ALBERT

Description: We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

Description: We examined the synthesis of lactoferrin, an iron binding protein that, among hematopoietic cells, is restricted to secondary granules of polymorphonuclear leukocytes. Lactoferrin biosynthesis was absent from leukemic myeloblasts and promyelocytes but abundant in normal bone marrow and both the bone marrow and peripheral blood of patients with chronic myelogenous leukemia (CGL) if the samples contained substantial numbers of myelocytes and metamyelocytes. Lactoferrin was present in the steady state in normal or CGL bands and polymorphonuclear leukocytes, but no lactoferrin biosynthesis was detectable in these samples. Taken together, these results suggest that lactoferrin accumulation begins with the onset of biosynthesis at the myelocyte stage and is largely complete by the beginning of the band stage of maturation. HL-60 cells, a permanent promyelocytic leukemia cell line, synthesized no lactoferrin. Translation of messenger RNA in Xenopus laevis oocytes revealed that mRNA from patients with chronic myelogenous leukemia and abundant myelocytes and metamyelocytes directed the synthesis of readily detectable amounts of lactoferrin, whereas HL-60 cells contained no translatable lactoferrin mRNA. We thus hypothesize that lactoferrin is a useful marker of gene expression restricted to the terminal stages of granulocyte maturation. Biosynthesis of this protein appears to be mediated by appearance of translatable mRNA at the myelocyte stage, coincident with development of secondary granules. Absence of lactoferrin production by HL-60 cells is due to absence of translatable lactoferrin mRNA, either because of lineage infidelity of these transformed cells or because of arrest before the developmental stage at which secondary granules appear.

Description: Lactoferrin is a member of the transferrin family of iron-binding proteins. It is found in several glandular epithelial tissues and human neutrophils, where it is localized to secondary granules. To examine the mechanisms controlling lactoferrin gene expression in neutrophils and defects in its expression in acute leukemia, we have cloned a lactoferrin cDNA from a chronic myelogenous leukemia library, and used it to obtain genomic clones representing the chromosomal lactoferrin gene. Using polymerase chain reaction, primer extension, and S1 analysis, we have identified the 5′ end of the lactoferrin mRNA. We have defined a putative promoter region for the gene, and characterized its first two exons. In addition, we have examined the structure of these regions in DNA from HL60 cells. HL60 is a leukemic cell line that undergoes phenotypic neutrophil maturation on exposure to dimethyl sulfoxide (DMSO). However, the cells cannot be induced to express any secondary granule protein genes. We have shown that the 5′ end of the lactoferrin gene, including the putative promoter region, is entirely normal in HL60. By Northern analysis, nuclear run-on studies, and primer extension assays we have shown that the gene is not transcribed in DMSO-induced HL60 cells. This supports the hypothesis that the defect in HL60 is an abnormality in the production or activity of a transacting regulator of lactoferrin gene expression.

Description: The human leukemia cell line K562 was derived from a patient with chronic granulocytic leukemia. This cell line has subsequently been shown to possess phenotypic markers typical of erythroid and myeloid cells. Using a rabbit antiserum directed against purified platelet glycoproteins (PGPs), we have obtained evidence for the constitutive expression of PGPs on the surface of K562 cells. PGPs expressed have been tentatively identified as IIa and III based on their apparent migration in a 7% sodium dodecylsulfate polyacrylamide gel. K562 may become an important tool for the study of early events involved in megakaryocytic differentiation.

Description: The gamma-globin genes from a patient homozygous for a deletion form of hereditary persistence of fetal hemoglobin (HPFH-1) have been cloned and sequenced. The DNA sequence of the patient's gamma-globin genes corresponds to a previously identified sequence framework (chromosome A) with the exception of 10 base changes. Seven of these base changes can be attributed to normal allelic variation generated by small gene conversion events. The remaining three base changes are present in a 0.76 kb HindIII fragment containing a putative enhancer located 3′ to the A gamma-globin gene. The same three base changes have also been described in the Seattle variant of nondeletion HPFH. We have analyzed 16 alleles from non-HPFH individuals and five alleles from individuals with nondeletion or deletion HPFH for the presence of these base changes by polymerase chain reaction amplification of cloned or chromosomal DNA and hybridization to allele-specific oligonucleotide probes. Although these base changes were found in an individual with HPFH-2, they were not found in the DNA from two patients with nondeletion HPFH. More importantly, all three base changes were detected in DNA from five non-HPFH individuals and appear to be common in blacks. We conclude that these base changes do not correlate with an HPFH phenotype and that the significant mutation in HPFH-1 is the deletion of over 100 kb of genomic DNA.

Description: Lactoferrin is a major constituent of polymorphonuclear leukocyte granules and is present in mature neutrophils but not in blasts or promyelocytes. We have isolated a cDNA probe for lactoferrin and used it to study the synthesis of lactoferrin mRNA by normal and leukemic granulocyte precursors. The probe pHL-41 has been subcloned in phage m13 and characterized by restriction endonuclease analysis and nucleic acid sequencing. pHL-41 contains approximately 40% of the coding sequence of the lactoferrin gene. The 3′ untranslated region includes a stop codon and a possible polyadenylation signal. There is a greater than 98% agreement between the cDNA-deduced amino acid sequence and that determined by analysis of the protein. Myeloid cells from normal bone marrow and circulating leukocytes from patients with chronic granulocytic leukemia contain lactoferrin mRNA transcripts that are indistinguishable in size and relative quantity. The human promyelocytic leukemia cell line HL-60 contains no lactoferrin mRNA. Induction of monocytic or granulocytic differentiation fails to induce the synthesis of detectable lactoferrin message. Similarly, studies with the human myeloblastic leukemia cell line PLB-985 reveal the inability of these cells to produce lactoferrin mRNA even under conditions that bring about morphologically demonstrable granulocytic differentiation. These data suggest that granulocytic differentiation in the leukemic cell lines is incomplete or defective. The presence of lactoferrin may play a role in the orderly expression of the genetic program leading to the development of the normal mature granulocyte.

Description: Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.

Description: Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, andone patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.

Description: The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3–4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3–5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Description: Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.