The aldo-keto reductase AKR1C3 contributes to 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol mediated oxidative DNA damage in myeloid cells: Implications for leukemogenesis

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Abstract

The aldo-keto reductase AKR1C3, has been shown to regulate myelopoiesis via its ability to metabolise prostaglandin D2 (PGD2). Other studies have demonstrated the oxidative activation of polycyclic aromatic hydrocarbon (PAH) procarcinogens by AKR1C3 in cell-free systems. This is the first study that addresses whether AKR1C3 mediates carcinogen activation within intact living cells following manipulation of AKR1C3 by molecular intervention. Quantitative RT-PCR identified AKR1C3 as the predominant AKR1C isoform expressed in acute myeloid leukemia (AML). Exposure of K562 and KG1a myeloid cell lines to the known AKR1C3 substrate 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol) resulted in both single strand DNA breaks and oxidative DNA damage as measured using conventional and FPG-modified comet assays respectively. PGD2-keto reductase activity was shown to be correlated with relative AKR1C3 expression and together with quantitative real time PCR was used to validate the RNAi-knockdown of AKR1C3 in K562 cells. Knockdown of AKR1C3 did not alter single strand DNA breaks following 7,12-DMBA-3,4-diol exposure but significantly decreased oxidative DNA damage. A similar interrelationship between AKR1C3 activity and 7,12-DMBA-3,4-diol mediated oxidative DNA damage but not single strand breaks was observed in KG1a cells. Finally, AKR1C3 knockdown also resulted in spontaneous erythroid differentiation of K562 cells. Since K562 cells are a model of AML blast crisis of chronic myeloid leukemia (CML) the data presented here identify AKR1C3 as a novel mediator of carcinogen-induced initiation of leukemia, as a novel regulator of erythroid differentiation and paradoxically as a potential new target in the treatment of CML.

Introduction

Links between cigarette smoke and leukemia have been reported [1], [2], [3], [4]; however the mechanisms underlying this association remain poorly understood. We have previously identified the aldo-keto reductase, AKR1C3, as a regulator of myeloid differentiation and demonstrated that it represents a potential therapeutic target in acute myeloid leukemia (AML) [5]. AKR1C3 belongs to a human sub-family of four aldo-keto reductases (AKR1C1-4). Each of these enzymes have steroid dehydrogenase activity; AKR1C1 is a 20α(3α)-HSD; AKR1C2 is a type III 3α-HSD; AKR1C3 is a type II 3α-HSD as well as a type V 17β-HSD; AKR1C4 is a type I 3α-HSD [6]. However, unlike AKR1C1, -2 and -4, AKR1C3 is also able to reduce prostaglandin D2 (PGD2) to 9α,11β-prostaglandin F (11-epi PGF) and also perform the reverse oxidation [7]. It is this activity that we have proposed to be important in regulating myelopoiesis [5]. However, all the AKR1C isoforms also share the capacity to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiol carcinogens as demonstrated in cell-free systems [8], [9], [10]. Since cigarette smoke contains many PAHs of the type that can be activated by AKR1C enzymes, we have considered whether these enzymes may provide a plausible link between smoking and leukemia by assessing PAH activation in live myeloid cells.

The PAH substrates of AKR1C type aldo-keto reductases include trans-dihydrodiol, 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol), a proximate genotoxic product of the smoke borne procarcinogen 7,12-dimethylbenz(a)anthracene (7,12-DMBA). In vitro studies have shown that (in addition to the known activation of various PAHs into reactive diol epoxides by cytochrome P450 [11]) AKR1C type aldo-keto reductases mediate NADP+ dependent oxidation of 7,12-DMBA-3,4-diol to form a ketol which undergoes spontaneous rearrangement to form a catechol [8], [9], [10]. It has been suggested that once the catechol is formed, a process of redox cycling can begin, cycling from catechol to the corresponding ortho-quinone (o-quinone), creating DNA damaging reactive oxygen species (ROS). The unstable catechol undergoes two consecutive one-electron transfers to create first an o-semiquinone radical anion and then the fully oxidized o-quinone. Aside from the ROS created from this pathway, o-quinones are highly reactive Michael acceptors capable of forming stable and depurinating DNA adducts (reviewed in [9]). A recent detailed study showed the activation of polycyclic aromatic trans-dihydrodiol in intact lung cells to the products and DNA damage known to be produced by AKR [12]. However because no manipulation of AKR1C enzyme expression was made it cannot be certain that activation was mediated by AKR1C activity. A previous study overexpressed rat AKR1C9 in MCF-7 breast carcinoma cells and observed conversion of the proximate carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) to benzo[a]pyrene-7,8-dione (BPQ) with associated increased toxicity [13]. However, this is the first in which levels of a human AKR1C have been depleted in cell lines and in which the effect on PAH-dependent DNA damage has been examined. This is a particularly important question with respect to PAH trans-dihydrodiols since the enzyme can operate in both reductive and oxidative pathways.

Here we have analyzed AKR1C1-4 expression in AML cells and identified AKR1C3 as the predominant isoform. Similar high AKR1C3 expression was detected in normal CD34+ve myeloid progenitors. Previous studies using cell-free systems identified 7,12-DMBA-3,4-diol as a substrate of AKR1C3. Since 7,12-DMBA-3,4-diol is an environmental proximate carcinogen, representative of PAH carcinogens formed by incomplete combustion and thus of pro-leukemic carcinogens, it is important to determine whether AKR1C3 activity contributes to 7,12-DMBA-3,4-diol activation in living cells. In order to test this hypothesis, in this study we use shRNA to knockdown expression of AKR1C3 in K562 cells, and we describe the effect on DNA damage.

Section snippets

Cell culture

Cell lines were sourced as follows: K562 and U937 were from ATCC; KG1a and HL-60 were from ECACC and NB4, a gift from Prof. A. Zelent (Institute of Cancer Research, London). Cell lines were maintained in exponential growth in RPMI 1640 + glutamine (Invitrogen Ltd.) supplemented with 10% (v/v) heat-inactivated FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen Ltd.) and treated as described. All cultures were incubated at 37 °C in a fully humidified atmosphere with 5% CO2.

AKR1C3 knockdown vector

The shRNA

Analysis of AKR1C expression in AML cells and normal myeloid progenitors

In initial experiments it was important to characterize the cell lines to be utilized with respect to the AKR enzymes expressed and their respective catalytic activities. A range of human cell lines were screened for expression of AKR1C1, -1C2, -1C3 and -1C4 by RT-PCR: KG1a (acute myeloid leukemia); HL-60 (acute myeloid leukemia); NB4 (acute promyelocytic leukemia); K562 (chronic myeloid leukemia in erythroid blast crisis); U937 (histiocytic lymphoma). AKR1C4 was not detected in any of the cell

Discussion

Penning and co-workers [8], [9] have shown that PAH activation by AKRs requires oxidation. In the second of these studies they used A549 cell lysates supplemented with NADP+ to demonstrate oxidative activation. However, the experiment did not study intact cells without cofactor modulation. Consequently it remained unknown whether such reactions would occur in intact cells at a level that would result in consequent DNA damage. Furthermore since the experiments neither manipulated AKR1C enzyme

Conflict of interest

None.

Acknowledgements

This work was supported by one programme grant and one Gordon Pillar Ph.D. studentship (JB) from the Leukaemia Research Fund. CP is the recipient of a BBSRC Ph.D. studentship.

Contributions: JB has contributed in experimental design, experimentation, intellectual input and manuscript preparation. REH and FLK have equally contributed in intellectual input, experimentation and manuscript preparation. RG and NJD have contributed towards intellectual input. CP and NW have helped in experimentation.

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