A pentamer transcriptional complex including tal-1 and retinoblastoma protein downmodulates c-kit expression in normal erythroblasts.

Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb(-) SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1-E12-Lmo2 and tal-1-E12-Lmo2-Ldb1 complexes but not that of a tal-1-E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1-E12-Lmo2-Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.

Enforced TAL-1 expression stimulates primitive, erythroid and megakaryocytic progenitors but blocks the granulopoietic differentiation program.

In human adult hematopoiesis, the TAL-1 gene is up- and down-modulated in erythropoiesis and granulopoiesis, respectively [G. L. Condorelli et al., Blood, 86: 164-175, 19951. Here, it is shown that, in a hematopoietic progenitor cell (HPC) unilineage differentiation culture, tal-1 is induced and then expressed, in a sustained manner, in the megakaryopoietic lineage, whereas it is barely or not detected in the monocytopoietic series. We have investigated the role of enforced tal-1 expression by retroviral transfer into HPCs [erythroid burst-forming units and megakaryocytic and granulomonocytic colony-forming units (CFUs)], primitive HPCs (high proliferative potential colony-forming cells), and putative hematopoietic stem cells (HSCs), assayed as long-term culture initiating cells. TAL-1 overexpression induces an increase of erythroid burst-forming unit colony number and size and megakaryocytic CFU colony number and an inhibition of granulomonocytic CFU and granulocytic CFU (CFU-G) but not monocytic CFU colony number; conversely, TAL-1 mutants with defective heterodimerizing or DNA-binding domains do not exert these effects at a significant level. Although it does not affect long-term culture initiating cells, exogenous TAL-1 causes a significant proliferative stimulus on primary and secondary high proliferative potential colony-forming cells. In conclusion, exogenous tal-1 exerts differential and stage- and lineage-specific effects on the HPC/HSC differentiation/proliferation gene programs. Thus, it induces a stimulatory effect at the level of erythroid and megakaryocytic HPCs, while exerting a selective proliferative action on downstream erythropoiesis. Furthermore, it induces differential effects on the myeloid series: the partial blockade of CFU-G differentiation is possibly linked to the sharp down-modulation of endogenous TAL-1 expression at the level of the CFU-G-to-granulopoietic precursor differentiation step; in contrast, no significant effect is observed on monocytic CFU colony formation. Finally, the stimulatory effect on primitive HPCs but not putative stem cells suggests subtle differences in the effects exerted by tal-1 overexpression on primitive HPC/HSC subsets in adult life.

Enforced expression of HOXB7 promotes hematopoietic stem cell proliferation and myeloid-restricted progenitor differentiation.

Hematopoietic progenitor/stem cells (HPCs/HSCs) purified from human adult peripheral blood (PB) were triggered into cycling, retrovirally transduced with HOXB7 and then functionally assayed in vitro. HPCs were assayed in multi- and unilineage differentiation cultures in either liquid phase or semisolid medium, primitive HPCs in the high proliferative potential colony-forming cell (HPP-CFC) evaluation system and putative HSCs in Dexter type long-term culture (LTC) as LTC initiating cells (LTC-ICs). Control experiments ensured that the exogenous HOXB7 gene was constantly expressed, while the endogenous one was barely or not transcribed. Enforced expression of the gene markedly modulated the proliferation/differentiation program of the entire HSC/HPC population. Enforced HOXB7 expression exerted a potent stimulatory effect on the proliferation of the primitive HPC and putative HSC subsets, assayed as HPP-CFCs and LTC-ICs respectively. While not modifying the total number of HPCs, exogenous HOXB7 induced an increase of the number of granulo-monocytic (GM) HPCs [colony-forming unit GM (CFU-GM) CFU-GM, CFU-G and CFU-M, as evaluated by clonogenic assays] and markedly amplified the progeny of both CFU-G and CFU-M, which showed a sustained proliferation through at least 1-2 months (as evaluated in liquid suspension culture). The prolonged proliferative stimulus induced by HOXB7 transfer into LTC, primitive and GM oriented HPC culture was characterized by persistent proliferation of a discrete population of blast cells and a large pool of differentiated myeloid precursors. Altogether, these results suggest the hypothesis that the proliferative stimulus exerted by exogenous HOXB7 in primitive and GM-oriented HPCs may represent a preleukemic immortalization step. Consistent with the functional role of HOXB7 in the initial ontogenetic phase, these studies indicate that ectopic HOXB7 expression in early HPCs and HSCs from adult PB stimulates their self renewal, sustained proliferation and myeloid differentiation.

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis.

The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105.

The subjective feeling of burden in caregivers of elderly with dementia: how to intervene?

The aim of this study was to know how caregiver burden was influenced by caregivers' and patients' characteristics and by social support; the intention was to use these elements in order to suggest how to optimize the interventions which may reduce caregivers' subjective feeling of burden. A sample of 99 caregivers was studied using a descriptive-correlational design. Caregiver burden was assessed by using the caregiver burden inventory (CBI). The burden was due to restrictions on their personal time and to the sense of failure regarding their hopes and expectations. These results suggest that it could be useful to intervene in two ways. On one hand, improving all those interventions targeted to reduce restrictions on the personal caregivers' time, making respite care and day care units more accessible to elderly with dementia. On the other hand, giving caregivers the opportunity to benefit from interventions oriented to cope the sense of failure and the physical stress, to say, individual counseling or continued informal support.

Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate.

Mechanisms underlying fetal hemoglobin (HbF) reactivation in adult life have not been elucidated; particularly, the role of growth factors (GFs) is controversial. Interestingly, histone deacetylase (HD) inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF. We developed a novel model system to investigate HbF reactivation: (1) single hematopoietic progenitor cells (HPCs) were seeded in serum-free unilineage erythroid culture; (2) the 4 daughter cells (erythroid burst-forming units, [BFU-Es]), endowed with equivalent proliferation/differentiation and HbF synthesis potential, were seeded in 4 unicellular erythroid cultures differentially treated with graded dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated in terminal erythroblasts by assay of F cells and gamma-globin content (control levels, 2.4% and 1.8%, respectively, were close to physiologic values). HbF was moderately enhanced by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor treatment (up to 5%-8% gamma-globin content), while sharply reactivated in a dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The stimulatory effects of KL on HbF production and erythroid cell proliferation were strictly correlated. A striking increase of HbF was induced by combined addition of KL and NaB or TSA (40%-43%). This positive interaction is seemingly mediated via different mechanisms: NaB and TSA may modify the chromatin structure of the beta-globin gene cluster; KL may activate the gamma-globin promoter via up-modulation of tal-1 and possibly FLKF transcription factors. These studies indicate that KL plays a key role in HbF reactivation in adult life. Furthermore, combined KL and NaB administration may be considered for sickle cell anemia and beta-thalassemia therapy.

High plasma insulin and triglyceride concentrations and blood pressure in offspring of people with impaired glucose tolerance.

The plasma glucose and insulin response to an oral glucose challenge, fasting plasma lipid concentration, and blood pressure were compared in 13 offspring of parents previously diagnosed as having impaired glucose tolerance (IGT) and 13 offspring of parents previously shown to have normal glucose tolerance. The parents with IGT had higher plasma glucose, insulin and triglyceride concentration, and blood pressure than parents with normal glucose tolerance. The two groups of offspring were young and non-obese, and similar in terms of age, gender distribution, and body mass index. However, the total integrated plasma insulin response during a 75 g oral glucose tolerance test was significantly higher (p less than 0.05, Student's t-test) in offspring of parents with IGT (718 +/- 71 pmol l-1 h) than in the subjects whose parents had normal glucose tolerance (524 +/- 47 pmol l-1 h). In addition, serum triglyceride concentration was somewhat higher in offspring of parents with IGT (1.17 +/- 0.11 vs 0.92 +/- 0.08 mmol l-1, 0.10 greater than p greater than 0.05), as were both systolic (132 +/- 5 vs 118 +/- 3 mmHg, p less than 0.05) and diastolic (79 +/- 3 vs 70 +/- 2 mmHg, p less than 0.05) blood pressure. Demonstration of similar abnormalities in plasma insulin response to glucose and blood pressure regulation in patients with IGT and in their offspring is consistent with the view that these changes have a genetic component.

Habitual leisure-time physical activity is associated with differences in various risk factors for coronary artery disease.

Various risk factors for coronary artery disease (CAD) were compared in 100 healthy, male factory workers, divided into two groups of 50 each on the basis of their leisure-time activity. The two groups, designated as sedentary and physically active, were similar in terms of age and body mass index, but the physically active group had a significantly slower pulse rate. In addition, the plasma glucose and insulin responses to an oral glucose load were significantly lower in physically active individuals. Furthermore, fasting plasma triglyceride concentration was significantly lower and the high-density lipoprotein-cholesterol concentration was higher in the physically active individuals. Thus, substantial benefits in terms of CAD risk were associated with increased leisure-time physical activity.

Risk factors for coronary artery disease in healthy persons with hyperinsulinemia and normal glucose tolerance.

We studied the relation of serum insulin levels to plasma lipid levels and blood pressure in two groups drawn from among 247 healthy, normotensive nonobese subjects with normal glucose tolerance. One group of 32 subjects was defined as having hyperinsulinemia (serum insulin, greater than 2 SD above the mean) and then compared with 32 normoinsulinemic subjects (serum insulin within 1 SD of the mean) matched for age (mean, 39 years), sex (22 men and 10 women), and body-mass index (24.7). The two groups had similar patterns of smoking, drinking, and physical exercise. Plasma glucose levels after an oral glucose challenge were significantly higher (P less than 0.05) in the hyperinsulinemic group. In addition, the mean (+/- SEM) fasting plasma triglyceride levels in subjects with hyperinsulinemia were significantly higher (1.73 +/- 0.2 vs. 1.24 +/- 0.1 mmol per liter) and the plasma high-density lipoprotein cholesterol concentrations were lower (1.21 +/- 0.06 vs. 1.43 +/- 0.06 mmol per liter) than in subjects with normoinsulinemia. Both systolic (126 vs. 119 mm Hg; P less than 0.05) and diastolic (85 vs. 78 mm Hg; P less than 0.01) blood pressures were significantly elevated in the group with hyperinsulinemia. We conclude that healthy persons with hyperinsulinemia and normal glucose tolerance have an increase in risk factors for coronary artery disease, as compared with a well-matched group of healthy subjects with normal insulin levels.

PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype.

The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RAR alpha fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RAR alpha mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RAR alpha alters the early hematopoietic program through N-CoR-dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells. (Blood. 2000;96:1531-1537)

Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors.

We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem cells (HPCs/HSCs) stringently purified from adult peripheral blood and grown in different culture systems. In these experiments HPCs/HSCs were treated with FL +/- kit ligand (KL) +/- monocyte colony-stimulatory factor (M-CSF). In clonogenetic HPC culture supplemented with interleukin-3 (IL-3)/granulomonocyte-CSF (GM-CSF)/erythropoietin (Epo), FL potentiates colony-forming unit (CFU)-GM proliferation in terms of colony number and size, but exerts little effect on burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythroid megakaryocyte macrophage (CFU-GEMM) growth, whereas KL enhances the proliferation of all HPC types; combined FL+KL +/- M-CSF treatment causes a striking shift of CFU-GM colonies from granulocytic to monocytic differentiation. In liquid suspension HPC culture, FL alone induces differentiation along the monocytic and to a minor extent the basophilic lineages, whereas M-CSF alone stimulates prevalent monocytic differentiation but little cell proliferation: combined M-CSF+FL treatment causes both proliferation and almost exclusive monocytic differentiation (97% monocytes in fetal calf serum-rich (FCS+) culture conditions, mean value). At primitive HPC level, FL potentiates the clonogenetic capacity of colony-forming units-blast (CFU-B) and high proliferative potential colony-forming cells (HPP-CFC) in primary and secondary culture; KL exerts a similar action, and additive effects are induced by FL combined with KL. More important, addition of FL alone causes a significant amplification of the number of long-term culture-initiating cells (LTC-ICs), ie, putative repopulating HSCs, whereas this effect is not induced by KL. The FL effects correlate with flt3 mRNA expression in HPCs differentiating throught the erythroid or GM pathway in liquid suspension culture: (1) flt3 mRNA is expressed in freshly purified, resting HPCs; after growth factor stimulus the message (2) is abruptly down-modulated in HPC erythroid differentiation, but (3) is sustainedly expressed through HPC GM differentiation and abolished in GM precursor maturation. This pattern contrasts with the gradual downmodulation of c-kit through both erythroid and GM HPC differentiation. The results indicate that FL exerts a stimulatory action on primitive HPCs, including a unique expanding effect on putative stem cells, whereas its distal proliferative/differentiative action is largely restricted to CFU-GM and monocytic precursors. The latter effect is potentiated by KL and M-CSF, thus suggesting that the structural similarities of FL, KL, M-CSF, and their tyrosine kinase receptors may mediate positive interactions of these growth factors son monocytic differentiation.

Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis.

The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.

Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program.

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.