Primary breast tumor levels of suspected molecular determinants of cellular sensitivity to cyclophosphamide, ifosfamide, and certain other anticancer agents as predictors of paired metastatic tumor levels of these determinants. Rational individualization of cancer chemotherapeutic regimens.

PURPOSE: Cyclophosphamide is one of the most frequently used agents in the neoadjuvant, adjuvant, and high-dose chemotherapeutic treatment of breast cancers. Preclinical models indicate that cellular sensitivity to cyclophosphamide and other oxazaphosphorines, e.g., ifosfamide, is inversely related to the cellular content of two aldehyde dehydrogenases, viz ALDH1A1 and ALDH3A1, and glutathione. Breast tumor levels of these "determinants of cellular sensitivity to the oxazaphosphorines" are known to vary widely, and the decision as to whether or not to use an oxazaphosphorine as part of the therapeutic strategy to treat breast cancer in any given patient is likely to depend, in large part, on the levels of these determinants in that cancer. ALDH1A1, ALDH3A1, and glutathione levels can be easily quantified in primary breast tumors and in detectable metastatic breast tumors present in axillary lymph nodes because the amounts of tissue required for the desired analysis can be readily obtained, whereas these levels cannot be quantified in residual metastatic breast cancer cell populations, i.e., those that escape detection and/or that are inaccessible to surgical harvest. The inability to directly quantify residual metastatic breast cancer cell ALDH1A1, ALDH3A1, and glutathione levels would not preclude a rational decision with regard to the inclusion/exclusion of an oxazaphosphorine as part of the chemotherapeutic strategy intended to eradicate residual metastatic breast cancer cells if primary breast tumor levels of these determinants reliably predicted those in metastatic breast cancer cells. METHODS: ELISAs and spectrophotometric assays were used to quantify enzyme and glutathione levels in paired human primary and locally advanced metastatic breast tumor samples. RESULTS: Primary breast tumor ALDH1A1 and ALDH3A1 levels were highly predictive of their respective levels in paired metastatic breast tumors present in axillary lymph nodes (r2 = 0.80 and 0.85, respectively). On the other hand, those of glutathione were relatively poorly predictive of its levels in paired metastatic offshoots (r2 = 0.35). Primary breast tumor levels of some additional enzymes known to catalyze the detoxification/toxification of various anticancer agents, though not of cyclophosphamide, were poorly predictive (DT-diaphorase and glutathione S-transferases alpha, mu, and pi) or not predictive (cytochrome P450 1A1) of their respective levels in paired metastatic offshoots. CONCLUSION: Since ALDH1A1, ALDH3A1 and, to a lesser extent, glutathione levels in primary breast tumors reliably predicted those in detectable and easily accessible metastatic breast cancer cell populations, viz those in axillary lymph nodes, they are also likely to be predictive of these levels in undetectable and/or relatively inaccessible metastatic breast cancer cell populations. Thus, quantification of primary breast tumor ALDH1A1, ALDH3A1 and, to a lesser extent, glutathione levels prior to the initiation of not only neoadjuvant but also adjuvant and high-dose breast cancer chemotherapy is likely to be of value in the rational design of individualized chemotherapeutic regimens intended to eradicate breast cancer cells with a minimum of untoward effects.

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-hydroperoxycyclophosphamide and other oxazaphosphorines. Constitutive ALDH3A1 levels, as well as those of certain other drug-metabolizing enzymes, e.g. NQO1 and CYP1A1, are relatively low in cultured, relatively oxazaphosphorine-sensitive, human breast adenocarcinoma MCF-7 cells. However, transient cellular insensitivity to the oxazaphosphorines can be brought about in these 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 common an electrophile responsive element (EpRE) in their 5'-upstream regions; or (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. ALDH3A1, NQO1 and CYP1A1, having in common a xenobiotic responsive element (XRE) in their 5'-upstream regions. Further, MCF-7 sublines that are constitutively, i.e. when grown in the absence of the original selecting pressure, relatively oxazaphosphorine-insensitive as a consequence of constitutively relatively elevated cellular ALDH3A1 levels evolved when MCF-7 cells were: (1) continuously exposed for several months to gradually increasing concentrations of 4-hydroperoxycyclophosphamide or benz(a)pyrene; or (2) briefly exposed (once for 30 min) to a high concentration (1 mM) of mafosfamide. Each of these three stable sublines is constitutively relatively cross-insensitive to benz(a)pyrene and other polycyclic aromatic hydrocarbons. Cellular levels of NQO1, but not of CYP1A1, are also constitutively relatively elevated in each of the three sublines. RT-PCR-based experiments established that ALDH3A1 mRNA levels are constitutively elevated ( approximately 5- to 8-fold) in each of the three sublines. The elevated ALDH3A1 mRNA levels are not the consequence of gene amplification, hypomethylation of a relevant regulatory element, or ALDH3A1 mRNA stabilization. Collectively, these observations suggest that constitutively elevated levels of ALDH3A1 and certain other enzymes in the three stable sublines are probably the consequence of a constitutive change in the cellular concentration of a key component of the EpRE signaling pathway, such that the cellular concentration of the relevant ultimate transactivating factor is constitutively elevated, i.e. gene transcription promoted by transactivated EpREs is constitutively upregulated. Further, constitutively upregulated gene transcription mediated by transactivated EpREs can be relatively easily induced, whereas that mediated by transactivated XREs cannot, at least in MCF-7 cells. Still further, the three sublines may facilitate study of the signaling pathway that leads to transactivation of the EpREs present in the 5'-upstream regions of ALDH3A1, NQO1 and other gene loci.

Inhibition of human class 3 aldehyde dehydrogenase, and sensitization of tumor cells that express significant amounts of this enzyme to oxazaphosphorines, by chlorpropamide analogues.

In some cases, acquired as well as constitutive tumor cell resistance to a group of otherwise clinically useful antineoplastic agents collectively referred to as oxazaphosphorines, e.g. cyclophosphamide and mafosfamide, can be accounted for by relatively elevated cellular levels of an enzyme, viz. cytosolic class 3 aldehyde dehydrogenase (ALDH-3), that catalyzes their detoxification. Ergo, inhibitors of ALDH-3 could be of clinical value since their inclusion in the therapeutic protocol would be expected to sensitize such cells to these agents. Identified in the present investigation were two chlorpropamide analogues showing promise in that regard, viz. (acetyloxy)[(4-chlorophenyl)sulfonyl]carbamic acid 1,1-dimethylethyl ester (NPI-2) and 4-chloro-N-methoxy-N-[(propylamino)carbonyl]benzenesulfonamide (API-2). Each inhibited NAD-linked benzaldehyde oxidation catalyzed by ALDH-3s purified from human breast adenocarcinoma MCF-7/0/CAT cells (IC50 values were 16 and 0.75 microM, respectively) and human normal stomach mucosa (IC50 values were 202 and 5 microM, respectively). The differential sensitivities of stomach mucosa ALDH-3 and breast tumor ALDH-3 to each of the two inhibitors can be viewed as further evidence that the latter is a subtle variant of the former. Human class 1 (ALDH-1) and class 2 (ALDH-2) aldehyde dehydrogenases were much less sensitive to NPI-2; IC50 values were >300 microM in each case. API-2, however, did not exhibit a similar degree of specificity; IC50 values for ALDH-1 and ALDH-2 were 7.5 and 0.08 microM, respectively. Each sensitized MCF-7/0/CAT cells to mafosfamide; the LC90 value decreased from >2 mM to 175 and 200 microM, respectively. Thus, the therapeutic potential of combining NPI-2 or API-2 with oxazaphosphorines is established.

Relative contribution of human erythrocyte aldehyde dehydrogenase to the systemic detoxification of the oxazaphosphorines.

Detoxification of cyclophosphamide is effected, in part, by hepatic class 1 aldehyde dehydrogenase (ALDH-1)-catalyzed oxidation of aldophosphamide, a pivotal aldehyde intermediate, to the nontoxic metabolite, carboxyphosphamide. This enzyme is found in erythrocytes as well. Detoxification of aldophosphamide may also be effected by enzymes, viz. certain aldo-keto reductases, that catalyze the reduction of aldophosphamide to alcophosphamide. Such enzymes are also found in erythrocytes. Not known at the onset of this investigation was whether the contribution of erythrocyte ALDH-1 and/or aldo-keto reductases to the overall systemic detoxification of circulating aldophosphamide is significant. Thus, NAD-linked oxidation and NADPH-linked reduction of aldophosphamide catalyzed by relevant erythrocyte enzymes were quantified. ALDH-1-catalyzed oxidation of aldophosphamide (160 microM) to carboxyphosphamide occurred at a mean (+/- SD) rate of 5.0 +/- 1.4 atmol/min/rbc (red blood cell). Aldo-keto reductase-catalyzed reduction of aldophosphamide (160 microM) to alcophosphamide occurred at a much slower rate, viz. 0.3 +/- 0.2 atmol/min/rbc. Thus, at a pharmacologically relevant concentration of aldophosphamide, viz. 1 microM, estimated aggregate erythrocyte ALDH-1-catalyzed aldophosphamide oxidation, viz. 2.0 micromol/min, was only about 3% of estimated aggregate hepatic enzyme-catalyzed aldophosphamide oxidation, viz. 72 micromol/min; however, this rate is greater than the estimated flow-limited rate of aldophosphamide delivery to the liver by the blood, viz. 1.5 micromol/min. These observations/considerations suggest an important in vivo role for erythrocyte ALDH-1 in systemic aldophosphamide detoxification. Erythrocyte ALDH-1-effected oxidation of other aldehydes to their corresponding acids, e.g. retinaldehyde to retinoic acid, may also be of pharmacological and/or physiological significance since a wide variety of aldehydes are known to be substrates for ALDH-1.

Cellular levels of class 1 and class 3 aldehyde dehydrogenases and certain other drug-metabolizing enzymes in human breast malignancies.

Molecular determinants of cellular sensitivity to cyclophosphamide, long the mainstay of chemotherapeutic regimens used to treat metastatic breast cancer, include class 1 and class 3 aldehyde dehydrogenases (ALDH-1 and ALDH-3, respectively), which catalyze the detoxification of this agent. Thus, interindividual variation in the activity of either of these enzymes in breast cancers could contribute to the wide variation in clinical responses that are obtained when such regimens are used to treat these malignancies. Consistent with this notion, ALDH-1 levels in primary and metastatic breast malignancies were found to range from 1-276 and 8-160 mIU/g tissue, respectively, and those of ALDH-3 range from 1-242 and 6-97 mIU/g tissue, respectively. ALDH-1 and ALDH-3 levels in normal breast tissue predicted the levels of these enzymes in primary and metastatic breast malignancies present in the same individuals. Confirming and extending the observations of others, levels of glutathione, a molecular determinant of cellular sensitivity to various DNA cross-linking agents including cyclophosphamide, and of DT-diaphorase, glutathione S-transferases, and cytochrome P450 1A1, each of which is known to catalyze the detoxification/toxification of one or more anticancer agents (although not of cyclophosphamide), also varied widely in primary and metastatic breast malignancies. Given the wide range of ALDH-1, ALDH-3, and glutathione levels that were observed in malignant breast tissues, measurement of their levels in normal breast tissue and/or primary breast malignancies prior to the initiation of chemotherapy is likely to be of value in predicting the therapeutic potential, or lack thereof, of cyclophosphamide in the treatment of metastatic breast cancer, thus providing a rational basis for the design of individualized therapeutic regimens when treating this disease.

Over-expression of glutathione S-transferases, DT-diaphorase and an apparently tumour-specific cytosolic class-3 aldehyde dehydrogenase by Warthin tumours and mucoepidermoid carcinomas of the human parotid gland.

Cytosolic class-3 aldehyde dehydrogenase (ALDH-3) may help to protect organisms from certain environmental aldehydes by catalysing their detoxification. Consistent with this notion are the reports that relatively high levels of this enzyme are present in tissues, e.g. stomach mucosa and lung, that are so-called ports of entry for such agents. Further, it is found in human saliva. The present investigation revealed that small amounts of this enzyme are also present in human salivary glands; mean values for ALDH-3 activities (NADP-dependent enzyme-catalysed oxidation of benzaldehyde) in cytosolic fractions prepared from submandibular and parotid glands were 52 (range: 29-92) and 44 (range: 13-73) mIU/g tissue, respectively. Essentially identical or slightly lower levels of this enzyme activity were found in pleomorphic adenomas, an undifferentiated carcinoma, and an adenocystic carcinomas, of the parotid gland. On the other hand, Warthin tumours, and mucoepidermoid carcinomas of the parotid gland exhibited relatively elevated levels of ALDH-3 activity; mean values were 1200 (range: 780-1880) and 810 (range: 580-1200) mIU/g tissue, respectively. The ALDH-3 found in normal salivary glands was, as judged by physical, immunological and kinetic criteria, identical to human stomach mucosa ALDH-3 whereas the ALDH-3 present in Warthin tumours, and mucoepidermoid carcinomas, of the parotid gland appeared to be a subtle variant thereof. Qualitatively paralleling the relatively elevated ALDH-3 levels in mucoepidermoid carcinomas and Warthin tumours were relatively elevated levels of glutathione S-transferase (alpha and pi) and DT-diaphorase. As was the case with ALDH-3 levels, glutathione S-transferase (alpha and pi) and DT-diaphorase levels were not elevated in pleomorphic adenomas. Glutathione S-transferase mu was not detected in the two normal parotid gland samples, or in the single pleomorphic adenoma sample, tested. It was found in the single mucoepidermoid carcinoma sample, and in one of the two Warthin tumour samples tested. Cellular levels of ALDH-3, glutathione S-transferases and/or DT-diaphorase could be useful criteria when the decision to be made is whether a salivary gland tumour is a mucoepidermoid carcinoma. ALDH-3 and glutathione S-transferases are known to catalyse the detoxification of two agents that are used to treat salivary gland tumours, viz. cyclophosphamide and cisplatin, respectively. Thus, elevated levels of these enzymes in the mucoepidermoid carcinomas must account for, or at least contribute to, the relative ineffectiveness of these agents when used to treat this tumour.

Interleukin-1 and tumor necrosis factor alpha induce class 1 aldehyde dehydrogenase mRNA and protein in bone marrow cells.

Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) protect normal human hematopoietic progenitors from the toxicity of 4-hydroperoxycyclophosphamide (4-HC). Aldehyde dehydrogenase Class 1 (ALDH-1) is the enzyme that inactivates 4-HC. Diethylaminobenzaldehyde (DEAB), a competitive inhibitor of ALDH-1, was shown to prevent the protective effects of IL-1 and TNF alpha. In this study, we examined the effect of IL-1 and TNF alpha on the expression of ALDH-1 in normal bone marrow as well as malignant cells. ALDH-1 mRNA and protein were quantified using Northern and Western blotting, respectively. In addition, the ALDH-1 enzyme activity in untreated as well as IL-1 and TNF alpha treated bone marrow cells was determined spectrophotometrically. The role of glutathione (GSH) in the protection against 4-HC toxicity was also studied. The results show that pretreatment with IL-1 and TNF alpha for 6 h or 20 h increase the expression of ALDH-1 mRNA and protein, respectively, in human bone marrow cells. In contrast, IL-1 and TNF alpha treatment did not affect the ALDH-1 expression in several leukemic and solid tumor cell lines, regardless of whether or not ALDH-1 is expressed constitutively. Furthermore, the ALDH-1 enzyme activity was significantly induced in bone marrow cells after 20 h pre-treatment with IL-1 and TNF alpha. Finally, the depletion of or inactivation of GSH did not affect the protection against 4-HC toxicity. In conclusion, inhibition of the protection from 4-HC toxicity by DEAB, together with the increase in ALDH-1 expression and activity, provide strong evidence that IL-1 and TNF alpha mediate their protective action, at least partially, through ALDH-1.

Human breast adenocarcinoma MCF-7/0 cells electroporated with cytosolic class 3 aldehyde dehydrogenases obtained from tumor cells and a normal tissue exhibit differential sensitivity to mafosfamide.

The cytosolic class aldehyde dehydrogenase (ALDH-3) present in human normal tissues/secretions is apparently much less able to catalyze the oxidation aldophosphamide to carboxyphosphamide than is the ALDH-3 present in human tumor cells/tissues, suggesting that the former may be less able to protect cells from the cytotoxic action of cyclophosphamide, mafosfamide, and other oxazaphosphorines. To test this notion, relatively large and approximately equal amounts of human normal stomach mucosa ALDH-3 and catechol-induced human breast adenocarcinoma MCF-7/0 ALDH-3 were first electroporated into cells (MCF-7/0) that constitutively express only very small amounts of the enzyme. The resultant preparations were then tested for sensitivity to mafosfamide. ALDH-3 activities (NADP-dependent catalysis of benzaldehyde oxidation) were 1.7, 212, and 183 mlU/10(7) cells in sham-electroporated MCF-7/0 cells, and MCF-7/0 cells electroporated with stomach mucosa ALDH-3 and catechol-induced MCF-7/0 ALDH-3, respectively. LC90 values (concentrations of mafosfamide required to effect a 90% cell kill) were 62, 417, and >1,000 microM, respectively. The three preparations were equisensitive to phosphoramide mustard (LC90 = approximately 850 microM). Inclusion of benzaldehyde in the drug exposure medium fully restored the sensitivity of MCF-7/0 cells electroporated with either enzyme to mafosfamide. These observations support the notions that 1) cellular sensitivity to the oxazaphosphorines decreases as the cellular content of ALDH-3 increases, 2) the foregoing is the consequence of ALDH-3-catalyzed oxidation (thus detoxification) of aldophosphamide, and 3) the ALDH-3 present in at least some tumor cells/tissues is a slight variant of the ALDH-3 present in normal tissues/secretions. Furthermore, they illustrate the utility of electroporation used as a tool to determine whether a given enzyme, or even more generally, protein or other macromolecule, is a determinant of cellular sensitivity to a given cytotoxic agent.

Identification of a class 3 aldehyde dehydrogenase in human saliva and increased levels of this enzyme, glutathione S-transferases, and DT-diaphorase in the saliva of subjects who continually ingest large quantities of coffee or broccoli.

Human saliva was tested for the presence of cytosolic class 3 aldehyde dehydrogenase, glutathione S-transferases alpha, mu, and pi, and DT-diaphorase, enzymes that are known to catalyze the biotransformation of many xenobiotics, including some that are carcinogens and some that are antineoplastic agents. Each of these enzymes was found to be present in this fluid. Inducers of these enzymes are known to be abundantly present in the human diet, especially in certain vegetables and fruits. Further investigation revealed that the salivary content of these enzymes rapidly, coordinately, and markedly increased upon daily consumption of relatively large amounts of coffee or broccoli. The enzyme activities of interest rapidly returned to basal levels when these substances were removed from the diet. Given the important role that cytosolic class 3 aldehyde dehydrogenase, the glutathione S-transferases, and DT-diaphorase are thought to play in determining the carcinogenic potential of some cancer-producing agents as well as the cytotoxic potential of some antineoplastic agents, and assuming that their salivary levels reflect their tissue levels, quantification of the salivary content of one or more of these enzymes, a noninvasive and relatively easy undertaking, could be useful in: (a) preliminarily assessing the chemopreventive potential of various diets and drugs; (b) establishing the optimal dose and schedule in Phase I clinical trials for any putatively chemopreventive diets or drugs of interest; and (c) the rational selection and use of chemotherapeutic agents, since several are inactivated, and a few are activated, by these enzymes; alternatively, the antineoplastic agent could be selected first and then a diet that enables the agent to achieve its full therapeutic potential would be selected based on whether high or low enzyme activity would be favorable in that regard. Such measurements may also be useful as an indicator when exposure to carcinogenic/teratogenic/otherwise toxic environmental/industrial/dietary agents that induce these enzymes is suspected.

Phenolic antioxidant-induced overexpression of class-3 aldehyde dehydrogenase and oxazaphosphorine-specific resistance.

High-level cytosolic class-3 aldehyde dehydrogenase (ALDH-3)-mediated oxazaphosphorine-specific resistance (> 35-fold as judged by the concentrations of mafosfamide required to effect a 90% cell-kill) was induced in cultured human breast adenocarcinoma MCF-7/0 cells by growing them in the presence of 30 microM catechol for 5 days. Resistance was transient in that cellular sensitivity to mafosfamide was fully restored after only a few days when the inducing agent was removed from the culture medium. The operative enzyme was identified as a type-1 ALDH-3. Cellular levels of glutathione S-transferase and DT-diaphorase activities, but not of cytochrome P450 IA1 activity, were also elevated. Other phenolic antioxidants, e.g. hydroquinone and 2,6-di-tert-butyl-4-hydroxytoluene, also induced ALDH-3 activity when MCF-7/0 cells were cultured in their presence. Thus, the increased expression of a type-1 ALDH-3 and the other enzymes induced by these agents was most probably the result of transcriptional activation of the relevant genes via antioxidant responsive elements present in their 5'-flanking regions. Cellular levels of ALDH-3 activity were also increased when a number of other human tumor cell lines, e.g. breast adenocarcinoma MDA-MB-231, breast carcinoma T-47D and colon carcinoma HCT 116b, were cultured in the presence of catechol. These findings should be viewed as greatly expanding the number of recognized environmental and dietary agents that can potentially negatively influence the sensitivity of tumor cells to cyclophosphamide and other oxazaphosphorines.

Intrinsic cellular resistance to oxazaphosphorines exhibited by a human colon carcinoma cell line expressing relatively large amounts of a class-3 aldehyde dehydrogenase.

A cultured human colon carcinoma cell line, viz. colon C, exhibiting intrinsic cellular resistance to mafosfamide mediated by relatively elevated levels of a cytosolic class-3 aldehyde dehydrogenase was identified. Colon C cells were found to be much less sensitive/more resistant (about 10-fold as judged by LC90 values) to mafosfamide than were two other cultured human colon carcinoma cell lines, viz. RCA and HCT 116b, and, as compared to the barely detectable aldehyde dehydrogenase activity (NADP-dependent enzyme-catalyzed oxidation of benzaldehyde to benzoic acid) in RCA and HCT 116b cells, that in colon C cells was about 200-fold greater. The three cell lines were equisensitive to phosphoramide mustard. Aldehyde dehydrogenase activity was confined to the cytosol in colon C cells (as well as in the other two cell lines) and, on the basis of its physical, immunological and catalytic characteristics, the operative enzyme was judged to be a Type-1 ALDH-3 identical to the Type-1 ALDH-3 expressed in methylcholanthrene-treated human breast adenocarcinoma MCF-7/0 cells and very nearly identical to the Type-1 ALDH-3 expressed in human normal stomach mucosa. Class-1 and class-2 aldehyde dehydrogenases were not found in these cells. The relative insensitivity to mafosfamide on the part of colon C cells was not observed when exposure to mafosfamide was in the presence of benzaldehyde or 4-(diethylamino)benzaldehyde, each a relatively good substrate for ALDH-3, whereas it was retained when exposure to mafosfamide was in the presence of acetaldehyde, a relatively poor substrate for this enzyme. Sensitivity to mafosfamide on the part of HCT 116b and RCA cells, and to phosphoramide mustard on the part of all three cell lines, was unaffected when drug exposure was in the presence of any of the three aldehydes. Together with earlier reports from our laboratory, these observations demonstrate that intrinsic, as well as stable and transient acquired, resistance to oxazaphosphorines, such as mafosfamide and cyclophosphamide, can be mediated by relatively increased levels of cytosolic class-3 aldehyde dehydrogenases.

Identification of the class-3 aldehyde dehydrogenases present in human MCF-7/0 breast adenocarcinoma cells and normal human breast tissue.

Affinity column chromatography was used to semipurify the very small amounts of class-3 aldehyde dehydrogenase (ALDH-3) present in human MCF-7/0 breast adenocarcinoma cells and human normal breast tissue. Characterization of the semipurified enzymes revealed that each was a type-1 ALDH-3 rather than a type-2 ALDH-3 as previously reported. Although clearly a type-1 ALDH-3, the MCF-7/0 enzyme, as well as the type-1 ALDH-3 constitutively present in cultured colon C cells and induced in cultured MCF-7/0 cells by methylcholanthrene, does, however, differ from the prototypical human stomach mucosa type-1 ALDH-3 in one, perhaps pharmacologically important, way, viz. when the ability to catalyse the oxidation of aldophosphamide is normalized by the ability to catalyse the oxidation of benzaldehyde, each of these enzymes, as well as the type-2 ALDH-3 found in MCF-7/OAP cells, exhibits greater ability to catalyse the oxidation of aldophosphamide than does stomach mucosa type-1 ALDH-3; hence, although not type-2 ALDH-3s, they may be slight variants of the prototypical type-1 ALDH-3.

A novel approach for the identification of isozymes using inhibitors.

Carboxylesterase isozymes of a termite and a fungus, have been identified by exploiting their differential sensitivity towards organophosphate inhibitors. The inhibition curves obtained by plotting pI (negative logarithm of inhibitor concentration) versus per cent inhibition are used to distinguish the isozymes. The termite and its associated fungus possess 2 and 4 isozymes respectively. Each of the purified isozyme gave a single sigmoidal inhibition curve with its characteristic I50 value. The pattern of the inhibition curve of the crude extract is found to mimic that of the mixture of the purified isozymes in both termite and fungus. This method provides not only identification of isozymes but also serves as a criterion of homogeneity.

Identification of a methylcholanthrene-induced aldehyde dehydrogenase in a human breast adenocarcinoma cell line exhibiting oxazaphosphorine-specific acquired resistance.

The class-3 aldehyde dehydrogenase that is overexpressed (> 100-fold) in human breast adenocarcinoma MCF-7/0 cells made resistant (> 30-fold as judged by LC90s) to oxazaphosphorines, such as mafosfamide, by growing them in the presence of polycyclic aromatic hydrocarbons, e.g., methylcholanthrene (3 microM for 5 days), was isolated and characterized. Its physical and catalytic properties were identical to those of the prototypical human stomach mucosa cytosolic class-3 aldehyde dehydrogenase, type-1 ALDH-3, except that it catalyzed, though not very rapidly, the oxidation of aldophosphamide, whereas the stomach mucosa enzyme essentially did not; hence, it was judged to be a slight variant of the prototypical enzyme. Carcinogens that are not ligands for the Ah receptor, barbiturates known to induce hepatic cytochrome P450s, steroid hormones, an antiestrogen, and oxazaphosphorines did not induce the enzyme or the largely oxazaphosphorine-specific acquired resistance. Whereas methylcholanthrene induced (a) resistance to mafosfamide and (b) class-3 aldehyde dehydrogenase activity, as well as glutathione S-transferase and DT-diaphorase activities, in the estrogen receptor-positive MCF-7/0 cells, it did not do so in two other human breast adenocarcinoma cell lines, MDA-MB-231 and SK-BR-3, each of which is estrogen receptor negative. Expression of the class-3 aldehyde dehydrogenase and the loss of sensitivity to mafosfamide by polycyclic aromatic hydrocarbon-treated MCF-7/0 cells were transient; each returned to essentially basal levels within 15 days when the polycyclic aromatic hydrocarbon was removed from the culture medium. Insensitivity to the oxazaphosphorines on the part of polycyclic aromatic hydrocarbon-treated MCF-7/0 cells was not observed when exposure to mafosfamide (30 min) was in the presence of benzaldehyde or octanal, each a relatively good substrate for cytosolic class-3 aldehyde dehydrogenases, whereas it was retained when exposure to mafosfamide was in the presence of acetaldehyde, a relatively poor substrate for these enzymes. These observations demonstrate that ligands for the Ah receptor can induce a transient, largely oxazaphosphorine-specific, acquired cellular resistance, and they are consistent with the notion that elevated levels of a cytosolic class-3 aldehyde dehydrogenase nearly identical to the prototypical type-1 class-3 aldehyde dehydrogenase expressed by human stomach mucosa account for the Ah receptor ligand-induced oxazaphosphorine-specific acquired resistance, most probably by catalyzing the detoxification of aldophosphamide.

Isolation and properties of carboxylesterases of the termite gut-associated fungus, Xylaria nigripes. K., and their identity from the host termite, Odentotermes horni. W., mid-gut carboxylesterases.

1. The termite, Odentotermes horni. W., houses three fungal species, viz. Xylaria nigripes, Termitomyces microcorpus, and Trichoderma (species not identified), in its gut. X. nigripes was found to possess higher esterase activity levels than the other two. 2. Four esterase enzymes, viz. FE-I, -II, -III and -IV, with pI values 5.1, 5.25, 5.4 and 5.6, respectively, were identified, isolated and purified to apparent homogeneity from the fungus X. nigripes, their biochemical and enzymological properties were determined, and compared with those of the previously characterized host termite mid-gut enzymes, TE-I and -II. 3. The M(r) of FE-I and -II was 85.1 kDa and those of FE-III and -IV was 87.5 kDa. However, TE-I and -II were relatively smaller (M(r) approximately 78.5 kDa). Each of the fungal enzymes, viz. FE-I to -IV, was a homodimer with subunits associated non-covalently. The subunit M(r) were 42.6 kDa for FE-I and -II, and 43.7 kDa for FE-III and -IV. On the other hand, the termite mid-gut enzymes, TE-I and -II, were also homodimeric, but the subunits were associated covalently (subunit M(r) = 40 kDa). Immunologically the fungal esterase enzymes, viz. FE-I to -IV, were different from those of the host termite mid-gut esterases, viz. TE-I and -II. 4. The substrate specificity and inhibitor sensitivity studies classify these enzymes, i.e. FE-I to -IV, as carboxylesterases (EC 3.1.1.1). Steady-state product inhibition kinetics suggested; an ordered release of products, i.e. alcohol followed by acid, and a Uni-Bi kinetic reaction mechanism. 5. The two preliminary studies, i.e. the confinement of most esterase activity to the gut-tissue free from microorganisms and starvation of termites not leading to complete loss of esterase activity in the gut of the termites, suggested that there may not be any symbiotic relationship between termite, O. horni, and its gut associated microorganisms with regard to ester metabolism. Though the enzymes from the two sources were carboxylesterases, several of their properties were different and hence, they are different entities.

Identification and characterization of a novel class 3 aldehyde dehydrogenase overexpressed in a human breast adenocarcinoma cell line exhibiting oxazaphosphorine-specific acquired resistance.

Associated with the oxazaphosphorine-specific acquired resistance exhibited by a human breast adenocarcinoma subline growing in monolayer culture, viz. MCF-7/OAP, was the overexpression (> 100-fold as compared with the very small amount expressed in the oxazaphosphorine-sensitive parent line) of a class 3 aldehyde dehydrogenase, viz. ALDH-3, judged to be so because it is a polymorphic enzyme (pI values ca. 6.0) present in the cytosol that is heat labile, is insensitive to inhibition by disulfiram (25 microM), much prefers benzaldehyde to acetaldehyde as a substrate and, at concentrations of 4 mM, prefers NADP to NAD as a cofactor. No other aldehyde dehydrogenases were found in these cells. As compared with those of the prototypical class 3 human ALDH-3, viz. constitutive human stomach mucosa ALDH-3, the physical and catalytic properties of the MCF-7/OAP enzyme differed somewhat with regard to pI values, native M(r), subunit M(r), recognition of the subunit by anti-stomach ALDH-3 IgY, pH stability, cofactor influence on catalytic activity, and the ability to catalyze, albeit poorly, the oxidation of an oxazaphosphorine, viz. aldophosphamide. Hence, the MCF-7/OAP ALDH-3 was judged to be a novel class 3 aldehyde dehydrogenase. Small amounts of a seemingly identical enzyme are also present in normal pre- and post-menopausal breast tissue. None could be detected in human liver, kidney or placenta, suggesting that it may be a tissue-specific enzyme.