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Volume 271, Number 20, Issue of May 17, 1996 pp. 11884-11890
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
De Novo Expression of Transfected Human Class 1 Aldehyde Dehydrogenase (ALDH) Causes Resistance to Oxazaphosphorine Anti-cancer Alkylating Agents in Hamster V79 Cell Lines
ELEVATED CLASS 1 ALDH ACTIVITY IS CLOSELY CORRELATED WITH REDUCTION IN DNA INTERSTRAND CROSS-LINKING AND LETHALITY (*)

(Received for publication, September 15, 1995; and in revised form, February 20, 1996)

Kevin D. Bunting (§),  ,  Alan J. Townsend (¶)

From the Biochemistry Department, Bowman Gray School of Medicine, Wake Forest, University Comprehensive Cancer Center, Winston-Salem, North Carolina 27157

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.

INTRODUCTION

The aldehyde dehydrogenase (ALDH) (^1)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(^2)(15) , or both class 1 and class 3 ALDH.^2

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.

EXPERIMENTAL PROCEDURES

Materials

Agarose was obtained from FMC Corp. Restriction endonucleases, T4 DNA ligase, and calf intestinal alkaline phosphatase were obtained from Promega. Taq DNA polymerase and dNTPs were obtained from Boehringer-Mannheim. Acrylamide and phenol were obtained from Life Technologies, Inc. The aminoglycoside antibiotic hygromycin B was obtained from Calbiochem. Mafosfamide, phosphoramide mustard, 4-hydroperoxycyclophosphamide, 4-hydroperoxyifosfamide, and isophosphoramide mustard were generously provided by Dr. P. Hilgard and Dr. J. Pohl of Asta Medica Inc. (Frankfurt, FRG), and by Dr. O. M. Colvin (Duke University, Durham, NC). alpha-[P]dCTP was obtained from DuPont NEN. All other reagents were reagent grade or higher and were obtained from Sigma or Fisher.

ALDH Expression Vector Construction

The cDNA for human class 1 ALDH was originally cloned by polymerase chain reaction from human liver cDNA and generously provided by Dr. Henry Weiner(16) . Because the cDNA provided was cropped for use in a bacterial expression vector, having only the coding region but lacking 5`- or 3`-untranslated regions, we added sequences to both the cDNA and the expression vector to create a 5`-UTR that would support translation in a mammalian system. The mammalian expression vector pCEP4 (Invitrogen, Inc.) was first modified to remove the sequences required for episomal replication in mammalian cells, in order to allow for selection of clonal transfectant cell lines expressing hygromycin resistance with the vector stably integrated into cellular DNA. The 4,280-bp sequence containing the Epstein-Barr nuclear antigen and oriP viral replication origin was released by digestion with ClaI and EcoRV, followed by blunt ligation of the remaining 6,130-bp gel-purified vector fragment. This nonepisomal parent vector, designated ``DeltapCEP4,'' retained the Hyg^R and AMP resistance genes, the multiple cloning site, the cytomegalovirus immediate early promoter, and the 3`-polyadenylation signal sequence.

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 DeltapCEP4 vector. The resulting vector, designated ``DeltapCEP4Delta,'' 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. (^3)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 DeltapCEP4Delta expression vector. The final product is henceforth referred to as the DeltapCEP4Delta/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).

Culture and Transfection of V79/SD1 Cells

Chinese hamster lung fibroblast cells (V79/SD1), previously transfected with a cytochrome P450IIB1 expression vector, were generously provided by Dr. Johannes Doehmer(21) . These cells were chosen as the recipient line in order to allow for activation of labeled CPA by cytochrome P450 IIB1 in situ. However, because of concerns about the nonpharmacologically high concentrations of CPA required to induce cytotoxicity in control cells and especially in high activity ALDH-expressing clones, we elected to utilize the hydrolytically activated OAP analogs MAF, 4-hp-CPA, and 4-hp-IF so that meaningful comparisons of IC values could be drawn. Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Life Technologies, Inc.) at 37 °C in a 5% CO(2) atmosphere. Cells (5 times 10^5) were transfected with the DeltapCEP4Delta/hALDH-1 expression vector (10 µg) by a calcium phosphate precipitation procedure(22) , and selected for resistance to hygromycin B (0.7 mg/ml) conferred by the vector Hyg resistance gene. Another population of V79/SD1 cells was similarly transfected with the empty DeltapCEP4Delta vector and selected for hygromycin resistance to serve as a transfected control cell line. This cell line, designated SD1/Hyg-1, had very low ALDH activity equal to the parental SD1 cell population. Colonies representing clonal transfectant lines were picked, expanded, characterized (see below), and cryogenically stored at -80 °C. The doubling times were determined to be similar (11.7-12.9 h) in all cell lines at early passage (data not shown). Since different clonal lines exhibited variable fractions of the ALDH expression that was stable in the absence of hygromycin selection, cells were grown under continuous selective pressure in order to maintain optimal activity. In order to control for this stress, the SD1/Hyg-1 empty vector-transfected cells were also selected and maintained in hygromycin. The lack of significant differences in growth (not shown) or IC for MAF between the SD1/Hyg-1 and V79/SD1 parental cell lines suggests that this selection did not affect sensitivity to the OAP analogs. Nevertheless, cells were cultured without hygromycin for 24 h prior to experiments, and cell stocks were replaced with a fresh aliquot of cryopreserved early-passage cells every 3-4 weeks in order to minimize any effect of hygromycin selection.

Biochemical Analysis

Forty-eight clones that survived hygromycin selection were expanded, and the cytosolic fractions were prepared and assayed for enzyme activity and protein by a modification of the method described by Manthey and Sladek(23) , as described previously(24) . One additional change in the assay was the time over which the activity rate was determined. We found that ALDH-1 activity measured repeatedly in the same reaction mixture gradually increased by 1.5-2-fold over a 30-min period before leveling off, possibly reflecting slow elimination of an inhibitor or exhaustion of an opposing reaction that reoxidized the NADH chromophore measured in the ALDH kinetic assay. Thus, all components of the reaction mixture were routinely preincubated for 30 min at 25 °C prior to spectrophotometric determination of the DeltaA/min. Under these assay conditions, less than 10% of substrates were converted to product. One milliunit of activity was defined as the amount of activity that oxidized 1 nmol of substrate/min at 25 °C.

DNA Slot Blot Analysis

Transfected cells growing in 0.7 mg/ml hygromycin were harvested by scraping into cold phosphate-buffered saline, and cell pellets were stored at -80 °C. Cells were lysed in buffer containing 100 mM NaCl, 10 mM Tris/HCl, pH 8.0, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K. This lysate mixture was incubated for 18 h at 50 °C followed by phenol/chloroform extraction, chloroform/butanol extraction, and ethanol precipitation. Digestion of RNA with 1 µg/ml DNase-free RNase was carried out for 1 h at 37 °C followed by an additional phenol/chloroform extraction. DNA (10 µg/slot) was denatured in 0.35 N NaOH for 1 h at 65 °C, then neutralized with 1 M ammonium acetate, pH 7.0. DNA was blotted onto a MagnaGraph nylon membrane (Micron Separations, Inc.) using a MilliBlot-S vacuum blotter (Millipore Systems, Inc.), and cross-linked to the blot by exposure to 254-nm UV light (Stratalinker, Stratagene Inc.). Gel-purified human class 1 ALDH cDNA was P-labeled by the random priming method using the Prime-a-Gene kit (Promega), denatured, and hybridized to the blot according to the manufacturer's instructions, with two final washes in 0.1 times SSC, 1% SDS at 60 °C and 65 °C for 1 h each. The blot was exposed to autoradiography film (DuPont) for 90 h.

Northern Blot Analysis

Transfected cells were grown and harvested as described above. Total RNA was isolated using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer's instructions. The RNA (25 µg/lane) was fractionated on a 1% agarose gel in 20 mM MOPS buffer, pH 7.0, 5 mM sodium acetate, 0.22 M formaldehyde, and 1 mM EDTA. The gel was stained with ethidium bromide (5 µg/ml), photographed to verify even loading of undegraded RNA, and blotted to MagnaGraph Nylon membrane by capillary blotting. The blot was then hybridized to P-labeled human class 1 ALDH cDNA as described above for DNA analysis but with a single high stringency 60 °C wash for 1 h. The blot was exposed to autoradiography film for 17 days.

Western Blot Analysis

Cytosolic protein (100 µg/lane) was prepared as for enzyme activity assay, electrophoresed on a 14% SDS-PAGE gel, transferred by semidry electroblotting at 200 mA for 1 h onto a nitrocellulose membrane, and probed as described previously (24) with a 1:2000 dilution of anti-rat class 1 ALDH antisera, generously provided by Dr. Ronald Lindahl (University of South Dakota). This antisera was cross-reactive with the human ALDH-1 homolog. A 1:2000 dilution of a biotinylated goat anti-rabbit antibody (Bio-Rad) was used as secondary antibody, and blots were developed with nitro blue tetrazolium + 5-bromo-4-chloro-3-indoyl phosphate chromophore (Bio-Rad), as described previously(24) . For determination of the percent of cytosolic protein expressed as ALDH, a separate gel stained with Coomassie Blue G-250 was scanned with a digital densitometer (PDI, Inc.). The percent of total protein expressed in the 56-kDa hALDH-1 protein band was calculated by subtracting the percent total protein in the SD1/Hyg-1 control lane from that in the hALDH1-28 lane in the same selected (55 kDa) regions to eliminate the background absorbance due to similar molecular weight proteins.

Cytotoxicity Assay

The protocol for the clonogenic survival assay was modified from that of Sreerama and Sladek(14) . Cells were subcultured without hygromycin 24 h prior to drug treatment. The ALDH activities determined from cells growing in hygromycin versus cells growing in hygromycin-free medium for 24 h were not significantly different (data not shown). On the day of the experiment, cells were trypsinized, resuspended in fresh medium, counted, and diluted into 5 ml of drug exposure medium (neutral 0.9% NaCl + 10% fetal bovine serum) in sterile tubes at a concentration of 50,000 cells/ml. The drug was then added to separate tubes at the concentrations indicated. Cells were incubated for 30 min in a 37 °C water bath with inversion of the tube every 5 min, then chilled on ice for 5 min and pelleted by centrifugation at 500 times g. Cells were resuspended in 37 °C medium and diluted to 10,000 and 1000 cells/ml. One-ml aliquots of cells were added to 60-mm^2 plates containing 3 ml of medium (10,000 cells/plate for the two highest concentrations, and 1000 cells/plate for all concentrations). Cells were allowed to grow for 6-7 days, stained, and counted as described previously(24) . The cloning efficiency under these conditions was approximately 60-70%. The relative survival was expressed as the percentage of the colonies (geq50 cells) formed in the presence of the drug compared to the colonies formed in control wells containing no drug. For ALDH inhibition studies, 25 µM DEAB was preincubated with the cell suspension for 10 min followed by drug incubation also in the presence of 25 µM DEAB for 30 min. This DEAB concentration was completely nontoxic (data not shown) but well above the K(i) (0.04 µM) (6) of this inhibitor for class 1 ALDH.

Alkaline Elution

Transfected cells were trypsinized, suspended (6 times 10^5 cells/ml), and treated as described above for cytotoxicity. Following a 30-min exposure to MAF, cells were washed and replated in 60-mm dishes for 5 h to allow accumulation of DNA damage. Cells were then placed on ice, irradiated with 400 rad of radiation from a cesium-137 source, and loaded onto the membrane manifold of each channel in saline A solution (per liter, NaCl 8 g, KCl 0.4 g, D-glucose 1.0 g, NaHCO(3) 0.35 g, Na(2)EDTA 1.86 g, pH 7.3). Following lysis in Sarkosyl solution (0.04 M Na(4)EDTA, 2 M NaCl, 0.2% Sarkosyl, pH 10) and proteinase K digestion (0.5 mg/ml, 1 h), DNA was eluted in 2% tetrapropylammonium hydroxide solution (pH 12.3) for 15 h through a 75-mm diameter, 2-µm pore size polycarbonate filter (Nucleopore). The DNA in eluted fractions and filter fractions was quantitated by a fluorometric assay as described by Cesarone et al.(25) . Proteinase K-resistant DNA cross-linking was determined from a plot of DNA remaining on the filter versus elution volume at an elution volume of approximately 28 ml. Rad equivalents of DNA cross-links were calculated according to the formula: cross-linking coefficient (p(c)) = k(c)bulletpbullet(SQR ((1 - R(0))/(1 - R(1)) - 1) where p = 400 rad, k(c) = constant (=1), R(0) = the retention of DNA in control cells treated only with 400 rad, and R(1) = the retention of DNA in cells treated with 400 rad + MAF. For DEAB reversal studies cells were treated with 75 µM DEAB for 10 min prior to and during a 30-min exposure to 100 µM MAF.

RESULTS

Transfection of the Human Class 1 ALDH Expression Construct

The human class 1 ALDH expression vector was constructed by subcloning a human class 1 ALDH cDNA (16) with added translation initiation concensus sequence (19) into the DeltapCEP4Delta mammalian expression vector, which was modified to allow stable integration into cellular DNA. This expression vector or a control vector without any cDNA insert was transfected into the Chinese hamster lung fibroblast V79/SD1 cell line. The transfection efficiency with the DeltapCEP4Delta expression vector in V79/SD1 cells was very high. Initial screening of 48 clonal cell lines revealed that almost every transfected clonal cell line expressed class 1 ALDH, spanning a broad range of activity levels. Three cell lines were chosen for further analysis representing the low and the high limits of ALDH expression obtained, and one intermediate level. Using propionaldehyde as substrate, a range of class 1 ALDH activity from 1.5 to 13.6 milliunits/mg was detected in the ALDH-transfected cell lines, compared to very low activity (<1 milliunit/mg) in the parental and the empty vector transfected lines (Table 1). Thus the cytomegalovirus promoter upstream of the hALDH-1 cDNA in the modified DeltapCEP4Delta mammalian expression vector appears to be highly active in V79 cells, consistent with a previous report of efficient transcription driven by this promoter in rodent cells(26) . The doubling time for all cell lines was similar (12 h), indicating that the growth rate was not a factor in any of their differences in alkylating agent sensitivity.

Characterization of Transfected Cells

The ALDH-transfected cell lines growing in the presence of 0.7 mg/ml hygromycin and the parent V79/SD1 line were analyzed to determine the amount of class 1 ALDH DNA, RNA, and protein present. The DNA slot blot analysis under high stringency conditions demonstrated the presence of the transfected DNA in each of the transfected cell lines (Fig. 1, lanes 3, 4, and 5) and only weak hybridization in SD1/parental cells (lane 1) or in the control transfected V79/SD1 cell line (SD1/Hyg-1, lane 2). The ALDH-1 DNA content appears to increase linearly with increasing ALDH-1 activity for these cell lines as determined by densitometric quantitation with a scanning densitometer (PDI, Inc.) (data not shown). This does not necessarily have to be the case, since the site of incorporation may have a greater effect on expression than the number of copies(18) .

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 times 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).


Cytotoxicity Studies

These cell lines were then analyzed for sensitivity to a range of oxazaphosphorine and non-oxazaphosphorine antineoplastic alkylating agents by colony forming assay. Mafosfamide (MAF) is an analog of CPA that spontaneously hydrolyzes to yield 4-hyroxycyclophosphamide and the thiol-containing reductant MESNA(24, 27) . The average cytotoxicity curves in all five cell lines for MAF are shown in Fig. 4A. The survival curves were similar in V79/SD1 parental and SD1/Hyg-1 cells, with IC values of 29 and 31 µM, respectively. The IC values were greatly increased as ALDH activity increased in the three transfected cell lines, from 112 µM for hALDH1-13, to 292 µM for hALDH1-5, to 640 µM for hALDH1-28. The fold-resistance relative to the SD1/Hyg-1 control line increased commensurately and linearly with activity (Fig. 4B), from 3.6-fold in hALDH1-13, to 9.4-fold in hALDH1-5, to 20.6-fold in hALDH1-28 (Table 1). These studies demonstrate that the resistance conferred to MAF by these cells is tightly linked to the ALDH activity in several clonal transfectant lines (Fig. 4B; r = 0.99 by linear regression analysis), and is not due to random clonal variability in hygromycin-selected cell lines. Thus, expression of human ALDH-1 alone is sufficient to confer a high level of resistance to MAF.

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 (bullet) and SD1/Hyg-1 (circle) 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^2 = 0.982). The data points are the activity and IC values from Table 1for control (circle) or hALDH-1 transfected (bullet) 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.

Inhibitor Studies

The SD1/Hyg-1 and hALDH1-28 cell lines were further examined to determine whether the resistance could be reversed by inhibition of ALDH activity. Cells were pretreated with the potent class 1 ALDH inhibitor DEAB, which completely reversed resistance, making ALDH-1-transfected cells as sensitive to MAF as control SD1/Hyg-1 cells (Table 3). However, DEAB did not enhance the cytotoxicity of MAF toward SD1/Hyg-1 cells which have very low ALDH activity. Furthermore, DEAB did not affect sensitivity to PM in either SD1/Hyg-1 or hALDH1-28 cells (Table 3). Control experiments demonstrate that DEAB is not cytotoxic in V79 cells at concentrations up to 500 µM, which is 20-fold greater than that used for these experiments (data not shown). Thus, these results provide very strong evidence that the catalytic activity of the class 1 ALDH protein is essential for the observed protection from MAF cytotoxicity.

Alkaline Elution Studies

The relative levels of proteinase K-resistant DNA interstrand cross-links were analyzed following MAF treatment in SD1/Hyg-1 cells and hALDH1-28 cells (Fig. 5). cross-link formation in control cells was linear at low mafosfamide concentrations but deviated from linearity at higher concentrations possibly due to acrolein-induced DNA strand breaks, which would reduce the apparent cross-linking index. Protection from DNA interstrand cross-link formation (24-fold at 30-rad equivalents) was significant and proportional to the fold resistance in hALDH1-28 cells, indicating that hALDH-1 expression likely reduces phosphormide mustard formation. Reversal of class 1 ALDH-mediated protection against cross-linking was seen when cells were pretreated with the ALDH inhibitor DEAB.

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 (bullet) cells was 24-fold at 30-rad equivalents.


DISCUSSION

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 ^3H-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 beta-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.^2

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.

FOOTNOTES

*
This work was supported by a grant from the Leukemia Research Foundation. Tissue culture medium was obtained from the Tissue Culture Core Laboratory of the Comprehensive Cancer Center of Wake Forest University, which is supported in part by National Institutes of Health Grants CA-12197 and RR-04869, as well as by a grant from the North Carolina Biotechnology Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Experimental Hematology, St. Jude Children's Hospital, 332 N. Lauderdale, Memphis, TN 38105.

To whom correspondence should be addressed: Biochemistry Dept., Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 910-716-7658; Fax: 910-716-7671.

(^1)
The abbreviations used are: ALDH, aldehyde dehydrogenase; hALDH-3, human class 3 aldehyde dehydrogenase; hALDH-1, human class 1 aldehyde dehydrogenase; CPA, cyclophosphamide; 4-OH-CPA, 4-hydroxy-cyclophosphamide; CBP, carboxyphosphamide; 4-hp-CPA, 4-hydroperoxy-cyclophosphamide; 4-hp-IF, 4-hydroperoxyifosfamide; MAF, mafosfamide; PM, phosphoramide mustard; ALDO, aldophosphamide; OAP, oxazaphosphorine; DEAB, diethylaminobenzaldehyde; MOPS, 3-(N-morpholino)propanesulfonic acid; UTR, untranslated region; MESNA, 2-mercaptoethanesulfonic acid; bp, base pair(s).

(^2)
K. Bunting and A. Townsend, manuscript in preparation.

(^3)
W. Fields and A. Townsend, manuscript in preparation.

ACKNOWLEDGEMENTS

The authors thank Dr. Johannes Doehmer for providing the V79/SD1 parent cell line, and Dr. Charles Morrow for helpful discussions and critical reading of manuscripts during the course of this work.

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