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Post-transcriptional inhibition of protein synthesis by rifampicin in rat hepatoma cells
Lüe Sciences Vol . 12, Part II, pp . 289-295, 1979 Printed in Great Britain
Pergamon Press
PO6T-TRANSCRIPTIONAL INHIBITION OF PRLYPEIN SYNTHESIS BY RIFAMPICIN TN RAT AEPATOMA CELTS Albert Groasmaa and Aural Boctor Department of Pharmacology, New York University School of Medicine, 550 Firnt Aveirue, New York, New York 10016 (Received 20 September 1972 ; in final form 1 February 1979) sUrn~nRY RIFAMPICTN (rifampin) has been ahowa to inhibit induction of tyrosine amiaotraasferaee by cortisol is rat hepatoma cells (Reuber H-35) grown is culture but not in rat liver. This affect was independent of nay action on RNA polymerase since m change is either the ~1-F- or Mn~/ (Nf~)2S04-activated enzymes of hepatoma cells was noted, Since a 30~ decrease in L[ 14C] leucine incorporation into protein of hepatoma celle was observed is the presence of rifampicin (166 Wg/ml), it ie likely that inhibition of tyrosine aminotraasferase induction occurred at a Bite related to translation rather than to transcription, Introduction Rat hepatoma cells grown in culture have been used ae e model eyatem for studying enzyme regulation in mammalian tissue (1) .
Tyrosine amiaotransferaee
is particularly well suited for such studies since induction of this enzyme with glucocorticoide occurs in these cells in a manner similar to that observed in intact rat liver (2),
Although there are several advantages of
a tissue culture system, one moat always keep in mind that the hapatocyte and hepatoma call do differ .
Two instances of dissimilarity were observed as a
result of our studies concerned with tyrosine aminotraasferaee regulation. While comparing the effects of several RNA polymerise inhibitors oa tyrosine aminotransferase induction by cortieol, it was observed that a-amsaitia, a bicyclic octapeptida derived from the mushroom Amanita phalloidee (3), was e potent inhibitor of tyroaiae aminotraaeferaae induction in rat liver, but was without effect is rat hepatoma cello,
Resistance to
a-amanitin was determined to be due to lack of penetration of this drug into 289
290
Effect of Rifampicin on Protein Synthesis
the hepatoma cell
(4),
Vol . 12, No. 7
Apparently, in the process of traneformatioa the
hepatoma cell membrane was altered so that permeability to a-amanitin (MW 916) was prevented, while permeability to actinomycin-D (MW 1254) remained about the same, An opposite effect, namely, inhibition of tyrosine aminotransferase induction by cortisol in hepatoma cells but not in rat liver, was observed with rifampicin (rifomycin), an antibiotic obtained from Streptomyces mediterranei (5),
This drug was demonstrated to be a potent inhibitor of
bacterial DNA dependent RNA polymerise (6), but was ineffective in inhibiting mammalian RNA polymerise
(7-10),
Transformation in this instance resulted in
either a mutated RNA polymerise that is sensitive to rifampicin, or a change at some other cellular site, previously insensitive to rifampicin, thereby rendering the hepatoma cell sensitive to this drug,
The data presented
demonstrate that rifampicin can inhibit L~ 14 C~leucine incorporation into protein without affecting either the polymerise,
~++-
or Mn~/(NFi4)2 S04-activated RNA
These observations suggest that a post-transcriptional site in
hepatoma cells has become sensitive to rifampicin,
Materials and Methods At the time this work began the hepatoma cell line (derived from Reuber H-35 rat hepatoma cells) had been subcultured weekly for over three years, The growth characteristics and procedures cells have been described previously activity
(11),
for culturing and harvesting these Tyrosine amiaotransferase
(12 ), DNA (13), and protein (14) were determined by standard
procedures, Determination of RNA polymerise activity ,
RNA polymerise was assayed in
cell nuclei obtained by combining the cells of 10-12 similarly treated cultures,
At the time of harvest each culture bottle contained about 25 mg
cells wet weight,
After washing the hepatoma cells 3 times with 0,1 M
sodium phosphate buffer
(pH 7,5) and once with a 0,32 M sucrose solution
Effect ad Rifampicin on Protein Synthesis
Vol. 12, No . 7 containing
0 .003 M MgC12 ,
~ad eoaicated for
5
they were suspended in
2
29 1
ml of the latter solution
sec with a Bronson sonifiar. The broken cells (no
unbroken calls were noted by light microscopy) ware centrifuged for
5
min at
about 4,000 a, g, The residue which was considered the hepatoma call miclear fraction was euspeadad is appropriate reaction mixtures and assayed for I~~and Mn~/(NH4 ) 2 S04 -activated RNA polymerise as outlined by Widaell and Tat~ (15). In the present experiments incorporation of radioactive uridiae triphosphate was measured (~II- 14CJUTP Na4 , specific activity 16 .6 WCi/mole, 2 .5
~Ci/ml obtained from New England Nuclear, Boston), Bach incubation
miaure contained
0,25 ~,Ci
in a total w lame of
0,5
ml. Assays for Mg~-
and Mn~/(NH4 ) 2 S0 4 -activated RNA polymarese were carried out for 20 and
45
min, respectively . Initially all tubes were kept at 0° for 5 min to allow for adequate mizing of the incubation components, After this period (zero time) some tubes were placed is a Dubnoff incubator at
37°
at 0° C,
2
Reactions ware terminated by the addition of
ml 0,5 N parchloric
min at 0° C the precipitates were collected by centrifugation
acid . After
30
sad washed
times with
3
sad others kept
were dissolved is
0,2
3
ml of
0 .25 N
parchloric acid, The final residues
ml NCS (Amereham/Searle) sad quantitatively transferred
to cotmtiag vials using a toluene-alcohol scintillation system (16), Cortisôl (solu-Cortef, hydrocortisone semi-succinate) was obtained from the Uphohn Co . Results and Discussion The data illustrated in Figure 1 indicate that induction of tyrosine
amiaotraneferasa was inhibited 50~ at a concentration of 80 ug rifampicin/ml culture medium . It should be stressed that this effect occurred within 7
hours sad is sot associated with degenerative changes is these cultures .
Rifampicia
(100-150
appear
days after drug exposure (17) . This effect of rifampicin is sot
3-4
Wg/ml) does cause general cell toxicity but these effects
due to say direct action on tyrosine amiaotransferase since concentrations
29 2
Effect of Rifa.mpicin on Protein Synthesis
Vol . 12, No . 7
Inhibition by Rifampicin of Tyrosine Aminotransferase Induction by Cortisol is Rat Hepatoma Celle Grown in Culture loo
1
SO
~91D
100
400
rif~mpicln (yp/ml)
FIG, 1 A aeries of culture bottles containing about 3 x 10~ cells each were ezpoaed to va~ious concentrations of rifampicia, Thirty minutes later cortisol (10- I~ was added to these cultures and incubation continued for as additional 7 hrs, At this time the cells were harvested and analysed for tyrosine aminotransferase activity and DNA content, Tyrosine aminotraneferase activity was expressed as ~molee p-hydroxypheaylpyruvate formed per hour per 100 F+g DNA, On the ordinate, 100X control induction ie considered that activity is the cortisol treated cultures minis enzyme activity is cultures not treated with hormone, Activity in the rifampicin treated cultures, expressed as a per cent of control induction, was calculated as follows : (rifampicin plus cortisol activity control activity/cortisol activity control activity) a 100, Each point represents the average activity of duplicate cultures, as high ae 500 Wg/ml do not alter enzyme activity is vitro , In a series of e:perimente using hepatoma cell nuclei, neither the t~~nor Mn~/(NH4)2504activated RNA polymerasa was inhibited by rifampicin at concentrations as high as 166 Wg/ml (Table 1),
Also, despite a statistically
significant enhancement of ~2-14Clruracil uptake (HC104 soluble counts, Table 2), whole cell studies showed that rifampicin did not inhibit ~2- 14~uracil incorporation into hepatoma RNA.
Then, by what machaaism did
rifampicia inhibit induction of tyrosine aminotransferase by cortisol?
Vol. 12, No . 7
Effect of Rifampicin on Protein Synthesis
29 3
TABLE 1 Effect of Rifampicia on Rat Hapatoma Mgt- and Mn~/(NH4)2S04-Activated RNA Polymerise Treatment _in viw (in culture)
Treatment in vitro
rifampicia (166 Wg/ml) cortieol
(lÔ6M)
cortieol (10-6 M) + rifampicin (166 wg/ml)
~C
Mgt Mn~/(NH4)2304 ,moles UTP incorporated Per ~ DNA_
Relative TA activity
37 0
1,080 60
2,040 190
37
1,020
1,960
37 0
1,070 50
1,820 220
5,7
37 0
930 40
1,870 230
2,6
1,0
Rifampicia was added to cultures 30 min prior to cortieol, The cells ware harvested 7 hrs after cortieol administration, The supernatant of the nuclear fraction was centrifuged at 18,000 x g and assayed for tyrosine aminotraasferase activity, Each value represents the average of duplicate assays, These results are representative of three such eaperiments,
TABLE 2 Effect of Rifampicin oa 2-L
14 ~
-Uracil Iacorporation Into Rat Hepatoma RNA
TA activity moles/hr per 100 N, g DNA Control Cortisol
(106M)
Rifampicin (166 Wg/ml) + cortiso1~10 -6M) -
HC104 soluble cpm/100 Wg DNA
HC104 insoluble cpm/100 wg DNA
0,8 ± 0,05
490 ± 10
139 ±
3,0 ± 0,05
450 ± 65
137 ± 12
1,4 _+ 0,08
735 ± 45
135 ±
7
4
Triplicate hapatoma cultures were ea~loyed for each of the experimental conditions listed, Cortical (10-6 M) was added to the cultures 30 mia after rifampicin treatment, Three hours after cortieol addition 2 p,Ci of 2-[14C] uracil was added (33,3 WCi/Wmole, 50 ~Ci/ml) aid incubation continued for as additional 30 min, At this time the calls were harvested, washed three times with 5 ml wlumes of 0,1 N sodium phosphate buffer, pH 7,5, and sonicated for 5 sec in 1 ml saline, To a 0,5 ml aliquot was added 0,5 ml of 1 N perchloric acid, After 30 min at 0° the precipitate was separated by centrifugation . A 0,2 ml aliquot of the supernatant was aouated (SC104 soluble counts) and the precipitate washed three times with 5 ml volumes of 0,25 N perchloric acid, The final residue was dissolved in Protocol (Nee England Nuclear Carp,) and counted (HC104 insoluble counts), Deviations are expressed se ± standard error,
Vol. 12, No . 7
Effect of Rüampicin on Protein Synthesis
29 4
The data in Table 3 indicate that rifampicin inhibited tyrosine aminotraaeferase induction by about 70% and L~ 14C~leucine incorporation into total protein (TCA insoluble counts) by about 30%,
It should be emphasized
once again that these effects occurred within 7 hrs after rifampicin administration when no change is RNA polymerise or RNA synthesis was detectable,
Inhibition of protein synthesis did not occur as a result of
diminished amino acid uptake since the TCA soluble counts, which reflect amino acid penetration, were the same in both the treated and control groups of cells, TABLE 3 Inhibition by Rifampicin of L[ 14 C~Leucine Incorporation Into Protein Of Rat Hepatoma Cells Grown in Culture TA activity ,moles/hr
TCA TCA insoluble soluble cpm/mg protein
Protein DNA mg
Wg
Cortisol, 10-6 M (4)
3,7+ 0,30
2,04
131
7,200±350
3,660±490
Cortisol, 10-6 M (4) + rifampicin (166 Wg/ml)
1,7±0,07*
2,30
142
5,100±450*
3,300±470
0,4 ~tCi L[14C]leucine (specific activity 250 Ci/mole) was added to the cultures one hour prior to harvesting of the cells (which took place 7 hrs after cortisol addition), The figures is parentheses indicate the number of cultures employed for each experimental procedure, The methods used to determine trichloracetic acid (TCElinsoluble and soluble counts were described previously (14), Deviations are expressed as ± standard error, * indicates a significant difference between the rifampicin treated any control cultures, It is likely that the disproportionate inhibition of tyrosine aminotraasferase as compared to total protein is related to the very short halflife (3-7 hrs) of this en:yma is hepatoma cells (18),
If all protein
synthesis is inhibited equally by rifampicin, those proteins which turn over moat rapidly would be the first to decline is activity (assuming that the processes responsible for inactivation are unaffected), To our knowledge this is the first demonstration that rifampicin can
Vol. 12, No . 7
Effect of Rifampicin on Protein Synthesis
295
inhibit induction of enzyme synthesis at a poet-tranecriptional site in mammalian derived cells, This work was supported by USPHS Graata GM 13728 and GM 07213, References 1,
H,C, Pitot, C, Peraino, P,A, Morse, and V,R, Potter, Nat, Cancer Last , MOnogr , 13, 229-245 (1964),
2,
E,B . Thompson, G,M, Tomkins, and J,F, Cutten, Proc, Nat, Acad, Sc i, USA 56, 296-303 (1966),
3,
T, Wieland, Science 159, 946-952 (1968),
4.
A, Boctor and A, Grossuran, Biochem, Pharmacol , (in press),
5,
N, Maggi, V, Arioli, and P. Sens i, J. Med, Chem . 8, 790-793 (1965),
6,
G, Iiartman, R,O, Honikel, F, Rnusel, and J, Nuesch, Biochim, Biophve, Acta 145, 843-844 (1967),
7,
W, Wehrli, J, Nuesch, F, Kausal, and M, Stechahn, Biochim, Biophve , Acta 157, 215-217 (1968),
8,
J,J, Furth and G,S, Austin, Cold Spring Harbor Sump . Quant, Biol , 35, 641-648 (1970), -
9.
B, Sugden and J, Sambrook, Cold Spring Harbor Symp, Quant, Biol , 35, 663-669 (1970),
10,
S,T, Jacob, S,M, Sajdel, and H,N, Mnnro, Biochem. Biophve, Res . Common , 32, 831-838 (1968),
11,
D, Mendelson, A. Grossman, and A, Boctor, Sur, J. Biochem, 24, 140-148 (1971), -
12,
E,C .C, Lin, B,M, Pitt, M, Civen, and W,S . Knox, J. Biol . Chem, 233, 668-673 (1958),
13,
A, Grosaman and C, Mavrides, J, Biol, Chem , 243,
14,
O,H, Lowry, N,J. Roaebrough, A,L, Fart, and R,J, Randall, J. Biol . Chem, 193, 265-275 (1951) .
15,
C,C, Widnell and J,R, Tata, Biothun, Biophve, Acta 123, 478-492 (1966),
16,
A, Boctor aad A, Grossman, J, Biol, Chem, 245, 6337-6345 (1970),
17 .
J,H, Subak-Sharpe, M,C, Timbury, and J,F, Williams, Nature 222, 341-345 (1969),
18,
F, Auricchio, D. Martin, and G. Tomkias, Natura 224. 806-808 (1969),
1398-1405 (1967),
Pergamon Press
PO6T-TRANSCRIPTIONAL INHIBITION OF PRLYPEIN SYNTHESIS BY RIFAMPICIN TN RAT AEPATOMA CELTS Albert Groasmaa and Aural Boctor Department of Pharmacology, New York University School of Medicine, 550 Firnt Aveirue, New York, New York 10016 (Received 20 September 1972 ; in final form 1 February 1979) sUrn~nRY RIFAMPICTN (rifampin) has been ahowa to inhibit induction of tyrosine amiaotraasferaee by cortisol is rat hepatoma cells (Reuber H-35) grown is culture but not in rat liver. This affect was independent of nay action on RNA polymerase since m change is either the ~1-F- or Mn~/ (Nf~)2S04-activated enzymes of hepatoma cells was noted, Since a 30~ decrease in L[ 14C] leucine incorporation into protein of hepatoma celle was observed is the presence of rifampicin (166 Wg/ml), it ie likely that inhibition of tyrosine aminotraasferase induction occurred at a Bite related to translation rather than to transcription, Introduction Rat hepatoma cells grown in culture have been used ae e model eyatem for studying enzyme regulation in mammalian tissue (1) .
Tyrosine amiaotransferaee
is particularly well suited for such studies since induction of this enzyme with glucocorticoide occurs in these cells in a manner similar to that observed in intact rat liver (2),
Although there are several advantages of
a tissue culture system, one moat always keep in mind that the hapatocyte and hepatoma call do differ .
Two instances of dissimilarity were observed as a
result of our studies concerned with tyrosine aminotraasferaee regulation. While comparing the effects of several RNA polymerise inhibitors oa tyrosine aminotransferase induction by cortieol, it was observed that a-amsaitia, a bicyclic octapeptida derived from the mushroom Amanita phalloidee (3), was e potent inhibitor of tyroaiae aminotraaeferaae induction in rat liver, but was without effect is rat hepatoma cello,
Resistance to
a-amanitin was determined to be due to lack of penetration of this drug into 289
290
Effect of Rifampicin on Protein Synthesis
the hepatoma cell
(4),
Vol . 12, No. 7
Apparently, in the process of traneformatioa the
hepatoma cell membrane was altered so that permeability to a-amanitin (MW 916) was prevented, while permeability to actinomycin-D (MW 1254) remained about the same, An opposite effect, namely, inhibition of tyrosine aminotransferase induction by cortisol in hepatoma cells but not in rat liver, was observed with rifampicin (rifomycin), an antibiotic obtained from Streptomyces mediterranei (5),
This drug was demonstrated to be a potent inhibitor of
bacterial DNA dependent RNA polymerise (6), but was ineffective in inhibiting mammalian RNA polymerise
(7-10),
Transformation in this instance resulted in
either a mutated RNA polymerise that is sensitive to rifampicin, or a change at some other cellular site, previously insensitive to rifampicin, thereby rendering the hepatoma cell sensitive to this drug,
The data presented
demonstrate that rifampicin can inhibit L~ 14 C~leucine incorporation into protein without affecting either the polymerise,
~++-
or Mn~/(NFi4)2 S04-activated RNA
These observations suggest that a post-transcriptional site in
hepatoma cells has become sensitive to rifampicin,
Materials and Methods At the time this work began the hepatoma cell line (derived from Reuber H-35 rat hepatoma cells) had been subcultured weekly for over three years, The growth characteristics and procedures cells have been described previously activity
(11),
for culturing and harvesting these Tyrosine amiaotransferase
(12 ), DNA (13), and protein (14) were determined by standard
procedures, Determination of RNA polymerise activity ,
RNA polymerise was assayed in
cell nuclei obtained by combining the cells of 10-12 similarly treated cultures,
At the time of harvest each culture bottle contained about 25 mg
cells wet weight,
After washing the hepatoma cells 3 times with 0,1 M
sodium phosphate buffer
(pH 7,5) and once with a 0,32 M sucrose solution
Effect ad Rifampicin on Protein Synthesis
Vol. 12, No . 7 containing
0 .003 M MgC12 ,
~ad eoaicated for
5
they were suspended in
2
29 1
ml of the latter solution
sec with a Bronson sonifiar. The broken cells (no
unbroken calls were noted by light microscopy) ware centrifuged for
5
min at
about 4,000 a, g, The residue which was considered the hepatoma call miclear fraction was euspeadad is appropriate reaction mixtures and assayed for I~~and Mn~/(NH4 ) 2 S04 -activated RNA polymerise as outlined by Widaell and Tat~ (15). In the present experiments incorporation of radioactive uridiae triphosphate was measured (~II- 14CJUTP Na4 , specific activity 16 .6 WCi/mole, 2 .5
~Ci/ml obtained from New England Nuclear, Boston), Bach incubation
miaure contained
0,25 ~,Ci
in a total w lame of
0,5
ml. Assays for Mg~-
and Mn~/(NH4 ) 2 S0 4 -activated RNA polymarese were carried out for 20 and
45
min, respectively . Initially all tubes were kept at 0° for 5 min to allow for adequate mizing of the incubation components, After this period (zero time) some tubes were placed is a Dubnoff incubator at
37°
at 0° C,
2
Reactions ware terminated by the addition of
ml 0,5 N parchloric
min at 0° C the precipitates were collected by centrifugation
acid . After
30
sad washed
times with
3
sad others kept
were dissolved is
0,2
3
ml of
0 .25 N
parchloric acid, The final residues
ml NCS (Amereham/Searle) sad quantitatively transferred
to cotmtiag vials using a toluene-alcohol scintillation system (16), Cortisôl (solu-Cortef, hydrocortisone semi-succinate) was obtained from the Uphohn Co . Results and Discussion The data illustrated in Figure 1 indicate that induction of tyrosine
amiaotraneferasa was inhibited 50~ at a concentration of 80 ug rifampicin/ml culture medium . It should be stressed that this effect occurred within 7
hours sad is sot associated with degenerative changes is these cultures .
Rifampicia
(100-150
appear
days after drug exposure (17) . This effect of rifampicin is sot
3-4
Wg/ml) does cause general cell toxicity but these effects
due to say direct action on tyrosine amiaotransferase since concentrations
29 2
Effect of Rifa.mpicin on Protein Synthesis
Vol . 12, No . 7
Inhibition by Rifampicin of Tyrosine Aminotransferase Induction by Cortisol is Rat Hepatoma Celle Grown in Culture loo
1
SO
~91D
100
400
rif~mpicln (yp/ml)
FIG, 1 A aeries of culture bottles containing about 3 x 10~ cells each were ezpoaed to va~ious concentrations of rifampicia, Thirty minutes later cortisol (10- I~ was added to these cultures and incubation continued for as additional 7 hrs, At this time the cells were harvested and analysed for tyrosine aminotransferase activity and DNA content, Tyrosine aminotraneferase activity was expressed as ~molee p-hydroxypheaylpyruvate formed per hour per 100 F+g DNA, On the ordinate, 100X control induction ie considered that activity is the cortisol treated cultures minis enzyme activity is cultures not treated with hormone, Activity in the rifampicin treated cultures, expressed as a per cent of control induction, was calculated as follows : (rifampicin plus cortisol activity control activity/cortisol activity control activity) a 100, Each point represents the average activity of duplicate cultures, as high ae 500 Wg/ml do not alter enzyme activity is vitro , In a series of e:perimente using hepatoma cell nuclei, neither the t~~nor Mn~/(NH4)2504activated RNA polymerasa was inhibited by rifampicin at concentrations as high as 166 Wg/ml (Table 1),
Also, despite a statistically
significant enhancement of ~2-14Clruracil uptake (HC104 soluble counts, Table 2), whole cell studies showed that rifampicin did not inhibit ~2- 14~uracil incorporation into hepatoma RNA.
Then, by what machaaism did
rifampicia inhibit induction of tyrosine aminotransferase by cortisol?
Vol. 12, No . 7
Effect of Rifampicin on Protein Synthesis
29 3
TABLE 1 Effect of Rifampicia on Rat Hapatoma Mgt- and Mn~/(NH4)2S04-Activated RNA Polymerise Treatment _in viw (in culture)
Treatment in vitro
rifampicia (166 Wg/ml) cortieol
(lÔ6M)
cortieol (10-6 M) + rifampicin (166 wg/ml)
~C
Mgt Mn~/(NH4)2304 ,moles UTP incorporated Per ~ DNA_
Relative TA activity
37 0
1,080 60
2,040 190
37
1,020
1,960
37 0
1,070 50
1,820 220
5,7
37 0
930 40
1,870 230
2,6
1,0
Rifampicia was added to cultures 30 min prior to cortieol, The cells ware harvested 7 hrs after cortieol administration, The supernatant of the nuclear fraction was centrifuged at 18,000 x g and assayed for tyrosine aminotraasferase activity, Each value represents the average of duplicate assays, These results are representative of three such eaperiments,
TABLE 2 Effect of Rifampicin oa 2-L
14 ~
-Uracil Iacorporation Into Rat Hepatoma RNA
TA activity moles/hr per 100 N, g DNA Control Cortisol
(106M)
Rifampicin (166 Wg/ml) + cortiso1~10 -6M) -
HC104 soluble cpm/100 Wg DNA
HC104 insoluble cpm/100 wg DNA
0,8 ± 0,05
490 ± 10
139 ±
3,0 ± 0,05
450 ± 65
137 ± 12
1,4 _+ 0,08
735 ± 45
135 ±
7
4
Triplicate hapatoma cultures were ea~loyed for each of the experimental conditions listed, Cortical (10-6 M) was added to the cultures 30 mia after rifampicin treatment, Three hours after cortieol addition 2 p,Ci of 2-[14C] uracil was added (33,3 WCi/Wmole, 50 ~Ci/ml) aid incubation continued for as additional 30 min, At this time the calls were harvested, washed three times with 5 ml wlumes of 0,1 N sodium phosphate buffer, pH 7,5, and sonicated for 5 sec in 1 ml saline, To a 0,5 ml aliquot was added 0,5 ml of 1 N perchloric acid, After 30 min at 0° the precipitate was separated by centrifugation . A 0,2 ml aliquot of the supernatant was aouated (SC104 soluble counts) and the precipitate washed three times with 5 ml volumes of 0,25 N perchloric acid, The final residue was dissolved in Protocol (Nee England Nuclear Carp,) and counted (HC104 insoluble counts), Deviations are expressed se ± standard error,
Vol. 12, No . 7
Effect of Rüampicin on Protein Synthesis
29 4
The data in Table 3 indicate that rifampicin inhibited tyrosine aminotraaeferase induction by about 70% and L~ 14C~leucine incorporation into total protein (TCA insoluble counts) by about 30%,
It should be emphasized
once again that these effects occurred within 7 hrs after rifampicin administration when no change is RNA polymerise or RNA synthesis was detectable,
Inhibition of protein synthesis did not occur as a result of
diminished amino acid uptake since the TCA soluble counts, which reflect amino acid penetration, were the same in both the treated and control groups of cells, TABLE 3 Inhibition by Rifampicin of L[ 14 C~Leucine Incorporation Into Protein Of Rat Hepatoma Cells Grown in Culture TA activity ,moles/hr
TCA TCA insoluble soluble cpm/mg protein
Protein DNA mg
Wg
Cortisol, 10-6 M (4)
3,7+ 0,30
2,04
131
7,200±350
3,660±490
Cortisol, 10-6 M (4) + rifampicin (166 Wg/ml)
1,7±0,07*
2,30
142
5,100±450*
3,300±470
0,4 ~tCi L[14C]leucine (specific activity 250 Ci/mole) was added to the cultures one hour prior to harvesting of the cells (which took place 7 hrs after cortisol addition), The figures is parentheses indicate the number of cultures employed for each experimental procedure, The methods used to determine trichloracetic acid (TCElinsoluble and soluble counts were described previously (14), Deviations are expressed as ± standard error, * indicates a significant difference between the rifampicin treated any control cultures, It is likely that the disproportionate inhibition of tyrosine aminotraasferase as compared to total protein is related to the very short halflife (3-7 hrs) of this en:yma is hepatoma cells (18),
If all protein
synthesis is inhibited equally by rifampicin, those proteins which turn over moat rapidly would be the first to decline is activity (assuming that the processes responsible for inactivation are unaffected), To our knowledge this is the first demonstration that rifampicin can
Vol. 12, No . 7
Effect of Rifampicin on Protein Synthesis
295
inhibit induction of enzyme synthesis at a poet-tranecriptional site in mammalian derived cells, This work was supported by USPHS Graata GM 13728 and GM 07213, References 1,
H,C, Pitot, C, Peraino, P,A, Morse, and V,R, Potter, Nat, Cancer Last , MOnogr , 13, 229-245 (1964),
2,
E,B . Thompson, G,M, Tomkins, and J,F, Cutten, Proc, Nat, Acad, Sc i, USA 56, 296-303 (1966),
3,
T, Wieland, Science 159, 946-952 (1968),
4.
A, Boctor and A, Grossuran, Biochem, Pharmacol , (in press),
5,
N, Maggi, V, Arioli, and P. Sens i, J. Med, Chem . 8, 790-793 (1965),
6,
G, Iiartman, R,O, Honikel, F, Rnusel, and J, Nuesch, Biochim, Biophve, Acta 145, 843-844 (1967),
7,
W, Wehrli, J, Nuesch, F, Kausal, and M, Stechahn, Biochim, Biophve , Acta 157, 215-217 (1968),
8,
J,J, Furth and G,S, Austin, Cold Spring Harbor Sump . Quant, Biol , 35, 641-648 (1970), -
9.
B, Sugden and J, Sambrook, Cold Spring Harbor Symp, Quant, Biol , 35, 663-669 (1970),
10,
S,T, Jacob, S,M, Sajdel, and H,N, Mnnro, Biochem. Biophve, Res . Common , 32, 831-838 (1968),
11,
D, Mendelson, A. Grossman, and A, Boctor, Sur, J. Biochem, 24, 140-148 (1971), -
12,
E,C .C, Lin, B,M, Pitt, M, Civen, and W,S . Knox, J. Biol . Chem, 233, 668-673 (1958),
13,
A, Grosaman and C, Mavrides, J, Biol, Chem , 243,
14,
O,H, Lowry, N,J. Roaebrough, A,L, Fart, and R,J, Randall, J. Biol . Chem, 193, 265-275 (1951) .
15,
C,C, Widnell and J,R, Tata, Biothun, Biophve, Acta 123, 478-492 (1966),
16,
A, Boctor aad A, Grossman, J, Biol, Chem, 245, 6337-6345 (1970),
17 .
J,H, Subak-Sharpe, M,C, Timbury, and J,F, Williams, Nature 222, 341-345 (1969),
18,
F, Auricchio, D. Martin, and G. Tomkias, Natura 224. 806-808 (1969),
1398-1405 (1967),