Vaccine implementation factors affecting maternal tetanus immunization in low- and middle-income countries: Results of the Maternal Immunization and Antenatal Care Situational Analysis (MIACSA) project.

OBJECTIVES: To examine the characteristics of existing maternal tetanus immunization programmes for pregnant women in low- and middle-income countries (LMICs) and to identify and understand the challenges, barriers and facilitators associated with maternal vaccine service delivery that may impact the introduction and implementation of new maternal vaccines in the future. DESIGN: A mixed methods, cross sectional study with four data collection phases including a desk review, online survey, telephone and face-to-face interviews and in country visits. SETTING: LMICs. RESULTS: The majority of countries (84/95; 88%) had a maternal tetanus immunization policy. Countries with high protection at birth (PAB) were more likely to report tetanus toxoid-containing vaccine (TTCV) coverage targets > 90%. Less than half the countries included in this study had a TTCV coverage target of > 90%. Procurement and distribution of TTCV was nearly always the responsibility of the Expanded Programme on Immunization (EPI), however planning and management of maternal immunization was often shared between EPI and Maternal, Newborn and Child Health (MNCH) programmes. Receipt of TTCV at the same time as the antenatal care visit correlated with high PAB. Most countries (81/95; 85%) had an immunization safety surveillance system in place although only 11% could differentiate an adverse event following immunization (AEFI) in pregnant and non-pregnant women. CONCLUSIONS: Recommendations arising from the MIACSA project to strengthen existing services currently delivering maternal tetanus immunization in LMICs include establishing and maintaining vaccination targets, clearly defining responsibilities and fostering collaborations between EPI and MNCH, investing in strengthening the health workforce, improving the design and use of existing record keeping for immunization, adjusting current AEFI reporting to differentiate pregnant women and endeavoring to integrate the provision of TTCV within ANC services where appropriate.

Resistance training in patients with schizophrenia: Concept and proof of principle trial.

Resistance training has been shown to contribute to the prevention and management of cardiovascular diseases, which is why it can help reducing morbidity and mortality in schizophrenia patients. Moreover, positive effects on different schizophrenia symptom domains have been proposed. However, a specific resistance training tailored to the needs of schizophrenia patients and its evaluation is still lacking. The objective in this proof of principle trial was to evaluate the feasibility and efficacy of a newly developed 12-week resistance program according to current recommendations of the WHO and the American College of Sports Medicine. We employed a single blind, parallel assignment clinical trial design with participants randomized to attend either a resistance training including three 50min units per week or a balance and tone program as control condition. The primary outcome was the impact on health-related difficulties assessed with the World Health Organization Disability Assessment Schedule (WHO-DAS). Secondary outcome parameters included the level of functioning, schizophrenia symptoms, selected cognitive parameters as well as risk factors for cardiovascular diseases. In our proof of principle trial, we could not find significant time or group effects of resistance training on the WHO-DAS. However, we could observe significant positive effects on the level of functioning assessed with the Global Assessment of Functioning Scale (GAF) over the course of time, which were more pronounced in the intervention group. Our findings indicated that patients with schizophrenia could safely participate in resistance training with relevant improvements in their level of functioning. Well-powered replication trials are needed to provide more efficacy data.

DPP4 truncated GM-CSF and IL-3 manifest distinct receptor-binding and regulatory functions compared with their full-length forms.

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony-stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated (T) factors (T-granulocyte-macrophage-CSF (T-GM-CSF)) and T-interleukin-3 (T-IL-3)) on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and -IL-3 had enhanced receptor binding, but decreased CSF activity, compared with their FL forms. Importantly, T-GM-CSF and -IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and -IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster-forming cells from patients with acute myeloid leukemia regardless of cytogenetic or molecular alterations and in vivo using animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared with their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.

Steel factor regulates cell cycle asymmetry.

Asymmetric segregation of cell-fate determinants during mitosis (spatial asymmetry) is an essential mechanism by which stem cells are maintained while simultaneously giving rise to differentiated progenitors that ultimately produce all the specialized cells in the hematopoietic system. Temporal cell cycle asymmetry and heterogeneity are attributes of cell proliferation that are also essential for maintaining tissue organization. Hematopoietic stem cells (HSCs) are regulated by a complex network of cytokines, some of which have very specific effects, while others have very broad ranging effects on HSCs. Some cytokines, like steel factor (SLF), are known to synergize with other cytokines to produce rapid expansion of progenitor cells. Using the human growth factor-dependent MO7e cell line as a model for synergistic proliferation, we present evidence that links proliferation asymmetry to SLF synergy with GM-CSF, and suggests that temporal asymmetry and cell cycle heterogeneity can be regulated by SLF in vitro. We also show that CDK-inhibitor and cell cycle regulator, p27kip-1, may be involved in this temporal asymmetry regulation. We propose that SLF/GM-CSF synergy is, in part, due to a shift in proliferation pattern from a heterogeneous and asymmetric one to a more synchronous and symmetric pattern, thus contributing dramatically to the rapid expansion that accompanies SLF synergy observed in MO7e cells. This kinetic model of asymmetry is consistent with recent evidence showing that even though SLF synergy results in a strong proliferative signal, it does not increase primary HSC self-renewal, which is believed to be highly dependent on asymmetric divisions. The factor-dependent MO7e/SCF- synergy/asymmetry model described here may therefore be useful for studies of the effects of various cytokines on cell cycle asymmetry.

Blocking of c-FLIP(L)--independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction.

Chemokines play a pivotal role in regulating leukocyte migration as well as other biological functions. CC chemokine receptor 9 (CCR9) is a specific receptor for thymus-expressed CC chemokine (TECK). It is shown here that engagement of CCR9 with TECK leads to phosphorylation of Akt (protein kinase B), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase--3 beta (GSK-3 beta), and a forkhead transcription factor, FKHR, in a human T-cell line, MOLT4, that naturally expresses CCR9. By means of chemical inhibitors, it is shown that phosphoinositide-3 kinase (PI-3 kinase), but not MAPK, is required for CCR9-mediated chemotaxis. Akt, GSK-3 beta, FKHR, and MAPK have been previously implicated in cell survival signals in response to an array of death stimuli. When MOLT4 cells, which expressed Fas as well as CXCR4, were stimulated with cycloheximide (CHX), an agonistic anti-Fas antibody, or a combination of these, the cells rapidly underwent apoptosis. However, costimulation of MOLT4 cells with TECK or stromal derived factor--1 significantly blocked CHX-mediated apoptosis, whereas stimulation only with TECK partially blocked Fas-mediated apoptosis. Concomitant with this blocking, cleavage of poly (adenosine 5'-diphosphate--ribose) polymerase and activation of caspase 3 were significantly attenuated, but the expression level of FLICE inhibitory protein c-FLIP(L), which had been shown to be regulated by CHX, was unchanged. This demonstrates that activation of CCR9 leads to phosphorylation of GSK-3 beta and FKHR and provides a cell survival signal to the receptor expressing cells against CHX. It also suggests the existence of a novel pathway leading to CHX-induced apoptosis independently of c-FLIP(L). (Blood. 2001;98:925-933)

Chemokine regulation of hematopoiesis and the involvement of pertussis toxin-sensitive G alpha i proteins.

Chemokines have been implicated in regulation of various aspects of hematopoiesis, including negative regulation of the proliferation of immature subsets of myeloid progenitor cells (MPCs), chemotaxis of MPCs, and survival enhancement of MPCs after delayed growth factor addition. Since chemokine receptors are seven-transmembrane-spanning G-protein-linked receptors and the chemotactic effect in vitro of the CXC chemokine SDF-1 is pertussis toxin (PT)-sensitive, implying the involvement of G alpha i proteins as mediators of SDF-1-induced chemotaxis, we evaluated the effects of PT on other chemokine actions influencing MPCs. While the in vitro survival-enhancing effects of SDF-1 on GM-CSF and steel factor-dependent mouse bone marrow granulocyte macrophage progenitors (CFU-GM) were pertussis toxin-sensitive, the suppressive effects of the CC chemokine MIP-1 alpha and the CXC chemokine IL-8 on colony formation by GM-CSF and steel factor-sensitive CFU-GM were insensitive to pertussis toxin. These results suggest that not all chemokine-mediated effects on MPCs are necessarily mediated through pertussis toxin-sensitive G alpha i proteins.

The interphase microtubule damage checkpoint defines an S-phase commitment point and does not require p21(waf-1).

Cell cycle checkpoints ensure orderly progression of events during cell division. A microtubule damage (MTD)-induced checkpoint has been described in G(1) phase of the cell cycle (G(1)MTC) for which little is known. The present study shows that the G(1)MTC is intact in activated T lymphocytes from mice with the p21(waf-1) gene deleted. However, p21(waf-1) gene deletion does affect the ratio of cells that arrest at the G(1)MTC and the spindle checkpoint after MTD. The G(1)MTC arrests T lymphocytes in G(1) prior to cdc2 up-regulation and prior to G(1) arrest by p21(waf-1). Once cells have progressed past the G(1)MTC, they are committed to chromosome replication and metaphase progression, even with extreme MTD. The G(1)MTC is also present in a human myeloid cell line deficient in p21(waf-1) gene expression. The p21-independent G(1)MTC may be important in cellular responses to MTD such as those induced by drugs used to treat cancer.

Transcriptional and ERK1/2-dependent synergistic upregulation of p21(cip1/waf1) associated with steel factor synergy in MO7e.

Steel factor (SLF) plus GM-CSF induces proliferative synergy in myeloid progenitors and factor-dependent cell line MO7e. We previously reported that the protein level of cyclin-dependent kinase inhibitor p21(cip1/waf1) (p21) increased synergistically when MO7e cells were stimulated with SLF plus GM-CSF and that p21 induction was required for SLF synergistic responses. Here we show that this p21 induction is regulated at the transcriptional level. Based on use of a multiprobe RNase protection assay, the synergistic increase of p21 mRNA was unique among many cell cycle regulators. While STAT5A and 5B were activated after stimulation with GM-CSF alone or SLF plus GM-CSF, there was no difference in activation between the groups. p44/42 MAP kinase (ERK1/2) was synergistically activated by SLF plus GM-CSF, but SAPK/JNK and p38 MAP kinase were not. Synergistic induction of p21 was significantly decreased with a MEK1 inhibitor, suggesting that the ERK1/2 pathway is involved in the synergistic increase of p21 after GM-CSF plus SLF stimulation.

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2.

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34(+) cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)-derived erythroblasts from a 7-day culture of CD34(+) cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2. (Blood. 2000;96:3985-3987)

Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation.

Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9-induced erythroid cell proliferation and differentiation.

Chemokines, chemokine receptors and hematopoiesis.

Hematopoiesis during steady state conditions is regulated and finely tuned by a network of cytokines and their effects on hematopoietic stem and progenitor cells and on accessory cells that influence the stem and progenitor cells. Amongst the numerous cytokines implicated in this regulation are members of the CC, CXC and C family of chemokines. Twenty-five chemokine members have been demonstrated to have the capacity to suppress the proliferation of myeloid progenitor cells. Three chemokines have been implicated in the chemotaxis of these stem and progenitor cells, and one has been linked to their survival after growth factor withdrawal. This review focuses on the proliferation-suppressing, chemotaxis-induced, and cell survival effects of different chemokine family members on myeloid progenitor cells. This is placed in the context of what we know and don't know about the intracellular signaling events mediating these effects. This information and what is yet to be learned in this area could have important clinical implications for treatment of disease.

Flt3 signaling involves tyrosyl-phosphorylation of SHP-2 and SHIP and their association with Grb2 and Shc in Baf3/Flt3 cells.

Flt3 ligand (FL) is an early-acting potent co-stimulatory cytokine that regulates proliferation and differentiation of a number of blood cell lineages. Its receptor Flt3/Flk2 belongs to class III receptor tyrosine kinases that also include the receptors for colony-stimulating factor 1, Steel factor, and platelet-derived growth factor. Using CSF-1 receptor/Flt3 chimeras, two groups have characterized some of the post-receptor signaling events and substrate specificity of murine Flt3 receptor. However, there are few studies on the signaling pathway through human Flt3. We examined human Flt3 signaling pathways in a murine IL-3-dependent hematopoietic cell line Baf3, which stably expresses full-length human Flt3 receptor. This subline proliferates in response to human FL. Like the chimeric murine Flt3, human Flt3 undergoes autophosphorylation, associates with Grb2, and leads to tyrosine phosphorylation of Shc on ligand binding. We found that SHP-2, but not SHP-1, is tyrosine-phosphorylated by FL stimulation. SHP-2 does not associate with Flt3, but binds directly to Grb2. SHIP is also tyrosine-phosphorylated and associates with Shc after FL simulation. We further examined the downstream signaling pathway. FL transiently activates MAP kinase. This activation could be blocked by PD98059, a specific MEK inhibitor. PD98059 also blocked cell proliferation in response to FL. These results demonstrate that SHP-2 and SHIP are important components in the human Flt3 signaling pathway and suggest that SHP-2 and SHIP, by forming complexes with adapter proteins Grb2 and Shc, may modulate MAP kinase activation, which may be necessary for the mitogenic signaling of Flt3.

p21(cip-1/waf-1) deficiency causes deformed nuclear architecture, centriole overduplication, polyploidy, and relaxed microtubule damage checkpoints in human hematopoietic cells.

A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21(cip-1/waf-1). Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21(cip-1/waf-1) deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21(cip-1/waf-1) expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21(cip-1/waf-1) in p21(cip-1/waf-1) antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.

H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation.

The proto-oncogene product, p21(ras), has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)-induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; alpha4beta1 integrins) and VLA-5 (alpha5beta1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras-transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras-transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras-transfected Baf3 cells. Anti-beta1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras-transfected Baf3 cells as much as the other types of H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti-phospho-MAPK antibody, but not adhesion of any type of H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3-induced VLA-4 and VLA-5 activation in Baf3 cells.

Interleukin-3 and Flt3-ligand induce adhesion of Baf3/Flt3 precursor B-lymphoid cells to fibronectin via activation of VLA-4 and VLA-5.

Adhesion of hematopoietic cells to extracellular matrix components is important for blood cell development. However, little is known regarding the potential influence of IL-3 on this process for precursor B cells and Flt3-ligand has not yet been implicated in induction of adhesion of any blood cell types to extracellular matrix components. Therefore, we examined the characteristics of cytokine-induced cell adhesion to fibronectin (FN), using as a model the murine precursor B cell line, Baf3, a factor-dependent cell line requiring IL-3 for both growth and survival. Since factor-dependent hematopoietic cell lines expressing Flt3 receptor are extremely rare, we also studied Baf3/Flt3, a subline of Baf3 transduced with the Flt3 receptor gene. IL-3 induced adhesion of Baf3 and Baf3/Flt3 cells to FN, while Flt3-ligand induced adhesion of Baf3/Flt3 cells only. Whereas both Baf3 and Baf3/Flt3 cells expressed VLA-4 and -5 integrins as FN receptors, expression levels of VLA-4 and -5 were not affected by IL-3 or Flt3-ligand treatment. However, blocking experiments using anti-integrin antibodies showed that cytokine-induced adhesion of cells depended on both VLA-4 and -5 suggesting that IL-3 and Flt3-ligand activated these integrins. PI-3 kinase inhibitor wortmannin, PKC inhibitor H-7, or PKA inhibitor HA1004 did not suppress adhesion induced by IL-3 or Flt3-ligand; in contrast, PLC inhibitor U-73122 did suppress adhesion, suggesting the possibility that PLC, but not PI-3 kinase, PKC, or PKA, may be involved in this process. Since it is known that IL-3 and Flt3-ligand receptors are expressed on precursor B cells, and these receptors are downregulated during B cell maturation of primary cells, the induction of precursor B cell adhesion to FN by IL-3 and Flt3-ligand may contribute a mechanism by which precursor B cells adhere to bone marrow stroma, thereby influencing their development.

A positive effect of p21cip1/waf1 in the colony formation from murine myeloid progenitor cells as assessed by retroviral-mediated gene transfer.

p21cip1/waf1 is a cyclin dependent kinase inhibitor. We have previously reported stimulation of p21cip1/waf1 by steel factor and GM-CSF in a factor dependent cell line and of p21cip1/waf1 involvement in hematopoiesis in vivo in p21cip1/waf1 gene knockout (-/-) mice. To further assess a role for increased p21cip1/waf1 in hematopoietic progenitor cells, we developed the retroviral vector L(p21cip1)SN to transcriptionally regulate p21cip1/waf1 from the Mo-MLV LTR. L(p21cip1)SN and the control vector LXSN were used to transduce murine bone marrow progenitor cells from p21cip1/waf1 (-/-) and littermate control (+/+) mice, as well as from other mouse strains. Hematopoietic colony formation by transduced cells was assessed in semi-solid culture medium with multiple growth factors. Myeloid colony formation by bone marrow cells from p21cip1/waf1 (-/-) mice was significantly lower than that by (+/+) mouse cells. Transduction of cells with LXSN had no effect on colony formation: however, (-/-) cells transduced with L(p21cip1)SN formed significantly greater numbers of colonies than either LXSN-transduced (-/-) or (+/-) cells. Moreover, L(p21cip1)SN-transduced (+/+) cells formed significantly more colonies than LXSN-transduced (+/+) cells. Increased cloning efficiency of progenitors from normal strains of mice induced by L(p21cip1)SN compared to LXSN-transduced cells was seen whether unseparated or highly purified populations of Sca1+ Lin- marrow cells were used. Gene transfer of L(p21cip1)SN increased the size and number of cells per colony, as well as the number of colonies compared to LXSN gene transfer. No colonies grew from non-transduced, LXSN-, or L(p21cip1)SN-transduced cells when no growth factors were added to the cultures. These results document the positive effect of p21cip1/waf1 in the proliferation and/or differentiation of the murine myeloid progenitor cells that lead to colony formation.

Biology and mechanisms of action of synergistically stimulated myeloid progenitor cell proliferation and suppression by chemokines.

A number of cytokines can act together to stimulate/enhance the proliferation of hematopoietic stem and progenitor cells in a greater than additive fashion. An example of this is the combination of a colony-stimulating factor with a potent costimulating molecule such as steel factor. Certain members of the chemokine family of cytokines can suppress this synergistically enhanced proliferation. This review focuses on cytokines involved in these stimulating/enhancing/suppressing effects with regard to biological activity and what is beginning to be learned about the intracellular signal transduction events that may be mediating these effects. Examples of intracellular mediators involved include, but are not limited to, the Raf-1/ MAP kinase pathway and cyclin-dependent kinase inhibitors p21cip-1 and p27kip-1 for cell proliferation, and eukaryotic initiation factor-4E and 4E binding protein 1 for protein synthesis.

Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo.

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip-1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip-1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.