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.