Olfactomedin 4 deletion induces colon adenocarcinoma in ApcMin/+ mice.

Colon carcinogenesis is a multiple-step process involving the accumulation of a series of genetic and epigenetic alterations. The most commonly initiating event of intestinal carcinogenesis is mutation of the adenomatous polyposis coli (APC) gene, which leads to activation of the Wnt/ß-catenin pathway. Olfactomedin 4 (OLFM4) has emerged as an intestinal stem-cell marker, but its biological function in the intestine remains to be determined. Here we show that Olfm4 deletion induced colon adenocarcinoma in the distal colon of ApcMin/+ mice. Mechanistically, we found that OLFM4 is a target gene of the Wnt/ß-catenin pathway and can downregulate ß-catenin signaling by competing with Wnt ligands for binding to Frizzled receptors, as well as by inhibition of the Akt-GSK-3ß (Akt-glycogen synthase kinase-3ß) pathway. We have shown that both Wnt and nuclear factor-κB (NF-κB) signaling were boosted in tumor tissues of Apc Olfm4 double-mutant mice. These data establish OLFM4 as a critical negative regulator of the Wnt/ß-catenin and NF-κB pathways that inhibits colon-cancer development initiated by APC mutation. In addition, Olfm4 deletion significantly enhanced intestinal-crypt proliferation and inflammation induced by azoxymethane/dextran sodium sulfate. Thus, OLFM4 has an important role in the regulation of intestinal inflammation and tumorigenesis, and could be a potential therapeutic target for intestinal malignant tumors. Unlike the human colonic epithelium, the mouse colonic epithelium does not express OLFM4, but nevertheless, systemic OLFM4 deletion promotes colon tumorigenesis and that loss from mucosal neutrophils may have a role to play.

Expression of hGC-1 is correlated with differentiation of gastric carcinoma.

AIMS: The human G-CSF-stimulated clone-1 (hGC-1) gene encodes a 510-amino acid olfactomedin-related glycoprotein whose exact in vivo localization and function still remain elusive. The aim of this study was to demonstrate hGC-1 protein localization in the normal human gastrointestinal tract and to explore further a potential relationship between hGC-1 expression and gastric carcinoma. METHODS AND RESULTS: A specific hGC-1 polyclonal antibody raised against purified hGC-1 protein was developed and characterized. Using immunohistochemistry, it was demonstrated that hGC-1 is expressed in the oesophagus, stomach, small intestine and colon. The expression pattern of hGC-1 protein in 173 cases of gastric carcinoma was investigated and a striking correlation was demonstrated between hGC-1 expression and histological type and differentiation of gastric carcinoma. Enhanced hGC-1 expression was more frequently seen in intestinal-type adenocarcinoma, whereas loss of expression tended to occur in the diffuse type. hGC-1 was highly expressed in well or moderately differentiated cancers and was remarkably reduced or lost in poorly differentiated or undifferentiated tumours. CONCLUSIONS: These investigations have defined for the first time the expression pattern of hGC-1 in the normal human gastrointestinal tract and provide a novel and sensitive marker for the differentiation of gastric carcinoma.

Nitric oxide transport on sickle cell hemoglobin: where does it bind?

We have recently reported that nitric oxide inhalation in individuals with sickle cell anemia increases the level of NO bound to hemoglobin, with the development of an arterial-venous gradient, suggesting delivery to the tissues. A recent model suggests that nitric oxide, in addition to its well-known reaction with heme groups, reacts with the beta-globin chain cysteine 93 to form S-nitrosohemoglobin (SNO-Hb) and that SNO-Hb would preferentially release nitric oxide in the tissues and thus modulate blood flow. However, we have also recently determined that the primary NO hemoglobin adduct formed during NO breathing in normal (hemoglobin A) individuals is nitrosyl (heme)hemoglobin (HbFeIINO), with only a small amount of SNO-Hb formation. To determine whether the NO is transported as HbFeIINO or SNO-Hb in sickle cell individuals, which would have very different effects on sickle hemoglobin polymerization, we measured these two hemoglobin species in three sickle cell volunteers before and during a dose escalation of inhaled NO (40, 60, and 80 ppm). Similar to our previous observations in normal individuals, the predominant species formed was HbFeIINO, with a significant arterial-venous gradient. Minimal SNO-Hb was formed during NO breathing, a finding inconsistent with significant transport of NO using this pathway, but suggesting that this pathway exists. These results suggest that NO binding to heme groups is physiologically a rapidly reversible process, supporting a revised model of hemoglobin delivery of NO in the peripheral circulation and consistent with the possibility that NO delivery by hemoglobin may be therapeutically useful in sickle cell disease.

Pathophysiology of sickle cell disease: role of cellular and genetic modifiers.

Sickle hemoglobin (HbS), caused by a point mutation in the beta-globin gene of hemoglobin, polymerizes when deoxygenated. The pathophysiology of sickle cell disease results from cellular defects caused directly by the hemoglobin mutation interacting with the environment and many other gene products--a few known, but most yet unidentified--a typical example of epistasis. How normal tissue perfusion is interrupted is complex and why the phenotype of sickle cell disease differs from patient to patient is poorly understood. We review the "classic" aspects of the pathophysiology of sickle cell disease and focus on known and potential modulators of the phenotype of this disorder.

Rapid differentiation of five common alpha-thalassemia genotypes by polymerase chain reaction.

The alpha-thalassemias are common genetic disorders that arise from reduced synthesis of the alpha-globin chains. At present, large-scale carrier screening and clinically valuable antenatal detection programs have not been established for the congenital disorder alpha-thalassemia (alpha-thal). We have developed a simple nonradioactive polymerase chain reaction (PCR) approach that can detect and differentiate several common alpha-globin gene deletional alpha-thals regardless of the break points. When three primer sets were used--two gene-specific sets for the alpha1- and alpha2-globin genes and one set for the beta-actin gene (serving as an internal control)--PCR products from genomic DNA were simultaneously amplified and analyzed after coamplification and gel electrophoresis. The number of alpha-globin genes present in the subjects was determined by the intensity of alpha1 and alpha2 bands normalized with that of beta-actin when using densitometry. Our results demonstrate that five common genotypes of deletional alpha-thal are differentiated by the ratios of alpha1/beta-actin and alpha2/beta-actin. We also examined the feasibility of coupling this allele-specific amplification to a color-complementary assay. This easy and reproducible PCR assay is suitable for identifying alpha-thal carriers in screenings of large populations and improving genetic counseling.

Clinical and hematological responses to hydroxyurea in Sicilian patients with Hb S/beta-thalassemia.

Although, several reports have detailed that hydroxyurea can ameliorate the clinical course of adult and pediatric patients with sickle cell anemia (Hb S or beta(S)), few clinical studies have been carried out in patients with beta(S)/beta-thalassemia. In a two-year clinical study, we evaluated the efficacy of hydroxyurea in a group of 22 adult Sicilian patients with beta(S)/beta-thalassemia with severe phenotypes. Among the 20 patients evaluated during 2 years of treatment, we observed a very good clinical response with a 93% reduction of the annual number of crises (median 7 versus 0.5 crises per year; P < 0.001) and of days in hospital (mean 22+/- 21.9 versus 1.2 +/- 2.3; P < 0.001), a significant increase in Hb F (7.5 +/- 5.3% versus 25.2 +/- 5.2%; P < 0.001) and in MCV (73.1 +/- 4.8 fL versus 96.4 +/- 7.2 fL; P < 0.001), and no significant modifications in Hb (9.6 +/- 1.3 g/dL versus 10.0 +/- 1.5 g/dL; P > 0.05) and in WBC (11.4 +/- 3.9 x 10(9)/L versus 10.2 +/- 3.9 x 10(9)/L; P > 0.05). Twelve patients had no crises from the first month of treatment; 16 patients showed a 2-3-fold increase over baseline in Hb F. During the study no severe complications and no important side effects of hydroxyurea were observed. Our data suggest that hydroxyurea efficacy in patients with beta(S)/beta-thalassemial may be greater than that described in patients with sickle cell disease. This pattern and durability of response will need to be confirmed in a larger, randomized, clinical trial.

Advances in experimental treatment of beta-thalassaemia.

Beta-thalassaemia is highly prevalent and world wide in its distribution. The gene to modify the clinical course of patients with transfusion-dependent thalassaemia (thalassaemia major), the gamma-globin gene, is already present in these patients but silenced in the course of development. During erythropoiesis, progenitors are believed to go through a phase where the milieu favours gamma-globin production. One pharmacological strategy to increase gamma-globin production is directed at recruiting such early progenitors through the use of cytotoxic agents (+/- erythropoietin) that presumably deplete more mature progenitors. Another promising strategy is to use chromatin-modifying agents that prevent the silencing of the gamma-globin gene that occurs during development. These agents, the methyl-transferase inhibitors and histone deacetylase inhibitors, either alone or in combination, may be able to produce the robust increase in gamma-globin and hence fetal haemoglobin and total haemoglobin, needed to successfully treat thalassaemia major. Studies of these agents, which are already available for clinical trials, should be encouraged.

Enrichment and detection of fetal erythroid cells from maternal peripheral blood using liquid culture.

Isolating fetal cells from maternal blood for prenatal genetic analysis is the least invasive method currently being investigated. In order to enrich these fetal erythroid cells we employed a two-phase liquid culture system that supports the growth and differentiation of erythroid progenitor cells. Mononuclear cells were separated from ten maternal blood samples at 8-14(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the non-adherent cells were harvested and recultured with erythropoietin for another 3-4 days. The mean number of total erythroid cells reached approximately 0.77x10(6)/ml with an average purity of 90.5%. Hb F stain disclosed fetal erythroid cells averaging approximately 5.7%, therefore the mean number of fetal erythroid cells isolated was 0.49x10(5)/ml. Female karyotypes were only observed while counting 5-66 metaphases. However, male DNA (SRY or DYZ1) was detected by PCR in 7/10 cases; in four of these cases and in one SRY-negative pregnancy the 46,XY karyotype was found by amniocentesis performed during the second trimester. This liquid culture system provided an enriched source of fetal cells without the need for more complicated separation or sorting procedures.

Polymorphisms within the angiotensinogen gene (GT-repeat) and the risk of stroke in pediatric patients with sickle cell disease: a case-control study.

Stroke is one of the most devastating complications of patients with sickle cell disease (SCD). Currently, there are no known molecular or genetic markers that can be used to assess the risk of stroke in this population. We have previously shown that relative hypertension may be one risk factor for stroke in SCD. In a case-control study, we investigated the association between GT-repeat polymorphism within the angiotensinogen (AGT) gene and the risk of stroke in pediatric patients with SCD. After informed consent was obtained, 63 patients (21 stroke subjects and 42 nonstroke control subjects matched according to age and sex) with SCD followed at local pediatric hematology clinics were genotyped to test the association of specific GT-repeat alleles of the AGT gene and occurrence of stroke. There were statistical differences in the distribution of the genotypes among stroke and nonstroke SCD patients (chi(2) = 10.82, df = 11, P < 0.05). We also found GT-repeat alleles A3 and/or A4 of the AGT gene conferred a four-fold increase in the risk of stroke (odds ratio [OR] = 4, P < 0.05). The attributable odds ratio for allele A3 and A4 is 2.24 and 4.33, respectively (P < 0.005). Our results suggest that GT-repeat within the AGT gene may be associated with risk of stroke in pediatric SCD. The relative risk of stroke in the presence of alleles A3 and/or A4 is fourfold greater than in the absence of these alleles. If these data are substantiated in a larger cohort of patients, our results indicate that the determination of GT-repeat of AGT gene may be a useful genetic marker to assess the risk for stroke of patients with SCD. Am. J. Hematol. 68:164-169, 2001. Published 2001 Wiley-Liss, Inc.

Effect of nitric oxide and nitric oxide donors on red blood cell oxygen transport.

A mechanism has been proposed in which nitric oxide (NO) may bind to cysteine beta93 and be transported by haemoglobin from the lungs to the tissues and modify vascular tone. In addition, it has been reported that treatment of sickle cell anaemia blood with 80 p.p.m. NO gas in air shifts the oxygen affinity, as measured by P50 to the left. We exposed normal and sickle cell anaemia blood to 80 p.p.m. NO in air for 1 h in vitro and found no change in P50 of either normal or sickle cell blood. In addition, we exposed normal and sickle cell blood in buffer to aqueous NO (NO gas dissolved in buffer) at varying concentrations and found that the induced left shift in P50 correlates strongly and linearly with methaemoglobin formation. We also treated normal and sickle cell blood with other nitric oxide donors, such as sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxodinitrate (OXINO, or Angeli's salt). In all cases, we found a dose-dependent increase in methaemoglobin that was strongly correlated with the dose-dependent P50 reduction. Our data do not support the report that low NO concentrations can selectively increase the oxygen affinity of sickle cell blood without affecting methaemoglobin levels significantly. NO, however, may have benefit in sickle cell disease by other mechanisms.

Hydroxyurea induces a senescence-like change of K562 human erythroleukemia cell.

Hydroxyurea, a differentiation-inducing agent of human erythroleukemia K562 cells, is commonly used to treat some types of leukemia. However, the mechanism for its therapeutic effect is not clearly understood yet. In this study, we have observed an interesting effect of hydroxyurea on tumor cells: an induction of senescence-like changes. Human erythroleukemia K562 cells, when treated with hydroxyurea for 7 days or more, underwent a change into phenotypically senescent cells together with a reduction of hemoglobin generation, a differentiation marker. The hydroxyurea-treated cells showed positive senescence associated-beta-galactosidase staining, a senescence index, and the accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16INK4a, p21Waf1, and p27Kip1, implicated in cellular senescence. Nonetheless, these changes were not accompanied by DNA fragmentation. Taken together, we summarize that the long-term treatment of cancer cells with hydroxyurea can induce cellular senescence different from differentiation or programmed cell death.

The role of trans-acting factors and DNA-bending in the silencing of human beta-globin gene expression.

The molecular mechanisms which govern the develop-mental specificity of human beta-globin gene transcription have been studied in K562 cells, a human eyrthroleukemia line that expresses minimal beta-globin. Protein-binding analysis reveals that the 5' region contains three elements bound by trans-acting factors, beta-protein 1 (BP1) and beta-protein 2 (BP2). In vitro mutagenesis of each individual element in a beta-globin vector containing chloramphenicol acetyl-transferase (pCAT) followed by transient transfection into K562 cells increased levels of CAT activity 5. 5-fold higher than wild-type (wt) betaCAT, consistent with their silencing role. Mutagenesis of all three elements, however, resulted in activity significantly lower than wt betaCAT. BP1 and BP2 motifs have overlapping binding sites for high mobility group proteins (HMG1+2), DNA-bending factors, shown here to extrinsically bend the beta-globin promoter. Theoretically, mutations in all beta-protein binding sites could affect the binding of HMG1+2 sufficiently to impede DNA-protein and/or protein-protein interactions needed to facilitate constitutive gene expression. Placing two turns of DNA between BP1 and BP2 motifs also increased expression 3-fold, indicative of spatial constraints required for optimal silencing. However, insertion of the HMG1+2 DNA-bending motif (also equivalent to two turns) facilitates beta-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specific transcriptional factors appear to be involved in beta-globin silencing in the embryonic/fetal erythroid stage.

Two-phase liquid culture system models normal human adult erythropoiesis at the molecular level.

We have studied the patterns of expression of various genes during maturation of normal human adult erythroid precursors cultured in a two-phase liquid culture method. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not erythropoietin (Epo). In Phase II, Epo is included in the medium. Cell samples were taken throughout phase II, and expression of globins, transcription factors, and cytokine receptors was assayed by RT-PCR and quantified by phosphor imaging. We have divided phase II into stages: early (days 0-5), intermediate (days 6-10) and late (days 11-15) and measured maximum expression of each gene. During early phase II, gamma-globin, Spl, and GATA-2 mRNAs were expressed at their highest levels. As the cells matured during the intermediate period, GATA-2 levels remained high, and then declined, while the transcription factors GATA-1, EKLF, NF-E2, and the Epo receptor (EpoR) reached maximum expression. In late phase II, beta-globin increased and reached its maximum level of expression. This erythroid culture system appears to recapitulate normal adult erythropoiesis at the molecular level, and thus may be a suitable model to examine the molecular basis of severe congenital or acquired disorders of erythropoiesis.