(Received for publication, September 15, 1995; and in revised form, February 20, 1996)
From the
Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize
aldophosphamide, a key aldehyde intermediate in the activation pathway
of cyclophosphamide and other oxazaphosphorine (OAP) anti-cancer
alkylating agents. Overexpression of class 1 ALDH (ALDH-1) has been
observed in cells selected for survival in the presence of OAPs. We
used transfection to induce de novo expression of human ALDH-1
in V79/SD1 Chinese hamster cells to clearly quantitate the role of
hALDH-1 expression in OAP resistance. Messenger RNA levels correlated
well with hALDH-1 protein levels and enzyme activities (1.5-13.6
milliunits/mg with propionaldehyde/NAD substrate,
compared to < 1 milliunit/mg in controls) in individual clonal
transfectant lines, and slot blot analysis confirmed the presence of
the transfected cDNA. Expressed ALDH activity was closely correlated (r = 0.99) with resistance to mafosfamide, up to
21-fold relative to controls. Transfectants were cross-resistant to
other OAPs but not to phosphoramide mustard, ifosfamide mustard,
melphalan, or acrolein. Resistance was completely reversed by
pretreatment with 25 µM diethylaminobenzaldehyde, a potent
ALDH inhibitor. Alkaline elution studies showed that expression of
ALDH-1 reduced the number of DNA cross-links commensurate with
mafosfamide resistance, and this reduction in cross-links was fully
reversed by the inhibitor. Thus, overexpression of human class 1 ALDH
alone is sufficient to confer OAP-specific drug resistance.
The aldehyde dehydrogenase (ALDH) ()multigene family
of enzymes is presumed to function as an important component of
cellular defenses against toxic
aldehydes(1, 2, 3) . The multiple ALDH
isoforms are classified according to their amino acid sequence homology
as either class 1 (cytosolic), class 2 (mitochondrial), or class 3
(cytosolic and microsomal)(2) . The class 1 ALDH isozyme has
also been implicated in the metabolic inactivation of activated
metabolites of the widely used anti-cancer and immunosuppressive agent
cyclophosphamide (CPA) and other members of the oxazaphosphorine (OAP)
class of DNA alkylating agents(4) . The inactive prodrug CPA
requires activation to 4-hydroxycyclophosphamide (4-OH-CPA) by
cytochrome P450-2B6 in humans(5) , primarily in the
liver(4) . Aldophosphamide (ALDO) is a membrane-permeable
ring-opened tautomer of the activated 4-OH-CPA that is the major
metabolite present in the blood of patients treated with
CPA(4) . This unstable aldehyde species undergoes spontaneous
cleavage to yield the cytotoxic DNA cross-linking agent phosphoramide
mustard (PM), and also the highly reactive side product
acrolein(4, 6, 7) . An alternative fate for
ALDO formed during CPA metabolism is irreversible oxidation to
carboxyphosphamide (CBP), a potential detoxification reaction that has
been demonstrated by in vitro enzymology studies using yeast
ALDH (8) and using purified murine and human class 1
ALDH(9, 10, 11, 12) . Overexpression
of either class 1 or class 3 ALDH isozymes has been observed in cell
lines following cytotoxic drug selection with OAP
analogs(13, 14) , and intrinsic OAP-specific
resistance has been found in cell lines that express high levels of
class 3 ALDH(
)(15) , or both class 1 and class 3
ALDH.
In order to quantitatively examine the effect of variable ALDH expression on OAP-specific resistance and also to clearly establish that expression of ALDH-1 alone is sufficient to cause resistance, we have developed transgenic cell lines that express a broad range of activities of either class 1 or class 3 ALDH, via transfection with mammalian expression vector constructs that contain the respective cDNAs encoding each isozyme. This report details direct testing of the ability of the cytosolic class 1 ALDH to confer oxazaphosphorine-specific resistance in transgenic cell lines expressing transfected human ALDH-1. This approach has an inherent advantage over comparison of drug-selected and parental cells, or comparison of different cell lines, in that expression of the transfected gene product should be the sole variable. In contrast, cell lines subjected to cytotoxic drug selection may have multiple phenotypic differences from control cells, and this would also be true for comparison of different cell lines.
We have demonstrated in the present study that fold-resistance to mafosfamide (up to 21-fold in the highest activity clone) was linearly correlated with ALDH activity in several clonal transfectant lines that express low, intermediate, or high levels of class 1 ALDH. Resistance was completely reversed in the ALDH-expressing transfectant lines by the pretreatment of cells with the potent ALDH inhibitor diethylaminobenzaldehyde (DEAB), which had little effect on the drug sensitivity of the control (empty vector-transfected) cell line. Interestingly, the ALDH-1-expressing transfectant lines exhibited lower levels of resistance to other OAPs (4-hp-CPA, 4-hp-IF). However, no resistance was conferred to the non-OAP alkylating agents PM, isophosphoramide mustard, L-phenylalanine mustard, or acrolein. Finally, the degree of protection from cytotoxicity was shown to be quantitatively correlated with both class 1 ALDH activity and the reduction in levels of DNA interstrand cross-links.
A second modification to
the vector was the introduction from position -29 to -47 bp
(relative to the ATG start of translation) of a 19-bp sequence derived
from the proximal 5`-UTR of the human GSTM1-1 cDNA (17) . This
was the shortest 5`-leader sequence that we knew to be capable of
supporting high level translation of the downstream cDNA (18) .
Two oligonucleotides, consisting of a 28-mer and a 20-mer,
(5`-AGCTTGGTTGGTGCGGATTCCGCGGTAC-3`) and (5`-CGCGGAATCCGCACCAACCA-3`),
were annealed to form an insert with KpnI- and HindIII-compatible overhangs, with a 19-bp sequence
corresponding to bases no. 1-19 of the 5`-UTR from the human GSTM1-1
cDNA (pGTH4) (17) in between. The annealed insert was ligated
at high vector, insert ratio into a KpnI- and HindIII-digested, gel-purified pCEP4 vector. The
resulting vector, designated ``
pCEP4
,'' was
transformed, amplified, and purified on ion-exchange columns (Qiagen).
Prior to incorporation into the vector, the cDNA was modified by
polymerase chain reaction amplification to include a translation
initiation leader sequence CCACC (19) immediately 5` to the ATG
start codon, since this sequence is present in the 5`-UTR of the human
GSTP1-1 cDNA(20) , and is known to support high levels of
expression of GSTP1-1 in V79 cells. ()An XhoI
restriction endonuclease cleavage site was included in the UTR of the
5`-primer, and a BamHI cleavage site in the UTR of the
3`-primer. The primers were, amino-terminal end:
5`-TTTCTCGAGCCACCATGTCATCCTCAGGCACG-3`, and carboxyl-terminal end:
5`-GAGGGATCCTTATGAGTTCTTCTGAGAGAT-3` (restriction sites and the ATG
start codon are underlined). Cycle parameters for amplification were:
94 °C/60 s denaturation, 50 °C/30 s annealing, and 72
°C/120 s extension, for 30 cycles. The 1.5-kilobase pair cDNA
product was gel-purified, digested with XhoI and BamHI restriction endonucleases and directionally subcloned
into the XhoI/BamHI digested and dephosphorylated
pCEP4
expression vector. The final product is henceforth
referred to as the
pCEP4
/hALDH-1 expression vector. Partial
DNA sequence analysis confirmed that the cDNA was identical to the
reported human class 1 ALDH sequence and also confirmed insertion of
the human GSTM1-1 5`-untranslated region into the the vector (data not
shown).
Figure 1:
DNA slot
blot analysis for ALDH-1 cDNA in transfected V79 cells. Genomic DNA was
prepared as described under ``Experimental Procedures'' and
cross-linked to a nylon blotting membrane using a stratalinker UV
source (Stratagene, Inc.) set at 1200 joules. Blots were probed with P-labeled hALDH-1 cDNA and washed extensively with the
final two washes in 0.1
SSC, 1% SDS at 60 °C and 65 °C,
for 1 h each. The blot was exposed to autoradiography film for 90 h.
The result demonstrates that the ALDH-1 cDNA sequence is present and
quantitatively correlated with ALDH activity in the transfected cell
lines (lanes 3-5) but is not present in V79/SD1 parental
or SD1/Hyg-1-transfected control (empty vector-transfected) cell lines (lanes 1 and 2).
The human ALDH-1 mRNA levels were determined
by Northern blot analysis of total RNA from each of the five cell lines (Fig. 2). The results demonstrate the presence of an
approximately 1.9-kilobase pair mRNA band that hybridized to P-labeled human class 1 ALDH cDNA in all three transfected
cell lines (lanes 4, 6, and 8). As expected,
neither the V79/SD1 parental nor the SD1/Hyg-1 control cell lines
exhibited a band of this size (lanes 1 and 2). The
mRNA levels analyzed by densitometry also correlate well with the
relative levels of propionaldehyde/NAD
ALDH activity (r = 0.98) (data not shown).
Figure 2:
Northern blot analysis of ALDH-1 mRNA
levels in transfected V79 cell lines. Total RNA was isolated and
fractionated on a 1% agarose gel containing 0.22 M formaldehyde, and transferred by capillary blotting and
cross-linked to a nylon membrane as described under ``Experimental
Procedures.'' The blot was then probed with P-labeled
hALDH-1 cDNA. The results demonstrate expression of ALDH-1 mRNA in
transfected cell lines (lanes 3-5) but not in V79/SD1
parental or SD1/Hyg-1 control (empty vector-transfected) lines (lanes 1 and 2).
The expression of the 55-kDa human ALDH-1 protein was confirmed by Western (immuno-) blot analysis, using antisera raised against rat class 1 ALDH (Fig. 3). The parental SD1 cells and SD1/Hyg-1 cells did not express detectable levels of the class 1 ALDH protein (lanes 1 and 2) and thus were good recipient cell lines for these studies. A wide range of ALDH protein expression was seen in the three cell lines with elevated ALDH activity (lanes 3, 4, and 5). Densitometry of a separate Coomassie Blue-stained gel (corrected for control background density) indicated that in the highest activity cell line obtained, the hALDH-1 band accounted for 0.72% of the total cytosolic protein expressed (data not shown).
Figure 3: Western blot analysis of human ALDH-1 protein expression in transfected V79 cell lines. Cytosolic protein (100 µg/lane) was loaded onto a 14% polyacrylamide gel, electrophoresed overnight, and protein was electroblotted onto nitrocellulose, blocked with 5% milk, and probed with anti-rat PB-ALDH antisera as described under ``Experimental Procedures.'' The ALDH-1 protein expression increased in a manner commensurate with activity in each of the three ALDH-1-transfected cell lines (lanes 3-5) but was undetectable in V79/SD1 parental or SD1/Hyg-1 control (empty vector-transfected) cell lines (lanes 1 and 2).
Figure 4:
A, concentration-response graph for MAF
cytotoxicity in class 1 ALDH-expressing V79 cells. Cells were treated
with MAF for 30 min as described under ``Experimental
Procedures.'' Following drug exposure, cells were pelleted by low
speed centrifugation, washed, and plated in 60-mm dishes. After
6-8 days, colonies were counted following staining with methylene
blue. Clonogenic survival was expressed as the percent of colonies in
drug-treated plates relative to the number of colonies in control
untreated plates. The survival curves demonstrate increasing levels of
resistance in the three transfected cell lines expressing hALDH-1, in
contrast to V79/SD1 parental () and SD1/Hyg-1 (
) control
(empty vector-transfected) cell lines which lack ALDH activity. B, correlation between ALDH-1 enzyme activity and resistance
to mafosfamide in transfected V79/SD1 cells. A plot of ALDH-1 activity
(milliunit/mg) versus MAF IC
(µM)
indicates a direct correlation between ALDH-1 activity and drug
resistance. A correlation coefficient of 0.99 obtained by linear
regression analysis provides strong evidence supporting the role of
ALDH-1 as an OAP-detoxifying ALDH isoform (linear regression equation: y = 80.8 + 41.75x, R
= 0.982). The data points are the activity and IC
values from Table 1for control (
) or hALDH-1
transfected (
) clonal lines, representing the average of at least
three independent experiments each. The line was fitted to the data
using the interpolation function of the Cricket Graph program (Cricket
Software, Inc.) on a Macintosh IIci
computer.
Cross-resistance to other oxazaphosphorine alkylating agents was also examined in the SD1/Hyg-1 (control) and hALDH1-28 (highest activity) cell lines (Table 2). A lower but clearly significant level of resistance was seen with both 4-hp-CPA and 4-hp-IF. Both of these compounds are activated by rapid spontaneous hydrolysis to yield 4-OH-CPA or 4-OH-IF and hydrogen peroxide. Resistance to these agents was 2.2-fold for 4-hp-CPA and 4-fold for 4-hp-IF, significantly less than the resistance exhibited to MAF (20.6-fold). These two cell lines were also analyzed for sensitivity to non-OAP alkylating agents, but no significant resistance was seen to the alkylating agents phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. Thus, resistance conferred by transfected hALDH-1 is OAP-specific.
Figure 5:
Alkaline elution analysis of DNA
cross-link formation in control versus hALDH-1 expressing V79/SD1 cell
lines. Cells were treated as described under ``Experimental
Procedures,'' followed by alkaline elution overnight and
quantitation of DNA in fractions by fluorometric assay. Following a
30-min exposure to MAF and 5 h of further incubation to allow
accumulation of DNA damage, cells were irradiated with 400 rad of
radiation on ice, and then analyzed for DNA cross-linking. Results
indicate that hALDH-1 can confer protection against proteinase
K-resistant DNA interstrand cross-linking by MAF. The fold-resistance
in hALDH1-28 (
) relative to empty vector-transfected control
SD1/Hyg-1 (
) cells was 24-fold at 30-rad
equivalents.
Cytosolic class 1 and class 3 ALDH isozymes have been
implicated in resistance to the oxazaphosphorine class of drugs in cell
lines selected for resistance to 4-hydroperoxy-cyclophosphamide. The
class 1 ALDH has been shown to be overexpressed in the mouse leukemia
L1210/OAP cell line selected in vitro for the ability to
survive drug exposures that are supralethal for the parent cell
line(4, 13) , and in an in vivo rat model for
acquired CPA resistance in acute myeloid leukemia cells(28) .
This detoxification reaction may also be a key factor in the relative
hematopoietic stem cell-sparing effect of CPA(29) , since class
1 ALDH expression is relatively high in CD34 hematopoietic progenitor cells(30) . The human class 3
ALDH, which as a purified enzyme has much less activity for ALDO
oxidation in vitro, has also been found to be overexpressed in
OAP-resistant cell lines, following drug selection or induction by
catechol or antioxidants(31, 32, 33) .
We have constructed human ALDH-expressing transgenic cell lines for use as in vitro model systems for the systematic study of the role of class 1 and class 3 ALDH isoforms in their ability to confer drug resistance. Results from a previous study indicated that transfected rat class 3 ALDH could play a significant role in oxazaphosphorine-specific resistance even at low levels of expression (24) , and despite the fact that only marginal activity was previously reported with purified human class 3 ALDH using ALDO as substrate. The studies described in this report address important questions regarding the capacity and mechanism of the hALDH-1-mediated resistance, and examine reversibility by an ALDH inhibitor of high level resistance at elevated expression of ALDH. A companion study in this issue focuses on the role in resistance of the human class 3 ALDH(41) .
The close correlations observed between relative
gene copy number, mRNA levels, hALDH-1 expression, ALDH activity, and
MAF resistance provides the strongest evidence to date that hALDH-1
expression alone is sufficient to confer high level resistance to
oxazaphosphorines. Furthermore, the OAP specificity and reversal of
high levels of MAF resistance with 25 µM DEAB argue
strongly that the catalytic activity of hALDH-1 is both necessary and
sufficient for mediation of its protective effects. Only the OAP
analogs give rise to intermediates containing an aldehyde function that
can be oxidized by hALDH-1 (4) and conversely, resistance is
not conferred to the non-OAP alkylating agents that do not generate
this moiety. Similarly, the OAP-specific reversal of resistance and
restoration of DNA cross-linking to control levels in
hALDH-1-expressing cells by the ALDH inhibitor DEAB also supports
catalytic inactivation as the sole mechanism of resistance. Comparison
of resistance to cytotoxicity (21-fold) with resistance to DNA
cross-linking (24-fold) in hALDH-1-28 cells also suggests that
resistance is fully accountable by reduced cross-linking, and is
consistent with DNA cross-linking as the overriding cause of
cytotoxicity. Finally, we have shown hALDH-1-dependent formation of H-labeled carboxyphosphamide from labeled cyclophosphamide
by TLC analysis of a co-incubation assay, in which activation by rat
liver microsomes was coupled with metabolism by cytosol from
transfected cell lines (41) .
An interesting aspect of the
OAP resistance conferred was that the hALDH1-28 clone exhibited much
lower resistance to 4-hp-CPA (2.2-fold) or 4-hp-IF (4-fold) than to MAF
(20.6-fold). The reason for this difference is presently not clear, but
cannot be explained solely on the basis of hALDH-1 substrate
specificity, since MAF and 4-hp-CPA both give rise to the same
intermediate, aldophosphamide(4) . A potentially important
factor may relate to the release of MESNA from MAF, whereas the
hydroperoxy compounds instead generate hydrogen peroxide in
stoichiometric amounts upon hydrolytic activation. The nucleophilic
thiol group in MESNA, which readily reacts with acrolein, has made it a
useful adjuvant agent in CPA treatment to relieve bladder toxicity (34) which is believed to be caused largely by
acrolein(35) , produced by -elimination from
aldophosphamide to form phosphoramide mustard. Thus, the released MESNA
might also play an important role in allowing ALDH isozymes to more
effectively protect cells from MAF cytotoxicity, since conjugation of
the thiol group with acrolein would sequester a potent hALDH-1
inhibitor(36) . Decreased ALDH inhibition by acrolein could
thus explain the differences seen in resistance conferred by hALDH-1 to
the OAP class of alkylating agents. This possibility has been recently
supported by the observation that extracellular GSH or MESNA can in
fact increase the fold-resistance to 4-hp-CPA in the hALDH-1-expressing
cells.
Another key distinction regarding the hydroperoxy prodrugs is that they generate an oxidant, hydrogen peroxide, during spontaneous hydrolysis to ALDO, whereas MAF releases the nontoxic thiol-containing antioxidant compound MESNA(4, 27) . Thus, a second reason for the weaker protection against the hydroperoxy compounds could be that the released peroxide contributes to toxicity by a mechanism that is unaffected by hALDH-1 expression. In addition to direct toxicity by peroxide, another mechanism may involve an acrolein-glutathione conjugate that has recently been proposed to generate oxygen radicals as a byproduct of oxidation of the conjugate by ALDH(37) . In cells with high ALDH activity, sufficient oxygen radicals could be formed to react with hydrogen peroxide and generate highly reactive hydroxyl radicals. The resulting toxicity would also antagonize protection by transfected ALDH against 4-hp-CPA treatment.
The studies presented herein clearly show that hALDH-1 expression confers OAP-specific resistance, and that even high level resistance is fully reversible by a potent ALDH inhibitor, DEAB. Thus, resistance conferred by ALDH expression could in certain cases represent a potential target for adjuvant therapeutic intervention with ALDH inhibitors. Either class 1 or class 3 ALDH can be elevated in cells exhibiting OAP-specific resistance following cytotoxic selection by OAP agents. Furthermore, preliminary results have been presented that indicate that both isozymes are also expressed to varying degrees in breast tumors, at generally higher levels than the adjacent normal tissue(38) . However, the class 1 isoform may not be as commonly expressed in tumors as is the class 3 ALDH. Moreover, it is important to remember that high class 1 ALDH expression appears to be a major factor in the relative resistance of normal hematopoietic stem cells to CPA cytotoxicity(29) . Thus, inhibition of class 1 ALDH might reduce rather than enhance the therapeutic index, since high class 1 ALDH expression in stem cells may be principally responsible for the relative stem cell sparing effect of CPA.
Alternatively, it may be possible to augment the natural resistance in normal stem cells by enhancing class 1 ALDH detoxification, in order to allow more intensive dosing of CPA. This could be accomplished by increasing endogenous ALDH-1 expression with inducers (gene regulation) or by enhancing the metabolic capacity at the existing expression level by other biochemical manipulations. This possibility was supported by experiments showing that pretreatment of normal human hematopoietic precursor cells with interleukin-1 plus tumor necrosis factor a resulted in protection against OAP toxicity, and this effect was blocked by the ALDH inhibitor DEAB(39) . Another approach could involve transduction of ALDH-1 expression into hematopoietic stem cells by insertion of an ALDH-1 expression vector, as we have done with cultured fibroblastic cells in the present study. We have shown that such an approach could result in fold-resistance of greater than an order of magnitude. Although ALDH-1 is already expressed at relatively high levels in the stem cell population(40) , this activity appears to decline with differentiation(29) . Thus, stable induction of constitutively high ALDH-1 expression in hematopoietic stem cells by gene therapy in conjunction with bone marrow transplantation could provide a substantial benefit for cancer patients who require high dose cyclophosphamide chemotherapy.