(Received for publication, March 23, 1995; and in revised form, July 12, 1995)
From the
Recent in vitro work with Golgi-enriched membranes showed that 3`-azidothymidine-5`-monophosphate (AZTMP), the primary intracellular metabolite of 3`-azidothymidine (AZT), is a potent inhibitor of glycosylation reactions (Hall et al.(1994) J. Biol. Chem. 269, 14355-14358) and predicted that AZT treatment of whole cells should cause similar inhibition. In this report, we verify this prediction by showing that treatment of K562 cells with AZT inhibits lipid and protein glycosylation. AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 µM), and suppresses the incorporation of both sialic acid and galactose into proteins. Control experiments demonstrate that these changes do not result from nonspecific effects on either the secretory apparatus or protein synthesis. On the other hand, studies using isolated nuclei as a model system for chromosomal DNA replication show that AZTTP is a very weak inhibitor of DNA synthesis. These observations strongly suggest that the myelosuppressive effects of AZT in vivo are due to inhibition of protein and/or lipid glycosylation and not to effects on chromosomal DNA replication.
3`-Azidothymidine (AZT) ()is one of the primary
chemotherapeutic agents used in the treatment of HIV infection (1) . This drug is effective because the triphosphate form of
AZT, AZTTP, is a potent and somewhat selective inhibitor of HIV reverse
transcriptase(2) . Unfortunately, AZT therapy is often
accompanied by side effects such as severe anemia and neutropenia due
to inhibition of the maturation of blood stem cells, especially in the
late stages of the disease(3) .
The current paradigm to
explain AZT's hematologic toxicity focuses on DNA replication.
AZT is proposed to impede growth or development of stem cells through
incorporation of the analog into chromosomal DNA (3) . This
hypothesis is consistent with the rapid proliferation of blood stem
cells and their general sensitivity toward inhibitors of DNA
replication (for example cancer chemotherapeutics). However, AZTTP is a
remarkably weak inhibitor of the three nuclear replicative DNA
polymerases, ,
, and
(4, 5) . Under
physiological nucleotide concentrations, the amount of AZTTP needed to
inhibit these enzymes is much higher than the concentrations that
accumulate in treated cells (6) and raises the possibility
that the myelosuppressive effects of AZT are not related to inhibition
of chromosomal DNA replication.
We recently demonstrated that the primary intracellular metabolite of AZT, AZTMP, is a potent competitive inhibitor of pyrimidine nucleotide sugar import into Golgi-enriched membrane fractions(7) . Consequently, the glycosylation reactions that occur within the Golgi lumen were almost completely inhibited. Since AZTMP is known to accumulate to millimolar levels in several cell types(8) , these observations suggested a novel mechanism for AZT toxicity, namely selective inhibition of lipid and protein glycosylation.
Several lines of evidence indicate that inhibition of glycosylation could indeed lead to cytotoxicity. Small changes in glycosphingolipid synthesis can profoundly affect signal transduction, differentiation, and cell-cell interactions. Ganglioside synthesis varies in a characteristic manner during growth and differentiation(9, 10) , and subtle changes in glycolipid composition dramatically alter the properties of many receptors and enzymes(10) . For example, variations in ganglioside composition as small as 15% can block activation of some growth factor receptors(11, 12) . Alterations in glycosylation pattern of proteins could also contribute to cytotoxicity since previous studies have established that inhibition of N-linked protein glycosylation is toxic and can block development(13, 14) .
We therefore examined the effects of AZT on lipid and protein glycosylation in whole cells and found that treatment with AZT did inhibit these reactions at clinically relevant concentrations (0.5-5 µM). In particular, AZT treatment dramatically altered the pattern of glycosphingolipid biosynthesis in the human blood cell line K562. In contrast, the AZT metabolites AZTMP and AZTTP were extremely weak inhibitors of DNA synthesis by isolated K562 nuclei. The significance of these results with respect to the side effects associated with AZT therapy are discussed.
The extent of H incorporation
into proteins and lipids was determined by collecting cells onto glass
fiber filters (G4) quenched in 1% milk solution and equilibrated with
5% trichloroacetic acid and then washing the precipitate nine times
with 3 ml of cold 5% trichloroacetic acid. Following four washes with
H
O, the amount of radiolabel retained on the dried filters
was determined by scintillation counting. To measure incorporation into
proteins specifically, lipids were extracted sequentially with
1-2 ml each of 1:2, 1:1, and 2:1 CHCl
:MeOH (v/v), and
the filters were air-dried prior to scintillation counting. Background
was determined by addition of radiolabel to cells immediately prior to
filtration and trichloroacetic acid precipitation. All measurements
were carried out in triplicate.
To verify the nature of the
radioactive label incorporated into proteins, washed cell pellets were
extracted twice with 60% EtOH, as described(23) . The insoluble
material was treated with 1 N HCl for 4 h at 100 °C to
release carbohydrates. The hydrolysates were then lyophilized and
resuspended in HO for analysis by descending paper
chromatography in EtOAc/pyridine/HOAc/H
O (5:5:1:3).
In assays containing 10 µM dNTPs, AZTTP poorly inhibited DNA synthesis in nuclei obtained
from K562 and CEM cells, with IC values greater than 500
µM (Table 1). The presence of 1 mM AZTMP
slightly enhanced inhibition in K562 nuclei, but the AZTTP
concentration needed for significant inhibition remained several
hundred-fold higher than the AZTTP concentration reported in
AZT-treated cells (about 1 µM(6) ). In contrast,
two other antiviral nucleotide analogs, ddCTP and ganciclovir
triphosphate, inhibited DNA synthesis in isolated nuclei much more
potently than AZTTP, in agreement with the fact that those are more
potent inhibitors of purified nuclear DNA
polymerases(15, 25) . These results suggest that
inhibition of nuclear DNA replication is unlikely to account for the
side effects associated with AZT therapy.
Figure 1:
AZT treatment dramatically alters the
synthesis of glycosphingolipids in K562 cells. A, K562 cells
were labeled with [C]galactose for 24 h in the
presence of the indicated AZT concentrations, and glycolipids were
analyzed by TLC as described under ``Experimental
Procedures.'' The phosphorimage of a representative chromatogram
obtained under neutral solvent conditions is shown. The mobilities of
standards are indicated, and the origin is marked with an arrow. B and C, K562 cells were labeled with
[
C]galactose for 24 h either with or without 20
µM AZT. Following extraction, glycolipids were separated
into neutral and acidic fractions and analyzed by HPTLC under various
solvent conditions listed in Table 2. Phosphorimages of
chromatograms of the acidic and neutral fractions obtained under basic
solvent conditions are shown in B and C,
respectively. Cerebr, cerebroside; LacCer,
lactosylceramide; Gb3, globotriosylceramide; Gb4,
globotetraosylceramide.
Fig. 1shows that AZT treatment dramatically altered the pattern of glycosphingolipid biosynthesis in a dose-dependent manner. Quantitative analysis of the data demonstrated that AZT treatment nearly abolished synthesis of GD1a, GM2, and SPG and had the opposite effect on lactosylceramide and material that comigrated with GM3 and PG standards (Fig. 2, leftpanel). Since AZT treatment typically results in 2-4 µM AZT in the serum of patients(28) , these results demonstrate that AZT can cause a dramatic remodeling of the glycolipid composition of cells at clinically relevant concentrations. Identical results were obtained in several separate experiments.
Figure 2:
Quantitative analysis of the effects of
AZT on glycosphingolipid synthesis. Leftpanel, K562
cells were labeled with [C]galactose for 24 h in
the presence of various AZT concentrations. The amount of radioactivity
present in each of the selected glycolipid was expressed as a fraction
of the total present at each AZT concentration and then normalized to
the fraction observed in the absence of AZT. The average of two to four
independent experiments is shown (standard deviation <6%). The
lipids analyzed and their corresponding plot symbols are as follows:
, cerebroside;
, lactosylceramide;
, PG/GM3;
,
GM2;
, SPG;
, GD1a. Rightpanel, K562
cells were labeled with [
C]serine for 24 h in
the presence of various AZT concentrations. Glycolipids were analyzed
and quantified as described for the leftpanel.
The analysis of total sphingolipids presented in Fig. 1A suggests that the synthesis of GM3, unlike that of the other acidic lipids, is stimulated by AZT. However, this analysis is complicated by the comigration of GM3 and PG, the immediate precursor to SPG, under standard chromatography conditions. To investigate the effects of AZT on PG and GM3 production, neutral and acidic lipid fractions were isolated by ion exchange chromatography and separately analyzed by HPTLC (Fig. 1, B and C). Analysis of the acidic glycolipids revealed that the synthesis of GM3, in contrast to that of GD1a, SPG, and GM2, increases in the presence of AZT (Fig. 1B). Note that HPTLC under basic conditions resolves GM3 from PG. Quantitation of several independent experiments shows that 20 µM AZT stimulates GM3 synthesis by 50 ± 10%.
The observed decrease
in SPG synthesis could have resulted either from inhibition of the
synthesis of its precursor, PG, or from a block in the conversion of PG
to SPG. Analysis of the neutral glycosphingolipids from AZT-treated
cells shows that relatively little PG normally accumulates in K562
cells but that treatment with 20 µM AZT resulted in a
2.2-fold increase in PG levels (Fig. 1C). ()The species identified as PG cochromatographed with
authentic PG under several solvent conditions, thereby confirming its
identification. The observed increase in PG accounts for nearly 60% of
the decrease in SPG production, which suggests that the decrease in SPG
synthesis results in great part from inhibition of the sialylation of
PG.
Figure 3:
AZT treatment dramatically alters the
synthesis but not the breakdown of glycosphingolipids in K562 cells.
Cells were metabolically labeled with
[C]galactose in the absence of AZT for 24 h.
Labeled cells were then washed and chased in media lacking
[
C]galactose for 0, 7, or 24 h in the presence
or absence of 20 µM AZT. Remaining labeled
glycosphingolipids were analyzed by TLC. Cerebr, cerebroside; LacCer, lactosylceramide.
To verify that the effects of AZT did not result from changes in the
incorporation of [C]galactose into the precursor
pool, we repeated our analysis of glycosphingolipid synthesis using a
non-carbohydrate metabolic label, [
C]serine,
that is readily incorporated into the sphingosine backbone of all
sphingolipids. As shown in Fig. 2, AZT treatment reduced
ganglioside synthesis and caused accumulation of neutral species to
levels very similar to those observed when using
[
C]galactose as a label. In contrast, the
synthesis of sphingomyelin, a non-glycosylated sphingolipid, was
completely unaffected by AZT. These results strongly suggest that the
effects of AZT are not mediated by changes in sugar uptake or
nucleotide sugar precursor pools. The small quantitative differences
probably arise because only one [
C]serine is
incorporated per sphingolipid, whereas different numbers of
[
C]galactose can be incorporated as either
glucose or galactose into each lipid species. In addition, direct
measurements of nucleotide sugar pools established that treatment with
5 µM AZT did not alter intracellular levels of either
hexosyl or N-acetylhexosyl-nucleotides. (
)
The
possibility that AZT treatment interferes with the secretory pathway
was excluded by measuring the synthesis and transport of a well
characterized glycoprotein of K562 cell membranes, the Tf receptor.
Cells were labeled with [S]methionine for 20
min, and movement of the Tf receptor to the cell surface in the absence
or presence of 20 µM AZT was monitored. Fig. 4shows that maximal accumulation of the Tf receptor at the
cell surface occurred in about 60 min, as previously observed. (
)AZT affected neither the amount nor the rate at which the
Tf receptor was incorporated into the plasma membrane, demonstrating
that AZT does not act as a general inhibitor of the secretory pathway.
The lack of effect of AZT on transport despite clear effects on protein
glycosylation (see below), is in agreement with previous work showing
that inhibition of complex carbohydrate synthesis does not affect
transport of the Tf receptor(21) . Incorporation of
[
S]methionine into bulk proteins was also used
to measure the effects of AZT on protein synthesis. AZT concentrations
as high as 50 µM had no effect on
[
S]methionine incorporation in trichloroacetic
acid-precipitable material. This lack of effect on the synthesis and
transport of proteins is consistent with previous work showing that
these low concentrations of AZT do not reduce the growth rate of K562
cells(30) .
Figure 4:
AZT treatment does not inhibit the
secretory pathway. K562 cells were labeled with
[S]methionine and chased for various lengths of
times in the presence of either 0 or 20 µM AZT, as
indicated. The synthesis and transport of the Tf receptor was monitored
as described under ``Experimental Procedures.'' The mobility
of the receptor was the same at all chase times, both in the presence
and absence of AZT. The amount of Tf receptor present at the cell
surface after various chase times is shown. Similar results were
obtained in two separate experiments.
In the lipid-labeling experiments presented in Fig. 1and Fig. 2, cells were treated and labeled for 24 h
prior to analysis. To test the possibility that effects of AZT were due
to alterations in the biosynthesis of the glycosylation machinery
(sugar transferases, etc.), short treatment and labeling times were
also examined. After treating cells with AZT for 60 min to allow
accumulation of AZTMP, [C]galactose was added,
and newly synthesized glycolipids were analyzed following an additional
2-h incubation. In this short term labeling, 20 µM AZT
inhibited the synthesis of GM2, SPG, and GD1a by 82, 54, and 65%,
respectively, and stimulated the synthesis of lactosylceramide and
GM3/PG by 92 and 43%, respectively, values similar to those obtained in
the 24-h labeling experiments shown in Fig. 2. These results
indicate that changes in glycolipid synthesis are rapid and unlikely to
result from AZT-induced alterations in the levels of proteins involved
in glycosylation.
We first assayed sialic acid
incorporation into total lipids and proteins by incubating cells for 24
h with [6-H]N-acetylmannosamine, a
specific precursor of sialic acid(32) , and measuring the
amount of [
H]sialic acid present in
acid-insoluble material. Fig. 5shows that AZT reduced sialic
acid incorporation at concentrations as low as 0.5 µM. The
effects of AZT were examined over a wide range of labeling times
(3-24 h) with identical results.
Figure 5:
AZT inhibits incorporation of
[H]N-acetylmannosamine in K562 cells. K562 cells
were metabolically labeled with
[
H]N-acetylmannosamine in the presence
of the indicated concentrations of AZT. A number of independent
experiments (up to 10) were performed under different conditions.
Varying the length of labeling from 3 to 26 h or increasing the total
glucose concentration from 0.075 g/liter to 2 g/liter had no effect on
the extent of inhibition by AZT. The average of these independent
determinations is shown.
Galactosylation of lipids and
proteins was similarly examined by incubating K562 cells with
[H]galactose. As shown in Fig. 6, AZT
reduced incorporation of galactose into total lipids and proteins at
concentrations as low as 1 µM. Since
[
H]galactose can be catabolized to
non-carbohydrate precursors upon conversion to glucose and transfer of
the [
H] to NADP
, we tested the
effect of altering the amount of glucose in the labeling medium.
Increasing the glucose concentration from 0.075 g/liter to 2 g/liter
decreased the rate of
H incorporation by 70% but did not
alter the extent of inhibition by AZT (Fig. 6).
Figure 6:
AZT inhibits incorporation of
[H]galactose in K562. K562 cells were
metabolically labeled with [
H]galactose for 24 h
in the presence of the indicated concentrations of AZT. Labeling was
carried out in medium containing either 0.075 (
) or 2 g/liter
(
) glucose.
We then
verified that AZT suppressed protein glycosylation by extracting lipids
from the trichloroacetic acid precipitates with CHCl:MeOH
prior to scintillation counting. Less than 15% of the radiolabeled
material was extracted by the organic solvent, indicating that most of
the label incorporated into biomolecules was present in proteins and that the results in Fig. 5and Fig. 6reflected effects on protein glycosylation. Analysis of
the CHCl
:MeOH insoluble material produced results identical
to those presented in Fig. 5and Fig. 6, thereby
confirming that AZT inhibited protein glycosylation. Further control
experiments established that >95% of the radioactivity incorporated
into protein during labeling with [
H]galactose
comigrates with galactose by paper chromatography and that this
fraction is unchanged by AZT (data not shown). No
[
H]glucose was detected in this analysis because
glucose is not permanently incorporated into proteins during N- and O-linked glycosylation.
We previously demonstrated that AZTMP potently inhibits the
import of pyrimidine nucleotide sugars into the lumen of Golgi-enriched
membrane fractions(7) . Since K562 cells treated with only 10
µM AZT accumulate greater than 1 mM AZTMP(8) , these data suggested that AZT should
selectively inhibit glycosylation reactions. Here, we have verified
this prediction and established that AZT treatment dramatically alters
glycosphingolipid synthesis and suppresses protein glycosylation in
K562 cells at clinically relevant concentrations. AZT may be a general
modulator of glycosphingolipid synthesis, since we have found that AZT
alters glycolipid synthesis in HEL and A431 cells. ()
Several observations support competitive inhibition of the import and accumulation of nucleotide sugars in the Golgi apparatus as the most likely molecular mechanism for these effects of AZT: (i) AZT treatment does not alter lipid degradation, protein biosynthesis, or protein secretion; (ii) AZT affects both lipid and protein glycosylation; and (iii) the onset of AZT's effects are rapid. Additionally, ddC and ddI, two nucleosides that do not accumulate as monophosphates(6) , did not affect glycosylation.
AZT treatment of K562 cells inhibited the synthesis of the SPG, GD1a, and GM2, and caused accumulation of GM3, PG, and lactosylceramide. While AZT treatment generally inhibited the synthesis of complex, acidic glycosphingolipids, this result cannot be explained by a simple model in which the import of a single nucleotide sugar is affected (see Table 2). Whereas the decrease in SPG and compensatory increase in PG demonstrate that import of CMP-sialic acid was inhibited, the increase in GM3 and decrease in GD1a and GM2 indicate that AZT also blocked the import of UDP-GalNAc.
The selective effects of AZT treatment on different glycosylation reactions could result from several causes. Since AZTMP inhibits the import reaction competitively with respect to the nucleotide sugar(7) , AZTMP will have greater impact on the import of those nucleotide sugars whose concentrations are lowest and whose transporters are most sensitive to inhibition. In addition, AZTMP could affect the various subcompartments of the ER and Golgi complex to different extents if each compartment had distinct numbers of transporters and/or unique requirements for nucleotide sugars. While the exact compartments in which the different glycosylation reactions occur have not been identified, studies using Brefeldin A suggest that the synthesis of lactosylceramide and GM3 occur in the cis-Golgi, whereas more complex acidic glycolipids are synthesized in later compartments(33) . The varying sensitivities of different reactions involving CMP-sialic acid (GM3 versus SPG) could be explained if the import of CMP-sialic acid were more rate-limiting, and thus more susceptible to inhibition by AZTMP, in later compartments of the Golgi complex.
The inhibitory activity of AZT toward complex acidic lipids probably causes accumulation of GM3, PG, and lactosylceramide. These species are all precursors for those glycosphingolipids whose synthesis was inhibited, such that their accumulation may reflect a precursor-product relationship. Furthermore, cellular feedback mechanisms may attempt to compensate for the lack of specific glycosphingolipids by increasing the synthesis of their precursors.
The effects of AZT were not limited to lipid glycosylation reactions since AZT inhibited addition of both sialic acid and galactose to proteins. Importantly, this observation provides further support for the hypothesis that nucleotide sugar import, a target common to both lipid and protein glycosylation reactions, is being affected. The moderate effects of AZT on total incorporation of sugars into proteins, a maximal inhibition of only about 30%, may mask more severe effects on specific classes of glycosylation reaction (O-linked versusN-linked, etc.). Experiments to test this possibility are in progress.
In contrast, the effects of AZT on protein and lipid glycosylation would readily account for the cytotoxicity of AZT. As described in the Introduction, changes in ganglioside content as small as 15% can compromise the function of membrane receptors(11, 12) . It is therefore quite likely that the dramatic changes in ganglioside synthesis reported here will interfere with the function of proteins such as the erythropoietin receptor. Whereas such changes may have minimal impact on the growth of most cultured cell lines, they will most likely be toxic to those cell types that are dependent on extracellular signals for growth or differentiation. For example, rapidly growing erythrocyte precursor cells may show such sensitivity, since erythropoietin-dependent cells undergo apoptosis when deprived of erythropoietin(35) . Direct evidence that inhibition of glycosylation can lead to anemia is provided by the observation that congenital dyserythropoietic anemia type II is due to defective poly-N-acetyllactosamine addition to proteins in erythrocyte precursors(36) .
Our demonstration that AZT inhibits glycosylation may not only facilitate development of methods to better control AZT toxicity but will also open up new avenues to study the role of gangliosides in regulating receptor function and cell-cell interactions. The lack of methods to depress ganglioside biosynthesis has limited previous studies to examining the effect of adding exogenous lipids. In the future, suppression of ganglioside synthesis by AZT treatment followed by selective incorporation of different glycosphingolipids should allow more control over membrane composition to better define the role of this important class of molecules.