MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27(KIP1). Generation of Mad1/p27(KIP1) double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27(KIP1), including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27(KIP1) double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27(KIP1) single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27(KIP1) operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.     

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.     

Osteoclast differentiation is associated with transient upregulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1)

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.     

Function of an inhibitor of cyclin-dependent kinase p27/Kip in cells transformed by E1A + E1B19 kDa + E1A + cHa-Ras, differing in their ability to realize a G1-block during serum starvation

We studied the capability of E1A + cHa-ras and E1A + E1B19kDa transformants to undergo the G1/S arrest of the cell cycle following depletion of serum growth factors. It has been shown that serum starvation induced the G1/S arrest both in normal rat embryo fibroblasts (REF) and in E1A + E1B19kDa transformants, whereas E1A + cHa-ras transformed cells lost this feature. To analyse the mechanisms underlying these differences, we studied the expression of p27/KIP, its intracellular distribution and association with E1A oncoproducts. The content of the p27/KIP inhibitor of cyclin-dependent kinases was found to change a little upon transformation by two complementary oncogene pairs. However, serum starvation for 24 h led to a significant increase in the content of p27/KIP in E1A + E1B19kDa transformants, while E1A + cHa-ras cells accumulated p27/KIP less markedly. According to the immunofluorescence study, the p27/KIP inhibitor is located in the nucleus of both normal and transformed cells. Moreover, serum starvation did not lead to its inhibition due to redistribution to the cytoplasm in both cell lines. Also, we were unable to detect association of p27/KIP with E1A oncoproducts in immunoprecipitated complexes. The obtained data indicate that, in contrast to E1A + cHa-ras transformants, in E1A + E1B19kDa cells the p27/KIP inhibitor is functional and it is capable of inducing the G1/S block after serum starvation.     

Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells

Activation of ERK1 and ERK2 protein kinases has been implicated in diverse cellular processes, including the control of cell proliferation and cell differentiation (Marshall [1995] Cell 80:179). In human myeloblastoid leukemia HL60 cells rapid (ca. 15 min) but transient activation of ERK1/2 has been reported following induction of macrophage/monocyte differentiation by phorbol esters, or by very high (10(-6) M) concentrations of 1,25-dihydroxyvitamin D(3) (1,25D3), while retinoic acid-induced granulocytic differentiation was accompanied by sustained activation of ERK1/2. We report here that monocytic differentiation of HL60 cells induced by moderate (10(-9) to 10(-7) M) concentrations of 1,25D3 could be divided into at least two stages. In the first phase, which lasts 24-48 h, the cells continued in the normal cell cycle while expressing markers of monocytic phenotype, such as CD14. In the next phase the onset of G1 cell cycle block became apparent and expression of CD11b was prominent, indicating a more mature myeloid phenotype. The first phase was characterized by high levels of ERKs activated by phosphorylation, and these decreased as the cells entered the second phase, while the levels of p27/Kip1 increased at that time. Serum-starved or PD98059-treated HL60 cells had reduced growth rate and slower differentiation, but the G1 block also coincided with decreased levels of activated ERK1/2. The data suggest that the MEK/ERK pathway maintains cell proliferation during 1,25D3-induced monocytic differentiation of HL60 cells, but that ERK1/2 activity becomes suppressed during the later stages of differentiation, and the consequent G1 block leads to "terminal" differentiation.     

Critical role of both p27KIP1 and p21CIP1/WAF1 in the antiproliferative effect of ZD1839 ('Iressa'), an epidermal growth factor receptor tyrosine kinase inhibitor,in head and neck squamous carcinoma cells

High expression of the epidermal growth factor receptor (EGFR) has been implicated in the development of squamous-cell carcinomas of head and neck (SCCHN). ZD1839 ('Iressa') is an orally active, selective EGFR-TKI (EGFR-tyrosine kinase inhibitor) that blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. We have demonstrated that ZD1839 induces growth arrest in SCCHN cell lines by inhibiting EGFR-mediated signaling. Cell cycle kinetic analysis demonstrated that ZD1839 induces a delay in cell cycle progression and a G1 arrest together with a partial G2/M block; this was associated with increased expression of both p27(KIP1) and p21(CIP1/WAF1) cyclin-dependent kinase (CDK) inhibitors. The activity of CDK2, the main target of CIP/KIP CDK inhibitors, was reduced in a dose-dependent fashion after 24 h of ZD1839 treatment and this effect correlated to the increased amount of p27(KIP1) and p21(CIP1/WAF1) proteins associated with CDK2-cyclin-E and CDK2-cyclin-A complexes. In addition, ZD1839-induced growth inhibition was significantly reduced in cell transfectants expressing p27(KIP1) or p21(CIP1/WAF1) antisense constructs. Overall, these results as well as the timing of the effect of ZD1839 on G1 arrest and p27(KIP1) and p21(CIP1/WAF1) upregulation, suggest a mechanistic connection between these events.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

Mapping of CIP/KIP inhibitors,G1 cyclins D1, D3, E and p53 proteins in the rat term placenta

As cell cycle regulation is fundamental to the normal growth and development of the placenta, the aim of the present study was to determine the immunolocalizations of cell cycle related proteins, which have key roles in proliferation, differentiation and apoptosis during the development of the rat placenta. Here immunohistochemistry has been used to localize G1 cyclins (D1, D3, E), which are major determinants of proliferation, CIP/KIP inhibitors (p21, p27, p57), p53 as a master regulator and proliferating cell nuclear antigen in all cell types of the rat term placenta. The proportion of each cell type immunolabeled was counted. Cyclin D1 and cyclin D3 were present mostly in cells of the fetal aspect of the placenta, whereas the G1/S cyclin E was present only in the spongio- and labyrinthine trophoblast populations. Among the CIP/KIP inhibitors, p21 was present only in cells of the fetal aspect whereas p27 and p57 were found in all cell types studied. p53 was only found in a small proportion of cells with no co-localization of p53 and p21. The data suggest that the cells of the fetal side of the rat placenta still have some proliferation potential which is kept in check by expression of the CIP/KIP cell cycle inhibitors, whereas cells of the maternal aspect have lost this potential. Apoptosis is only marginal in the term rat placenta. In conclusion, proliferation and apoptosis in rat placental cells appears controlled mostly by the CIP/KIP inhibitors in late pregnancy.     

Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A^b) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G₁ to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27^KIP1. Saponins also increased stability of p27^KIP1 in Th cells after antigenic stimulation.     

Modulation of CD157 expression in multi-lineage myeloid differentiation of promyelocytic cell lines

CD157/BST-1 is expressed on mature myeloid cells but not on their precursors in vivo. Also CD38, a homologous gene to CD157, is upregulated in promyelocytic HL-60 cells by the monocyte and granulocyte differentiation-inducing 1alpha,25dihydroxyvitamin D3 (VD3) and all-trans retinoic acid (ATRA), respectively. We have examined whether CD157 expression is upregulated when the promyeloid HL-60 and/or U937 cells are induced to differentiate into mature phenotypes in vitro. VD3 treatment irreversibly upregulated the expression of CD157 in HL-60 cells but not in U937 cells in a time- and concentration-dependent manner when analyzed by flow cytometry, immunoblotting and/or RT-PCR. Different monocyte and granulocyte lineage inducers induced CD157 expression to varying extents while the macrophage differentiation-inducing phorbol 12-myristate 13-acetate (PMA) induced its down-regulation. Time-kinetics of VD3 treatment of HL-60 cells showed that the appearance of CD157 and CD11b (a differentiation marker) antigens were not substantial up to 24 hours but increased subsequently although the appearance of CD38 became significant within 6 hours. Two-color staining of VD3-treated HL-60 cells displayed an apparently linear correlation between CD157 and CD11b expression. Dibutyryl cAMP (cAMP agonist) and forskolin (cAMP-increasing agent) augmented the VD3-dependent induction of CD157 and CD11b expression while PGE1 (cAMP-decreasing agent) inhibited it, suggesting the involvement of a cAMP-dependent mechanism in VD3-induced CD157 upregulation. Co-treatment of HL-60 cells with VD3 plus TNF-alpha or ara-C produced an additive effect on CD157 upregulation. The upregulated CD157 in the VD3-differentiated HL-60 cells was able to activate CD157-dependent tyrosine kinase signal when cross-linked with anti-CD157 antibody.     

Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expression associated with progression in B-CLL

B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activation level, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradual accumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase is impaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2, p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately control the proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral blood CLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of disease was accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearly statistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53 and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.     

p57(KIP2) regulates radial glia and intermediate precursor cell cycle dynamics and lower layer neurogenesis in developing cerebral cortex

During cerebral cortex development, precise control of precursor cell cycle length and cell cycle exit is required for balanced precursor pool expansion and layer-specific neurogenesis. Here, we defined the roles of cyclin-dependent kinase inhibitor (CKI) p57(KIP2), an important regulator of G1 phase, using deletion mutant mice. Mutant mice displayed macroencephaly associated with cortical hyperplasia during late embryogenesis and postnatal development. Embryonically, proliferation of radial glial cells (RGC) and intermediate precursors (IPC) was increased, expanding both populations, with greater effect on IPCs. Furthermore, cell cycle re-entry was increased during early corticogenesis, whereas cell cycle exit was augmented at middle stage. Consequently, neurogenesis was reduced early, whereas it was enhanced during later development. In agreement, the timetable of early neurogenesis, indicated by birthdating analysis, was delayed. Cell cycle dynamics analyses in mutants indicated that p57(KIP2) regulates cell cycle length in both RGCs and IPCs. By contrast, related CKI p27(KIP1) controlled IPC proliferation exclusively. Furthermore, p57(KIP2) deficiency markedly increased RGC and IPC divisions at E14.5, whereas p27(KIP1) increased IPC proliferation at E16.5. Consequently, loss of p57(KIP2) increased primarily layer 5-6 neuron production, whereas loss of p27(KIP1) increased neurons specifically in layers 2-5. In conclusion, our observations suggest that p57(KIP2) and p27(KIP1) control neuronal output for distinct cortical layers by regulating different stages of precursor proliferation, and support a model in which IPCs contribute to both lower and upper layer neuron generation.     

Expression of surface antigen and mRNA for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family in hematopoietic cells

We characterized the surface antigen and mRNA expression for the CD11c (alpha X, p150) subunit of the human leukocyte adherence receptor family during hematopoietic cell differentiation. The CD11c subunit antigen and mRNA are constitutively expressed in undifferentiated HL-60 promyelocytic leukemia cells, and levels increase markedly with differentiation along the monocyte/macrophage pathway using phorbol myristate acetate. Human monocyte-derived macrophages and human alveolar macrophages express elevated levels of the CD11c subunit antigen and mRNA, indicating that the changes observed in vitro are present in vivo. Dot blot analysis of immature and mature lymphoid and myeloid cells and cell lines demonstrate equivalent levels of CD11c mRNA expression. We conclude that CD11c gene expression is selectively increased during hematopoietic cell differentiation along the monocyte/macrophage pathway.     

UCN-01 (7-hydroxystauorsporine) blocks PMA-induced maturation and reciprocally promotes apoptosis in human myelomonocytic leukemia cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21(CIP1) induction, p27(KIP1) cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21(CIP1) mRNA antisense construct, suggesting that p21(CIP1) expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program.     

Rab7b, a novel lysosome-associated small GTPase, is involved in monocytic differentiation of human acute promyelocytic leukemia cells

Rab7 is a small Rab GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Here we report the cloning and characterization of a novel Rab7-like GTPase, which shares highest homology with Rab7 and thus is designated as Rab7b. Northern blot analysis shows that Rab7b mRNA is expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte. RT-PCR or Western blot analysis of Rab7b expression shows that Rab7b is selectively expressed in monocytes, monocyte-derived immature dendritic cells (DCs), and promyeloid or monocytic leukemia cell lines. In the peripheral blood, Rab7b is specifically detected in CD14(+) cells, but not in CD4(+), CD8(+), CD19(+) or CD56(+) cells. When immature DCs are matured with lipopolysaccharide (LPS), Rab7b expression is gradually downregulated, while Rab7b is upregulated when monocytes are activated by LPS treatments. In acute promyelocytic leukemia (APL) HL-60 and NB4 cell lines, Rab7b expression is upregulated after phorbol myristate acetate (PMA)-induced monocytic differentiation. By immunofluorescence confocal microscopy, we demonstrate that Rab7b is associated with lysosomal organelles. Our data suggest that Rab7b is a lysosome-localized monocytic cell-specific small GTPase, and is involved in PMA-induced APL cell differentiation and possibly in regulation of monocyte functions.     

The multiple roles of the cyclin-dependent kinase inhibitory protein p57(KIP2) in cerebral cortical neurogenesis

The members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitory proteins (CKIs), including p57(KIP2), p27(KIP1), and p21(CIP1), block the progression of the cell cycle by binding and inhibiting cyclin/CDK complexes of the G1 phase. In addition to this well-characterized function, p57(KIP2) and p27(KIP1) have been shown to participate in an increasing number of other important cellular processes including cell fate and differentiation, cell motility and migration, and cell death/survival, both in peripheral and central nervous systems. Increasing evidence over the past few years has characterized the functions of the newest CIP/KIP member p57(KIP2) in orchestrating cell proliferation, differentiation, and migration during neurogenesis. Here, we focus our discussion on the multiple roles played by p57(KIP2) during cortical development, making comparisons to p27(KIP1) as well as the INK4 family of CKIs.