Antisense TGF-beta Research Articles

Evidence for Control of Nitric Oxide Synthesis by Intracellular Transforming Growth Factor-β1 in Tumor Cells: Implications for Tumor Development

Transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate NO synthesis in a variety of normal cells. In the present study, we investigated the influence of TGF-beta1 upon NO production in tumor cells and its consequences for tumor development. During the growth of PROb colon carcinoma cells intraperitoneally injected in syngeneic BDIX rats, intratumoral concentration of TGF-beta1 increases while NO concentration stays very low. Tumor regression induced by intraperitoneal injections of a lipid A is associated with a decrease in TGF-beta1 and an increase in NO intratumoral concentration. In these tumors, PROb tumor cells are the NO- and TGF-beta1-secreting cells. Using PROb cells transfected with an expression vector coding for TGF-beta1 antisense mRNA, we demonstrate in vitro that there is an inverse correlation between the amount of TGF-beta1 secreted and the ability of PROb cells to secrete NO. As the same results were obtained in the presence of an anti-TGF-beta type II receptor neutralizing antibody, and as exogenous TGF-beta1 is without any effect on NO secretion by PROb cells, TGF-beta1 apparently down-regulates NO synthesis in PROb cells by an intracellular mechanism. These results suggest that endogenous TGF-beta1 constitutes a potential target in a search for new antitumoral agents.

Alpha2-macroglobulin reduces paracrine- and autocrine-stimulated matrix synthesis of cultured rat hepatic stellate cells.

Transforming growth factor beta1 (TGF-beta1) is considered to represent a major fibrogenic mediator in the liver. The aim of the present study was to investigate whether alpha2-macroglobulin (alpha2M) might reduce paracrine- and autocrine-stimulated matrix synthesis of cultured rat hepatic stellate cells (HSCs) by scavenging TGF-beta. Using native agarose electrophoresis, we demonstrated that alpha2M binds [125I]-TGF-beta1 within minutes. Preincubation of transiently acidified supernatants of cultured Kupffer cells, secondary cultured (activated) HSC and platelet lysate with, respectively, 500 and 2000 microg mL-1 alpha2M significantly reduced the concentration of active TGF-beta1 in these media. As a consequence of TGF-beta scavenging by alpha2M, paracrine-stimulated proteoglycan synthesis of primary cultured HSCs was reduced significantly. Furthermore, addition of 200 microg mL-1 alpha2M to passaged (activated) HSCs resulted in (a) a reduction in autocrine-stimulated extracellular matrix synthesis (proteoglycan -52%, fibronectin -55%) and (b) increased cell proliferation. A similar reduction in matrix synthesis was observed after the addition of 5 micromol L-1 TGF-beta1 antisense oligonucleotide to activated HSCs. We conclude that alpha2M reduces paracrine-and autocrine-stimulated extracellular matrix synthesis of cultured HSCs by scavenging TGF-beta. These mechanisms might restrict liver fibrogenesis.

Interferon gamma and antisense transforming growth factor beta transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma.

Transforming growth factor beta (TGFbeta) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFbeta production by EMT6 murine mammary tumor cells expressing an antisense TGF-beta transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFbeta production coupled with interferon gamma (IFNgamma) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFbeta were transduced with the murine IFNgamma gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8+ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity.

The Transcription Factor EGR-1 Suppresses Transformation of Human Fibrosarcoma HT1080 Cells by Coordinated Induction of Transforming Growth Factor-β1, Fibronectin, and Plasminogen Activator Inhibitor-1

Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054-5062) owing in part to up-regulation of the transforming growth factor (TGF)-beta1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831-11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (RPEARSON = 0.971 and 0. 985, respectively). Addition of authentic TGF-beta1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-beta antibody or lipofection with specific antisense TGF-beta1 oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions -75/-52 and -4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment ("oikis" factor) and normal growth control.

Suppression of TGF-beta1 in human gliomas by retroviral gene transfection enhances susceptibility to LAK cells.

Human glioma cell line, Onda 10 produces TGF-beta1. TGF-beta1 has a biological role for the immunosuppression of the host. We have investigated whether suppression of TGF-beta1 on human glioma cell enhanced the susceptibility to lymphokine-activated killer (LAK) cells. In vitro, susceptibility to LAK cells on Onda 10 cell is augmented by retroviral gene transfection with antisense TGF-beta1. Nude mice bearing Onda 10 cells transduced with antisense TGF-beta1 gene has a longer life span compared to mice carrying that of sense TGF-beta1 gene or vector alone. The cytotoxic activity of LAK cells induced from spleen cells of mice carrying antisense TGF-beta1 gene transduced cells is higher against Onda 10 cell than that of LAK cells from mice carrying vector alone transduced cells. Also, antisense TGF-beta1 gene transduced cells are much more sensitive to LAK cells compared to Onda 10. These suggest that the augmented host systemic immunity in mice is one of the mechanisms of the reduced tumorigenicity of antisense TGF-beta1 gene transduced cells and that the increased systemic immunity could be ascribed to the increased immunogenicity of the tumor cells. The gene therapy for malignant glioma with antisense TGF-beta1 gene is expected to be promising.

Costimulation of Fibroblast Collagen and Transforming Growth Factor β1 Gene Expression by Monocyte Chemoattractant Protein-1 via Specific Receptors

Recent studies indicate potential roles of monocyte chemotactic protein-1 (MCP-1) in recruitment of monocytes to sites of inflammation. However, their increased expression does not always correlate with monocyte influx, suggesting other possible biological activities for this member of the C-C chemokine family. In view of its potential role in regulating extracellular matrix expression in fibrotic disorders, the effects of MCP-1 on lung fibroblast collagen expression were evaluated. Isolated rat lung fibroblasts were treated with increasing doses of MCP-1 for variable periods of time and examined for effects on collagen synthesis and expression of procollagen alpha1(I) mRNA expression. The results show that MCP-1 was able to stimulate collagen expression in these cells in a dose-dependent manner but required over 24 h for significant elevation to occur. In view of this delayed time course, the possibility of mediation via endogenous transforming growth factor beta (TGFbeta) was tested by the ability of anti-TGFbeta antibody to inhibit this MCP-1 stimulation of collagen expression. Significant but incomplete inhibition by this antibody was observed. Pretreatment of the cells with antisense but not by sense or missense TGFbeta1 oligodeoxyribonucleotides caused essentially complete inhibition of this MCP-1 stimulatory effect. Furthermore, MCP-1 treatment was found to also stimulate TGFbeta secretion and mRNA expression, which was also abolished by pretreatment with antisense TGFbeta1 oligodeoxyribonucleotides. The kinetics of TGFbeta expression indicates that significant increase preceded that for collagen expression. Binding studies using 125I-labeled MCP-1 indicated the presence of specific and saturable binding sites with a dissociation constant consistent with the dose response curves for stimulation of fibroblast collagen synthesis and TGFbeta activity by MCP-1. These results taken together suggest that MCP-1 stimulates fibroblast collagen expression via specific receptors and endogenous up-regulation of TGFbeta expression. The latter then results in autocrine and/or juxtacrine stimulation of collagen gene expression.

Glucocorticoid-induced tropoelastin expression is mediated via transforming growth factor-beta 3.

Glucocorticoids play a central role in fetal lung maturation. As fibroblasts have been shown to be an important target cell for glucocorticoids in fetal lung, we have recently cloned several cDNAs representing genes induced by cortisol in fetal rat lung fibroblasts. Approximately 30% of the cDNAs were identified as transforming growth factor (TGF)-beta 3. Here, we selected and sequenced another cDNA. Analysis of DNA sequence homology indicated that this cDNA encodes the rat tropoelastin (TE). Using this cDNA as a TE probe, we confirmed that TE mRNA was expressed in a developmental, organ- and cell type-specific fashion. Exogenous glucocorticoids indeed increased the number of TE transcripts in fetal lung fibroblasts. Blockage of endogenous TGF-beta 3 production with antisense TGF-beta 3 oligonucleotides abrogated the stimulatory effect of cortisol on TE mRNA production by fetal lung fibroblasts. Also, neutralizing antibodies to TGF-beta 3 blocked the cortisol-stimulated TE mRNA expression. Fetal lung fibroblasts expressed both TGF-beta type I and II receptors. Cortisol did not influence the expression of either receptor mRNA. Antisense TGF-beta type I receptor oligonucleotides inhibited cortisol-induced TE mRNA expression. Cortisol activated the transcription of stable transfected cDNA for TGF-beta 3 under the control of glucocorticoid-inducible long terminal repeat (LTR) promoter in RFL-6 fibroblasts. Induction of TGF-beta 3 in the transfectants was accompanied by a marked increase in TE mRNA. These findings suggest that glucocorticoids mediate their stimulatory effect on TE mRNA expression in fetal lung fibroblasts via an autocrine action of glucocorticoid-induced TGF-beta 3.

Control of scarring in adult wounds using antisense transforming growth factor‐β1 oligodeoxynucleotides

Adult wounds heal with scar formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. We reasoned that a strategy employing antisense oligodeoxynucleotides (ODN) complementary to transforming growth factor (TGF)-beta-1 mRNA might decrease the scarring of dermal wounds in the mouse. To evaluate this concept, we tested the effects of antisense ODN targeted to TGF-beta 1 mRNA by topical application of the ODN on the skin wound. Phosphorothioation of ODN to retard their degradation. When antisense TGF-beta 1 ODN were applied on the wound site, there was marked reduction of scarring compared with a control wound site. This effect of antisense TGF-beta 1 ODN on scar formation was associated with decreased expression of the TGF-B1 gene. However, sense TGF-beta 1 ODN had no effect on the expression of the TGF-beta 1 gene. In addition, control wounds healed with excessive fibrosis compared with the antisense-treated wounds. In conclusion, our results indicate that antisense TGF-beta 1 ODN could be used for ameliorating scar formation during wound healing.

Autocrine transforming growth factor-beta (TGF beta) modulates the 120 kD and 105 kD retinoblastoma (RB) protein species in CBS colon carcinoma cells as revealed by an anti-sense TGF beta 1 expression vector

Specific RE protein species modulated by autocrine TGF beta are unknown. CBS NEO (with autocrine TGF beta) and antisense TGF beta 1 transfected CBS RSVAS cells were compared for mitogenesis and RE species modulation. Significantly, high MW (120, 130 kD) were abundant relative to low MW (116, 110, 105 kD combined) RE species. CBS NEO required growth factors (GF) for maximal DNA synthesis (14-fold). 120 kD and 105 kD were significantly modulated in CBS NEO-GF. Nutrients (N) were an sufficient to maximally stimulate CBS RSVAS (8-fold). 130 kD and 116-110 low MW species were moderately induced by both N (CBS RSVAS-N) and GF (CBS NEO-GF), independent of autocrine TGF beta. In conclusion, autocrine TGF beta (CBS) has a role in modulation of 120 kD and 105 kD RE species which may play a role in the inhibitory action of autocrine TGF beta pathway.

Quercetin Inhibits the Growth of Leukemic Progenitors and Induces the Expression of Transforming Growth Factor-β1 in These Cells

We previously showed that quercetin (3,3',4',5,7 pentahydroxyflavone) inhibits in a dose-dependent manner the growth of acute leukemias and is able to enhance the antiproliferative activity of cytosine arabinoside. We show here that quercetin inhibits the clonogenic activity of 20 of 22 acute leukemias (AL; 4 M1-AML, 3 M2-AML, 2 M3-AML, 3 M4-AML, 3 M5-AML, and 7 ALL). In the present report, we show that the induction of transforming growth factor-beta 1 (TGF-beta 1) in leukemic blasts is one of the growth-inhibitory mechanisms of quercetin in these cells. This observation was supported by the following data. (1) Quercetin-sensitive leukemic blasts, when treated with quercetin, secrete large amounts of TGF-beta 1 in the medium and show positivity for TGF-beta 1-immunoreactive material in the cytoplasm. (2) At a concentration of 8 mumol/L, antisense TGF-beta 1 oligonucleotides prevent the growth-inhibitory action of quercetin. (3) Anti-TGF-beta 1 neutralizing monoclonal antibodies can prevent almost completely the growth-inhibitory activity of quercetin. The analysis of quercetin-resistant cases confirmed as well the central role of TGF-beta 1 in the growth-inhibitory activity of quercetin. In conclusion, quercetin can act as a cytostatic agent for leukemic cells by modulating the production of TGF-beta 1.

Gene transfer for transplantation. Prolongation of allograft survival with transforming growth factor-beta 1.

The authors tested the ability of plasmid gene transfer to express transforming growth factor-beta 1 (TGF-beta 1), prolong allograft survival, and evaluate promoter effects on gene expression. Delivery of immunosuppressants directly to allografts using gene transfer and gene therapy approaches may inhibit immune activation while avoiding the systemic toxicity of conventional immunosuppression. Candidate genes include soluble cytokines, which could be expressed at low levels throughout the graft while inducing a local immunosuppressive effect. Transforming growth factor-beta 1 is a soluble cytokine that has pleiotropic immunosuppressive effects. Cardiac grafts from syngeneic (CBA/J, H-2k) or allogenic (C57BL/6, H-2b) donors were placed into CBA/J recipients. Purified plasmid DNA-encoding murine TGF-beta 1 or beta-galactosidase (Lac Z) under the control of RSV, SV40, MMTV, or pancreatic elastase promoters was injected into grafts at surgery. The Lac Z expression was determined by histologic examination and TGF-beta 1 expression by graft survival. Cytotoxic T lymphocyte and flow cytometric analyses were performed to evaluate the immunosuppressive effects of TGF-beta 1 in vitro. Plasmid DNA-encoding TGF-beta 1 prolonged survival from 12.6 +/- 1.1 days to 26.3 +/- 2.5 days (p < 0.02, Student's t test). The SV40 promoter was superior to the MMTV promoter in its ability to prolong survival. The effects of the plasmids were specific because Lac Z, antisense TGF-beta 1 inserts, or pancreatic elastase promoter did not prolong allograft survival. Histologic examination demonstrated Lac Z expression at least 14 days post-transplant in myocardial cells. Both RSV and SV40 promoters were effective in this respect, while a control null promoter was not. Toxicity testing showed that gene transfer of TGF-beta 1 did not alter survival or histology of syngeneic grafts. In addition, plasmids and purified TGF-beta 1 protein were not toxic to myoblasts in vitro. Recombinant TGF-beta 1 inhibited cytotoxic T lymphocyte generation and altered T cell surface receptor expression and subset expansion in vitro. Gene transfer/therapy with plasmid DNA encoding TGF-beta 1 in vivo achieves immunologic effects that prolong allograft survival. Multiple promoters effectively induce plasmid expression, which is achieved in cardiac myocytes for at least 2 weeks without toxicity or adverse systemic effects. Transforming growth factor-beta 1 inhibits immune responses by different mechanisms, revealed by in vitro analysis of T cell cytolytic function, subset distribution, and receptor display.

Antisense S-oligonucleotide against transforming growth factor-beta 1 inhibits proteoglycan synthesis in arterial wall.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent stimulator of proteoglycan (PG) synthesis by cultured smooth muscle cells. To test the hypothesis that TGF-beta 1 stimulates PG synthesis in whole artery wall we have investigated the effect of blocking endogenous TGF-beta 1 using an antisense S-oligonucleotide (ASO) directed against the first 7 codons for the N-terminal region of active TGF-beta 1. This sequence reduced TGF-beta 1 secretion by cultured endothelial cells by 40-55%. To determine the effect of the ASO on PG synthesis in whole vessel we chose the rat carotid artery (RCA) maintained in organ culture, a model in which we previously documented endothelial-dependent increases in PG synthesis over time in culture. We report here that the increases in PG in the inner layers of the vessel wall are matched by similar increases in TGF-beta 1 mRNA. To test for the effect of ASO on PG synthesis, small segments of RCA, maintained in organ culture (Medium 199, supplemented with 1% FCS), were exposed on day 6 to either control media, antisense TGF-beta 1, sense TGF-beta 1, or a non-sense sequence at 5, 10 and 20 microM concentrations. Following 24 h exposure to the oligonucleotides, cultures were labelled for a further 24 h with [3H]glucosamine and processed for autoradiography. Antisense TGF-beta 1 at 10 and 20 microM produced a 60% reduction in PG synthesis in the endothelium and adjacent first layer of the media.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of transforming growth factor-beta isoforms (beta 2 and beta 3) in the mouse uterus: analysis of the periimplantation period and effects of ovarian steroids.

Expression of beta-type transforming growth factor genes (TGF beta 2 and TGF beta 3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17 beta-estradiol (E2) and progesterone (P4) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGF beta 2 or TGF beta 3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGF beta 2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGF beta 2 immunostaining was also detected in decidual cells. In contrast, TGF beta 3 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (D1-D8). Antisense TGF beta 2 and TGF beta 3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF beta 2 transcripts (approximately 6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)+ RNA on D1-D6 and in poly(A)+ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF beta 2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGF beta 3 transcript in total uterine poly(A)+ RNA on D1-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGF beta 2 and TGF beta 3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF beta 2 or TGF beta 3 mRNA persisted in ovariectomized mice. However, an injection of E2 induced a rapid (6 h), but transient, induction (approximately 3- to 4-fold) of TGF beta 2 mRNA. An injection of P4 had no effect on TGF beta 2 mRNA levels, and coinjection of P4 with E2 did not antagonize the E2-stimulated transient accumulation of TGF beta 2 mRNA. In comparison, neither an injection of E2 nor one of P4 exerted significant effects on TGF beta 3 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of transforming growth factor-beta isoforms (beta 2 and beta 3) in the mouse uterus: analysis of the periimplantation period and effects of ovarian steroids

Expression of beta-type transforming growth factor genes (TGF beta 2 and TGF beta 3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17 beta-estradiol (E2) and progesterone (P4) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGF beta 2 or TGF beta 3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGF beta 2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGF beta 2 immunostaining was also detected in decidual cells. In contrast, TGF beta 3 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (D1-D8). Antisense TGF beta 2 and TGF beta 3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF beta 2 transcripts (approximately 6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)+ RNA on D1-D6 and in poly(A)+ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF beta 2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGF beta 3 transcript in total uterine poly(A)+ RNA on D1-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGF beta 2 and TGF beta 3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF beta 2 or TGF beta 3 mRNA persisted in ovariectomized mice. However, an injection of E2 induced a rapid (6 h), but transient, induction (approximately 3- to 4-fold) of TGF beta 2 mRNA. An injection of P4 had no effect on TGF beta 2 mRNA levels, and coinjection of P4 with E2 did not antagonize the E2-stimulated transient accumulation of TGF beta 2 mRNA. In comparison, neither an injection of E2 nor one of P4 exerted significant effects on TGF beta 3 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Sense and antisense TGF beta 3 mRNA levels correlate with cardiac valve induction.

The formation of the valves in the heart is a spatially and temporally controlled process. A tissue interaction between the endothelium and its adjacent myocardium initiates the transformation of the endothelium into the mesenchymal precursors of the heart valve. One or more of the molecules implicated as critical for valve formation are members of the transforming growth factor beta family of molecules. Presented here is a spatial and temporal analysis of TGF beta 2 and TGF beta 3 in the chick heart during valve formation. We show that TGF beta 3 mRNA is concentrated in AV canal tissue where valve formation will occur, consistent with previous observations that TGF beta 3 production is critical during valve formation. Additionally, an RNA complementary to TGF beta 3 encoding mRNA is present in the heart. The temporally controlled appearance of RNA complementary to TGF beta 3 suggests that this molecule may play a role in the regulation of TGF beta 3 production in the heart.

Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-beta is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.