Short-chain fatty acid derivatives induce fetal globin expression and erythropoiesis in vivo

Orally bioactive compounds that induce gamma globin gene expression at tolerable doses are needed for optimal treatment of the beta-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce gamma globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce gamma globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which gamma globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce gamma globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human beta globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, gamma globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate gamma globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the beta hemoglobinopathies and useful for the oral treatment of other anemias.     

Hydroxamide derivatives of short-chain fatty acid have erythropoietic activity and induce gamma gene expression in vivo

OBJECTIVE: The hydroxamic acid derivatives of short-chain fatty acids, butyryl and propionyl hydroxamate, subericbishydoxamic acid, and suberoylanilide hydroxamic acid, are potent inhibitors of histone deacetylase (HDAC) and have been shown to induce fetal hemoglobin in vitro. In this study we examined whether these compounds have erythropoietic activity and can induce gamma globin gene expression in vivo. MATERIALS AND METHODS: Transgenic mice heterozygous for a deletion beta thalassemia and hemizygous for a human gamma globin transgene were treated with these compounds and hematologic responses as well as the induction of gamma gene expression were evaluated. Hematologic studies included measurement of reticulocytes, hematocrit, and the in vivo levels of BFU-E. Effects on globin gene expression were assessed by measuring F reticulocytes and the human gamma/murine alpha globin mRNA ratios by RNAse protection assay. RESULTS: Propionyl and butyryl hydroxamate increased reticulocytes by 71% and 139%, the in vivo BFU-E counts by 75% and 51%, and the in vivo gamma gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. CONCLUSION: Hydroxamic acid derivatives can stimulate the in vivo erythropoiesis and fetal globin production in a thalassemic murine model. The combined effect of certain histone deacetylase inhibitors on erythropoiesis and on gamma gene expression make these compounds desirable targets for development of therapeutics for beta chain hemoglobinopathies.     

mda-7/IL-24 inhibits the proliferation of hematopoietic malignancies in vitro and in vivo

OBJECTIVE: Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) has been consistently shown to exert growth inhibitory effects on various tumor types. However, the majority of these reports were limited to solid tumors. The purpose of this study was to investigate the antitumor activity of mda-7/IL-24 and the underlying mechanism in hematopoietic malignancies. MATERIALS AND METHODS: We determined the expression of mda-7/IL24 and its heterodimeric receptors in hematopoietic tumor cell lines and then stably transfected mda-7/IL-24 into K562 (leukemia) and Namalwa (lymphoma) cell lines to assess the effects of mda-7/IL-24 on cell proliferation, cell cycle, apoptosis, colony-forming ability, and tumor growth in vivo. Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells. RESULTS: Expression of mda-7/IL-24 or its intact receptor pairs was not detected in the 11 cell lines tested. Ectopic expression of mda-7/IL-24 induced significant (p < 0.05) inhibition of cell growth and colony formation in both K562 and Namalwa cells, and the growth inhibition in K562 cells was associated with G(0)/G(1) cell-cycle arrest. Results of in vivo studies showed good correlation with in vitro inhibition of tumor cell proliferation in both the cell lines. We also showed that the increase in p21(WAF-1) and BCCIP and decrease in cdk6, smurf2, and phosphorylated pRb, which are regulators of cell-cycle progression, might account for G(0)/G(1) cell-cycle arrest in K562 cells. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of mda-7/IL-24 in chronic myelogenous leukemia and lymphoma.     

Enhancement of growth and survival and alterations in Bcl-family proteins in beta-thalassemic erythroid progenitors by novel short-chain fatty acid derivatives

Accelerated apoptosis of erythroid progenitors is a characteristic of beta-thalassemia which presents a significant barrier to definitive therapeutic approaches utilizing induction of endogenous fetal globin gene expression. gamma-globin gene expression may not be inducible in, or may not be able to rescue, erythroid cells in which programmed cell death is initiated early in erythroblast development. In this report, short-chain fatty acid derivatives (SCFADs) which induce fetal globin gene expression were tested for their ability to promote proliferation and survival of erythroid progenitors cultured from beta-thalassemic subjects, and of cytokine-dependent erythroid cell lines. Certain SCFADs promoted thalassemic Bfu-e growth and cytokine-independent growth and survival of erythroid cell lines. A 40-80% increase in erythroid Bfu-e colony number was observed in cultures established with any of five mitogenic SCFADs, compared to control or butyrate-treated cultures from the same subjects. Immunoblot analysis demonstrated that these same SCFADs also regulated the expression of specific protein inhibitors of apoptosis. Anti-apoptotic ratios of the proteins Bcl-xL/Bcl-xS in thalassemic Bfu-e were increased by 30-120% with exposure to the SCFDs, compared to the ratios in the same cells cultured under control conditions. Similar anti-apoptotic increases in Mcl-1L/Mcl-1S ratios were induced by the SCFADs. These findings suggest that select fetal globin-inducing SCFADs which enhance proliferation of beta-thalassemia progenitors may enhance survival of these progenitors by altering levels of Bcl-family protein members. This combination of effects should enhance erythroid cell survival in the beta-thalassemia syndromes, allowing fetal globin gene expression to be induced more effectively than currently available, growth-suppressing, fetal globin-inducing agents, such as the butyrates or chemotherapeutic agents.     

Transgenic mouse model of pharmacologic induction of fetal hemoglobin: Studies using a new ribonucleotide reductase inhibitor,didox

Evaluation of pharmacologic agents that stimulate fetal hemoglobin production has been done mainly in baboons and macaques. We investigated whether results in transgenic mice can predict the stimulation of fetal hemoglobin in primates, by testing γ globin induction in response to a new rlbonucleotide reductase inhibitor, Didox. A transgenic mouse line carrying the human Aγ gene linked to a locus control region cassette was used. Treatment of transgenic mice with Didox resulted in induction of γ gene expression as documented by an Increase in F reticulocytes and F cells and an elevation of γ/γ + β biosynthetic ratio. Similarly, administration of Didox to a baboon in the nonanemic and chronically anemic state resulted In induction of γ gene expression as shown by increases in F reticuiocytes, F cells, and Hb F. These results suggest that the μLR-Aγ transgenic mice can be used to screen new pharmacologic compounds for γ globin inducibillty. © 1994 Wiley-Liss, Inc.     

Metabolic persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) has typically been ascribed to mutations in the beta-globin gene cluster. Pharmacologic agents, including the short-chain fatty acid butyrate, have been shown to upregulate fetal and embryonic globin gene expression. In this report we investigate the possibility that metabolic derangements characterized by an inability to metabolize another short-chain fatty acid, propionate, could be associated with a persistence of fetal hemoglobin unrelated to alterations in the beta-globin cluster. Embryonic globin gene upregulation in a murine adult erythroid cell culture was shown by RNase protection after induction with three short- chain fatty acids (C2-C5). Chart reviews and measurement of fetal hemoglobin in five patients with abnormalities in propionate (C3) metabolism were undertaken; SSCP/dideoxy fingerprint analysis of the gamma-globin gene promoters was done in three of these five patients. Twelve patients with other metabolic derangements served as controls. Only the four patients with clinically severe abnormalities in propionate metabolism (ages 2 to 11), but without anemia, showed a sustained elevation in fetal hemoglobin (3% to 10%). The level of elevation of fetal hemoglobin in these patients, who lack erythropoietic stress, suggests that propionic acid and/or its metabolites are potent stimulators of fetal hemoglobin expression. Study of this group of patients should allow unique insights into the long-term effects of sustained exposure to elevations of short-chain fatty acid levels.     

Stimulation of fetal hemoglobin production by short chain fatty acids

Butyrate, a four-carbon fatty acid, and its two-carbon metabolic product, acetate, are inducers of gamma-globin synthesis. To test whether other short-chain fatty acids share this property, we first examined whether propionic acid, a three-carbon fatty acid that is not catabolized to acetate, induces gamma-globin expression. Sodium propionate increased the frequency of fetal hemoglobin containing erythroblasts and the gamma/gamma + beta mRNA ratios in adult erythroid cell cultures and F reticulocyte production in a nonanemic juvenile baboon. Short-chain fatty acids containing five (pentanoic), six (hexanoic), seven (heptanoic), eight (octanoic), and nine (nonanoic) carbons induced gamma-globin expression (as measured by increase in gamma-positive erythroblasts and gamma/gamma + beta mRNA ratios) in adult erythroid burst-forming unit cultures. There was a clear-cut relationship between the concentration of fatty acids in culture and the degree of induction of gamma-globin expression. Three-, four-, and five-carbon fatty acids were better inducers of gamma globin in culture as compared with six- to nine-carbon fatty acids. These results suggest that all short-chain fatty acids share the property of gamma-globin gene inducibility. The fact that valproic acid, a derivative of pentanoic acid, also induces gamma-globin expression suggests that short-chain fatty acid derivatives that are already approved for human use may possess the property of gamma-globin inducibility and may be of therapeutic relevance to the beta-chain hemoglobinopathies.     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

Myofibroblastic stromal cells isolated from human bone marrow induce the proliferation of both early myeloid and B-lymphoid cells

Normal human bone marrow stromal cells (BMSC) were isolated from Dexter- type long-term cultures according to their capacity to adhere to plastic and to their lack of hematopoietic antigens. The BMSC displayed a homogeneous appearance and a myofibroblastic phenotype in culture. The stromal cells (SC) were shown to support the proliferation of purified CD34+ hematopoietic progenitors and permitted us to maintain myeloid cells for several weeks in culture. In addition, the BMSC induced the proliferation of purified CD10+ s mu- fetal BM B-cell precursors (BCP). The capacity of the BMSC to induce the proliferation of early myeloid cells was shared by several other human fibroblastic- like cell types. In contrast, the BMSC were far superior to other adherent cells for induction of BCP proliferation. This capacity was largely mediated by endogenously produced interleukin-7 (IL-7), because it could be inhibited by anti-IL-7 antibody. In line with this finding, addition of IL-7 considerably enhanced BCP proliferation in cocultures with skin fibroblasts or synoviocytes. Thus, production of IL-7 appears to be a critical parameter that determines the ability of fibroblastic- like cells to induce BCP proliferation. Taken together, our data show that normal human myofibroblastic BMSC induce the proliferation of both early myeloid and B-lymphoid cells in the absence of accessory hematopoietic cells. The present system should constitute a model to study interactions between native human BM myofibroblastic stroma and various hematopoietic cell subsets.     

Expression of interleukin 6 and its specific receptor by untreated and PMA-stimulated human erythroid and megakaryocytic cell lines

Different human hematopoietic cell lines were analyzed for the presence of interleukin 6 (IL-6) and IL-6 receptor (IL-6-R). Both IL-6 mRNA and secreted active IL-6 protein were detectable in untreated cell lines with erythroid or megakaryoblastic features (K562, HEL, KU 812, MEG-01, and Dami), but they were not expressed constitutively in other leukemic cell lines (KG1, HL60, and U937). IL-6-R production, studied by the presence of its mRNA and specific binding sites for iodinated recombinant IL-6, was detected in most cell lines except K562, HEL, and Dami. Therefore, only KU 812 and MEG-01 coexpress both IL-6 and IL-6-R. After phorbol ester myristate acetate (PMA) treatment, all the cell lines studied expressed or overexpressed IL-6. In the erythroid K562 cell line, IL-6-R was not detectable before induction, but was promptly expressed after stimulation with PMA, suggesting that some of the new features of K562 cells induced by PMA may be mediated by IL-6. However, neutralizing antibodies against IL-6 did not block either the growth arrest or the loss of the erythroid phenotype induced by PMA. The presence of IL-6 and IL-6-R in erythroid and megakaryocytic leukemic cell lines suggests that their synthesis may occur during normal hematopoietic differentiation.     

Induction of fetal hemoglobin by propionic and butyric acid derivatives: correlations between chemical structure and potency of Hb F induction

Short-chain fatty acids (C2-C9) induce fetal hemoglobin synthesis in primary cell cultures, primates, and patients. We carried out experiments to test whether relationships exist between chemical structure and the Hb F-inducing potential of several short-chain fatty acid derivatives. BFUe cultures were performed in the presence of propionic and butyric congeners, covering the full spectrum of substitutions of these molecules, including polar and non-polar groups, esters, and double bonds. We found that the fetal hemoglobin inducibility is related to the chemical structure of the inducing compound. This structure-activity relation depends on the length of carbon chain, the nature of the substitutions, and the position of more potent substitutions on the carbon chain. It appears that substitutions enhancing the inducibility of these compounds are (with decreasing potency): methyl > phenyl > hydroxy > amino groups. Placement of these substitutions at a position distal to the carboxyl group enhances gamma-globin inducibility. Presence of the carboxyl group is prerequisite for gamma-globin inducibility.     

Accumulation of γ-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.     

In vitro effect on human leukemic K562 cells of co-administration of liposome-associated retinoids and cytosine arabinoside (Ara-C).

The administration of retinoids has been demonstrated to be of potential utility in the therapy of a wide spectrum of neoplastic pathologies due to the ability to induce differentiation in a large variety of primary tumor cells as well as in vitro cultured cell lines. Moreover, a number of compounds, including hemin, cytosine arabinoside, and 5-azacytidine are able to induce erythroid differentiation of the erythroleukemic cell line K562. In this paper we determined whether a combined treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids containing liposomes lead to a full expression of differentiated functions. Liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Cell growth kinetics studies and intracellular detection of hemoglobin by benzidine staining were performed. The results obtained showed that the combined treatment with liposomes containing retinoids and sub-optimal concentration of ara-C is an effective strategy to induce K562 cell differentiation, minimizing at the same time toxic effects. Control experiments aimed to determine possible selection of subpopulations of K562 cells suggest that the observed results are not related to toxicity and/or potential selection of induced cells. In conclusion, liposomally delivered retinoids could be proposed for differentiation therapy as an effective strategy in the treatment and management of malignancy. In addition, the finding that liposomally delivered retinoids increase the capacity of cytosine arabinoside to induce erythroid differentiation, could be of interest in studies aimed at the development of treatment able to reactivate fetal globin genes in beta-thalassemia patients.     

Effect of structural analogues of propionate and butyrate on colon cancer cell growth

The aim of this study was to evaluate the effects of natural short-chain fatty acids (butyrate, propionate, valerate, acetate) and structural analogues of butyrate and propionate on cell growth and apoptosis in three human colonic adenocarcinoma cell lines (HT-29, Colo-320, and SW-948). We have previously shown that mercapto- and bromo-analogues of butyrate and propionate compete with natural short-chain fatty acids for uptake in the colonocyte. Among naturally occurring short-chain fatty acids, butyrate was the most potent inhibitor of proliferation in all three cell lines. Propionate exhibited a weaker antiproliferative effect, while other short-chain fatty acids (valerate, acetate) were ineffective. Bromo-analogues of butyrate and propionate were more potent proapoptotic agents than butyrate. In contrast to butyrate, the analogues induced strand breaks on isolated supercoiled DNA, the effect being completely reversed by a DNA-protecting agent, spermine. We conclude that bromo-analogues of butyrate and propionate are more potent proapoptotic agents than butyrate in colon cancer cells in culture. Their effect may be a result of direct DNA damage.     

Proteasome inhibitors induced caspase-dependent apoptosis and accumulation of p21WAF1/Cip1 in human immature leukemic cells

The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.     

Oncostatin M suppresses generation of lymphoid progenitors in fetal liver by inhibiting the hepatic microenvironment

OBJECTIVE: Interaction between hematopoietic cells and stromal cells is important for regulation of hematopoiesis. Numerous soluble and membrane-bound factors directly regulating hematopoiesis have been documented, but little is known about how stromal cell activity is controlled. We previously reported that fetal hepatic cells in primary culture create the hematopoietic microenvironment and support expansion of blood cells from hematopoietic stem cells. In this study, we focused on lymphopoiesis reconstituted in our culture system and analyzed how stroma-mediated lymphopoiesis is regulated during embryonic development. MATERIALS AND METHODS: Subconfluent cultures of murine fetal hepatic cells were cocultured with hematopoietic stem cells derived from fetal liver in the presence of various cytokines. After 10 days of incubation, hematopoietic cells floating over the stromal layer were analyzed by various assays, including cell proliferation and FACS analysis. RESULTS: We found that oncostatin M, an inducer of hepatic development, strongly inhibited generation of B220(+) lymphocytic cells and colony-forming unit-interleukin-7 (CFU-IL-7) from hematopoietic stem cells in our coculture system. In contrast, oncostatin M did not directly inhibit proliferation of B cells in response to IL-7 and SCF in semisolid cultures. Analysis of antigen expression in lymphoid cells revealed that oncostatin M apparently did not arrest cells at a particular stage of B-cell development. CONCLUSIONS: The results suggest that oncostatin M inhibits lymphopoiesis by suppressing stromal activity of fetal hepatic cells to stimulate generation of CFU-IL-7 from their progenitors rather than by acting directly on lymphocytic cells.     

Effect of transforming growth factor-beta 1 on proliferation and induction of hemoglobin accumulation in K-562 cells

The effects of transforming growth factor-beta 1 (TGF-beta 1) on proliferation and hemoglobinization in K-562 cells, a human multipotential hematopoietic cell line, were studied. We found that TGF- beta 1 could induce hemoglobin accumulation in K-562 cells. Various clones were selected on the basis of the inducibility of hemoglobinization by TGF-beta 1. One high response clone (no. 1) and one low response clone (no. 8) were studied in detail. Hemoglobin accumulation peaked on day 5 of culture in the presence of TGF-beta 1 (0.5 ng/mL, 20 pmol/L), when 90% of clone 1 cells, 55% of parent line cells, and less than 10% of clone 8 cells contained hemoglobin. There was a concomitant reduction in proliferation of 60% for clone 1, 40% for the parent line, and 30% for the clone 8 on day 5 of culture. Quantitative analysis showed that the hemoglobin contents in clone 1 after 5-day induction by TGF-beta 1 and hemin were 1.0 pg/cell and 2.9 pg/cell, respectively. The hemoglobin induced by TGF-beta 1 showed the same electrophoretic characteristics as the hemoglobin induced by hemin. The expression of epsilon-globin mRNA was minimally detectable in control cells and was induced in both TGF-beta 1 and hemin treated cells. Other cytokines with potential effects on K-562 cell proliferation and differentiation were also studied. Interleukin-1, interleukin-3, interferon alpha, interferon gamma, and inhibin, tested as single agents, showed minimal effects on proliferation. None of these agents could induce hemoglobinization or inhibit the hemoglobinization induced by TGF-beta 1.