(Received for publication, December 13, 1994; and in revised form, March 8, 1995)
From the
Several ribonucleases serve as cytotoxic agents in host defense
and in physiological cell death pathways. Although certain members of
the pancreatic ribonuclease A superfamily can be toxic when applied to
the outside of cells, they become thousands of times more toxic when
artificially introduced into the cytosol, indicating that
internalization is the rate-limiting step for cytotoxicity. We have
used three agents that disrupt the Golgi apparatus by distinct
mechanisms, retinoic acid, brefeldin A, and monensin, to probe the
intracellular pathways ribonucleases take to reach the cytosol.
Retinoic acid and monensin potentiate the cytotoxicity of bovine
seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times
or more. Retinoic acid-mediated potentiation of ribonucleases is
completely blocked by brefeldin A. Ribonucleases appear to route more
efficiently into the cytosol through the Golgi apparatus disrupted by
monensin or retinoic acid. Intracellular RNA degradation by BS-RNase
increased more than 100 times in the presence of retinoic acid
confirming that the RNase reaches the cytosol and indicating that
degradation of RNA is the intracellular lesion causing toxicity. As
retinoic acid alone and Onconase are in clinical trials for cancer
therapy, combinations of RNases and retinoic acid in vivo may
offer new clinical utility.
Ribonucleases serve as selective cytotoxic agents in host
defense and physiological cell death pathways in bacteria, higher
plants, and mammals (reviewed in (1) and (2) ), and
they have potential use as therapeutic agents for human disorders
either alone (3, 4) or after conjugation to targeting
molecules(5, 6) . They appear to bind cell surface
receptors and enter the cytosol where they degrade RNA to kill the
target cell(1) .
Bovine seminal ribonuclease, a homodimer
purified from bull semen with 80% amino acid sequence homology to RNase
A, is toxic to certain mammalian cells in culture and expresses
anti-cancer activity in animal models(7, 8) .
Retinoic acid and monensin disrupt the Golgi
apparatus by distinct mechanisms, yet both potentiate the cytotoxicity
of ricin A chain immunotoxins(14) . These agents appear to
alter the intracellular trafficking of certain protein toxins and
facilitate their transport into the cytosol. Here we report an
investigation of the intracellular route ribonucleases take to the
cytosol.
Figure 1:
The sequence of the human
pancreatic RNase A gene synthesized using an E. coli codon
bias.
Figure 2:
Cytotoxicity of bovine seminal RNase to 9L
cells in the absence or presence of retinoic acid or monensin. 9L cells
(2
Figure 3:
Cell viability after treatment with
BS-RNase dimer in the absence or presence of retinoic acid. A,
cells in 96-well plates were treated with increasing concentrations of
BS-RNase for 6 h with (
Figure 4:
RNA
degradation in 9L cells by BS-RNase in the presence of retinoic acid versus in the absence of retinoic acid. 9L cells were cultured
in 75-cm
Figure 5:
Cytotoxicity of Onconase, human
angiogenin, and RNase A in the absence or presence of retinoic acid. 9L
cells in 96-well plates were incubated with increasing concentrations
of specified RNases in the absence or presence of 10 µM retinoic acid for 16 h in leucine-free RPMI 1640 medium. Then
cells were pulsed, harvested, and counted as described in Fig. 2.
Figure 6:
Brefeldin A blocks
all-trans-retinoic acid-potentiated cytotoxicity of BS-RNase
and Onconase. 9L cells were incubated with increasing concentrations of
either BS-RNase (A) or Onconase (B) at 37 °C with
or without 10 µM retinoic acid in the presence or absence
of 4 µg/ml brefeldin A. After 16 h, protein synthesis was assayed
as described for Fig. 2.
Ribonucleases have potential as therapeutic agents for a
number of human disorders including cancer and
HIV(1, 2, 3, 4, 5, 6) .
Certain members of the ribonuclease family can be used as a toxin
moiety to construct chimeric or fusion proteins for targeted therapy (5, 6, 12) and some members of the RNase
family, such as Onconase, are cytotoxic by themselves(18) . How
RNases kill cells is poorly understood. It has been proposed that the
mechanism of cytotoxic RNases resembles that of plant and bacterial
toxins to some degree whereby the RNases bind the mammalian cell
surface, enter into the cell cytosol, and degrade RNA resulting in cell
death(18) .
It has been demonstrated recently
that retinoic acid specifically disrupts the Golgi
apparatus(14) . Golgi-specific staining with mannosidase II (28) and NBD-ceramide (29, 30) was reduced by
treating cells with 10 µM retinoic acid; after removal of
the retinoic acid, the Golgi apparatus staining rapidly reappeared as
normal. By electron microscopy, the Golgi apparatus appeared swollen in
the presence of retinoic acid and rapidly assumed normal morphology
upon removal of retinoic acid. The morphology of the Golgi apparatus
under electron microscopy in the presence of retinoic acid resembled
that of monensin-treated cells in contrast to NBD-ceramide staining of
cells where retinoic acid-treated cells were markedly different from
monensin-treated cells. Retinoic acid and monensin increase the
cytotoxicity of ricin A chain immunotoxins by increasing the
intracellular routing of the toxins to the cytosol(14) . A
variety of data suggest that ricin immunotoxins route through the Golgi
apparatus on the way to the cytosol.
We examined the effect of
retinoic acid and monensin on the cytotoxicity of a series of
homologous ribonucleases. Retinoic acid and monensin increased the
cytotoxicity of all four members of the ribonuclease family examined.
This indicates that ribonucleases reach the cytosol after endocytosis
and do not cross directly across the plasma membrane. These results
also indicate that ribonucleases can reach the cytosol more efficiently
when routed through a disrupted Golgi apparatus, and that, normally,
transmembrane transport of the ribonucleases is rate-limiting for
cytotoxicity.
The sensitization of cells to ribonucleases by
retinoic acid is completely blocked by BFA. BFA blocks the vesicular
transport from the ER to the cis-Golgi apparatus causing a
collapse of the cis-Golgi and a termination of the retrograde
vesicular transport from the cis-Golgi back to the
ER(27, 28) . The block of the retinoic acid
potentiation by BFA suggests that retinoic acid stimulates transport of
the ribonucleases through the cis-Golgi, possibly to the ER,
en route to the cytosol. BFA has also been shown to affect endosomes,
lysosomes, and the trans-Golgi
network(9, 31) , and we cannot ascertain at this time
which of the cellular effects of BFA cause the blockage of ribonuclease
potentiation by retinoic acid. BFA, however, does not block the
cytotoxicity caused by Onconase itself, indicating that the pathway
through which Onconase goes to the cytosol in the absence of retinoic
acid is qualitatively different from that of the retinoic acid-induced
intracellular routing of ribonucleases.
Previous experiments
indicate that cytotoxic RNases can enter cells and cause RNA
degradation to effect cell cytotoxicity and death (18) .
It has been shown that dimerization of the bovine seminal
ribonuclease is necessary for its anti-cancer activity both in cell
culture and in animal models(23) . However, we found that two
monomers of the bovine seminal ribonuclease showed similar cytotoxicity
to 9L cells in the presence of retinoic acid compared with dimerized
BS-RNase. This indicates that monomer forms of bovine seminal
ribonuclease cannot efficiently get into the cytosol by themselves,
but, once in the cytosol, they can be almost as toxic as the dimer.
Perhaps the dimer form of BS-RNase functions to route the RNase through
the Golgi to effect more efficient entry into the cytosol compared to
the monomer. It is interesting that the human RNase A, another monomer
form of RNase that is 73% identical in sequence with BS-RNase monomer,
is 300 times less toxic in the presence of retinoic acid than are the
BS-RNase monomers. The reason for the large biological difference
resulting from the small sequence difference may be a key to further
understanding the mechanism of cytotoxicity of ribonucleases.
Onconase is in clinical trials for cancer therapy(3) , and
BS-RNase has interesting anti-cancer activity in animal
models(7, 8) . Retinoic acid is also in several
experimental trials for cancer therapy. Retinoic acid caused
potentiation of BS-RNase cytotoxicity results in greater protein
synthesis inhibition and cell death than in the absence of retinoic
acid. Combination of RNases and retinoic acid in vivo may
improve the clinical utility of ribonucleases.
We are grateful to Prof. Matteo Adinolfi for his
supervision in chemical synthesis, to Dr. Renata Piccoli for
discussions, and to Patricia Johnson for her technical
assistance.
Note Added in Proof-After completion of
this manuscript, an interesting study of BS-RNase toxicity was
published (32) .
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)Another member of the RNase A superfamily, Onconase,
isolated from frog eggs(10) , also expresses anti-tumor
activity in animals and is now in phase II clinical trials for cancer
therapy(3) . Angiogenin, originally purified based upon
angiogenesis activity(11) , is also homologous to ribonuclease
A and can be cytotoxic when fused with targeting
molecules(12) . Although these RNases can be toxic when applied
to the outside of cells, RNases become thousands of times more toxic
when artificially introduced into the cytosol(13) , indicating
that internalization is the rate-limiting step. The pathways
ribonucleases take to cross the membrane surrounding the cytosol
remains unknown.
Materials
All-trans-retinoic acid was
purchased from Calbiochem; brefeldin A was from Sigma; bovine seminal
ribonuclease was purified from bull semen or seminal vesicles as
reported(15) ; and Onconase was isolated from Rana pipiens eggs as described(10) ; alkylated Onconase was prepared as
described (16) with 98% of the ribonuclease activity being
inactivated; the catalytically active monomeric derivatives of
BS-RNase,(
)MSSR (monomeric Cys-31,
32-S-ethylamine-BS-RNase), and MCM (monomeric Cys-31,
32-S-carboxymethyl-BS-RNase) were prepared by stably blocking
the sulfhydryls exposed after selective reduction with dithiothreitol
of the intersubunit disulfides as described previously (17) .
Cell Line and Protein Synthesis Assay
9L (rat
glioma) cells were grown in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum, 2 mM glutamine, 1
mM sodium pyruvate, 0.1 mM nonessential amino acids,
and 10 µg/ml gentamicin. Protein synthesis inhibition by RNases was
determined as described previously(14, 18) . Briefly,
cells in 100 µl were plated at concentrations of 2 10
cells/ml in 96-well microtiter plates overnight in
Dulbecco's modified Eagle's complete medium. Retinoic acid
(15 mM in dimethyl sulfoxide), monensin (2 mM in
ethanol), and brefeldin A (BFA, 10 mg/ml in ethanol) stock solutions
were diluted into leucine-free RPMI 1640 medium without fetal calf
serum to the appropriate concentrations. The same amounts of dimethyl
sulfoxide and/or ethanol were added in the control solutions. After
removing the complete Dulbecco's modified Eagle's medium,
cells were incubated in the above leucine-free RPMI 1640 medium
containing increasing concentrations of ribonucleases with or without
retinoic acid or monensin and/or BFA for 16 h followed by a 1-h pulse
with 0.1 µCi of [
C]leucine. Cells were
harvested onto glass fiber filters using a PHD cell harvester, washed
with water, dried with ethanol, and counted. The results were expressed
as the percentage of [
C]leucine incorporation in
mock-treated control cells.
Cell Viability Assay with Trypan Blue
9L cells
cultured in 96-well plates were incubated with increasing
concentrations of BS-RNase in leucine-free RPMI 1640 medium with or
without 10 µM retinoic acid for either 6 h or 16 h. Cells
were then trypsinized by adding trypsin in each well without removing
the incubation medium, resuspended, mixed with trypan blue, and
counted. Cell viability was calculated as the percentage of trypan blue
excluding cells with respect to the total cell counts.
Total RNA Extraction and Electrophoresis
9L cells
(1 10
cells/ml) cultured in 75-cm
flasks were treated with increasing concentrations of BS-RNase in
leucine-free RPMI 1640 medium with or without 10 µM retinoic acid. After 6 h, cells were trypsinized, washed, and
processed for total RNA isolation using the RNAzol
method
supplied by TEL-TEST, Inc. Briefly, cells were homogenized in
RNAzol
(2 ml of RNAzol
per 1
10
cells), RNA was then extracted with 0.1 volume of chloroform,
precipitated with 1 volume of isopropyl alcohol, and finally washed
with 75% ethanol. Total RNA was analyzed either on a 1.4% agarose gel
or a polyacrylamide gel containing 7.5 M urea as
described(19) .
Gene Synthesis, Expression, and Purification of Human
RNase A
The human RNase A gene was synthesized using an Escherichia coli codon bias(20) . Twelve
oligonucleotides for assembling the synthetic human ribonuclease A gene
were synthesized on a Cyclone Plus DNA Synthesizer. After
being phosphorylated with T4 kinase, these oligonucleotides were
ligated together with DNA ligase. The ligated product was used as the
template for amplification with the polymerase chain reaction. The
amplified polymerase chain reaction product was then cloned into the
PET-11d plasmid using BamHI and XbaI restriction
sites and sequenced. The final sequence (Fig. 1) was that
desired to generate the human pancreatic RNase protein lacking the
leader sequence with an additional Met-1 residue. Human RNase A was
then expressed in BL21-DE3 E. coli cells with
isopropyl-1-thio-
-D-galactopyranoside as the inducing
agent. The fraction of inclusion bodies that contains the expressed
protein was isolated and treated as described(21) . The
refolded ribonuclease was then purified by ion exchange chromatography
on S-Sepharose followed by size exclusion chromatography on Sephacryl
S-100. The S-Sepharose column was developed with a linear sodium
chloride gradient (0.35-0.5 M) in 0.15 M sodium
acetate buffer, pH 5.0. The main peak was collected, concentrated by
ultrafiltration, and run on a Sephacryl column in 0.075 M ammonium bicarbonate. The pooled peak fractions were lyophilized.
The resulting preparation was homogeneous in polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate. Ribonuclease
activity of the recombinant (Met-1) human RNase was about 90% relative
to the specific activity of bovine pancreatic RNase A.
Expression and Purification of Human
Angiogenin
The human angiogenin cDNA gene (22) was
cloned into the PET-11d plasmid and expressed in BL21-DE3 E. coli by induction with
isopropyl-1-thio--D-galactopyranoside. The expressed
protein was purified using the same method as described under the
purification of human ribonuclease A and was homogeneous by Coomassie
staining of SDS-polyacrylamide gels.
Affect of Retinoic Acid and Monensin on BS-RNase
Cytotoxicity
When incubated with 9L glioma cells, BS-RNase
inhibited cellular protein synthesis only 30% at 1
10
M (Fig. 2A). In the
presence of either retinoic acid or monensin, however, the inhibition
of protein synthesis reached 98% at the same concentration of BS-RNase
and reached 50% at about 1
10
M.
Thus, these drugs increase the cytotoxicity of BS-RNase over 100-fold.
BS-RNase is a disulfide-linked dimer (17) which may be reduced
and alkylated to generate single chain monomers(17) . The dimer
form is much more potent than the monomer in a variety of biologic
activities of this protein (2) . Alkylation with ethyleneimine
results in a Cys-31, Cys-32-S-ethylamine derivative (MSSR),
whereas alkylation with iodoacetamide leads to a Cys-31,
Cys-32-carboxymethyl monomer of BS-RNase (MCM). Neither MSSR nor MCM
were toxic to 9L glioma cells within the employed concentration range (Fig. 2, B and C, respectively). However, they
both were quite potent in the presence of either retinoic acid or
monensin. The respective values of IC
were 5
10
M for MSSR and 1
10
M for MCM. The potency of the two monomers was within
one order of magnitude of the dimer in the presence of either retinoic
acid or monensin. Dimerization is not essential for BS-RNase toxicity
in the presence of Golgi disrupting drugs.
10
cells/ml) in 96-well plates were incubated
with increasing concentrations of BS-RNase dimer (A), MSSR
monomer (B), and MCM monomer (C) in the absence or
presence of 10 µM retinoic acid or monensin. After a 16-h
incubation at 37 °C in leucine-free RPMI medium, cells were pulsed
with [
C]leucine for 1 h and then harvested onto
glass fiber filters using a PHD cell harvester and
counted.
Cell Viability after Treatment with BS-RNase in the
Presence of Retinoic Acid
Fig. 3shows the cell viability
compared to protein synthesis inhibition after treatment of cells with
BS-RNase in the presence or absence of 10 µM retinoic
acid. Although protein synthesis was inhibited by 60% after a 6-h
incubation with BS-RNase in the presence of 10 µM retinoic
acid, the cells remained intact as measured by trypan blue exclusion (Fig. 3A). However, incubation of the cells with
BS-RNase in the presence of retinoic acid for 16 h resulted in the
complete inhibition of protein synthesis and cell lysis as shown in Fig. 3B. After 16 h of treatment, no cells were lysed
by BS-RNase alone at 10M, whereas, in the
presence of retinoic acid, 90% of the cells were lysed. Thus, the
potentiation of RNase inhibition of protein synthesis seen with
retinoic acid effects a large decrease in cell viability.
) or without (
) 10 µM retinoic acid. Trypsin was then added to the culture medium and
further incubated for 5 min. Suspended cells were then mixed with
trypan blue and counted in a hemocytometer. A percentage was obtained
by dividing trypan blue excluding cells with total cell counts. A
parallel of cells treated with the same concentrations of BS-RNase for
the same period of time were also done for protein synthesis assay as
described in the legend to Fig. 2and under ``Experimental
Procedures.'' Open circles, protein synthesis in the
absence of 10 µM retinoic acid; filled circles,
protein synthesis in the presence of 10 µM retinoic acid. B, all the same as described in the legend to A except that cells were treated for 16
h.
Retinoic Acid Caused an Increased RNA Degradation in
BS-RNase-treated 9L Cells
Although protein synthesis was
inhibited up to 60%, cells remained intact for at least 6 h after
treatment with BS-RNase in the presence of retinoic acid (Fig. 3A). We examined the status of RNA in the cells
treated with BS-RNase with or without retinoic acid for 6 h (Fig. 4). There was no detectable RNA degradation in cells
treated with BS-RNase up to a 100 nM concentration. At a
concentration of 1.0 µM BS-RNase, 28 S and 18 S rRNA began
to be degraded, whereas there was no detectable degradation of 5.8 S, 5
S, or tRNA (Fig. 4). Thus, 28 S and 18 S rRNA are the more
susceptible substrates for BS-RNase than 5.8 S, 5 S rRNA, and tRNA.
However, in the presence of retinoic acid, the 28 S, 18 S, 5.8 S, 5 S
rRNA, and tRNA were all degraded to some extent, even at BS-RNase
concentrations as low as 10 nM (Fig. 4). In retinoic
acid-treated cells, RNA is degraded at concentrations of BS-RNase 100
times lower than that needed for RNA degradation in cells not exposed
to retinoic acid. However, the 28 S and 18 S rRNA seem to be more
susceptible to BS-RNase than 5.8 S, 5 S, and tRNA both in the presence
and absence of BS-RNase. These results indicate that BS-RNase gets into
cytosol more efficiently in the presence than in the absence of
retinoic acid. As rRNA degradation correlates with cytotoxicity, both
in the presence and absence of retinoic acid, these results
substantiate the model that rRNA degradation after cytosolic entry of
BS-RNase is the mechanism of BS-RNase protein synthesis inhibition.
flasks and treated with varying concentrations of
BS-RNase in the presence of 10 µM retinoic acid (lanes
c-e) or in the absence of retinoic acid (lanes
f-h). After a 6-h incubation at 37 °C in leucine-free
RPMI 1640 medium, cells were trypsinized and washed twice with
phosphate-buffered solution pretreated with diethylpyrocarbonate. Total
RNAs were purified as described under ``Experimental
Procedures.'' The same volume of the purified total RNAs were
loaded on both a 1.4% agarose (A) and a polyacrylamide gel (B). Lane a, total RNAs from control cells; lane
b, total RNAs from retinoic acid-treated cells; lanes
c-e, total RNAs from cells incubated with 10, 100, and 1000
nM BS-RNase in the presence of 10 µM retinoic
acid; lanes f-h, total RNAs from cells incubated with
10, 100, and 1000 nM BS-RNase in the absence of retinoic
acid.
Retinoic Acid Potentiation of the Cytotoxicity of
Onconase, Angiogenin, and Human RNase A
A number of proteins
discovered based on a variety of biologic activities have recently been
found to be homologous to RNase A and to express RNase activity
(reviewed in Refs. 1, 2, and 23). We compared the cytotoxicity of
different RNases with and without Golgi apparatus selective drugs. In
addition to BS-RNase and its monomers, retinoic acid potentiated the
cytotoxicity of Onconase, angiogenin, and human RNase A as shown in Fig. 5. Onconase is a cytotoxic ribonuclease isolated from R. pipiens eggs and early embryos based upon its anti-cancer
activity both in vitro and in
vivo(10, 18, 24, 25, 26) .
Alone, Onconase has an IC of 10
M and in the presence of retinoic acid cytotoxicity increased
100-fold to around 10
M (Fig. 5A), quite close to that of BS-RNase.
Angiogenin, a ribonuclease originally purified because of its
angiogenesis activity(11) , is not detectably toxic alone, but,
in the presence of retinoic acid, has an IC
of 3
10
M (Fig. 5B). Recombinant
human pancreatic RNase A was also more toxic with retinoic acid (Fig. 5C), yet the potency was relatively low compared
to the other RNases. Although all four members of the RNase A
superfamily examined were more toxic in the presence of retinoic acid,
their potency varied 10,000-fold from 2
10
to 2
10
M. What molecular
features account for these large differences remain unknown.
As
shown in Table 1, Onconase ribonuclease activity is essential for
its cytotoxicity. Inactivation of Onconase ribonuclease activity by 98%
with iodoacetic acid abolished its cytotoxicity. Recombinant Onconase
which only contains 2% of the native Onconase ribonuclease activity
also lost its cytotoxicity, although the crystal structure showed that
recombinant Onconase has been properly folded.(
)
Brefeldin A Blocks the Retinoic Acid
Potentiation of BS-RNase and Onconase Toxicity
It has been
demonstrated that retinoic acid disrupts the Golgi apparatus and
potentiates the cytotoxicity of ricin A chain
immunotoxins(14) . To examine whether or not retinoic acid
affects the routing of RNases through the Golgi apparatus, the effect
of brefeldin A (BFA) on the retinoic acid potentiation of BS-RNase and
Onconase was examined (Fig. 6). BFA, by inhibiting vesicular
transport from the endoplasmic reticulum (ER) to the Golgi, results in
collapse of the cis-Golgi apparatus blocking the retrograde
vesicular transport of vesicles from the Golgi to the ER (27, 28) . BFA was incubated with 9L cells in the
presence of retinoic acid and BS-RNase or Onconase. Fig. 6shows
that BFA completely blocks the potentiation of toxicity of both
BS-RNase and Onconase by retinoic acid. BFA, however, has no effect on
the cytotoxicity caused by Onconase and BS-RNase themselves. This
indicates that BS-RNase and Onconase route through the Golgi apparatus
in the presence of retinoic acid. The results also indicate that
Onconase routes to the cytosol through a BFA insensitive compartment in
the absence of retinoic acid. Thus, Onconase likely routes to the
cytosol normally without going through the Golgi apparatus, and
addition of retinoic acid alters the intracellular routing through the
Golgi where Onconase accesses the cytosol 100-fold more efficiently.
BS-RNase is also routed more than 100 times more efficiently through
the Golgi to the cytosol in the presence than in the absence of
retinoic acid.
Retinoic Acid Potentiation of RNase Cytotoxicity Is
Independent of New Gene Expression
To test whether the RNase
affects cell death via some signaling pathway that can be enhanced by
retinoic acid through transcriptional controls, cells were incubated
with actinomycin D before exposure to retinoic acid and Onconase. Table 2showed that pretreatment with actinomycin D did not
prevent the potentiation of Onconase cytotoxicity by retinoic acid.
Thus, the well established transcription activation activity of
retinoic acid does not appear to be the mechanism by which retinoic
acid increases cell sensitivity to RNases.
How the RNases cross the membrane and
enter the cytosol remains unclear.
The finding that retinoic acid potentiated BS-RNase toxicity
orders of magnitude allowed a further test of this hypothesis. If
intracellular RNA degradation was the lesion that caused cell death,
then RNA degradation in retinoic acid-treated cells should occur at
orders of magnitude lower RNase concentrations, correlating with
protein synthesis inhibition. BS-RNase caused 28 S and 18 S rRNA
degradation only at 1.0 µM in the absence of retinoic
acid, without any detectable degradation of 5.8 S, 5 S rRNA, or tRNA.
In retinoic acid-treated cells, all RNA species showed some degradation
at a 10 nM concentration of BS-RNase, 100 times lower than the
concentration of BS-RNase needed for cells not exposed to retinoic
acid. This result indicates that BS-RNase gets into the cytosol more
efficiently in the presence of retinoic acid. In both retinoic
acid-treated and untreated cells, 28 S and 18 S rRNA are more readily
degraded by BS-RNase than 5.8 S, 5 S rRNA, or tRNA. The correlation
between the degradation of cytosolic RNA by BS-RNase and cytotoxicity
indicates that retinoic acid increases delivery of BS-RNase to the
cytosol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.