Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system.

Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5' untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5' splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 10(5)-10(6) transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.

Prevention of graft-versus-host disease by induction of immune tolerance with ultraviolet B-irradiated leukocytes in H-2 disparate bone marrow donor.

Transfusions (Tx) of Ultraviolet B (UVB)-irradiated peripheral blood mononuclear leukocytes (MNL) have been shown to induce humoral immune tolerance to major histocompatability complex (MHC) antigens (Blood 88:4375, 1996). To determine whether cellular immune tolerance to MHC antigens can be induced by the same approach, transplantation of bone marrow and spleen cells from tolerant donors across the H-2 barrier was conducted to study its effect on prevention of graft-versus-host disease (GVHD). After immune tolerance induction by four weekly Tx of UVB-irradiated BALB/c (H-2(d)) peripheral blood MNL into CBA/HT6 (H-2(k)) mice, bone marrow cells (BMC) and spleen MNL from tolerant or naive CBA mice were transplanted into lethally irradiated BALB/c mice. The transplanted mice were followed by measuring body weight, peripheral leukocyte counts, GVHD, survival, and cytokine response. All BALB/c recipient mice were fully engrafted with H-2(k) CBA donor cells after transplantation. The severity of GVHD was significantly attenuated in BALB/c mice transplanted with BMC and spleen MNL from tolerant CBA donor mice. The recovery of peripheral leukocyte and lymphocyte counts were faster and more complete in mice transplanted with cells from the tolerant donors. The serum cytokine profile after transplantation with tolerant donor cells showed increased interleukin-4 and reduced gamma interferon that are consistent with a polarized Th2 response. The results pooled from three separate experiments showed that BALB/c mice transplanted with 5 x 10(6) BMC and 4 x 10(5) spleen MNL from tolerant CBA donors had better overall survival than the control group (72% v 17%, P =.018). The findings show that transplantation with bone marrow and spleen cells from tolerant H-2 disparate donor mice is associated with significant attenuation of GVHD and better outcomes. The results also support that transfusions of UVB-irradiated leukocytes may induce cellular immune tolerance.

In vitro selection for K562 cells with higher retrovirally mediated copy number of aldehyde dehydrogenase class-1 and higher resistance to 4-hydroperoxycyclophosphamide.

Previously, we have reported the successful expression of human aldehyde dehydrogenase class-1 (ALDH-1) in K562 leukemia cells using a retroviral vector and demonstrated low expression that resulted in up to three-fold increase in resistance to 4-hydroperoxycyclophosphamide (4-HC), an active derivative to cyclophosphamide. The purpose of this study was to investigate whether in vitro treatment with 4-HC will allow selection of K562 cells expressing higher levels of ALDH-1, and whether these selected cells are more resistant to 4-HC. Stably transfected or transduced K562 cells with retroviral pLXSN vector containing ALDH-1 cDNA (ALDH-1 cells) were treated repeatedly with 4-HC and then allowed to grow to confluence in liquid culture. Subsequently, the resistance to 4-HC of ALDH-1 cells treated once (ALDH-1+) or twice (ALDH-1++) with 4-HC was compared to ALDH-1 cells or wild-type K562 cells (WT cells). The results show significant increase in 4-HC resistance of ALDH-1+ (2- to 16-fold, p < 0.005) over ALDH-1 or WT cells. No difference was detected between ALDH-1+ and ALDH-1++. In addition, higher ALDH-1 mRNA and enzyme activity were found in ALDH-1+ compared to ALDH-1 cells. Southern analysis of DNA extracted from the different experimental groups demonstrated an eight-fold increase in ALDH-1 cDNA in ALDH-1+ versus the ALDH-1 cells. This was confirmed by sequential FISH analysis using biotin labeled pLXSN/ALDH-1 vector. Positive signals consistently localized to the centromeric region of chromosome 9 and the long arm of chromosome 17 were demonstrated only in the ALDH-1+ cells and represented a fusion product of multiple copies of the pLXSN/ALDH-1 vector. In summary, we have demonstrated that in vitro treatment with 4-HC results in the selection of K562 cells with multiple copies of ALDH-1 gene that are clustered in two main integration sites. These cells demonstrate significantly higher resistance to 4-HC when compared to previously untreated cells. Such successful in vitro selection could have significant implications for future cancer gene therapy protocols.

Bleomycin-induced differentiation of bcl-2-transfected U937 leukemia cells.

In this study, the effect of bleomycin on myeloid leukemic U937 cells transfected with murine bcl-2 or vector alone (vector containing neomycin-resistant gene only; MNC) was investigated. Sublethal concentrations of bleomycin (1 microgram/ml) induced a decrease in cell growth in both vector-only and bcl-2-transfected U937 cells. In MNC-transfected U937 cells, loss of viable cells and colony-forming cells was observed following 4 days of bleomycin treatment. This was accompanied by accumulation of cells in the G0-G1 phase of the cell cycle and morphological changes as well as induced expression of markers associated with myeloid differentiation (i.e., increased granularity and CD11b expression). In contrast, bcl-2-transfected U937 cells maintained viable cell numbers and colony forming cells for up to 2 weeks in the presence of bleomycin. These cells did not show cell cycle accumulation in G0-G1 and in addition, displayed delayed expression of differentiation markers when compared with bleomycin-treated, vector-only transfected U937 cells. One day following a 5-day exposure to 1 microgram/ml bleomycin, a loss of differentiated cells by apoptosis, as demonstrated by dUTP and analyzed by flow cytometry, was observed in the MNC-transfected U937 cell population. In contrast, differentiated bcl-2-transfected U937 cells remained viable for 2 weeks following bleomycin treatment. The results of this study suggest that up-regulated Bcl-2 not only blocks apoptosis in proliferating myeloid cells but also delays or prevents apoptosis in differentiated myeloid cells.

Quantitation of resistance to cytosine arabinoside by myeloid leukemic cells expressing bcl-2.

The presence of bcl-2 in myeloid leukemias has been associated with a decrease in therapy-induced apoptosis, reduced patient survival and in vitro autonomous growth of leukemic cells. The present study focuses on the quantitation of resistance to increasing doses of 1-beta-d-arabinofuranosylcytosine (Ara-C) by using hematological tumors expressing different levels of bcl-2. Scanning densitometry of Western blots demonstrated that the myeloid U-937 cells express low levels of bcl-2 (RD = 0.008), whereas the follicular lymphoma RL-7 expressed very high levels (RD = 3.084). Colony formation was also examined following incubation with Ara-C and RL-7 cells demonstrated a higher clonogenic survival (LD50 = 0.5 microns) when compared with U-937 cells (LD50 = 0.005 microM). Similarly, the level of bcl-2 expression in each cell line was also related to apoptosis with U-937 cells demonstrating increased DNA fragmentation when compared with RL-7 cells. To further evaluate the effect of upregulated bcl-2 on Ara-C treatment, U-937 cells were transfected with a retroviral vector carrying the murine bcl-2 or vector alone. Upregulation of bcl-2 by myeloid leukemic cells increased the resistance by 3 logs to Ara-C when comparing LD50 values from clonogenic assays, and decreased apoptosis by at least 3 logs when measuring dUTP positive cells by flow cytometry.

Overexpression of the human aldehyde dehydrogenase class I results in increased resistance to 4-hydroperoxycyclophosphamide.

A correlation between overexpression of aldehyde dehydrogenase and resistance to oxazaphosphorines, widely used anticancer agents, has been shown. To investigate the direct role of the human aldehyde dehydrogenase class 1 (ALDH-1) in the resistance to one of these agents, 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide, neomycin-selectable plasmid or retroviral constructs harboring the wild-type ALDH-1 complementary DNA in the sense orientation were transfected into K562 leukemic cell lines. Polymerase chain reaction (PCR) analysis confirmed the presence of vector DNA in the stably transfected K562 cells. Reverse transcriptase PCR and Northern and Western blot analysis showed expression of ALDH-1 mRNA and protein in the cells transfected with ALDH-1 in the sense orientation but not in cells transfected with vector alone. The activity of the expressed ALDH-1 was demonstrated using spectrophotometric assay. Stably transfected K562 cells were subjected to various doses of 4-HC, and cell viability was assayed using clonogenic cell culture in semisolid medium. Results demonstrate that K562 cells transfected with ALDH-1 in the sense orientation display increased resistance to 4-HC compared with wild-type or vector-transfected K562 cells. Furthermore, the addition of diethylaminobenzaldehyde, a specific inhibitor for ALDH-1, restored the sensitivity of the ALDH-1-expressing K562 cells to 4-HC. Thus, the data pinpoint the direct role of ALDH-1 in the protection against 4-HC cytotoxicity.

Interleukin-1 and tumor necrosis factor alpha induce class 1 aldehyde dehydrogenase mRNA and protein in bone marrow cells.

Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) protect normal human hematopoietic progenitors from the toxicity of 4-hydroperoxycyclophosphamide (4-HC). Aldehyde dehydrogenase Class 1 (ALDH-1) is the enzyme that inactivates 4-HC. Diethylaminobenzaldehyde (DEAB), a competitive inhibitor of ALDH-1, was shown to prevent the protective effects of IL-1 and TNF alpha. In this study, we examined the effect of IL-1 and TNF alpha on the expression of ALDH-1 in normal bone marrow as well as malignant cells. ALDH-1 mRNA and protein were quantified using Northern and Western blotting, respectively. In addition, the ALDH-1 enzyme activity in untreated as well as IL-1 and TNF alpha treated bone marrow cells was determined spectrophotometrically. The role of glutathione (GSH) in the protection against 4-HC toxicity was also studied. The results show that pretreatment with IL-1 and TNF alpha for 6 h or 20 h increase the expression of ALDH-1 mRNA and protein, respectively, in human bone marrow cells. In contrast, IL-1 and TNF alpha treatment did not affect the ALDH-1 expression in several leukemic and solid tumor cell lines, regardless of whether or not ALDH-1 is expressed constitutively. Furthermore, the ALDH-1 enzyme activity was significantly induced in bone marrow cells after 20 h pre-treatment with IL-1 and TNF alpha. Finally, the depletion of or inactivation of GSH did not affect the protection against 4-HC toxicity. In conclusion, inhibition of the protection from 4-HC toxicity by DEAB, together with the increase in ALDH-1 expression and activity, provide strong evidence that IL-1 and TNF alpha mediate their protective action, at least partially, through ALDH-1.

Protective effects of human interleukin-1 on hematopoietic progenitor cells from L-phenylalanine mustard.

The effects of interleukin-1 (IL-1) on protecting both human and murine bone marrow cells were studied using in vitro clonogenic assays, long-term bone marrow cultures and in vivo mouse studies. Incubation with 100 ng/ml human recombinant IL-1 beta for 20 hours prior to a one hour exposure to L-phenylalanine mustard (L-PAM) provided significant protection of bone marrow colony forming cells when compared to bone marrow cells not exposed to IL-1. Complete inhibition of colony formation was observed above 40 mu M L-PAM in the absence of IL-1 preincubation; whereas, colonies were still detectable in cultures which were initiated with IL-1-treated bone marrow cells. Similar results demonstrating greater protection with IL-1 incubation from L-PAM were seen when murine bone marrow cells were assayed for long-term culture-initiating cells. Furthermore, IL-1 protects long-term repopulating hematopoietic stem cells from L-PAM when studied using an in vivo irradiated mouse assay. In contrast, incubation with IL-1 does not protect colony formation by K562, KG-1 or HL-60 leukemic cell lines implying that protection by IL-1 may be selective. Finally, the protection observed by IL-1 preincubation could be abrogated by incubation with 50 mu M L-buthionine sulfoximine (BSO). This result indicates that IL-1 may increase the amount of glutathione in hematopoietic cells and be responsible for the observed protection from L-PAM.

Role of manganese superoxide dismutase in radioprotection using gene transfer studies.

Overexpression of manganese superoxide dismutase (MnSOD) has been postulated as one possible mechanism of radioprotection for hematopoietic cells. In this study retroviral constructs having the human MnSOD gene in both the sense and antisense orientations and the Neo-R gene as a selectable marker were transfected into the human erythroleukemic cell line K562 and the human melanoma cell line A375 by electroporation. Stably transfected K562 and A375 cells selected in G418 for 3 weeks were subjected to various doses of irradiation, and cell viability was assayed using a colony assay system in semisolid medium. Results demonstrated that K562 cells transfected with MnSOD in the antisense orientation displayed increased sensitivity to irradiation compared to parental or vector-transfected K562 cells. In contrast, A375 cells transfected with the sense MnSOD gene demonstrated increased resistance to irradiation compared to parental or vector-transfected A375 cells. The expression of the MnSOD gene in these transfected cell lines correlates with the up- or down-modulation of radiosensitivity. Thus, increased MnSOD protein was seen in the A375 cells containing the sense MnSOD, whereas decreased MnSOD protein was seen in the K562 cells containing the antisense MnSOD. These data provide evidence for the direct role of MnSOD in radioprotection using antisense gene transfer/inhibition studies.

Mechanisms of protection of hematopoietic stem cells from irradiation.

Current therapies for the treatment of malignancies are associated with significant limitations to the hematopoietic system since chemotherapy and radiation therapy do not discriminate between normal and malignant cells. Since bone marrow depression occurs at low to midlethal doses of irradiation, approaches to improving the therapeutic index of treatment must include measures to enhance the sensitivity of the tumor relative to normal hematopoietic tissue or, by reducing toxicity to normal hematopoietic tissues leaving tumor resistance unchanged. Radioprotective agents have been proposed to unravel the fundamental processes by which radiation itself damages hematopoietic tissue. In radiotherapy, the importance of these agents is derived from their potential use as selective protectors against radiation damage to normal hematopoietic tissue such that higher doses of radiation can be delivered to tumors to achieve a therapeutic advantage. A variety of agents have been and are being evaluated as possible protectants. These include aminothiols, synthetic polysaccharides, vitamins and cytokines. This review attempts to summarize the role both chemical and biological response modifiers play as hematopoietic radioprotectors. In addition, possible mechanisms of protection of hematopoietic stem cells from irradiation are discussed.

Radioprotection of hematopoietic stem cells by interleukin-1.

Radioprotective agents such as interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha), when given prior to irradiation, protect animals from radiation damage. However, in vivo administration of these cytokines does not allow one to determine whether the protective effects act directly on the hematopoietic system. In the present study, we subjected male bone marrow cells to in vitro treatment with IL-1 prior to irradiation and bone marrow transplantation. We found that male bone marrow cells pretreated with IL-1 prior to irradiation increased the survival of irradiated female recipient mice when compared with nontreated irradiated marrow cells. In addition, irradiated female recipients that received IL-1-pretreated male donor bone marrow cells displayed an increased presence of male donor cells in their bone marrow, spleen, and thymus for up to 3 months posttransplant. Furthermore, serial transplantation studies revealed that male cells could only be detected in tertiary female recipients who received bone marrow from mice transplanted with IL-1-treated cells. These results indicate that IL-1 pretreatment protects both short-term and long-term repopulating stem cells from an irradiation insult and that these cells are capable of reconstituting the myeloid and lymphoid organs of recipient mice.

A role for manganese superoxide dismutase in radioprotection of hematopoietic stem cells by interleukin-1.

Pretreatment with interleukin-1 (IL-1) has been shown to protect mice from the myelotoxicity associated with irradiation via a mechanism potentially mediated through the induction of the antioxidant enzyme manganese superoxide dismutase (MnSOD). In this study, we have compared the ability of IL-1 to induce MnSOD mRNA in murine bone marrow cells and human cell lines with its ability to protect these cells against the damaging effects of ionizing radiation. Bone marrow cells obtained from mice 6 hours after a single injection of IL-1 demonstrate a dose-dependent increase in the expression of MnSOD RNA. In this same study, IL-1 was also shown to be radioprotective when given to mice 20 hours before lethal irradiation. Similarly, in vitro treatment with IL-1 of bone marrow cells isolated from 5-fluorouracil-treated mice results in elevated levels of MnSOD RNA. Pretreatment with IL-1 also protected bone marrow long-term culture-initiating cells capable of reconstituting irradiated stromal cultures from an irradiation insult. Furthermore, IL-1-treated human bone marrow cells display both elevated MnSOD RNA and protein levels when compared with media controls. The human A375 melanoma, A549 adenocarcinoma, and factor-dependent TF-1 leukemic cell lines demonstrate low basal MnSOD RNA levels that increase following treatment with IL-1. For the A375 cells, this correlates with increased MnSOD protein expression and radioprotection by IL-1 using a colony assay. In contrast, the chronic myelogenous leukemic cell line, K562, displays a high basal MnSOD RNA level, and this RNA expression is not further increased by IL-1 treatment. In addition, these cells are comparatively radioresistant and are not further protected by IL-1 treatment. Finally, the Mo-7 cell line displays a low basal level of MnSOD RNA that correlates with a high sensitivity to irradiation and IL-1 pretreatment has no effect on MnSOD RNA levels. Our results indicate that increased radioprotection by IL-1 correlates with the induction of the antioxidant enzyme MnSOD and this induction may be an important factor in IL-1 radioprotection.

Hematopoietic growth factor induction of gamma-glutamyl transferase in the KG-1 myeloid cell line.

The enzyme gamma-glutamyl transferase (GGT) is a multifunctional enzyme that participates in a number of metabolic processes, including the conversion of leukotriene C4(LTC4) to leukotriene D4(LTD4). LTD4 is necessary for normal myeloid proliferation and differentiation. We have examined the ability of hematopoietic growth factors (HGF) to induce GGT enzyme activity and mRNA content in a HGF-responsive cell line (KG-1). Incubation of KG-1 with recombinant human cytokines interleukin-1 beta (IL-1 beta), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF), but not interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) or monocyte colony-stimulating factor (M-CSF), results in significant increases in GGT enzyme activity. The increases in GGT activity are both dose- and time-dependent. In response to IL-1, increases in enzyme activity are seen by 6 hours and activity is maximal by 24 hours. GGT mRNA increases also occur and peak by 3 to 6 hours. These results indicate that induction of increases in GGT mRNA levels and enzyme activity occur in myeloid cells in response to HGFs. This induction, together with the requirement for LTD4 for normal granulopoiesis, supports a role for GGT in the cellular events occurring in myeloid cells in response to HGFs.

The therapeutic potential of interleukin-1 and tumor necrosis factor on hematopoietic stem cells.

Dose intensity is emerging as a crucial determinant of success in cytotoxic cancer therapy; however, myelosuppression presents as one of the major complications encountered with increased dose intensity. Therefore, investigators are looking at the use of cytokine administration in combination with cytotoxic therapy to overcome this problem. Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) have been shown to be beneficial in protecting the hematopoietic system from radiation and chemotherapy. In this report, we give an overview of studies using IL-1 and TNF-alpha as protective agents and discuss possible mechanisms involved in their protective action. Mice pretreated with IL-1 and/or TNF-alpha were shown to be protected from the lethal effects of radiation and it has been suggested that the mechanism for this protection may be through the production of the antioxidant enzyme manganese superoxide dismutase. Similarly, aldehyde dehydrogenase, an enzyme important in the metabolic pathway of cyclophosphamide compounds, has been implicated as being important in the protection of hematopoietic cells from 4-hydroperoxycyclophosphamide. While IL-1 and TNF-alpha stimulate both of these enzymes, other mechanisms are probably also operative for other forms of chemotherapy, i.e. IL-1 and TNF-alpha were shown to protect hematopoietic progenitors from phenylketophosphamide, a cyclophosphamide derivative that is not metabolized by the enzyme aldehyde dehydrogenase. Furthermore, malignant as well as normal cells may possess receptors for these cytokines; therefore, IL-1 and TNF-alpha will have to be selective in their protection. They must be capable of protecting normal hematopoietic cells while rendering malignant cells susceptible to the toxic actions of the chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of cells capable of reconstituting long-term bone marrow stromal cultures from 4-hydroperoxycyclophosphamide by interleukin 1 and tumor necrosis factor.

The cytokines interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have been implicated in protecting normal hematopoiesis from both irradiation and chemotherapy damage. The mechanism of action of these cytokines and which cells are protected is not known. In this study, we report on the ability of IL-1 and TNF-alpha to protect hematopoietic cells capable of repopulating irradiated long-term bone marrow stromal cultures from 4-hydroperoxycyclophosphamide (4-HC). Irradiated long-term bone marrow cultures recharged with hematopoietic cells pretreated with IL-1 and TNF-alpha prior to 4-HC were shown to give rise to greater numbers of colony-forming cells at 4-5 weeks of culture within both the nonadherent and adherent cell populations of the long-term cultures when compared to controls. These results suggest that IL-1 and TNF-alpha can protect human long-term culture-initiating cells, which are closely related to reconstituting stem cells.

Role of aldehyde dehydrogenase in the protection of hematopoietic progenitor cells from 4-hydroperoxycyclophosphamide by interleukin 1 beta and tumor necrosis factor.

Preincubation of human bone marrow cells with interleukin 1 beta (IL-1) and tumor necrosis factor alpha (TNF-alpha) for 20 h can protect early progenitor cells from 4-hydroperoxycyclophosphamide (4-HC) toxicity. In this report, we have studied the mechanism for such protection. We examined the effect of the length of incubation time and found that preincubation for at least 20 h with IL-1 and TNF-alpha is needed for significant protection. The addition of 2 micrograms/ml cycloheximide, a protein synthesis inhibitor, during the 20-h preincubation completely abolished the protection observed for all colony-forming cells. In order to study the role of aldehyde dehydrogenase (ALDH), an enzyme which inactivates 4-HC, we used diethylaminobenzaldehyde, an inhibitor of ALDH. Diethylaminobenzaldehyde was added during the last 10 min of the 20-h preincubation with IL-1 and TNF-alpha. Diethylaminobenzaldehyde prevented the protection of colony-forming cells from 4-HC. Finally, using the same protection assay system, we showed that a 20-h preincubation with IL-1 and TNF-alpha can also protect early progenitor cells from phenylketophosphamide, an analogue of 4-HC which is resistant to inactivation by ALDH. From these studies, we conclude that preincubation with IL-1 and TNF-alpha for at lest 20 h is required for the protection of early progenitor cells from 4-HC. During that time period, protein synthesis, specifically aldehyde dehydrogenase synthesis, is critical for the protection from 4-HC. Preincubation with IL-1 and TNF-alpha also protects early progenitors from phenylketophosphamide. Because phenylketophosphamide cannot be metabolized by ALDH, the reason for this protection must be due to other, as yet unidentified, mechanisms.

Effects in vivo of purified recombinant human activin and erythropoietin in mice.

Activin has been shown to act in vitro as an erythroid specific enhancing activity for erythropoietin (epo)-stimulated erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells. To evaluate effects in vivo, purified recombinant activin-A and epo were administered s.c. to hypertransfused polycythemic mice for analysis of iron (59Fe) uptake, and to previously untreated mice for effects on reticulocyte release and proliferation of bone marrow (BM) and spleen (Spl) hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) and BM stem (CFU-S) cells. Activin alone had no effect in polycythemic BDF1 mice, but synergised with epo to significantly enhance 59Fe-incorporation into erythrocytes. In untreated C3H/HeJ mice, a single dose of activin enhanced reticulocyte release in 24 h to the level seen with epo. Activin plus epo did not further enhance reticulocyte release. Reticulocyte release was still apparent at day 4 in mice given epo twice a day for 3 days, but not in mice given activin twice a day for 3 days. Activin or epo each significantly enhanced the percent cells in S-phase of BM and Spl CFU-GEMM, BFU-E and CFU-GM in C3H/HeJ, W/Wv and Sl/Sld mice and BM CFU-S in BDF1 mice. The combination of epo plus activin did not further enhance proliferation. These results demonstrate activin's erythropoietic enhancing activities in vivo, and also activin and epo induction of enhanced proliferation of non-erythroid, as well as erythroid progenitors.