Low Aldehyde Dehydrogenase Activity Research Articles

Proteomic profiling reveals an association between ALDH and oxidative phosphorylation and DNA damage repair pathways in human colon adenocarcinoma stem cells

Several members of the aldehyde dehydrogenase (ALDH) family, especially ALDH1 isoenzymes, have been identified as biomarkers of cancer stem cells (CSCs), a small subpopulation of oncogenic cells with self-renewal and multipotency capability. Consistent with this contention, cell populations with high ALDH enzymatic activity exhibit greater carcinogenic potential. It has been reported that ALDH1, especially ALDH1A1, serves as a valuable biomarker for colon CSCs. However, the functional roles of ALDHs in CSCs and solid tumors of the colon tissue is not fully understood. The aim of the present study was to identify molecular signature associated with high ALDH activity in human colorectal adenocarcinoma (COLO320DM) cells by proteomics profiling. Aldefluor™ assay was performed to sort COLO320DM cells exhibiting high (ALDHhigh) and low (ALDHlow) ALDH activity. Label-free quantitative proteomics analyses were conducted on these two cell populations. Proteomics profiling revealed a total of 229 differentially expressed proteins (DEPs) in ALDHhigh relative to ALDHlow cells, of which 182 were down-regulated and 47 were up-regulated. In agreement with previous studies, ALDH1A1 appeared to be the principal ALDH isozyme contributing to the Aldefluor™ assay activity in COLO320DM cells. Ingenuity pathway analysis of the proteomic datasets indicated that DEPs were associated with mitochondrial dysfunction, sirtuin signaling, oxidative phosphorylation and nucleotide excision repair. Our proteomics study predicts that high ALDH1A1 activity may be involved in these cellular pathways to promote a metabolic switch and cellular survival of CSCs.

Drug-Tolerant Persister Cells in Cancer Therapy Resistance.

One of the current stumbling blocks in our fight against cancer is the development of acquired resistance to therapy, which is attributable to approximately 90% of cancer-related deaths. Undercutting this process during treatment could significantly improve cancer management. In many cases, drug resistance is mediated by a drug-tolerant persister (DTP) cell subpopulation present in tumors, often referred to as persister cells. This review provides a summary of currently known persister cell subpopulations and approaches to target them. A specific DTP cell subpopulation with elevated levels of aldehyde dehydrogenase (ALDH) activity has stem cell-like characteristics and a high level of plasticity, enabling them to switch rapidly between high and low ALDH activity. Further studies are required to fully elucidate the functions of ALDH-high DTP cells, how they withstand drug concentrations that kill other cells, and how they rapidly adapt under levels of high cellular stress and eventually lead to more aggressive, recurrent, and drug-resistant cancer. Furthermore, this review addresses the processes used by the ALDH-high persister cell subpopulation to enable cancer progression, the ALDH isoforms important in these processes, interactions of ALDH-high DTPs with the tumor microenvironment, and approaches to therapeutically modulate this subpopulation in order to more effectively manage cancer.

Purification and Functional Characterization of CD34-Expressing Cell Subsets Following Ex Vivo Expansion of Umbilical Cord Blood-Derived Endothelial Colony-Forming Cells.

Fluorescent-activated cell sorting (FACS) remains a powerful tool to enrich blood-derived progenitor cells for the establishment of highly proliferative endothelial colony-forming cells (ECFC). Further investigation remains necessary to determine whether the retention of progenitor cell phenotypes after expansion can identify ECFC with enhanced proangiogenic and regenerative functions. This study employed FACS purification to segregate umbilical cord blood-derived ECFC using conserved provascular progenitor cell markers CD34 or aldehyde dehydrogenase (ALDH) activity. ECFC FACS purified for high versus low ALDH activity formed single cell-derived colonies and demonstrated tubule formation in Matrigel at comparable rates. Surprisingly, FACS purification of ECFC for CD34 enriched cells with enhanced colony-forming capabilities and tubule formation within the CD34- population. CD34 expression was enriched on early ECFC populations; however, steady-state expression of CD34 rapidly declined and stabilized on expanded ECFC after serial passage. CD34 expression on ECFC was shown to be cell density dependent and coincided with a loss of progenitor cell characteristics in vitro. Silica-bead surface membrane capture followed by proteomic analysis by label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) identified >100 distinctions (P < 0.05) associated with the plasma membrane of CD34- versus CD34+ ECFC, including a significant enrichment of CD143 (angiotensinogen converting enzyme) on CD34+ cells. Despite an enrichment for traditional endothelial cell markers on the CD34+ ECFC in vitro, implantation of both CD34+ and CD34- ECFC within Matrigel plugs in immunodeficient NOD.SCID mice promoted the formation of vessel-like structures with equivalent integration of human cells at 7 days post-transplantation. Although positive selection of CD34 enriched ECFC establishment before culture, FACS-purified CD34+ ECFC demonstrated reduced colony and tubule formation in vitro, yet demonstrated equivalent vessel formative function in vivo compared to CD34- counterparts. The knowledge will support future studies aiming to identify ECFC subsets with enhanced vessel forming functions for applications of regenerative medicine.

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

SummaryUterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

Age-associated antigen-presenting cell alterations promote dry-eye inducing Th1 cells.

Aging is a significant risk factor for dry eye. Here we used a murine aged mode to investigate the effects of aging on antigen presenting cells (APCs) and generation of pathogenic T helper (Th)1. Our results showed that APCs from aged mice accumulate at the conjunctiva, have higher levels of co-activation marker CD86 and lower aldehyde dehydrogenase activity. Using topical ovalbumin peptide as a surrogate antigen, we observed an increased number of antigen-loaded APCs in the draining cervical lymph nodes in the aged group and loss of tight junction protein occludin in the conjunctiva. Aged cervical lymph nodes APCs showed a greater generation of Th1 cells than young APCs in antigen-presentation assays in vitro. Aged lacrimal glands and draining nodes showed an accumulation of IFN-γ producing CD4+T cells, while Th17 cells were present only in aged draining nodes. There was also an age-related increase in CD4+CXCR3+IFN-γ+ cells in the conjunctiva, nodes and lacrimal glands while CD4+CCR6+IL-17A+ cells increased in the draining nodes of aged mice. Adoptive transfer of aged CD4+CXCR3+ donor cells into young, naive immunodeficient recipients caused greater goblet cell loss than young CD4+CXCR3+ cells. Our results demonstrate that age-associated changes in APCs are critical for the pathogenesis of age-related dry eye.

BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDHhi), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDHhi cells available for transplantation compared with untreated controls. Newly expanded ALDHhi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDHhi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of β-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating β-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDHhi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDHhi cells without the loss of islet regenerative functions.

Abstract 3880: Assessment of ALDH activity in neuroblastoma patient-derived xenograft cells as a potential marker of neuroblastoma stem cells

Abstract Neuroblastoma is a tumor derived from immature neuroblasts predestined to form the sympathetic nervous system. Children with aggressive neuroblastoma are at high-risk of disease relapse despite their initial response to treatment. The molecular mechanisms underlying these relapses are poorly understood, although clonal expansion and de novo mutations have been suggested as potential mechanisms. An alternative hypothesis is that neuroblastoma initiating capacity and resistance to chemotherapy reside in a subpopulation of tumor initiating cells or cancer stem cells, which are not genetically different from most tumor bulk cells but through unknown mechanisms differ phenotypically and functionally. Here we examine aldehyde dehydrogenase (ALDH) expression and activity in neuroblastoma patient-derived xenograft (PDX) cells, cultured under stem cell-promoting conditions, as a potential marker for neuroblastoma stem cells. We found that neuroblastoma PDX cells in general expressed higher levels of various ALDH isoforms as compared to an aggressive classical serum-grown neuroblastoma cell line. Also, a distinct subgroup with high ALDH activity was observed in PDX cells but not in the serum-grown cell line. Growing the PDX cells in serum induced neuronal differentiation accompanied with lower expression of ALDH1 isoforms and reduced ALDH activity. The change in ALDH expression and activity was reversible, implying phenotypic plasticity. Re-analyzing cells initially sorted on ALDH activity also confirmed an inter-conversion of cells with low and high ALDH activity. Furthermore, in vitro clonogenic capacity was significantly higher in cells with high ALDH activity. Serial in vivo passaging of PDXs allowed us to compare PDX cells isolated from different in vivo generations. Interestingly, cells derived from later in vivo generations contained a higher frequency of cells with high ALDH activity. Finally, orthotopic injection of cells with either low or high ALDH activity showed that both subgroups led to tumor formation, however a higher tumor take was observed when injecting cells with high ALDH activity. In conclusion, our results suggest that ALDH activity is associated with an immature phenotype, which is of clinical importance since less differentiated tumors are more aggressive and associated with poor prognosis. Also, cells with high ALDH activity showed higher in vivo tumor initiating capacity. We are currently pursuing a more extensive characterization of neuroblastoma cells with high ALDH activity. Citation Format: Camilla Persson, Kristoffer von Stedingk, Daniel Bexell, My Merselius, Noémie Braekeveldt, Sven Påhlman, Caroline Wigerup. Assessment of ALDH activity in neuroblastoma patient-derived xenograft cells as a potential marker of neuroblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3880. doi:10.1158/1538-7445.AM2017-3880

Microvesicles derived from Alde-Low EPCs support the wound healing capacity of AT-MSCs

Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation.In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing.We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45+ inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs.These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites.

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

Background:Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs.Methods:Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment.Results:None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not.Conclusions:PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

BMP-2 inhibits tumor-initiating ability in human renal cancer stem cells and induces bone formation.

We have previously shown that BMP-2 induces bone formation and inhibits tumorigenicity of cancer stem cells (CSCs) in a human osteosarcoma OS99-1 cell line. In this study, we sought to determine whether BMP-2 can similarly induce bone formation and inhibit the tumorigenicity of renal CSCs identified based on aldehyde dehydrogenase (ALDH) activity in renal cell carcinoma (RCC) cell lines and primary tumors. Using a xenograft model in which cells from human RCC cell lines ACHN, Caki-2, and primary tumors were grown in NOD/SCID mice, renal CSCs were identified as a subset of ALDH(br) cells. The ALDH(br) cells possessed a greater colony-forming efficiency, higher proliferative output, increased expression of stem cell marker genes Oct3/4A, Nanog, renal embryonic marker Pax-2, and greater tumorigenicity compared to cells with low ALDH activity (ALDH(lo) cells), generating new tumors with as few as 25 cells in mice. In vitro, BMP-2 was found to inhibit the ALDH(br) cell growth, down-regulate the expression of embryonic stem cell markers, and up-regulate the transcription of osteogenic markers. In vivo, all animals receiving a low number of ALDH(br) cells (5 × 10(3)) from ACHN, Caki-2, and primary tumor xenografts treated with 30 µg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation. These results suggest that BMP-2 inhibits the tumor-initiating ability of renal CSCs and induces osseous bone formation. BMP-2 may therefore provide a beneficial strategy for human RCC treatment by targeting the CSC-enriched population.

Abstract 3982: Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia

Abstract Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children while significantly less in older adults. For patients with relapsed AML refractory to chemotherapy, there is little chance for survival. Therefore, the need for novel treatment modalities continues to be critically important. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor that has been used for decades for the treatment of alcoholism without major side effects. While DS has been reported to have anticancer cytotoxicity that is in part copper (Cu2+)-dependent, the mechanisms are still not fully elucidated. The goal of the present study was to investigate whether DS has significant cytotoxicity against AML. Methods and Results In the present study the IC50 were determined for DS alone or in combination with 1 µM Cu2+ in a panel of 16 AML cell lines using Cell Titer Glo assay. DS/Cu2+ resulted in significant killing of all cell lines (IC50 between 20-75 nM) while the response to DS alone showed significant variation (IC50 from 20 nM - 7 µM). Cu2+ alone was not toxic. Cytarabine (Ara-C), a key drug in AML treatment, was compared with DS/Cu2+. The AML cell lines THP1 (acute monocytic leukemia), UT7-epo and CMY (acute megakaryoblastic leukemia) were profoundly resistant to Ara-C (IC50 between 1.2-2.4 µM) but showed extreme sensitivity to DS/Cu2+ (IC50 between 31-46 nM). Although DS is an inhibitor of ALDH, we demonstrated that the cytotoxicity of DS or DS/Cu2+ was not directly associated with the level of ALDH or its inhibition. AML cell lines were treated with different doses of DS or DS/Cu2+ and ALDH activity and cell death were determined by flow cytometry. The percentage of cell death was dose-dependent and occurred irrespective of ALDH activity in cell lines with high endogenous ALDH activity (MV-4-11, CMY, CMK and K562) compared to those with low or undetectable ALDH activity (Kasumi-1, Molm-13, NB4 and KG-1a). As DS-mediated cytotoxicity has been associated in previous reports with proteasome inhibition, the effects of DS and DS/Cu2+ were compared to Bortezumib. Western blot analysis showed that DS induced PARP cleavage similar to Bortezumib, suggesting that proteasome inhibition may be contributing to DS or DS/Cu2+ cytotoxicity. Conclusion These data demonstrate that multiple AML cell lines are sensitive to DS and to DS/Cu2+, including some that show extreme resistance to Ara-C, such as CMY from a patient with Down Syndrome. This DS sensitivity may provide a less toxic and efficacious treatment approach to be tested in this group of patients with poor outcome following relapse. The cytotoxic activity of DS does not appear to be solely dependent on ALDH inhibition. Ongoing studies are focusing on determining the mechanisms of cell death, and cytotoxic efficacy in in vitro and xenograft models using patient AML samples, with the goal of developing a clinical trial in patients with relapsed/refractory AML. Citation Format: Eiman A. Aleem, David O. Azorsa, Ranjan Bista, Oliver B. Pepper, David W. Lee, Daniel H. Wai, Robert J. Arceci. Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3982. doi:10.1158/1538-7445.AM2014-3982

Functional endothelial progenitor cells selectively recruit neurovascular protective monocyte-derived F4/80+/Ly6c+ macrophages in a mouse model of retinal degeneration

Retinitis pigmentosa is a group of inherited eye disorders that result in profound vision loss with characteristic retinal neuronal degeneration and vasculature attenuation. In a mouse model of retinitis pigmentosa, endothelial progenitor cells (EPC) from bone marrow rescued the vasculature and photoreceptors. However, the mechanisms and cell types underlying these protective effects were uncertain. We divided EPC, which contribute to angiogenesis, into two subpopulations based on their aldehyde dehydrogenase (ALDH) activity and observed that EPC with low ALDH activity (Alde-Low) had greater neuroprotection and vasoprotection capabilities after injection into the eyes of an rd1 mouse model of retinitis pigmentosa compared with EPC with high ALDH activity (Alde-High). Of note, Alde-Low EPC selectively recruited F4/80(+) /Ly6c(+) monocyte-derived macrophages from bone marrow into retina through CCL2 secretion. In addition, the mRNA levels of CCR2, the neurotrophic factors TGF-β1 and IGF-1, and the anti-inflammatory mediator interleukin-10 were higher in migrated F4/80(+) /Ly6c(+) monocyte-derived macrophages as compared with F4/80(+) /Ly6c(-) resident retinal microglial cells. These results suggest a novel therapeutic approach using EPC to recruit neuroprotective macrophages that delay the progression of neural degenerative disease.

Identification of a subpopulation of nasopharyngeal carcinoma cells with cancer stem‐like cell properties by high aldehyde dehydrogenase activity

Cancer stem cells have been reported as a new therapeutic target in many cancers, but their existence in nasopharyngeal carcinoma (NPC) is largely unknown. This study was conducted to determine cancer stem-like cells in NPC cell line. Basic science experimental study. Aldehyde dehydrogenase (ALDH) activity, a putative functional marker for cancer stem cells, was assessed in Epstein-Barr virus-associated NPC cell line C666-1 cells. The ability of cells with high and low ALDH activity to proliferate, resist therapy, and initiate tumor formation was compared. Enrichment of cancer stem-like cells (with high ALDH activity) in C666-1 was associated with a significantly greater ability to proliferate, be clonogenic, resist chemotherapy drugs and radiation, reconstitute a heterogeneous population, and express pluripotent markers. Furthermore, subcutaneous injection of these cells into immunodeficient nude mice resulted in a tendency of tumor formation at a higher rate as compared to cells with low ALDH activity. These results provide evidence for the existence of cancer stem-like cells in the NPC cell line C666-1 cells.

Aldehyde dehydrogenasehigh gastric cancer stem cells are resistant to chemotherapy

Cancer stem cells (CSCs) are known to influence chemoresistance, survival, relapse and metastasis. Aldehyde dehydrogenase (ALDH) functions as an epithelial CSC marker. In the present study, we investigated the involvement of ALDH in gastric CSC maintenance, chemoresistance and survival. Following screening for eight candidate markers (CD13, CD26, CD44, CD90, CD117, CD133, EpCAM and ALDH), five gastric cancer cell lines were found to contain small subpopulations of high ALDH activity (ALDH(high) cells). We also examined the involvement of ALDH(high) cell populations in human primary tumor samples. Immunodeficient NOD/SCID mice were inoculated with tumor tissues obtained from surgical specimens. ALDH(high) cells were found to persist in the xenotransplanted primary tumor samples. in the immunodeficient mice, ALDH(high) cells exhibited a greater sphere‑forming ability in vitro and tumorigenic potential in vivo, compared with subpopulations of low ALDH activity (ALDH(low) cells). Cell cultures treated with 5-fluoro-uracil and cisplatin exhibited higher numbers of ALDH(high) cells. Notch1 and Sonic hedgehog (Shh) expression was also found to increase in ALDH(high) cells compared with ALDH(low) cells. Therefore, it can be concluded that ALDH generates chemoresistance in gastric cancer cells through Notch1 and Shh signaling, suggesting novel treatment targets.

Can lower aldehyde dehydrogenase activity in saliva be a risk factor for oral cavity cancer?

Salivary ALDH3A1 protects the oral cavity from aromatic and medium-chain aliphatic aldehydes originating from food and air pollution and generated during oxidative stress. Due to their reactivity, aldehydes may exhibit an irritating effect as well as cytotoxic, genotoxic, mutagenic and even carcinogenic effects. The aim of this study was to verify whether lower ALDH3A1 activity is a risk factor for oral cavity cancer. Fasting saliva samples were collected one day before and about one week after surgery from patients with oral cancer (OCC) (n=59), other tumours (cysts, neoplasms) (n=108), gnathic defects and fractures (controls after the surgery) (n=63), and from healthy volunteers (n=116). Enzyme activity was measured using a fluorometric method. Total ALDH3A1 activity [Ug(-1) ] in patients with OCC was statistically lower than in patients with keratocystic odontogenic tumour (KCOT) (P=0.00697), odontogenic cysts (OC) (P<0.00001), neoplasms (P=0.03343) and the healthy volunteers up to and over 40years old (P<0.00001; P=0.00019). The activity in the saliva of OCC after surgery was lower than in the healthy volunteers (P<0.00001) and in the groups with fractures (P=0.00303) and gnathic defects (P=0.00538). Low salivary ALDH activity may be a risk factor for oral cancer development.

Canonical NF-κB Signalling Is a Potential Target in FLT3/ITD AML.

AbstractAbstract 2447Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor is common in acute myeloid leukemia (AML) and is associated with a dismal outcome. Despite initial response, FLT3/ITD AMLs often relapse early, suggesting a residual population of resistant leukemia stem cells (LSC). Clinically, FLT3 inhibitors asmonotherapy have yet to improve outcome significantly and therefore, targeting additional pro-survival pathways may be necessary for this group of AML patients. Mice genetically egineered to express a hemizygous FLT3/ITD mutation develop a progressive, fatal, myeloproliferative neoplasm. Lin−cells isolated from the bone marrow of FLT3/ITD or control mice were subjected to gel shift analysis using a radio-labeled NF-kB binding site. This analysis demonstrated high levels of nuclear activation of NF-kB in the FLT3/ITD-expressing cells, suggesting its activation downstream of mutant FLT3 signaling. MV4–11 is a human AML-derived cell line harboring a homozygous FLT3/ITD mutation. Cells expressing high levels of aldehyde dehydrogenase (ALDH) have been shown to be enriched for LSC in primary AML samples and cell lines. High ALDH expressing MV4–11 cells were isolated using FACS and analyzed for NF-kB activation. Western blot analysis demonstrated preferential phosphorylation of NF-kB p65 by activated IKK on Ser536 in this subpopulation, compared with cells with low ALDH activity. These findings indicate activation of NF-kB in MV4–11 LSCs.We wanted to next test the requirement for NF-kB signaling in transformation by FLT3/ITD mutations. NF-kB p65 null mice die in utero. We therefore established C57BL/6 p65(flox/flox);Mx1-Cre mice. Intra-peritoneal injection of pIpC every other day for 7 doses efficiently deletes the RelA/p65 gene, resulting in expression of <1% of the corresponding RNA or protein. Despite effective excision of p65, the mice survive. Bone marrow cells harvested from control or p65(del/del) mice were transduced with a FLT3/ITD-expressing lentivirus and seeded in methylcellulose without cytokines. Equal transduction rate was verified by measurement of GFP expression by flow cytometry. Reproducibly, p65(del/del) marrow transduced with FLT3/ITD was ineffective in forming cytokine independent colonies, in contrast to wild-type marrow (5 +/− 0.6 vs. 55 +/− 6 colonies per 1E5 cells, P<0.001), and the few p65(del/del) colonies that resulted were smaller than those from p65 expressing wild-type marrow cells. Cells transduced with a lentiviral vector expressing GFP but not FLT3/ITD did not form colonies without cytokines, and p65(del/del) marrow formed normal numbers of colonies of normal size and distribution in the presence of IL-3, IL-6, and SCF.Sorafenib inhibits FLT3 signaling and kills MV4–11 cells with an IC50 of approximately 10 nM. Reproducibly, a sub-toxic dose of sorafenib (5 nM) combined with sub-toxic levels of the IKKb inhibitor IMD-0354 (400 nM) resulted in synergistic cell killing as indicated by the calculated combination index of 0.55.Currently, clinical efforts in FLT3/ITD leukemia concentrate on FLT3 inhibition alone. Our data suggest that canonical NF-kB may be an important pathway in FLT3/ITD AML and that simultaneously targeting FLT3 and NF-kB in this disease may be an effective approach. Disclosures:No relevant conflicts of interest to declare.

Leucine accelerates blood ethanol oxidation by enhancing the activity of ethanol metabolic enzymes in the livers of SHRSP rats

Chronic ethanol consumption induces liver diseases, such as alcoholic hepatitis and cirrhosis. The enhancement of alcohol oxidation is important in the prevention of these liver diseases. Chronic supplementation with branched chain amino acids (BCAAs) prevents liver cirrhosis. Therefore, BCAAs may be associated with enhanced ethanol oxidation. To evaluate this hypothesis, we investigated the effect of the administration of individual BCAAs on ethanol oxidation and changes in alcohol-metabolizing enzyme activities following acute alcohol intake in rats. Blood ethanol concentrations and the activities of alcohol-metabolizing enzymes, such as alcohol dehydrogenase (ADH) and low and high Km aldehyde dehydrogenase (ALDH), were measured in the liver following acute ethanol administration in rats; the ethanol was administered 30 min after the treatment with amino acids [such as leucine (Leu), isoleucine (Ile), valine (Val) or alanine (Ala)]. Leu significantly decreased the blood ethanol concentration 1 h after ethanol administration compared to the water-treated control (C) [C 0.46 ± 0.09, Leu 0.18 ± 0.04, Ile 0.27 ± 0.09, Val 0.46 ± 0.1, Ala 0.43 ± 0.06, mean ± SEM (g/l), P < 0.05]. In addition, leucine significantly stimulated ADH activity 30 min after ethanol intake [C 0.042 ± 0.014, Leu 0.090 ± 0.016, Ile 0.042 ± 0.008, Val 0.022 ± 0.010, Ala 0.070 ± 0.016, mean ± SEM (unit/mg protein), P < 0.05] and low Km ALDH activity 15 min after ethanol intake [C 0.51 ± 0.63, Leu 3.72 ± 0.66, Ile 1.26 ± 0.89, Val: ND, Ala 1.86 ± 1.57, mean ± SEM (unit/mg protein), P < 0.05]. However, leucine and its metabolite α-keto-isocaproic acid did not enhance ethanol clearance in isolated rat hepatocytes. These results indicate that leucine accelerates ethanol oxidation by indirectly enhancing ADH and low Km ALDH activities in the liver.

Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration

We sought to investigate the stimulation of islet regeneration by transplanted human umbilical cord blood (UCB) cells purified according to high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), a conserved characteristic of multiple progenitor lineages. We hypothesised that direct intrapancreatic (iPan) delivery of ALDH(hi) progenitors would augment islet regeneration via timely and localised exposure to islet-regenerative stimuli. Cells were purified from UCB based on flow cytometry for low ALDH activity (ALDH(lo)) vs ALDH(hi). UCB ALDH(lo) or ALDH(hi) cells were compared for surface marker expression, as well as haematopoietic, endothelial and multipotent stromal progenitor content in vitro. UCB ALDH(lo) or ALDH(hi) cells were i.v. or iPan injected into streptozotocin-treated non-obese diabetic/severe combined immune-deficient mice temporally monitored for blood glucose, serum insulin and glucose tolerance. Human cell recruitment and survival in the pancreas, insulin content, islet-associated cell proliferation and islet vascularisation were documented in situ. UCB-derived ALDH(hi) cells were highly enriched for haematopoietic and endothelial progenitor frequency, and showed increased expression of progenitor and myeloid cell surface markers. Although i.v. transplantation of ALDH(hi) cells demonstrated low pancreas engraftment and only transient blood glucose lowering capacity, iPan injected ALDH(hi) cells reversed established hyperglycaemia, increased serum insulin and improved the response to a glucose challenge. iPan injected ALDH(hi) cells surrounded damaged islets at early time points and increased islet-associated cell proliferation, resulting in the recovery of beta cell mass. iPan delivery of UCB ALDH(hi) cells potentiated islet-associated cell proliferation, insulin production and islet revascularisation, resulting in the recovery of host islet function. Elucidation of the progenitor-specific pathways stimulated during islet regeneration may provide new approaches to promote islet expansion during diabetes.

A subpopulation of endothelial progenitor cells with low aldehyde dehydrogenase activity attenuates acute ischemic brain injury in rats

Previous studies have examined the therapeutic effect of endothelial progenitor cells (EPCs) during the chronic phase of cerebral infarction in rats; however, few studies have investigated the effects of EPCs during the acute phase of infarction. In this study, we evaluated the therapeutic effect of EPCs with low aldehyde dehydrogenase activity (Alde-Low EPCs) in rats with acute cerebral infarction, and our results provide insight that may help to identify a therapeutic mechanism of EPCs for acute cerebral infarction. The administration of Alde-Low EPCs into rats with acute cerebral infarction results in the accumulation and migration of the Alde-Low EPCs into the infarct area and the subsequent decrease of infarct volume. Moreover, we found that the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) signaling pathway may regulate the accumulation of Alde-Low EPCs. The transplantation of Alde-Low EPCs may represent a potential treatment strategy for acute cerebral infarction.

MicroRNA Expression Profiling of Hematopoietic and Leukemic Stem Cells in Acute Myeloid Leukemia,

Abstract 3999Treatment of acute myeloid leukemia (AML) patients with chemotherapy results in complete remission in up to 80% of patients. However, 50% of patients experience a relapse which is thought to be caused by the survival of a population of dormant leukemic stem cells (LSC) that are resistant to chemotherapy. These LSC are responsible for the initiation, maintenance and relapse of AML and eradication of these cells is therefore necessary to cure AML patients. LSC and normal hematopoietic stem cells (HSC) coexist in AML bone marrow and future LSC-specific therapy relies on identification of new targets differentiating normal from malignant stem cells. Our major aim is the development of novel LSC specific microRNA-based therapeutic strategies by identification of microRNAs differentially expressed and functioning in the AML LSC and HSC critical for self-renewal and/or therapy response. We want to use microRNA expression profiling of LSC and HSC within one AML bone marrow sample to identify LSC-specific targets.MicroRNAs are non-coding single stranded RNAs of 21–25 nucleotides. They regulate gene expression by promoting degradation of mRNA or repressing its translation. MicroRNAs are critical regulators involved in various cell processes, including differentiation, proliferation and apoptosis. Whereas a few studies have been performed comparing microRNA expression of LSC with healthy donor HSC, which is not identical to AML HSC, no such studies have been performed comparing LSC with HSC both present in the same AML bone marrow. This is now possible with the by us identified markers/parameters discriminating LSC from HSC present within one AML bone marrow sample.Recently we have shown that the small portion of healthy HSC in the CD34+CD38- compartment of AML patients can be identified and purified by using aldehyde dehydrogenase activity. CD34+CD38- stem cells with high aldehyde dehydrogenase activity have no molecular and immunophenotypical aberrancies suggesting a normal phenotype (HSC). Whereas CD34+CD38- stem cells with low aldehyde dehydrogenase activity, harbor leukemia associated aberrancies. This shows that normal HSC have high aldehyde dehydrogenase activity as compared to LSC residing in the same AML bone marrow. We have studied the microRNA expression profile of AML LSC and normal HSC, discriminated by aldehyde dehydrogenase activity, within the patient's bone marrow in order to find microRNAs differentially expressed in LSC and HSC. Besides this, we have studied the microRNA expression profile in the more differentiated blast population of the AML bone marrow.Cells from the bone marrow of AML patients were purified by flow cytometry based on surface marker expression and aldehyde dehydrogenase activity. Leukemic blasts by being CD34+, LSC by being CD34+CD38-ALDHlow and HSC by being CD34+CD38-ALDHhigh. RNA was isolated from the various cell populations and microRNA expression was studied using Agilent microRNA microarrays. Most microRNAs show similar levels of expression in the different cell compartments. However, expression of miR-126 was significantly enhanced while the tumorsupressors miR-451 and miR-34a were downregulated in LSC as compared to leukemic blasts. Comparison of the microRNA expression profile of LSC and HSC showed that expression of miR-21, miR-181a/b and the cluster miR-221/222 was significantly upregulated while others, such as miR-10a and miR-451 were downregulated.In conclusion, we have identified several microRNAs that are differentially expressed between leukemic blasts, LSC and HSC. Some of these microRNAs have already been linked to stem cell properties such as senescence or chemotherapy resistance while others have unknown functions. Our findings might give more insights in the mechanisms regulating LSC and are important for understanding processes like leukemogenesis and relapse. Furthermore, these microRNAs might in de future prove to be valuable targets for LSC directed therapy while saving the normal HSC. Disclosures:No relevant conflicts of interest to declare.