Liver injury is associated with mortality in sickle cell disease.

BACKGROUND: Increased life expectancy in sickle cell disease (SCD) has resulted in greater recognition of the consequences of repeated intravascular vaso-occlusion and chronic haemolysis to multiple organ systems. AIM: To report the long-term consequences of liver dysfunction in SCD. METHODS: A cohort of SCD patients was prospectively evaluated at the National Institutes of Health (NIH) Clinical Center. The association of mortality with liver enzymes, parameters of liver synthetic function and iron overload was evaluated using Cox regression. RESULTS: Exactly, 247 SCD patients were followed up for 30 months of whom 22 (9%) died. After controlling for predictors, increased direct bilirubin (DB), ferritin, alkaline phosphatase and decreased albumin were independently associated with mortality. In a multivariable model, only high DB and ferritin remained significant. Ferritin correlated with hepatic iron content and total blood transfusions but not haemolysis markers. Forty patients underwent liver biopsies and 11 (28%) had fibrosis. Twelve of 26 patients (48%) had portal hypertension by hepatic venous pressure gradient (HVPG) measurements. All patients with advanced liver fibrosis had iron overload; however, most patients (69%) with iron overload were without significant hepatic fibrosis. Ferritin did not correlate with left ventricular dysfunction by echocardiography. DB correlated with bile acid levels suggesting liver pathology. Platelet count and soluble CD14 correlated with HVPG indicating portal hypertension. CONCLUSIONS: Ferritin and direct bilirubin are independently associated with mortality in sickle cell disease. Ferritin likely relates to transfusional iron overload, while direct bilirubin suggests impairment of hepatic function, possibly impairing patients' ability to tolerate systemic insults.

Mechanisms of hemolysis-associated platelet activation.

BACKGROUND: Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias, and in other conditions, and is associated with hypercoagulable states. Hemolysis has been shown to potently activate platelets in vitro and in vivo, and several mechanisms have been suggested to account for this, including: (i) direct activation by hemoglobin (Hb); (ii) increase in reactive oxygen species (ROS); (iii) scavenging of nitric oxide (NO) by released Hb; and (iv) release of intraerythrocytic ADP. OBJECTIVE: To elucidate the mechanism of hemolysis-mediated platelet activation. METHODS: We used flow cytometry to detect PAC-1 binding to activated platelets for in vitro experiments, and a Siemens' Advia 120 hematology system to assess platelet aggregation by using platelet counts from in vivo experiments in a rodent model. RESULTS: We found that Hb did not directly activate platelets. However, ADP bound to Hb could cause platelet activation. Furthermore, platelet activation caused by shearing of red blood cells (RBCs) was reduced in the presence of apyrase, which metabolizes ADP to AMP. The use of ROS scavengers did not affect platelet activation. We also found that cell-free Hb enhanced platelet activation by abrogating the inhibitory effect of NO on platelet activation. In vivo infusions of ADP and purified (ADP-free) Hb, as well as hemolysate, resulted in platelet aggregation, as shown by decreased platelet counts. CONCLUSION: Two primary mechanisms account for RBC hemolysis-associated platelet activation: ADP release, which activates platelets; and cell-free Hb release, which enhances platelet activation by lowering NO bioavailability.

Near-infrared spectra absorbance of blood from sickle cell patients and normal individuals.

Limited data are available regarding the physicochemical dynamics of tissue hypoxia in sickle cell disease. Studies using near-infrared spectroscopy (NIRS) have reported that patients with sickle cell disease (SCD) have lower cerebral oxygen saturation values (rSO2) than normal individuals. The reason SCD patients have subnormal rSO2 values is not known. It may be related to the degree of anaemia, sickle haemoglobin, disease complications and the possibility of SCD different NIRS absorbance spectra than normal. This study compared NIRS absorbance spectra of blood with adult haemoglobin AA, sickle haemoglobin SS, and AS. Venous blood was collected from SCD (SS and AS) and non-SCD patients (AA). Whole blood, cell free haemoglobin samples were scanned through the wavelength range of 600-1000 nm. The results showed no different NIRS spectra absorbance between the haemoglobin's AA, SS. It thus appears that lower brain oxygen saturation in sickle cell anaemia patients is related to impaired oxygen carrying capacity or delivery by sickle haemoglobin.

Homozygous factor-V mutation as a genetic cause of perinatal thrombosis and cerebral palsy.

A 5-year old girl with cerebral palsy (CP), preterm birth, postnatal aortic thrombus, and cerebellar venous infarction who is homozygous for the thrombophilic factor-V Leiden (fVL) mutation is reported. The role of hereditary thrombophilic disorders in the development of perinatal vascular lesions such as aortic thrombi, renal-vein thrombosis, venous-sinus thrombosis, and cerebral infarction is unknown. This case report brings into question a potential association between fVL, perinatal vascular lesions, perinatal stroke, and CP.

Human genetic diseases of proteolysis.

Although in the past protein stability commonly has been considered an inherent property of a given protein, the truth is far more complex. Elaborate enzymatic systems exist in multiple intracellular compartments to hydrolyze proteins. These systems are capable of providing a sensitive mechanism to regulate protein expression, a mechanism that is complementary to the transcriptional and translational control mechanisms that influence protein synthesis. The power of regulated proteolysis has been well-demonstrated in the abrupt degradation of cyclins that underlies eukaryotic cell cycle progression. Coincidental with the recent rapid gains in understanding proteolysis at a biochemical level, several human diseases have been found to result from disordered proteolysis. This article reviews several examples of human disease resulting from mutations of genes encoding serine proteases, cysteine proteases, and their inhibitors. Examples are also presented of human diseases resulting from disorders in the highly intricate ubiquitin-proteasome pathway of protein degradation. It is certain that many more human diseases will be associated in the future with disorders of proteolysis.

The mUBC9 murine ubiquitin conjugating enzyme interacts with the E2A transcription factors.

The ubiquitin-mediated degradation of cellular proteins requires the sequential activity of E1, E2 and, in some cases, E3 enzymes. Using the yeast two-hybrid system, we have cloned 1.0- and 2.5-kb cDNAs encoding the identical murine E2, or ubiquitin conjugating enzyme by virtue of its interaction with the E2A transcription factor. This cDNA encodes the 158-amino-acid protein, mUBC9, which has considerable sequence homology to UBC9 from Saccharomyces cerevisiae and HUS5 from Schizosaccharomyces pombe and is identical to the human UBC9 protein. HUS5 is essential for DNA damage repair, whereas UBC9 is necessary for G2/M progression. The human protein has been shown to correct the UBC9 defect in yeast. Antisera raised against bacterially expressed mUBC9 fusion protein recognize a murine cellular protein of approximately 18 kDa, corresponding to the predicted mobility. Unlike E2A, the mUBC9 protein level is not regulated by serum growth factors. The activity of the apparent homologues UBC9 and HUS5 suggests that mUBC9 may be involved in the degradation of key nuclear proteins that regulate cell cycle progression.

Successful treatment of life-threatening acute chest syndrome of sickle cell disease with venovenous extracorporeal membrane oxygenation.

PURPOSE: We describe a pediatric patient with sickle cell disease and life-threatening acute chest syndrome who was successfully treated with venovenous extracorporeal membrane oxygenation (ECMO). PATIENT AND METHODS: An 8-year-old boy with sickle cell disease presented with vaso-occlusive crisis, which progressed to fulminant acute chest syndrome requiring a partial exchange transfusion and mechanical ventilation. Despite very high ventilator settings and significant barotrauma, hypoxia persisted and circulatory failure occurred. He was then successfully treated with venovenous ECMO for 11 days. One month after decannulation he had a seizure associated with abnormalities on magnetic resonance images (MRIs). His disease has been managed with a chronic transfusion program since then. Follow-up after 5 years reveals normal pulmonary function tests, a normal magnetic resonance angiogram (MRA), and above-average cognitive skills. CONCLUSION: This is the first report of a pediatric patient with acute chest syndrome successfully managed with venovenous ECMO. His course was complicated by a seizure associated with MRI abnormalities, although the outcome has been excellent. This case suggests that treatment with venovenous ECMO should be strongly considered for sickle cell patients with life-threatening acute chest syndrome, despite maximal conventional support.

E2A basic-helix-loop-helix transcription factors are negatively regulated by serum growth factors and by the Id3 protein.

Id3, a member of the Id multigene family of dominant negative helix-loop-helix transcription factors, is induced sharply in murine fibroblasts by serum growth factors. To identify relevant targets of Id3 activity, the yeast two-hybrid system was used to identify proteins that dimerize with Id3. Four murine cDNAs were identified in the screen, all of which encode helix-loop-helix proteins: E12, E47, ALF1 and Id4. Co-immunoprecipitation assays confirm that Id3 interacts with E12, E47 and two alternative splice products of ALF1 in vitro. Id3 disrupts DNA binding by these proteins in vitro and blocks transcriptional activation by these factors in cultured murine cells. Additionally, Id3 shows evidence of interacting with the related proteins E2-2 and MyoD, but not c-Myc. These results suggest that Id3 can function as a general negative regulator of the basic-helix-loop-helix family of transcription factors exemplified by the 'E' proteins and MyoD. Although it was previously suspected that E2A is constitutively expressed, our data indicate that E2A is induced in quiescent fibroblasts, by growth factor withdrawal but not by contact inhibition of cell proliferation. These observations extend the role of Id3 in the functional antagonism of E2A-class transcription factors, and suggest that E2A proteins may mediate growth inhibition.

High glucocorticoid receptor content of leukemic blasts is a favorable prognostic factor in childhood acute lymphoblastic leukemia.

We have previously shown that the number of glucocorticoid receptors (GR) per cell in malignant lymphoblasts from children with newly diagnosed pre-B- and early pre-B-cell acute lymphoblastic leukemia (ALL) has a positive correlation with the probability of successful remission induction (Quddus et al, Cancer Res, 45:6482, 1985). We report now on the long-term outcome for these patients treated on a single protocol with 3 different treatment arms, all of which included glucocorticoid pulses during maintenance therapy. GR were quantitated in leukemic cells from 546 children with ALL at the time of diagnosis. Immunophenotyping studies were performed on all specimens. Prior studies showed that in pre-B- and early pre-B-cell ALL, successful remission induction was associated with a median GR number of 9,900 sites/cell, whereas induction failure was associated with a median receptor number of 4,800 sites/cell. Long-term follow-up of these patients shows an association between higher GR number and improved prognosis. The 5-year event-free survival of 61.0% (SE 2.8%) for patients whose leukemic cells had greater than 8,000 receptors/cell and 47.3% (SE 3.3%) for those with less than 8,000 receptors/cell is significantly different (P < .001). This difference remains significant when adjusted multivariately for blast immunophenotype and clinical risk factors (P < .001) or for treatment type (P < .001). We conclude that GR number greater than 8,000 sites/leukemic cell is a favorable prognostic marker for children with acute lymphocytic leukemia. This finding offers deeper insights into molecular mechanisms of anti-leukemia therapy and suggests that manipulation of steroid receptor number might augment the antitumor response, thus opening new avenues for basic and clinical research.

Function of the c-Myc oncoprotein.

The c-Myc protein, the product of the c-myc proto-oncogene, is a nuclear phosphoprotein with DNA binding properties. Deregulated c-myc expression participates in the development of experimentally induced tumors, and its expression appears to be abnormal in many naturally occurring malignancies. Although the precise molecular mechanism of c-Myc activity in oncogenesis and in normal cell proliferation is unknown, recent advances have uncovered a series of molecular and cellular properties of c-Myc. These properties include nuclear localization, transcriptional activation, oligomerization nonspecific and specific DNA binding. Recently, the c-Myc protein was found to heterodimerize with Max, a protein that cooperates with c-Myc to bind specifically to a core DNA sequence, CAC(G/A)TG. These characteristics suggest that c-Myc participates in the regulation of gene transcription in normal and neoplastic cells.

Activation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells.

Members of the Myc family of proteins share a number of protein motifs that are found in regulators of gene transcription. Conserved stretches of amino acids found in the N-terminal transcriptional activation domain of c-Myc are required for cotransforming activity. Most of the Myc proteins contain the basic helix-loop-helix zipper (bHLH-Zip) DNA-binding motif which is also required for the cotransforming activity of c-Myc. L-Myc, the product of a myc family gene that is highly amplified in many human lung carcinomas, was found to cotransform primary rat embryo cells with an activated ras gene. However, L-Myc cotransforming activity was only 1 to 10% of that of c-Myc (M. J. Birrer, S. Segal, J. S. DeGreve, F. Kaye, E. A. Sausville, and J. D. Minna, Mol. Cell. Biol. 8:2668-2673, 1988). We sought to determine whether functional differences between c-Myc and L-Myc in either the N-terminal or the C-terminal domain could account for the relatively diminished L-Myc cotransforming activity. Although the N-terminal domain of L-Myc could activate transcription when fused to the yeast GAL4 DNA-binding domain, the activity was only 5% of that of a comparable c-Myc domain. We next determined that the interaction of the C-terminal bHLH-Zip region of L-Myc or c-Myc with that of a Myc partner protein, Max, was equivalent in transfected cells. A Max expression vector was found to augment the cotransforming activity of L-Myc as well as that of c-Myc. In addition, a bacterially synthesized DNA-binding domain of L-Myc, like that o c-Myc, heterodimerizes with purified Max protein to bind the core DNA sequence CACGTG. To determine the region of L-Myc responsible for its relatively diminished cotransforming activity, we constructed chimeras containing exons 2 (constituting activation domains) and 3 (constituting DNA-binding domains) of c-Myc fused to those of L-Myc. The cotransforming potencies of these chimeras were compared with those of full-length L-Myc of c-Myc in rat embryo cells. The relative cotransforming activities suggest that the potencies of the activation domains determine the cotransforming efficiencies for c-Myc and L-Myc. This correlation supports the hypothesis that the Myc proteins function in neoplastic cotransformation as transcription factors.

DNA binding by the Myc oncoproteins.

The c-Myc protein is a potential activator of transcription, with the ability to bind in a heterodimer form with Max to DNA sequences containing the core hexanucleotide sequence CAC(G/A)TG. These properties are shared with L-Myc, a homologous oncoprotein expressed in small cell lung carcinoma cells; with N-Myc, expressed in neuroblastoma cells; and with avian v-Myc, the c-Myc homolog expressed by a chicken retrovirus. The c-Myc, and probably v-Myc, proteins also have nonspecific DNA binding function, which may improve the kinetics of specific DNA binding. Curiously, this domain appears not to be conserved in L-Myc or N-Myc [22]. The data that have accumulated to date are consistent with a model in which a c-Myc/Max heterodimer positively regulates the transcription of growth-related genes, with Max homodimer functioning as a negative regulator of the same genes (Fig. 4) [55]. Max is expressed constitutively at low levels, whereas c-Myc is expressed at low levels in quiescent cells, but high levels of c-Myc are induced by mitogenic stimulation [56]. Thus, in proliferating cells c-Myc/Max heterodimers might bind to the regulatory elements of growth-related genes, where the c-Myc TAD might stimulate transcription. Conversely, in quiescent cells with little c-Myc present, Max homodimers might predominate. They might bind to exactly the same regulatory elements, but due to the apparent absence of a TAD in Max [36], transcription might be repressed. Validation of this model will require the demonstration of clear regulation of a physiological promoter of a growth-related gene by c-Myc/Max. Although it is widely believed that Myc proteins function as transcriptional activators, this hypothesis has only been conclusively supported recently [57, 58]. A theory that c-Myc plays a role in DNA replication is not as well substantiated at this point. It is even possible that Myc might be involved in both transcription and replication. Although the function of these fascinating proteins has been enigmatic for a decade, the rate of progress in our understanding of Myc function is accelerating. Such progress will undoubtedly lead to a deeper appreciation of this protein, which lies at the crossroads of cellular proliferation and oncogenesis.

Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins.

A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.

Max: functional domains and interaction with c-Myc.

The product of the c-myc proto-oncogene is a DNA-binding protein, the deregulated expression of which is associated with a variety of malignant neoplasms. The cDNA for the max gene was recently cloned as a result of the ability of its protein product to interact with the c-Myc protein. We studied bacterially produced Max, c-Myc, and a series of truncated c-Myc proteins. Full-length c-Myc alone cannot bind DNA. However, a truncated c-Myc protein comprising the basic, helix-loop-helix, and leucine zipper regions can bind specifically to DNA bearing the sequence GGGCAC(G/A)TGCCC. Max protein, either alone or in a heteromeric complex with full-length c-Myc, binds to the same core sequence. Using a novel combination of chemical and photo-cross-linking analysis, we demonstrate that either Max or a c-Myc/Max heteromeric complex binds to DNA virtually exclusively in a dimeric structure. Using fusion proteins in cultured cells, we establish a number of functional characteristics of Max. First, we show that Max can interact with c-Myc intracellularly in a manner dependent on the integrity of the helix-loop-helix and leucine zipper motifs. Second, a nuclear localization domain that contains the sequence PQSRKKLR is mapped to the carboxy-terminal region of Max. Third, Max lacks a transcriptional activation domain that is functional in Chinese hamster ovary cells when fused to a heterologous DNA-binding domain. These data suggest that Max may serve as a cofactor for c-Myc in transcriptional activation or, by itself, as a transcriptional repressor.

Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization.

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

A potential transcriptional activation element in the p53 protein.

The human p53 gene codes for a 393 amino acid nuclear phosphoprotein. p53 is most commonly described as a tumor suppressor, or anti-oncogene, although its role in vivo remains unclear. We report that GAL4-p53 fusion protein can activate transcription of a CAT reporter gene downstream of a GAL4-DNA binding site. We tested both the amino terminal 160 amino acids and the carboxyl terminal 233 amino acids of the p53 protein and found that the transcriptional activating (TA) region was restricted to the amino terminal fragment. These results imply that p53 may be a transcriptional activating factor (TAF); furthermore, these data lend support to the hypothesis of p53 as a positive regulator of transcription which might mediate its tumor suppressor role by inducing expression of a set of genes with a negative effect on cellular growth.

An amino-terminal c-myc domain required for neoplastic transformation activates transcription.

The product of the c-myc proto-oncogene is a nuclear phosphoprotein whose normal cellular function has not yet been defined. c-Myc has a number of biochemical properties, however, that suggest that it may function as a potential regulator of gene transcription. Specifically, it is a nuclear DNA-binding protein with a short half-life, a high proline content, segments that are rich in glutamine and acidic residues, and a carboxyl-terminal oligomerization domain containing the leucine zipper and helix-loop-helix motifs that serve as oligomerization domains in known regulators of transcription, such as C/EBP, Jun, Fos, GCN4, MyoD, E12, and E47. In an effort to establish that c-Myc might regulate transcription in vivo, we sought to determine whether regions of the c-Myc protein could activate transcription in an in vitro system. We report here that fusion proteins in which segments of human c-Myc are linked to the DNA-binding domain of the yeast transcriptional activator GAL4 can activate transcription from a reporter gene linked to GAL4-binding sites. Three independent activation regions are located between amino acids 1 and 143, a region that has been shown to be required for neoplastic transformation of primary rat embryo cells in cooperation with a mutated ras gene. These results demonstrate that domains of the c-Myc protein can function to regulate transcription in a model system and suggest that alterations of Myc transcriptional regulatory function may lead to neoplastic transformation.