Three different stable human breast adenocarcinoma sublines that overexpress ALDH3A1 and certain other enzymes, apparently as a consequence of constitutively upregulated gene transcription mediated by transactivated EpREs (electrophile responsive elements) present in the 5 '-upstream regions of these genes

ALDH3A1 catalyzes the detoxification of cyclophosphamide, mafosfamide, 4-hy droperoxycyclophosphamide and other oxazaphosphorines. Constitutive ALDK3A1 levels, as well as those of certain other drug-metabolizing enzymes, e.g. NQO1 and CYP1A1, are relatively low in cultured, relatively oxazaphosphorin e-sensitive, human breast adenocarcinoma MCF-7 cells. However, transient ce llular insensitivity to the oxazaphosphorines call be brought about in thes e cells by transiently elevating ALDH3A1 levels in them as a consequence of transient exposure to: (1) electrophiles such as catechol that induce the transcription of a battery of genes, e.g. ALDH3A1 and NQO1, having in commo n an electrophile responsive element (EpRE) in their 5'-upstream regions; o r (2) Ah-receptor agonists, e.g, indole-3-carbinol and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, that induce the transcription of a battery of genes, e.g. ALDN3A1, NQO1 and CYP1A1, having in common a xeno biotic responsive element (XRE) in their 5'-upstream regions. Further, MCF- 7 sublines that are constitutively, i.e. when grown in the absence of the o riginal selecting pressure, relatively oxazaphosphorine-insensitive as a co nsequence of constitutively relatively elevated cellular ALDH3A1 levels evo lved when MCF-7 cells were: (1) continuously exposed for several months to gradually increasing concentrations of 4-hydroyeroxycyclophosphamide or ben z(a)pyrene; or (2) briefly exposed (once for 30 min) to a high concentratio n (1 mM) of mafosfamide. Each of these three stable sublines is constitutiv ely relatively cross-insensitive to benz(a)pyrene and other polycyclic arom atic hydrocarbons. Cellular levels of NQO1, but not of CYP1A1, are also con stitutively relatively elevated in each of the three sublines. RT-PCR-based experiments established that ALDH3A1 mRNA levels are constitutively elevat ed (similar to 5- to 8-fold) in each of the three sublines. The elevated AL DH3A1 mRNA levels are not the consequence of gene amplification, hypomethyl ation of a relevant regulatory element, or ALDH3A1 mRNA stabilization. Coll ectively, these observations suggest that constitutively elevated levels of ALDH3A1 and certain other enzymes in the three stable sublines are probabl y the consequence of a constitutive change in the cellular concentration of a key component of the EpRE signaling pathway, such that the cellular conc entration of the relevant ultimate transactivating factor is constitutively elevated, i.e. gene transcription promoted by transactivated EpREs is cons titutively upregulated. Further, constitutively upregulated gene transcript ion mediated by transactivated EpREs can be relatively easily induced, wher eas that mediated by transactivated XREs cannot, at least in MCF-7 cells. S till further, the three sublines may facilitate study of the signaling path way that leads to transactivation of the EpREs present in the 5'-upstream r egions of ALDH3A1, NgOl and other gene loci. (C) 2001 Published by Elsevier Science Ireland Ltd. All rights reserved.