The APC gene product in normal and tumor cells.

The APC gene has been found to be mutated during the development of sporadic colorectal tumors as well as in the germ line of familial adenomatous polyposis patients. To facilitate the characterization of both normal and mutant APC protein, a series of monoclonal and polyclonal antibodies specific for the APC protein was produced. When lymphoblastoid cell lines derived from seven familial adenomatous polyposis patients with known mutations were analyzed by Western blot, an approximately 300-kDa protein corresponding to the predicted size of full-length APC was detected in all 7 cell lines. In addition, truncated APC proteins corresponding to the product of the known mutated alleles could be detected in 4 of the 7 lines. Similar analysis of 23 colon carcinoma and 9 adenoma cell lines revealed truncated proteins in 24 (75%) of the cell lines. Moreover, 26 (81%) of the colon tumor lines were totally devoid of the normal, full-length protein. In contrast, Western blot analysis of 40 cell lines derived from sporadic tumors of other organs detected only full-length APC. Immunohistochemical analysis of APC in normal colonic mucosa revealed cytoplasmic staining with more intense staining in the basolateral margins of the epithelial cell. This staining was markedly increased in the upper portions of the crypts, suggesting an increased level of expression with maturation. These studies provide some initial clues to the function of the cytoplasmic protein APC and demonstrate the feasibility of identifying APC mutations by direct analysis of the APC protein.     

Role of the microtubular system in morphological organization of normal and oncogene-transfected epithelial cells.

To understand better the role of the microtubular system in the development and maintenance of morphological organization of nonpolarized and polarized cells of the same origin we examined the effects of two microtubule-specific drugs, colcemid and taxol, on discoid cultured epithelial rat cells of the IAR-2 line and on polarized cells obtained from this line by transfection of mutated N-ras oncogene; morphometric, immunomorphologic, and videomicroscopic methods were used. Depolymerization of microtubules by colcemid did not cause major changes in the discoid shape of IAR cells but altered organization of actin cortex; in particular, it led to disappearance of circumferential bundle of actin microfilaments. Taxol reorganized the normal network of microtubules radiating from the perinuclear centers into numerous arrays of short microtubules not associated with any centers. Taxol-treated cells had wider circumferential bundles of microfilaments than control cells and morphometric analysis showed that their contours were closer to geometric circle than those of control or of colcemid-treated cells. These data show that function of the microtubular system is essential for maintenance of the characteristic morphological organization of discoid cells; we propose to name this function "contra-polarization." Contra-polarization is not prevented and is even promoted by taxol; this result suggests that a decentralized system of microtubules is sufficient for this function. In contrast, maintenance of polarized morphology of IAR-2 cells transfected by the N-ras oncogene is inhibited not only by colcemid but also by taxol and thus requires the presence of a normal centralized microtubular system.     

Retrovirus-mediated transfer of the erythropoietin gene in hematopoietic cells improves the erythrocyte phenotype in murine beta- thalassemia

Repeated injections of large doses of erythropoietin (Epo) have been shown to be of benefit in the treatment of murine and human beta- thalassemia. To determine whether Epo gene therapy could replace this treatment for long-term periods, lethally irradiated beta-thalassemic (Hbbd3th haplotype) and normal DBA/2J (Hbbd haplotype) mice were grafted with syngeneic bone marrow cells infected with a retroviral vector carrying the Epo cDNA. In normal mice, dysregulated Epo production induced elevated serum Epo levels (176 +/- 68 mU/mL), high hematocrit levels (73% +/- 8%), and elevated beta-minor globin chain synthesis. In contrast, in thalassemic mice, moderate increases in the hematocrit levels (from 33% +/- 1% to 43% +/- 9%), associated with limited increases in the initially elevated Epo levels (from 83 +/- 22 to 190 +/- 230 mU/mL), were recorded 2 months after transplantation. In mice in which the hematocrit increased most, from 33% +/- 1% before transplantation to 49% +/- 10%, the retroviral Epo gene expression induced a striking improvement of the beta-thalassemic syndrome. These mice exhibited normal or near-normal beta/alpha-globin chain synthesis ratios, induced by the activation of the beta-minor chain. This led to the elimination of the high amounts of unpaired alpha chains in erythrocytes and finally reduced the reticulocyte count despite the permanent Epo stimulation. These results show that efficient Epo gene expression corrects the erythrocyte phenotype of the mouse beta- thalassemic syndrome. However, the incidence of lethal polycythemia or of transient improvements indicates that the present strategy is only the first step toward such indirect gene therapy.     

Patterns of organization of actin and myosin in normal and transformed cultured cells.

The patterns of distribution of intracellular actin and myosin were examined by specific immunofluorescence in a series of normal, simian-virus-40-transformed, and revertant cell lines of rat and mouse origin. A consistent correlation was found between sensitivity to anchorage-dependent growth control and the presence of large, thick sheaths of actin-containing material. The presence of these sheaths was temperature-dependent in a rat line transformed by a temperature-sensitive mutant in the complementation group A of the oncogenic virus simian virus 40.     

The role of the polycomb complex in silencing alpha-globin gene expression in nonerythroid cells

Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human alpha- and beta-globin genes are silenced by fundamentally different mechanisms. The alpha-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the beta-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the alpha-cluster by sequences within the CpG islands associated with their promoters; the beta-globin promoters do not lie within such islands. Chromatin associated with the alpha-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The alpha- and beta-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.     

Association of α globin gene quadruplication and heterozygous β thalassemia in patients with thalassemia intermedia

Ten patients with thalassemia intermedia with variable severity and apparent simple heterozygosis for β⁰ 39 C>T nonsense mutation were submitted to clinical, hematologic and molecular studies. The presence of an unknown molecular defect (silent β-thalassemia) unlinked to the β cluster interacting with the heterozygous β thalassemia, was previously postulated in these families. Analysis of the α globin gene cluster with PCR-based methods (MLPA, GAP-PCR, digestion with restriction enzymes) detected complex rearrangements in the α cluster. A duplication of the α globin gene locus, including the upstream regulatory region, was present in all the patients, associated in some of them with deletion or non-deletion α thalassemia. The variability of the clinical phenotype correlates with the degree of the globin chain imbalance. The presence of α globin cluster duplication should be considered in patients heterozygote for β-thalassemia with thalassemia intermedia phenotype and in the carriers of suspected silent β thalassemia.     

The ovalbumin gene: cloning and molecular organization of the entire natural gene.

We report the analyses of recently cloned restriction fragments of the natural ovalbumin gene that overlap in part with previously cloned DNA fragments but extend further into the flanking sequences of the gene. These clones now permit us to identify the DNA sequence that codes for the 5' end of ovalbumin mRNA. Based on these and previous results, the molecular organization of the entire ovalbumin gene was established. The entire gene is composed of eight structural DNA sequences separated by seven intervening sequences that are not present in the mature mRNA. In addition, an ovalbumin gene clone has been obtained from a chicken gene library. Analysis of DNA isolated from this particular clone by molecular hybridization and electron microscopic mapping revealed that it contains the entire ovalbumin gene a single segment of DNA and its structure was consistent with that predicted from our physical map constructed from individually cloned fragments of the gene.     

Extracting the hierarchical organization of complex systems

Extracting understanding from the growing "sea" of biological and socioeconomic data is one of the most pressing scientific challenges facing us. Here, we introduce and validate an unsupervised method for extracting the hierarchical organization of complex biological, social, and technological networks. We define an ensemble of hierarchically nested random graphs, which we use to validate the method. We then apply our method to real-world networks, including the air-transportation network, an electronic circuit, an e-mail exchange network, and metabolic networks. Our analysis of model and real networks demonstrates that our method extracts an accurate multiscale representation of a complex system.     

Oxidative denaturation of red blood cells in thalassemia

We believe that on the basis of all available data, severe oxidative damage occurs in alpha- and beta-thalassemic RBCs, as depicted schematically in Fig 6. The differences in the severity and pattern of the oxidative damage may be related to the type and, perhaps, quantity of precipitated globin chains. The detrimental effect of the excess chains is multifold. In the process of globin-chain precipitation, free radicals are generated. The end product of the precipitated hemoglobin chains is heme, from which eventually iron and globin are liberated. Globin chains have been found to interact and disrupt the RBC membrane, damaging the cytoskeleton. The role of heme has not yet been studied in detail in thalassemic RBCs. However, there is some evidence that it participates in damaging RBCs in other types of hemoglobinopathies. Excess of iron is known to be a catalyst of peroxidation via the Fenton reaction, causing damage to the various RBC membrane components (lipids, proteins, etc). The denatured hemaglobin, in the form of hemichromes, aggregates with protein 3, forming Actual proof of excessive free radical production in thalassemia is still warranted. It will not be easy to document since the amount of superoxide dismutase in RBCs is above and beyond that required for neutralizing excess amount of superoxide. The more active radicals, particularly hydroxyl free radical, are difficult to measure because they are so active an interact immediately with any given substrate in their vicinity. In addition, we have to better understand the finding of excess membrane lipids in thalassemic RBCs and whether there are changes in the formation and propagation of lipid peroxidation in these cells compared with normal RBCs. Regarding the proteins, further understanding is required concerning the exact type and sites of oxidation that occurs in the beta-thalassemia 4.1 protein, and whether the damage found in alpha-thalassemia is due to oxidation of ankyrin itself or its entrapment within the complex of the precipitated hemichromes of beta chains. What is the role of the different globin chain oxidation and precipitation in generating such different cytoskeletal protein alterations? Another point that needs to be elucidated is the role of different kinds of antibodies that are attached to the newly exposed antigenic sites on the thalassemic RBC membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pseudo-Gaucher cells in Hb E disease and thalassemia intermedia

Abstract

Pseudo-Gaucher cells are morphologic curiosities described in a number of conditions. We report their presence in a bone marrow biopsy and a spleen from 2 patients with hemoglobin E disease and thalassemia intermedia, respectively. The pathogenesis of these cells and their multiple known associations are discussed.     

Activated peripheral blood and endothelial cells in thalassemia patients

BACKGROUND AND OBJECTIVES: Thalassemia patients have alterations in the expression of some activation and adhesion molecules on peripheral blood lymphocytes. We studied cell surface antigens on peripheral blood cells associated with the activation of these cells and soluble molecules produced by activated endothelium. DESIGN AND METHODS: We investigated the expression of CD11b, CD18, CD35, CD43, CD44, and CD69 on the peripheral blood monocytes, Cd11b, CD18, CD35, CD43, CD44, CD67 on peripheral blood neutrophils and CD38 and CD69 on peripheral blood lymphocytes. We studied 68 transfusion-dependent thalassemics (group A), 10 transfusion non-dependent thalassemics (group B), 18 beta-thalassemia carriers (group C), and 28 normal individuals. Relative fluorescence intensity was used to determine the antigen density. Analysis was performed with an EPICS ELITE flow cytometer. Furthermore, soluble intercelullar adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), and E-selectin, tumor necrosis factor (TNF) alpha, and interleukin (IL) 1beta were measured in the plasma of patients by enzyme-linked immunometric assay. Results: The expression of CD11b, CD18, and CD69 on the monocytes of group A was significantly greater than in groups B and C and in controls, while CD44 was significantly downregulated in group A. CD11b, CD18, CD35, CD44, and CD67 on the surface of neutrophils and CD38 and CD69 on the surface of lymphocytes were also overexpressed in group A. CD44 was downregulated on the monocytes and upregulated on the neutrophils of the patients compared to controls. The levels of sICAM-1, sVCAM-1, E-selectin, TNF-alpha, and IL-1beta in the serum of patients in groups A and B were higher than those in group C and the controls. CONCLUSION: Endothelial activation markers are significantly increased in thalassemia patients, and activated blood cells circulate in the peripheral blood. These may be related to the vascular complications in these patients and might be useful markers for the follow-up of the vascular disease.     

Pseudo-Gaucher cells in Hb E disease and thalassemia intermedia

Pseudo-Gaucher cells are morphologic curiosities described in a number of conditions. We report their presence in a bone marrow biopsy and a spleen from 2 patients with hemoglobin E disease and thalassemia intermedia, respectively. The pathogenesis of these cells and their multiple known associations are discussed.     

Iodide symporter gene expression in normal and transformed rat thyroid cells

OBJECTIVE: Decrease or loss of the Na+/I- symporter (NIS) activity profoundly affects the suitability of the use of radioiodine to detect or treat metastatic thyroid tissues. The aim of our study was to verify whether specific oncogene abnormalities were responsible for the alteration in NIS activity in thyroid cells. DESIGN AND METHODS: Expression of the NIS gene was investigated by Northern blot analysis in normal and in some oncogene-transformed cell lines with different degrees of malignancy which had lost the iodide uptake ability. RESULTS: NIS gene expression was up-regulated by TSH in a dose-dependent and time-dependent way in normal PC Cl 3 cells. The same effect was observed by activating the cAMP-dependent pathway by forskolin. Conversely, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a partial inhibitory effect on NIS gene expression. The oncogene-transformed cell lines PC v-erbA, PC HaMSV, PC v-raf, and PC E1A cells showed reduced NIS mRNA levels compared with the normal PC Cl 3 cells. Conversely, an almost complete absence of NIS gene expression was found in PC RET/PTC, PC KiMSV, PC p53(143ala), and PC PyMLV cell lines. CONCLUSIONS: Our data show that oncogene activation could play a role in affecting the iodide uptake ability in thyroid tumoral cells; different mechanisms are involved in the oncogene-dependent loss of NIS activity in transformed thyroid cells.     

Two arrangements of the human fetal globin genes may be responsible for high G gamma values in Chinese newborns: -G gamma-G gamma-delta-beta and -G gamma-A gamma G gamma-A gamma-delta-beta

High G gamma values (78% and higher) have been observed in about 3% of Chinese newborns from the Shanghai area. We describe here two arrangements different from the normal -G gamma-A gamma-delta-beta arrangement which have been characterized in the DNA from three of these babies. One baby was heterozygous for a chromosome with two linked G gamma globin genes (-G gamma-G gamma-delta-beta), which has also been observed in Black newborns [Powers et al, Nucleic Acids Res 12:7023, 1984], while the two other babies were heterozygous for a chromosome with triplicated gamma globin genes, presumably of the -G gamma-A gamma G gamma-A gamma-delta-beta arrangement. A similar triplication has been observed in natives of the New Hebrides [Trent et al, Nucleic Acids Res 9:6723, 1981].