Concise reviews: cancer stem cells: from concept to cure.

In 1953, noting a remarkable consistency between the agents causing mutations and those associated with cancer, Carl Nordling, a Finnish-born architect, proposed that cancer results from an accumulation of genetic mutations. It is now generally accepted that inherited mutations and environmental carcinogens can lead to the development of premalignant clones. After further mutations, one cell reaches a critical state which confers a survival or growth advantage over normal cells. Such cells have the ability to initiate a malignant tumour. They share many of the features of normal stem cells, including the capacity for self-renewal and differentiation, and are widely termed cancer stem cells (CSCs). Although CSCs have been well characterized in hematological malignancies, their existence in some other tissues has been questioned. Here, we review recent work in which stem cells and stem cell-like cells have been used to investigate the pathogenesis of cancer and potential anticancer treatment strategies, in the context of both hematological and somatic tissue disease.

Wnt-ß-catenin-Tcf-4 signalling-modulated invasiveness is dependent on osteopontin expression in breast cancer.

BACKGROUND: We have previously demonstrated that Tcf-4 regulates osteopontin (OPN) in rat breast epithelial cells, Rama37. In this report, we have examined the importance of this regulation in human breast cancer. METHODS: The regulatory roles of Tcf-4 on cell invasion and OPN expression were investigated. The mRNA expression of Tcf-4 and OPN, and survival of breast cancer patients were correlated. RESULTS: Tcf-4 enhanced cell invasion in both MCF10AT and MDA MB 231 breast cancer cells by transcriptionally activating OPN expression. Osteopontin was activated by Wnt signalling in MDA MB 231 cells. Paradoxical results on Tcf-4-regulated OPN expression in MCF10AT (activation) and Rama37 (repression) cells were shown to be a result of differential Wnt signalling competency in MCF10AT and Rama37 cells. High levels of OPN and Tcf-4 mRNA expression were significantly associated with survival in breast cancer patients. Most importantly, Tcf-4-positive patients had a poorer prognosis when OPN was overexpressed, while OPN-negative patients had a better prognosis when Tcf-4 was overexpressed. CONCLUSION: Our results suggest that Tcf-4 can act as a repressor or activator of breast cancer progression by regulating OPN expression in a Wnt-dependent manner and that Tcf-4 and OPN together may be a novel prognostic indicator for breast cancer progression.

Mutation of the von Hippel-Lindau gene alters human cardiopulmonary physiology.

Intracellular responses to hypoxia are coordinated by the von Hippel-Lindau--hypoxia-inducible factor (VHL-HIF) transcriptional system. This study investigated the potential role of the VHL-HIF pathway in human systems-level physiology. Patients diagnosed with Chuvash polycythaemia, a rare disorder in which VHL signalling is specifically impaired, were studied during acute hypoxia and hypercapnia. Subjects breathed through a mouthpiece and ventilation was measured while pulmonary vascular tone was assessed echocardiographically. The patients were found to have elevated basal ventilation and pulmonary vascular tone, and ventilatory, pulmonary vasoconstrictive and heart rate responses to acute hypoxia were greatly increased, as were heart rate responses to hypercapnia. The patients also had abnormal pulmonary function on spirometry. This study's findings demonstrate that the VHL-HIF signalling pathway, which is so central to intracellular oxygen sensing, also regulates the organ systems upon which cellular oxygen delivery ultimately depends.

Pyruvate kinase deficient hemolytic anemia in the Northern Irish population.

A common cause of hereditary nonspherocytic hemolytic anemia is pyruvate kinase deficiency, which is associated with lifelong chronic hemolysis. Pyruvate kinase deficiency has a worldwide distribution with a higher prevalence in the Caucasian population, and especially in Europe and North America. It is inherited in an autosomal fashion and over 180 different mutations have been described. Investigation of hemolytic anemia in Northern Ireland has uncovered 4 new cases of pyruvate kinase deficiency. Molecular investigation revealed a total of six different mutations. One mutation (p.Arg495Val) is reported here for the first time in a homozygous patient. Another mutant PKLR allele harbored a nonsense and frameshift mutation in cis: c.[721G>T; 826delG]. Considering the three previously described Irish cases of pyruvate kinase deficiency, this study raises the total number of pyruvate kinase-deficient Irish patients to seven in which a total of nine mutant PKLR alleles were identified. This indicates the absence of a founder pyruvate kinase mutation in the Northern Ireland population. Although pyruvate kinase deficiency is prevalent in the Caucasian population it is not reflected in the number of individuals diagnosed in Northern Ireland. Hence, many cases of pyruvate kinase deficiency may remain undetected possibly due to the resultant anemia being mild.

Induction of signalling in non-erythroid cells by pharmacological levels of erythropoietin.

Erythropoiesis is maintained by the hormone erythropoietin (Epo) binding to its cognate receptor (EpoR) on erythroid progenitor cells. The Epo-EpoR interaction initiates a signal transduction process that regulates the survival, growth and differentiation of these cells. Originally perceived as highly lineage-restricted, Epo is now recognised to have pleiotropic effects extending beyond the maintenance of red cell mass. Functional interactions between Epo and EpoR have been demonstrated in numerous cells and tissues. EpoR expression on neoplastic cells leads to concern that recombinant human erythropoietin, used to treat anaemia in cancer patients, may augment tumour growth. Here we demonstrate that EPO, at pharmacological concentrations, can activate three major signalling cascades, viz. the Jak2/STAT5, Ras/ERK and PI3K/Akt pathways in non-small cell lung carcinoma (NSCLC) cell lines. EpoR synthesis is normally under the control of GATA-1, but NSCLC cells exhibit decreased GATA-1 levels compared to GATA-2, -3 and -6, suggesting that GATA-1 is not essential for EpoR production. The increased Epo-induced signalling was not associated with a growth advantage for the NSCLC cells.

Pre-operative plasma erythropoietin concentration and survival following surgery for non-small cell lung cancer.

BACKGROUND: Suppression of the effect of the hormone erythropoietin (EPO) on the bone marrow, and an inadequate EPO response to anaemia have been shown to be factors in the genesis of cancer related anaemia. Low haemoglobin (Hb) concentration pre-operatively has been shown to have prognostic significance in patients with surgically resected NSCLC. This study investigates the relationship between pre-operative EPO and survival in patients having surgery for NSCLC. METHODS: Pre-operative plasma EPO concentration and haemoglobin concentration were analysed in patients undergoing surgery for NSCLC between April 1998 and January 1999. Full follow-up was available for all patients. RESULTS: Forty two patients were included. Median EPO concentration was 9.4 mIU/ml, range (3.7-56.4) with 17 patients (40.4%) having values above the normal range. Median haemoglobin concentration was 13.3g/dl (range 8.5-16.8) with 15 patients (26%) anaemic pre-operatively. Pathological staging revealed 17 (40.4%) patients with stage I, 6 (14.3%) with stage II, 19 (45.3%) with stage III disease. Ten patients had irresectable disease. There was a significant difference in median EPO but not haemoglobin concentration, between the different pathological stages. Survival was significantly lower in patients with pre-operative EPO >10.5 mIU. CONCLUSIONS: Raised pre-operative EPO is associated with reduced survival in patients having surgery for NSCLC. Its measurement should be considered in the pre-operative assessment of patients undergoing surgery for NSCLC. Further research is required to further investigate the biological relationship between EPO and NSCLC.

Recessive congenital methaemoglobinaemia: functional characterization of the novel D239G mutation in the NADH-binding lobe of cytochrome b5 reductase.

Type I recessive congenital methaemoglobinaemia (RCM), caused by the reduced form of nicotinamide adenine dinucleotide (NADH)-cytochrome b(5) reductase (cytb(5)r) deficiency, manifests clinically as cyanosis without neurological dysfunction. Two mutations, E255- and G291D, have been identified in the NADH-binding lobe of cytb(5)r in previously reported patients, and we have detected a further novel mutation, D239G, in this lobe in two unrelated Irish families. Although one family belongs to the genetically isolated Traveller Community, which separated from the general Irish population during the 1845-48 famine, the D239G mutation was present on the same haplotype in both families. Three known cytb(5)r mutations were also identified, including the R159- mutation, which causes loss of the entire NADH-binding lobe and had previously been reported in an individual with type II RCM. Characterization of the three NADH-binding lobe mutants using a heterologous expression system revealed that all three variants retained stoichiometric levels of flavin adenine dinucleotide with spectroscopic and thermodynamic properties comparable with those of native cytb(5)r. In contrast to the E255- and G291D variants, the novel D239G mutation had no adverse impact on protein thermostability. The D239G mutation perturbed substrate binding, causing both decreased specificity for NADH and increased specificity for NADPH. Thus cytb(5)r deficient patients who are heterozygous for an NADH-binding lobe mutation can exhibit the clinically less severe type I phenotype, even in association with heterozygous deletion of the NADH-binding lobe.

The pathophysiology of HOX genes and their role in cancer.

The HOM-C clustered prototype homeobox genes of Drosophila, and their counterparts, the HOX genes in humans, are highly conserved at the genomic level. These master regulators of development continue to be expressed throughout adulthood in various tissues and organs. The physiological and patho-physiological functions of this network of genes are being avidly pursued within the scientific community, but defined roles for them remain elusive. The order of expression of HOX genes within a cluster is co-ordinated during development, so that the 3' genes are expressed more anteriorly and earlier than the 5' genes. Mutations in HOXA13 and HOXD13 are associated with disorders of limb formation such as hand-foot-genital syndrome (HFGS), synpolydactyly (SPD), and brachydactyly. Haematopoietic progenitors express HOX genes in a pattern characteristic of the lineage and stage of differentiation of the cells. In leukaemia, dysregulated HOX gene expression can occur due to chromosomal translocations involving upstream regulators such as the MLL gene, or the fusion of a HOX gene to another gene such as the nucleoporin, NUP98. Recent investigations of HOX gene expression in leukaemia are providing important insights into disease classification and prediction of clinical outcome. Whereas the oncogenic potential of certain HOX genes in leukaemia has already been defined, their role in other neoplasms is currently being studied. Progress has been hampered by the experimental approach used in many studies in which the expression of small subsets of HOX genes was analysed, and complicated by the functional redundancy implicit in the HOX gene system. Attempts to elucidate the function of HOX genes in malignant transformation will be enhanced by a better understanding of their upstream regulators and downstream target genes.

A randomized trial investigating an iron-rich natural mineral water as a prophylaxis against iron deficiency in pregnancy.

Spatone Iron-Plus is a naturally occurring mineral water from Trefriw Wells Spa in Conwy County, North Wales, UK. It contains approximately 0.20 mg of iron per millilitre as ferrous sulphate and has been shown to provide iron in a highly bio-available form. A 24 ml sachet contains approximately 5 mg of iron. Iron deficiency is common in the obstetric population. However, compliance with traditional iron supplements is poor because of gastrointestinal side-effects. We designed a randomized, double-blind, placebo-controlled trial. A total of 102 low-risk antenatal patients, who were noncompliant with routinely prescribed ferrous sulphate tablets, were randomized to receive 48 ml of Spatone water or placebo. The study was conducted between 22 and 28 weeks gestation. Primary outcome measures were compliance, gastrointestinal side-effects and changes in ferritin levels during the trial period. Compliance in the intervention group was 57% compared with 67% in the control group, P = 0.22. Dyspepsia scores, as determined by a recognized and well-validated questionnaire, did not differ between the two groups. During the trial period, mean ferritin levels fell by 24% in the Spatone Iron-Plus group compared with a mean fall of 51% in ferritin levels among the control group, P = 0.016.

Homocysteine and methylmalonic acid as indicators of folate and vitamin B12 deficiency in pregnancy.

Deficiency of folate during pregnancy is associated with megaloblastic anaemia. Lower levels of folate and vitamin B12 have been reported in mothers whose offspring had neural tube defects compared to unaffected controls. Increased methylmalonic acid levels are a sensitive indicator of mild vitamin B12 deficiency and elevated homocysteine levels denote vitamin B12 or folate deficiency. We have investigated the relationship between serum concentration of total homocysteine, methylmalonic acid, vitamin B12 and folate in pregnancy. A significant inverse correlation was found between homocysteine and red cell folate and, to a lesser extent, serum folate. In addition, a significant inverse correlation was found between methylmalonic acid and vitamin B12. No significant relationship was found between homocysteine and vitamin B12. The relationship between red cell folate and serum folate and homocysteine may be useful for detecting borderline folate deficiency in pregnancy and indicate pregnancies at risk of neural tube defect. These sensitive assays are useful tools for the further investigation of folate vitamin B12 and metabolism in normal and abnormal pregnancy.

Cord blood transferrin receptors to assess fetal iron status.

OBJECTIVE: To study iron status at different gestational ages using cord blood serum transferrin receptors (STfRs). METHODS: STfRs, iron, ferritin, total iron binding capacity, haemoglobin, and reticulocytes were measured in 144 cord blood samples. The babies were divided into three groups according to gestation (26 very preterm (24-29 weeks); 50 preterm (30-36 weeks); 68 term (37-41 weeks)). RESULTS: Serum iron, ferritin, and total iron binding capacity were highest at term, whereas reticulocytes were highest in the very preterm. STfR levels were not influenced by gestation. Haemoglobin (r = 0.46; p < 0.0001) and reticulocytes (r = 0.42; p < 0.0001) were the only indices that independently correlated with STfR levels. CONCLUSIONS: STfR levels in cord blood are not directly influenced by gestation and probably reflect the iron requirements of the fetus for erythropoiesis.

Characterization and localization of expression of an erythropoietin-induced gene, ERIC-1/TACC3, identified in erythroid precursor cells.

Gene expression profiles during erythropoietin (Epo)-induced differentiation of erythroid progenitor cells derived from the Friend virus anaemia (FVA) and phenylhydrazine (PHZ) murine models have been examined using differential display polymerase chain reaction (PCR). Ten cDNA fragments upregulated by Epo were isolated. The ribonuclease protection assay confirmed differential expression between Epo-stimulated and Epo-deprived cells for one of these, provisionally named ERIC-1. Sequencing of the full-length cDNA predicted a protein of 558 amino acids, 17 amino acids longer than mTACC3, the third member of a novel family of proteins that contain a coiled-coil domain. The human homologue, cloned using rapid amplification of cDNA ends (RACE)-PCR, encodes a larger protein of 838 amino acids that is identical to hTACC3. In addition to erythroid precursor cells, ERIC-1/TACC3 is expressed at high levels in the testes, at moderate levels in the thymus and peripheral leucocytes, and at lower levels in the spleen and intestinal tissue. Immunohistochemical analysis using an antibody to a GST fusion product of the C-terminus of hERIC-1/TACC3 revealed that it is localized to Sertoli cells in the human testes. Confocal microscopy demonstrated hERIC-1/TACC3 protein concentrated in the perinuclear vesicles of dermal microvascular endothelial cells. Although ERIC-1/TACC3 is expressed in a wide range of tissues, its upregulation by Epo in erythroid progenitors implies that it has a role in terminal erythropoiesis.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Study of maternal influences on fetal iron status at term using cord blood transferrin receptors.

AIMS: To determine effects of maternal iron depletion and smoking on iron status of term babies using serum transferrin receptors (STfR) and their ratio to ferritin (TfR-F index) in cord blood. METHODS: Iron, ferritin, STfR, and haemoglobin (Hb) concentration were measured and TfR-F index calculated in 67 cord /maternal blood pairs. Twenty six mothers were iron depleted (ferritin <10 microg/l) and 28 were smokers. RESULTS: Maternal iron depletion was associated with decreased cord ferritin (113 v 171 microg/l) and Hb (156 v 168 g/l) but no change in STfR or TfR-F index. Smoking was associated with increased cord Hb (168 v 157 g/l) and TfR-F index (4.1 v 3.4), and decreased ferritin (123 v 190 microg/l). Cord TfR-F index and Hb were positively correlated (r = 0.48). CONCLUSIONS: Maternal iron depletion is associated with reduced fetal iron stores but no change in free iron availability. Smoking is associated with increased fetal iron requirements for erythropoiesis.

High bax expression is a good prognostic indicator in acute myeloid leukaemia.

Most cytotoxic drugs kill cells by instigating the process of apoptosis and it has been suggested that apoptotic markers may provide an indication of tumour chemosensitivity. The aim of this study was to determine if such a relationship exists in acute myeloid leukaemia (AML). The levels of spontaneous apoptosis, bcl-2 and bax were evaluated in 56 newly diagnosed AML patients to determine if they correlated with a response to cytotoxic therapy. Spontaneous apoptosis was lower, but bcl-2, bax and the bcl-2/bax ratio were higher in AML compared with normal individuals. AML patients with high bax expression at diagnosis had significantly better prognosis for disease-free survival, event-free survival and overall survival (P = 0.016). In the standard risk group, high bax expression was in keeping with significantly improved survival. Multivariate analysis revealed bax to be an independent predictor of survival. There was a significant reduction in bcl-2 and bax expression when AML patients entered complete remission and also in relapsed AML patients who entered a second remission. This study suggests that bax is a useful prognostic indicator in AML and may assist with therapeutic decision-making for patients in the standard risk category.

Erythroblasts from friend virus infected- and phenylhydrazine-treated mice accurately model erythroid differentiation.

The dynamics of gene expression during terminal erythroid differentiation have been examined in three murine models; the erythroleukaemia cell line HCD-57 and splenic erythroblasts isolated from mice treated with either the anaemia-inducing strain of Friend virus (FVA cells) or the haemolytic agent phenylhydrazine (PHZ cells). In response to erythropoietin (EPO) and haemin, HCD-57 cells proliferated and synthesized haemoglobin, but failed to complete terminal differentiation as indicated by lack of change in both gene expression and morphological appearance. In contrast, EPO-induced terminal differentiation in FVA and PHZ cells in vitro was accompanied by increases in haemoglobin positivity, morphological maturation and a shared pattern of gene expression. EPO receptor (EPO-R) mRNA levels peaked before globin gene expression which was maximal at 24 h. Peak GATA-1 and EKLF mRNA levels also preceded the globin gene peak, but the highest NF-E2 levels coincided with maximal globin levels, suggesting a role for NF-E2 in the maintenance, rather than the initiation of globin gene expression. Peak expression of delta-aminolaevulinic acid synthase (ALAS) coincided with peak globin expression. FVA and PHZ cells represent more effective models than the HCD-57 cell line for the investigation of erythroid gene expression during EPO-regulated terminal erythropoiesis.

Erythropoietin production: evidence for multiple oxygen sensing pathways.

The production of erythropoietin (Epo), the glycoprotein hormone which controls red blood cell formation, is regulated by feedback mechanisms sensing tissue oxygenation. The mechanism of the putative oxygen sensor has yet to be elucidated. There is evidence that at least two pathways participate in hypoxia signal transduction. One appears to involve a specific haem protein, and a second implicates reactive oxygen species (ROS). Iron catalyses the generation of intracellular ROS and therefore alters the cellular redox state. We have investigated the effect of modulating intracellular iron content on Epo production in Hep 3B cells. Iron chelation stimulates Epo production at 20% O2 and enhances Epo production at 1% O2, but it has no additive effect on cobalt-induced Epo production. Excess molar iron inhibited Epo production in response to hypoxia, desferrioxamine (DFO) and cobalt chloride and inhibited the DFO-enhancing effect of hypoxia-induced Epo production. We found that sulphydryl oxidising agents exert a differential inhibitory effect on hypoxia-induced versus DFO-induced Epo production, providing further evidence that multiple pathways of oxygen sensing exist.