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To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU- Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week. The decline of gamma/gamma + beta ratio with culture time was also noted in erythroid bursts composed of immature erythroblasts. The enhanced HbF formation in peripheral blood BFU-E cultures is thus most pronounced among the bursts that become hemoglobinized early, and there is a tendency for normalization of HbF synthesis in bursts that mature in late culture days. These results can be interpreted by several alternatives, including the possibility that the expression of high HbF levels in the early days of adult BFU-E cultures is a reflection of premature commitment to terminal differentiation of progenitors that possess an active HbF program. The present data indicate that the variation of HbF synthesis with culture time should be taken into consideration when the influence of various culture conditions of HbF synthesis is studied in BFU-E cultures.

The frequency of thalassemia was determined in a group of 541 healthy adult black males. Individuals with decreased mean corpuscular hemoglobin (MCH) values were evaluated further with hemoglobin analysis, iron studies, and globin chain synthesis. Of the males screened, 13.4% had MCH levels below 27.0 pg, while 1.4% had heterozygous beta thalassemia, 2.3% had iron deficiency, and 5.7% had globin synthesis findings compatible with the diagnosis of alpha thalassemia trait (alpha thalassemia-1). This study suggests that thalassemia is one of the most frequent hematologically depictable abnormalities in American blacks.

The erythroid phenotype of HEL cells, before and after the addition of a variety of inducers, was assessed at the cellular and biochemical level. Among 14 inducers used, delta-aminolevulinic acid (delta-ALA) was identified as the most optimal inducer of heme and globin synthesis in HEL cells. The relative synthesis of globin chains produced by HEL cells, mainly gamma and alpha chains with traces of epsilon and zeta chains, was not influenced by the majority of the inducers used. However, delta-ALA and bromodeoxyuridine did increase the relative synthesis of alpha and epsilon chains respectively. Subcloning experiments revealed heterogeneity in the constitutive expression of alpha globin; however, the latter was inducible in all clones by either hemin or delta-ALA. One rare clone of HEL cells was found to produce, in contrast to parental cells, significant amounts of epsilon globin. This clone differed from K562 cells by the absence of any zeta globin expression, thus demonstrating the independent regulation of the two embryonic chains, epsilon and zeta. Changes in the expression of several surface markers specific for erythroid cells were found to accompany the globin accumulation in these cells, and some of these changes appeared to be inducer specific. Thus, the unique globin and nonglobin phenotypic properties of HEL cells and their subclones make them valuable cellular models complementary to the existing K562 cells for studying regulatory aspects of erythroid-specific proteins.

Vinblastine was administered to anemic baboons to test whether stimulation of Hb F takes place following distortion of erythropoiesis by an M-stage-specific compound. The treatments elicited erythroid cell cytoreduction followed by regeneration. During the phase of reticulocyte reduction, gamma/gamma + beta biosynthetic ratios increased without increment in F reticulocytes, suggesting that there was increased production of Hb F per F cell. The phase of reticulocyte regeneration was associated with sharp increments in relative (percentage) and absolute F reticulocytes. These data suggest that perturbations of erythropoiesis underlie the stimulation of Hb F synthesis by vinblastine. Accelerated or abnormal precursor maturation may account for the release of shift F reticulocytes with higher Hb F content, during the reduction phase. Accelerated total erythroid differentiation/maturation may account for the increment in F reticulocyte numbers during the phase of regeneneration.

Fetal hemoglobin was studied in endogenous colonies produced in plasma clot and methylcellulose cultures of circulating progenitors from patients with polycythemia vera (PV). Analysis of globin chain synthesis showed that gamma chains constituted from 13% to 42% of the non-alpha chains produced in cultured cells, whereas from 27% to over 50% of the endogenous colonies contained Hb F, as indicated by the fluorescent antibody probe. Since the endogenous colonies in PV cultures originate from the abnormal PV clone, the findings provide direct evidence that a single pluripotent stem cell can have committed progeny that differ in their expressions of the Hb F production program.

The production of fetal hemoglobin was investigated in plasma clot cultures of adult bone marrow cells from normal donors and from individuals with homozygous HbS or HbC disease. Synthesis of gamma and beta chains was assessed either by 35S-methionine labeling of cultures and measurement of the radioactivity incorporated into the methionine- containing tryptic peptides of the gamma and beta subunits or by 3H- leucine labeling and measurement of the radioactivity incorporated into the globin chains of Hbs F0 and A0 isolated by ion-exchange chromatography. The cultures from all individuals responded with increased production of HbF. Cultured cells from subjects without a hemoglobinopathy produced an average of 8.2% gamma chains (range 3.1%– 20.3%), while cultured cells from subjects homozygous for HbS or HbC produced an average of 16.6% gamma chains (range 12.2%–20.4%). These findings indicate that fetal Hb production was regularly enhanced in adult bone marrow cells triggered in vitro to clonal growth in the plasma clot culture system.

Fetal hemoglobin was studied in endogenous colonies produced in plasma clot and methylcellulose cultures of circulating progenitors from patients with polycythemia vera (PV). Analysis of globin chain synthesis showed that gamma chains constituted from 13% to 42% of the non-alpha chains produced in cultured cells, whereas from 27% to over 50% of the endogenous colonies contained Hb F, as indicated by the fluorescent antibody probe. Since the endogenous colonies in PV cultures originate from the abnormal PV clone, the findings provide direct evidence that a single pluripotent stem cell can have committed progeny that differ in their expressions of the Hb F production program.

In order to further characterize the modulation of non-alpha-chain synthesis during the maturation of erythro- blasts, we studied globin chain synthesis in individual erythroid clones of different degrees of maturity. Cultures of neonatal and adult BFU-Es were inspected at desired intervals and clones classified according to maturity as young or mature were lifted for biosynthesis. Young clones appeared to be at the onset of hemoglobin formation and were composed of mostly early basophilic erythroblasts. Mature clones were at the peak of hemoglobin formation, had intense red color, and were composed of strongly acidophilic erythroblasts. Young clones had higher γ/γ + β chain synthetic ratios compared to the γ/γ + β ratios in the mature clones of the same culture day; furthermore, young clones had higher γ/γ β ratios compared to the γ/γ + β ratios in mature clones of later culture days. These data strongly suggest that there is an asynchrony in gamma and beta chain synthesis during maturation, with gamma chains preferentially being synthesized at the onset of hemoglobinization of the erythroblast. In these experiments, the relative synthesis of the two molecular species of gamma chains, the Gγ and Aγ chains, was also determined in individual clones. When the measurements in neonatal and adult erythroid bursts were analyzed together, a strong positive correlation of γ/γ + β to gγ/Gγ + Aγ ratios was obtained, in support of the concept of synchronous γ→β and GγAγ switching during development. However, at any given level of γ/γ + β ratio, the adult cells express a higher Aγ synthesis than the neonatal cells, suggesting that there is no strict quantitative relationship between γ/γ β and Gγ/ Gγ + Aγ synthesis. The developmental stage of the cells may influence the program of relative synthesis of Gγ and Aγ chains stronger than the ratio of γ to β chain synthesis. In both the neonatal and adult BFU-E cultures, the mean Gγ/Gγ + Aγ synthetic ratio in the immature erythroid bursts was almost identical to the mean Gγ/Aγ + Aγ ratio in their mature counterparts. This suggests that the program of Gγ and Aγ chain synthesis in the cell is not significantly affected by the change in relative expression of gamma and beta chains, which occurs during the maturation of the cell.

Detailed characterization of the composite phenotype of two newly established erythroleukemia lines (OCIM1, OCIM2) shows that these lines share many of their erythroid markers (ie, surface antigens and globin program) as well as several of their nonerythroid properties (myeloid/monocytic/megakaryocytic) with the two known erythroleukemia lines (K562, HEL). In addition, each displays novel and instructive features. We argue that the surface and globin phenotype of all erythroleukemia lines is nonrandom and that it may be of physiologic relevance; it could represent the most prevalent phenotype of cells transformed by leukemia in vivo, and it raises the possibility that cells with similar potentials exist transiently during normal hematopoietic differentiation before their irreversible commitment to a single lineage. As such, these cells demonstrate a greater phenotypic adaptability in vitro than do their single lineage-committed counterparts since they can differentiate toward more than one lineage.

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (approximately 50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.

In aiming to develop a gene therapy approach for hemophilia B, we expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression (0.84 and 3.6 micrograms per 10(6) cells per day for CECs and NIH 3T3 cells, respectively) as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX of 68 kilodaltons identical to purified plasma factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including gamma-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

Human inter-alpha-trypsin inhibitor (I alpha TI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that I alpha TI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of I alpha TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum I alpha TI. RNA blot analyses of liver RNAs with H- or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000. These results unambiguously establish that I alpha TI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiol-proteinase inhibitors. These data indicate that I alpha TI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.

An erythropoietic activity that exerts a profound effect on fetal Hb synthesis is present in fetal sheep sera and it attains a peak concentration at the end of the second to the middle of the third trimester of fetal life. The activity consistently inhibits the increased synthesis of fetal Hb in cultures of burst-forming units (BFUes) from normal adults. In cultures of BFUes from homozygous beta+-thalassemias the activity produces a striking decline in gamma chain synthesis, a decline in G gamma/A gamma chain synthesis ratio, and an increase in delta/gamma and alpha/non-alpha ratios--i.e., findings suggesting a genuine gamma-to-beta switch. The activity accelerates Hb F-to-Hb A switching in neonatal BFUe cultures but it has no effect on fetal Hb synthesis in cultures of BFUe obtained from human fetuses. These findings provide direct evidence that (a) humoral factors play a role in the regulation of the switch from fetal to adult Hb formation, and (b) progenitor cells from various stages of ontogeny respond differently to these factors. The results are compatible with the hypothesis that Hb switching during development is mediated through a change in a developmental program which controls the responsiveness of progenitor cells to "switching" activities in their environment.

Somatic cell hybridization of mouse erythroleukemia (MEL) cells and HEL cells, a human erythroleukemia line that produces fetal (gamma) but fails to express adult (beta) globin, was used to test whether the expression of the two human globin genes is regulated cis or trans. An experimental approach using anti-human globin monoclonal antibodies for detection, efficient cloning, and monitoring of hybrids of interest was employed. Further characterization of hybrids used isoelectric focusing for detection of human globins and S1 nuclease mapping. In contrast to the parental HEL line, all chromosome 11-retaining HEL-MEL hybrids expressed human beta-globin, suggesting that the HEL beta-globin genes (i) are transcriptionally competent, (ii) become activated in response to a positive trans-acting element within the MEL environment, and (iii) fail to express into the HEL environment because of either the absence of a positive trans-acting element or the presence of a trans-acting inhibitor of beta-globin gene expression. In addition to beta-globin, the primary HEL-MEL hybrids co-expressed gamma-globin; however, gamma-globin expression segregated by subcloning so that secondary and tertiary clones either expressed only beta-globin or co-expressed gamma- and beta-globin. The results of subcloning can be explained by assuming that gamma-globin gene expression is controlled by a HEL cell-derived transacting element encoded by a gene not syntenic to chromosome 11 or by postulating that the HEL gamma-globin genes become randomly modified during the continuous proliferation of hybrids.