Carol Swiderski

Preferential induction of apoptosis for primary human leukemic stem cells

Acute myelogenous leukemia (AML) is typically a disease of stem/progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G0 cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominant-negative allele of inhibitor of nuclear factor κB (IκBα) demonstrate that inhibition of nuclear factor κB (NF-κB) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis. Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.

Identification of OCT6 as a novel organic cation transporter preferentially expressed in hematopoietic cells and leukemias.

OBJECTIVE Human organic cation transporters (OCTs) play a critical role in the cellular uptake and efflux of endogenous cationic substrates and hydrophilic exogenous xenobiotics. We sought to identify OCT genes preferentially expressed in hematopoietic cells. MATERIALS AND METHODS We isolated a novel OCT, named OCT6, by data-mining human expressed sequence tag databases for sequences homologous to known OCT genes. We developed a quantitative reverse transcriptase polymerase chain reaction assay to determine the relative expression of this gene in 50 cancer cell lines and in tissues. RESULTS The two highest expressing cell lines were the leukemia cell lines HL-60 and MOLT4. Quantitative reverse transcriptase polymerase chain reaction analysis using a normal tissue cDNA panel demonstrated that this transport gene is highly expressed in testis and fetal liver, with detectable RNA levels in bone marrow and peripheral blood leukocytes. Unlike other OCT genes, RNA levels were not detectable in placenta, liver, or kidney. To further define the expression of OCT6 in hematopoietic tissues, we measured OCT6 RNA levels in sorted peripheral blood cell populations and found a clear enrichment of OCT6-expressing cells in purified CD34(+) cells. To determine if OCT6 was highly expressed in leukemias, we examined circulating leukemia cells from 25 patients and found high levels of OCT6 RNA in all specimens in comparison with liver, kidney, and placenta. CONCLUSIONS The results demonstrate the existence of a novel OCT preferentially expressed in human hematopoietic tissues, including CD34(+) cells and leukemia cells. Its narrow tissue distribution, potential for substrate specificity, and close homology to other cell membrane transporters make OCT6 an attractive target for the treatment of leukemia.

Novel mutations in the polyadenine tract of the transforming growth factor β type II receptor gene are found in a subpopulation of human pancreatic adenocarcinomas

In this study, we determined the incidence of microsatellite instability (MIN) in pancreatic adenocarcinoma and determined whether MIN might target, for mutations, the simple nucleotide repeats of the transforming growth factor β type II receptor (TGFBR2) gene. Forty‐eight surgically resected pancreatic tumor tissue samples and two normal pancreas tissue samples were analyzed in this study. Microsatellite analysis was performed for six loci in 14 of the 48 tumor specimens for which we had matching normal genomic DNA. Only four of the 14 tumors (29%) were MIN‐positive as determined by the presence of microsatellite variations in more than one locus. Interestingly, eight of the 14 specimens (57%) showed microsatellite variations or loss of heterozygosity at D18S34, suggesting that this locus may be a critical region of genetic instability in pancreatic tumorigenesis. Of the 48 tumors, only two (4%) showed mutations in the polyA region, one of the MIN‐targeted sites of the TGFBR2 gene. DNA sequence analysis of these two specimens showed the presence of a two‐base deletion in one tumor specimen and the other tumor specimen showed a base substitution in the polyA tract at codon 128 of the TGFBR2 gene. The fact that these mutations occurred in the polyA tract of some pancreatic tumors suggests that a subpopulation of these tumors may be susceptible to MIN‐targeted mutations. The incidence of these mutations are low and similar to that reported for nonhereditary, sporadic colon cancers. Genes Chromosomes Cancer 22:138–144, 1998.

Congenic interval of CD45/Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment.

The B6.SJL-Ptprc(d)Pep3(b)/BoyJ (B6.SJL) congenic mouse strain, a valuable and widely used tool in murine bone marrow transplantation studies, has long been considered equivalent to the parental C57B/L6 (B6) strain with the exception of a small congenic interval on chromosome 1 harboring an alternative CD45/Ly-5 alloantigen (Ly-5.1). In this study we compared functional properties of stem and stromal cells between the strains, and delineated the boundary of the B6.SJL congenic interval. We identified a 25% reduction in homing efficiency, 3.8-fold reduction in transplantable long-term hematopoietic stem cells (LT-HSCs), a 5-fold reduction in LT-HSCs capable of 24-hour homing, and a cell-intrinsic engraftment defect of 30% to 50% in B6.SJL-derived bone marrow cells relative to B6-derived cells. These functional differences were independent of stem cell number, cycling, or apoptosis. Genotypic analysis revealed a 42.1-mbp congenic interval in B6.SJL including 306 genes, and at least 124 genetic polymorphisms. Moreover, expression profiling revealed 288 genes differentially expressed between nonhematopoietic stromal cells of the 2 strains. These results indicate that polymorphisms between the B6 and SJL genotype within the B6.SJL congenic interval influence HSC engraftment and result in transcriptional variation within bone marrow stroma.

Characterization of a newly established human pancreatic carcinoma cell line, UK Pan-1.

A highly tumorigenic cell line designated as UK Pan‐1 was established in a surgically removed human pancreatic adenocarcinoma and characterized as having many of the genotypic and phenotypic alterations commonly found in pancreatic tumors.

Latexin Is Down-Regulated in Hematopoietic Malignancies and Restoration of Expression Inhibits Lymphoma Growth

Latexin is a negative regulator of hematopoietic stem cell number in mice. Its dysregulated expression in other tumors led us to hypothesize that latexin may have tumor suppressor properties in hematological malignancies. We found that latexin was down-regulated in a variety of leukemia and lymphoma cell lines as well as in CD34+ cells from the blood and marrow of patients with these malignancies. 5-aza-2′-deoxycytodine treatment and bisulfite sequencing revealed hypermethylation of latexin promoter in tumor cells. Retrovirus-mediated latexin overexpression in A20 mouse lymphoma cells inhibited their in vitro growth by 16 fold and in vivo tumor volume by 2 fold. Latexin caused growth inhibition of lymphoma cells by significantly increasing apoptosis through the down-regulation of anti-apoptotic genes Bcl-2 and Pim-2. The molecular mechanism underlying latexin-mediated tumor inhibition was not through its canonical carboxypeptidase inhibitor activity. These results are consistent with a tumor suppressor role for latexin and suggest that latexin may have clinical efficacy in the treatment of malignancies.

Mechanisms of Stem Cell Ageing

There are many definitions of the term “ageing” and perhaps even more theories that seek to explain its causes (Balcombe and Sinclair 2001). From a physiological standpoint, ageing beyond reproductive maturity is often viewed as a progression of multisystem deficits in tissue function. In adult mammals, tissue homeostasis is maintained by stem cell populations that reside in, or migrate between, a variety of adult tissues. These stem cells ensure proper tissue function by generating new cells to replace those lost or damaged over time. Despite the presence of adult stem cells in muscle, nervous, gastrointestinal, hematopoietic, and other tissues, each of these systems exhibit functional decline with age (Edwards et al. 2002, Campisi 2003, Kondo et al. 2003, Penninx et al. 2003, Pinto et al. 2003, Linton and Dorshkind 2004, Balducci and Ershler 2005, Fulle et al. 2005, Kamminga 2005, Bauer et al. 2006, Keller 2006, Theise 2006). It is compelling to consider that functional changes in the stem cell compartment of adult tissues precedes and perhaps contributes to the manifestation of ageing phenotypes. In this chapter we will review literature describing the effects of ageing on the well-characterized stem cells of the hematopoietic system. In this system, ageing is accompanied by immune compromise, anemia, and a dramatic increase in the incidence of malignancy (Edwards et al. 2002, Campisi 2003, Penninx et al. 2003, Pinto et al. 2003, Linton and Dorshkind 2004, Balducci and Ershler 2005). Several studies support the hypothesis that these ageing phenotypes stem from functional changes in hematopoietic stem cells (HSCs).

Stem Cell Aging: Potential Effects on Health and Mortality

Aging in a statistical sense is the increasing probability of death with increasing time of an organism’s existence (1, 2). Can we extrapolate this to self-regenerating tissues and most particularly to the stem cells that drive the replenishment of lost and damaged cells throughout life? To be succinct, how close is the linkage between the vitality of the stem cell population and organismal longevity? These questions are currently without clear answers and the nature of the linkage, if any, is likely to be complicated, but is nonetheless conceptually compelling. However, in the most straightforward and blunt analysis, limiting numbers of hematopoietic stem cells, for example, resulting in aplastic anemia is an infrequent cause of death (3). Moreover, the hallmark property that distinguishes stem cells from most other somatic cells, their ability to self-replicate, in theory should provide a life-long supply. It was shown many years ago that hematopoietic stem cells could be transplanted into myeloablated recipients and continue to produce large numbers of differentiated blood cells over a time period that greatly exceeded the lifespan of the donor mouse (4). Serial transplants, in which an original bone marrow graft is passaged through a series of recipients, put even greater demands on stem cell proliferation and differentiation and thus demonstrate the tremendous regenerative potential of these cells. However, the number of transplant iterations that may be carried out is limited using marrow from young mice (5, 6), and further reduced if donors are old (6, 7). In fact it is restricted to less than five, depending on mouse strain, and although it has been argued that the limitation is not so much a result of diminished stem cell potential as in the transplantation procedure itself (8), it is now clear that stem cells’ regenerative Chapter 1

EX VIVO Expansion of CD34+ Cells in Stemline™ II Hematopoietic Stem Cell Expansion Medium Generates a Large Population of Functional Early and Late Progenitor Cells

Abstract Hematopoietic stem cell replacement therapy is an area of research lacking an optimal culture system that allows for the ex vivo expansion of CD34+ cells for transplant. The necessity for expansion is due to the lack of sufficient material from umbilical cord blood (UCB), the preferred source of CD34+ cells. UCB transplantations are preferred because fewer patients suffer from graft-versushost disease compared to transplantations using CD34+ cells isolated from mobilized peripheral blood or bone marrow. In addition to the source of the starting material, the final composition of the expanded cells is also very important to a successful transplant. The expanded transplantation material must contain both early and late progenitor cells to ensure the long-term engraftment that is required in patients with various genetic disorders and those that have gone through ablative, high dose chemotherapy.