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Originally published online as doi:10.1189/jlb.1003474 on December 23, 2003

Published online before print December 23, 2003
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(Journal of Leukocyte Biology. 2004;75:569-578.)
© 2004 by Society for Leukocyte Biology

End-stage differentiation of neutrophil granulocytes in vivo is accompanied by up-regulation of p27kip1 and down-regulation of CDK2, CDK4, and CDK6

Pia Klausen1, Malene Digmann Bjerregaard, Niels Borregaard and Jack Bernard Cowland

The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, University of Copenhagen, Denmark

1 Correspondence: The Granulocyte Research Laboratory, Rigshospitalet, Department of Hematology 93.2.2, 20 Juliane Mariesvej, DK-2100 Copenhagen, Denmark. E-mail: pklausen{at}rh.dk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The in vivo expression profiles of cell-cycle proteins regulating G1-to-S-phase transition were determined in three neutrophil precursor populations from human bone marrow: myeloblasts (MBs) and promyelocytes (PMs); myelocytes (MCs) and metamyelocytes (MMs); and band cells (BCs) and segmented neutrophil cells (SCs) and in mature polymorphonuclear neutrophils (PMNs) from peripheral blood. Complete cell-cycle arrest was observed in BCs/SCs and PMNs. Cyclins D1, D2, and D3 were found to be down-regulated during granulopoiesis, whereas a slight increase of cyclin E was seen. In contrast, cyclin-dependent kinase (CDK)2, -4, and -6 were down-regulated from the MC/MM stages and onward. The transcript levels of CDK2, -4, and -6 were concurrently down-regulated. As the only CDK inhibitor, p27kip1 protein and mRNA expression were up-regulated in MCs/MMs and reached peak levels in PMNs. Protein expression of retinoblastoma protein and the related pocket proteins p107 and p130 was down-regulated from the MC/MM stages and onward. This is the first report to describe expression levels of cell-cycle proteins during granulopoiesis in vivo, and it strongly contrasts the observations made in cell-culture systems in vitro.

Key Words: granulopoiesis • cell cycle • bone marrow


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three fundamental processes are vital for development and maintenance of all tissues: the ability to reproduce, to differentiate, and to undergo apoptosis. This is also true during granulopoiesis, where hematopoietic stem cells are able to provide a life-long maintenance of circulating neutrophil granulocytes. Granulopoiesis is the result of a strictly regulated process by which the myeloid cell progenitor, the myeloblast (MB), divides and matures along a well-known path where phenotypically distinct intermediates, differing in their functional capacity and ability to divide, can be recognized [1 ]. The intermediates can be divided into six different stages: the MBs, promyelocytes (PMs), and myelocytes (MCs), which all have maintained the ability to divide, and the more mature metamyelocytes (MMs), band cells (BCs), and segmented neutrophil cells (SCs), which are unable to divide [1 ]. Thus, cell-cycle arrest is an intimate part of granulopoiesis. It is well established that the cell-cycle machinery can only be affected by extracellular signals in the G1 phase up to the restriction point in late G1 [2 ]. Beyond the restriction point, cells progress through the cell cycle in an autonomous manner [3 ]. Thus, to understand how the irreversible cell cycle stops during myeloid differentiation is exerted, examination of G1-phase protein expression during granulopoiesis is necessary. The enzymes that regulate G1-to-S-phase transition are the cyclin-dependent kinases (CDK)2, -4, and -6 [4 ]. CDK4 and CDK6, complexed with D-type cyclins, initially phosphorylate the retinoblastoma protein (pRb). Later in G1, CDK2 complexed with cyclin E additionally phosphorylates pRb [4 5 ]. This relieves the inhibitory binding of pRb to transcription factors of the E2F family, and transcription of genes necessary for S-phase progression is initiated [4 ]. The CDK inhibitors (CDKIs) [6 ], which can be divided into two structurally distinct families—the INK4 and CIP/KIP families—negatively regulate the activity of CDKs. Whereas the INK4 family (consisting of p15ink4b, p16ink4a, p18ink4c, and p19ink4d) specifically inhibits CDK4 and CDK6, the CIP/KIP family (consisting of p21cip1, p27kip1, and p57kip2) inhibits a broad range of CDKs [6 ]. Expression of cell-cycle proteins during differentiation of neutrophil granulocytes has been studied extensively in cell lines. However, a consistent pattern is not evident, as the regulation of cell-cycle proteins during differentiation varies between the different cell systems studied [7 8 9 10 11 12 13 14 15 16 ]. The most commonly used human cell models are the acute promyelocytic leukaemia (APL)-derived cell line NB4 and the acute myeloid leukaemia-derived cell line HL60. However, a major problem with these cell lines is that the cells are unable to faithfully reproduce all stages of neutrophil differentiation, as exemplified by their inability to express matrix proteins of peroxidase-negative granules [17 18 ]. Furthermore, great care must be imposed when interpreting cell-cycle regulation in these cell systems as a result of the immortalized nature of cell lines. Thus, to gain a true insight into the regulation of the cell cycle during granulopoiesis, in vivo populations of neutrophil precursors must be investigated.

In this work, we present a scan of cell-cycle proteins regulating G1-to-S transition in myeloid precursor populations and terminally differentiated neutrophils isolated from healthy individuals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and isolation of neutrophil precursor populations and polymorphonuclear neutrophils (PMNs)
Isolation of neutrophil precursor populations and PMNs was performed as described previously [19 ]. Briefly, bone marrow samples from healthy donors were used for isolation of neutrophil precursor populations by Percoll (Amersham Biosciences, Little Chalfont, UK) density centrifugation. Following centrifugation, non-neutrophil bone marrow cells were immunodepleted using magnetic cell sorting (MACS) columns (Miltenyi Biotech, Bergisch Gladbach, Germany), and the purified cells were used for protein and RNA preparation. Purity of the cell populations was analyzed on a FACScan flow cytometer (Becton Dickinson, San Diego, CA). NB4 and THP-1 cells were cultured in RPMI media (Invitrogen, San Diego, CA) containing 10% fetal calf serum (Invitrogen) and 100 U/mL penicillin/100 µg/mL streptomycin (Invitrogen). Cells were grown at 37°C in a humid atmosphere with 5% CO2.

Cell-cycle analysis
Cells/sample (105) were washed in ice-cold, phosphate-buffered saline and were lysed in 200 µL modified Vindeloev buffer [1% w/v sodium citrate, pH 7.4, 0.02 mg/mL RNase A, 0.3% v/v Nonidet P-40 (NP-40), 0.05 mg/mL propidium iodide (PI)] in the dark. The intensity of PI-labeled DNA was subsequently measured on a FACScan flow cytometer (Becton Dickinson).

RNA isolation and Northern blotting
Total RNA was isolated with Trizol (Invitrogen). The RNA was ethanol-precipitated and resuspended in 0.1 mM EDTA. For Northern blotting, 5 µg RNA was separated on a 1% agarose gel and transferred to a Hybond-N membrane (Amersham Biosciences) as described [19 ]. Filters were prehybridized for 30 min at 42°C in 6 mL ULTRAhyb (Ambion, Austin, TX) and were hybridized overnight at 42°C after addition of further 4 mL containing the 32P-labeled probe and sheared salmon sperm DNA (10 µg/mL). The membranes were washed as described [19 ] and developed by a Fuji BAS2500 Phosphor Imager (Fuji, Tokyo, Japan). Membranes were stripped by boiling in 0.1% sodium dodecyl sulfate (SDS) before rehybridization. Probes used for hybridization were radiolabeled with [{alpha}-32P]deoxycytidine 5'-triphosphate using the Random Primers DNA labeling system (Invitrogen). Sizes of the hybridizing bands were determined relative to 18S and 28S and were found to be in accordance with previous reports (Table 1 ).


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  		Table 1. Synthesis of cDNA Probes for Northern Blot Hybridization

 
Construction of probes for Northern blot
The probes were constructed by polymerase chain reaction (PCR) amplification with the use of a human bone marrow cDNA library (Clontech, Palo Alto, CA) or cDNA from HL60 cells as template (Table 1) and were cloned in pCRII (Invitrogen). Correctness of the inserts was confirmed by sequencing. Inserts were excised and gel-purified before use. Specificity of the probes was confirmed by a Blast search (www.ncbi.nlm.nih.gov/blast/).

Preparation of whole-cell lysates and Western blotting
Cells/mL (3x106–7.5x106) were preincubated in 0.7 µM diisopropyl fluorophosphate (DFP) for 5 min prior to lysis in 1 mL Trizol (Invitrogen)/107 cells. Lysates were added to 200 µL chloroform and were centrifuged for 15 min/12,000 g. DNA was removed by adding 300 µL ethanol to the organic phase and centrifuged for 5 min/7500 g. Isopropanol (1 mL) was added to the supernatant, and the lysate was incubated for 10 min at room temperature following centrifugation for 10 min/12,000 g. Precipitated protein was washed three times in 2 mL 0.3 M guanidine HCl/ethanol and once in 2 mL ethanol. Pellet was dissolved in 333 µL boiling 2x Laemmli buffer (0.125 M Tris-base, pH 6.8, 20% v/v glycerol, 4% w/v SDS, bromphenol blue, 2% v/v 2-mercaptoethanol). For Western blotting, 3 x 105 cells/population were run on SDS gels (between 7.5% and 14%, depending on the protein tested) and were transferred onto nitrocellulose membranes (Sartorius, Göttingen, Germany). Immediately after blotting, membranes were incubated in Ponceau S (Sigma-Aldrich, St. Louis, MO) for verification of successful blotting. The immune complexes were visualized by enhanced chemiluminescence (Amersham Biosciences), according to the manufacturer’s description. For the experiments using radio immunoprecipitation assay (RIPA) buffer, cells were lysed in ice-cold RIPA buffer (50 mM Tris-base, pH 7.4, 1% v/v NP-40, 0.25% v/v sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4) containing a broad cocktail of protease inhibitors (1836153; Roche, Mannheim, Germany) for 30 min on ice and subsequently centrifuged for 10 min/12,000 g. Supernatants were analyzed on Western blots as described above.

Antibodies and positive controls
Antibodies against CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), cyclin A (sc-751), cyclin D1 (sc-718), p16ink4a (sc-468), p18ink4c (sc-1064), p19ink4d (sc-1063), p57kip2 (sc-1040), p130 (sc-9963), signal transducer and activator of transcription (STAT)3 (sc-482; Santa Cruz Biotechnology, Santa Cruz, CA); {alpha}-tubulin (T9026; Sigma-Aldrich); cyclin D2 (AHF0122; Biosource, Camarillo, CA); cyclin D3 (610279), p21cip1 (C24420), p27kip1 (K25020), pRb (14001A; BD Biosciences-PharMingen, San Jose, CA); cyclin E (RB-012), p15ink4b (MS-1053), p107 (RB1414; Neomarkers, Fremont, CA); and neutrophil gelatinase-associated lipocalin (NGAL) [22 ] were used for Western blot analysis according to the manufacturers’ recommended concentrations. Horseradish peroxidase-linked rabbit anti-mouse immunoglobulin G (IgG; P0260, Dako, Glostrup, Denmark) or swine anti-rabbit IgG (P0217, Dako) was used as a secondary antibody. Whole-cell lysates from the following cell lines were used as positive controls for Western blot analysis: HepG2 (CDK2, CDK4, cyclins A, D1, E, p15ink4b, p27kip1, p57kip2), K562 (CDK6, p18ink4c, p19ink4d), HL60 (cyclin D2), Jurkat (cyclin D3), HeLa (p16ink4a, pRb), MCF-7 (p21cip1, p130), and NIH3T3 (p107).

Nitroblue tetrazolium (NBT) test
NBT test was performed as described previously [23 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
End-stage differentiated neutrophil granulocytes are arrested in G0/G1
Neutrophil precursor populations were isolated by Percoll density centrifugation as described previously [19 ]. Following centrifugation, the neutrophil populations were purified by a depletion protocol on a MACS column. PMNs isolated from peripheral blood were also depleted of contaminating eosinophils. As described previously, we were able to obtain neutrophil precursor populations and PMNs with a purity >95% [19 ].

We initially investigated the proportion of cells in G0/G1 and S/G2 + M phases. MBs/PMs, MCs/MMs, BCs/SCs, and PMNs (Fig. 1 A ) were lysed, and DNA was stained with PI. In the most immature cell fraction (MBs/PMs), 59% of the cells were in G0/G1, and 36% of the cells were in S/G2 + M phases (Fig. 1B and 1C) . The amount of cells in S/G2 + M was significantly reduced in the MC/MM fraction, only 12% of the cells were in S/G2 + M, and the amount of cells in G0/G1 was concordantly increased to 86% (Fig. 1B and 1C) . The two most mature cell populations, BCs/SCs and PMNs, were 99% G0/G1 cells (Fig. 1B and 1C) . None of the populations showed more than 9% apoptotic cells (Fig. 1B) .



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  		Figure 1. Morphology and DNA staining of the four neutrophil cell populations. Percoll-separated bone marrow populations: MBs/PMs, MCs/MMs, and BCs/SCs and PMNs from peripheral blood were purified using MACS columns. (A) Cytospin preparations of the four cell populations. Cells were stained with May-Grünwald-Giemsa. (B and C). Populations of 105 cells were lysed in modified Vindeloev buffer, and DNA was stained with PI. The extracts were analyzed by flow cytometry, and the percentages of cells in G0/G1 and S/G2 + M were calculated. The shown diagrams are representative of three independent experiments.

 
Inhibition of granule-derived proteases is necessary for preparation of whole-cell protein lysates
To investigate cell-cycle protein expression, whole-cell lysates were prepared. As a result of the high amount of granule-derived proteases in neutrophil granulocytes, the lysis process was optimized. PMNs, which contain the highest amount of granule-derived proteases [24 ], were lysed in RIPA buffer, boiled directly in 2x Laemmli buffer containing 2-mercaptoethanol, or lysed in Trizol buffer/guanidine HCl. Lysis in Trizol buffer/guanidine HCl was the method that allowed recovery of the largest amount of proteins as determined by Western blot, followed by boiling of the cells in 2x Laemmli buffer (Fig. 2 A ). Lysis in RIPA buffer caused loss of most proteins and only allowed recovery of trace amounts of {alpha}-tubulin (Fig. 2B) . To ensure that the lower amount of protein in the RIPA and Laemmli lysates was not a result of unsuccessful lysis, we also measured the amount of the granule-derived protein NGAL, which is very resistant to degradation by granule-derived proteases [25 ]. NGAL was fully recovered after lysis in RIPA, Laemmli, and Trizol buffers (Fig. 2A) . The active proteases stored in neutrophil granulocytes are predominantly serine proteases [26 ]. Thus, the effect of irreversible inhibition of serine proteases with DFP was therefore also tested. Preincubation of PMNs with DFP before lysis clearly improved the recovery of p27kip1, STAT3, and {alpha}-tubulin by all three methods. As expected, the effect was less prominent in the Trizol/guanidine HCl lysate, as this had the least spontaneous proteolysis (Fig. 2A) . Thus, for the following experiments, we decided to preincubate the cells with DFP before preparation of whole-cell lysates using Trizol/guanidine HCl.



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  		Figure 2. Comparison of three different lysis methods for recovery of proteins in PMNs (3x105), which were lysed in RIPA, 2x Laemmli, or Trizol buffer as described in Materials and Methods. Cells were preincubated with 0.7 µM DFP prior to lysis where indicated. (A) Western blots of STAT3 (a 91-kDa STAT3{alpha} form and a 90-kDa STAT3ß form), {alpha}-tubulin, p27kip1, and NGAL expression. Equal amounts of cells (3x105) were loaded on each lane. RIPA lysate of HepG2 cells is shown as a positive control for successful RIPA lysis. (B) Long-time exposure of the {alpha}-tubulin blot showing trace amounts of {alpha}-tubulin in the RIPA lysates.

 
Protein expression profiles of G1- and S-phase cyclins, CDKs, and pocket proteins during differentiation of neutrophil granulocytes
We next investigated protein expression profiles of the G1 phase regulating cyclins D1, D2, D3, and E and the S phase regulating cyclin A. Cyclins D1 and D2 proteins diminished at the MC/MM stage, whereas the protein level of cyclin D3 peaked at the MC/MM stage and almost disappeared at the BC/SC stage (Fig. 3A ). Cyclin E (~45-kDa form) was slightly up-regulated from the MC/MM stage and onward. Additionally, an ~50-kDa form of cyclin E appeared in the more mature populations of neutrophil granulocytes: BCs/SCs and PMNs (Fig. 3A) . The protein level of cyclin A was strongly decreased already at the MC/MM stage and was not present in BCs/SCs and PMNs (Fig. 3A) , as expected from the cell-cycle arrest pattern seen in Figure 1 .



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  		Figure 3. Protein expression profiles of cyclins, CDKs, and pocket proteins in the four neutrophil populations. MACS-purified neutrophil cell populations (3x105) were pretreated with 0.7 µM DFP prior to lysis in Trizol buffer. Western blots showing protein levels of (A) cyclins D1, D2, D3, E, and A, (B) the G1 phase CDK2, -4, and -6, and (C) the pocket proteins pRb and P-pRb (hyper-phosphorylated), p107, and p130. We have previously observed a decrease of ß-actin and glyceraldehyde-3-phosphatase dehydrogenase in the more mature populations of neutrophils [19 ]. Thus, we decided to use STAT3 expression as a loading control, as we found that the expression of this protein was concurrent with the cell number in all neutrophil populations. The shown results are representative of three independent experiments. See Materials and Methods for the positive controls used on the Western blots.

 
As we were only able to detect down-regulation of D-type cyclins, we next speculated whether cell-cycle arrest during terminal differentiation might be executed by down-regulation of the CDKs, regulating G1-to-S-phase transition. CDK2, -4, and -6 proteins were clearly expressed in the most immature populations—MBs/PMs (Fig. 3B) . The protein levels of the three CDKs were, however, strongly decreased at the MC/MM stage, and only a weak expression of CDK4 and -6 was detected in the BC/SC and PMN populations. We were unable to detect CDK2 in BCs/SCs and PMNs (Fig. 3B) .

To confirm cell-cycle arrest and low activity of CDK2, -4, and -6 in the more mature stages of neutrophil granulocytes, we investigated the phosphorylation status of pRb. In concordance with the decreased fraction of cells in S/G2 + M phases (Fig. 1B and 1C) and the lack of expression of S-phase cyclin A (Fig. 3A) , no hyperphosphorylated pRb could be detected in BCs/SCs and PMNs (Fig. 3C) . Hyperphosphorylation of pRb was most prominent in MBs/PMs and already diminished in the MCs/MMs (Fig. 3C) . The protein level of nonphosphorylated pRb was decreased in BCs/SCs and PMNs, compared with the less mature populations (Fig. 3C) . We next investigated protein expression levels of the two pRb-related pocket proteins p107 and p130, which were present in MBs/PMs but were down-regulated at the MC/MM stage an onward. Although p130 already diminished at the MC/MM stages and was not present in the more mature populations, p107 was down-regulated at a slower rate, allowing the detection of minor amounts of p107 in BCs/SCs and PMNs (Fig. 3C) .

Protein expression profiles of CDKIs during differentiation of neutrophil granulocytes
Up-regulation of CDKIs might also play a role in induction of cell-cycle arrest during terminal differentiation. We therefore investigated the expression pattern of the specific inhibitors of CDK4 and –6, the INK4 CDKIs. Although we were unable to detect p15ink4b and p16ink4a proteins, the protein level of p18ink4c was constant through all stages of differentiating neutrophil granulocytes (Fig. 4 A ). Following prolonged exposure of the p19ink4d Western blot, we were also able to detect a constant level, comparable with the control lysate, of p19ink4d in all four populations (Fig. 4A) . We next investigated the generic CIP/KIP CDKIs. The protein level of p57kip2 remained unchanged during granulopoiesis, and p21cip1 was undetectable. In sharp contrast, the protein level of p27kip1 clearly increased from the MC/MM stages and onward (Fig. 4B) . These data are in contrast to those previously reported, where an induction of p21cip1 mRNA and protein was seen following differentiation of the promyelocytic APL cell line NB4 with all-trans retinoic acid (ATRA) [10 11 27 ]. To determine whether we could reproduce the induction of p21cip1 under the same assay conditions, we incubated NB4 cells with 1 µM ATRA for 6 days, which induced differentiation of the cells into the granulocytic lineage as determined by morphology, NBT reduction, and down-regulation of CD49d expression (data not shown). Following MACS depletion of not fully differentiated cells still expressing CD49d [28 ], protein expression of p27kip1 and p21cip1 was investigated. In concordance with the literature, we too observed an increase of p21cip1 and p27kip1 protein in the differentiated NB4 cells (Fig. 4C) . This demonstrates differences in cell-cycle regulation between in vivo and in vitro systems. Whereas p21cip1 seems to be important in the NB4 cell system, it is apparently not involved in differentiation of neutrophils in vivo.



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  		Figure 4. Protein expression profiles of CDKIs in the four neutrophil populations. Cell lysates were prepared as in Figure 3 . Western blots showing protein levels of (A) the INK4 family of CDKIs, p15ink4b, p16ink4a, p18ink4c, and p19ink4d, and of (B) the CIP/KIP family of CDKIs, p21cip1, p27kip1, and p57kip2. (C) Protein expression levels of p21cip and p27kip1 were assessed on Western blots in undifferentiated NB4 cells (–) and in NB4 cells differentiated with 1 µM all-trans retinoic acid (ATRA) for 6 days (+). STAT3 expression was used as a loading control. The shown results are representative of three independent experiments. See Materials and Methods for the positive controls used on the Western blots.

 
CDK4, CDK6, CDK2, and p107 mRNAs disappear, and p27kip1 mRNA increases during differentiation of neutrophil granulocytes
We next wished to investigate whether the decrease of CDKs, p107, and p130 proteins and the increase of p27kip1 protein could be executed by changes in levels of corresponding mRNAs, which would indicate that the protein level was regulated at the transcriptional level. We found that the transcript profiles for CDK2, -4, and -6 followed the same pattern as the protein expression profiles (Fig. 5 A ). Previously, it has been reported that three different CDK6 transcripts can be detected in human cells [20 ], but we only observed the largest of these CDK6 mRNAs, sized around 11 kb (Fig. 5A) . The transcript profile for p107 was also in concordance with the protein expression profile (Fig. 5B) . In contrast, the transcript level for p130 was unchanged during differentiation (Fig. 5B) , and the protein level of p130 was clearly diminished in MCs/MMs and undetectable in BCs/SCs and PMNs (Fig. 3C) . As a control, we investigated the transcript profile for pRb, for which the protein level also decreased during granulopoiesis, although some nonphosphorylated pRB was still present in PMNs. As for p130, we observed a constant level of pRb transcript in all populations (Fig. 5B) . The only CDKI for which we observed a change in quantity during granulopoiesis was p27kip1. We therefore wished to investigate whether p27kip1, like the CDKs, had concurrent transcript and protein level profiles. This was indeed the case as p27kip1 mRNA was up-regulated in MCs/MMs and reached its maximal level in PMNs (Fig. 5C) . To substantiate our findings on p21cip1 protein expression, we also decided to examine the transcript profile of p21cip1. As for the protein data (Fig. 4B) , no p21cip1 transcript (Fig. 5C) could be detected in any of the neutrophil cell populations. As a control of the specificity of our probe, we tested mRNA from a variety of tissues and cell lines. As shown in Figure 5C , we were clearly able to detect p21cip1 mRNA in lung and colon tissues and A549, NHEK, and NHBE cell lines. The reason for the discrepancy regarding p21cip1 expression in PMNs is not known, but in our hands p21cip1 expression appears to be insignificant during granulopoiesis.



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  		Figure 5. Transcript profiles of the CDKs, the pocket proteins, p27kip1, and p21cip1. Northern blots of total RNA from the four MACS purified neutrophil cell populations. The blots were hybridized with probes against (A) CDK2, -4, and -6, (B) pRb, p107, and p130, and (C) p21cip1 and p27kip1. 18S expression was used as a loading control. Total RNA extracted from lung, colon, and brain tissues, and A549, normal human bronchial epithelial (NHBE), normal human epidermal keratinocytes (NHEK), and HL60 cell lines were used as positive controls for p21cip1 mRNA expression.

 
Disappearance of p107, p130, and CDK proteins in the BCs/SCs and PMNs is not caused by degradation of the proteins during preparation of whole-cell lysates
As presented above, CDK2, -4, and -6, p27kip1, pRb, and p107 had concurrent transcript and protein expression profiles (Figs. 3 4 5) . In contrast, the level of p130 mRNA was constant in all stages of differentiating granulocytes, and p130 protein was absent in the BC/SC and PMN populations (Figs. 3 and 5) . As shown in Figure 2 , proteases must be inhibited to recover cell-cycle-regulating proteins when preparing whole-cell lysates. The disappearance of p130 protein in the mature populations of neutrophil granulocytes might therefore be explained by a higher susceptibility of p130 for degradation during lysis than the other proteins investigated. THP-1 cells express CDK2, -4, and -6, p107, and p130. To rule out the possibility that lack of CDK2, -4, and -6, p107, and p130 expression in the PMN lysate was a result of protease activity, we mixed DFP-treated THP-1 cells and PMNs before lysis in Trizol/guanidine HCl. As shown in Figure 6 , CDK2, -4, and -6, p107, and p130 proteins from THP-1 cells were not degraded by the PMN-derived proteases, demonstrating that our lysis procedure is able to fully recover these proteins. Combined, these data indicate that p130 and pRb proteins might have an increased turnover rate in BCs/SCs and PMNs compared with the more immature neutrophil precursors, whereas the levels of CDK2, -4, and -6, and p107 proteins reflect their cognate mRNA levels.



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  		Figure 6. Protein from THP-1 cells is fully recovered in PMN lysates. THP-1 cells or PMNs were preincubated with 0.7 µM DFP prior to lysis in Trizol buffer. For the double lysate, equal amounts of THP-1 cells and PMNs were mixed immediately following DFP treatment and were subsequently lysed in Trizol buffer. Lysates from 3 x 105 cells were loaded on lanes 1 (THP-1) and 2 (PMN), and a lysate from 6 x 105 cells was loaded on lane 3 (THP-1+PMN) to ensure that the protein contribution from THP-1 cells was as in lane 1. The Western blots show the recovery of CDK2, -4, and -6, p107, and p130 proteins.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well established that granulopoiesis is accompanied by irreversible cell-cycle arrest [1 ] (Fig. 1) . Here, we show that expression of several cell-cycle proteins is altered during differentiation of human neutrophils, as summarized in Figure 7 . We observed down-regulation of all three D-type cyclins at the BC/SC stage, whereas the protein expression of cyclin E was slightly up-regulated during differentiation, and an ~50-kDa form of cyclin E emerged in the BC/SC and PMN populations. Of the CDKIs, only p27kip1 was extensively up-regulated from the MC/MM stage and onward. It is striking that all three G1-phase CDKs were strongly down-regulated from the MC/MM stages and onward on transcript and protein levels. Down-regulation of CDKs is a common theme in end-stage, differentiated cells [29 30 31 32 ]. However, simultaneous down-regulation of all three G1-phase CDKs is not the general picture. Although CDK2 is down-regulated, protein levels of CDK4 and -6 are unchanged in differentiating mouse keratinocytes [32 ] and during renal development [31 ]. The expression pattern of G1-phase proteins during neuronal differentiation, conversely, resembles the one shown in granulocytic differentiation. Cell-cycle arrest and down-regulation of CDK4 and CDK2 [29 30 ] accompanied differentiation of oligodendrocyte progenitor cells in vitro and a concomitant up-regulation of p27kip1 [30]. Combined, these data indicate that the cell-cycle arrest that accompanies end-stage differentiation of neutrophils is executed through two different mechanisms: by turning off the cell-cycle accelerators via down-regulation of all three G1 CDKs and by inducing a generic CDK brake via up-regulation of p27kip1. These events seem to be regulated by changes of transcript levels and possibly expression activity of the cognate genes. Regulation of p27kip1 has previously been shown to occur at the post-translational level [33 34 ]. CDK2 complexed with cyclin E is known to down-regulate p27kip1 protein by phosphorylation, which tags p27kip1 for degradation via the ubiquitin proteasome pathway [35 36 37 ]. However, during granulocytic differentiation, p27kip1 transcript is also up-regulated, rendering it unlikely that lack of CDK2 alone and thus merely stabilization of p27kip1 protein is causing the increase of p27kip1 protein in BCs/SCs and PMNs. Oligodendrocyte progenitor cells obtained from p27kip1-/- mice differentiate poorly [29 ]. Likewise, p27kip1-/- mice produce a reduced number of mature neutrophils [16 ]. These experiments indicate that p27kip1 deficiency alone is not sufficient to block cell-cycle arrest and differentiation. Speculatively, the concomitant down-regulation of the CDKs and up-regulation of p27kip1 might be two differently regulated mechanisms to induce cell-cycle stop. If one of these fails, the other will compensate to some degree, ensuring that the neutrophil is able to differentiate. This hypothesis has been tested in vitro, as inhibition of CDK2 restores the ability of Mad3/p27kip1 double-null granulocyte cell lines to differentiate in response to ATRA [16 ]. The existence of such back-up systems might explain why a single mutation often is insufficient to cause the formation of a malignant myeloid clone [38 39 ].



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  		Figure 7. Hypothetical distribution scheme of neutrophil cell-cycle proteins during granulopoiesis. Hypothetical distribution scheme of cell cycle proteins during granulopoiesis based on the data presented in Figures 3 and 4 .

 
We were surprised to find that the pocket protein p130 was down-regulated during granulocytic differentiation, as p130 has been described to be the predominant E2F regulator in quiescent cells [40 41 42 ]. Regulation of p130 is exerted mainly at the post-translational level [41 ]. In line with this, we did not observe any changes in the transcript level of p130 (Fig. 5) , indicating that p130 protein degradation is accelerated in the mature populations of neutrophils. p130 is degraded via the ubiquitin proteasome pathway, and CDK4 and -6 have been shown to tag p130 for degradation by phosphorylation [43 ]. As we only detected trace amounts of CDK4 and -6 protein in BCs/SCs and PMNs, other kinases may be involved in priming p130 for degradation. In agreement with our data, p107 has been shown to be regulated at the transcriptional level and to accumulate only at the stage of differentiation where the cell progresses through the cell cycle [41 42 ]. It has recently been observed that p130 and p107 are up-regulated during granulocytic differentiation in mice [16 ]. However, these experiments were performed in in vitro cultures and also differed from our results by showing unaltered levels of CDK2, -4, and -6 [16 ]. It has been demonstrated that p107 and p130 can bind and inhibit CDKs [44 45 ], and it could therefore be speculated that the need for p130 and p107 during differentiation may only be required if the CDKs are expressed. Accordingly, during in vivo granulopoiesis, where the CDKs are down-regulated, there is no need for p107 and p130 at the terminal stages of differentiation.

When aligning the protein expression profiles of cell-cycle proteins during granulopoiesis (Fig. 7) , it becomes clear that the major alterations take place at the MC/MM stage. Also, this is the point where the proliferative myelocyte turns into a nonproliferative metamyelocyte [1 ]. Hypothetically, several events need to be coordinated in the myelocyte to propagate to the metamyelocyte stage. The transcription factor c-myc is a likely candidate to control these events, as c-myc is rapidly down-regulated during granulocytic differentiation and disappears at the MM/MC stage [16 ] (unpublished). Concordantly, it is well known that c-myc down-regulates p27kip1 by suppressing its transcription and stability [46 47 48 ] and up-regulates CDK4 and cyclin D2 [49 50 ]. Other transcription factors, which may be involved in cell-cycle regulation during granulopoiesis are members of the CCAAT/enhancer-binding protein (C/EBP) family. We have recently described an individual expression pattern of the six C/EBP members during granulopoiesis [19 ], and a role in cell-cycle regulation and granulocytic differentiation has been ascribed to many of these proteins [51 ]. The forkhead transcription factor Foxo3a has also been shown to regulate transcription of the p27kip1 gene and thereby induce cell-cycle arrest [52 53 54 ]. However, Kops et. al. [53] showed that in human colon carcinoma cells, conditional activation of Foxo3a increases p27kip1 and p130 protein expression. Our finding that p130 is down-regulated during granulopoiesis indicates that p130 is not regulated by Foxo3a in granulocytes, or Foxo3a is not involved in regulating cell-cycle arrest during granulopoiesis. Another likely candidate is STAT3, which also has been shown to regulate p27kip1 on a transcriptional level [55 56 ]. Granulocyte-colony stimulating factor is a well-known inducer of granulocytic differentiation, and this has been shown to be followed by a strong STAT3 activation and up-regulation of p27kip1 [55 ].

Cell-cycle regulation during myeloid differentiation has been studied extensively in vitro. However, two major problems exist when comparing the in vitro models and the presented in vivo data: the mechanisms by which cell-cycle arrest is executed using various agents and differences between model cell lines and neutrophil precursor cells. ATRA is a commonly used agent to differentiate granulocytes in vitro [11 27 ]. Furthermore, ATRA is used to exert end-stage differentiation of promyelocytes in APL patients [57 58 ]. No concise picture is evident regarding which cell-cycle proteins are influenced by ATRA in model cell systems. However, it seems to be that ATRA is acting on the brakes, especially the INK4s and p21cip1 [7 10 11 59 ] (Fig. 4C) and through down-regulation of cyclins [9 60 ] rather than influencing CDK levels [12 60 ]. This resembles more the pattern seen in the reversible withdrawal of cells from the cell cycle, which is regulated by cytokines [4 ], than the irreversible cell-cycle stop seen, e.g., in terminally differentiated neurons [61 ]. The second problem lies in the model cell systems used. As mentioned in Introduction, the human cell lines commonly used as models for granulocytic differentiation are unable to express matrix proteins of peroxidase-negative granules [17 18 ]. Expression of the genes encoding matrix proteins of specific granules (such as lactoferrin, NGAL, and hCAP18) is coordinately regulated and initiated in the myelocyte [62 ]. The observation that human myeloid cell lines such as NB4 and HL60 are unable to express these genes [17 18 62 ] indicates that a major transcriptional regulator(s), acting on the myelocyte stage, is missing or is nonfunctional in these cells. As transcription of the genes encoding specific granule matrix proteins occurs at the same differentiation stage as the up-regulation of p27kip1 mRNA and down-regulation of CDK2, -4, and -6 mRNA are observed in vivo, a concern is that the transcriptional regulation of the genes encoding the cell-cycle regulatory proteins also is corrupted in the model cell lines. One difference in the mechanisms by which cell-cycle arrest, e.g., of NB4 cells and human bone marrow neutrophils, is executed may be the role of p21cip1. In concordance with the literature, we detected up-regulation of p21cip1 protein in NB4 cells during differentiation, and neither p21cip1 mRNA nor protein was present in any of the neutrophil populations. Thus, ATRA-induced differentiation of human neutrophil cell lines does not faithfully reflect granulopoiesis in vivo.

In conclusion, cell-cycle arrest during granulocytic differentiation in vivo is mediated by a coordinated down-regulation of the G1 CDKs and pocket proteins and up-regulation of p27kip1. As regulation of p27kip1 and the CDKs appears to be controlled at the transcriptional level, delineation of the regulation of CDK and p27kip1 promoters in granulocytes is likely to give valuable information of how cell-cycle arrest is exerted during differentiation, which in turn might give rise to development of new therapeutic drugs.


   ACKNOWLEDGEMENTS
 
Grants from the Danish Medical Research Council, Danish Cancer Society, Carlsberg Foundation, Danish Foundation for Cancer Research, Danish Medical Association Research Fund, Copenhagen University Hospital (H:S), and the Novo Nordisk Foundation supported this work. The expert technical assistance of Inge Kobbernagel and Katja Maj Nielsen is greatly appreciated. Mikkel Faurschou, M.D., is thanked for helpful comments about the manuscript.

Received October 13, 2003; revised November 14, 2003; accepted November 19, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bainton, D. F., Ullyot, J. L., Farquhar, M. G. (1971) The development of neutrophilic polymorphonuclear leukocytes in human bone marrow J. Exp. Med. 134,907-934[Abstract]
  2. Sherr, C. J. (1996) Cancer cell cycles Science 274,1672-1677[Abstract/Free Full Text]
  3. Ferreira, R., Naguibneva, I., Pritchard, L. L., Ait-Si-Ali, S., Harel-Bellan, A. (2001) The Rb/chromatin connection and epigenetic control: opinion Oncogene 20,3128-3133[CrossRef][Medline]
  4. Bartek, J., Bartkova, J., Lukas, J. (1997) The retinoblastoma protein pathway in cell cycle control and cancer Exp. Cell Res. 237,1-6[CrossRef][Medline]
  5. Harbour, J. W., Luo, R. X., Dei, S. A., Postigo, A. A., Dean, D. C. (1999) Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1 Cell 98,859-869[CrossRef][Medline]
  6. Sherr, C. J., Roberts, J. M. (1999) CDK inhibitors: positive and negative regulators of G1-phase progression Genes Dev 13,1501-1512[Free Full Text]
  7. Navakauskiene, R., Treigyte, G., Gineitis, A., Magnusson, K. E. (2002) Assessment of p16, p21, and p27 in granulocytic differentiation of human promyelocytic HL-60 cell line Ann. N. Y. Acad. Sci. 973,284-286[Free Full Text]
  8. Schwaller, J., Pabst, T., Koeffler, H. P., Niklaus, G., Loetscher, P., Fey, M. F., Tobler, A. (1997) Expression and regulation of G1 cell-cycle inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D) in human acute myeloid leukemia and normal myeloid cells Leukemia 11,54-63[CrossRef][Medline]
  9. Burger, C., Wick, M., Muller, R. (1994) Lineage-specific regulation of cell cycle gene expression in differentiating myeloid cells J. Cell Sci. 107,2047-2054[Abstract]
  10. Bocchia, M., Xu, Q., Wesley, U., Xu, Y., Korontsvit, T., Loganzo, F., Albino, A. P., Scheinberg, D. A. (1997) Modulation of p53, WAF1/p21 and BCL-2 expression during retinoic acid-induced differentiation of NB4 promyelocytic cells Leuk. Res. 21,439-447[CrossRef][Medline]
  11. Liu, T. X., Zhang, J. W., Tao, J., Zhang, R. B., Zhang, Q. H., Zhao, C. J., Tong, J. H., Lanotte, M., Waxman, S., Chen, S. J., Mao, M., Hu, G. X., Zhu, L., Chen, Z. (2000) Gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells Blood 96,1496-1504[Abstract/Free Full Text]
  12. Shimizu, T., Awai, N., Takeda, K. (2000) Complex regulation of CDKs and G1 arrest during the granulocytic differentiation of human myeloblastic leukemia ML-1 cells Oncogene 19,4640-4646[CrossRef][Medline]
  13. Rane, S. G., Mangan, J. K., Amanullah, A., Wong, B. C., Vora, R. K., Liebermann, D. A., Hoffman, B., Grana, X., Reddy, E. P. (2002) Activation of the Jak3 pathway is associated with granulocytic differentiation of myeloid precursor cells Blood 100,2753-2762[Abstract/Free Full Text]
  14. Mori, A., Higashi, H., Hoshikawa, Y., Imamura, M., Asaka, M., Hatakeyama, M. (1999) Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue Oncogene 18,6209-6221[CrossRef][Medline]
  15. Kato, J. Y., Sherr, C. J. (1993) Inhibition of granulocyte differentiation by G1 cyclins D2 and D3 but not D1 Proc. Natl. Acad. Sci. USA 90,11513-11517[Abstract/Free Full Text]
  16. McArthur, G. A., Foley, K. P., Fero, M. L., Walkley, C. R., Deans, A. J., Roberts, J. M., Eisenman, R. N. (2002) MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity Mol. Cell. Biol. 22,3014-3023[Abstract/Free Full Text]
  17. Johnston, J. J., Rintels, P., Chung, J., Sather, J., Benz, E. J., Jr, Berliner, N. (1992) Lactoferrin gene promoter: structural integrity and nonexpression in HL60 cells Blood 79,2998-3006[Abstract/Free Full Text]
  18. Khanna-Gupta, A., Kolibaba, K., Zibello, T. A., Berliner, N. (1994) NB4 cells show bilineage potential and an aberrant pattern of neutrophil secondary granule protein gene expression Blood 84,294-302[Abstract/Free Full Text]
  19. Bjerregaard, M. D., Jurlander, J., Klausen, P., Borregaard, N., Cowland, J. B. (2003) The in vivo profile of transcription factors during neutrophil differentiation in human bone marrow Blood 101,4322-4332[Abstract/Free Full Text]
  20. Cover, C. M., Hsieh, S. J., Tran, S. H., Hallden, G., Kim, G. S., Bjeldanes, L. F., Firestone, G. L. (1998) Indole-3-carbinol inhibits the expression of cyclin-dependent kinase-6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling J. Biol. Chem. 273,3838-3847[Abstract/Free Full Text]
  21. Yan, W., Kero, J., Suominen, J., Toppari, J. (2001) Differential expression and regulation of the retinoblastoma family of proteins during testicular development and spermatogenesis: roles in the control of germ cell proliferation, differentiation and apoptosis Oncogene 20,1343-1356[CrossRef][Medline]
  22. Kjeldsen, L., Koch, C., Arnljots, K., Borregaard, N. (1996) Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils J. Immunol. Methods 198,155-164[CrossRef][Medline]
  23. Le Cabec, V., Calafat, J., Borregaard, N. (1997) Sorting of the specific granule protein, NGAL, during granulocytic maturation of HL-60 cells Blood 89,2113-2121[Abstract/Free Full Text]
  24. Borregaard, N., Cowland, J. B. (1997) Granules of the human neutrophilic polymorphonuclear leukocyte Blood 89,3503-3521[Free Full Text]
  25. Le Cabec, V., Cowland, J. B., Calafat, J., Borregaard, N. (1996) Targeting of proteins to granule subsets is determined by timing and not by sorting: The specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells Proc. Natl. Acad. Sci. USA 93,6454-6457[Abstract/Free Full Text]
  26. Owen, C. A., Campbell, E. J. (1999) The cell biology of leukocyte-mediated proteolysis J. Leukoc. Biol. 65,137-150[Abstract]
  27. Jing, Y., Xia, L., Waxman, S. (2002) Targeted removal of PML-RAR{alpha} protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia Blood 100,1008-1013[Abstract/Free Full Text]
  28. Lund-Johansen, F., Terstappen, L. W. (1993) Differential surface expression of cell adhesion molecules during granulocyte maturation J. Leukoc. Biol. 54,47-55[Abstract]
  29. Casaccia-Bonnefil, P., Tikoo, R., Kiyokawa, H., Friedrich, V., Jr, Chao, M. V., Koff, A. (1997) Oligodendrocyte precursor differentiation is perturbed in the absence of the cyclin-dependent kinase inhibitor p27Kip1 Genes Dev 11,2335-2346[Abstract/Free Full Text]
  30. Tikoo, R., Casaccia-Bonnefil, P., Chao, M. V., Koff, A. (1997) Changes in cyclin-dependent kinase 2 and p27kip1 accompany glial cell differentiation of central glia-4 cells J. Biol. Chem. 272,442-447[Abstract/Free Full Text]
  31. Park, S. K., Kang, S. K., Lee, D. Y., Kang, M. J., Kim, S. H., Koh, G. Y. (1997) Temporal expressions of cyclins and cyclin-dependent kinases during renal development and compensatory growth Kidney Int 51,762-769[Medline]
  32. Martinez, L. A., Chen, Y., Fischer, S. M., Conti, C. J. (1999) Coordinated changes in cell cycle machinery occur during keratinocyte terminal differentiation Oncogene 18,397-406[CrossRef][Medline]
  33. Hengst, L., Reed, S. I. (1996) Translational control of p27Kip1 accumulation during the cell cycle Science 271,1861-1864[Abstract]
  34. Klausen, P., Pedersen, L., Jurlander, J., Baumann, H. (2000) Oncostatin M and interleukin 6 inhibit cell cycle progression by prevention of p27kip1 degradation in HepG2 cells Oncogene 19,3675-3683[CrossRef][Medline]
  35. Montagnoli, A., Fiore, F., Eytan, E., Carrano, A. C., Draetta, G. F., Hershko, A., Pagano, M. (1999) Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation Genes Dev 13,1181-1189[Abstract/Free Full Text]
  36. Morisaki, H., Fujimoto, A., Ando, A., Nagata, Y., Ikeda, K., Nakanishi, M. (1997) Cell cycle-dependent phosphorylation of p27 cyclin-dependent kinase (Cdk) inhibitor by cyclin E/Cdk2 Biochem. Biophys. Res. Commun. 240,386-390[CrossRef][Medline]
  37. Shirane, M., Harumiya, Y., Ishida, N., Hirai, A., Miyamoto, C., Hatakeyama, S., Nakayama, K., Kitagawa, M. (1999) Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing J. Biol. Chem. 274,13886-13893[Abstract/Free Full Text]
  38. Castilla, L. H., Garrett, L., Adya, N., Orlic, D., Dutra, A., Anderson, S., Owens, J., Eckhaus, M., Bodine, D., Liu, P. P. (1999) The fusion gene Cbfb-MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia Nat. Genet. 23,144-146[CrossRef][Medline]
  39. Rhoades, K. L., Hetherington, C. J., Harakawa, N., Yergeau, D. A., Zhou, L., Liu, L. Q., Little, M. T., Tenen, D. G., Zhang, D. E. (2000) Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model Blood 96,2108-2115[Abstract/Free Full Text]
  40. Trimarchi, J. M., Lees, J. A. (2002) Sibling rivalry in the E2F family Nat. Rev. Mol. Cell Biol. 3,11-20[CrossRef][Medline]
  41. Grana, X., Garriga, J., Mayol, X. (1998) Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth Oncogene 17,3365-3383[CrossRef][Medline]
  42. Classon, M., Harlow, E. (2002) The retinoblastoma tumour suppressor in development and cancer Nat. Rev. Cancer 2,910-917[CrossRef][Medline]
  43. Tedesco, D., Lukas, J., Reed, S. I. (2002) The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2) Genes Dev 16,2946-2957[Abstract/Free Full Text]
  44. Woo, M. S., Sanchez, I., Dynlacht, B. D. (1997) p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity Mol. Cell. Biol. 17,3566-3579[Abstract]
  45. Castano, E., Kleyner, Y., Dynlacht, B. D. (1998) Dual cyclin-binding domains are required for p107 to function as a kinase inhibitor Mol. Cell. Biol. 18,5380-5391[Abstract/Free Full Text]
  46. Yang, W., Shen, J., Wu, M., Arsura, M., FitzGerald, M., Suldan, Z., Kim, D. W., Hofmann, C. S., Pianetti, S., Romieu-Mourez, R., Freedman, L. P., Sonenshein, G. E. (2001) Repression of transcription of the p27(Kip1) cyclin-dependent kinase inhibitor gene by c-Myc Oncogene 20,1688-1702[CrossRef][Medline]
  47. O’Hagan, R. C., Ohh, M., David, G., de Alboran, I. M., Alt, F. W., Kaelin, W. G., Jr, DePinho, R. A. (2000) Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression Genes Dev 14,2185-2191[Abstract/Free Full Text]
  48. Nasi, S., Ciarapica, R., Jucker, R., Rosati, J., Soucek, L. (2001) Making decisions through Myc FEBS Lett 490,153-162[CrossRef][Medline]
  49. Hermeking, H., Rago, C., Schuhmacher, M., Li, Q., Barrett, J. F., Obaya, A. J., O’Connell, B. C., Mateyak, M. K., Tam, W., Kohlhuber, F., Dang, C. V., Sedivy, J. M., Eick, D., Vogelstein, B., Kinzler, K. W. (2000) Identification of CDK4 as a target of c-MYC Proc. Natl. Acad. Sci. USA 97,2229-2234[Abstract/Free Full Text]
  50. Bouchard, C., Thieke, K., Maier, A., Saffrich, R., Hanley-Hyde, J., Ansorge, W., Reed, S., Sicinski, P., Bartek, J., Eilers, M. (1999) Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27 EMBO J 18,5321-5333[CrossRef][Medline]
  51. Lekstrom-Himes, J., Xanthopoulos, K. G. (1998) Biological role of the CCAAT/enhancer-binding protein family of transcription factors J. Biol. Chem. 273,28545-28548[Abstract/Free Full Text]
  52. Stahl, M., Dijkers, P. F., Kops, G. J., Lens, S. M., Coffer, P. J., Burgering, B. M., Medema, R. H. (2002) The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2 J. Immunol. 168,5024-5031[Abstract/Free Full Text]
  53. Kops, G. J., Medema, R. H., Glassford, J., Essers, M. A., Dijkers, P. F., Coffer, P. J., Lam, E. W., Burgering, B. M. (2002) Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors Mol. Cell. Biol. 22,2025-2036[Abstract/Free Full Text]
  54. Medema, R. H., Kops, G. J., Bos, J. L., Burgering, B. M. (2000) AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1 Nature 404,782-787[CrossRef][Medline]
  55. de Koning, J. P., Soede-Bobok, A. A., Ward, A. C., Schelen, A. M., Antonissen, C., van Leeuwen, D., Lowenberg, B., Touw, I. P. (2000) STAT3-mediated differentiation and survival and of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27(Kip1) Oncogene 19,3290-3298[CrossRef][Medline]
  56. Kortylewski, M., Heinrich, P. C., Mackiewicz, A., Schniertshauer, U., Klingmuller, U., Nakajima, K., Hirano, T., Horn, F., Behrmann, I. (1999) Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1 Oncogene 18,3742-3753[CrossRef][Medline]
  57. Collins, S. J. (2002) The role of retinoids and retinoic acid receptors in normal hematopoiesis Leukemia 16,1896-1905[CrossRef][Medline]
  58. Warrell, R. P., Jr, Frankel, S. R., Miller, W. H., Jr, Scheinberg, D. A., Itri, L. M., Hittelman, W. N., Vyas, R., Andreeff, M., Tafuri, A., Jakubowski, A. (1991) Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid) N. Engl. J. Med. 324,1385-1393[Abstract]
  59. Yang, W. L., Zeng, Y. X., el Deiry, W. S., Nason-Burchenal, K., Dmitrovsky, E., Chin, K. V. (1999) Transcriptional activation of the cyclin-dependent kinase inhibitor p21 by PML/RAR{alpha} Mol. Cell Biol. Res. Commun. 1,125-131[CrossRef][Medline]
  60. Shimizu, T., Oka, Y., Awai, N., Takeda, K. (1999) Hypophosphorylation of pRB and repression of cyclin D3 and cdc25A during the granulocytic differentiation of human myeloblastic leukemia ML-1 cells Leuk. Res. 23,901-907[CrossRef][Medline]
  61. Nguyen, M. D., Mushynski, W. E., Julien, J. P. (2002) Cycling at the interface between neurodevelopment and neurodegeneration Cell Death Differ 9,1294-1306[CrossRef][Medline]
  62. Cowland, J. B., Borregaard, N. (1999) The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules J. Leukoc. Biol. 66,989-995[Abstract]



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