Molecular mechanism of transforming growth factor beta-mediated cell-cycle modulation in primary human CD34(+) progenitors

Abstract

The mechanisms by which transforming growth factor beta (TGF-beta) exerts a negative effect on cell-cycle entry in primary human hematopoietic stem/progenitor cells were examined at the molecular and cellular levels. After treatment of primary human CD34+ progenitors with TGF-beta there was a decrease in the levels of cyclin D2 protein and an increase in levels of the cyclin-dependent kinase inhibitor (CDKI) p15 as compared to the levels in untreated cells. The converse was true after addition of neutralizing anti-TGF-beta antibody. Administration of TGF-beta to CD34+ cells in the presence of cytokines prevented retinoblastoma protein (pRb) phosphorylation, which occurred in the same cells treated with cytokines alone or cytokines and anti-TGF-beta antibody. Neutralization of TGF-beta during 24 to 48 hours of culture with cytokines significantly increased the number of colony-forming progenitors, but did not modulate the human stem cell pool, as measured in 6- to 12-month xenotransplantation assays. Equivalent numbers of human B, T, and myeloid cells were obtained after transplantation of cells treated with or without neutralization of TGF-beta.

Figure 1. Cell-cycle analysis of CD34⁺ progenitors incubated with soluble TGF-β1 or anti–TGF-β antibody

After 48 hours of incubation, CD34⁺ cells isolated from UCB were collected, fixed, permeabilized, and stained with Ki67-FITC. Cells were then stained with 7-AAD overnight before acquisition analysis by FACS Calibur flow cytometry. Controls for the G0 population were peripheral blood T cells starved overnight in serum-free medium. Controls for the cycling cell population were peripheral blood T cells stimulated with PHA and IL-2 overnight. White bars indicate cells incubated with the cytokines IL-3, IL-6, and SCF alone. Black bars indicate cells incubated in the same cytokine mixture with addition of soluble TGF-β1. Gray bars indicate cells incubated in the same cytokine mixture with addition of anti–TGF-β–neutralizing antibody. *P < .05.

Figure 2. Optimizing the amount of anti–TGF-β antibody to decrease p15 protein levels

UCB CD34⁺ progenitors were incubated for 18 hours in serum-free media alone or in the presence of increasing concentrations of anti–TGF-β antibody (ng/mL). Cell lysates were prepared and subjected to 15% SDS-PAGE, followed by immunoblotting with a polyclonal antibody to p15Ink4b.

Figure 3. Addition of soluble TGF-β1 alters the levels of cyclin D2, not cyclin D3, cdk2, cdk4, or cdk6

After incubating with and without soluble TGF-β1 for 18 hours, CD34⁺ progenitors were lysed in 1% NP-40, loaded onto a 10% or 15% SDS-PAGE gel, and transferred to Immunobilon PSQ membrane. Immunoblotting with antibodies to cdk4 (SC-749), cdk6 (SC-7961), cyclin D2 (SC-754), cyclin D3 (D-7), and p15 (SC-613) was then performed.

Figure 4. Immunocomplexes between cdk4 and cyclin D2 were detected only in the presence of anti–TGF-β antibody

After starvation in methionine-free DMEM for 4 hours, CD34⁺ progenitors were metabolically labeled for 18 hours with ³⁵S-methionine/cysteine in the presence or absence of soluble TGF-β1 or anti–TGF-β antibody. Cell lysates were first immunoprecipitated with agarose-coated polyclonal CDK4 antibody. Subsequent serial immunoprecipitation was then carried out further using agarose-coated polyclonal cyclin D2 antibody. Samples were run on a 10% SDS-PAGE gel, dried, and exposed to autoradiogram film.

Figure 5. Soluble TGF-β1 specifically decreased the level of pRb phosphorylation

CD34⁺ progenitors were incubated in the presence or absence of soluble TGF-β1 or neutralizing antibody to TGF-β1 (a-TGFB) for 48 hours. Total lysates were run on 7.5% SDS-PAGE gel and transferred onto Immunobilon membrane. (A) Immunoblotting with an antibody that recognizes underphosphorylated, hypophosphorylated, and hyperphosphorylated forms of pRb. (B) Immunoblotting with an antibody specific to Ser811 pRb. Membranes were also stained in amido black staining to ensure equal protein loading in all lanes.