Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay.

Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.

Optimizing donor selection for public cord blood banking: influence of maternal, infant, and collection characteristics on cord blood unit quality.

BACKGROUND: Banked unrelated donor umbilical cord blood (CB) has improved access to hematopoietic stem cell transplantation for patients without a suitably matched donor. In a resource-limited environment, ensuring that the public inventory is enriched with high-quality cord blood units (CBUs) addressing the needs of a diverse group of patients is a priority. Identification of donor characteristics correlating with higher CBU quality could guide operational strategies to increase the yield of banked high-quality CBUs. STUDY DESIGN AND METHODS: Characteristics of 5267 CBUs donated to the Carolinas Cord Blood Bank, a public bank participating in the National Cord Blood Inventory, were retrospectively analyzed. Eligible CBUs, collected by trained personnel, were processed using standard procedures. Routine quality and potency metrics (postprocessing total nucleated cell count [post-TNCC], CD34+, colony-forming units [CFUs]) were correlated with maternal, infant, and collection characteristics. RESULTS: High-quality CBUs were defined as those with higher post-TNCC (>1.25 × 10(9)) with CD34+ and CFUs in the upper quartile. Factors associated with higher CD34+ or CFU content included a shorter interval from collection to processing (<10 hr), younger gestational age (34-37 weeks; CD34+ and CFUs), Caucasian race, higher birthweight (>3500 g), and larger collection volumes (>80 mL). CONCLUSIONS: We describe characteristics identifying high-quality CBUs, which can be used to inform strategies for CBU collection for public banks. Efforts should be made to prioritize collections from larger babies born before 38 weeks of gestation. CBUs should be rapidly transported to the processing laboratory. The lower quality of CBUs from non-Caucasian donors highlights the challenges of building a racially diverse public CB inventory.

The Cord Blood Apgar: a novel scoring system to optimize selection of banked cord blood grafts for transplantation (CME).

BACKGROUND: Engraftment failure and delays, likely due to diminished cord blood unit (CBU) potency, remain major barriers to the overall success of unrelated umbilical cord blood transplantation (UCBT). To address this problem, we developed and retrospectively validated a novel scoring system, the Cord Blood Apgar (CBA), which is predictive of engraftment after UCBT. STUDY DESIGN AND METHODS: In a single-center retrospective study, utilizing a database of 435 consecutive single cord myeloablative UCBTs performed between January 1, 2000, to December 31, 2008, precryopreservation and postthaw graft variables (total nucleated cell, CD34+, colony-forming units, mononuclear cell content, and volume) were initially correlated with neutrophil engraftment. Subsequently, based on the magnitude of hazard ratios (HRs) in univariate analysis, a weighted scoring system to predict CBU potency was developed using a randomly selected training data set and internally validated on the remaining data set. RESULTS: The CBA assigns transplanted CBUs three scores: a precryopreservation score (PCS), a postthaw score (PTS), and a composite score (CS), which incorporates the PCS and PTS values. CBA-PCS scores, which could be used for initial unit selection, were predictive of neutrophil (CBA-PCS ≥ 7.75 vs. <7.75, HR 3.5; p < 0.0001) engraftment. Likewise, CBA-PTS and CS scores were strongly predictive of Day 42 neutrophil engraftment (CBA-PTS ≥ 9.5 vs. <9.5, HR 3.16, p < 0.0001; CBA-CS ≥ 17.75 vs. <17.75, HR 4.01, p < 0.0001). CONCLUSION: The CBA is strongly predictive of engraftment after UCBT and shows promise for optimizing screening of CBU donors for transplantation. In the future, a segment could be assayed for the PTS score providing data to apply the CS for final CBU selection.

Luteolin Regulation of Estrogen Signaling and Cell Cycle Pathway Genes in MCF-7 Human Breast Cancer Cells.

cRNA microarray and real-time PCR (qPCR) studies identified a number of Estrogen Signaling Pathway (ESP) genes (GTF2H2, NCOR1, TAF9, NRAS, NRIP1, POLR2A, DDX5, NCOA3) and Cell Cycle Pathway genes (CCNA2, PCNA, CDKN1A, CCND1, PLK1) in MCF-7 breast cancer cells that are regulated by the bioflavonoid luteolin. Chromatin immunoprecipitation (ChIP) studies revealed that luteolin modified histone H4 acetylation at the PLK-1 promoter suggesting that this bioflavonoid controls gene transcription via an epigenetic mechanism involving histone H4 acetylation. These findings are consistent with the anti-estrogenic and anti-proliferative properties of luteolin in normal and malignant cells.

Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood.

BACKGROUND AIMS: Oligodendrocyte precursor cells (OPC) hold promise as a cellular therapy for demyelinating diseases. The feasibility of using OPC-based therapies in humans depends upon a reliable, readily available source. We have previously described the isolation, expansion and characterization of oligodendrocyte-like cells from fresh human umbilical cord blood (UCB). We now describe the isolation and expansion of OPC from thawed, cryopreserved UCB. METHODS: We thawed cryopreserved UCB units employing a standard clinical protocol, then isolated and plated mononuclear cells under previously established culture conditions. All OPC cultures were trypsinized at 21 days, counted, then characterized by flow cytometry after fixation, permeablization and labeling with the following antibodies: anti-oligodendrocyte marker 4 (O4), anti-oligodendrocyte marker 1 (O1) and anti-myelin basic protein (MBP). OPC were also placed in co-culture with shiverer mouse neuronal cells then stained in situ for beta tubulin III (BT3) and MBP as a functional assay of myelination. RESULTS: The average OPC yield per cryopreserved UCB unit was 64% of that seen with fresh UCB. On flow cytometric analysis, 74% of thawed UCB units yielded cells with an O4-expression level of at least 20% of total events, compared with 95% of fresh UCB units. We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials. CONCLUSIONS: Our results demonstrate that OPC can be derived reliably from thawed, cryopreserved UCB units, and support the feasibility of using these cells in human clinical trials.

Luteolin and gefitinib regulation of EGF signaling pathway and cell cycle pathway genes in PC-3 human prostate cancer cells.

cRNA microarray and real-time PCR (qPCR) studies from our lab identified five Cell Cycle Pathway (CCP) genes (CCNA2, CCNE2, CDC25A, CDKN1B, and PLK-1) as targets for luteolin in PC-3 prostate cancer cells [Shoulars et al., J. Steroid Biochem. Mol. Biol. 118 (2010) 41-50]. In this paper, Ingenuity Pathway Analysis of the microarray data identified 7 luteolin-regulated genes (EGFR, c-Fos, SOS, GRB2, JNK1, MKK4 and RasGAP) in the Epidermal Growth Factor Signaling Pathway (EGFSP) potentially involved in luteolin regulation of CCP genes and cell proliferation. To address these possibilities, we compared the response profiles (RNA and protein) of these EGFSP and CCP genes to luteolin and gefitinib by real-time PCR (qPCR) and Western blot analyses. Luteolin and gefitinib are known antagonists of EGFR-associated tyrosine protein kinase. Thus, the response profiles of EGFR regulated EGFSP or CCP genes should be very similar if genes in both pathways are controlled through this common mechanism of action. Treatment of PC-3 cell with luteolin for 24h caused a 4-fold stimulation of c-Fos gene expression, significant inhibition (p<0.001) of the CCP genes and G2/M arrest. Treatment of PC-3 cells with gefitinib also inhibited most of the CCP genes in a fashion similar to that of luteolin, however, the EGFR antagonist inhibited c-Fos gene expression, stimulated CDKN1B (p27) and arrested the cells in G0/G1. Thus, although the response patterns of most of the CCP genes to luteolin or gefitinib were similar, the effects of the two compounds on EGFSP gene expression and cell cycle arrest were clearly different. Combination studies revealed that the response of EGFSP genes to luteolin was not affected by gefitinib, even though the two compounds were additive with respect to their abilities to inhibit CCNA2, CCNE2, CDC25A and PCNA. These findings suggest that luteolin and gefitinib regulate CCP gene expression through a common mechanism involving EGFR-associated tyrosine kinase. Conversely, luteolin regulates PC-3 cell proliferation through an EGFR-tyrosine kinase independent mechanism(s), likely involving the epigenetic control of gene EGFSP gene expression through histone H4 binding interactions resulting in the upregulation of c-Fos and p21 gene expression.

Regulation of cell cycle and RNA transcription genes identified by microarray analysis of PC-3 human prostate cancer cells treated with luteolin.

Prostate cancer is the second leading cause of cancer-related deaths in men in the United States. Our previous studies have shown that ligands for the nuclear type II [(3)H]estradiol binding site such as luteolin significantly inhibit prostate cancer cells in vitro and in vivo; however, the role of these ligands in cell growth and proliferation is poorly understood. In order to further elucidate the molecular mechanism through which luteolin exerts its effects on PC-3 cells, cRNA microarray analyses was performed on 38,500 genes to determine the genes altered by luteolin treatment. The expression of 3331 genes was changed greater than 1.2-fold after luteolin treatment. Analysis of the altered genes identified two pathways that were significantly affected by luteolin. The Cell Cycle Pathway contained 22 down-regulated genes (including polo-like kinase 1, cyclin A2, cyclin E2 and proliferation cell nuclear antigen) and one up-regulated gene (cyclin-dependent kinase inhibitor 1B). In addition, 13 genes were down-regulated by luteolin in the RNA Transcription Pathway. Real-time polymerase chain reactions and western blots verified the observations from the microarray. In addition, two synthetic, chemically distinct type II ligands, ZN-2 and BMHPC, mimicked the effects of luteolin on gene expression at the mRNA and protein level in PC-3 cells. Finally, chromatin immunoprecipitation assays indicated that luteolin exerts its effects on genes by altering the acetylation state of promoter-associated histones. Taken together, the data suggest that type II ligands inhibit cell growth and proliferation through epigenetic control of key genes involved in cell cycle progression and RNA transcription.

Regulation of the nitric oxide pathway genes by tetrahydrofurandiols: microarray analysis of MCF-7 human breast cancer cells.

THF-diols (9,12-oxy-10,13-dihydroxyoctadecanoic and 10,13-oxy-9,12-dihydroxyoctadecanoic acids) are endocrine disrupters in rats and mitogens in breast cancer cells. Microarray analyses and real-time PCR analyses on RNA from THF-treated MCF-7 cells revealed a number of genes (caveolin 1, heat shock protein 90 alpha and 90 beta, vascular endothelial growth factor, ATPase, Ca++ transporting, ubiquitous) in the nitric oxide pathway (NOP) were targets for THF-diols. Chromatin immunoprecipitation studies suggest THF-diols modify of histone H4 acetylation at the caveolin 1 promoter via an epigenetic mechanism. These findings are consistent with the well-known involvement of NOP genes in cell proliferation and sexual behavior.

Tetrahydrofurandiol stimulation of phospholipase A2, lipoxygenase, and cyclooxygenase gene expression and MCF-7 human breast cancer cell proliferation.

BACKGROUND: We characterized an endocrine disruptor from ground corncob bedding material that interferes with male and female sexual behavior and ovarian cyclicity in rats and stimulates estrogen receptor (ER)-positive and ER-negative breast cancer cell proliferation. The agents were identified as an isomeric mixture of tetrahydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxy-octadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid). Synthetic THF-diols inhibited rat male and female sexual behavior at oral concentrations of 0.5-1 ppm, and stimulated MCF-7 human breast cancer cell proliferation in vitro. OBJECTIVES: Because THF-diols are derived from lipoxygenase and cyclooxygenase pathways, we suspected that these compounds may regulate cell proliferation by modulating specific enzymatic sites involved in linoleic acid metabolism including phospholipase A(2) (PLA2), lipoxygenases (LOX-5 and LOX-12), cyclooxygenases (COX-1 and COX-2), and closely coupled enzymes including aromatase (AROM). METHODS: MCF-7 human breast cancer cells were treated with inhibitors for PLA2 (quinacrine), lipoxygenases (LOX-5 and LOX-12; baicalein, REV-5091, nordihydroguaiaretic acid), cyclooxygenases (COX-1, COX-2, indomethacin), and AROM (formestane). The effects of these enzyme inhibitors on cell proliferation in response to THF-diols or estradiol (E(2)) were assessed. THF-diol modulation of the expression (RNA and protein) of these enzymes was also evaluated by quantitative real-time PCR (QPCR) and Western blot analyses. RESULTS: The enzyme inhibition and gene expression (RNA and protein) studies identified PLA2, LOX-5, LOX-12, COX-2, and perhaps AROM as likely sites of THF-diol regulation in MCF-7 cells. COX-1 was not affected by THF-diol treatment. DISCUSSION: THF-diol stimulation of MCF-7 cell proliferation is mediated through effects on the expression of the PLA2, COX-2, LOX-5, and LOX-12 genes and/or their respective enzyme activities. The products of these enzymes, including prostaglandins, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecenoic acids (HODEs), are well-established mitogens in normal and malignant cells. Therefore, it is likely that these compounds are involved in the mechanism of action of THF-diols in breast cancer cells. Although the formestane inhibition studies suggested that AROM activity might be modulated by THF-diols, this was not confirmed by the gene expression studies.

Nuclear type II [3H]estradiol binding site ligands: inhibition of ER-positive and ER-negative cell proliferation and c-Myc and cyclin D1 gene expression.

These studies assessed the effects of 3,4-dihydroxybenzalacetone (ZN-1) and 1-(3,4-dihydroxyphenyl)-2-propanol (ZN-2) on MCF-7 cell proliferation. The compounds blocked [3H]estradiol binding to nuclear type II sites, but did not compete for [3H]estradiol binding to recombinant ERalpha or ERbeta. ZN-1 and ZN-2 inhibited the proliferation of ERalpha and ERbeta positive (MCF-7) and negative (MCF-10A) breast cells, further ruling out direct binding to ER in the mechanism of action of these compounds. Pre-loading type II sites with ZN-1 or ZN-2 reduced [3H]estradiol exchange, strongly suggesting the drugs were binding covalently. ZN-1 treatment resulted in complete occupancy of type II sites and sustained (9 days) inhibition of MCF-7 cell proliferation following its removal from the tissue culture medium. This cell growth inhibition was not due to non-specific toxicity, as the numbers of viable, attached cells per dish (determined by trypan blue dye exclusion) remained constant throughout this 9-day period and eventually reversed by day 19. ZN-2 effects on cell proliferation reversed more rapidly following discontinuation of treatment, a response consistent with the inability of the compound to totally block type II binding. Both ZN-1 and ZN-2 blocked estradiol stimulation of c-Myc and cyclin D1 gene expression in MCF-7 cells, two events that are clearly coupled to cell cycle progression. We suspect this may occur through ZN-1 or ZN-2 modification of nucleosome function and/or chromatin remodeling since nuclear type II sites are localized to a complex of histones H3 and H4 (Shoulars et. al, J Steroid Biochem. Mol. Biol. 96: 19-30, 2005).

Reconstitution of the type II [3H]estradiol binding site with recombinant histone H4.

Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd approximately 20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient approximately 4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.

Leukotoxin diols from ground corncob bedding disrupt estrous cyclicity in rats and stimulate MCF-7 breast cancer cell proliferation.

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (iso-leukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [3H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (>> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents.

Nuclear type II [3H]estradiol binding sites: a histone H3-H4 complex.

[(3)H]luteolin covalently labels two forms (11kDa and 35kDa proteins) of type II binding sites in rat uterine nuclear extracts [K. Shoulars, T. Brown, M. Alejandro, J. Crowley, B. Markaverich, Identification of rat uterine nuclear type II [(3)H]estradiol binding sites as histone H4, Biochem. Biophys. Res. Commun. 296 (2002) 1083-1090]. The 11kDa protein was identified as histone H4. Levels of the 35kDa protein were insufficient for sequencing; however, this protein was recognized by anti-histone H4 antibodies. Histones H3 and H4 exist as dimers in vivo (mw>>35kDa) and we suspected the 35kDa [(3)H]luteolin-labeled protein in uterine nuclear extracts might be a complex of histones H3 and H4. This manuscript describes methods for the purification of commercially available calf thymus core histones that retain [(3)H]luteolin binding activity and are of sufficient purity for recombination studies. Mixing experiments with pure H3 and H4 from calf thymus demonstrate that a 35kDa H3-H4 dimer capable of binding [(3)H]luteolin is generated and this protein appears equivalent to the 35kDa [(3)H]luteolin binding protein in rat uterine nuclear extracts. If this is the case, type II site ligands including MeHPLA, luteolin, and other bioflavonoids and phytoestrogens may control histone-dependent gene transcription and cellular proliferation via binding to and modulating core histone/nucleosome function.

Identification of nuclear type II [(3)H]estradiol binding sites as histone H4.

[3H]Luteolin binds covalently to uterine nuclear type II sites [B. Markaverich, K. Shoulars, M.A. Alejandro, T. Brown, Steroids 66 (2001) 707] and was used to identify this protein(s). SDS-PAGE analyses of [3H]luteolin-labeled type II site preparations revealed specific binding to 11- and 35-kDa proteins. The 11-kDa protein was identified as histone H4 by amino acid sequencing. Western blotting confirmed that the 11- and 35-kDa proteins were acetylated forms of histone H4. Anti-histone H4 antibodies (but not H2A, H2B, or H3 antibodies) quantitatively immunoadsorbed type II binding sites from nuclear extracts. Binding analyses by [3H]estradiol exchange, using luteolin as a competitor, detected specific type II binding activity to histone H4 (but not histones H2A, H2B, or H3) generated in a rabbit reticulocyte lysate translation system and confirmed that histone H4 is the type II site.