Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors"

Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.     

Distinct Specificities of pRb Phosphorylation by CDK4 Activated by Cyclin D1 or Cyclin D3: Differential Involvement in the Distinct Mitogenic Modes of Thyroid Epithelial Cells

Two distinct mitogenic modes coexist in the physiologically relevant model ofprimary cultures of dog thyroid epithelial cells. The differentiation-associated mitogenicstimulation by TSH and cAMP specifically requires the assembly and activation of cyclin D3-cyclin-dependent kinase (CDK)4 associated to p27kip1, while the dedifferentiatingproliferation induced by growth factors is associated with induction of cyclin D1. Here, wesuggest that the related CDK “inhibitors” p21cip1 and p27 are differentially utilized as positiveCDK4 regulators in these mitogenic stimulations. p21 was induced by EGF+serum, butrepressed by TSH, which, as previously shown, up-regulates p27. In response to EGF+serum,p21 supported the nuclear localization, phosphorylation and pRb-kinase activity of CDK4.Unexpectedly, partly different site-specificities of pRb-kinase activity, leading to similardifferences in the phosphorylation pattern of pRb in intact cells, were associated with cyclinD3-CDK4 bound to p27 in TSH-stimulated cells, or with CDK4 bound to p21 in growthfactor-stimulated cells. These differences were ascribed to the predominant association of thelatter complex to cyclin D1. Indeed, in different cell types and species, cyclin D1 varied fromcyclin D3 by more efficiently driving the phosphorylation of pRb at sites (Ser807/811 andThr826) required for its electrophoretic mobility shift. Therefore, different D-type cyclinscould differently impact some pRb functions, which should be considered not only in theunderstanding of the relationships between cell cycle and differentiation expression in thedistinct mitogenic modes of thyroid cells, but also in various development or differentiationmodels associated with dramatic switches in the expression of individual D-type cyclins.     

CDK4 activity in mouse embryos expressing a single D-type cyclin

D-type cyclins (D1, D2, and D3) are components of the cell cycle machinery. Their association with cyclin-dependent kinase 4 (CDK4) and CDK6 causes activation of these protein kinases and leads to phosphorylation and inactivation of the retinoblastoma protein, pRb. Using embryos expressing single D-type cyclin ('cyclin D1-only', 'cyclin D2-only' and 'cyclin D3-only'), we tested whether each of D-type cyclin plays the same role in CDK activation and phosphorylation of pRb during mouse embryonic development. We found that the level of CDK4 activity was similar in wild-type embryos and those expressing only cyclin D3 or cyclin D2. However, we did not detect CDK4 activity in embryos expressing only cyclin D1, despite the fact that this cyclin was able to form complexes with CDK4 and p27(kip1) in wild-type as well as in mutant embryos. Analysis of the expression pattern of mRNA encoding cyclin D1 revealed that the expression of this RNA is regulated temporally during embryogenesis. These data and results from other laboratories indicate that cyclin D1-dependent CDK4 activity is dispensable for normal development of the mouse embryo.     

Levels and interactions of p27, cyclin D3, and CDK4 during the formation and maintenance of the corpus luteum in mice

The cyclin-dependent kinase (CDK) inhibitor p27, the regulator of the cell cycle, is required for proper functioning of luteinizing/luteinized cells in vivo. Since different members of the CDK family may be targeted by p27 during luteinization-associated cell cycle exit, this in vivo study further analyzed the organization of the network of cell cycle regulators that may underlie both the establishment and maintenance of the luteal phenotype. Most importantly, it shows that the luteinization process is associated with down-regulation of CDK2 and cyclin D1, and up-regulation of p27 and cyclin D3. Both p27 and cyclin D3 proteins not only accumulated during initial phases of luteinization, but they remained elevated until termination of the luteal function. Along with its accumulation, p27 lost physical contact with CDK2 and instead became associated with CDK4. In fully luteinized cells, all cyclin D3 was incorporated into complexes with p27, some complexes being p27/cyclin D3/CDK4 trimers. Despite the significant amounts of CDK4 and CDK6, only nonphosphorylated forms of retinoblastoma protein were detectable in fully luteinized cells. Together, our data indicate that while inhibition of proliferation is underlaid by the progressive loss of positive regulators of the cell cycle, including cyclins and CDK2, maintenance of the luteal phenotype is driven by up-regulated levels of p27 and cyclin D3, at least partially owing to formation of p27/cyclin D3/CDK4 trimers.     

Cyclin D- and E-Dependent Kinases and the p57KIP2 Inhibitor: Cooperative Interactions In Vivo

This study examines in vivo the role and functional interrelationships of components regulating exit from the G₁ resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57^KIP2 inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57^KIP2 inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G₁, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57^KIP2-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57^KIP2 function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57^KIP2 to regulate the G₁/S transition in a cell type highly dependent upon pRb.     

Unscheduled expression of cyclins D1 and D3 in human tumour cell lines

D-type cyclins are involved in regulation of cell traverse through G₁ primarily by activating the cyclin-dependent kinase 4 (CDK4) and targeting it to the retinoblastoma tumour suppressor protein. There is a vast body of evidence that defective expression of D-type cyclins is associated with tumour development and/or progression. Immunocytochemical detection of D cyclins combined with multiparameter flow cytometry makes it possible to measure the expression of these proteins in individual cells in relation to their cell cycle position without the need for cell synchronization. This approach was used in the present study to compare the cell cycle phase specific expression of cyclins D3 and D1 in human normal proliferating lymphocytes and fibroblasts, respectively, with nine tumour cell lines of different lineage. During exponential, unperturbed growth, expression of cyclin D1 in fibroblasts from donors of different age, or cyclin D3 in lymphocytes, was limited to mid-G₁ cells: Less than 7% of the cells entering S phase or progressing through S and G₂ were cyclin D positive. In contrast, expression of either cyclin D1 or cyclin D3 in tumour cell lines of different lineage was not limited to G₁ phase. Namely, over 80% of the cells in S and G₂+M were cyclin D positive in eight of the nine cell lines studied. The data indicate that while expression of cyclin D1 or D3 in normal cells is discontinuous, occurring transiently in G₁, these proteins are expressed in some tumour lines persistently throughout the cell cycle. This suggests that the partner kinase CDK4 is perpetually active throughout the cell cycle in these tumour lines.     

The cyclin D3-CDK4-p27kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFbeta, and phosphorylations of its subunits demonstrated by two-dimensional gel electrophoresis

The cAMP-dependent mitogenic stimulation elicited by thyroid-stimulating hormone (TSH) in primary cultures of canine thyroid epithelial cells is unique as it upregulates the cyclin-dependent kinase (CDK) inhibitor p27kip1 but not D-type cyclins. TSH and cAMP promote the assembly of required cyclin D3-CDK4 complexes and their nuclear import. Here, the nuclear translocation of these complexes strictly correlated in individual cells with the enhanced presence of nuclear p27. p27, like cyclin D3, supported the TSH-stimulated pRb-kinase activity of the CDK4 complex and, as demonstrated using the high-resolution power of the two-dimensional (2D) gel electrophoresis, the phosphorylation of CDK4, presumably by the nuclear CDK-activating kinase. In the presence of TSH, transforming growth factor beta (TGFbeta) did not affect the assembly of cyclin D3-CDK4, but it strongly inhibited the pRb-kinase activity associated with both cyclin D3 and p27, not only by preventing the nuclear import of cyclin D3-CDK4 and its binding to p27, but also by inhibiting CDK4 phosphorylation within residual p27-bound cyclin D3-CDK4 complexes. No alterations of the relative abundance of multiple (un)phosphorylated forms of cyclin D3 and p27 demonstrated by 2D-gel electrophoresis were associated with these processes. This study suggests a crucial positive role of p27 in the TSH-stimulated nuclear import, phosphorylation, and catalytic activity of cyclin D3-bound CDK4. Moreover, it demonstrates a technique to directly assess the in vivo phosphorylation of endogenous CDK4, which might appear as a last regulated step targeted by the antagonistic cell cycle effects of TSH and TGFbeta.     

Cyclin D3 expression in melanoma cells is regulated by adhesion-dependent phosphatidylinositol 3-kinase signaling and contributes to G1-S progression

D-type cyclins regulate G1 cell cycle progression by enhancing the activities of cyclin-dependent kinases (CDKs), and their expression is frequently altered in malignant cells. We and others have previously shown that cyclin D1 is up-regulated in melanoma cells through adhesion-independent MEK-ERK1/2 signaling initiated by mutant B-RAF. Here, we describe the regulation and role of cyclin D3 in human melanoma cells. Cyclin D3 expression was enhanced in a cell panel of human melanoma cell lines compared with melanocytes and was regulated by fibronectin-mediated phosphatidylinositol 3-kinase/Akt signaling but not MEK activity. RNA interference experiments demonstrated that cyclin D3 contributed to G1-S cell cycle progression and proliferation in melanoma cells. Overexpression of cyclin D1 did not recover the effects of cyclin D3 knockdown. Finally, immunoprecipitation studies showed that CDK6 is a major binding partner for cyclin D3, whereas CDK4 preferentially associated with cyclin D1. Together, these findings demonstrate that cyclin D3 is an important regulator of melanoma G1-S cell cycle progression and that D-type cyclins are differentially regulated in melanoma cells.     

Expression of cyclin D1 and CDK4 causes hypertrophic growth of cardiomyocytes in culture: a possible implication for cardiac hypertrophy

Differentiated cardiomyocytes have little capacity to proliferate and show the hypertrophic growth in response to alpha1-adrenergic stimuli via the Ras/MEK pathway. In this study, we investigated a role of cyclin D1 and CDK4, a positive regulator of cell cycle, in cultured neonatal rat cardiomyocyte hypertrophy. D-type cyclins including cyclin D1 were induced in cells stimulated by phenylephrine. This induction was inhibited by MEK inhibitor PD98059 and the dominant negative RasN17, but mimicked by expression of the constitutive active Ras61L. Over-expression of cyclin D1 and CDK4 using adenovirus gene transfer caused the hypertrophic growth of cardiomyocytes, as evidenced by an increase of the cell size as well as the amount of cellular protein and its rate of synthesis. However, the cyclin D1/CDK4 kinase activity was not up-regulated in cells treated by hypertrophic stimuli or in cells over-expressing the cyclin D1 and CDK4. Furthermore, a CDK inhibitor, p16, did not inhibit the hypertrophic growth of cardiomyocytes. These results clearly indicated that cyclin D1 and CDK4 have a role in hypertrophic growth of cardiomyocytes through a novel mechanism(s) which appears not to be related to its activity required for cell cycle progression.     

The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes

CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.     

Cyclins D2 and D1 are essential for postnatal pancreatic beta-cell growth

Regulation of adult beta-cell mass in pancreatic islets is essential to preserve sufficient insulin secretion in order to appropriately regulate glucose homeostasis. In many tissues mitogens influence development by stimulating D-type cyclins (D1, D2, or D3) and activating cyclin-dependent kinases (CDK4 or CDK6), which results in progression through the G(1) phase of the cell cycle. Here we show that cyclins D2 and D1 are essential for normal postnatal islet growth. In adult murine islets basal cyclin D2 mRNA expression was easily detected, while cyclin D1 was expressed at lower levels and cyclin D3 was nearly undetectable. Prenatal islet development occurred normally in cyclin D2(-/-) or cyclin D1(+/-) D2(-/-) mice. However, beta-cell proliferation, adult mass, and glucose tolerance were decreased in adult cyclin D2(-/-) mice, causing glucose intolerance that progressed to diabetes by 12 months of age. Although cyclin D1(+/-) mice never developed diabetes, life-threatening diabetes developed in 3-month-old cyclin D1(-/+) D2(-/-) mice as beta-cell mass decreased after birth. Thus, cyclins D2 and D1 were essential for beta-cell expansion in adult mice. Strategies to tightly regulate D-type cyclin activity in beta cells could prevent or cure diabetes.     

Differential utilization of cyclin D1 and cyclin D3 in the distinct mitogenic stimulations by growth factors and TSH of human thyrocytes in primary culture

Two distinct mitogenic modes coexist in thyroid epithelial cells. TSH via cAMP induces proliferation and differentiation expression, whereas growth factors including epidermal growth factor (EGF) induce proliferation and dedifferentiation. Divergent models of TSH/cAMP-dependent mitogenesis have emerged from different thyroid cell culture systems. In the FRTL-5 rat cell line, cAMP cross-signals with transduction pathways of growth factors to induce cyclin D1 and p21(cip1) and down-regulate p27(kip1). By contrast, in canine primary cultures, mitogenic pathways of cAMP and growth factors are fully distinct. cAMP does not induce D-type cyclins and p21, it up-regulates p27, and it stimulates the formation and activity of cyclin D3-cyclin-dependent kinase (CDK) 4 complexes. In primary cultures of normal human thyrocytes, EGF + serum increased cyclin D1 and p21 accumulation, and it stimulated the assembly and activity of cyclin D1-CDK4-p21 complexes. By contrast, TSH repressed or did not induce cyclin D1 and p21, and it rather up-regulated p27. TSH did not increase cyclin D1-CDK4 activity, but it stimulated the activating phosphorylation of CDK4 and the pRb-kinase activity of preexisting cyclin D3-CDK4 complexes. As recently demonstrated in dog thyrocytes and other systems, cyclin D1 and cyclin D3 differently oriented the site specificity of CDK4 pRb-kinase activity, which might differently impact some pRb functions. Cyclin D1 or cyclin D3 are thus differentially used in the distinct mitogenic stimulations by growth factors or TSH, and potentially in hyperproliferative diseases generated by the overactivation of their respective signaling pathways. At variance with dog thyroid primary cultures, rat thyroid cell lines might not be valid models of TSH-dependent mitogenesis of human thyrocytes.     

Cell type-specific induction of cyclin D and cyclin-dependent kinase inhibitor p27(kip1) expression by estrogen in rat endometrium

Cyclins, cyclin-dependent kinases (CDKs) and the CDK inhibitor p27(kip1) are known to be involved in the regulation of G(1)/S phase transition by estrogen in the rodent endometrium. Little is known, however, of the cell-specific location and regulation of these proteins during this process, or the way they mediate the differential effect of estrogen in the epithelium and stroma of the endometrium. Here we studied the cell-specific regulation of D-type cyclin (D(1-3)), of cyclin A and E, of CDK(2) and p27(kip1) by 17beta-estradiol in the endometrium of ovariectomized rats. Time-course changes in these proteins in the endometrium of ovariectomized rats were examined by immunohistochemistry at 2, 4, 8, 12, 20, 28 and 32 h after estrogen stimulation. The expression of proliferation cell nuclear antigen (PCNA) was also studied as a marker of proliferating cells. As expected from previous studies, all the proteins investigated were up-regulated by estrogen, with peak times from 8 to 32 h. The induction of cyclin D(1) is predominant in the glandular epithelium, whereas cyclin D(3) increases mainly in the luminal epithelium. The up-regulation of p27(kip1) is restricted to stromal cells with a 'gradient-like' expression pattern, in which the sub-epithelial (functional) layer showed stronger staining than the basal layer. The differential regulation of cyclins and p27(kip1) in the epithelium and stroma of the endometrium appear indicative of distinct actions of estrogen in different cell types in the uterus, as D-type cyclins mediate the proliferative effect of estrogen in epithelial cells while p27(kip1) might help prevent the same effect in the stroma.     

Cyclic AMP inhibits the proliferation of thyroid carcinoma cell lines through regulation of CDK4 phosphorylation

How cyclic AMP (cAMP) could positively or negatively regulate G1 phase progression in different cell types or in cancer cells versus normal differentiated counterparts has remained an intriguing question for decades. At variance with the cAMP-dependent mitogenesis of normal thyroid epithelial cells, we show here that cAMP and cAMP-dependent protein kinase activation inhibit S-phase entry in four thyroid carcinoma cell lines that harbor a permanent activation of the Raf/ERK pathway by different oncogenes. Only in Ret/PTC1-positive TPC-1 cells did cAMP markedly inhibit the Raf/ERK cascade, leading to mTOR pathway inhibition, repression of cyclin D1 and p21 and p27 accumulation. p27 knockdown did not prevent the DNA synthesis inhibition. In the other cells, cAMP little affected these signaling cascades and levels of cyclins D or CDK inhibitors. However, cAMP differentially inhibited the pRb-kinase activity and T172-phosphorylation of CDK4 complexed to cyclin D1 or cyclin D3, whereas CDK-activating kinase activity remained unaffected. At variance with current conceptions, our studies in thyroid carcinoma cell lines and previously in normal thyrocytes identify the activating phosphorylation of CDK4 as a common target of opposite cell cycle regulations by cAMP, irrespective of its impact on classical mitogenic signaling cascades and expression of CDK4 regulatory partners.     

Mouse development and cell proliferation in the absence of D-cyclins

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.     

Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules.     

Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation

Using theconditionally immortalized human cell line tsFHI, we have investigatedthe role of cyclin-dependent kinase inhibitors (CKIs) in intestinalepithelial cell differentiation. Expression of cyclins,cyclin-dependent kinases (Cdk), and CKIs was examined under conditionspromoting growth, growth arrest, or expression of differentiatedtraits. Formation of complexes among cell cycle regulatory proteins andtheir kinase activities were also investigated. The tsFHI cells expressthree CKIs: p16, p21, and p27. With differentiation, p21 and p27 werestrongly induced, but with different kinetics: the p21 increase wasrapid but transient and the p27 increase was delayed but sustained. Ourresults suggest that the function of p16 is primarily to inhibit cyclinD-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 forpRb phosphorylation and G₁/Sprogression. Furthermore, they indicate that p21 is the main CKIinvolved in irreversible growth arrest during the early stages of celldifferentiation in association with D-type cyclins, cyclin E, and Cdk2,whereas p27 may induce or stabilize expression of differentiated traitsacting independently of cyclin-Cdk function.

     

Differential role of D cyclins in the regulation of cell cycle by influencing Ki67 expression in HaCaT cells

D-type cyclins are important regulatory proteins of the G1/S phase of the cell cycle however, their specific functions are only partially understood. We show that silencing of individual D-type cyclins has no effect on the proliferation and morphology of Immortalized non-tumorigenic human epidermal (HaCaT) cells, while double and triple D cyclin silencing results in the failure of the cytokinesis leading to the appearance of large multinucleated cells. Both CDC20 and Ki67 mRNA is downregulated in these cells. Ki67 mRNA silenced cells show similar multinucleated cellular phenotype as double or triple D cyclin silenced cells without affecting D cyclin expression, suggesting that Ki67 is necessary for normal G2/M transition. Our data have revealed that cyclin D1 may have a leading role in G1/S phase regulation and suggest an incomplete functional overlap among D cyclins. Our results indicate that beside their well-known functions during the G0-G1/S phase, D-type cyclins play a pivotal role in the regulation of mitosis via influencing Ki67 expression in a downstream manner probably through E2F1 activation in HaCaT cells.