Activation of Adriamycin by the Ph-Dependent Formaldehyde- Releasing Prodrug Hexamethylenetetramine1

Vol. 2, 189–198, February 2003 Molecular Cancer Therapeutics 189

Activation of Adriamycin by the pH-dependent Formaldehyde- releasing Prodrug Hexamethylenetetramine1

Lonnie P. Swift, Suzanne M. Cutts, Ada Rephaeli, factors, including a dose-limiting cardiotoxicity and MDR3 (1, Abraham Nudelman, and Don R. Phillips2 2). Adriamycin undergoes metabolic reduction and in the Department of Biochemistry, La Trobe University, Bundoora, Victoria presence of oxygen leads to the production of reactive ox- 3086, Australia [L. P. S., S. M. C., D. R. P.]; Felsenstein Medical ygen species (3, 4), and these species are particularly dam- Research Center, Sackler School of Medicine, Tel Aviv University, Beilinson Campus, Petach Tikva 49100, Israel [A. R.]; and Chemistry aging to cardiac tissue that do not possess enzymes to Department, Bar Ilan University, Ramat Gan 52900, Israel [A. N.] detoxify the resulting radicals (1). MDR occurs as a result of up-regulation of the membrane-associated P-glycopro- tein efflux pump (5). MDR therefore results in a diminished Abstract response to anthracycline anticancer agents such as Previous studies have shown that Adriamycin can Adriamycin. react with formaldehyde to yield an activated form The mechanism of action of Adriamycin and other anthra- of Adriamycin that can further react with DNA cyclines appears to involve impairment of topoisomerase II to yield Adriamycin-DNA adducts. Because activity (6–10) as well as the formation of DNA adducts hexamethylenetetramine (HMTA) is known to hydrolyze (11–14). Initial investigations of Adriamycin-DNA adducts us- under cellular conditions and release six molecules of ing an in vitro transcription footprinting assay suggested that formaldehyde in a pH-dependent manner, we examined the formation of adducts was enhanced in the presence of this clinical agent for its potential as a formaldehyde- iron (11). However, it was later shown that the Fe(III)/DTT/Tris releasing prodrug for the activation of Adriamycin. In buffer resulted in the production of formaldehyde that me- IMR-32 neuroblastoma cells in culture, increasing diated the formation of the drug-DNA adducts (3). levels of HMTA resulted in enhanced levels of Following the realization that formaldehyde mediated the Adriamycin-DNA adducts. These adducts were formed formation of Adriamycin-DNA adducts, studies were under- in a pH-dependent manner, with 4-fold more detected taken to fully understand this drug-DNA interaction in vitro.It at pH 6.5 compared with pH 7.4, consistent with the has been shown that formaldehyde supplies a methylene known acid lability of HMTA. The resulting drug-DNA group that links the 3Ј amino of Adriamycin to the 2-amino of lesion was shown to be cytotoxic, with combined deoxyguanosine residues of DNA via Schiff base chemistry Adriamycin and prodrug treatment resulting in a 3-fold (3, 13–16) and that the formaldehyde-conjugated complex is lower IC50 value compared with that of Adriamycin the active form of the drug (17). Although the adducts are alone. Given the acidic nature of solid tumors and the attached covalently to only one strand of DNA and are there- preferential release of formaldehyde from HMTA in fore mono-adducts, they stabilize the local region of DNA acidic environments, HMTA therefore has some sufficiently that they are resistant to thermal denaturation potential for localized activation of Adriamycin in solid and can therefore be detected in denaturation-based cross- tumors. linking assays (12–14, 17). It has been demonstrated that the presence of the amino group on the sugar moiety is critically Introduction important for the formation and stabilization of the drug-DNA Chemotherapeutic drugs are important in most types of cur- interaction (18–20). A preactivated form of Adriamycin rent cancer treatment protocols. One of the most widely (doxoform) has been shown to be taken up by cells at an used agents, Adriamycin (doxorubicin), exhibits a wide ac- accelerated rate, is retained longer in the nucleus, and is tivity against leukemias and breast, lung, thyroid, and ovarian substantially more cytotoxic than Adriamycin (21). carcinomas, as well as Hodgkin’s and non-Hodgkin’s lym- An alternative approach to enhance the activity of Adria- phomas (1, 2). Despite the remarkable efficiency with which mycin is to increase the intracellular level of formaldehyde, Adriamycin kills cells, treatment is complicated by many leading to enhanced formation of Adriamycin-DNA lesions. This approach has been investigated recently using a combination of Adriamycin and the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate (AN-9; Ref. 22). The prodrug is cleaved intracellularly by esterases, liberating formalde- Received 3/6/02; revised 8/12/02; accepted 11/27/02. hyde, which reacts with Adriamycin and results in a dramatic The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked increase of the number of Adriamycin-DNA adducts, to- advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. 1 Supported by the Australian Research Council (D. R. P. and S. M. C.), La Trobe University Postgraduate Scholarship (to L. P. S.), The Marcus Center for Pharmaceutical and Medicinal Chemistry, and the Bronia and Samuel 3 The abbreviations used are: MDR, multidrug resistance; HMTA, hexa- Hacker Fund for Scientific Instrumentation at Bar Ilan University (A. N.). methylenetetramine; TE, 10 mM Tris (pH 8)-1 mM EDTA; mtDNA, mito- 2 To whom requests for reprints should be addressed. E-mail: chondrial DNA; DHFR, dihydrofolate reductase; dsDNA, double-stranded [email protected]. DNA; ssDNA, single-stranded DNA.

gether with a synergistic cytotoxic response. However, AN-9 Worthington Chemical Corp. Tris-saturated ultrapure phenol also releases butyric acid, which inhibits histone deacetylase was purchased from Life Technologies, Inc., and formalde- and, as such, potentially modulates the chromatin accessi- hyde was purchased from BDH. ProbeQuant G-50 columns, bility of Adriamycin, thus contributing to the observed syn- [␣- 32P]dCTP, [␣- 32P]dATP, [␣- 32P]UTP (3000 Ci/mmol) and ergistic response. [14-14C]Adriamycin hydrochloride were obtained from Am- HMTA is a tertiary amine that hydrolyzes under acidic ersham Pharmacia Biotech. The plasmid containing the conditions to yield ammonia and formaldehyde (23). We have DHFR probe, pBH31R1.8 (36), was a gift from Dr. V. A. Bohr therefore examined it as a pH-dependent formaldehyde- (National Institute on Aging, NIH, Baltimore, MD), whereas releasing prodrug, thus enabling us to study the effects of the plasmid with a mitochondrial insert (pCRII-H1) was a gift formaldehyde release independently of butyric acid release. from Dr. C. A. Filburn (National Institute on Aging, NIH). HMTA has been examined as an antiseptic for the treatment Qiagen Plasmid Maxi Kits and QIAamp blood kit were from of urinary tract infections (24), as well as being studied in Qiagen. The Klenow fragment of DNA polymerase, glycogen, patients with maxillofacial phelegmons (25) and as a prophy- and the random primed labeling kit were from Roche Molec- lactic agent against recurrent acute cystitis (26). It has also ular Biosciences. Lambda exonuclease was purchased from been tested in water systems at a concentration of 1 mg/liter Life Technologies, Inc. All other enzymes were purchased (27) for its antiseptic properties (25). It is particularly well from New England Biolabs. tolerated by humans, even at high doses of up to 5 g/kg/day (28). The degradation of HMTA produces six molecules of Methods formaldehyde for every molecule of drug hydrolyzed, sug- In Vitro Detection of Drug-DNA Adducts. The plasmid gesting that it may be a more efficient prodrug than AN-9, pCCI (37) was linearized with HindIII and end-labeled with which releases only one molecule of formaldehyde when [␣-32P]dCTP or [␣-32P]dATP in the presence of the Klenow degraded by esterases (29). fragment of Escherichia coli DNA polymerase I. Unincorpo- Warburg (30) suggested that tumors were acidic because rated label was removed using G-50 ProbeQuant columns. of the production of lactic acid, due to the metabolism of The labeled DNA was resuspended in calf thymus DNA to a tumor cells in an anaerobic environment. However, more final concentration of 400 ␮⌴ bp in TE buffer. recently it has been shown that the intracellular pH in tumors End-labeled DNA (25 ␮M bp) was reacted with drugs at tends more toward neutral or alkaline values (31, 32), 37°C for defined times in PBS (adjusted to the desired pH). whereas the extracellular pH was shown to be acidic (33–35). Unreacted drugs were extracted with phenol and chloroform, Due to the acidic extracellular pH of solid tumors, it would be and the DNA was precipitated in ethanol (using glycogen as expected that HMTA would be preferentially hydrolyzed to an inert carrier). The pellet was washed with 70% ethanol, release formaldehyde in the tissues adjacent to such tumors. dried, and resuspended in TE. Samples were denatured in a It has been shown that the dose of Adriamycin required to final concentration of 66% loading dye (60% formamide, 6.6 induce a cytotoxic response can be diminished when coad- mM EDTA, 0.07% xylene cyanol, and 0.07% bromphenol ministered with a formaldehyde-releasing prodrug (22). It blue) at 65°C for 5 min. Samples were quenched on ice and was therefore expected that a similar response would also be then loaded onto a 0.8% agarose gel (40 cm), and the DNA achieved with the coadministration of HMTA with Adriamy- was separated electrophoretically in TAE buffer [40 mM Tris- cin. Moreover, the fact that formaldehyde is released from acetate, 1 mM EDTA (pH 8.0)] at 45 V for 16 h. The gels were HMTA in acidic environments (such as those associated with dried, and all image analysis was performed on a Molecular solid tumors) raises the possibility of a tumor-localized re- Dynamics model 400B PhosphorImager using ImageQuaNT lease of formaldehyde and hence a tumor-localized response software (Molecular Dynamics). to Adriamycin. DNA containing drug-DNA adducts stabilized the DNA HMTA-mediated Adriamycin-DNA lesions were there- sufficiently to resist denaturation at 65°C and therefore mi- fore investigated at various pH values in vitro to establish grated as dsDNA, whereas DNA that lacked adducts was the potential for tumor-localized responses to formalde- denatured under the same conditions and migrated as hyde-releasing prodrugs and Adriamycin. The cytotoxicity ssDNA. Relative drug-DNA adduct levels were determined as of Adriamycin and prodrug as single agents and in com- the percentage of DNA that migrated as dsDNA and calcu- bination was investigated in tumor cells in culture, to- lated using ImageQuaNT software. gether with the ability of the drug combination to form Exonuclease Studies. A 188-bp fragment was excised drug-DNA adducts in cellular DNA. from pCC1 using EcoRI and PvuII. This fragment was separated electrophoretically and collected using a Biotrap elec- Materials and Methods troeluter (Schleicher and Schuell). The DNA fragment was Materials subjected to 3Ј end-labeling with the Klenow fragment and Adriamycin, daunomycin, and epirubicin were gifts from [␣-32P]dATP. The labeled DNA was resuspended in calf thy- Farmitalia Carlo Erba (Milan, Italy). Idarubicin hydrochloride mus DNA to a final concentration of 400 ␮M bp and used for was purchased from Pharmacia and Upjohn. HMTA (Aldrich, exonuclease studies. Milwaukee, WI) was freshly dissolved in Milli-Q water as a 50 End-labeled DNA was incubated in the presence of Adria- mM stock solution. The anthracyclines were dissolved in mycin and either HMTA or formaldehyde to induce the for- Milli-Q water to a stock concentration of approximately 1 mM mation of drug-DNA adducts. The samples were precipitated and stored at Ϫ20°C. Calf thymus DNA was purchased from with ethanol and resuspended in ␭ exonuclease buffer [67

␮ mM glycine-KOH (pH 9.4), 2.5 mM MgCl2, and 50 g/ml BSA] before being digested for 2 h with 5 units of ␭ exonuclease at 37°C. The digestion was terminated by the addition of an equal volume of 90% formamide (containing 0.1% xylene cyanol and 0.1% bromphenol blue) in TE buffer. Sequence identification was performed with a Maxam-Gilbert G sequencing lane (38). Cell Culture. IMR-32 neuroblastoma cells were main-

tained at 37°C, 5% CO2 in DMEM (pH 6.8–7.4) obtained from Trace Scientific and supplemented with 10% fetal calf se- rum, 0.1 mg/ml streptomycin, and 100 U/ml penicillin. Growth Inhibition. IMR-32 cells (1 ϫ 104) were seeded into individual 96-well plates in 100 ␮l of DMEM (10% FCS) and allowed to adhere overnight. Drug treatment consisted of either HMTA, Adriamycin, or a combination of both. After a 3-day incubation period, the cells were treated with 300 ␮g/ml 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme- thoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (39, 40) and incubated for 4 h, and the absorbance was measured at 490 nm using a Molecular Devices Spectra Max 250 micro plate Fig. 1. Dependence of pH on adduct formation. A, linearized pCC1 DNA reader. The IC50 was determined as the concentration at (25 ␮M bp) was incubated with 1 ␮M Adriamycin and 1 mM HMTA for 6 h which only 50% of cells survived. at 37°C at varying pH values. The DNA was then subjected to phenol/ Gene-specific Detection of Adducts. Specific probes chloroform extraction and ethanol precipitation. The pellet was resus- were used to detect adducts in both mtDNA and nuclear pended in TE and denatured at 65°C in 60% formamide for 5 min. C1 is a non-drug-treated ssDNA control. C2 is a double-strand control (nonde- DNA as outlined previously (41). Briefly, a 1.8-kb probe natured DNA). B, the percentage of dsDNA (i.e., DNA containing one or specific for the DHFR gene was isolated from pBH31R1.8 more adducts) is shown as a function of pH. and used to assess nuclear DNA adducts. The 1.8-kb fragment (coding for exons 1 and 2 of the DHFR gene) was labeled using a random primed labeling kit, incorporating Results [␣-32P]dATP. To probe mtDNA, a strand-specific probe was made by generating run-off transcripts from the T7 Because it has been shown that formaldehyde activates promoter and incorporating [␣-32P]UTP. Cells (106) were Adriamycin, leading to the formation of Adriamycin-DNA ad- seeded in 10-cm Petri dishes approximately 14 h before ducts, and that this process is also facilitated intracellularly the addition of drug. Cells were treated with HMTA and by formaldehyde-releasing prodrugs, it was likely that HMTA, Adriamycin at varying concentrations and for the desired which is known to dissociate to formaldehyde and ammonia, times. Cells were then harvested, and media were washed may also facilitate the formation of Adriamycin-DNA ad- from the pellet with chilled PBS. DNA was extracted using ducts. the QIAamp blood kit, digested with BamH1 (to linearize the mitochondrial genome) or HindIII (to release the 1.8-kb DHFR fragment), and separated electrophoretically on a Dependence of pH on Formation of Drug-DNA Lesions Mediated by HMTA 0.5% agarose gel. DNA was transferred to nylon membranes and probed with the DHFR (nuclear DNA) and To establish the optimal pH for the release of formaldehyde mtDNA probes. Adducts were quantitated, and the fre- from HMTA and hence the optimal pH for the formation of quency was calculated as described previously using the drug-DNA adducts, linearized end-labeled pCC1 DNA was ␮ Poisson distribution (42). reacted with Adriamycin (1 M) and HMTA (1 mM)for6hata Detection of 14C-labeled Adducts. IMR-32 cells were range of pH values from 5.3 to 7.7 (Fig. 1). After a cleanup seeded at 7.5 ϫ 105 cells/3.5-cm Petri dish. The cells were and denaturation procedure, DNA adducts (migrating as incubated with varying concentrations of HMTA and dsDNA) were separated from DNA devoid of adducts (mi- [14C]Adriamycin for the desired times and then harvested. grating as ssDNA) by gel electrophoresis. The genomic DNA was isolated as described for the gene- It was seen that the more acidic the reaction conditions, specific detection of adducts and subjected to two phenol the greater the number of adducts detected. This was most extractions and one chloroform extraction before being pre- pronounced at pH values Ͻ6.3 (Fig. 1). At pH 7.7, the per- cipitated in ammonium acetate. Pellets were resuspended in centage of DNA containing adducts was approximately 30%, 100 ␮M TE buffer, and DNA concentration was calculated at compared with essentially 100% at pH 5.3. Because of the 260 nm. A 50-␮l aliquot was added to 1 ml of Optiphase pronounced dependence of adduct formation on pH, the Hisafe scintillation mixture, and the incorporation of optimal HMTA concentration and rates of formation were [14C]Adriamycin into DNA was quantitated in a Wallac 1410 examined at physiologically relevant pH extremes of pH 6.4 Liquid Scintillation Counter and calculated as Adriamycin and 7.4 to reflect typical scenarios for tumor and normal cell adducts/10 kb. environments, respectively.

Fig. 2. Adducts formed at pH 6.4 and 7.4. A, DNA was incubated with 500 nM Adriamycin and varying concentrations of HMTA (0, 0.5, 1, 1.5, and 2mM)for6hat37°C at pH 6.4 and 7.4, and the extent of drug-DNA lesions Fig. 3. Rate of release of formaldehyde. DNA was incubated with 0.5 ␮M was determined by electrophoretic analysis. C1 is a dsDNA control, and Adriamycin and 500 ␮M HMTA at 37°C, pH 6.4 (A) and pH 7.4 (B). Aliquots C2 is a ssDNA control. B, the percentage of dsDNA (i.e., DNA containing were taken at 0-, 1-, 2-, 4-, 6-, and 8-h intervals at each pH, and the one or more adducts) is shown at each HMTA concentration. F, pH 7.4; amount of adducts was assessed. C, the percentage of dsDNA formed f, pH 6.4. over time of incubation is shown at pH 7.4 (F) and pH 6.4 (f).

HMTA Dependence The extent of formation of adducts at the two pH extremes was investigated to establish the optimal HMTA concentration required for additional studies of this reaction. Concen- trations that resulted in moderate to high levels of adducts (50–80% dsDNA) were optimal. At very low levels, quantitation of adducts is less reliable, whereas at concentrations that resulted in Ͼ90% lesions, multiple adducts can contribute to one “cross-linked” DNA duplex, also making quantitation less reliable. DNA was incubated with Adriamycin (0.5 ␮M) and HMTA (0–2mM)for6hat37°C. HMTA-mediated Adriamycin-DNA adduct formation was clearly concentration dependent (Fig. 2), with increasing HMTA resulting in a near linear increase of adduct formation. At pH 6.4, the formation of adducts was more pronounced, with 3–4-fold more lesions formed compared with pH 7.4. The maximal enhancement (4-fold) of adducts (pH 6.4 compared with pH 7.4) was at a HMTA concentration of 1 mM, and this level was therefore used for all subsequent analysis of adduct formation at these pH values, unless stated otherwise. Fig. 4. HMTA is required for the formation of adducts in vitro. A, DNA was incubated for6hinthepresence and absence of 2 mM HMTA and Release Rate of Formaldehyde varied concentrations of Adriamycin (0, 0.2, 0.4, 0.6, 0.8, and 1 ␮M)atpH Because maximal drug-DNA adduct formation is dependent 6.4. C1 represents a double-stranded control, and C2 represents a single- stranded control. B, the percentage of dsDNA is shown at each Adriamy- on the availability of formaldehyde, it was useful to establish cin concentration in the presence (f) and absence (F) of HMTA. the time frame for complete hydrolysis of HMTA at the two pH extremes of interest. DNA was incubated with 0.5 ␮M HMTA and 0.5 ␮M Adriamycin over8hatpH6.4and7.4. It Absolute Requirement of HMTA for the Formation can be seen that up to 5-fold more adducts were formed at of Drug-DNA Adducts in Vitro pH 6.4 than at pH 7.4 (Fig. 3), but only after an extensive It was important to assess the requirement for HMTA in reaction time of 8 h. This indicates that at pH 6.4, HMTA is drug-DNA adduct formation and to confirm that Adriamycin degraded much more than at pH 7.4, albeit slowly, leading to formed few adducts with DNA in the absence of HMTA at pH greater release of formaldehyde. For convenience, an incu- 6.4. Varying concentrations of Adriamycin (0–1 ␮M) were bation time of 6 h was selected for all additional studies. incubated with DNA in the presence and absence of 2 mM

similar basic structure, the small changes in chemical com- position resulted in significant clinical differences. Adriamy- cin and daunomycin induced adducts to a greater degree at pH 7.4 compared with idarubicin, whereas at pH 6.4, Adria- mycin, daunomycin, and idarubicin all formed approximately the same amount of adducts (Ϯ5%) at all drug concentrations and at a much greater level that that detected at pH 7.4 (Fig. 6). Idarubicin (1 ␮M) formed approximately 5-fold more adducts at pH 6.4 compared with pH 7.4, whereas daunomycin formed approximately 3-fold more adducts at pH 6.4. In contrast, epirubicin exhibited a dramatically reduced ability to form lesions at both pH values.

Sequence Specificity of HMTA-mediated Drug-DNA Adducts A 188-bp fragment of DNA was 3Ј end-labeled and reacted with Adriamycin and either HMTA or formaldehyde, resulting in the formation of drug-DNA adducts. This DNA was then subjected to 5Ј exonuclease digestion using ␭ exonuclease, and the residual fragments were resolved electrophoretically (Fig. 7A). The location of blockages was compared with a

Fig. 5. Stability of Adriamycin-DNA adducts. A, DNA (25 ␮M bp) was Maxam-Gilbert G sequencing lane. The blockages were cal- incubated with Adriamycin and either formaldehyde or HMTA (as a source culated as the mole fraction of fragments, with the DNA of formaldehyde) until approximately 70% dsDNA resulted at 37°C. Ali- quots were then taken at 0, 1, 2, 4, 6, 8, 12, 24, and 36 h, and the residual fragment length indicating the site of blockage (Fig. 7B). All lesions were determined by electrophoretic analysis. B, the remaining blockages were observed 1–2 bases before a GpC se- dsDNA is shown with increasing time of incubation at 37°C and as a quence. The frequency of blockages was as high as 0.052 first-order kinetic decay (inset). mole fraction (i.e., 5.2% drug occupancy) for the lesion associated with the blockage at the cytosine residue at position 111. For clarity, only lesions with a fractional occupancy of HMTA for 6 h (Fig. 4). There was a concentration-dependent Ն1% have been shown. It was found that blockages for the increase in DNA adducts formed in the presence of HMTA at HMTA-mediated adduct were the same as those observed pH 6.4, but no lesions were detected in the absence of HMTA for the formaldehyde-mediated adducts (Fig. 7A). In both under these conditions. A concentration of approximately 0.6 cases, the lesions were specific to GpC regions. Because ␮M Adriamycin was required to form 50% dsDNA under it has been shown previously that formaldehyde-mediated these conditions, and lesions were detected at drug levels Adriamycin-DNA adducts are specific to GpC sites (43), this above 0.2 ␮M. result confirms that HMTA and formaldehyde induce the same drug-DNA lesion and are in fact the same lesion. Stability of HMTA-mediated Drug-DNA Adducts To establish whether the HMTA-mediated drug-DNA Cellular Response to Adriamycin and HMTA adducts were similar to the formaldehyde-mediated Once the relationship between formaldehyde release from Adriamycin-DNA adducts, the half-life of these lesions HMTA and the formation of Adriamycin-DNA adducts had was determined. The loss of adducts was measured over been established in vitro, it was of interest to investigate this 36hat37°C, and from the first-order kinetic decay (Fig. 5, phenomenon in cells. IMR-32 neuroblastoma cells were used Ϯ inset), the half-lives were found to be 12.3 1.3 h for the for this study because they represent a model of solid tumors Ϯ HMTA-mediated lesion, compared with 13.2 0.5 h for and are one of the tumors that exhibit good clinical re- the formaldehyde-mediated lesion (Fig. 5). The stabilities sponses to Adriamycin (1). The IC50 values of Adriamycin and are therefore essentially identical and correlate with the HMTA were established alone and in combination (Table 1) Ϯ previously determined half-life of 14.4 1.9 h for formal- and revealed a progressive increase of activity of Adriamycin dehyde-mediated DNA adducts (19), and this suggests with increasing levels of HMTA. that the HMTA-mediated lesion is the same as the form- Dependence on HMTA Concentration. Cells were incu- aldehyde-mediated lesion. bated with Adriamycin and HMTA at varying concentrations, and drug-DNA adducts were detected using a gene-specific Formation of Other Anthracycline-DNA Adducts assay (41). It was seen that drug-DNA lesions increased with To investigate the potential of other widely used anthracy- increasing levels of HMTA. This relationship was evident in clines to form DNA adducts, varying concentrations of these both nuclear and mtDNA, with approximately the same anthracyclines (0–1 ␮M Adriamycin, daunomycin, and idaru- amount of lesions being formed in both genomes (Fig. 8). The bicin and 0–50 ␮M epirubicin) were examined at a HMTA control reactions (absence of prodrug) confirm the absolute concentration of 1 mM. Although each of these drugs has a necessity for HMTA to be present to induce detectable levels

Fig. 6. Enhancement of anthracycline- DNA adducts by HMTA. DNA was incubated with HMTA (1 mM) and 0–1 ␮M Ad- riamycin (f), daunomycin ( ), idarubicin (F), or 0–50 ␮M epirubicin ()for6hat37°Cat a pH of either 6.4 (A) or 7.4 (B). The resulting adducts formed are shown in terms of percentage dsDNA detected with increasing anthracycline concentration.

Fig. 7. Sequence specificity of HMTA-mediated lesions. A, a3Ј-labeled 188-bp fragment was incubated with 5 ␮M Adriamycin and either HMTA (5 mM) or formaldehyde (5 mM)for6htoallow adducts to form. The fragment was then subjected to phenol/chloroform extraction and ethanol precipitation. The pellet was resuspended in a ␭-exonuclease digestion buffer and incubated with ␭-exonuclease for 2 h, and then the DNA fragment was separated on a 10% denaturing polyacrylamide gel. C1 is a non-exonuclease-digested control, C2 is a digested untreated control, C3 is an Adriamycin (5 ␮M) control, and C4 is a HMTA (5 mM) control. G represents Maxam- Gilbert G sequences; H is a HMTA- and Adriamycin-treated fragment; and C is a formaldehyde- and Adriamycin-treated fragment. B, the mole fraction of adducts (i.e., relative occupancy) is shown at each blockage site. The sequence is from 5Ј to 3Ј, and GpC locations are indicated as shaded boxed regions.

14 of drug-DNA adducts. At 15 ␮M Adriamycin and 1 mM HMTA, Total Cellular [ C]Adriamycin-DNA Adducts. The 1.9 and 1.8 lesions/10 kb were formed in mtDNA and nuclear use of [14C]Adriamycin enabled the detection of lesions DNA, respectively, after exposure to these agents for 8 h. at decreased drug concentrations compared with that Rate of Release of Formaldehyde from HMTA. IMR-32 required for the gene-specific method described above. cells were incubated with 10 ␮M Adriamycin and 1 mM HMTA The formation of Adriamycin-DNA adducts was concen- 14 for 0–8 h. The longer the incubation period, the greater the tration and time dependent (Fig. 10). At 2 ␮M [ C]Adria- extent of formaldehyde released and hence the more drug- mycin and 0–2.5 mM HMTA, an exponential increase in DNA adducts that were formed. Adduct levels were approx- lesions was observed, with 23 lesions formed/10 kb at 2.5 14 imately the same for both nuclear and mtDNA, with slightly mM HMTA. After an 8-h exposure to 2 ␮M [ C]Adriamycin more lesions observed in nuclear DNA (Fig. 9). At 10 ␮M and1mM HMTA, approximately 4 lesions/10 kb were Adriamycin and 1 mM HMTA, there were 0.48 and 0.40 le- detected. These levels of lesions are substantially greater sions formed/10 kb in the nuclear DNA and mtDNA, respec- than those detected using the gene-specific assay, due tively. To ensure that the concentrations of drug-prodrug to the additional procedures required to isolate and used in the gene-specific assays did not result in high levels prepare the genomic DNA in the gene-specific assay, of nonviable cells in the time frame investigated, the viability and the resulting effects these procedures have on the of the cells (at maximal time points of Figs. 8 and 9) was labile Adriamycin-DNA adducts has been reported previ- assessed and found to be in excess of 95%. ously (22).

Table 1 Cytotoxicity of Adriamycin/HMTA combinations IMR-32 (104) cells were seeded and allowed to adhere overnight. Cells were treated with varied concentrations of Adriamycin, HMTA, and a combination of both drug and prodrug at 37°C, 5% CO2 for 72 h. The IC50 was calculated as the concentration of drug, prodrug, or drug/prodrug combination that inhibited 50% cell growth.

Drug IC50 (M) Adriamycin 9.1 Ϯ 0.3 ϫ 10Ϫ9 HMTA 504.9 Ϯ 5.6 ϫ 10Ϫ6 Ϫ9 Adriamycin ϩ HMTA (50 ␮M) 8.5 Ϯ 0.3 ϫ 10 Ϫ9 Adriamycin ϩ HMTA (100 ␮M) 6.2 Ϯ 0.4 ϫ 10 Ϫ9 Adriamycin ϩ HMTA (200 ␮M) 2.9 Ϯ 0.2 ϫ 10

Fig. 9. Release of formaldehyde from HMTA in IMR-32 cells. Cells (1 ϫ 106) were seeded and allowed to attach overnight and then treated with 10 ␮M Adriamycin and 1 mM HMTA for 0–8 h. Once harvested, DNA was extracted as described in Fig. 8 and probed for the nuclear DHFR gene (A) and mtDNA (B). The adducts were calculated as lesions/10 kb and plotted (C) against time for nuclear DNA (f) and mtDNA (F).

Formaldehyde Release by HMTA Is Optimal at Low pH. The potential for HMTA to release formaldehyde suggests that it would serve as an efficient formaldehyde-releasing prodrug when coadministered with Adriamycin and that it would function similar to other formaldehyde-releasing pro- Fig. 8. Formation of Adriamycin-DNA adducts in cells. IMR-32 cells (1 ϫ 106) were seeded into 10-cm Petri dishes and allowed to adhere over- drugs such as AN-9 (22). The hydrolysis constant of HMTA is night. Cells were then treated with 15 ␮M Adriamycin and treated 4 h later favored by acidic conditions (44), and this indicates possible with 0, 0.5, 1, 1.5, 2, and 2.5 mM HMTA. Cells were harvested after 8 h, and the DNA was extracted, restriction-digested, and separated electro- selective release of formaldehyde in low pH necrotic areas, phoretically on a 0.5% agarose gel. The gel was transferred to a nylon hence providing the potential for selective enhancement of membrane and probed for the nuclear DHFR gene (A) and for the mito- Adriamycin activity in solid tumors. Increased release of chondrial genome (B). C1 is an untreated control, C2 is a positive control using a known formaldehyde-releasing drug, C3 isa15␮M Adriamycin formaldehyde was clearly observed within the physiological control, and C4 is a 2.5 mM HMTA control. The adducts were calculated pH range of 6.4–7.4, with up to 4-fold more drug-DNA ad- as lesions/10 kb and are shown at each HMTA concentration (C) for ducts forming at pH 6.4 (Fig. 1). This suggests that a pH as nuclear DNA (f) and mtDNA(F). low as 6.4 may result in selective hydrolysis of HMTA near tumor cells (compared with normal cells), indicating that Discussion HMTA may serve as a useful prodrug for the site-specific Current treatment of tumors with Adriamycin results in many delivery of formaldehyde. This acid-dependent release of dose-limiting side effects. The ability to increase the cyto- formaldehyde from HMTA has been reported in humans (45), toxicity of Adriamycin by using a formaldehyde-releasing where up to 9% of the total HMTA dose was detected as prodrug lends to the possibility of reducing the concentration formaldehyde in the urine (pH range, 5.5–6.8) with even of Adriamycin required for similar levels of cell kill. HMTA is greater release (up to 30%) in the acidic stomach environ- a potential solid tumor-specific activator of Adriamycin be- ment (46). cause it degrades preferentially in acidic environments to HMTA-mediated Drug-DNA Adducts Are Identical to produce six molecules of formaldehyde and four molecules Formaldehyde-mediated Adducts. The essentially identi- of ammonia (44). cal stability of the formaldehyde-mediated adduct with that of HMTA-mediated adducts indicates that these lesions are (CH ) N ϩ 4Hϩ ϩ 6HO 3 6CHO ϩ 4NHϩ (Reaction 1) 2 6 4 2 2 4 the same. The half-lives of the formaldehyde-mediated lesion This hydrolysis is also catalyzed by the presence of an active and HMTA lesions were essentially identical (Fig. 5). HMTA- formaldehyde acceptor (44) such as Adriamycin. mediated adducts were specific for GpC sequences, also

anthracycline-DNA adduct (21). The formation of the oxazo-

line does not take place in epirubicin because the NH2 and OH groups are found in equatorial positions, whereas in

Adriamycin, daumomycin, and idarubicin, the NH2 group is found in an equatorial position, and the OH is in an axial position, favorable for five-membered ring cyclization. HMTA Increases the Cytotoxicity of Adriamycin. The potential use of HMTA is not compromised by any known toxicity, and it has been used previously as an antibacterial

agent for the treatment of urinary infections (48). The IC50 of Ϯ ␮ HMTA in IMR-32 cells is 504.9 5.6 M, whereas the IC50 of Adriamycin alone is 9.1 Ϯ 0.3 nM. When fixed concentrations

of HMTA were used in combination with Adriamycin, the IC50 of Adriamycin was decreased, and this was more pronounced at higher HMTA concentrations (Table 1). This suggests that by combining Adriamycin treatment of cells with HMTA, the dose of Adriamycin required to achieve sufficient cytotoxicity could be reduced and may lead to reduced side effects. These data also suggest that HMTA is an efficient prodrug for the activation of Adriamycin. The use of cell culture experiments established that the formation of Adriamycin-DNA adducts was enhanced by Fig. 10. HMTA-dependent formation of [14C]Adriamycin-DNA adducts. HMTA in both nuclear DNA and mtDNA (Fig. 8). At 1 mM 6 14 IMR-32 cells (1 ϫ 10 ) were exposed to 2 ␮M [ C]Adriamycin and 0–2.5 HMTA and 15 ␮M Adriamycin, 0.28 and 0.24 lesions/10 kb mM HMTA for8h(A)or1mM HMTA for 0–8h(B). Genomic DNA was isolated, and incorporation of radiolabeled drug was determined by scin- were formed in mtDNA and nuclear DNA (measured in the tillation counting. The level of DNA-Adriamycin adducts was calculated as DHFR gene), respectively. The similar amounts of lesions lesions/10 kb. formed in both of these genomes suggest that the uptake of both drug and prodrug is not impeded by either the mitochondrial or nuclear membranes. identical to that known for the formaldehyde-mediated Ad- The release of formaldehyde from HMTA was also seen to riamycin lesion (Ref. 47; Fig. 6, A and B). Collectively, these be time dependent. With increasing incubation times, cells two independent results confirm that both lesions (formalde- treated with both Adriamycin and HMTA formed increasing hyde-mediated and HMTA-mediated lesions) are actually the levels of drug-DNA adducts (Fig. 9). The relative time of same lesion. Formaldehyde directly activates Adriamycin, addition of Adriamycin and HMTA did not significantly affect resulting in drug-DNA adducts, whereas release of formal- the amount of lesions formed over the time interval examined dehyde from the hydrolysis of HMTA activates Adriamycin, (IMR-32 cells were exposed to 2 ␮M Adriamycin for 8 h, and resulting in formation of the same formaldehyde-mediated 1mM HMTA was added up to 4 h before or after the addition Adriamycin-DNA adducts. of Adriamycin; data not shown). This is in contrast to that Other Anthracyclines. The preferential release of formal- observed with the Adriamycin/AN-9 combination, where ad- dehyde at pH 6.4 (compared with pH 7.4) resulted in an increase in adduct formation of Adriamycin, daunomycin, duct levels were maximal when Adriamycin was adminis- and idarubicin at the lower pH value. The methoxy group of tered 2 h before AN-9 (22). The present results suggest that Adriamycin and daunomycin therefore appears to enhance formaldehyde may be released much more slowly from drug activation at pH 7.4 compared with idarubicin. The HMTA than from AN-9, thereby possibly avoiding the initia- addition of the hydroxyl group on Adriamycin (absent on tion of formaldehyde detoxification mechanisms. daunomycin) does not significantly contribute to formation of To ensure that adduct levels detected in the gene-specific ␮ drug-DNA adducts at either pH or to the stability of the assay (requiring short-term exposure to 15 M Adriamycin) adducts (the half-lives of the daunomycin and Adriamycin- were representative of more clinical concentrations, the ab- DNA adducts were essentially identical; Ref. 19). Idarubicin solute level of lesions formed was also determined at lower 14 exhibited an approximate 9-fold increase in adduct formation drug levels (2 ␮M) using [ C]Adriamycin, and the trends at pH 6.4 compared with pH 7.4, but it is not clear why the observed mimicked that observed using higher drug levels in methoxy group should have any influence on Schiff base the Southern-based gene-specific assay (compare Figs. 8 and 9 with Fig. 10). However, 8-fold more adducts were chemistry or formation of the drug-DNA aminal (-N-CH2-NH-) linkage. Epirubicin does not appear to form a significant level detected by scintillation analysis of 14C-labeled adducts (2 of drug-DNA adducts, and this relates to the fact that in the ␮M Adriamycin) than by gene-specific Southern-based anal- presence of formaldehyde, an initial cyclization to a five- ysis (10 ␮M Adriamycin) after exposure to drug for 8 h. This membered oxazoline ring forms, similar to that present in difference reflects the loss of the unstable adducts during the doxoform (21), which subsequently undergoes a nucleophilic additional processing steps required for the electrophoretic ring opening, leading to the aminal bridge found in the procedure and has been discussed previously (41).

The above-mentioned data suggest that the total time of this treatment could also be applicable to those tumors with exposure with HMTA is important. It is not known whether apHofϽ6.8 (e.g., glioblastomas, mammary carcinomas, this is due to the formaldehyde that is released from HMTA and adenocarcinomas; Ref. 31). being more susceptible to time-dependent detoxification processes or whether this is due to other mechanisms. Be- References cause formaldehyde is toxic to cells, it is not surprising that 1. DeVita, V. T., Hellman, S., and Rosenberg, S. A. Cancer, Principles and it has a limited availability once released within the cell. For Practice of Oncology, 5th ed. Philadelphia: Lippincott-Raven, 1997. clinical purposes, further investigation would be required in 2. Pratt, W. B., Ruddon, R. W., Ensiminger, W. D., and Maybaum, J. The animal models to define the toxicity and optimal times and Anticancer Drugs, 2nd ed., pp. 1–165. 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Lonnie P. Swift, Suzanne M. Cutts, Ada Rephaeli, et al.

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