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A localized stimulation of lens protein synthesis by actinomycin D
42~
PRELIMINARY NOTES
BBA 91099
A Iocolized stimulotion of lens protein synthesis by octinomycin D It has been reported that low concentrations of actinomycin D stimulate the rate of penicillinase 1 and ribonuclease 2 formation in Bacillus subElis. The selectivity of this effect is indicated by the observation that neither fi-galactosidase nor amylase formation were stimulated under the same conditions. Similar observations have been made for enzymes in animal cells. Treatment of young mice with actinomycin D resulted in an increase in intestinal alkaline phosphatase activity a and this same enzyme was stimulated in mammalian cells in tissue culture a. In addition, the cortisolinducible rat-liver enzymes, alanine transaminase, tyrosine transaminase, serine dehydrase and tryptophan pyrrolase have all been shown to be induced ~ or stimulated by actinomycin D (ref. 6). During our studies of protein synthesis in differentiating lens cells, it was observed that actinomycin D inhibits the synthesis of lens proteins in the epithelial cells and stimulates the synthesis of these same proteins in the fiber cellsV,8. The final stage of lens-cell differentiation involves an elongation of the epithelial cells to form fiber cells. The proteins referred to in these studies are the ~-, //- and y-crystallins which are groups of structural proteins found in the lens. Intact calf lenses were incubated at 37 ° for 2 h in the presence of 14C-labeled algal protein hydrolysate, with and without actinomycin D (Ioffg/ml). The epithelial
L
"'
,38 6£~
Q
&O c 3'T
0
G
8 4.c
/l o12
/ . . . . .
, 60
120 180 240 Effluent (ml)
300
360
60
120 18(3 240 Effluent (ml)
300
360
Fig. I. The fractionation of calf lens epithelial cell crystallins. The regions in which the crystallins are eluted from the c o l u m n are designated b y the arrows. - - - - - , total p r o t e i n (mg)/fraction; , c o u n t s / m i n per fraction in control cells; - - -, c o u n t s / m i n per fraction in actinomycint r e a t e d cells. The p r o t e i n s were eluted b y a stepwise addition of the following buffers: I. 5 ° ml o.005 M s o d i u m p h o s p h a t e (pH 7.0); I I . 50 ml o.oo75 M s o d i u m p h o s p h a t e (pH 6.5); I I I . 50 nil o.oi M s o d i u m p h o s p h a t e (pH 6.0); IV. 75 ml 0.02 M s o d i u m p h o s p h a t e (pH 5.7); V. 5 ° m l 0.02 M s o d i u m p h o s p h a t e (pH 5.7) + o . i M NaC1; VI. 5 ° ml o.i M s o d i u m p h o s p h a t e (pH 5.7) + + o . i M NaC1; V I I . 5° ml o.i M s o d i u m p h o s p h a t e (pH 5.7) + 0 . 3 M NaC1. F r a c t i o n s were collected in 3-mi aliquots. A o.5-ml aliquot was dissolved in a h y a m i n e - t o l u e n e - f l u o r s solution and counted in a liquid-scintillation c o u n t e r (2.5 ml of a 1.5 M s o l u t i o n i n m e t h a n o l of diisobutyl-cresoxye t h o x y - d i m e t h y l - b e n z y l - a m m o n i u m chloride m o n o h y d r a t e (hyamine- i oN q- 12.5 ml of a solution in toluene of 4 g/1 2,5-diphenyloxazole (PPO) and 5 ° g/1 p-bis-(5-phenyloxazolyl-2)-benzene (POPOP)). Fig. 2. The fractionation of calf lens fiber cell crystallins. F r a c t i o n a t i o n and c o u n t i n g conditions are identical to those described in Fig. i.
Biochim. Biophys. dcla, 114 (1966) 428-43 °
PRELIMINARY NOTES
429
cells, which adhere tightly to the external, non-cellular lens capsule, were separated from the fiber cells by removing this capsule. The ~-, /3- and 7-crystallins were fractionated on DEAE-cellulose columns. Procedures for their identification in column eluates have been reported in a previous paper 9. An elution diagram showing the separation of ~-, /3- and 7-crystallins from untreated epithelial cells is shown in Fig. I. Since the elution pattern for proteins from actinomycin treated cells is the same as that for proteins from untreated cells it was not shown in Fig. I. This also holds true for the fiber-cell protein pattern (Fig. 2). The incorporation of [14C?amino acids into these proteins is also shown in Fig. I. Incorporation of amino acids into epithelial cell lens proteins could be extensively inhibited by treatment with Io~,g/ml actinomycin D (c]. Fig. I). The specific activity data (Table I) show that incorporation of amino acids into eTABLE I THE
EFFECT
OF ACTINOMYCIN
D
ON LENS
PROTEIN
SYNTHESIS
Epithelial cells (counts~rain per mg protein)
Fiber cells (counts~rain per mg protein)
Control
Actinomycin Inhibition D (io ktg/ml) (%)
Control
Actinomycin Stimulation D (zo tzg/ml) (%)
389 314 166 572
240 235 99 34 °
318 396 165 69o
Homogenate 2597 T-Crystallins 159o ~-Crystallins 963 ~-Crystallins 198o
85 80 83 71
33 68 66 lO3
crystallins was inhibited by 71%,/3-crystallins by 83 % and y-crystallins by 80 %. The same experiments have been performed with the lens fiber cells. An elution pattern of a-, /3- and ),-crystallins from untreated lens-cortex fiber cells is seen in Fig. 2. The incorporation of amino acids into proteins from treated and control fiber cells is also shown. The incorporation of amino acids into lens proteins is significantly greater in the actinomycin-treated cells. A comparison of the specific activity of the e-,/3- and y-crystallins from control and actinomycin-treated lenses shows that there is a significant stimulation of protein synthesis by the antibiotic which ranges from 66 % for the/3-crystallins to lO3 % for the ~-crystallins (Table I). In an earlier report from this laboratory it was shown that lens epithelial cells do not contain ),-crystallins and that the synthesis of this group of proteins is specifically associated with fiber-cell differentiation1°, 11. A similar observation was made in regenerating salamander lens TM. The elution diagram (Fig. I) shows detectable quantities of y-crystallins in the calf lens epithelial cells. However, during the incubation period it was noted that some of the peripheral cells which have entered the stage of elongation to form fiber cells come loose and are removed with the epithelial cell layer. The presence of small amounts of, y-crystallins in epithelial cell homogenates (Fig. i) therefore, appears to be due to the presence of elongating epithelial cells where the y-crystallins first appear. A comparison of the specific activity of actinomycin-treated cells shows an 85 % inhibition of 7-crystallin synthesis in the elongating epithelial cells and a 68 o, /o Biochim. Biophys. dcta, 114 (1966) 428-43 °
430
PRELIMINARY NOTES
stimulation of this same group of proteins in the fiber cells. Thus, at the time of ?J-crystallin appearance the synthesis of this protein as well as of the ~- and flcrystallins, is still sensitive to inhibition by actinomycin D whereas in the completed fiber cell the synthesis of these same proteins is stimulated. The mechanism by which actinomycin D stimulates protein synthesis is unknown. The mechanisms which have been proposed for this effect are as follows: Firstly, the stimulation might be attributed to the availability of more ATP for protein synthesis as a result of the inhibition of RNA synthesis by actinomycin 1. Thus, in the fiber cell the ATP normally used for RNA synthesis could be channeled into the synthesis of the proteins being formed on stable RNA templates. Secondly, this stimulation might be attributed to the inhibition of the synthesis of a repressor protein by actinomycin 6. It has been shown that the actinomycin D stimulation of t r y p t o p h a n pyrrolase and tyrosine transaminase occurs after these enzymes have been induced by hydrocortisone, when their m R N A is relatively stable. By inhibiting the m R N A responsible for the synthesis of repressor protein, the level of this repressor is decreased and a stimulation of these enzymes results. Finally, the lens epithelial cells are essential for the active transport of nutrients into the lens fiber cells. It is therefore possible that actinomycin alters these properties such that there is an increase in the transport of amino acids into the fiber cell layer, resulting in a stimulation of protein synthesis on stable RNA templates. Experiments are now in progress to determine the mechanism of this stimulatory phenomenon. Actinomycin D was a gift from Merck, Sharpe and Dohme, Inc., Rahway, N.J. This work was supported b y a grant from the National Foundation and from the U.S. Public Health Service. One of the authors (J.A.S.) was supported by a predoctoral fellowship of the Institute of Cellular Biology; P.V.K. was supported b y a post-doctoral fellowship of the Institute of Cellular Biology.
The Department o[ Zoology, Institute o/ Cellular Biology, The University o[ Connecticut, Storrs, Conn. (U.S.A.) I 2 3 4 5 6 7 8 9 io II i2
J O H N PAPACONSTANTINOU JAMES A . STEWART PAUL V. KOEHN
1R. POLLOCK, Biochim. Biophys. Acta, 76 (1963) 80. COLEMAN AND W. H. ELLIOTT, Nature, 202 (1964) lO83. M o o a , Science, 144 (1964) 414 . NITOWSKY. S. GELLER AND R. CASPER, Federation Proc., 23 (I964) 556. ROSEN, P. N. RAINA, I~. J. MILHOLLAND AND C. A. NICHOL, Science, 146 (1964) 661. D. GARREN, R. I{. HOWELL, G. ~V[. TOMKINS AND R. M. CROCCO, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1121. J. PAPACONSTANTINOU, P. V. KOEHN AND J. A. STEWART, Am. Zool., 4 (1964) 321. J. PAPACONSTANTINOU, P. V. KOEHN AND J. A. STEWART, Abstr. I48th Meeting Am. Chem. Sot., 4 (1964) 39C. J. PAPACONSTANTINOU, R. A. I~ESNIK AND E. SAITO, Biochim. Biophys. Acta, 60 (1962) 205. J. PAPACONSTANTINOU, Am. Zool., 4 (1964) 279. J. PAPACONSTANTINOU, Biochim. Biophys. Acta, lO 7 (1965) 81. C. TAKATA, J. F. ALBRIGHT AND T. YAMADA, Science, 147 (1965) 1299.
M. G. F. H. F. g.
Received October I l t h , 1965 Biochim. Biophys. Acta, 114 (1966) 428-43o
PRELIMINARY NOTES
BBA 91099
A Iocolized stimulotion of lens protein synthesis by octinomycin D It has been reported that low concentrations of actinomycin D stimulate the rate of penicillinase 1 and ribonuclease 2 formation in Bacillus subElis. The selectivity of this effect is indicated by the observation that neither fi-galactosidase nor amylase formation were stimulated under the same conditions. Similar observations have been made for enzymes in animal cells. Treatment of young mice with actinomycin D resulted in an increase in intestinal alkaline phosphatase activity a and this same enzyme was stimulated in mammalian cells in tissue culture a. In addition, the cortisolinducible rat-liver enzymes, alanine transaminase, tyrosine transaminase, serine dehydrase and tryptophan pyrrolase have all been shown to be induced ~ or stimulated by actinomycin D (ref. 6). During our studies of protein synthesis in differentiating lens cells, it was observed that actinomycin D inhibits the synthesis of lens proteins in the epithelial cells and stimulates the synthesis of these same proteins in the fiber cellsV,8. The final stage of lens-cell differentiation involves an elongation of the epithelial cells to form fiber cells. The proteins referred to in these studies are the ~-, //- and y-crystallins which are groups of structural proteins found in the lens. Intact calf lenses were incubated at 37 ° for 2 h in the presence of 14C-labeled algal protein hydrolysate, with and without actinomycin D (Ioffg/ml). The epithelial
L
"'
,38 6£~
Q
&O c 3'T
0
G
8 4.c
/l o12
/ . . . . .
, 60
120 180 240 Effluent (ml)
300
360
60
120 18(3 240 Effluent (ml)
300
360
Fig. I. The fractionation of calf lens epithelial cell crystallins. The regions in which the crystallins are eluted from the c o l u m n are designated b y the arrows. - - - - - , total p r o t e i n (mg)/fraction; , c o u n t s / m i n per fraction in control cells; - - -, c o u n t s / m i n per fraction in actinomycint r e a t e d cells. The p r o t e i n s were eluted b y a stepwise addition of the following buffers: I. 5 ° ml o.005 M s o d i u m p h o s p h a t e (pH 7.0); I I . 50 ml o.oo75 M s o d i u m p h o s p h a t e (pH 6.5); I I I . 50 nil o.oi M s o d i u m p h o s p h a t e (pH 6.0); IV. 75 ml 0.02 M s o d i u m p h o s p h a t e (pH 5.7); V. 5 ° m l 0.02 M s o d i u m p h o s p h a t e (pH 5.7) + o . i M NaC1; VI. 5 ° ml o.i M s o d i u m p h o s p h a t e (pH 5.7) + + o . i M NaC1; V I I . 5° ml o.i M s o d i u m p h o s p h a t e (pH 5.7) + 0 . 3 M NaC1. F r a c t i o n s were collected in 3-mi aliquots. A o.5-ml aliquot was dissolved in a h y a m i n e - t o l u e n e - f l u o r s solution and counted in a liquid-scintillation c o u n t e r (2.5 ml of a 1.5 M s o l u t i o n i n m e t h a n o l of diisobutyl-cresoxye t h o x y - d i m e t h y l - b e n z y l - a m m o n i u m chloride m o n o h y d r a t e (hyamine- i oN q- 12.5 ml of a solution in toluene of 4 g/1 2,5-diphenyloxazole (PPO) and 5 ° g/1 p-bis-(5-phenyloxazolyl-2)-benzene (POPOP)). Fig. 2. The fractionation of calf lens fiber cell crystallins. F r a c t i o n a t i o n and c o u n t i n g conditions are identical to those described in Fig. i.
Biochim. Biophys. dcla, 114 (1966) 428-43 °
PRELIMINARY NOTES
429
cells, which adhere tightly to the external, non-cellular lens capsule, were separated from the fiber cells by removing this capsule. The ~-, /3- and 7-crystallins were fractionated on DEAE-cellulose columns. Procedures for their identification in column eluates have been reported in a previous paper 9. An elution diagram showing the separation of ~-, /3- and 7-crystallins from untreated epithelial cells is shown in Fig. I. Since the elution pattern for proteins from actinomycin treated cells is the same as that for proteins from untreated cells it was not shown in Fig. I. This also holds true for the fiber-cell protein pattern (Fig. 2). The incorporation of [14C?amino acids into these proteins is also shown in Fig. I. Incorporation of amino acids into epithelial cell lens proteins could be extensively inhibited by treatment with Io~,g/ml actinomycin D (c]. Fig. I). The specific activity data (Table I) show that incorporation of amino acids into eTABLE I THE
EFFECT
OF ACTINOMYCIN
D
ON LENS
PROTEIN
SYNTHESIS
Epithelial cells (counts~rain per mg protein)
Fiber cells (counts~rain per mg protein)
Control
Actinomycin Inhibition D (io ktg/ml) (%)
Control
Actinomycin Stimulation D (zo tzg/ml) (%)
389 314 166 572
240 235 99 34 °
318 396 165 69o
Homogenate 2597 T-Crystallins 159o ~-Crystallins 963 ~-Crystallins 198o
85 80 83 71
33 68 66 lO3
crystallins was inhibited by 71%,/3-crystallins by 83 % and y-crystallins by 80 %. The same experiments have been performed with the lens fiber cells. An elution pattern of a-, /3- and ),-crystallins from untreated lens-cortex fiber cells is seen in Fig. 2. The incorporation of amino acids into proteins from treated and control fiber cells is also shown. The incorporation of amino acids into lens proteins is significantly greater in the actinomycin-treated cells. A comparison of the specific activity of the e-,/3- and y-crystallins from control and actinomycin-treated lenses shows that there is a significant stimulation of protein synthesis by the antibiotic which ranges from 66 % for the/3-crystallins to lO3 % for the ~-crystallins (Table I). In an earlier report from this laboratory it was shown that lens epithelial cells do not contain ),-crystallins and that the synthesis of this group of proteins is specifically associated with fiber-cell differentiation1°, 11. A similar observation was made in regenerating salamander lens TM. The elution diagram (Fig. I) shows detectable quantities of y-crystallins in the calf lens epithelial cells. However, during the incubation period it was noted that some of the peripheral cells which have entered the stage of elongation to form fiber cells come loose and are removed with the epithelial cell layer. The presence of small amounts of, y-crystallins in epithelial cell homogenates (Fig. i) therefore, appears to be due to the presence of elongating epithelial cells where the y-crystallins first appear. A comparison of the specific activity of actinomycin-treated cells shows an 85 % inhibition of 7-crystallin synthesis in the elongating epithelial cells and a 68 o, /o Biochim. Biophys. dcta, 114 (1966) 428-43 °
430
PRELIMINARY NOTES
stimulation of this same group of proteins in the fiber cells. Thus, at the time of ?J-crystallin appearance the synthesis of this protein as well as of the ~- and flcrystallins, is still sensitive to inhibition by actinomycin D whereas in the completed fiber cell the synthesis of these same proteins is stimulated. The mechanism by which actinomycin D stimulates protein synthesis is unknown. The mechanisms which have been proposed for this effect are as follows: Firstly, the stimulation might be attributed to the availability of more ATP for protein synthesis as a result of the inhibition of RNA synthesis by actinomycin 1. Thus, in the fiber cell the ATP normally used for RNA synthesis could be channeled into the synthesis of the proteins being formed on stable RNA templates. Secondly, this stimulation might be attributed to the inhibition of the synthesis of a repressor protein by actinomycin 6. It has been shown that the actinomycin D stimulation of t r y p t o p h a n pyrrolase and tyrosine transaminase occurs after these enzymes have been induced by hydrocortisone, when their m R N A is relatively stable. By inhibiting the m R N A responsible for the synthesis of repressor protein, the level of this repressor is decreased and a stimulation of these enzymes results. Finally, the lens epithelial cells are essential for the active transport of nutrients into the lens fiber cells. It is therefore possible that actinomycin alters these properties such that there is an increase in the transport of amino acids into the fiber cell layer, resulting in a stimulation of protein synthesis on stable RNA templates. Experiments are now in progress to determine the mechanism of this stimulatory phenomenon. Actinomycin D was a gift from Merck, Sharpe and Dohme, Inc., Rahway, N.J. This work was supported b y a grant from the National Foundation and from the U.S. Public Health Service. One of the authors (J.A.S.) was supported by a predoctoral fellowship of the Institute of Cellular Biology; P.V.K. was supported b y a post-doctoral fellowship of the Institute of Cellular Biology.
The Department o[ Zoology, Institute o/ Cellular Biology, The University o[ Connecticut, Storrs, Conn. (U.S.A.) I 2 3 4 5 6 7 8 9 io II i2
J O H N PAPACONSTANTINOU JAMES A . STEWART PAUL V. KOEHN
1R. POLLOCK, Biochim. Biophys. Acta, 76 (1963) 80. COLEMAN AND W. H. ELLIOTT, Nature, 202 (1964) lO83. M o o a , Science, 144 (1964) 414 . NITOWSKY. S. GELLER AND R. CASPER, Federation Proc., 23 (I964) 556. ROSEN, P. N. RAINA, I~. J. MILHOLLAND AND C. A. NICHOL, Science, 146 (1964) 661. D. GARREN, R. I{. HOWELL, G. ~V[. TOMKINS AND R. M. CROCCO, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1121. J. PAPACONSTANTINOU, P. V. KOEHN AND J. A. STEWART, Am. Zool., 4 (1964) 321. J. PAPACONSTANTINOU, P. V. KOEHN AND J. A. STEWART, Abstr. I48th Meeting Am. Chem. Sot., 4 (1964) 39C. J. PAPACONSTANTINOU, R. A. I~ESNIK AND E. SAITO, Biochim. Biophys. Acta, 60 (1962) 205. J. PAPACONSTANTINOU, Am. Zool., 4 (1964) 279. J. PAPACONSTANTINOU, Biochim. Biophys. Acta, lO 7 (1965) 81. C. TAKATA, J. F. ALBRIGHT AND T. YAMADA, Science, 147 (1965) 1299.
M. G. F. H. F. g.
Received October I l t h , 1965 Biochim. Biophys. Acta, 114 (1966) 428-43o