Department of Chemistry and Biochemistry, University of Texas at
Arlington, Arlington, Texas (S.A., S.S.S.); Department of Human
Biological Chemistry and Genetics, University of Texas Medical Branch,
Galveston, Texas (R.S., Y.C.A.); and Department of Internal Medicine
and Department of Biochemistry and Molecular Biology, University of
Arkansas for Medical Sciences, and Central Arkansas Veterans Healthcare
System, Little Rock, Arkansas (P.Z.)
Transport of xenobiotics and their metabolites by ATP-binding
cassette (ABC) transporters particularly P-glycoprotein (Pgp) and the multidrug resistance associated protein (MRP1) has been extensively studied during last decade. Our recent studies demonstrate that RLIP76, a previously known GTPase-activating protein catalyzes ATP-dependent, uphill transport of anionic glutathione conjugates as
well as of weakly cationic anthracyclines including doxorubicin (Adriamycin), a widely used drug in cancer chemotherapy. RLIP76 has
inherent ATPase activity, which is stimulated by doxorubicin and
glutathione conjugates. RLIP76 does not meet the criteria for classical
ABC proteins such as MRP1 or Pgp, but similar to ABC proteins, it has
two ATP-binding sequences, 69GKKKGK74 and
418GGIKDLSK425. Mutations in these sequences
abrogate its ATP-binding, ATPase activity, and transport function.
Purified RLIP76 when reconstituted in proteoliposomes mediates
ATP-dependent saturable transport of doxorubicin and glutathione
conjugates. Transfection of K562 cells with RLIP76 confers these cells
resistance to doxorubicin and 4-hydroxynonenal. Cells enriched with
RLIP76 also acquire resistance to radiation toxicity. RLIP76 also
catalyzes the transport of physiologic ligands such as leukotrienes
(LTC4) and the conjugate of 4-hydroxynonenal and glutathione. In some
cells (e.g., erythrocytes and lung cancer cells), the majority of
transport activity for Adriamycin and glutathione conjugates including
LTC4 is accounted for by RLIP76. These studies strongly suggest that
RLIP76-mediated transport of organic ions has physiological and
toxicological relevance and that it may play an important role in the
mechanism of drug resistance.
 |
Introduction |
Reactions
leading to the biotransformation of xenobiotics are traditionally
classified into two phases (Williams, 1959
). In phase I, reactions
catalyzed by enzymes including cytochromes P450, epoxide hydrolases,
esterases, and amidases introduce/expose reactive groups in
xenobiotics, so that these bioactivated metabolites can be conjugated
to hydrophilic compounds such as glutathione (GSH1), glucuronate, sulfate, etc., by phase II
enzymes. The phase II reaction products must eventually be transported
to complete the detoxification process because accumulation of these
products can cause not only toxicity but can also inhibit the phase II reactions. These transport mechanisms, designated as phase III of the
detoxification process (Ishikawa, 1992), are therefore an essential
component of cellular defense mechanisms against toxic chemicals (shown
schematically in Fig. 1).
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Fig. 1.
Schematic representation of the pathway of
detoxification mechanisms of xeno- and endobiotics showing the role of
transporters.
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Both phase I and phase II biotransformation enzymes occur as members of
multiple gene "superfamilies", which have been extensively characterized. For example, the structures, functions, and roles of the
members of the gene superfamilies of CYP450s and glutathione S-transferases are well understood. In contrast to phase I
and phase II enzymes, relatively little is known about the transporters comprising phase III of the detoxification process, but it is now clear
that these transporters also belong to several superfamilies (Saier and
Paulsen, 2001). In recent years, these transporters have attracted a
great deal of interest because of their involvement in multidrug
resistance of bacteria, parasites, and human cancer cells (Gottesman
and Pastan, 1993; Bambeke et al., 2000; Johnstone et al., 2000; Renes
et al., 2000; Leslie et al., 2001; Saier and Paulsen, 2001). Based on
the analyses of the available genome sequences in organisms from
bacteria to man, at least five superfamilies of transporters along with
a small family specific to eukaryotic organisms have been identified
(Saier and Paulsen, 2001). Only limited information on the
physiological/pharmacological roles of these predicted transporters is
currently available, and only a few of these have been assigned
specific transport functions (Johnstone et al., 2000; Leslie et al.,
2001; Saier and Paulsen, 2001).
Among transporters involved in detoxification, the members of the
ATP-binding cassette (ABC) family (Higgins, 1992; Holland and Blight,
1999) are most widely studied. In the present Minireview, we summarize
evidence showing that a previously described protein RLIP76
(Jullien-Flores et al., 1995), which is a nonABC, GTPase-activating protein functions as an alternative transporter of the end products of
detoxification pathways (Fig. 1). As detailed below, RLIP76 may act in
parallel with ABC transporters, or it may be the predominant pump in
certain cell types or situations. Since many excellent reviews on ABC
transporters have been published, we will only briefly summarize the
most salient characteristics of these proteins. Against this backdrop,
we will present our data on RLIP76-mediated transport and its
physiological significance.
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Transporters of the ABC Family |
ABC transporters use the free energy of ATP hydrolysis to
translocate substrates or allocrites across the membrane, and have Walker motifs (ATP-binding sites) and transmembrane domains in their
sequences. Overexpression of ABC transporters has been clearly linked
with the drug resistance of certain bacteria, parasites, and human
cancer cells (Gottesman and Pastan, 1993; Ruetz et al., 1996; Bambeke
et al., 2000; Johnstone et al., 2000; Leslie et al., 2001; Saier and
Paulsen, 2001). Two ABC transporter family members, P-glycoprotein (Pgp
or MDR1) and multidrug resistance associated protein (MRP1) best
characterized with respect to this function, are often referred to as
the drug efflux pumps. Overexpression of Pgp, MRP1, or both is observed
in many cancer cell lines exhibiting the multidrug resistance phenotype
(Gottesman and Pastan, 1993; Johnstone et al., 2000; Leslie et al.,
2001). Pgp-overexpressing cancer cells exposed to drugs such as
Adriamycin, vinblastine, and colchicine show decreased accumulation of
these drugs (Gottesman and Pastan, 1993; Ambudkar et al., 1999).
MRP, now designated as MRP1 (first characterized member of the MRP
family) or ABCC1 (http://www.med.rug.nl/mdl/tab3.htm) was originally
cloned from a drug resistant line selected for doxorubicin resistance
(Cole et al., 1992). MRP1-mediated transport of the conjugates of GSH,
glucuronate, and sulfate has been clearly demonstrated (reviewed by
Leslie et al., 2001 and references cited therein). MRP1 also mediates
the transport of physiological GSH conjugates, such as leukotrienes and
GS-HNE, and the GSH conjugate of lipid peroxidation end product,
4-hydroxynonenal (4-HNE), which suggests a physiological role of MRP1
in the normal cells (Renes et al., 2000). Transport of vincristine by
MRP1-rich membrane vesicles has been demonstrated, and this transport
has been suggested to be linked to GSH cotransport (Loe et al., 1998).
Despite the identification of multiple families of drug transporters in
the human genome, including at least 48 sequences of putative proteins
having characteristics of ABC transporters (Higgins and Linton, 2001),
the functional characterization of the majority of these is lacking. To
fill this gap, we have focused the effort of our laboratories on
functional studies of transporters, which are involved in the primary
active transport of xenobiotics, their conjugates, and the
chemotherapeutic agents relevant to drug resistance. In the following
section, we highlight these efforts, which have led to the
characterization of the transport function of a Ral-binding
GTPase-activating protein, RLIP76, first reported by Jullien-Flores et
al. (1995). Our findings that RLIP76 parallels in function to the well
known transport proteins associated with drug resistance mechanisms
provide evidence for a link between cell-signaling mechanisms and
transport of exogenous and endogenous toxicants.
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DNP-SG ATPase, a Transporter for Anionic as well as Cationic
Xenobiotics |
Our earlier studies identified a protein in membranes of human
cells, which catalyzed ATP hydrolysis in the presence of GSH conjugates
(LaBelle et al., 1988). This protein was designated as DNP-SG ATPase
because S-(2,4-dinitrophenyl)glutathione (DNP-SG) stimulated
its ATPase activity. The presence of DNP-SG ATPase was demonstrated in
all human tissues examined including liver, heart, lung, muscle,
kidneys, erythrocytes, leukocytes, and various human cell lines of
diverse tissue origin (LaBelle et al., 1988; Sharma et al., 1990;
Awasthi et al., 1994; Awasthi et al., 1998a,b). Surprisingly, DNP-SG
ATPase-mediated ATP hydrolysis was stimulated not only by the organic
anions (e.g., DNP-SG), but also by weak cations such as doxorubicin
(DOX) and its metabolites (Awasthi et al., 1994, 1998b). Furthermore,
our studies with membrane vesicles (Awasthi et al., 1994) as well as
with reconstituted proteoliposomes (Awasthi et al., 1998a) demonstrated
that DNP-SG ATPase catalyzed transport of anionic GSH conjugates as
well as of weakly cationic drugs such as DOX and colchicine (Awasthi et
al., 1994; 1998a,b; 1999). ATP-dependent transport of both DNP-SG and
DOX against a concentration gradient was demonstrated in
proteoliposomes reconstituted with highly purified DNP-SG ATPase
(Awasthi et al., 1998a). The transport was temperature-dependent and
sensitive to the osmolarity of the assay medium. ATP hydrolysis was
required for the transport because when ATP was replaced by its
nonhydrolyzable analog, methylene-ATP, the transport activity
was abolished. This suggested that transport was directly coupled to
ATP hydrolysis and that DNP-SG ATPase was a primary active transporter.
Antibodies raised against DNP-SG ATPase inhibited the transport of DOX
and DNP-SG in inside-out vesicles prepared from erythrocyte membranes
suggesting that the transport was specifically catalyzed by DNP-SG
ATPase (Awasthi et al., 1994). On the other hand, antibodies against
MRP1or Pgp neither recognized DNP-SG ATPase in Western blots nor
affected its transport activity, establishing that DNP-SG ATPase was
distinct from these transporters (Awasthi et al., 1998a,b).
We also identified a transport protein related to DNP-SG ATPase in
rodents (Zimniak et al., 1992; Pikula et al., 1994a,b). Antibodies
against human DNP-SG ATPase recognized a protein in rat canalicular
membranes (Zimniak et al., 1992). This protein, when purified and
reconstituted in proteoliposomes, catalyzed concentrative transport of
DNP-SG with kinetic parameters similar to those of human DNP-SG ATPase
(Pikula et al., 1994a). Interestingly, the
Vmax of the rat transporter for DNP-SG
increased by about 3-fold upon phosphorylation by protein kinase C,
without a change in the KM. The
biochemical characteristics of the rat transporter and human DNP-SG
ATPase were clearly distinct from MRP2, a well characterized ABC
transporter present in human and rat canalicular membranes (Paulusma et
al., 1996). These results clearly demonstrate that in rat canalicular
membranes in which MRP2 has been shown to transport anionic conjugates,
other transporter(s) besides MRP2 is/are present. This conclusion is
consistent with the fact that rat and human mutants that lack
functional MRP2 (GY/TR
rats and patients with
the Dubin-Johnson syndrome, respectively) retain a residual capability
to transport organic anions across the canalicular membrane (Takenaka
et al., 1995).
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Cloning of DNP-SGATPase and Its Identity with RLIP76 |
The molecular identity of DNP-SG ATPase remained elusive for over
a decade because of the inherent difficulties in its purification. The
protein was prone to degradation, and depending on the conditions of
purification, peptides of varying chain lengths were observed in SDS
gels of purified preparations. In these preparations, a 38 kDa peptide
fragment was, however, consistently observed. This initially led us to
an erroneous conclusion that this peptide was the intact DNP-SG ATPase
because purified preparations highly enriched in this peptide mediated
ATP-dependent, uphill transport of DNP-SG as well as DOX in
reconstituted proteoliposomes (Awasthi et al., 1998a).
Immunoscreening of a human bone marrow cDNA library using the
polyclonal antibodies against the 38 kDa DNP-SG ATPase peptide surprisingly yielded RLIP76 (Awasthi et al., 2000), a previously known
Ral-binding, GTPase-activating protein (GAP), which is believed to
bridge the Ral, Rac, Cdc42 pathways (Jullien-Flores et al., 1995). More
recently, involvement of RLIP76 has been suggested in the assembly and
activity of the exocyst, a multisubunit complex required for the
vectorial targeting of secretory vesicles (Moskalenko et al., 2002).
When we expressed RLIP76 in Escherichia coli, the recombinant protein readily underwent degradation and yielded peptide
patterns in SDS gel, which were dependent on the conditions of
purification, a feature reminiscent of the behavior of tissue purified
DNP-SG ATPase. The authors who originally described RLIP76 and its rat
and mouse orthologs, RalBP1 and RIP respectively (Jullien-Flores et
al., 1995; Cantor et al., 1995; Park and Weinberg, 1995), also noted
the aberrant behavior of these proteins in SDS gels. These proteins
migrated as a major band in the range of molecular weight values 95 to
110 kDa which was higher than their predicted molecular weights from
the sequences. Several peptides with lower molecular weights also
appeared in these gels suggesting proteolytic degradation of the parent
protein. Our preparations of recombinant RLIP76 also showed the 95 kDa
band along with several smaller molecular weight peptides in which a 38 kDa fragment was consistently prominent. Bands corresponding to
molecular weights higher than those predicted for RLIP76 were also
observed, which suggested aggregation of the peptides. All these
fragments were recognized by antibodies raised against DNP-SG ATPase
and had internal sequences of RLIP76 (Fig.
2), demonstrating that these fragments
originated from RLIP76, due to proteolytic processing (Awasthi et al.,
2000). The major fragments among these were
C-RLIP76410-654 and
N-RLIP1-367 derived from the C- and
N-terminus of RLIP76, respectively (Awasthi et al., 2001b).
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Fig. 2.
Various motifs in the primary structure of
RLIP76.
The method of cloning RLIP76 from human bone marrow cDNA Lamda gt11
expression library using antibody against human DNP-SG ATPase,
expression and purification of recombinant RLIP76, isolation and
sequencing of its internal peptides has been described previously
(Awasthi et al., 2000). The deduced amino acid sequences were compared
with published sequences generated by the Blast Program available as a
network service from the National Center of Biotechnology Information,
National Institutes of Health, and analyzed with the help of Wisconsin
Genetics Computer Group (Madison WI). Red, experimentally determined
sequences of RLIP76 peptides obtained during purification;
greenish-blue, leucine zipper pattern; black, ATP-binding sites; green,
trypsin cut site; purple, chymotrypsin site; yellow, protein kinase C
phosphorylation site; blue, tyrosine kinase phosphorylation site;
brown, N-glycosylation site; gray, cAMP-dependent
protein kinase site; chartreuse, cGMP-dependent protein kinase site;
lavender, casein kinase II phosphorylation site.
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RLIP76 Mediates ATP-dependent Transport or Organic Anions as Well
As Cations |
The striking similarity in the behavior of DNP-SG ATPase and
RLIP76, that it could be purified to homogeneity by the same DNP-SG
affinity chromatography protocol used to purify DNP-SG ATPase, and that
we had cloned RLIP76 by immunoscreening with an antibody against DNP-SG
ATPase indicated that DNP-SG ATPase and RLIP76 were identical and
prompted us to explore its involvement in drug transport. Similar to
the preparations of DNP-SG ATPase purified from human tissues,
rec-RLIP76 showed constitutive ATPase activity that was stimulated by
anionic (e.g., DNP-SG) as well as cationic (e.g., DOX) ligands (Awasthi
et al., 1998a,b; 2000). The KM
for the ATPase activity of RLIP76 for ATP, DNP-SG, DOX, colchicine, and
GS-HNE were similar to those of DNP-SG-ATPase. Purified rec-RLIP76,
reconstituted in proteoliposome either with asolectin or phospholipids
of defined composition, catalyzed ATP-dependent uphill transport of the
anionic conjugates including DNP-SG, GS-HNE, as well as the weakly
cationic amphiphilic drugs such as DOX and daunomycin (Awasthi et al.,
2000; Singhal et al., 2001), which are used in cancer chemotherapy.
ATP-dependent transport of DOX and other amphiphilic cationic drugs has
been demonstrated in proteoliposomes reconstituted with Pgp (review by
Ambudkar et al., 1999 and references cited therein), but Pgp does not
catalyze the transport of anionic conjugates. MRP1-mediated transport
of the anionic conjugates such as DNP-SG, leukotrienes, GS-HNE, and glucuronides has been clearly demonstrated (Leslie et al., 2001). However, MRP1-mediated transport of vincristine and daunomycin requires
GSH cotransport, and direct evidence for MRP1-mediated transport of DOX
is lacking. Our results suggest that the mechanism through which RLIP76
transports anthracyclines or vincristine is distinct from that of MRP1
and that the allocrite spectrum of RLIP76 is broader than that of
either Pgp or MRP1 because RLIP76 can transport organic anions as well
as organic cations without the requirement of GSH cotransport. Table
1 summarizes the comparative structural
characteristics, chromosomal location, tissue localization, and
substrate profiles of RLIP76, MRP1, and Pgp. These characteristics clearly indicate that RLIP76 has overlapping functional similarities both with MRP1 and Pgp but does not share their structural attributes.
Physiological significance of the ATP-dependent transport of DOX and
GSH conjugates by RLIP76 was further confirmed by the results of
transfection experiments in which RLIP76-overexpressing cells showed
increased efflux of DOX as well as GS-HNE (Awasthi et al., 2000) and
acquired resistance to both DOX and 4-HNE-induced cytotoxicity (Awasthi
et al., 2000). These results taken together with the ability of RLIP76
to transport leukotrienes (Sharma et al., 2001) and its ubiquitous
expression in human tissues indicate that its transport function has
toxicological as well as physiological relevance.
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Structure of RLIP76 |
Primary structure of RLIP76 reveals several interesting features.
It can be divided into four regions of which two central domains carry
a Rac1/CDC42 GAP (aa 210-357) activity and a Ral-binding domain (aa
391-499) (Jullien-Flores et al., 1995). The C-terminal (aa 500-647)
domain has been shown to bind Reps1 and POB1 proteins linking RLIP76 to
EGFR, insulin receptor and TGF
receptor (Yamaguchi et al., 1997;
Ikeda et al., 1998). The amino acid sequence of RLIP76 (Fig. 2)
indicates the presence of N-glycosylation site (aa
341-344), cAMP (aa113-116), cGMP-dependent protein kinase phosphorylation sites (aa 650-653), tyrosine kinase phosphorylation site (aa 308-315), N-mysristolation sites (aa 21-26, aa
40-45, aa 191-196), leucine zipper pattern (aa 547-578), and several protein kinase C phosphorylation, casein kinase II phosphorylation, trypsin, and chymotrypsin cut sites. The presence of these motifs in
the primary structure of RLIP76 and its facile proteolytic degradation
may suggest that in addition to its as yet known functions, RLIP76 may
be involved in other cellular processes and that the proteolytic
processing of RLIP76 may be required for its multiple functions. The
peptide fragments of RLIP76 individually or in association with other
fragments may catalyze various functions. This idea is consistent with
the studies showing that RLIP76 functions in Ral-mediated regulation of
endocytosis of EGF receptor, insulin receptor, and TGF receptor
(Yamaguchi et al., 1997; Ikeda et al., 1998). Additional support for
this postulate is provided by our studies showing that the
N-terminal and C-terminal fragments of RLIP76, which are
individually incapable of mediating ATP-dependent transport, can
catalyze the transport of DOX as well as of colchicine when
reconstituted together in proteoliposomes (Awasthi et al., 2001b).
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RLIP76 Contains Two ATP-binding Sites |
RLIP76 expressed in cultured cells or in E. coli
undergoes facile proteolysis during purification (Awasthi et al.,
2000). We have studied the characteristics of the two most prominent peptides, N-RLIP761-367 and
C-RLIP76410-655, arising from the N-and
C-termini of RLIP76, respectively. These bands appear as 49 and 38 kDa
in SDS-gels. Both these peptides had constitutive ATPase activity,
which was stimulated in the presence of the anionic or cationic ligands
transported by RLIP76. Both peptides did bind ATP, as shown by
photoaffinity labeling, which increased in the presence of vanadate,
indicating the trapping of a reaction intermediate in the ATP-binding
site (Awasthi et al., 2001b). Neither of the two fragments catalyzed
transport when reconstituted alone in proteoliposomes. However, when
both of these fragments were reconstituted together, ATP-dependent transport of DNP-SG as well as DOX was observed with kinetic parameters similar to those for RLIP76 or DNP-SG ATPase (Awasthi et al., 2001b).
The ATP-binding sites in N-RLIP761-367 and
C-RLIP76410-655 were identified to be
69GKKKGK74 and
418GGIKDLSK425, respectively (Awasthi et al.,
2001b). Mutations of K74 and K425 in the
N-and C-terminal peptides, respectively, abrogated their ATPase activity, ATP-binding capacity, and transport function. The
sequence of these ATP-binding sites was similar but not identical with
the consensus for the P-loop (Walker motif). The
N-terminal ATP-binding site,
69GKKKGK74, resembled that of ABC proteins,
whereas the C-terminal site, 418GGIKDLSK425,
had similarity with the motif found in phosphoglycerate kinases (Saraste et al., 1990).
Surprisingly, unlike the ABC transporters, no transmembrane 
-helices
were evident in the RLIP76 sequence. Its association with membranes
has, however, been demonstrated by immuno-histochemical studies using
specific antibodies (Awasthi et al., 2001c, 2002). Its role in
endocytosis of EGFR, TGF, and insulin receptors (Yamaguchi et al.,
1997; Ikeda et al., 1998; Matsuzaki et al., 2002) exocytosis, and membrane ruffling (Moskalenko et al.2002) is also consistent with
membrane association. Furthermore, the extraction of DNP-SG ATPase/RLIP76 from cell lysates requires detergent, suggesting membrane
association, a feature essential for transport. These findings suggest
that a greater diversity in transporters exists, in terms of structural
elements defining ATP binding and mode of membrane insertion than is
currently accepted. In addition, the distinction between transporters
for organic anions as opposed to neutral or cationic substrates appears
to be blunted since RLIP76 can catalyze the transport of both, and, in
contrast to MRP1, does so without cotransporting GSH. Another
intriguing aspect of RLIP76 function is that it undergoes facile
proteolytic fragmentation, and at least some of the resulting peptides
can be reconstituted into an active transport complex (Awasthi et al.,
2001b). The processing of RLIP76 may be crucial for its transport
function, but this speculation needs to be substantiated through
further studies. The physiological significance of the hypothetical
processing of RLIP76 into a number of peptides is currently not
understood, but it is possible that it may be relevant to the functions
of RLIP76 as a GAP protein in pathways regulating endocytosis,
exocytosis, and membrane ruffling (Moskalenko et al., 2002).
| |
RLIP76-Mediated Transport of GS-HNE and Its Physiological
Significance |
Our studies have shown that a mild transient heat shock or
oxidative stress induces RLIP76 prior to inducing heat shock proteins or the antioxidant enzymes, which constitute the typical stress response (Cheng et al., 2001). In these studies, when K562 cells were
exposed to a mild heat shock (42°C, 30 min) or oxidative stress (50 µM H2O2, 20 min) and
allowed to recover for 2 h, enhanced lipid peroxide and 4-HNE
formation were observed in stressed cells as compared with the control
cells. There was a 3-fold induction of a GST isozyme hGST5.8, which
catalyzes the conjugation of 4-HNE and GSH to GS-HNE, and a 3.7-fold
induction of RLIP76, which was shown to mediate ATP-dependent transport
of GS-HNE from cells (Cheng et al., 2001). As shown in Fig.
3, the cells preconditioned with stress
shock transported GS-HNE at 3-fold higher rate as compared with the
untreated controls. This was consistent with more than 3-fold induction
of RLIP76 in the preconditioned cells. To confirm that RLIP76 did
indeed transport the GS-HNE and not its degradation products or
metabolites, the transported allocrite, hemiacetal of
3-(4-hydroxynonanyl) glutathione (Fig. 3, inset), was isolated from
media and characterized by mass spectral analysis. The increased efflux
of GS-HNE could be blocked by coating the cells with antibodies against
RLIP76, confirming that GS-HNE was transported by RLIP76 (Cheng et al.,
2001). More importantly, the stress-preconditioned cells with induced
hGST5.8 and RLIP76 acquired resistance to
H2O2-mediated cytotoxicity
and apoptosis (Fig. 4). The activation of
c-jun N-terminal kinase (JNK)-signaling pathway under
cellular stress conditions has been implicated in the apoptotic process
(Yang et al., 2001). Our studies indicated that this activation of JNK
was suppressed in the stress preconditioned cells, therefore rendering
them more resistant to the
H2O2-mediated apoptosis.
Figure 4b shows the protective effect both heat shock and oxidative
stress preconditioning on the
H2O2-induced apoptosis. The
protective effect of stress preconditioning against
H2O2 or 4-HNE-induced
apoptosis was abrogated by coating the cells with anti-RLIP76 IgG (Fig.
5), which inhibited the efflux of GS-HNE from cells suggesting a link between RLIP76-mediated efflux of 4-HNE
and apoptosis. Induction of hGST5.8 and RLIP76 by mild transient stress
and the resulting resistance of stress-preconditioned cell to apoptosis
appears to be a general phenomenon, since it was not limited to K562
cells but was also evident in lung cancer cells (H69 and H226), human
leukemia cells (HL60), and human retinal pigmented epithelial cells
(Cheng et al., 2001). The results of these studies strongly suggest
that the transport activity of RLIP76 in various cells regulates the
intracellular levels of 4-HNE, a lipid peroxidation product which is
known to be involved in apoptosis signaling, differentiation, and
perhaps cell proliferation at relatively lower concentrations (Ruef et
al., 1998; Cheng et al., 1999; Dianzani et al., 1999; Uchida et al.,
1999).
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Fig. 3.
Effect of heat shock and
H2O2 exposure on GS-HNE transport in K562
cells.
K562 cells (5 × 107 cells) were exposed to 42°C for
30 min and allowed to recover for 2 h in medium at 37°C. The
cells were pelleted and reincubated for 10 min at 37°C in 2 ml of
medium containing 20 µM [3H]4-HNE. The cells were
pelleted and washed twice with 2 ml of PBS. The supernatants and
washings were discarded, and the cells were incubated at 37°C for
2 h in 2 ml of 4-HNE free medium after which radioactivity was
determined in the medium. Radioactivity was found to be associated with
the hemiacetal of 3-(4-hydroxynonanyl) glutathione (inset), which was
isolated from the medium by high performance liquid chromatography and
characterized by mass spectral analysis. For
H2O2 treatment, the cells were incubated for 20 min at 37°C in media containing 50 µM H2O2.
After incubation, the cells were pelleted, washed free of
H2O2, incubated in H2O2
free medium at 37°C for 2 h, after which radioactivity was
measured in the medium. For treatment with antibodies, the cells after
heat shock treatment were allowed to recover for 1 h, and
respective IgGs were added (20 µg/ml medium) and incubated at 37°C
for additional 1 h. The cells were pelleted and
[3H]GS-HNE transport was measured as described above.
Values are means ± S.D. (n = 3 separate
experiments); *, indicates statistically significant differences
between treated and control cells evaluated by the Student's
t test (P < 0.05). **,
indicates the significant difference between heat shock preconditioned
cells treated with preimmune IgG and cells treated with RLIP76 IgG.
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Fig. 4.
a, effect of heat shock on the
H2O2-mediated cytotoxicity in K562 cells; b,
protective effect of heat shock and H2O2
pretreatment on H2O2 induced apoptosis in K562
cells.
In panel a, aliquots (40 µl) containing 2 × 104
control or heat shock-treated cells were washed with PBS and plated
into eight replicate wells in a 96-well plate.
H2O2 (50 µM) in 10 µl of PBS was added, and
the plates were incubated at 37°C for 2 h, after which 200 µl
of growth medium was added to each well. Following 72 h of
incubation at 37°C, the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was
performed. The OD590 values of sample subtracted from those
of respective blanks (no cells) were normalized with control values (no
H2O2). Averages and standard deviations from
three separate determinations of cytotoxicity of 4-HNE and
H2O2 are presented. In panel b, K562 cells
(2.5 × 106) in 5 ml of medium were treated with heat
shock at 42°C for 30 min, or 50 µM H2O2
(final concentration in medium) for 20 min and allowed to recover for
2 h in normal growth medium at 37°C. The cells, preconditioned
with heat shock or H2O2 treatment, were treated
with 100 µM H2O2 for 2 h. DNA (1 µg)
extracted from the cells was electrophoresed on 2% agarose gels
containing 10 µg/ml ethidium bromide. Lane 1, marker; lane 2, control; lane 3, H2O2 (100 µM, 2h); lane 4, heat shock pretreatment; lane 5, heat shock pretreatment + H2O2 (100 µM, 2h); lane 6, H2O2 pretreatment; lane 7, H2O2 pretreatment + H2O2 (100 µM, 2 h).
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Fig. 5.
Effect of anti-RLIP76 IgG on 4-HNE-mediated
apoptosis in heat shock preconditioned cells.
Aliquots (50-100 µl) containing 1 to ~2 × 106
cells were fixed onto poly-L-lysine-coated slides by
cytospin at 500g for 5 min and the terminal
deoxynucleotidyl transferase dUTP nick-end labeling apoptosis assay was
performed. The slides were analyzed by fluorescence microscope (Nikon
Eclipse 600; Tokyo, Japan) using a standard fluorescein filter (EX
450-490, DM 505, BA 520, B-2A). Photomicrographs at ×400
magnification are presented. Apoptotic cells showed characteristic
green fluorescence. Panel 1, control cells, without heat shock
pretreatment, incubated with 20 µM 4-HNE for 2 h; panel 2, control K562 cells pretreated with heat shock (42°C, 30 min) and
allowed to recover for 2 h at 37°C. Panel 3, cell pretreated
with heat shock, allowed to recover for 2 h at 37°C, followed by
incubation in medium containing 20 µM 4-HNE for 2 h at 37°C;
panel 4, heat shock pretreated cells, allowed to recover for 1 h
at 37°C, anti-RLIP76 IgG was added to medium (20 µg/ml final
concentration) and incubated for additional 1 h. Cells were then
incubated for 2 h at 37°C in medium containing 20 µM 4-HNE.
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The physiological significance of RLIP76-mediated transport of
endogenously generated GS-E (e.g., conjugate of 4-HNE) is further indicated by results of our studies showing that RLIP76-enriched cells
are resistant to radiation toxicity. In these studies, lung cancer
cells (H82) were loaded with RLIP76 by incubating with RLIP76
encapsulated in artificial liposomes. As shown in Fig. 6, cells enriched with RLIP76 were
remarkably resistant to radiation as compared with controls. These
results suggest that the electrophilic products of lipid peroxide
caused by reactive oxygen species (ROS) generated during radiation may
account for, at least partly, the cell killing by radiation and that
RLIP76-mediated transport of GSH conjugates of these electrophiles
provides protection from radiation. Proposed physiological significance
of the transport functions of RLIP76 including the ATP-dependent efflux
of xenobiotics and GS-E of exogenous as well as the endogenous
electrophiles is summarized in Fig. 7.
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Fig. 6.
Effect of RLIP76 on radiation sensitivity.
Small cell lung cancer cells, H82 were irradiated at 500 cGy with
high-energy photon (6 × 106 volt photon/min) for 1.25 min. at the Texas Cancer Center, Arlington. Cells were serially
passaged daily by inoculating 0.5 × 107 trypan blue
dye excluding cells/ml in fresh Roswell Park Memorial Institute medium.
The cell density measured each day was normalized to cell density in
respective unirradiated controls. Results presented are the mean and
S.D. of values from the following three groups: without treatment with
liposomes (circle), treatment with liposomes without RLIP76 (square),
and treatment with liposomes with RLIP76 (triangle).
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Fig. 7.
Physiological significance of RLIP76.
Xenobiotics, radiation, mitochondrial electron transport and metal ions
generate ROS, which cause membrane lipid peroxidation.
4-Hydroxynonenal, the toxic end product of lipid peroxidation has been
implicated in causing DNA damage leading to mutagenesis,
carcinogenesis, and apoptosis. 4-HNE also modulates the stress-mediated
signaling pathways. RLIP76 is capable of mediating ATP-dependent efflux
of a wide variety of amphiphilic drugs, GSH conjugates (GS-E) of both
xeno- and endobiotics including GS-HNE and leukotrienes from human
cells. The transport of GS-E is crucial for maintaining functionality
of GSTs and GR because these enzymes are inhibited by GS-E. RLIP76
regulates the intracellular concentrations of 4-HNE by a coordinated
mechanism with cellular GSTs. GR, glutathione reductase; CYP 450, cytochrome P450; GST, glutathione S-transferase; GPX,
glutathione peroxidase; LOH, reduced lipid hydroperoxides; GSSG,
glutathione disulfide.
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RLIP76 and Multidrug Resistance |
Our studies on the transport functions of RLIP76 strongly
suggest that it may be involved in the mechanism of multidrug
resistance of cancer cells. RLIP76 mediates ATP-dependent primary
active transport of not only anionic compounds (e.g., GSH conjugates) but also of the cationic chemotherapeutic drugs such as DOX,
daunomycin, and colchicine (Awasthi et al., 2000, 2001b; Singhal et
al., 2001). RLIP76 does not have significant sequence homology with ABC
transporters, known to be involved in the mechanisms of multidrug
resistance. RLIP76 differs from the ABC transporters in several other
important aspects, including 1) lack of any close homologs in humans,
2) ubiquitous expression in tissues, 3) lack of a classical nucleotide binding Walker domains, 4) integral membrane association without clearly defined transmembrane domains, and, most importantly, 5) its
role as a direct link to Ras/Ral/Rho and EGFR signaling through
its multifunctional nature including GAP activity and Ras/Ral/Rho-regulated effector function involved in receptor-mediated endocytosis. Its multifunctional nature perhaps derives from the presence of multiple motifs including Rho/Rac-GAP-domain, Ral-effector domain binding motif, two distinct ATP-binding domains, protein kinase
C and tyrosine kinase phosphorylation sites, and its proteolytic processing into multiple smaller peptides, which may participate as
components of macromolecular functional complexes.
RLIP76 overexpression confers resistance to both DOX and alkylating
toxins such as 4-HNE by increasing their efflux from cells (Awasthi et
al., 2000). Our studies (Cheng et al., 2001) also show that besides its
known GTPase-activating, Ral GTP-binding activity, RLIP76 can also
modulate stress signaling by regulating intracellular concentrations of
4-HNE, which is known to be involved in stress signaling (Ruef et al.,
1998; Dianzani et al., 1999; Uchida et al., 1999). Antibodies against
RLIP76 can block the transport of drugs (Awasthi et al., 2001c, 2002;
Cheng et al., 2001) and enhance cytotoxicity of DOX to cancer cells.
The higher resistance to DOX of nonsmall cell lung cancer (NSCLC) cells
as compared with the small cell lung cancer cells correlates with a
higher RLIP76-mediated efflux of DOX in NSCLC (Awasthi et al., 2001a,c;
2002). Coating with RLIP76 antibodies sensitizes NSCLC to DOX by
blocking their RLIP76-mediated transport. Taken together, these results
demonstrate that RLIP76, besides its physiological role as a GAP
protein, modulates drug sensitivity of cancer cells. RLIP76 is
expressed in all human tissues and cell lines examined so far, and it
can catalyze the transmembrane movement of physiologically relevant
ligands as well as a wide variety of xenobiotics irrespective of their
net charge.
The significance of RLIP76-mediated transport to the mechanisms of
multidrug resistance may go beyond the protection of cells through drug
efflux. Instead, RLIP76 could also impact on signaling mechanisms via
the modulation of the intracellular concentration of GS-HNE and its
precursor, 4-HNE, which is known to cause cell cycle arrest and promote
differentiation and apoptosis in cancer cell lines (Ruef et al., 1998;
Cheng et al., 1999; Dianzani et al., 1999; Uchida et al., 1999). Recent
studies suggest that the effects of 4-HNE on cell cycle signaling may
be concentration-dependent as it can have the opposite effect at lower
concentrations where proliferation is observed in the presence of low
4-HNE levels (Cheng et al., 1999). The level of 4-HNE is likely to
reflect the redox (or, more generally, stress) status of the cell and to convey the corresponding signal to the cell cycle and/or apoptosis machinery. Induction of RLIP76, perhaps by oxidative or chemical stress
due to anticancer drugs, would deplete 4-HNE and thus promote the
proliferation of cancer cells. RLIP76 could therefore have a
two-pronged effect in MDR. In addition to drug transport analogous to
that catalyzed by Pgp or MRP, RLIP76 could shift the signaling balance
in favor of cell proliferation.
Over the last two decades, a great deal of effort has been
devoted to the role of the conventional ABC proteins Pgp and MRP in the
mechanisms of MDR. A multitude of significant novel findings refining
the existing concepts in the mechanisms of xenobiotic transport have
emerged from these studies (see reviews Ambudkar et al., 1999; Leslie
et al., 2001). However, it may be worthwhile to broaden the scope of
the field to explore the putative role(s) of RLIP76 in the mechanisms
of MDR, not only because of its distinct transport properties but also
especially because of its potential role in modulating, directly or via
expression of regulatory genes, signaling that affects cell
proliferation and cell death. Furthermore, studies into the mechanism
of RLIP76-mediated transport of both organic anions and cations may
result in novel concepts that would broaden the understanding of
transmembrane movement of physiological/pharmacological ligands.
Supported in part by National Institutes of Health Grants GM 32304 (Y.C.A.), CA 77495 (S.A.) and Veterans Affairs Merit Review (P.Z.)
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Abstract
Introduction
