MONOAMINE OXIDASE ACTIVITY I N NORMAL AND LESCH-NYHAN FIBROBLASTS SL~SAN B. EDELSTEIN, CARMELAM. CASTIGLIONE and XANDRA0. BREAKEFIELD Department of Human Genetics. Yale University School of Medicine, New Haven, CT 06510. U.S.A. ( R e w i r e d 13 December 1977. Accepted 16 Mu! 1978)

Abstract-Monoamine oxidase ( M A O ) activity was studied in cultured skin fibroblasts from 10 LeschNyhan patients. a L e s c h ~Nyhan tariant and 11 controls matched for age. sex and race. Activity (predominantly type A) was measured in cell homogenates using tryptamine as the substrate. For each line activity varied with the conditions of culture. Activity increased 3-10 fold as cultures went from logarithmic to stationary phase of growth. When cultures were confluent. activity was lowered by frequent feedings or the use of fresh medium and serum. Activity for each line remained fairly stable during successive passages. but rose 3-8 fold as cultures became senescent. When comparing activity between control and Lesch-Nyhan lines. cells were cultured under standardized conditions. The mean value of M A 0 activity in Lesch-Nyhan lines was approximately one fourth of the mean acticity in control lines ( P i0.012). In the control population. the distribution of activity appeared to be bimodal. Activities in the Lesch-Nyhan lines fell completely within the lower portion of the control distribution. Cells from a Lesch-Nyhan patient who lacked several of the neurologic symptoms of the disease (including self-mutilation) had a n M A 0 activity 6 fold greater than the control mean. although his hypoxanthine phosphoribosyltransferase activity was < 3",, of control levels. I t appears that: ( I ) M A 0 actility is low in fibroblasts from typical Lesch-Nyhan patients: (2) the severity of neurologic symptoms may be correlated with levels of M A 0 activity: and (3) some interaction between purine and catecholamine metabolism can affect nerve function.

THE LESCHNYHANsyndrome (LESCH & NYHAN, 1964) is a rare X-linked recessive disorder characterized by an excessive production of uric acid and a severe deficiency of the enzyme hypoxanthine phosphoribosyltransferase (HPRT. IMP: pyrophosphate phosphoribosyltransferase EC 2.4.2.8.) (SEEGMILLER ef nl., 1967). Although it is not clear why this deficiency leads to neurologic dysfunction. patients with this disease exhibit mental retardation, spasticity, choreoathetosis and a compulsive self-mutilation of the lips and fingers. The levels of HPRT activity in Lesch-Nyhan individuals vary from zero to a few per cent of normal. Although some studies have reported a correlation between the extent of the en7yme deficiency and the severity of neurologic dysfunction (GREENE,1972: SEEGMILLER. 1976). other studies have not found this (EMMERSON & THOMPSON. 1973: DE BRUYN.1976). Moreover, individuals have been described who have undetectable HPRT activity in blood cells and fibroblasts and d o not exhibit the typical neurologic symptoms of the Lesch-Nyhan syndrome (NYHAN.1968: GEERDINK et (11.. 1973: MANZKE,1976: SWEETMAN rf a/., 1978). Conversely. individuals with fully normal HPRT activity have been found t o exhibit symptoms

Abhreiiurions used: MAO, monoamine oxidase: HPRT. hypoxanthine phosphoribosyltransferase.

suggestive of the Lesch-Nyhan syndrome (NYHAN, 1968: DE BRUYN,1976). Several findings suggest that catecholamine metabolism may be altered in this disease. Potentiation of catecholamine pathways in the brains of experimental animals by administration of dopamine (CosTALL & NAYLOR,1975). apomorphine (a dopamine agonist : ERNST,1967) or clonidine (an z-adrenergic agonist: RAZZAKet ul., 1975) can induce choreic movements, compulsive gnawing and self-mutilation. Hypoxanthine phosphoribosyltransferase activity is normally highest in the basal ganglia (ROSENBLOOM et nl., 1967), an area of the brain which receives catecholaminergic input and controls involuntary movements. It is possible t h a t a combination of HPRT deficiency and altered catecholamine metabolism in this area of the brain could lead to some of the neurologic dysfunction in Lesch-Nyhan individuals. In previous studies cultured murine neuroblastoma cells were used as a model system to explore the interaction between HPRT deficiency and catecholamine metabolism (BREAKEFIELD et a / . , 1976). Activities of synthetic and degradative enzymes involved in catecholamine metabolism were compared in four parental lines and 12 HPRT deficient clones derived from them. Only monoamine oxidase (MAO, monoamine: 0, oxidoreductase EC 1.4.3.4), an enzyme important in the degradative deamination of biogenic amines. had altered activity. A decrease was observed in 10

1247

1248

$CS.t.!

B EDFLSTEIN.CARMFLA M CASTICLIONE and

XANDRA

0 BRtAKtkltLD

of 3&35 days. Cell numbers were determined using a hemacytometer following resuspension with Pancreatin. The confluency of cultures was monitored by phase contrast microscopic examination. To test the effect of different feeding schedules on M A 0 activity, cells were inoculated in parallel onto IOOmm dishes. Cultures were fed once prior to and again at confluency. Plates then were fed at scheduled times during the 7 day interval before harvesting using either medium and serum incubated at 37-C for 2-3 weeks ('incubated medium') or medium and recently thawed serum incubated at 37'C overnight ('fresh medium'). The same batch of medium and lot of serum was used for feeding throughout the course of an experiment. For all other determinations of M A 0 activity. cultures on 150mm dishes were fed at 3-9 day intenals and on the day before harvesting with medium and serum incubated at 37'C for 1--10 days. Cultures here hanested 1-8 days after becoming confluent. Cell lines were found free of mycoplasma contamination when stained with a DNA-binding fluorescent dye. 4 - 6 diamindino-2'-phenylindole(425: Bioassay Systems Corp., Cambridge, MA). Preparation of homoyenates. Fibroblasts on dishes were rinsed quickly 3 4 times with isotonic phosphate buffered saline. After draining 45 s, cells were scraped off the surface of the plate with a Teflon-coated straight edge. Cells in flasks were treated with 1X Pancreatin for 15min. resuspended by trituration with medium containing lo", serum and pelleted by low speed centrifugation. The pellet surface MATERIALS AND METHODS was rinsed with a small amount of isotonic buffer. FollowMaterials. Tissue culture dishes and flasks were obtained ing either procedure, the cells were resuspended in a small from Falcon or Corning: the Dulbecco-Vogt modification volume of ice cold homogenate buffer (0.1 M-potassium of Eagle's medium (DMEM. No. H-21) and 4X Pancreatin phosphate, pH 7.4) to a concentration of 1-6 mg protein (pancreatic hydrolases) from Grand Island Biological Co., ml. Homogenates were frozen on dry ice and stored in fetal calf serum from Flow Labs: [G-3H]tryptamine hy- the vapor phase of liquid nitrogen ( - 70-C). Prior to assay drochloride (1- 1.65 Ci, mrnol) from Amersham Searle they were thawed, held on ice and homogenized 20 strokes (stored in M-ascorbic acid): Liquifluor from New Eng- by hand in a 2 ml Teflon-glass tissue grinder (Potter Elvehland Nuclear: tryptamine hydrochloride from Sigma: and jem Type). clorgyline from Dr. SABATGABAY.V.A. Hospital, BrockAssays. Monoamine oxidase activity in cell homogenates ton, MA. All other chemicals used were of reagent grade. was measured by a modification of the method of WURTC d l lines. Cultured lines of human fibroblasts grown M A N & AXELROD(1963). The reaction mixture contained M 115 mM-pOtaSSiUm phosphate buffer. 3.C5.0 x from skin biopsies of Caucasian males were obtained from the American Type Culture Collection, Bethesda, M D [G-3H]tryptamine (1 pCi, final specific activity 0.17-0.27 (names designated by letters): the Institute for Medical Ci/mmol), 0.1 mM-aSCorbiC acid and 0-60 pl of homogenate Research, Camden, NJ (prefixed with GM): the Depart- in a final volume of 100pl (final pH 7.4). Reactions were ment of Human Genetics. Yale University School of Medi- initiated by addition of labelled substrate and incubated cine (designated by numbers): Dr. SAMUELGOLDSTEIN, at 37'C for 30min. Reactions were stopped by addition McMaster University, Hamilton, Ontario ( A l . A2, R-ELL) of 200 p l 2 N-HCI. Deaminated products were extracted University of California School directly into 6 ml scintillation fluid (lo00 ml toluene plus and Dr. UTA FKANCKE, of Medicine. La Jolla. CA (LN BUR). Viable frozen stocks 42 ml Liquifluor). Four milliliters of the toluene phase were removed and counted in a Beckman LS 250 liquid scintillaof all lines were maintained in the vapor phase of liquid tion spectrometer. Monoamine oxidase activities were nitrogen ( - 70'C). Tissue culture. Cells were grown as monolayers on poly- assayed in duplicate or triplicate within the range of linearstyrene tissue culture flasks (75cm') or dishes (100mm or ity for protein and time, up to 750 pg and 40 min. respect150mm) in medium supplemented with 104.: (vjv) serum, ively. Final substrate concentrations were near saturation without antibiotics. Cultures were maintained at 37'C in for the enzyme and 3-8 fold higher than K , values for a humidified atmosphere of 5",, COz and 9504 air. Stock tryptamine (6-13 x 1 0 - 6 ~in six lines tested). Buffer flasks were fed at 4-8 day intervals and subcultured (pas- blanks were used routinely to establish background kalues. saged) every 1&20 days in a ratio of 1 : 5, following resus- and were comparable to blanks in which acid was added, or homogenates were preincubated with clorgyline M. pension by treatment with 1X Pancreatin for 5-10min. 15 min on ice), before addition of labelled substrate. Pancreatin was diluted in isotonic phosphate buffered Protein was determined by a modification of the method saline (HAWKINS & BREAKEFIELD, 1978). Growth curves were established by innoculating 75 cm2 of LOWRYet al. (1951) using bovine serum albumin as flasks in parallel with 4-5 x LO5 cells/flask. Flasks were a standard. fed twice a week and harvested periodically over a period Statistical analyses were performed using a one-tailed H P R T deficient clones derived from three of four parental n e u r o b l a t o m a lines. Low M A 0 activity associated with H P R T deficiency also has been observed in rat glioma ( S K A P E R & SEEGMILLER,1976) and mouse melanoma. but not rat hepatoma cells (HAWKINS & BREAKEFIELD, 1978). These findings led us to examine M A 0 activity in fibroblasts cultured from skin biopsies of LeschNyhan patients. Monoamine oxidase activity in fibroblasts was found t o be predominantly type A (ROTH ut a/.. 1976: GROSHONG et a/.. 1977). Six Lesch--Nyhan lines exhibited one third the mean M A 0 activity of six control lines (BREAKEFIELD et a/.. 1976) but no difference was seen in catechol methyltransferase (5-adenosyl-~-methionine: catechol O-methyltransferase EC 2.1.1.6) activity (ROTHef a/.. 1976). To extend these findings, we examined the effect of varying culture conditions on M A 0 activity and increased the number of fibroblast lines studied. We have included a fibroblast line from a Lesch-Nyhan patient with atypical symptoms. This patient ( L N BUR) has very low HPRT activity (< 37; of control), hyperuricemia and choreoathetosis, but n o mental retardation or self-mutilation ( U t a Francke. personal communication: CATEL& SCHMIDT,1959: MANZKE,1976).

0

O

L 5

10

I

15

J

1 - 1 ---

25

30

20

0

40

20 -

5 10

15

20

25

'F. '

/, I

,

5 10 30 : Days in c u l t u r e

SAL MAT

15

I

1

TO SER

,

I

20

1

25

0

30

?

5

10

15

20

I

25

I

P

in triplicate over a linear range of protein concentration. Homogenates were prepared from parallel cultures plated o n day 0. fed at 2-3 day intervals and harvested periodically over 30- 35 days. Cell numbers were determined using ii hemacytometer and percent confluency was estimated by phase microscopic examination.

:

L

H

--

;

G.

FIG. 1. Variation in M A 0 activity with stage of growth. Monoamine oxidase activity was measured in cell homogenates incubated 30 min at 3 7 C in 115 M-potassium phosphate buffer, pH 7.4, with [G-'H]-tryptamine as substrate. Deaminated products were extracted into toluene and counted in a liquid scintillation spectrometer. Each point represents the mean value of one homogenate assayed

20

RID MOR

50 -

100-

C. 200-

N W P

1250

S U S A ~8

EutLbitiN,

CARMELA M. CASTIGLIONE and X A ~ U R 0 A BKEAKLFIFLD

Student‘s r-test. Values with P < 0.05 were considered significant. RESULTS We examined the effects of the stage of growth, feeding schedule and passage number on M A 0 activity. I n all lines examined, M A 0 activity is lowest right after cells are plated at low density and rises rapidly during logarithmic growth (Fig. 1). In one control line (Rid Mor, Fig. 1B) and two Lesch-Nyhan lines (Sal Mat, Fig. 1D: To Ser. Fig. lF), M A 0 activity levelled off at maximal values approx 1 week after the cultures became confluent. This phenomenon was also observed for two other control lines (data not shown). In the Lesch-Nyhan variant line ( L N BUR, Fig. lH), M A 0 activity did reach a plateau as the cultures reached confluency. but decreased several days later. During the transition from logarithmic to stationary phase, M A 0 activity increased threefold for Rid Mor and L N BUR, and 7-10 fold for Sal Mat and T o Ser. Differences in feeding schedules and the age of medium and serum also affected M A 0 activity. In one schedule, cultures were fed at regular intervals until the time of confluency, then once more at varying times between confluency and harvesting, using ‘incubated medium’ (see Methods). In three lines examined, activity did not vary with this schedule (Fig. 2). However, M A 0 activity did decrease with frequent feedings or addition of fresh medium and serum (Table 1). The two lines with lower M A 0 activity were decreased to a greater extent than the two lines with higher M A 0 activity. Frequent feedings with ‘incubated medium’ appeared to decrease M A 0 activity more than a single feeding with ‘fresh medium’. but the difference was not statistically significant. TABLF1

VARIATION OF

MA0

-

r

?

0

n--P-D -D -.-

a-.

a

Fibroblasts proliferate rapidly in culture for only a limited number of passages and then become senescent. Senescence is characterized by increased time necessary to reach confluency, lower cell density at confluency, flatter cell morphology and debris present in the culture media. The effect of increasing passage number on M A 0 activity is shown in Fig. 3. Passage numbers were used to indicate the number of times a fibroblast line was subcultured, usually at a ratio of 1: 5. From the growth curves (Fig. l), each passage corresponds to approximately four population doublings, as defined by HAYFLICK & MOORHEAD(1961). Monoamine oxidase activity remained fairly constant

ACTI\ITY WITH U l F F t R t N T FEEDING SCHEDULES

Schedules* Standard Cell line Control Ro Be1 Rid Mor Al

Lesch Nyhan To Ser

Passage No.

A

B

(pmoliminlmg protein)t

7 20: 12

33.31 f 2.04(4) 38.75 f 2.77(3) 4.64 f 0.37(4)

29.47 f 1.75(4) 32.70 f 2.51(3) 3.24 f 0.38(3)

25.34 1.12(3) 31.71 F 3.68(3) 1.96 f 0.26(3)

13

1.29 f 0.23(3)

0.56 f 0.21(2)

0.47 f 0.17(3)

* Homoyenates were prepared from parallel cultures plated on 100mm dishes.

All cultures were fed once prior and again at confluency with medium and serum incubated at 37°C for 2-3 weeks (‘incubated medium’). In the standard schedule. cultures were fed for the last time on the day before harvesting with ‘incubated medium’. In Schedule A. cultures were fed similarly except fresh medium and serum was used for the last feeding. In Schedule B, cultures were fed ebery other day from the time of confluency until the day before harvesting using ‘incubated medium’. Before statistical comparisons were made, values within each line were normalized to the homogenate with the highest MA0 activity. Differences between the feeding schedules were tested by a one-tailed Student’s (-test. Activities in Schedules A and B were significantly lower than the standard at P < 0.005 and P < 0.0005, respectively. t MA0 activity was measured in cell homogenates using [G-3H]tryptamine as described in the footnote to Fig. I . Values are expressed as means f S.E.M. Numbers in parentheses refer to the number of separate homogenates assayed in triplicate over a linear range of protein concentration. Note the passage of Rid Mor was near senescence (Fig. 3). to

I

I

I 1

SAL MAT

,

I

'

'

I

F. 10 -

I

I

I

M I TEN

I

I

50

10

l0 ot

19

,

1-

20

21

22

13 21

O ' C

Passage number

17

4 E.

'

I

1'0

'

T O SER

I

12 '

'

2 4