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Keywords:

  • p107;
  • dendritic cell;
  • retinoblastoma protein;
  • cell cycle;
  • E2F transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

A member of the retinoblastoma family of cell cycle regulatory proteins, p107, is not normally expressed in non-cycling cells. We demonstrate here that p107 is expressed in monocytes as they differentiate into dendritic cells under the influence of granulocyte-macrophage colony-stimulating factor and interleukin 4, a process shown not to involve cellular proliferation. We show that p107 is localized to the nucleus of these cells and is active, in that it binds an E2F-DNA binding site, together with E2F transcription factors. These findings suggest a hitherto unknown role for p107 in non-proliferating dendritic cells that warrants further investigation.

The retinoblastoma family of cell cycle regulatory proteins comprises pRb, p107 and p130. In their hypophosphorylated forms, the proteins bind to the E2F family of transcription factors, repressing their activity. As pRb, p107 and p130 become hyperphosphorylated, the E2F transcription factors are released and become active as transcription factors, resulting in the expression of many genes that are required for progression through the cell cycle (Thomas (1999).

The phosphorylation status of the protein, p107, alters as cells progress through the cell cycle. Non-dividing cells, including peripheral blood T and B lymphocytes and CD34+ progenitor cells, do not express p107 (Williams et al, 1997a; Thomas et al, 1998). When cells enter the cell cycle, p107 is produced in mid/late G1 and becomes phosphorylated at the G1/S border, remaining in this form throughout the remainder of the cycle. p107 is first dephosphorylated and then depleted during cell cycle exit by negative growth factors or differentiating agents (Thomas et al, 1998). Depletion occurs through calpain-mediated and proteasomal-targeted destruction (Jang & Choi, 1999).

Immature dendritic cells (MoDCs) can be generated from monocytes by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). We and others have shown that this process does not involve cellular proliferation (Ardeshna et al, 2000).

In this report, we present data demonstrating the presence of p107–E2F complexes in the nuclei of these non-proliferating MoDCs. Our findings suggest a hitherto unknown role for p107 in some non-dividing haemopoietic cell types.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cell culture Monocytes were positively selected from the peripheral blood of normal volunteers and cultured with GM-CSF and IL-4 for 7 d to generate immature DCs (Ardeshna et al, 2000). Lipopolysaccharide (LPS; 100 ng/ml) was added for 48 h to mature the cells. T lymphocytes were isolated from peripheral blood (Thomas et al, 1998).

Antibodies Anti-pRb (PMG3-245, Pharmingen), anti-p107 (C18), anti-E2F4 (C20) and anti-p21Cip1 (C19) (Santa Cruz Biotech) tubulins (Roche) were used.

Western blot and DNA pull-down assays These were performed as described previously with an E2F DNA-binding site (E2FWT: 5′-GATCTAGTTTTCGCGCTTAAATTTGA-3′) or a mutant site (E2FMUT: 5′-GATCTAGTTTTCGataTTAAATTTGA-3′) (Thomas et al, 1998).

Immunocytochemistry This was performed as described elsewhere (Williams et al, 1998).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The presence of p107 during the differentiation of monocytes to MoDCs was determined using Western blot analysis. p107 was present in freshly isolated monocytes and remained as the cells differentiated into immature MoDCs (Fig 1A). p107 also remained as the MoDCs matured in response to LPS. The same samples were probed for the cdk inhibitor, p21Cip1, which is induced during myeloid differentiation and acts as an inhibitor of apoptosis (Asada et al, 1999). p21Cip1 was present in monocytes on longer exposure (not shown), but was further induced after the addition of GM-CSF and IL-4.

image

Figure 1. (A) Western blot analysis. Monocytes were cultured in medium containing GM-CSF and IL-4 for 7 d to generate immature dendritic cells. At this point, lipopolysaccharide (LPS) was added for 48 h to mature the DCs. At varying time intervals cells were removed from culture and lysates made. Western blotting was perfomed on these samples which were subsequently probed with antibodies directed against p107 (top), p21 (middle) and tubulin (bottom). Lysates from non-proliferating T cells (NP) and proliferating Daudi cells (P) were run as controls. p107 was present in d 0 monocytes, remained present throughout the culture period and continued to be present in LPS-matured DCs. The cyclin-dependent kinase inhibitor p21 was detected in these cells early in the culture period, but was further induced by d 4. (B) ‘DNA pull-down assays’. Nuclear extracts were made from MoDCs (and quiescent T cells and proliferating Daudi cells used as controls). These were incubated with oligonucleotides bearing a wild-type or mutant E2F DNA binding site. Proteins that bound to the oligonucleotides (i.e. E2F transcription factors and any proteins bound to them) were then separated using Western blotting and probed with antibodies against p107 and pRb. p107 bound to a E2Fwildtype DNA site but not to a E2Fmutant DNA site. Hypophosphorylated pRb also bound to a E2Fwildtype DNA site. Go T cell, non-proliferating T cells; prolif Daudi, proliferating Daudi cells; WT, wild type; MUT, mutant. The numbers 2 and 10 refer to the number of cells ( × 106) that were used to make the nuclear extracts. In the final two lanes, whole cell lysates were used.

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In order to determine whether the p107 found in the MoDCs was bound to E2F transcription factors and, hence, active in these cells, ‘DNA pull-down’ assays were performed. Cell extracts were incubated with wild-type or mutant E2F DNA binding sites and the bound proteins analysed using Western blot analysis. p107 bound to an E2Fwildtype DNA site but not to an E2Fmutant DNA site, indicating that the p107 in the MoDCs was active. Hypophosphorylated pRb, which we showed previously was present in MoDCs (Ardeshna et al, 2000), also bound to the E2Fwildtype DNA site (Fig 1B).

The subcellular localization of p107 and E2F4 was determined using immunocytochemistry. In d 7 MoDCs, both p107 and E2F4 were evenly distributed throughout the nucleus (Fig 2). These signals were abolished when the primary antibodies were blocked with their cognate peptides, demonstrating the specificity of the antibody staining.

image

Figure 2. MoDCs that had been permeabilized were incubated with antibodies directed against p107 and E2F4. These in turn were detected using immunoenzymatic staining. p107 and E2F4 were evenly distributed throughout the nuclei of these cells. Preincubation of the primary antibodies with their cognate peptides abolished this pattern of staining, indicating the specificity of the antibodies (inserts).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

p107 was present in monocytes and remained detectable during differentiation into dendritic cells. This differentiation process does not involve cell proliferation, as determined by several criteria (Ardeshna et al, 2000). The presence of p107 in these non-cycling cells is of interest because p107 is not normally expressed in other non-proliferating cells. For example, p107 is undetectable or present at low levels in quiescent CD34+ haemopoietic progenitor cells, T or B lymphocytes and is depleted during the cell cycle arrest of Daudi B cells by α-interferon or HL60 cells with retinoic acid (Thomas et al, 1998). In non-haemopoietic cells, p107 is present in terminally differentiated rat lens fibre cells (Rampalli et al, 1998) and human granulosa lutein cells (Green et al, 2000).

We have shown here that p107 and E2F4 bind to an E2F DNA-binding site, indicating that the p107 is active and may repress E2F transcriptional activity. We used the same E2F DNA-binding site in a previous study in which we showed that the predominant E2F complex in monocytes was comprised of p130–E2F4–DP1 (Williams et al, 1997b). The study used an electrophoretic mobility shift assay which was not sensitive enough to pick up the p107–E2F complex that we have now shown to be present using DNA pull-down/Western blot assays. p107 only binds and represses E2F complexes containing E2F4 or E2F5 (Thomas et al, 1998). This, together with the finding that p107 and E2F4 were localized to the nucleus of dendritic cells, raises the possibility that p107–E2F4 complexes may play an important, hitherto undescribed, role in maintaining their differentiated state. Indeed, the closely related molecule, pRb, maintains the post-mitotic state in some mouse cell types (Lipinski & Jacks, 1999).

Monocytes can differentiate into either MoDCs or macrophages depending on the cytokine stimuli and, upon removal of growth factors, immature DCs revert to monocytes (Banchereau & Steinman, 1998). It is thus tempting to ascribe the presence of p107–E2F complexes to the ability of such cells to retain their phenotypic plasticity. However the phenotype of mature DCs, which also have p107–E2F complexes, is stable upon growth factor withdrawal and is not known to change under the influence of cytokines.

In conclusion, we have demonstrated for the first time that p107 is present in non-proliferating MoDCs and is localized to the nucleus. Taken together with our previous study, we have shown that each member of the pRb family is active in MoDCs, although to what extent they each regulate specific processes and why p107 is specifically required remain to be determined.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

K.A. was supported by a grant from the Leukaemia Research Fund and N.S.T.B. by the Kay Kendall Leukaemia Trust.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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