New amber mutation in a beta-thalassemic gene with nonmeasurable levels of mutant messenger RNA in vivo.

We have identified a beta-thalassemia gene that carries a novel nonsense mutation in a Chinese patient. This mutation, a G to T substitution at the first position of codon 43, changes the glutamic acid coding triplet (GAG) to a terminator codon (TAG). Based on oligonucleotide hybridization studies of 78 Chinese and Southeast Asian beta-thalassemia chromosomes, we estimate that this mutation accounts for a small minority of the beta-thalassemia mutations in that population. Study of the expression of this cloned gene in a transient expression system demonstrated a 65% decrease in levels of normally spliced mutant beta-globin mRNA. However, the study of reticulocyte RNA isolated from an individual heterozygous for this mutation demonstrated a total absence of this mutant mRNA in vivo. The basis for this big discrepancy between the level of accumulated mRNA in vivo and in vitro is probably the result of differences in the stabilities of the mutant mRNA in erythroid cells.

A silencer element from the alpha-globin gene inhibits expression of beta-like genes.

We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.

Antisense RNA inhibition of phosphoprotein p18 expression abrogates the transformed phenotype of leukemic cells.

Phosphoprotein p18 was identified originally on the basis of its very high level of expression in leukemic cells of different lineages. Changes in the level of p18 accumulation and phosphorylation associated with induction of differentiation of leukemic cells suggested a potential role for this phosphoprotein in cellular proliferation and differentiation and possibly in malignant transformation. Recent studies have demonstrated that p18 plays an important role in cell cycle progression by serving as a substrate for p34(cdc2) kinase. These studies showed that inhibition of p18 expression in leukemic cells results in growth retardation and accumulation of cells in G(2)-M. In this study, we explore the potential role of p18 in cellular transformation by investigating the effects of inhibition of p18 expression on the malignant phenotype of K562 erythroleukemia cells. These studies show that antisense inhibition of p18 expression in leukemic cells results in growth arrest at a lower saturation density, loss of serum independence, and loss of anchorage-independent growth in vitro. In addition, inhibition of p18 expression results in a marked inhibition of tumorigenicity of leukemic cells in vivo in the severe combined immune deficiency mouse model. These studies demonstrate that the high level of p18 expression in leukemic cells is necessary for the maintenance of the transformed phenotype and suggest p18 as a potential target for antileukemic interventions.

Taxol and anti-stathmin therapy: a synergistic combination that targets the mitotic spindle.

Stathmin is an abundant cytosolic phosphoprotein that plays an important role in the regulation of cellular proliferation. Its major function is to promote depolymerization of the microtubules that make up the mitotic spindle. Taxol is an effective chemotherapeutic agent whose activity is mediated through stabilization of the microtubules of the mitotic spindle. We demonstrate that antisense inhibition of stathmin expression chemosensitizes K562 leukemic cells to the antitumor effects of Taxol and results in a synergistic inhibition of their growth and clonogenic potential. In the presence of stathmin inhibition, exposure to Taxol results in more severe mitotic abnormalities (hypodiploidy and multinucleation). This, in turn, results in increased apoptosis of the aneuploid cells during subsequent cell division cycles. This novel molecular-based therapeutic approach may provide an effective form of cancer therapy that would avoid the severe toxicities associated with the use of multiple chemotherapeutic agents with overlapping toxicity profiles.

Expression of the leukemia-associated gene, p18, in normal and malignant tissues; inactivation of expression in a patient with cleaved B cell lymphoma/leukemia.

p18 is a well conserved gene coding for an 18 kDa cytosolic phosphoprotein. Although the function of p18 is unknown, it is suspected of playing a role in regulation of cell proliferation or the proliferation-differentiation switch. Here we have found p18 mRNA expression highest in testis, brain, thymus and a multipotent hematopoietic stem cell line and lowest in liver. p18 was also expressed vigorously in all but one of 85 diverse tumor cell lines and primary human malignant specimens examined. In five primary tumors, expression was substantially elevated with respect to expression in contiguous normal tissue. Expression in chronic phase chronic myelogenous leukemia cells was far greater than in normal blood cells and increased with progression of disease. In liver material, the highest level of p18 was found in a primary hepatoblastoma, a stem cell tumor, whereas a benign adenoma demonstrated very low level expression. Cells from a cleaved B cell lymphoma/leukemia failed to express p18 whereas 18 specimens from other B lymphoid malignancies, including a second cleaved cell malignancy, expressed p18 at substantial levels. These data are consistent with p18 playing a role in control of cell proliferation in at least certain tissues. The questions arise if high level p18 expression in certain malignancies may play a primary role in driving cell proliferation or, based on chromosomal localization and inactivation of p18 expression in one lymphoma, if p18 may act as a tumor suppressor.

The tumor suppressor gene WT1 inhibits ras-mediated transformation.

Wilms' tumor belongs to a small group of pediatric neoplasms that have served as paradigms of human cancers in which recessive mutations play a primary role in tumorigenesis. WT1 is a candidate tumor suppressor gene that is mutationally inactivated in a proportion of both familial and sporadic Wilms' tumors. Recent studies demonstrated that WT1 can partially suppress growth of a Wilms' tumor cell line in vitro and in vivo. We investigated the ability of WT1 to inhibit the expression of the transformed phenotype in non-Wilms' tumor cells. The expression of WT1 cDNA in ras-transformed NIH3T3 cells yielded large, flat cells that exhibited complete contact-inhibition. These morphologic changes were associated with decreased proliferation, suppression of clonogenicity in soft agar and inhibition of tumor growth in nude mice. Moreover, expression of WT1 in non-transformed NIH3T3 cells resulted in similar morphologic changes and profound resistance to transformation by an activated ras oncogene. These studies suggest that tumor inhibition by WT1 in these cells may be achieved by interference with the ras-mediated signalling pathway.

WT1 expression induces features of renal epithelial differentiation in mesenchymal fibroblasts.

The WT1 tumor suppressor gene, implicated in hereditofamilial and sporadic Wilms' tumor, is required for normal renal development and is up-regulated during the mesenchymal-epithelial transition. NIH3T3 fibroblasts overexpressing WT1 were less proliferative, larger in size and more firmly attached to tissue culture plastic, suggesting an alteration of their state of differentiation. These cells were studied in vivo by subcutaneous injection into nude mice. The resulting tumors exhibited epithelioid histopathology and formed desmosome-like structures. Molecular analyses of these WT1 expressing fibroblasts grown in culture and in nude mice revealed significant alterations in the expression of many kidney epithelial markers. These studies indicate that WT1 expression can initiate features of a program of epithelial differentiation consistent with a prominent role for WT1 in the mesenchymal epithelial transition that occurs during renal development. Through this work we identified a number of novel target genes for the WT1 transcription factor, including uvomorulin, integrin alpha8 and perlecan, and suggest that WTI may activate the IGF-II gene, also implicated in the development of Wilms' tumor.

Pharmacological induction of fetal hemoglobin in sickle cell disease and beta-thalassemia.

A number of pharmacological agents are currently available for the induction of fetal hemoglobin (HbF) in patients with sickle cell disease and beta-thalassemia. Here we review the development of this new class of therapeutics and summarize the clinical trials that investigate their efficacy in patients with hemoglobin disorders. Hydroxyurea is the first of these drugs to be approved by the Food and Drug Administration for the treatment of sickle cell disease. Currently, the major focus is the development of safer agents and combinations of drugs that can increase HbF to levels high enough to prevent all complications of the disease. Progress in adapting the same strategy to the treatment of thalassemic disorders has been much slower. Although all the agents that are effective in sickle cell disease have similar HbF-inducing activity in beta-thalassemia, their use has rarely resulted in significant amelioration of the anemia. More research and more effective agents will be needed to make a significant impact on thalassemia. Nonetheless, success in this relatively young field has been very gratifying; before the end of this decade, clinically meaningful induction of HbF may become an achievable goal in most patients with hemoglobin disorders.

Intracranial solitary extraskeletal plasmacytoma resembling meningioma.

A patient had an intracranial solitary extraskeletal plasmacytoma (SEP). To date, only 12 similar cases have been reported in the English literature. This report includes the first description of computed tomographic findings in a patient with intracranial SEP. There is a striking similarity between the features of intracranial SEP and meningiomas. A detailed comparative study of the features of these two neoplasms was done. The findings indicate that the age and sex distribution, clinical features, location of the tumors, roentgenographic findings, and even the gross pathologic nature of the two neoplasms are very similar. The course and prognosis, however, are probably different. Intracranial SEP, although rare, should be included in the differential diagnosis of brain tumors that arise in areas where meningiomas commonly arise.

Stathmin expression in immortalized and oncogene transformed cells.

Stathmin is a major cytosolic phosphoprotein that is highly expressed in leukemic cells and many other malignant cell types. The level of stathmin expression in a wide variety of cancer cells suggested that this phosphoprotein may play a role in the process of malignant transformation. We compared the levels of stathmin expression in low passage primary embryonic fibroblasts and in their immortalized counterparts. The immortalized cells had a markedly higher rate of proliferation that was associated with a four fold increase in the level of stathmin expression. In contrast, transformation of the immortalized fibroblasts with seven different activated oncogenes did not result in significant changes in the level of stathmin expression nor in a consistent increase in their rate of proliferation. These observations do not support a direct role for the activation of stathmin gene expression in the transformed phenotype. We suggest that the increased levels of stathmin in transformed cells compared to non-transformed cells may be a reflection of differences in their rate of proliferation and state of differentiation.

Resolving conflicts: misconceptions and myths in the care of the patient with sickle cell disease.

Sickle cell disease is an autosomal recessive disease that primarily affects persons of African ancestry. The hallmark of the disease is hemolytic anemia and vaso-occlusive crisis. Patients often have recurrent and severely painful episodes that necessitate the use of opioids. The reluctance of some health care providers to prescribe narcotics has resulted in adversarial relationships with some patients. The socio-cultural disparity between patients and providers may play a role. However, the lack of knowledge and understanding of the underlying pathophysiology of the disease and pain are the key issues. Education, research and hands-on experience, resulting in changes in attitudes and behaviors, will ultimately lead to a more empathic approach to the sickle cell patient.

Identification of a beta-thalassemia mutation associated with a novel haplotype of RFLPs.

The study of the molecular defects that result in beta-thalassemia in Mediterraneans has uncovered a large number of unique mutations. This information is already being utilized for prenatal diagnosis of pregnancies at risk. Here, we report the definitive identification, by molecular cloning, of the beta-thalassemia mutation associated with a Mediterranean chromosome bearing a novel haplotype of restriction fragment length polymorphisms (RFLPs) in the beta gene cluster that has been previously designated as haplotype X. The thalassemia mutation was identified as a T----C base substitution at IVS-1 position 6, a mutation previously described in association with haplotype VI. We describe the use of the restriction enzyme SfaNI for the detection of this mutation and point out a possible pitfall that should be avoided if such an approach is used for the detection of this mutation, which appears to be a common cause of mild beta+-thalassemia in some populations.

Pharmacologic induction of fetal hemoglobin: raising the therapeutic bar in sickle cell disease.

The favorable effects of high levels of fetal hemoglobin (Hb F) in sickle cell disease have been recognized for several decades. This has been an important incentive for the development of therapeutic agents that increase Hb F production. 5-Azacytidine, the first such agent in clinical use, was proposed based on a molecular understanding of the role of DNA methylation in globin gene regulation. Controversy over the mechanism of Hb F induction by 5-azacytidine led to the identification of hydroxyurea as another agent that can increase Hb F production. Although the clinical benefit of hydroxyurea has been demonstrated in a randomized clinical trial, greater increases in Hb F are clearly needed for optimal therapeutic effect. Butyrates also increase Hb F levels, and their use in combination with hydroxyurea appears to be synergistic. Now that multiple therapeutic agents are available for Hb F induction, the use of combination therapy to increase Hb F levels sufficiently to prevent all the complications of sickle cell disease has become a realistic goal.

Stathmin inhibition enhances okadaic acid-induced mitotic arrest: a potential role for stathmin in mitotic exit.

Stathmin is a microtubule-destabilizing phosphoprotein that plays a critical role in the regulation of mitosis. The microtubule-depolymerizing activity of stathmin is lost upon phosphorylation in mitosis. Although the role of phosphorylation of stathmin by p34(cdc2) kinase in the assembly of the mitotic spindle is well established, the role of dephosphorylation of stathmin in mitosis is unknown. In this study, we tested the hypothesis that dephosphorylation of stathmin may be critically important for the depolymerization of the mitotic spindle and the exit from mitosis. We compared the effects of okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, on different parameters of mitotic progression in the presence or absence of stathmin deficiency. Because okadaic acid prevents dephosphorylation of stathmin and results in accumulation of the inactive phosphorylated form, exposure to okadaic acid would be expected to have a more profound effect on mitosis in the presence of relative stathmin deficiency. We found that inhibition of stathmin expression results in increased sensitivity to the antimitotic effects of okadaic acid. This was reflected by increased growth inhibition associated with mitotic arrest. A vast majority of the stathmin-inhibited cells were found to be arrested in late metaphase/anaphase and had severe mitotic spindle abnormalities. Exposure to okadaic acid also resulted in a bigger ratio of polymerized/unpolymerized tubulin in stathmin-inhibited cells relative to control cells. Because the only difference between the control and the stathmin-inhibited cells is the deficiency of stathmin in the latter, the increased susceptibility of the stathmin-inhibited cells to okadaic acid-induced mitotic arrest implies a role for stathmin in the later stages of mitosis.

Clinical and molecular correlations in the sickle/beta+-thalassemia syndrome.

Sickle/beta thalassemia is a sickling disorder of varying severity which results from compound heterozygosity for sickle cell trait and beta-thalassemia trait. Clinical and genetic studies have shown an inverse correlation between the level of hemoglobin A and the severity of the disease. It has been suggested that the level of hemoglobin A may be a function of the severity of the beta-thalassemia defect. In this study, we use molecular biological techniques to test this hypothesis. We show that the interaction of the mildest of the beta+-thalassemia genes with the sickle gene results in a high level of hemoglobin A. However, the interaction in this case resulted in a severe sickling disorder in the absence of significant anemia. We hypothesize that a mild beta+-thalassemia gene may have two opposite effects on the clinical course of sickle/beta+ thalassemia: (1) A high level of hemoglobin A which probably confers a favorable antisickling effect and (2) decreased hemolysis leading to increased numbers of total circulating red cells, thereby increasing the blood viscosity and the propensity for sickling. The inheritance of heterozygous alpha thalassemia 2 in conjunction with the mild beta+-thalassemia gene and sickle gene in this patient may have further enhanced the latter effect and resulted in a severe sickling disorder.

The major regulatory element upstream of the alpha-globin gene has classical and inducible enhancer activity.

A major positive regulatory element has recently been identified 40 kb upstream from the human zeta 2-globin gene. This regulatory element increases the expression of a linked alpha-globin gene in mouse erythroleukemia cells and in transgenic mice. This element has been shown to share many of the structural and functional features of the locus control region (LCR) of the beta-globin gene cluster. We have examined the activity of a small fragment from this regulatory domain (alpha LCR) in a transient expression system. We show that this element is active as an enhancer in the erythroid environment of K562 cells. It is somewhat less effective as an enhancer in the nonerythroid environment of HeLa cells. This alpha LCR fragment does not exhibit promoter specificity because it can activate both the promoter of its endogenous target gene and the heterologous promoter of the SV40 early genes. Although the major activity of this element is mediated by its interaction with the promoter of the alpha-globin gene, some increase in activity is seen when structural elements from the 5' end of the alpha-globin gene are included with the target promoter. In addition, we show that the enhancing activity of the alpha LCR is potentiated by hemin-induction of K562 cells. Whereas phorbol esters that induce megakaryocytic differentiation of K562 cells markedly decrease alpha-globin messenger RNA accumulation, they do not seem to have a negative effect on the activity of the alpha LCR. These studies suggest a role for the alpha LCR in the basal activity of the alpha-globin gene in erythroid cells and in its increased expression seen with erythroid differentiation. The mechanism of negative regulation of alpha-globin gene expression in phorbol-differentiated K562 cells does not appear to be mediated through the action of the alpha LCR.

A novel regulatory element of the human alpha-globin gene responsible for its constitutive expression.

The alpha-globin gene is expressed at a constitutively high level upon gene transfer into both erythroid and nonerythroid cells. The beta-globin gene, on the other hand, is dependent on the presence of a linked viral enhancer for its efficient expression upon transfer into heterologous cells. In this report, we describe a novel regulatory element within the structural alpha-globin gene which can activate its own promoter to result in a high level of expression in both erythroid and non-erythroid cells. This regulatory element does not appear to have the properties of a classical enhancer. While this element exerts a positive effect on its own promoter, we have demonstrated in a previous study that the same element exerts a negative effect on heterologous genes such as the beta- and gamma-globin genes. In this study, we localize this element to a 259 nucleotide fragment immediately downstream from the translation initiation codon which is partially overlapped by a DNase I hypersensitive domain only in erythroid cells. We propose that this element may activate the alpha-globin gene promoter in all cell types in vivo as it does in vitro. The specificity of erythroid expression of the alpha-globin gene in vivo is probably determined by a "permissive" chromatin configuration in erythroid cells and a "nonpermissive" configuration in non-erythroid cells.

Transcriptional regulation of phosphoprotein p18 during monocytic differentiation of U937 leukemic cells.

Differentiation of leukemic cells is frequently associated with downregulation of expression of genes that are important for cell proliferation and differentiation. The p18 gene encodes a major cytosolic phosphoprotein that appears to play a role in transducing signals that control the proliferation and differentiation of normal and leukemic cells. Recent reports have shown that p18 expression and phosporylation by p34cdc2 kinase is essential for progression through the cell cycle. It was previously shown that the level of p18 gene expression is markedly reduced when several different leukemic cell lines are induced to differentiate by exposer to a variety of chemical inducers. The mechanism of this downregulation of p18 mRNA expression has not been elucidated. We have explored the mechanism(s) of p18 mRNA downregulation in U937 promonocytic leukemia cells that are induced with phorbol esters to differentiate along a monocyte/macrophage pathway. We find that the half-life of p18 mRNA that is exceptionally stable in uninduced U937 cells does not change significantly with induced differentiation. We also determined that the stability of the p18 mRNA in these cells does not depend on the synthesis of a labile protein. Direct comparison of the transcription of this gene in induced and uninduced U937 cells showed that transcription is the predominant level of regulation of the activity of this gene in leukemic cells.

A novel basis for delta beta-thalassemia in a Chinese family.

We have studied a Chinese family in which beta-thalassemia and delta beta-thalassemia were found in simple and compound heterozygous states. The delta beta-thalassemia heterozygote (the mother) had 22.3% hemoglobin F, of which 40% was G gamma and 60% A gamma; globin chain studies showed an alpha/beta + gamma ratio of 1.36. The compound heterozygote for delta beta-thalassemia and beta-thalassemia (the child) had the clinical picture of thalassemia intermedia and an alpha/beta + gamma ratio of 4.44. Gene mapping studies were performed using DNA from the affected child. Seventy kilobases of DNA in the beta-globin gene cluster starting upstream from the epsilon-globin gene and ending downstream from the beta-globin gene were mapped, and no detectable deletions or rearrangements were detected. In addition, heterozygosity was detected at multiple polymorphic restriction sites in and 3' to the beta-globin gene, which excludes the possibility of a deletion of the entire beta-globin gene cluster. This is the first example of a nondeletion delta beta-thalassemia associated with increased expression of both G gamma and A gamma genes.