diego, La Jolla. CA 92093, U.S.A. (Receioed 22 October 1976. Accepted 28 January 1977)
Abstract---The intracellular concentrations of a number of amino acids were measured in a normal clone of rat glioma cells, and in several independently derived clones selected for gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). A significant, approx 2-fold increase in the concentration of free glycine was observed in both mutagenized and non-mutagenized HGPRT deficient clones. The increase in glycine was independent of the phase of cell growth. A similar increase did not occur in HGPRT deficient lymphoblasts.
THELesch-Nyhan syndrome (LESCH& NYHAN, 1964) is a rare X-linked recessive form of cerebral palsy, which is characterized by hyperuricemia, marked overproduction of uric acid. and severe neurological dysfunction which includes a compulsive self-mutilation, spasticity, chorcoathetosis, and mental retardation. A virtually complete deficiency of the purine reutilization enzyme hypoxanthine-guanine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.8; HGPRT) (SEEGMILLERet a/., 1967) represents the primary biochemical defect in these patients. Subjects with a partial HGPRT deficiency exhibit hypcruricemja, excessive production of and a tendency to develop acid, gouty 1967, calculi of uric acid (KELLEY et with1969)'occasional mild neurologic may Occur in this latter group Of Out patients (KELLEYrt al., 1967, 1969). The b*~&emicalsequence responsible for the mwrologic and behavioral manifestations present in the Lesch-Nyhan patients remains unknown. Recently we reported an increased intracellular concentration of the putative transmitter amino acid glycine in several HGPRT deficient clones of neuroblastoma cells (SUPER & SEEGMILLER, 1976a). A similar Of phenomenon could not be demonstrated in non-neural origin. Since these studies were carried out using only one type of cultured neural cell, the
bility could not be ruled out that the observations were characteristic of a specific cell type. Our studies have now been extended to include another cell type of neural origin, namely, glioma cells. A significant increase in the intracellular glycine concentration was observed in the several HGPRT deficient clones tested, thus supporting the neural-specific nature of this phenomenon. MATERIALS AND METHODS
Marerials. Dulbecco's modified Eagle's medium was obtained from Grand Island Biological (Grand Island, NY) and fetal calf serum from Irvine Scientific Sales (Irvine, CA). All other chemicals were of the highest purity obtainable from commercial sources. Cell culture, The rat glioma clone (3 (BEND*et a/., 1968) and HGPRT deficient clones selected from ethyl methanesulfonate mutagenized (Cb16) and non-mutagenized (TgRCI,) C6 cells showed less than 1% of normal activity 1976b). and have been described (SKAPER& SEEGMILLER, Cells were grown at 37°C in monolayer culture in sealed plastic culture flasks (Falcon, Oxnard, CA) purged with 10% CO,-W"/, air, in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 m ~ - ~ - g l u t a mine (SUPER & SEEGMILLER. 1976a). All cultures tested negative for Mycoplasma contamination (LEVINE, 1972). Lymphoblast lines were established from peripheral blood samples obtained from a normal adult male and a Lesch-Nyhan patient (CHOI& BLOOM,1970) and cultured as described previously (SKAPERet al., 19764. ' This work was supported in part by grants AM-13622, Amino acid analysis. Glial cells were grown and harAM-05646, and GM-17702 from the United States Public vested as described for neuroblastoma (SKAPER& SEEGHealth Service, Veterans Administration grant M.R.I.S. MILLER, 1976a). Culture medium was replaced with fresh No. 3181 to Dr. Nathan Gochman, and grants from the medium 24h before harvesting. Cold acid-soluble amino acids were extracted from the pelleted cells (2-4 x lo6 National Foundation and the Kroc Foundation. Present Address: Department of Pediatrics, Case Wes- cells) using a slight modification of an earlier method & SEEGMILLER, 1976a). Cells were lysed by resustern Reserve University, Cleveland Metropolitan General (SKAPER pending the cell pellet directly in 0.4 N perchloric acid Hospital, Cleveland, OH 44109, U.S.A. (freeze-thaw step omitted). After 10 min at 5°C. the acid To whom reprint requests should be directed. Abbreliiation used: HGPRT, hypoxanthine-guanine was neutralized with KOH and the KCIO4 precipitate and denatured protein were removed by centrifugation. phosphoribosyltransferase.
'
83
S. D. SKAPER and J. E.
84
TABLE1,
SEEC;MII.L~R
~NTRACEI.I.l;IAR C0NCI:NTRATION OF SELECTED AMlh’O ACIDS IN OLIOMA CELLS AT EARLY LOG PHASE CROWTll
Cell line ( m u ) HGPRT deficient ~~
Amino acid
Wild type (C6) (n = 8)
Spontaneous (Tg RCI 2 1 (n = 6)
Mutagenized (C616) (n = 6)
Taurine Aspartic acid Clutamic acid Clutamine Glycine Serine Alanine Tyrosine Phenylalanine
13.0 f 0.7 5.7 f 0.7 22.2 f 0.7 24.1 f 2.0 10.2 f 0.6 3.5 f 0.2 1.9 f 0.8 0.7 f 0.1 1.2 f 0.1
9.9 f 1.4 4.5 & 0.4 22.2 f 0.8 20.1 f 1.4 15.6 f 1.0* 2.5 f 0.2 6.4 f 0.4 0.5 f 0.1 0.8 f 0.1
17.6 f 1.2t 4.4 f 1.1 27.3 f 1.5 20.1 f 0.6 16.8 f 0.3. 2.4 f 0.3 8.0 f 0.2 0.8 0.1 1.1 f 0.2
Early log: 2 4 x lo4 cells/cm2; cell volume 1.62.0 pl/cell (amino acid concentrations calculated on the basis of 80”, cell water). Values given represent the mean of six or more determinations f 1 S.E.M. Significant difference from wild type: P < 0.05; t P < 0.02. Lymphoblasts in suspension culture were harvested and deficient clones with and without mutagenesis prior extracted for their amino acids as described earlier (SKAPERto selection; and (3) the elevation of glycine in ri a/., 1976). HGPRT deficient glioma is independent of the Amino acid analyses were performed on a Durrum growth stage of the cells. D-500 amino acid analyzer. The details of this procedure. In the early logarithmic phase of growth (Table 1) as well as for measurement of cell volumes. have been puban approximatc 1.7-fold increase in the free glycine & SEEGMILLER, 1976~). lished (SKAPER
RESULTS
The intracellular amino acid concentrations for normal C6 and HGPRT deficient (showing less than ;1: of normal activity) glioma clones are summarized in Tables 1 3. Several features are evident: (1) the concentration of the putative neurotransmitter glycine is significantly increased in cells deficient in HGPRT activity; (2) this increase is seen in HGPRT
concentration of the mutagenized HGPRTdeficient glioma clone C6-16 was observed, when compared to the parental clone C6 (P < 0.05). The difference increased to 1 .&fold (P < 0.02) for mid log phase cultures (Table 2). and to 2-fold (P < 0.03) for the confluent culture (Table 3). Among the other putative transmitter amino acids, only taurine showed a significant increase (P < 0.02) from the corresponding value in normal cells, and only in early log growth (Table 1). Serine, a possible precursor of glycine, never showed any consistent alterations in the mutant clone
TAHLI~ 2. INTRAC-ELLULARCONCNTRATION AT M I D I
OF SELECTED A M I N O ACIDS I N GI-IOMA CELLS X PHASE GROWlW
\
Cell line (mM) ~
HGPRT deficient Wild type Amino acid Taurine Aspartic acid Glutamic acid Glutamine Glycinc Scrine Alanine Tyrosine Phrnykddnine
(W
(n
=
8)
8.4 f 1.3 5.4 f 0.8 17.0f 1.6 16.2 f 2.9 9.0 f 0.4 2.7 f 0.4 9.0 2.0 1.1 f 0.1 1.1 f 0.2
Spontaneous (TgRCIJ (n = 6)
Mutagenized (C616) (n = 6)
6.6 f 0.8 3.8 f 0.3 16.1 f 1.2 13.9 f 1.2 15.9 f 0.4. 2.4 f 0.2 6.6 f 0.4 0.7 f-0.1 0.8 f 0.1
4.1 19.7 f 1.3 14.8 f 0.7 16.3 & 1.2* 4.1 f 0.3 8.7 0.8 1.0 0.2 1.4 f 0.1
*
Mid log: 6.5 8.0 x lo4 cells/cmz; cell volumc 1.9-2.0 pl/cell (amino acid concentrations calculated on the basis of 80% cell water). Values given represent the mean of 6 or more determinations f 1 S.E.M. Significant difference from wild type: * P < 0.02.
lntracellular glycine in mutant glioma cells TABLE 3.
85
INTRACELLULAR CONCENTRAIIOK OF SELECTII) AMINO ACIDS IN CONFLLENT CULTURES OF GLIOMA CELLS
Cell line (mM) HGPRT deficient ~~
Amino acid Taurine Aspartic acid Glutamic acid Glutamine Glycine Serine Alanine Tyrosine Phenylalanine
Wild type
Spontaneous
M utagenized
(C6)
(TgRC12)
(C6-16)
5.6 f 0.3 3.8 f 0.5 13.7 f 1.3 7.6 f 0.8 6.0 f 0.3 1.8 f 0.2 4.6 0.3 0.4 f 0.1 0.6 f 0.1
9.7 k 2.0 5.4 f 1.2 15.6 k 2.5 7.4 f 1.2 12.9 f 0.8* 2.5 f 0.3 5.3 f 1.5 0.3 f 0.1 0.6 f 0.1
9.2 k 0.4 3.7 f 0.4 17.7 k 0.3 6.2 f 0.2 12.4 f 0.6* 1.7 f 0.1 6.0 f 0.2 0.3 f 0.1 0.5 f 0.1
Confluent: 35-50 x lo4 cells/cm*; cell volume 0.9-1.1 pl/cell (amino acid concentrations calculated on the basis of 807, cell water). Values given represent the mean of 6 determinations f 1 SEW. Significant dilference from wild type: * P = 0.03. C6-16, although the lower value in early log cultures bordered on being statistically significant. The catecholamine precursors tyrosine and phenylalanine showed no significant variations. None of the other naturally occurring amino acids were increased or decreased in C 6 1 6 cells (not shown). Data for the spontaneous HGPRT deficient glial clone TgRClz were quite similar to the mutagenized clone C6-16. The concentration of free glycine was increased 1.6-fold ( P < 0.05) in early logarithmic growth phase (Table l), 1.8-fold ( P < 0.02) in mid log phase (Table 2) and 2.1-fold ( P < 0.03) in confluent cultures when compared to normal clone C6. No significant increases or decreases were observed for any other amino acids in TgRCI2 cells, with the possible exception of serine in early log cultures. The free intracellular amino acid concentrations measured in glioma cells were remarkably close to the values determined previously for neuroblastoma & SEEGMILLER, 1976a), at each growth cells (SKAPER stage. The high content of taurine in glioma was interesting, although not surprising as brain contains large amounts of taurine (PERRYet a/., 1971a,b).
Corresponding data for cultured lymphoblast lines established from the peripheral blood of a normal adult male (HGPRT+) and a Lesch--Nyhan patient (HGPRT-) are given in Table 4. The intracellular concentration of free glycine did not increase in either log phase or confluent cultures of these HGPRT deficient lymphoblasts. Similar data were obtained for clones of human splenic lymphoblasts selected for HGPRT deficiency from an established parental cell line, WI L2 (LEVERef a/., 1974), and for fibroblasts from Lesch-Nyhan patients (not shown). DISCUSSION The activity of HGPRT has been found markedly deficient in brain tissue from Lesch-Nyhan patients at autopsy (ROSENRLOOM ef a/., 1967; BOYLEet a/., 1970). Despite severe neurological impairment, no specific morphological or histological changes in the CNS of these patients have been observed. The role of HGPRT activity in normal neurological function remains to be defined. Recently we reported an increased concentration (2-fold) of free intracellular glycine associated with
TARLE 4. INTRACELLULAR
CONCEKTRATIOK OF SILI:CTED AMINO ACIDS I N EARLY LOG PHASli AND CONFI.lJC:NT CULTURES OF HUMAN LYYPHOBLASTS
Early log (mW
Confluent (mM)
Amino acid
HGPRT'
HGPRT-
HGPRT'
HGPRT-
Aspartic acid Glutamic acid Glutamine Glycine Serine Alanine Tyrosine Phenylalanine
2.9 f 0.2 19.1 f 0.1 15.5 f 0.9 3.6 f 0.1 1.2 f 0.1 6.2 f 0.1 0.5 f 0.1 0.5 f 0.1
4.0 f 0.4 18.6 f 1.2 15.6 f 1.2 3.4 f 0.2 0.7 f 0.1 5.2 f 0.5 0.5 f 0.1 0.4 f 0.1
1.4 f 0.1 14.3 f 0.4 12.0 f 0.4 3.6 0.1 0.3 f 0.1 4.7 f 0.5 0.7 f 0.1 0.7 f 0.1
1.5 f 0.1 12.9 f 0.4 13.2 f 0.2 2.6 f 0.1 0.4 f 0.1 4.5 f 0.2 0.5 f 0.1 0.5 f 0.1
Early log: 1-4 x lo5 cellsjml; cell volume 1.3--1.4pl/cell. Confluent: 2 20 x lo5 cells/ml; cell volume 1.1-1.3 pl/cell (amino acid concentrations calculated on the basis of 807: cell water). Values represent the mean of 4 determinations + 1 S.E.M.
86
S. D. S K A P I ~and K J . E. SEE(;MII.I.ER
HGPRT deficiency in neuroblastoma cclls grown iri uifro (SKAPEK & SEEGMILLER. 1976~).with the glycinc concentration returning to normal in HGPRT' revertant cells selected from an HGPRT deficient line. The present study documents the occurrence of a similar phenomcnon in another type of cultured cell of neural origin, i.e. glioma cells. Free intracellular glycinc was increased approx 2-fold in several independently derived HGPRT deficient clones of C6 glioma cells selected without and with mutagenesis. Thcse mutant glial clones had biochemical characteristics of purine metabolism very similar to fibroblasts and lymphoblasts derived from Lesch Nyhan patients and to HGPRT deficient neuroblastoma cells. as dis1L SEEGYILLER. 1976b). cussed earlier (SKAPER The importance of the above data is the extension of earlier observations with ncuroblastoma to other neural cell types. Especially significant is the absence of similar increases in glycine in cells of non-neural origin. e g lymphoblasts and fibroblasts from LcsohNyhan patients and lymphoblasts selected for HGPRT deficiency from wild-type cells. These alterations in intracellular glycine concentration are of particular interest in light of the evidence iiccumulated for a neurotransmitter role for glycinc in certain regions of the nervous system. and the association of a primary disorder of glycine catabolism, nonketotic hyperglycinemia, with mental and developmental & retardation, seizures. and convulsions (re. SKAYER SEEGMILLER. 1 9 7 6 ~for detailed discussion). Glycinc levels in cerebrospinal fluid and autopsied brain from Lesch- Nvhan naticnts are n o t available. but would be of importaniC i n validating the physio~ogica~ significance of this observation in cultured cells. The above studies are not intended to imply a ncurophysiological function for glycinc in neuroblastorna or glioma cclls in culture. e.g. the ability to clicit an action potential. Electrophysiological studies of this nature havc not yct k e n carried out. The reason for this association of increased intracellular glycinc with HGPRT deficiency in cultured cells is not yet clear. A n increased glycine concentration may represent a form of metabolic regulation of purine biosynthesis (SKAPER & SEEGMII.I.I:K, 1976a). Serinc, a potential glycine precursor, was not consistently altered in the HGPRT deficient cells. The general decline in intracellular free amino acids as cultures reached confluency was probably not a result of depletion of amino acids from the culture medium or excretion into the medium of a substance inhibiting amino acid uptake by the cclls. Culture medium was normally replaced with fresh medium 24 h before harvesting cells, and omission of this procedure did not influence the values for intracellular amino acids. Also, earlier experiments with other types of cells in culturc showcd that amino acids in the medium wcrc not depleted as the cultures became confluent, with the exception of glutamine (unpublished observations). Association of HGPRT dcficiency with a defect in
neuronal properties has yet to be definitely established. Reccnt studies have shown a reduction of monoamine oxidase activity in certain neuroblastoma (RKEAKEFIELD ef ul., 1976) and glioma (SKAPER& SEEGMILLER, 1976~)cell lines deficient in HGPRT, suggesting a relation between the regulation of purine and biogcnic amine metabolism. Other studies with HCIPKT deficient neuroblastoma cells have shown normal electrical excitability, neurite formation, and acctylcholinesterase activity (MINNA er a/., 1971, 1972). These observations, and data which provide evidence for a link between the activity of HGPRT and the level of a putative transmitter amino acid in neural cells. may be indicative of more than one secondary metabolic perturbation underlying the neurological dysfunction of the Lesch--Nyhan syndrome. Ackno~vletlgrnlerirs The authors wish to thank Dr. N . GOCIIMAN of the San Diego Veterans Administration Hospital for use of the amino acid analyzer. and Mr. S. SMITH Tor performing the amino acid analyses. REFERENCES
BENDA P.. LIGHIHODY J.. SATO G., LEVINEL. & SWEET W.(1968)Sciericr, N . Y . 161, 37@371. BOYLCJ . A,, RAIVIO K . 0.. ASTRIN K . H., SCIiIJLMAN J. D.. G R A FM . L., SEEGMILLER J. E. & JACORSEN C. B. ( I 970)Scicwcc,, N . Y 169. 688-689. BRFAKEFIELD X. 0.. CASTIOLK)NE C. M . & EDELSTEINS. B. (1976)Science. N . Y 192, 1018-1020. CHOI K.W.& BLOOMA. D. (1970)Science, N . b 170, 89. KELIXYW. N., GREENE M. L., ROSENBLOOM I-', M., HENDERSON J. F. & SEFGMILLER J. E. (1969)Ann. intern. Med. 7"'
KELLI~Y W. N., ROSENRLWMF. M., HENDERSONJ. F. & S E E G M I L LJ ~. RE. (1967)Proc. riafn. Acad. Sci., U.S.A. 5,, 735 739, LEsCH M , & Nyl,AN W, I-, (1964)Am, J . Med, 36, 561 .S7o, LEVERJ, E,, N~~~~G. & s ~ ~J , E. ~(1974)~proc,~ tiuftl. Actrtl. Sci., U.S.A. 71. 2679 2683. LEVINEE. M. (1972) E x p . Cell Rex 74, 99-109. M I N N AJ., GLAZER D. & NIRENRERC M . (1972)Narure New B i d . 235, 225-231. MINNAJ.. Nrirso~P.. PEACOCK P., GLAZERD. & NIRENnliRc; M. (1971) Proc. natri. Acud. Sci., U.S.A. 68, 2W.239. PERRY T. L.. BERRYK.. HANSENS., DIAMOND S. & MOK C. (1971~) J. Nrurochrm. IS, 513 519. P E R R Y T. L., HANSENS.. B E R R Y K., MOK C . & LESK D. (1971h)J . Nrrrrocliern. 18, 521-528. ROSENBL(X)MF. M., KELLEYW . N., MILLER J.. HENDERSON J. F. & SEEGMILLER J. E. (1967)J. Am. mrd. Ass. 202, 175-177. SI;IX;MII,I.ER J . E., KCISENBLOOM F. M. & KI
ELEVATED INTRACELLULAR GLYCINE ASSOCIATED WITH HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE DEFICIENCY IN GLIOMA CELLS'
s. D. SKAPER' and J. E. SEEGMILLER3 Department of Medicine, University of California, San Diego, La Jolla. CA 92093, U.S.A. (Receioed 22 October 1976. Accepted 28 January 1977)
Abstract---The intracellular concentrations of a number of amino acids were measured in a normal clone of rat glioma cells, and in several independently derived clones selected for gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). A significant, approx 2-fold increase in the concentration of free glycine was observed in both mutagenized and non-mutagenized HGPRT deficient clones. The increase in glycine was independent of the phase of cell growth. A similar increase did not occur in HGPRT deficient lymphoblasts.
THELesch-Nyhan syndrome (LESCH& NYHAN, 1964) is a rare X-linked recessive form of cerebral palsy, which is characterized by hyperuricemia, marked overproduction of uric acid. and severe neurological dysfunction which includes a compulsive self-mutilation, spasticity, chorcoathetosis, and mental retardation. A virtually complete deficiency of the purine reutilization enzyme hypoxanthine-guanine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.8; HGPRT) (SEEGMILLERet a/., 1967) represents the primary biochemical defect in these patients. Subjects with a partial HGPRT deficiency exhibit hypcruricemja, excessive production of and a tendency to develop acid, gouty 1967, calculi of uric acid (KELLEY et with1969)'occasional mild neurologic may Occur in this latter group Of Out patients (KELLEYrt al., 1967, 1969). The b*~&emicalsequence responsible for the mwrologic and behavioral manifestations present in the Lesch-Nyhan patients remains unknown. Recently we reported an increased intracellular concentration of the putative transmitter amino acid glycine in several HGPRT deficient clones of neuroblastoma cells (SUPER & SEEGMILLER, 1976a). A similar Of phenomenon could not be demonstrated in non-neural origin. Since these studies were carried out using only one type of cultured neural cell, the
bility could not be ruled out that the observations were characteristic of a specific cell type. Our studies have now been extended to include another cell type of neural origin, namely, glioma cells. A significant increase in the intracellular glycine concentration was observed in the several HGPRT deficient clones tested, thus supporting the neural-specific nature of this phenomenon. MATERIALS AND METHODS
Marerials. Dulbecco's modified Eagle's medium was obtained from Grand Island Biological (Grand Island, NY) and fetal calf serum from Irvine Scientific Sales (Irvine, CA). All other chemicals were of the highest purity obtainable from commercial sources. Cell culture, The rat glioma clone (3 (BEND*et a/., 1968) and HGPRT deficient clones selected from ethyl methanesulfonate mutagenized (Cb16) and non-mutagenized (TgRCI,) C6 cells showed less than 1% of normal activity 1976b). and have been described (SKAPER& SEEGMILLER, Cells were grown at 37°C in monolayer culture in sealed plastic culture flasks (Falcon, Oxnard, CA) purged with 10% CO,-W"/, air, in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 m ~ - ~ - g l u t a mine (SUPER & SEEGMILLER. 1976a). All cultures tested negative for Mycoplasma contamination (LEVINE, 1972). Lymphoblast lines were established from peripheral blood samples obtained from a normal adult male and a Lesch-Nyhan patient (CHOI& BLOOM,1970) and cultured as described previously (SKAPERet al., 19764. ' This work was supported in part by grants AM-13622, Amino acid analysis. Glial cells were grown and harAM-05646, and GM-17702 from the United States Public vested as described for neuroblastoma (SKAPER& SEEGHealth Service, Veterans Administration grant M.R.I.S. MILLER, 1976a). Culture medium was replaced with fresh No. 3181 to Dr. Nathan Gochman, and grants from the medium 24h before harvesting. Cold acid-soluble amino acids were extracted from the pelleted cells (2-4 x lo6 National Foundation and the Kroc Foundation. Present Address: Department of Pediatrics, Case Wes- cells) using a slight modification of an earlier method & SEEGMILLER, 1976a). Cells were lysed by resustern Reserve University, Cleveland Metropolitan General (SKAPER pending the cell pellet directly in 0.4 N perchloric acid Hospital, Cleveland, OH 44109, U.S.A. (freeze-thaw step omitted). After 10 min at 5°C. the acid To whom reprint requests should be directed. Abbreliiation used: HGPRT, hypoxanthine-guanine was neutralized with KOH and the KCIO4 precipitate and denatured protein were removed by centrifugation. phosphoribosyltransferase.
'
83
S. D. SKAPER and J. E.
84
TABLE1,
SEEC;MII.L~R
~NTRACEI.I.l;IAR C0NCI:NTRATION OF SELECTED AMlh’O ACIDS IN OLIOMA CELLS AT EARLY LOG PHASE CROWTll
Cell line ( m u ) HGPRT deficient ~~
Amino acid
Wild type (C6) (n = 8)
Spontaneous (Tg RCI 2 1 (n = 6)
Mutagenized (C616) (n = 6)
Taurine Aspartic acid Clutamic acid Clutamine Glycine Serine Alanine Tyrosine Phenylalanine
13.0 f 0.7 5.7 f 0.7 22.2 f 0.7 24.1 f 2.0 10.2 f 0.6 3.5 f 0.2 1.9 f 0.8 0.7 f 0.1 1.2 f 0.1
9.9 f 1.4 4.5 & 0.4 22.2 f 0.8 20.1 f 1.4 15.6 f 1.0* 2.5 f 0.2 6.4 f 0.4 0.5 f 0.1 0.8 f 0.1
17.6 f 1.2t 4.4 f 1.1 27.3 f 1.5 20.1 f 0.6 16.8 f 0.3. 2.4 f 0.3 8.0 f 0.2 0.8 0.1 1.1 f 0.2
Early log: 2 4 x lo4 cells/cm2; cell volume 1.62.0 pl/cell (amino acid concentrations calculated on the basis of 80”, cell water). Values given represent the mean of six or more determinations f 1 S.E.M. Significant difference from wild type: P < 0.05; t P < 0.02. Lymphoblasts in suspension culture were harvested and deficient clones with and without mutagenesis prior extracted for their amino acids as described earlier (SKAPERto selection; and (3) the elevation of glycine in ri a/., 1976). HGPRT deficient glioma is independent of the Amino acid analyses were performed on a Durrum growth stage of the cells. D-500 amino acid analyzer. The details of this procedure. In the early logarithmic phase of growth (Table 1) as well as for measurement of cell volumes. have been puban approximatc 1.7-fold increase in the free glycine & SEEGMILLER, 1976~). lished (SKAPER
RESULTS
The intracellular amino acid concentrations for normal C6 and HGPRT deficient (showing less than ;1: of normal activity) glioma clones are summarized in Tables 1 3. Several features are evident: (1) the concentration of the putative neurotransmitter glycine is significantly increased in cells deficient in HGPRT activity; (2) this increase is seen in HGPRT
concentration of the mutagenized HGPRTdeficient glioma clone C6-16 was observed, when compared to the parental clone C6 (P < 0.05). The difference increased to 1 .&fold (P < 0.02) for mid log phase cultures (Table 2). and to 2-fold (P < 0.03) for the confluent culture (Table 3). Among the other putative transmitter amino acids, only taurine showed a significant increase (P < 0.02) from the corresponding value in normal cells, and only in early log growth (Table 1). Serine, a possible precursor of glycine, never showed any consistent alterations in the mutant clone
TAHLI~ 2. INTRAC-ELLULARCONCNTRATION AT M I D I
OF SELECTED A M I N O ACIDS I N GI-IOMA CELLS X PHASE GROWlW
\
Cell line (mM) ~
HGPRT deficient Wild type Amino acid Taurine Aspartic acid Glutamic acid Glutamine Glycinc Scrine Alanine Tyrosine Phrnykddnine
(W
(n
=
8)
8.4 f 1.3 5.4 f 0.8 17.0f 1.6 16.2 f 2.9 9.0 f 0.4 2.7 f 0.4 9.0 2.0 1.1 f 0.1 1.1 f 0.2
Spontaneous (TgRCIJ (n = 6)
Mutagenized (C616) (n = 6)
6.6 f 0.8 3.8 f 0.3 16.1 f 1.2 13.9 f 1.2 15.9 f 0.4. 2.4 f 0.2 6.6 f 0.4 0.7 f-0.1 0.8 f 0.1
4.1 19.7 f 1.3 14.8 f 0.7 16.3 & 1.2* 4.1 f 0.3 8.7 0.8 1.0 0.2 1.4 f 0.1
*
Mid log: 6.5 8.0 x lo4 cells/cmz; cell volumc 1.9-2.0 pl/cell (amino acid concentrations calculated on the basis of 80% cell water). Values given represent the mean of 6 or more determinations f 1 S.E.M. Significant difference from wild type: * P < 0.02.
lntracellular glycine in mutant glioma cells TABLE 3.
85
INTRACELLULAR CONCENTRAIIOK OF SELECTII) AMINO ACIDS IN CONFLLENT CULTURES OF GLIOMA CELLS
Cell line (mM) HGPRT deficient ~~
Amino acid Taurine Aspartic acid Glutamic acid Glutamine Glycine Serine Alanine Tyrosine Phenylalanine
Wild type
Spontaneous
M utagenized
(C6)
(TgRC12)
(C6-16)
5.6 f 0.3 3.8 f 0.5 13.7 f 1.3 7.6 f 0.8 6.0 f 0.3 1.8 f 0.2 4.6 0.3 0.4 f 0.1 0.6 f 0.1
9.7 k 2.0 5.4 f 1.2 15.6 k 2.5 7.4 f 1.2 12.9 f 0.8* 2.5 f 0.3 5.3 f 1.5 0.3 f 0.1 0.6 f 0.1
9.2 k 0.4 3.7 f 0.4 17.7 k 0.3 6.2 f 0.2 12.4 f 0.6* 1.7 f 0.1 6.0 f 0.2 0.3 f 0.1 0.5 f 0.1
Confluent: 35-50 x lo4 cells/cm*; cell volume 0.9-1.1 pl/cell (amino acid concentrations calculated on the basis of 807, cell water). Values given represent the mean of 6 determinations f 1 SEW. Significant dilference from wild type: * P = 0.03. C6-16, although the lower value in early log cultures bordered on being statistically significant. The catecholamine precursors tyrosine and phenylalanine showed no significant variations. None of the other naturally occurring amino acids were increased or decreased in C 6 1 6 cells (not shown). Data for the spontaneous HGPRT deficient glial clone TgRClz were quite similar to the mutagenized clone C6-16. The concentration of free glycine was increased 1.6-fold ( P < 0.05) in early logarithmic growth phase (Table l), 1.8-fold ( P < 0.02) in mid log phase (Table 2) and 2.1-fold ( P < 0.03) in confluent cultures when compared to normal clone C6. No significant increases or decreases were observed for any other amino acids in TgRCI2 cells, with the possible exception of serine in early log cultures. The free intracellular amino acid concentrations measured in glioma cells were remarkably close to the values determined previously for neuroblastoma & SEEGMILLER, 1976a), at each growth cells (SKAPER stage. The high content of taurine in glioma was interesting, although not surprising as brain contains large amounts of taurine (PERRYet a/., 1971a,b).
Corresponding data for cultured lymphoblast lines established from the peripheral blood of a normal adult male (HGPRT+) and a Lesch--Nyhan patient (HGPRT-) are given in Table 4. The intracellular concentration of free glycine did not increase in either log phase or confluent cultures of these HGPRT deficient lymphoblasts. Similar data were obtained for clones of human splenic lymphoblasts selected for HGPRT deficiency from an established parental cell line, WI L2 (LEVERef a/., 1974), and for fibroblasts from Lesch-Nyhan patients (not shown). DISCUSSION The activity of HGPRT has been found markedly deficient in brain tissue from Lesch-Nyhan patients at autopsy (ROSENRLOOM ef a/., 1967; BOYLEet a/., 1970). Despite severe neurological impairment, no specific morphological or histological changes in the CNS of these patients have been observed. The role of HGPRT activity in normal neurological function remains to be defined. Recently we reported an increased concentration (2-fold) of free intracellular glycine associated with
TARLE 4. INTRACELLULAR
CONCEKTRATIOK OF SILI:CTED AMINO ACIDS I N EARLY LOG PHASli AND CONFI.lJC:NT CULTURES OF HUMAN LYYPHOBLASTS
Early log (mW
Confluent (mM)
Amino acid
HGPRT'
HGPRT-
HGPRT'
HGPRT-
Aspartic acid Glutamic acid Glutamine Glycine Serine Alanine Tyrosine Phenylalanine
2.9 f 0.2 19.1 f 0.1 15.5 f 0.9 3.6 f 0.1 1.2 f 0.1 6.2 f 0.1 0.5 f 0.1 0.5 f 0.1
4.0 f 0.4 18.6 f 1.2 15.6 f 1.2 3.4 f 0.2 0.7 f 0.1 5.2 f 0.5 0.5 f 0.1 0.4 f 0.1
1.4 f 0.1 14.3 f 0.4 12.0 f 0.4 3.6 0.1 0.3 f 0.1 4.7 f 0.5 0.7 f 0.1 0.7 f 0.1
1.5 f 0.1 12.9 f 0.4 13.2 f 0.2 2.6 f 0.1 0.4 f 0.1 4.5 f 0.2 0.5 f 0.1 0.5 f 0.1
Early log: 1-4 x lo5 cellsjml; cell volume 1.3--1.4pl/cell. Confluent: 2 20 x lo5 cells/ml; cell volume 1.1-1.3 pl/cell (amino acid concentrations calculated on the basis of 807: cell water). Values represent the mean of 4 determinations + 1 S.E.M.
86
S. D. S K A P I ~and K J . E. SEE(;MII.I.ER
HGPRT deficiency in neuroblastoma cclls grown iri uifro (SKAPEK & SEEGMILLER. 1976~).with the glycinc concentration returning to normal in HGPRT' revertant cells selected from an HGPRT deficient line. The present study documents the occurrence of a similar phenomcnon in another type of cultured cell of neural origin, i.e. glioma cells. Free intracellular glycinc was increased approx 2-fold in several independently derived HGPRT deficient clones of C6 glioma cells selected without and with mutagenesis. Thcse mutant glial clones had biochemical characteristics of purine metabolism very similar to fibroblasts and lymphoblasts derived from Lesch Nyhan patients and to HGPRT deficient neuroblastoma cells. as dis1L SEEGYILLER. 1976b). cussed earlier (SKAPER The importance of the above data is the extension of earlier observations with ncuroblastoma to other neural cell types. Especially significant is the absence of similar increases in glycine in cells of non-neural origin. e g lymphoblasts and fibroblasts from LcsohNyhan patients and lymphoblasts selected for HGPRT deficiency from wild-type cells. These alterations in intracellular glycine concentration are of particular interest in light of the evidence iiccumulated for a neurotransmitter role for glycinc in certain regions of the nervous system. and the association of a primary disorder of glycine catabolism, nonketotic hyperglycinemia, with mental and developmental & retardation, seizures. and convulsions (re. SKAYER SEEGMILLER. 1 9 7 6 ~for detailed discussion). Glycinc levels in cerebrospinal fluid and autopsied brain from Lesch- Nvhan naticnts are n o t available. but would be of importaniC i n validating the physio~ogica~ significance of this observation in cultured cells. The above studies are not intended to imply a ncurophysiological function for glycinc in neuroblastorna or glioma cclls in culture. e.g. the ability to clicit an action potential. Electrophysiological studies of this nature havc not yct k e n carried out. The reason for this association of increased intracellular glycinc with HGPRT deficiency in cultured cells is not yet clear. A n increased glycine concentration may represent a form of metabolic regulation of purine biosynthesis (SKAPER & SEEGMII.I.I:K, 1976a). Serinc, a potential glycine precursor, was not consistently altered in the HGPRT deficient cells. The general decline in intracellular free amino acids as cultures reached confluency was probably not a result of depletion of amino acids from the culture medium or excretion into the medium of a substance inhibiting amino acid uptake by the cclls. Culture medium was normally replaced with fresh medium 24 h before harvesting cells, and omission of this procedure did not influence the values for intracellular amino acids. Also, earlier experiments with other types of cells in culturc showcd that amino acids in the medium wcrc not depleted as the cultures became confluent, with the exception of glutamine (unpublished observations). Association of HGPRT dcficiency with a defect in
neuronal properties has yet to be definitely established. Reccnt studies have shown a reduction of monoamine oxidase activity in certain neuroblastoma (RKEAKEFIELD ef ul., 1976) and glioma (SKAPER& SEEGMILLER, 1976~)cell lines deficient in HGPRT, suggesting a relation between the regulation of purine and biogcnic amine metabolism. Other studies with HCIPKT deficient neuroblastoma cells have shown normal electrical excitability, neurite formation, and acctylcholinesterase activity (MINNA er a/., 1971, 1972). These observations, and data which provide evidence for a link between the activity of HGPRT and the level of a putative transmitter amino acid in neural cells. may be indicative of more than one secondary metabolic perturbation underlying the neurological dysfunction of the Lesch--Nyhan syndrome. Ackno~vletlgrnlerirs The authors wish to thank Dr. N . GOCIIMAN of the San Diego Veterans Administration Hospital for use of the amino acid analyzer. and Mr. S. SMITH Tor performing the amino acid analyses. REFERENCES
BENDA P.. LIGHIHODY J.. SATO G., LEVINEL. & SWEET W.(1968)Sciericr, N . Y . 161, 37@371. BOYLCJ . A,, RAIVIO K . 0.. ASTRIN K . H., SCIiIJLMAN J. D.. G R A FM . L., SEEGMILLER J. E. & JACORSEN C. B. ( I 970)Scicwcc,, N . Y 169. 688-689. BRFAKEFIELD X. 0.. CASTIOLK)NE C. M . & EDELSTEINS. B. (1976)Science. N . Y 192, 1018-1020. CHOI K.W.& BLOOMA. D. (1970)Science, N . b 170, 89. KELIXYW. N., GREENE M. L., ROSENBLOOM I-', M., HENDERSON J. F. & SEFGMILLER J. E. (1969)Ann. intern. Med. 7"'
KELLI~Y W. N., ROSENRLWMF. M., HENDERSONJ. F. & S E E G M I L LJ ~. RE. (1967)Proc. riafn. Acad. Sci., U.S.A. 5,, 735 739, LEsCH M , & Nyl,AN W, I-, (1964)Am, J . Med, 36, 561 .S7o, LEVERJ, E,, N~~~~G. & s ~ ~J , E. ~(1974)~proc,~ tiuftl. Actrtl. Sci., U.S.A. 71. 2679 2683. LEVINEE. M. (1972) E x p . Cell Rex 74, 99-109. M I N N AJ., GLAZER D. & NIRENRERC M . (1972)Narure New B i d . 235, 225-231. MINNAJ.. Nrirso~P.. PEACOCK P., GLAZERD. & NIRENnliRc; M. (1971) Proc. natri. Acud. Sci., U.S.A. 68, 2W.239. PERRY T. L.. BERRYK.. HANSENS., DIAMOND S. & MOK C. (1971~) J. Nrurochrm. IS, 513 519. P E R R Y T. L., HANSENS.. B E R R Y K., MOK C . & LESK D. (1971h)J . Nrrrrocliern. 18, 521-528. ROSENBL(X)MF. M., KELLEYW . N., MILLER J.. HENDERSON J. F. & SEEGMILLER J. E. (1967)J. Am. mrd. Ass. 202, 175-177. SI;IX;MII,I.ER J . E., KCISENBLOOM F. M. & KI
