Erythropoietic protoporphyria in the house mouse. A recessive inherited ferrochelatase deficiency with anemia, photosensitivity, and liver disease.

A viable autosomal recessive mutation (named fch, or ferrochelatase deficiency) causing jaundice and anemia in mice arose in a mutagenesis experiment using ethylnitrosourea. Homozygotes (fch/fch) display a hemolytic anemia, photosensitivity, cholestasis, and severe hepatic dysfunction. Protoporphyrin is found at high concentration in erythrocytes, serum, and liver. Ferrochelatase activity in various tissues is 2.7-6.3% of normal. Heterozygotes (+/fch) are not anemic and have normal liver function; they are not sensitive to light exposure; ferrochelatase activity is 45-65% of normal. Southern blot analysis using a ferrochelatase cDNA probe reveals no gross deletion of the ferrochelatase gene. This is the first spontaneous form of erythropoietic protoporphyria in the house mouse. Despite the presence in the mouse of clinical and biochemical features infrequent in the human, this mutation may represent a model for the human disease, especially in its severe form.

Identification of a novel cDNA, encoding a cytoskeletal associated protein, differentially expressed in diffuse large B cell lymphomas.

Diffuse large B-cell lymphomas (DLBL) constitute an heterogeneous clinico-pathological entity. To characterize molecular events related to histological subtypes, clinical presentation or outcome, we compared the mRNAs expressed in a limited series of DLBL by Differential display-reverse transcription (DDRT) and cloned a differential cDNA, that we called LB1. LB1 open reading frame encodes a 683 amino-acid polypeptide that does not show significant homology upon comparison to protein databases, nor any structural domain relating LB1 to an already known protein family. Immunofluorescence analysis of transfected COS cells showed a cytoplasmic filamentous staining, indicating that LB1 protein is tightly associated with cytoskeletal fibers. Two LB1 transcripts, a major 3.6-3.9 Kb and a minor 2.2 Kb transcripts, were detected among human haematopoietic and non-haematopoietic lines and tissues. LB1 transcripts were abundant in testis, thymus and in tumour derived cell lines, while barely detectable in liver, prostate and kidney. Concerning DLBL, LB1 expression was high in two cases of DLBL, and low or undetectable in four others, confirming the differential expression previously observed in the DDRT experiment. Furthermore, LB1 gene mapped to chromosome 13q14, a region that has been involved as a chromosomal breakpoint in DLBL. The cellular function of LB1 and its relationship with B cell maturation and/or oncogenesis remain to be established.

Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.

The fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.

Improvement of mouse beta-thalassemia by recombinant human erythropoietin.

Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines.

The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters.

Mouse beta thalassemia, a model for the membrane defects of erythrocytes in the human disease.

The present study describes the pathophysiology, at the cellular level, of the mouse beta thalassemia and shows the pertinence of this model for the human disease. The homozygous state of mouse beta thalassemia is characterized by a clinical syndrome similar to the human beta thalassemia intermedia, but it cannot be explained by the small deficiency in beta chain synthesis. The small pool of unpaired and soluble alpha chains present in mouse reticulocytes contrasts with the large amount of insoluble alpha chains in erythrocytes which is induced by the high instability of mouse alpha chains and the absence of significant proteolysis. The amount of insoluble alpha chains associated with red cell ghosts is similar in human and mouse disease of similar severity. The study of membrane protein defects showed a decreased amount of spectrin (alpha and beta chains) and dramatic changes in the distribution of the most reactive thiol groups of membrane proteins. These results were similar to that previously described in the human disease (Rouyer-Fessard, P., Garel, M. C., Domenget, C., Guetarni, D., Bachir, D., Colonna, P., and Beuzard, Y. (1989) J. Biol. Chem. 264, 19092-19098). Abnormal density distribution curves of erythrocytes and oxidant-induced lysis of red blood cells used as functional tests were similar in the human and mouse beta thalessemia. We conclude from the present study that 1) mouse beta thalassemia is an excellent model for the membrane defects occurring in the human disease; 2) disease expression is not the reflection of the globin chain unbalance only nor of the soluble pool of alpha hemoglobin chain but mainly is a reflection of insoluble alpha chains; and 3) the rate of proteolysis and instability of alpha chains are important factors which must be taken into consideration in the pathophysiology and the clinical heterogeneity of the disease.

Covalent binding of human thrombin to a human endothelial cell-associated protein.

Binding of 125I-thrombin to endothelial cells derived from human umbilical vein was studied in tissue culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed covalent binding of thrombin in a 72-kDa complex. This binding is specific and requires the catalytically active site of the enzyme. Formation of the complex could be detected as early as 3 min after addition of thrombin or with a thrombin concentration as low as 0.5 nM. This irreversible binding exhibits thrombin dose-dependence and reaches maximum levels at a concentration of 50 nM (10 fmol/10(5) cells). Some characteristics of the 72-kDa complex were compared to those of the complexes formed between thrombin and protease nexin originating from fibroblasts or platelets: (i) its electrophoretic mobility on SDS-PAGE is identical to that of the thrombin-platelet protease nexin complex, (ii) heparin prevents the appearance of the complex on the cell surface, (iii) plasmin in a 100-fold molar excess prevents the covalent linkage of thrombin, suggesting that the protease specificity of the endothelial component involved in the complex might not be restricted to thrombin. Yet no release, nor any secretion of the endothelial protein, could be detected. These results indicate that active thrombin binds covalently to a specific endothelial protein that is in several respects similar to fibroblast or platelet protease nexin and provides a thrombin binding site distinct from thrombomodulin and glycosaminoglycans.

Expression of TAL-1 proteins in human tissues.

Rearrangement of the tal-1 gene (also known as SCL or TCL-5) occurs in at least 25% of T-cell acute lymphoblastic leukemias (T-ALLs) and results in the aberrant expression of tal-1 mRNA in the neoplastic cells. Also, tal-1 mRNA is constitutively expressed in erythroid precursors and megakaryocytes. This report describes a direct immunocytochemical study of the distribution and localization of TAL-1 protein in normal human tissues and cell lines using four monoclonal antibodies raised against recombinant TAL-1 proteins. One of these reagents recognizes a protein of 41 kD molecular weight in in vitro-translated TAL-1 proteins, two others recognize proteins of 39 and 41 kD molecular weight, and the fourth antibody also recognizes a TAL-1 protein of 22 kD in addition to the 39- and 41-kD proteins. These anti-TAL-1 antibodies label the nuclei of erythroid precursor cells and megakaryocytes in fetal liver and adult bone marrow. The punctate pattern of nuclear labeling suggests that TAL-1 may comprise part of a novel nuclear structure, similar to that recently found for the PML protein. The nuclei of T cell lines known to express mRNA encoding the full-length TAL-1 protein (eg, CCRF-CEM, RPMI 8402, and Jurkat) are also labeled. A study of normal human tissues (including thymus) showed labeling of smooth muscle, some tissue macrophages, and endothelial cells. TAL-1 protein is undetectable in other cell types. These reagents may play an important role in the diagnosis of T-ALL and could also be used in the context of lymphoma diagnosis on routinely fixed material.

Loss of TAL-1 protein activity induces premature apoptosis of Jurkat leukemic T cells upon medium depletion.

Transcriptional activation of the tal-1 gene occurs in -30% of patients with T cell Acute Lymphoblastic Leukemia and is therefore likely to be involved in human T cell leukemogenesis. However, the TAL-1 protein functional properties involved in this process have not been assessed so far. We have derived a clonal subline of the Jurkat T cell line which produced solely a mutant truncated form of TAL-1 protein. Sequencing of genomic DNA and cDNAs showed that the only transcribed tal-1 allele of this mutant subline harbored a G nucleotide insertion at codon 270. The resulting frameshift modifies TAL-1 residues 272-278 and creates a stop at codon 279. Although the deletion of the 53 carboxy-terminal residues of the TAL-1 protein did not directly affect the TAL-1 basic helix-loop-helix domain (residues 185-243), it had drastic effects on TAL-1 functional properties, since the mutant subline exhibited a dramatic decrease of protein binding activity to the TAL-1 DNA consensus sequence. Growth curves indicated that the mutant subline exhibited premature apoptosis upon medium depletion or serum reduction when compared with the parental cells. However, no difference between Jurkat and the mutant subline was observed in etoposide- or Fas/APO-1-triggered apoptosis. Stable expression of the mutant TAL-1 protein in Jurkat cells resulted in a phenotype that was similar to that of the mutant Jurkat subline, indicating that the TAL-1 mutant protein behaved like a dominant negative mutant and that the premature apoptosis of the mutant subline upon medium depletion was the consequence of the loss of TAL-1 protein activity.

Low level of interleukin 10 synthesis during liver allograft rejection.

Interleukin 10 (IL-10) is a macrophage and T-cell-derived cytokine with potent immunosuppressive properties. To assess its role in liver allograft rejection, we evaluated the plasma level and in situ production of IL-10 after liver transplantation and designed in vitro studies to asses the effects of IL-10 on the allogeneic response. Normal controls and liver transplant recipients with acute rejection, chronic rejection, other complications (recurrent hepatitis C, biliary complications), or no complications were evaluated. The plasma IL-10 level was measured by an immunoenzymatic technique. IL-10 expression in the liver was detected on frozen liver biopsies by in situ hybridization and immunohistochemistry. Plasma IL-10 levels were not elevated during acute or chronic rejection, when compared with liver recipients with uncomplicated transplants. IL-10 mRNA and protein expressions in the liver graft were restricted to rare scattered sinusoidal cells of transplant recipients with acute or chronic rejection, as well as in those with no complications. In mixed lymphocyte cultures performed with peripheral blood mononuclear cells (PBMC) from normal subjects, IL-10 decreased the cell proliferation in a dose-dependent manner, and this immunosuppression was synergistic with that of cyclosporine or FK506. These findings indicate that IL-10 production is low during allograft rejection. Thus, IL-10 therapy in association with cyclosporine or FK506 might be proposed after liver transplantation.

Human interleukin-10 expression in T/natural killer-cell lymphomas: association with anaplastic large cell lymphomas and nasal natural killer-cell lymphomas.

Several cytokines have been implicated in the pathogenesis of human lymphomas. Among them, interleukin-10 (IL-10) is a pleiotropic cytokine with various biological effects on B and T lymphocytes. Its expression has been essentially studied in B-cell lymphomas, where it appears to act as an autocrine growth factor. BCRF1 (also called viral IL-10), an open reading frame of Epstein-Barr virus, exhibits extensive sequence and functional homologies with human IL-10. Some entities belonging to T- or natural killer (NK)-cell lymphomas are characterized by a frequent association with Epstein-Barr virus. We analyzed 39 cases of peripheral T-cell lymphoma, as well as 7 cases of nasal NK-cell lymphoma, for the presence of IL-10 transcripts by in situ hybridization, to see whether this cytokine was expressed in these tumors and whether its expression could be related to their histological subtype and to the presence of Epstein-Barr virus. Because the riboprobe used for in situ hybridization recognizes both human and viral IL-10, 12 cases were also analyzed by reverse transcription-polymerase chain reaction to verify the human or viral origin of IL-10. It was found that 8 of 11 (73%) anaplastic large cell lymphomas (ALCLs), 2 of 11 (18%) pleomorphic T-cell lymphomas, and 3 of 7 (43%) nasal NK-cell lymphomas exhibited a large number of IL-10-expressing cells, whereas only rare scattered cells were detected in angioimmunoblastic (11 of 11) and in gammadelta T-cell lymphomas (6 of 6). In ALCLs, the pattern of IL-10 mRNA-expressing cells showed an overlapping with the CD30 staining and preferential localization in sinusal and perifollicular areas, thereby suggesting that IL-10-expressing cells were tumor cells. Furthermore, IL-10 transcripts were detected in the SU-DHL-1 anaplastic lymphoma cell line. No correlation with Epstein-Barr virus profile was found, because all cases of ALCL were negative for EBER 1 and 2 genes by in situ hybridization. We confirmed the presence of human IL-10 mRNA by reverse transcription-polymerase chain reaction in ALCLs as well as in NK-cell lymphomas, whereas viral IL-10 was not detected. Thus, human and not viral IL-10 is frequently expressed by tumor cells in ALCLs and nasal NK-cell lymphomas. In view of its function in the proliferation and the differentiation of cytotoxic T and NK cells, and its immunosuppressive properties, IL-10 may have a role in the pathogenesis of these lymphomas.

Correlations between p53-protein accumulation, serum antibodies and gene mutation in colorectal cancer.

Only half of colorectal-cancer patients elicit serum antibodies in response to intratumoral p53-gene mutations. Our study was designed to compare cellular events (p53-protein accumulation and gene mutations) with the presence of circulating anti-p53 antibodies (p53-Ab). Thirty-five colorectal-cancer patients were studied for their intratumoral p53-protein accumulation and circulating p53-Ab. Tumour DNA was analyzed for genomic mutations in a sub-set of 28 patients. In all, 18 tumours (51.4%) were positive by immunohistochemistry, and 17 tumour extracts were shown to contain "mutant" conformation p53 protein, 16 of them being were concordant by both methods. Of the 28 tumours tested by DGGE, 16 contained alterations in p53 exons 5 to 8 (57.1%). Of 12 tumours without detectable mutations, 10 were "mutant"-conformation-negative by immunohistochemistry and ELISA. Paradiploid tumors presented more frequently wild-type p53 genes and were significantly less frequently immunohistochemistry- or p53-Ab-positive than polyploid tumors. Circulating p53-Ab were detected in the serum of 11 patients (31%). In 9/11 cases, a gene mutation was found in the corresponding tumour. Three of four mutations in exon 8 and 3/3 mutations in exons 5-6 were associated with p53-Ab, in contrast with only 3/9 mutations in exon 7. We found good agreement in the detection of p53-gene alterations by different methods. However, our data suggest that all gene mutations may not be equivalent in term of immunogenicity.