(Received for publication, September 15, 1995; and in revised form, February 20, 1996)
From the
Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been
associated with acquired or inherent resistance to oxazaphosphorine
(OAP) antineoplastic alkylating agents (e.g. cyclophosphamide). We previously demonstrated that expression of
transfected rat ALDH-3 can confer OAP-specific resistance in human
MCF-7 cells (Bunting, K. D., Lindahl, R., and Townsend, A. J. (1994) J. Biol. Chem. 269, 23197-23203). However, the
aldophosphamide intermediate inactivated by human class 1 ALDH
(hALDH-1) has not proven to be a good substrate for the purified
hALDH-3. We have examined the ability of transfected human or rat
ALDH-3 to confer OAP resistance in V79/SD1 cells. Clones expressing
elevated human (386-5938 milliunits/mg) or rat (4-597
milliunits/mg, benzaldehyde/NADP substrate) ALDH-3
activity were 1.3- to 12-fold resistant to mafosfamide relative to
control cells (<1 milliunit/mg). Resistance was correlated with
hALDH-3 activity, and was reversed by pretreatment with the ALDH
inhibitor diethylaminobenzaldehyde. Transfectants were cross-resistant
to 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide but not to
phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. DNA
interstrand cross-links were reduced commensurately with the fold
resistance to mafosfamide in the highest activity clone. A key finding
was the detection of a metabolite, most likely carboxyphosphamide, that
is formed only by cytosols from cells expressing either class 3 or
class 1 ALDH.
Increased expression of either class 1 or class 3 aldehyde
dehydrogenase (ALDH) ()isoforms has been found in tumor
cells surviving cytotoxic selection for OAP resistance (1, 2, 3) . High de novo expression
of one or both has also been associated with intrinsic OAP resistance
in tumor cell lines(
)(4) , and transient induction
of the class 3 ALDH isozyme with methylcholanthrene (5) or with
phenolic antioxidants (6) coincided with the development of
resistance in a human breast cancer cell line. Irreversible enzymatic
oxidation of aldophosphamide (ALDO), an aldehyde-containing activated
intermediate of CPA, to the inactive metabolite carboxyphosphamide (7) has been shown to be catalyzed by purified
yeast(8) , murine(9, 10) , or human (11, 12, 13) ALDH-1 in vitro. In
contrast, the role of class 3 ALDH in OAP resistance is still unclear
due to the lack (or low level) of ALDO oxidation by purified human
class 3 ALDH (hALDH-3) in vitro(14, 15) . In
particular, the class 3 ALDH normally expressed in human stomach
exhibited poor activity with ALDO as
substrate(15, 16) . However, a low activity for ALDO
oxidation was reported with human class 3 ALDH purified from
OAP-selected or methylcholanthrene-induced tumor
cells(4, 14) .
Although purified human ALDH-3 had weak catalysis with ALDO as a substrate in enzymology studies, we have previously shown that MCF-7 cells induced to express rat class 3 tumor-associated ALDH (tALDH-3) by stable transfection of a plasmid expression vector were resistant to OAP analogs(17) . The resistance was OAP-specific and closely correlated with tALDH-3 activity, and it was reversible by pretreatment with the ALDH inhibitor diethylaminobenzaldehyde (DEAB). Since the rat and human class 3 ALDH share significant sequence homology (81%) and similar substrate preferences, and because expression of a human class 3 ALDH has been strongly implicated as a causative factor in OAP-resistant cell lines, it was important to determine whether expression of the human cytosolic ALDH-3 would also be sufficient to confer resistance.
We have utilized transfection to produce genetically modified clonal derivative cell lines that should differ only in their levels of expression of ALDH activity. Using this transgenic model system, we were able to assess the capacity and activity dependence of this resistance mechanism over a very broad range of ALDH expression and to compare the rat and human class 3 ALDH with the human class 1 ALDH. The results indicated that the rat and human class 3 ALDH confer comparable levels of OAP-specific resistance at similar expression levels, but the absolute resistance with the class 3 isozymes was less than that conferred in this model system by the transfected human class 1 ALDH (28) . The resistance was correlated with class 3 ALDH activity, but the relationship was hyperbolic rather than the linear correlation observed with class 1 ALDH-mediated resistance. Importantly, the degree of protection from cytotoxicity was shown to be quantitatively similar to the reduction in levels of DNA interstrand cross-links. Protection against both cytotoxicity and DNA cross-links was abolished by preincubation with the ALDH competitive inhibitor DEAB. Finally, we were able to directly demonstrate metabolism of labeled activated cyclophosphamide by cytosol extracted from transfectant lines expressing hALDH-3 or hALDH-1, but not by extract from nonexpressing control cells.
The rat class 3 ALDH cDNA was excised from the pMTP-3H expression
vector previously used to transfect MCF-7 cells (17) by
digestion with EcoRI and BamHI, and then gel purified
and ligated into an EcoRI and BamHI digested and
dephosphorylated pGEM-7Z(-) plasmid (Promega). The rat class 3
cDNA was then released with XhoI and BamHI,
gel-purified, and directionally ligated into an XhoI and BamHI-digested and dephosphorylated pCEP4
vector.
Expression vectors were amplified in Escherichia coli DH5
(Life Technologies, Inc.) with ampicillin selection and purified using
Qiagen Maxi Prep ion exchange columns.
Western blot analysis, and densitometric analysis of a parallel Coomassie-stained SDS-polyacrylamide gel electrophoresis gel were performed essentially as described in our companion study(28) . Rabbit anti-rat class 3 ALDH antisera (generously provided by Dr. Ronald Lindahl, University of South Dakota) that was cross-reactive with human ALDH-3 was used as primary antibody to probe the blots at 1:3000 dilution.
Transfected cell lines exhibited elevated ALDH activity relative to SD1 parental and SD1/Hyg-1 (empty vector-transfected) control cell lines (<1 mU/mg) (Table 1). Rat class 3 ALDH transfectants (designated tALDH-X) showed increased activity ranging from 4 to 597 milliunits/mg and human class 3 ALDH transfectants (designated hALDH-X) showed increased activity ranging from 386 to 5938 milliunits/mg (Table 1).
Expressed mRNA levels were compared in rat and
human ALDH-3-transfected cell lines by Northern blot analysis with
specific P-labeled ALDH cDNA probes (data not shown). The
V79/SD1 parental and SD1/Hyg-1 cells contained no detectable class 3
ALDH mRNA, consistent with the undetectable activity in these lines. In
clonal lines expressing rat or human class 3 ALDH, the range of mRNA
transcripts detected was well correlated with ALDH-3 activities, as
quantitated by densitometry (r = 0.96; data not shown).
Western blot analysis revealed a broad range of expression of rat ALDH-3 (Fig. 1A) and human ALDH-3 (Fig. 1B) in the three transfected clones of each (lanes 3-5) while no expression is detectable in any of the control cell lines (lanes 1 and 2). Densitometric analysis of a separate SDS-polyacrylamide gel electrophoresis gel stained with Coomassie G250 indicated that human class 3 ALDH accounted for 2.3% of the cytosolic protein expressed in hALDH-3-26 cells (data not shown).
Figure 1: Western blot analysis of rat and human ALDH-3 protein expression in transfected V79 cell lines. Cytosolic protein (100 µg/lane) was loaded onto a 14% polyacrylamide gel and electrophoresed overnight. Protein was transferred to nitrocellulose by semidry electroblotting and probed with a 1:3000 dilution of rat anti-tALDH antisera as described under ``Experimental Procedures.'' Expression of class 3 ALDH was detected in each of the three high activity transfected cell lines (lanes 3-5), but was undetectable in V79/SD1 parental or SD1/Hyg-1 control (empty vector-transfected) cell lines.
Figure 2:
A,
sensitivity to MAF cytotoxicity of V79/SD1 cells expressing transfected
human ALDH-3. Cells were treated with mafosfamide for 30 min as
described under ``Experimental Procedures.'' Clonogenic
survival was determined as the number of colonies in drug-treated
plates as a percent of the number of colonies in control untreated
plates. The symbols denote: () control V79/SD1 parental and
(
), SD1/Hyg-1 (empty vector-transfected) cell lines; (
)
hALDH3-31; (
) hALDH3-35; and (
) hALDH3-26. B,
correlation between enzyme activity and resistance to mafosfamide in
V79/SD1 cells expressing transfected rat or human class 3 ALDH. A plot
of rat and human ALDH-3 activity (milliunits/mg) versus mafosfamide IC
(µM) indicates that
resistance correlates linearly with enzyme activity at activities below
about 2000 milliunits/mg. Above this level further increases in ALDH-3
activity have a progressively decreasing effect on resistance to
mafosfamide. Symbols are: (
) control cell lines; (
) rat
class 3 tALDH-transfected cell lines; (
) human class 3
ALDH-transfected cell lines.
The highest activity rat and human class 3 ALDH transfected cell lines were chosen to determine sensitivity to a range of OAP and non-OAP drugs (Table 2). A lower level of resistance was seen to 4-hydroperoxy CPA analogs (4-hydroperoxy-CPA, = 2.7-fold; 4-hydroperoxyifosamide, = 5.1-fold) in hALDH-3-26 cells. No resistance was seen in either tALDH-3 or hALDH-3-26 to non-oxazaphosphorines phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. These data indicate that resistance conferred by class 3 ALDH expression is OAP-specific.
Figure 3:
Reduction in DNA cross-link formation in
V79/SD1 cell lines expressing transfected human ALDH-3. Cells were
treated with mafosfamide for 30 min, and then incubated a further 5 h
to allow accumulation of DNA damage. Cells were irradiated with 400 rad
of gamma radiation on ice, and then analyzed for DNA cross-linking by
alkaline elution and quantitation of DNA in fractions by fluorometric
assay as described under ``Experimental Procedures.'' Results
indicate that the resistance in hALDH3-26 () relative to SD1/Hyg-1
(
) cells was 13-fold at 30-rad
equivalents.
Figure 4:
Metabolism of activated cyclophosphamide
by human cytosolic class 3 ALDH. Chloroethyl-H-labeled cyclophosphamide was
preincubated at 25 °C for 10 min with a washed rat microsome
reaction mixture, and then for another hour after addition of cytosol.
Metabolites were extracted, concentrated, and separated by TLC as
described under ``Experimental Procedures,'' and radiolabel
was detected using a
H-sensitive screen on a PhosphorImager
(Molecular Dynamics). Results clearly show formation of a metabolite
(see arrow) that migrates with an R
value of 0.29, similar to the R
value of 0.26 previously reported for
carboxyphosphamide(26) , in the samples containing added
hALDH-1 or hALDH-3.
An important role for the class 1 aldehyde dehydrogenase isozyme in resistance to the anticancer drug cyclophosphamide has been well supported by both cellular expression and enzymology studies. Elevation of either class 1 or class 3 ALDH have been found in OAP-selected resistant cell lines(1, 2, 3) . The ALDH-1-mediated resistance was accompanied by increased formation of carboxyphosphamide(1) , the inactivated product of ALDH oxidation of aldophosphamide, and decreased DNA cross-linking was also found(24) . The role of ALDH-1 in OAP resistance has also been directly supported by enzymologic studies that demonstrated favorable kinetics for ALDO oxidation by the purified enzyme(9, 10, 13, 16) . The validity and high capacity of resistance conferred by hALDH-1 has been firmly established our companion report (28) in which we demonstrated that expression of transfected class 1 ALDH indeed confers high level OAP-specific resistance.
The ability of the class 3 ALDH to confer
OAP resistance has been less clear than that of the class 1 isozyme.
Several studies indicated association of increased hALDH-3 expression
with OAP resistance(3, 4, 6, 14) ,
and we have previously shown that expression of rat class 3 ALDH
conferred OAP-specific resistance in MCF-7 cells(17) . Although
most purified human class 3 ALDH isozymes have consistently exhibited
low or absent activity for ALDO
oxidation(3, 4, 6, 16) , evidence
has been presented that class 3 ALDH purified from human tumor cells
exhibited a measurable, albeit low ALDO oxidation
activity(4, 5, 15) . Since class 3 ALDH is
expressed de novo in rat hepatoma
cells(25, 26) , and is elevated in some human primary
breast carcinoma samples (27) and carcinoma cell
lines(4) , the intriguing possibility exists that
ALDH-3 expression may constitute a tumor-associated intrinsic
resistance mechanism in a subset of malignancies.
A limitation of the above-mentioned correlative studies is the use of drug selection to obtain OAP-resistant cell lines, an approach that can result in cells that differ from drug sensitive parental cells in other respects in addition to increased expression of class 3 ALDH. Similarly, while studies with inducers or inhibitors have shown a correlation between ALDH-3 expression and OAP-specific resistance, such agents are often pleiotropic and can affect the expression or efficacy of resistance mechanisms other than, or in addition to ALDH. With these considerations in mind, we have attempted to address the role of ALDH isozymes in OAP resistance by utilizing transfected cell lines that should differ only in the level of expression of ALDH isozymes. The control lines used included both the original parent cell lines and empty vector-transfected and hygromycin-selected clonal lines to be sure that selection for antibiotic resistance did not affect OAP sensitivity.
The results of these studies clearly demonstrate that elevated expression of human or rat class 3 ALDH can confer relatively high-level OAP-specific resistance in V79 cells. The capacity of increased ALDH-3 activity to confer increasing levels of MAF resistance is limited, however, approaching an asymptotic limit with diminishing increments in resistance above approximately 2000 milliunits/mg activity. While the mechanism is not yet fully understood, reversal of resistance by the competitive inhibitor DEAB strongly suggests that catalysis of ALDO oxidation is the mechanism of resistance. The OAP specificity of human ALDH-3-mediated resistance and reversibility with ALDH inhibitors is consistent with results of analogous studies using high ALDH-3-expressing drug-selected (3, 15) or ALDH-3-transfected (17) cell lines, and those results also supported direct oxidation of ALDO as a likely mechanism of protection. Indeed, the specificity of class 3 ALDH-mediated resistance only for OAP analogs that can give rise to an aldehyde-containing intermediate has provided the best evidence to date that catalytic oxidation of ALDO to carboxyphosphamide is the most likely mechanism of resistance.
The analysis by TLC of metabolites formed by hALDH-3 from activated labeled CPA has provided new and compelling evidence in support of direct catalysis of ALDO oxidation as the primary mechanism of OAP-specific resistance conferred by this isozyme. A metabolite corresponding to the expected Rf value for carboxyphosphamide (23) was detected in reaction mixtures that contained cytosols derived from either the class 1 or class 3 ALDH-expressing cells (but not with control cell cytosol). The relative amount of this metabolite produced by cytosol containing hALDH-3 was nearly half of that produced by cytosol containing hALDH-1, which is consistent with the higher level of resistance conferred by expression of transfected hALDH-1 (21-fold versus 12-fold for hALDH-3).
Although the human
cytosolic ALDH-3 appears to catalyze ALDO oxidation, it remains to be
explained why the measured activity with this substrate was absent with
enzyme preparations purified from human
stomach(15, 16) . One possible reason for the lack of
activity toward ALDO with purified stomach ALDH could be a selective
modification during purification that abrogates hALDH-3 activity with
ALDO but leaves intact activity with the standard assay substrate
benzaldehyde. This could be due to chemical modification,
conformational changes, or loss of activity modulators during
purification. We have found that hALDH-3 activity is much more labile
than hALDH-1 activity in crude cytosol, with most of the
benzaldehyde/NADP activity lost within 10 min at 37
°C (data not shown).
A second potential reason for the poor ALDO
activity with purified hALDH-3 may relate to the extreme sensitivity of
the enzyme to inhibition by acrolein, a secondary product of the
spontaneous -elimination of PM from ALDO. The hALDH-3 isozyme may
be relatively well protected in intact cells, where acrolein would
likely react rapidly and nonspecifically with many competing
nucleophiles such as glutathione and other non-protein or protein
sulfhydryls. However, in a purified enzyme preparation the ALDH would
be the sole target protein and hence would potentially be much more
vulnerable to inhibition and/or covalent modification by acrolein.
Consistent with this possibility, we have found that the human class 3
ALDH was also inherently severalfold more sensitive to acrolein
inhibition than the human class 1 isoform, when measured with a
constant amount of cytosolic protein in the assay mixture.
The greater sensitivity of the hALDH-3 to inhibition by
acrolein may also play a role in the nonlinear relationship between the
degree of resistance and the level of expression of this isozyme.
Whereas resistance increased linearly with hALDH-1 expression in
transfected V79/SD1 cells(28) , the relationship was asymptotic
in cells expressing transfected hALDH-3, and thus indicates that some
factor is limiting for protection by this isozyme. The greater
sensitivity to acrolein inhibition is one possible reason, since this
by-product would be present in increasing amounts at the higher
IC concentrations of OAPs required to kill the higher
expressing clones. Since glutathione is protective against acrolein,
progressive depletion of this thiol pool at higher MAF concentrations
might also result in a nonlinear resistance response by increasing the
susceptibility of hALDH-3 to inhibition by acrolein. Another possible
reason for the nonlinearity of resistance could relate to the
sensitivity of hALDH-3 to inhibition by other OAP metabolites at high
drug concentrations, including carboxyphosphamide, the product of ALDH
oxidation of ALDO.
We have shown that both the hALDH-1 (28) and hALDH-3 isoforms are potentially major determinants of cellular sensitivity to cyclophosphamide during chemotherapy treatment. The class 3 isoform represents a potentially tumor-specific target for adjuvant chemotherapy treatment due to its increased expression in certain types of neoplasia. However, targeting by the use of an inhibitor would need to be selective for class 3 and not class 1 ALDH, since the relatively modest marrow toxicity of CPA appears to be at least partly due to the high levels of hALDH-1 in hematopoietic progenitor cells. An alternative gene therapy approach, that would take advantage of the OAP resistance conferred by class 1 or class 3 ALDH expression, could be to use expression vectors to engender constitutive ALDH expression in marrow stem cells. While ALDH-1 activity normally decreases with differentiation, stable expression of high hALDH-1 activity could enhance the ability of cells to survive CPA chemotherapy throughout the hematopoietic cascade, rather than only in stem cells. Alternatively, the resistance could be augmented in both stem cells and their downstream progeny by transfection of class 3 ALDH, which is normally low in hematopoietic cells. The studies described in this report and in our companion study (28) provide a rational basis for these strategies to take therapeutic advantage of the effect of ALDH expression on cellular sensitivity to oxazaphosphorine anticancer alkylating agents.