Aldehyde dehydrogenase (ALDH) is an enzyme that is expressed in the liver and is required for the conversion of retinol (vitamin A) to retinoic acids. ALDH is also highly enriched in hematopoietic stem cells (HSCs) and is considered a selectable marker of human HSCs, although its contribution to stem cell fate remains unknown. In this study, we demonstrate that ALDH is a key regulator of HSC differentiation. Inhibition of ALDH with diethylaminobenzaldehyde (DEAB) delayed the differentiation of human HSCs that otherwise occurred in response to cytokines. Moreover, short-term culture with DEAB caused a 3.4-fold expansion in the most primitive assayable human cells, the nonobese diabetic͞severe combined immunodeficiency mouse repopulating cells, compared with day 0 CD34 ؉ CD38 ؊ lin ؊ cells. The effects of DEAB on HSC differentiation could be reversed by the coadministration of the retinoic acid receptor agonist, all-trans-retinoic acid, suggesting that the ability of ALDH to generate retinoic acids is important in determining HSC fate. DEAB treatment also caused a decrease in retinoic acid receptor-mediated signaling within human HSCs, suggesting directly that inhibition of ALDH promotes HSC self-renewal via reduction of retinoic acid activity. Modulation of ALDH activity and retinoid signaling is a previously unrecognized and effective strategy to amplify human HSCs.retinoic acid ͉ self-renewal ͉ diethylaminobenzaldehyde ͉ long-term repopulating cells H ematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all mature lymphohematopoietic progeny throughout the lifetime of an individual (1, 2). Several molecular pathways that regulate HSC self-renewal have now been identified, including Notch (3), HOXB4 (4), Wnt (5), and bone morphogenetic protein signaling pathways (6). The osteoblastic niche for HSCs within the bone marrow (BM) has also been characterized (7,8). Despite these advances in understanding HSC biology, clinical methods to amplify human HSCs have yet to be realized, and characterization of the pathways that regulate HSC self-renewal continues to evolve.Two decades ago, Colvin et al. (9,10) demonstrated that the intracellular enzyme, aldehyde dehydrogenase (ALDH), protected BM progenitors from the cytotoxic effects of cyclophosphamide by deactivation of its metabolite, 4-hydroxycyclophosphamide (9, 10). Several isoforms of ALDH have been identified, with ALDH1 being the primary isoform expressed within human hematopoietic progenitors (11,12). Recent studies have shown that human and murine hematopoietic progenitors can be isolated by using a fluorescently labeled dye specific for ALDH activity (13-16) and cord blood (CB) ALDH br lin Ϫ cells are enriched for nonobese diabetic͞severe combined immunodeficiency (NOD͞SCID) mouse repopulating cells [SCID-repopulating cells (SRCs)] (15, 16). Although these data demonstrate that ALDH is a selectable marker for human stem͞progenitor cells, the HSC-specific function of ALDH remains unknown. In the liver, ALDH1 contributes prima...

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Human breast tumors often exist in an acidic and hypoxic microenvironment, which can promote resistance to radiation and chemotherapies. A tumor-selective pH gradient arises in these tumors which favors uptake and retention of drugs like camptothecin that are weak acids. We evaluated the effect of alkyl substitutions at the 7 position in seven CPTs with varying groups at the 10 position on modulation by acidic extracellular pH in three human breast cancer cell lines. Growth inhibition was assessed by propidium iodide staining of nucleic acids in human breast cancer cells cultured at either extracellular pH 6.8 or 7.4 that were (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc). Over 10-fold pH modulation was observed in 7-halomethyl analogs of methylenedioxy-CPT and in 7-alkyl analogs of 10-amino-CPT. Of 39 analogs tested, the overall pattern of activity across breast tumor cell lines was similar with some notable exceptions. For example, 7-propyl-10-amino-CPT was modulated 16- to 20-fold by acidic extracellular pH in the MCF-7 cell lines, but only 6-fold in MDA-MB-231 cells. One mechanism that can contribute to pH modulation is enhanced cellular drug uptake and retention. In MCF-7/wt cells, uptake of 10-amino-CPT increased 4-fold, while retention increased over 10-fold at acidic extracellular pH. In addition, gene expression analysis of MCF-7/wt cells indicated that expression of a number of genes changed under acidic culture conditions, including down-regulation of the CPT efflux protein pump breast cancer resistance protein (BCRP). Interestingly, expression of topoisomerase I, the molecular target of CPT, was not affected by acidic growth conditions. These results highlight the importance of maintaining key features of tumor physiology in cell culture models used to study cancer biology and to discover and develop new anticancer drugs. While several substitutions at the 7 and 10 positions enhance potency, 7-halomethyl and 10-amino CPT analogs show selective activity at the acidic pH common to the microenvironment of most solid tumors.

Except for the risk of infection inherent in reoperation, this tk-GCV paradigm was both feasible and safe. Pathological studies indicated that limited dissemination of VPCs and vector from the infusion site and failure to transduce tumor cells with the tk gene are major barriers to efficacy.

The alkylating agent cyclophosphamide (CYP) 1 may suppress or enhance both B and T cell-mediated immune responses in vivo but requires microsomal enzyme activation for in vitro efficacy. 4-hydroperoxycyclophosphamide (4-HC) is a synthetic compound spontaneously hydrolyzed in aqueous solution to 4-hydroxycyclophosphamide, the initial metabolite formed by microsomal activation, and which subsequently yields the several active metabolites formed in vivo. It has been demonstrated that CYP activated by liver microsomes in vitro as well as 4-HC will mimic the immunosuppressive or immunoaugmenting effects observed in the intact organism (1-4).By using 4-HC as a probe of immunoregulatory T subset function in vitro, Diamantstein et al. (4) recently demonstrated in a murine system that the enhancement of the cellular immune response to sheep erythrocytes by the compound is a result of selective inactivation of suppressor T cells or their precursors at low concentrations (3). These data provide support for the hypothesis that the enhancement of cellular responses by CYP administered before antigenic challenge in vivo (5-9) is a result of selective elimination of precursors of suppressor T cells. Diamantstein et al. (2,4) have further demonstrated that the murine inducer T cell subset mediating humoral immunity is relatively more sensitive to lower concentrations of 4-HC than are differentiated suppressor T cells for humoral responses or effector T cells mediating delayed-type hypersensitivity.This selective property of differential concentrations of 4-HC has been employed in the studies described here to investigate functional immunoregulatory subsets of human T cells involved in a pokeweed mitogen (PWM)-driven polyclonal immuno-* Supported by grants

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Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O(6)-benzylguanine; BG) potentially useful in the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[(18)F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [(131)I]7 in about 70% overall radiochemical yield. The IC(50) values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [(18)F]6 and [(131)I]7 to purified AGT was specific and saturable with both exhibiting similar IC(50) values (5-6 microM).

The genetic transfer of drug resistance to hematopoietic cells is an attractive approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors. We constructed a retroviral vector containing both a human cytosolic ALDH-1 cDNA and a human doubly mutated DHFR cDNA (Phe22/Ser31; termed F/S in the description of constructs) to generate increased resistance to both CTX and MTX. Infection of NIH3T3 cells resulted in increased resistance to both 4-hydroperoxycyclophosphamide (4HC) (1.9 +/- 0.1-fold) and MTX (73 +/- 2.8-fold). Transduced human CD34(+) enriched hematopoietic progenitor cells were also resistant to both 4HC and MTX by CFU-GM readout. Lethally irradiated mice transplanted with SFG-ALDH-IRES-F/S or mock-transduced bone marrow cells were treated with high-dose pulse CTX or high-dose CTX/MTX. Animals receiving marrow not transduced with ALDH-1 or mutated DHFR cDNA died from CTX or CTX/MTX toxicity, whereas mice transduced with ALDH-1 and mutated DHFR cDNA-containing marrow were able to tolerate the same doses of CTX or CTX/MTX treatment posttransplant. These data taken together indicate that ALDH-1 overexpression and mutant DHFR increased both 4HC and MTX resistance in vitro and in the in vivo mouse model. This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression.