Inhibition of D1, D2, and a cyclin expression in HL-60 cells by the lipid peroxydation product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a product of lipid peroxidation, is an highly reactive aldehyde that, at concentration similar to those found in normal cells, blocks proliferation and induces a granulocytic-like differentiation in HL-60 cells. These effects are accompained by a marked increase in the proportion G0/G1 cells. The mechanisms of HNE action were investigated by analyzing the expression of the cyclins and cyclin-dependent protein kinases (CDKs), controlling the cell cycle progression. Data obtained by exposing cells to dimethyl sulfoxide (DMSO) were used for comparison. 4-Hydroxynonenal downregulated both mRNA and protein contents of cyclins D1, D2, and A until 24 h from the treatments, whereas DMSO inhibited cyclin D1 and D2 expression until the end of experiment (2 days) and induces an increase of cyclin A until 1 day. Cyclins B and E, and protein kinase CDK2 and CDK4 expressions were not affected by HNE, whereas DMSO induced an increase of cyclin E, B, and CDK2 from 8 h to 1 day. These data are in agreement with previous results indicating a different time-course of accumulation in G0/G1 phases of cells treated with HNE and DMSO and suggest that the HNE inhibitory effect on proliferation and cell cycle progression may depend by the downregulation of D1, D2, and A cyclin expression.     

4-Hydroxynonenal affects pRb/E2F pathway in HL-60 human leukemic cells

4-Hydroxynonenal (HNE), a highly reactive product of lipid peroxidation, has an antiproliferative effect in several tumor cell lines and provokes alteration of cell cycle progression in HL-60 cells. HNE down-regulates c-myc expression in K562, HL-60, and MEL cells. This prompted us to study the cascade of phenomena that, starting from the CKIs expression and the phosphorylation of pRb, arrives at the E2F binding to consensus sequence in the P2 promoter of the c-myc gene. Treatment of HL-60 cells with HNE (1 microM) causes a p53-independent increase of p21(WAF1/CIP1) expression, pRb dephosphorylation, a decrease of low molecular weight E2F complexes and an increase of high molecular weight E2F complexes bound to P2 c-myc promoter. E2F4 expression is reduced by HNE treatment as well as the amount of pRb/E2F4 complexes, whereas the amount of pRb/E2F1 complexes is increased. In conclusion, HNE can affect the pRb/E2F pathway by modifying the expression of several genes involved in the control of cell proliferation.     

Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression

The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.     

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.     

Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G₁ phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ~120 and ~200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.     

Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.     

Cyclin D2 and the CDK substrate p220NPAT are required for self‐renewal of human embryonic stem cells

Self‐renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB‐dependent growth control. We investigated whether self‐renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220^NPAT/HiNF‐P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220^NPAT causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220^NPAT, decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220^NPAT are principal cell cycle regulators that determine competency for self‐renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220^NPAT/HiNF‐P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. J. Cell. Physiol. 222: 456–464, 2010. © 2009 Wiley‐Liss, Inc.     

Differential regulation of retinoblastoma protein by hormonal and antihormonal agents in T47D breast cancer cells

Phosphorylation of the tumor suppressor protein, retinoblastoma (pRb), regulates the progression of the cell cycle. Previous work from this laboratory had shown that estradiol (E(2)) regulates tumor suppressor proteins, p53 and retinoblastoma in breast cancer cells. In the present study, we have examined the phosphorylation of pRB in T47D breast cancer cells following treatments with R5020 and antiprogestins. In growth medium containing serum depleted of endogenous steroids by charcoal treatment, pRb appeared mainly in its hypophosphorylated form. Addition of 10 nM R5020 to the culture medium caused hyperphosphorylation of pRb within 24 h, but the hypophosphorylated form of pRb began to accumulate after 72 h. Upon prolonged R5020 treatment (72-96 h), pRb was detected exclusively in its hypophosphorylated form. While treatment of cells with R5020 caused a transient increase in the level of cyclin D1, E(2) addition caused a sustained increase in the level of cyclin D1 consistent with its role in stimulating pRb phosphorylation. Antagonists of both estrogen receptor (ER) and progesterone receptor (PR) blocked the E(2) and R5020-induced pRb phosphorylation, respectively. These results suggest that R5020 induces pRb phosphorylation via a transient increased expression of cyclin D1, whereas E(2) treatment results in sustained expression of cyclin D1 and increased pRb phosphorylation. Furthermore, R5020 effects on pRb phosphorylation appear PR-mediated as no cross-antagonism of pRb phosphorylation was observed: the R5020 effects were blocked by RU486 and ZK98299, but not by the pure ER antagonist, ICI 182, 780 (ICI).     

DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.     

Regulation of the cell cycle in response to inhibition of mitochondrial generated energy

Cell cycle control is regulated through the temporal action of both cyclin-dependent kinases and cyclin binding partners. Previously, we have demonstrated that low doses of oligomycin result in a cell cycle arrest of HL-60 cells in G(1) [S. Sweet, G. Singh, Accumulation of human promyelocytic leukemic (HL-60) cells at two energetic cell cycle checkpoints, Cancer Res. 55 (1995) 5164-5167]. In this study, we provide the molecular mechanisms for the observed G(1) arrest following mitochondrial ATPase inhibition. Protein expression of cyclin E and CDK2, the kinase activity of complexed cyclin E/CDK2, and protein expression of p16, p21, and p27 were all unaffected by oligomycin administration. While CDK4 levels were unchanged following oligomycin treatment, a dramatic reduction in cyclin D(1) was observed. Moreover, increased amounts of hypo-phosphorylated retinoblastoma protein (Rbp) and Rbp bound E2F were observed following mitochondrial ATP synthase inhibition. These data provide further evidence that surveillance of available energy occurs during G(1) and ATP deprivation results in cell cycle arrest via a reduction in cyclin D.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

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.     

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.     

The role of p27(Kip1) in maintaining the levels of D-type cyclins in vivo

This in vivo study employs p27-deficient mice to investigate the significance of p27 for the metabolism of D-type cyclins in differentiated cells. The absence of p27 results in decreased levels of cyclins D2 and/or D3 in some organs. As demonstrated on Leydig cells of testis, such dependency is only restricted to certain cell types including terminally differentiated ones, and the absence of p27 in these cells can interfere with their differentiation. The decrease of cyclin D caused by the absence of p27 equals the amount of cyclin D physically associated with p27 in non-mutant animals. The data indicate that it is the proportion of p27-associated cyclin D that determines the response to p27 deficiency. Cells in which the level of D-type cyclin is dependent on p27 do not up-regulate the activity of their CDK2 and CDK4 upon loss of p27, and these cells have a negligible amount of p27 bound to CDK2 and/or cyclin A/E under normal conditions. Together, the findings suggest the existence of a dual role for p27, one being a classical regulation of cell cycle via inhibition of cyclin-dependent kinases (CDK), and the other being participation in the establishment and/or maintenance of differentiated status that is realized in conjunction with D-type cyclins.     

CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes

INTRODUCTION/OBJECTIVES: Cell cycle progression is driven by the coordinated regulation of cyclin-dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G(1) phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. METHODS AND RESULTS: In this study, we have explored the role of CDK4 activity during G(1) progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. CONCLUSIONS: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.