Cellular levels of aldehyde dehydrogenases (ALDH1A1 and ALDH3A1) as predictors of therapeutic responses to cyclophosphamide-based chemotherapy of breast cancer: a retrospective study. Rational individualization of oxazaphosphorine-based cancer chemotherapeutic regimens.

PURPOSE: In preclinical models, established molecular determinants of cellular sensitivity to cyclophosphamide, long a mainstay of chemotherapeutic regimens used to treat breast cancers, include the aldehyde dehydrogenases that catalyze the detoxification of this agent, namely, ALDH1A1 and ALDH3A1. As judged by bulk quantification of relevant catalytic activities, as well as of relevant proteins (ELISAs), tissue levels of these enzymes vary widely in primary and metastatic breast malignancies. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses obtained when such regimens are used to treat these malignancies. Direct evidence for this notion was sought in the present investigation. METHODS: Cellular levels of ALDH1A1 and ALDH3A1 in 171 repository human breast tumor (122 primary and 49 metastatic) samples were semiquantified using immunocytochemical staining. Clinical responses were retrieved from the archived medical records of each of 48 metastatic breast cancer sample donors, 26 of whom had been treated with a cyclophosphamide-based chemotherapeutic regimen subsequent to tumor sampling and 22 of whom had not. The premise that cellular levels of ALDH1A1 and/or ALDH3A1 predict clinical responses to cyclophosphamide-based chemotherapeutic regimens was submitted to statistical analysis. RESULTS: Confirming an earlier report, ALDH1A1 and ALDH3A1 levels varied widely in both primary and metastatic breast tumor cells. When measurably present, each of the enzymes appeared to be evenly distributed throughout a given tumor cell population. Retrospective analysis indicated that cellular levels of ALDH1A1, but not those of ALDH3A1, were (1) significantly higher in metastatic tumor cells that had survived exposure to cyclophosphamide than in those that had not been exposed to this drug, and (2) significantly higher in metastatic tumors that did not respond (tumor size did not decrease or even increased) to subsequent treatment with cyclophosphamide-based chemotherapeutic regimens than in those that did respond (tumor size decreased) to such regimens. The therapeutic outcome of cyclophosphamide-based chemotherapy corresponded to cellular ALDH1A1 levels in 77% of cases. The frequencies of false-positives (cyclophosphamide-based chemotherapy not effective when a low level of ALDH1A1 predicted it would be) and false-negatives (cyclophosphamide-based chemotherapy effective when a high level of ALDH1A1 predicted it would not be) were 0.00 and 0.43, respectively. Thus, partial or complete responses to cyclophosphamide-based chemotherapy occurred 2.3 times more often when the ALDH1A1 level was low than when it was high. CONCLUSIONS: Given (1) the wide range of ALDH1A1 levels observed in malignant breast tissues, (2) that ALDH1A1 levels in primary breast tumor tissue, as well as those in normal breast tissue, directly reflect ALDH1A1 levels in metastatic breast tumor cells derived therefrom, and (3) the findings reported here, measurement of ALDH1A1 levels in primary breast malignancies and/or normal breast tissue prior to the initiation of chemotherapy is likely to be of value in predicting the therapeutic potential, or lack of potential, of cyclophosphamide and other oxazaphosphorines, e.g. ifosfamide, in the treatment of primary, as well as metastatic, breast cancer, thus providing a rational basis for the design of individualized therapeutic regimens for this disease. Failure to observe the expected inverse relationship between clinical responses to cyclophosphamide-based chemotherapeutic regimens and ALDH3A1 levels was probably because even the highest breast tumor tissue ALDH3A1 level thus far reported appears to be below the threshold level at which ALDH3A1-catalyzed detoxification of oxazaphosphorines becomes pharmacologically meaningful. However, ALDH3A1 levels in certain other malignancies, e.g. those of the alimentary tract and lung, may be of a sufficient magnitude in that regard.

Transient induction of increased aldehyde dehydrogenase 3A1 levels in cultured human breast (adeno)carcinoma cell lines via 5'-upstream xenobiotic, and electrophile, responsive elements is, respectively, estrogen receptor-dependent and -independent.

Transient up-regulation of ALDH3A1, CYP1A1 and CYP1B1 transcription by transient exposure to aryl hydrocarbon receptor (AhR) ligands, e.g. 3-methylcholanthrene, is via transient transactivation of xenobiotic responsive elements (XRE) present in the 5'-upstream regions of these genes. Others have shown that AhR ligand-mediated induction of increased CYP1A1 levels in cultured human breast (adeno)carcinoma cell lines is apparently estrogen receptor (ER)-dependent, i.e. it was observed in ER(+) cell lines but not in ER(-) cell lines, whereas AhR ligand-mediated induction of increased CYP1B1 levels is ER-independent, i.e. it was observed in both ER(+) and ER(-) cell lines. The present investigation established that transient, AhR ligand/XRE-mediated induction of increased ALDH3A1 levels in human breast (adeno)carcinoma cell lines was, like that of CYP1A1 and unlike that of CYP1B1, apparently ER-dependent. Thus, transient exposure to 3-methylcholanthrene induced increased levels of ALDH3A1 in five cultured human breast (adeno)carcinoma cell lines that were documented as being ER(+), viz., MCF-7/0, MCF-7/OAP, T-47D, ZR-75-1 and MDA-MB-468, but failed to induce increased levels of this enzyme in four cultured human breast (adeno)carcinoma cell lines that have been historically viewed as being ER(-), viz., MDA-MB-231, SK-BR-3, HS-578-T and MDA-MB-435. Somewhat at odds with the foregoing, transient exposure to 3-methylcholanthrene also induced increased levels of ALDH3A1 and CYP1A1 in cultured, essentially ER(-), human breast epithelial MCF-10A cells. These cells, like cultured human breast (adeno)carcinoma cells, are immortal, but unlike the latter, are not tumorigenic. Transient induction of increased ALDH3A1 levels can also be effected by agents that are not AhR ligands, viz., electrophiles such as catechol, and thus, cannot up-regulate ALDH3A1 transcription via transactivation of a 5'-upsteam region XRE. Rather, they are thought to up-regulate ALDH3A1 transcription via transient transactivation of an electrophile responsive element (EpRE) that is putatively also present in the 5'-upstream region of this gene. Electrophile-initiated/EpRE-mediated induction of increased ALDH3A1 levels was found to be ER-independent. Thus, catechol transiently induced increased levels of ALDH3A1 in the five ER(+) human breast (adeno)carcinoma cell lines, the four ER(-) human breast (adeno)carcinoma cell lines, and the ER(-), immortal but not tumorigenic, human breast epithelial cell line.

Ultraviolet radiation decreases expression and induces aggregation of corneal ALDH3A1.

Substantial reduction in corneal ALDH3A1 enzymatic activity associated with eye pathology was previously reported in C57BL/6J mice subjected to ultraviolet radiation (UVR). The aim of this study was to examine whether UVR diminishes corneal ALDH3A1 expression through modifications at the transcriptional, translational, or post-translational level. Adult C57BL/6J mice were subjected to UVR exposure (302 nm peak wavelength) for various periods of time, and corneal ALDH3A1 mRNA and protein levels were monitored by Northern and Western blot analysis, respectively. In addition, ALDH3A1 enzymatic activity was determined as a measure of post-translational modification. Mice exposed to 0.2 J/cm(2) UVB radiation demonstrated an extensive decrease, approximately 80%, in mRNA and protein levels, as well as enzymatic activity of corneal ALDH3A1. Significant reductions in corneal ALDH3A1 enzymatic activity were detected in mice 96 h after exposure to 0.05 and 0.1 J/cm(2) UVB radiation; no significant changes were observed in mRNA and protein levels. These data suggest that UVB down-regulates corneal ALDH3A1 expression at the transcriptional and/or post-translational level depending on the dose of UVB. Reduction in gene transcription requires UVB doses greater than or equal to 0.2 J/cm(2). In vitro experiments with human corneal epithelial cell lines stably transfected with human ALDH3A1 cDNA, and with purified recombinant human ALDH3A1 protein, indicated that ALDH3A1 undergoes post-translational modifications after UVR exposure. These modifications result in both covalent and non-covalent aggregation of the protein with no detectable precipitation. Such conformational changes may be associated with the function of ALDH3A1 as a chaperone-like molecule in the cornea.

Aldehyde dehydrogenases: measurement of activities and protein levels.

Seventeen enzymes are currently viewed as belonging to the human aldehyde dehydrogenase superfamily, and all of them catalyze the pyridine nucleotide-dependent oxidation of aldehydes to acids. Depending on the specific aldehyde dehydrogenase, lack of sufficient catalytic activity (1) results in a gross pathological phenotype in the absence of any insult, or (2) is ordinarily of no consequence with respect to gross phenotype, but is of consequence when the organism is subjected to a relevant insult. Described in this unit are eight assays that can be used to (semi)quantify various in vitro aldehyde dehydrogenase protein and/or catalytic activity levels, and two that can be used to semiquantify various in situ aldehyde dehydrogenase protein and/or catalytic activity levels. Aldehyde dehydrogenases also catalyze the hydrolysis of esters; this unit includes an assay that can be used to quantify that catalytic activity as well. Preparation of test materials and of antibodies to the aldehyde dehydrogenases are described in three support protocols.

Human aldehyde dehydrogenases: potential pathological, pharmacological, and toxicological impact.

Aldehyde dehydrogenases catalyze the pyridine nucleotide-dependent oxidation of aldehydes to acids. Seventeen enzymes are currently viewed as belonging to the human aldehyde dehydrogenase superfamily. Summarized herein, insofar as the information is available, are the structural composition, physical properties, tissue distribution, subcellular location, substrate specificity, and cofactor preference of each member of this superfamily. Also summarized are the chromosomal locations and organization of the genes that encode these enzymes and the biological consequences when enzyme activity is lost or substantially diminished. Broadly, aldehyde dehydrogenases can be categorized as critical for normal development and/or physiological homeostasis (1). even when the organism is in a friendly environment or (2). only when the organism finds itself in a hostile environment. The primary, if not sole, evolved raison d'être of first category aldehyde dehydrogenases appears to be to catalyze the biotransformation of a single endobiotic for which they are relatively specific and of which the resultant metabolite is essential to the organism. Most of the human aldehyde dehydrogenases for which the relevant information is available fall into this category. Second category aldehyde dehydrogenases are relatively substrate nonspecific and their evolved raison d'être seems to be to protect the organism from potentially harmful xenobiotics, specifically aldehydes or xenobiotics that give rise to aldehydes, by catalyzing their detoxification. Thus, the lack of a fully functional first category aldehyde dehydrogenase results in a gross pathological phenotype in the absence of any insult, whereas the lack of a functional second category aldehyde dehydrogenase is ordinarily of no consequence with respect to gross phenotype, but is of consequence in that regard when the organism is subjected to a relevant insult.