Cyclin E-associated Kinase Activity Research Articles

Loss of Cyclin-Dependent Kinase 2 (CDK2) Inhibitory Phosphorylation in a CDK2AF Knock-In Mouse Causes Misregulation of DNA Replication and Centrosome Duplication

Cyclin-dependent kinase 1 (CDK1) inhibitory phosphorylation controls the onset of mitosis and is essential for the checkpoint pathways that prevent the G(2)- to M-phase transition in cells with unreplicated or damaged DNA. To address whether CDK2 inhibitory phosphorylation plays a similar role in cell cycle regulation and checkpoint responses at the start of the S phase, we constructed a mouse strain in which the two CDK2 inhibitory phosphorylation sites, threonine 14 and tyrosine 15, were changed to alanine and phenylalanine, respectively (CDK2AF). This approach showed that inhibitory phosphorylation of CDK2 had a major role in controlling cyclin E-associated kinase activity and thus both determined the timing of DNA replication in a normal cell cycle and regulated centrosome duplication. Further, DNA damage in G(1) CDK2AF cells did not downregulate cyclin E-CDK2 activity when the CDK inhibitor p21 was also knocked down. We were surprised to find that this was insufficient to cause cells to bypass the checkpoint and enter the S phase. This led to the discovery of two previously unrecognized pathways that control the activity of cyclin A at the G(1) DNA damage checkpoint and may thereby prevent S-phase entry even when cyclin E-CDK2 activity is deregulated.

A novel interaction between HER2/neu and cyclin E in breast cancer

HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. Since both are involved in cell cycle regulation we investigated whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P < .0001) In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 siRNA or treatment with trastuzumab. Cyclin E expression levels were determined, and functional effects investigated using kinase assays, MTT assays to assess cell viability as a marker of proliferation, and FACS analysis to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional effects, including decreased cyclin E-associated kinase activity and decreased proliferation, due to increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies demonstrate that HER2-mediated signaling effects LMW cyclin E expression, which in turn effects cell cycle regulation. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.

CDK2 is required for LMW-cyclin E mediated murine mammary tumorigenesis.

Abstract Abstract #2007 Cyclin E is a regulatory protein with multiple functions that activates CDK2, controls centrosome duplication, and regulates histone gene transcription. Our previous studies have shown that the presence of Low-molecular-weight (LMW) forms of cyclin E correlates strongly with decreased survival in patients with breast cancer. Transgenic mice overexpressing LMW cyclin E had increased incidence of mammary tumors and distant metastasis when compared to full length cyclin E. The critical role of LMW cyclin E in mammary tumorigenesis may be the result of the ability of LMW cyclin E to bind and activate the cyclin-dependent kinase 2 (CDK2). To specifically test the importance of LMW cyclin E-associated CDK2 kinase activity in mammary tumorigenesis, we generated double and triple transgenic mice LMW cyclin E-T1;CDK2-/-, and LMW cyclin E-T1;p53+/-;CDK2-/-. We found that: (i) The development of the mammary glands proceeds relatively normally in these animals demonstrating that CDK2 kinase activity is largely dispensable for mammary gland development; (ii) These mice were resistant to breast cancers initiated by LMW cyclin E overexpression. Whereas LMW-cyclin E-T1 mice in CDK2+/- or CDK2+/+ background succumb to mammary tumors with mean latencies of 16 and 19.5 months respectively, their LMW cyclin E-T1; CDK2-/- littermates do not form mammary tumors up to 24 months (P=0.05 CDK2+/+ versus CDK2-/-; P=0.0252 CDK2+/- versus CDK2-/-; P=0.1457 CDK2+/+ versus CDK2+/-, Log rank test). Similarly, while MMTV-LMW cyclin E-T1/p53+/- transgenic mice in CDK2+/- or CDK2+/+ background succumb to mammary gland tumors between 7 and 13 months with 100% penetrance, their MMTV-LMW cyclin E-T1;p53+/-; CDK2-/- littermates are resistant to tumor formation (P&amp;lt;0.01 CDK2+/+ versus CDK2-/-; P=0.011 CDK2+/- versus CDK2-/-, P=0.5072 CDK2+/+ versus CDK2+/-, Log rank Test). In fact none of the CDK2-/- mice developed mammary tumors when crossed with cyclin E LMW transgenic mice. However, 2 out of the 12 MMTV-LMW cyclin E; p53+/-; CDK2-/- mice generated salivary gland tumors. Hence, these LMW cyclin E; p53+/-; CDK2-/- are susceptible to LMW cyclin E induced salivary tumors in the absence of CDK2. We concluded that CDK2 is critically required for LMW cyclin E induced mammary tumorigenesis and consequently, the loss of CDK2 renders CDK2-/- mice resistant to breast cancer induced by LMW cyclin E expression. In clear contrast to mammary epithelial cells, we found that in salivary gland cells LMW cyclin E continued to elicit malignant transformation even in the absence of CDK2. Our work suggests that due to the requirement of CDK2 for LMW-cyclin E mediated mammary tumorigeneis, these tumors are prime candidates for anti-CDK2 therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2007.

G1 control in yeast and animal cells.

In budding yeast, Saccharomyces cerevisiae, the cell cycle is controlled at the G1/S phase transition by regulating the activity of the CDC28 protein kinase. This is the budding yeast homologue of the cdc2 protein kinase associated in most organisms with control of mitosis. In budding yeast CDC28 controls both the G1/S phase transition and the G2/M phase transition by being differentially activated by two distinct classes of positive regulatory subunits known as G1 cyclins or CLNs and B-type cyclins or CLBs, respectively. To establish whether a similar dual role for Cdc2-related kinases exists in animal cells, we and others have sought human homologues of yeast G1 cyclins. Of several candidates, cyclin E is the most promising in that it accumulates prior to S phase and is associated with a pre-S phase protein kinase activity. The kinetics of accumulation of cyclin E-associated protein kinase activity is consistent with a role at the mammalian cell cycle restriction point.

Safe from Degradation

DNA replication is initiated through the assembly of prereplication complexes (pre-RCs); replication does not occur in quiescent cells and is limited to once per cell cycle because, outside of the G 1 phase of the cell cycle, inhibitors prevent assembly of the pre-RC. During G 1 , APC Cdh1 (the form of the anaphase-promoting complex E3 ubiquitin ligase that contains Cdh1) targets the pre-RC assembly inhibitors geminin and cyclin A for degradation. Paradoxically, however, APC Cdh1 targets Cdc6, which is essential to pre-RC assembly, for destruction as well (see Ayad). Mailand and Diffley noted that, in cultured human cells treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine, Cdc6 abundance was reduced, whereas the abundance of geminin and cyclin A was unaffected. Knockdown of Cdk2 with small interfering RNA (siRNA) also decreased Cdc6 abundance. Roscovitine accelerated Cdc6 degradation, as did depletion of Cdk2 or cyclins E1 and E2, whereas overexpression of cyclin E (which forms an active CDK complex with Cdk2) promoted Cdc6 accumulation. Mutational analysis indicated that the effects of cyclin E on Cdc6 stability depended on Cdc6 phosphorylation. Cdc6 phosphorylation inhibited its APC Cdh1 -dependent ubiquitination and degradation; further, whereas wild-type Cdc-6 or an unphosphorylatable mutant coimmunoprecipitated with Cdh1, a phospho-mimicking Cdc6 mutant did not. In serum-starved (noncycling) cells, Cdc6, geminin, and cyclin A were undetectable. After serum stimulation, and reentry into the cell cycle, Cdc6 abundance increased 3 to 4 hours before the other APC Cdh1 targets became detectable. When Cdc6 was detected, it was phosphorylated, and cyclin E-associated kinase activity appeared simultaneously with Cdc6. Thus, the authors propose that, after entry into the cell cycle, cyclin E-Cdk2-dependent phosphorylation of Cdc6 allows it to accumulate (and promote pre-RC assembly) before inactivation of APC Cdh1 and the ensuing accumulation of pre-RC assembly inhibitors such as geminin and cyclin A. N. Mailand, J. F. X. Diffley, CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122 , 915-926 (2005). [PubMed] N. G. Ayad, CDKs give Cdc6 a license to drive into S phase. Cell 122 , 825-827 (2005). [PubMed]

The SV40 Large T Antigen Contains a Decoy Phosphodegron That Mediates Its Interactions with Fbw7/hCdc4

Cell transformation by simian virus 40 (SV40) results mostly from the highly oncogenic activities of the large T antigen (LT), which corrupts the cellular checkpoint mechanisms that guard cell division and the transcription, replication, and repair of DNA. The most prominent LT targets are the retinoblastoma protein (pRb) and p53. Here we report that LT binds directly to Fbw7, the substrate recognition component of the SCF(Fbw7) ubiquitin ligase and a human tumor suppressor. LT binding mislocalizes the nucleolar Fbw7gamma isoform to the nucleoplasm. Interestingly, the binding of LT to Fbw7 occurs via a decoy phospho-epitope within the C terminus of LT that closely mimics the consensus Cdc4 phospho-degron found within Fbw7 substrates. We demonstrate that, using this mode of interaction, LT can interfere with Fbw7-driven cyclin E turnover in vivo and causes increased cyclin E-associated kinase activity. Our data suggest that LT competes with cellular proteins for Fbw7 binding in a substrate-like fashion.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

JC virus small tumor antigen promotes S phase entry

A number of studies revealed the detection of human polyomavirus, JC virus (JCV), in a variety of human tumors. Viral large tumor antigen (T-Ag) is known to play a major role in cell proliferation and transformation but the role of JCV small t-Ag in such processes remains completely unknown. Several studies from monkey polyomavirus, SV40, small tumor antigen (tAg) suggest that it significantly contributes to the large T-Ag-mediated cell transformation. Here, we investigated the effects of JCV small t-Ag on cell cycle progression and cell proliferation. Examination of subcellular distribution of small t-Ag by means of immunocytochemistry demonstrated that it mostly localizes to the cytoplasmic compartment of the cell. Cell cycle progression analysis of a human glioblastoma cell line, U-87MG, demonstrated that small t-Ag expressing cells enter S phase of the cell cycle at a significantly higher rate than controls when released from G0/G1 arrest. In addition, the growth rate of these cells is consistent with the rate of cell cycle progression and is significantly enhanced compared to controls. Moreover, analysis of the mechanism through which small t-Ag accelerates the cell cycle progression showed that JCV small t-Ag targets a major serine/threonine phosphatase, PP2A, and its interaction domain with this enzyme is mapped toward the middle portion of the protein, residues 82 to 124. Furthermore, examination of the cell cycle stage specific expression profiles of selected cyclins and cyclin-dependent kinases, particularly those active in G1/S and G2/M transition states demonstrated that the rate of the appearance of cyclin E, cyclin B, and cyclin E-dependent kinase 2 (Cdk2) substantially increased in small t-Ag positive cells as compared to controls when the cells were released from G0/G1 arrest. In parallel, in vitro analysis of cyclin E-associated H1 kinase activity of clones demonstrated that small t-Ag positive clones exhibited significantly higher activity than controls. Additionally, results from reporter gene assays showed that small tAg has the ability to activate the cyclin E promoter which is consistent

인체 신경아세포종에서 cAMP 처리에 의한 pRB의 인산화 억제 및 p21WAF1/CIP1의 유도

인체 신경아세포종의 성장에 미치는 cAMP의 영향을 조사하기 위하여 Ewing's sarcoma 세포주인 CHP-100 세포에 dibutyry1-cAMP 및 8-bromo-cAMP를 처리하였다. 두 종류의 cAMP analog처리 시간 증가에 따라 CHP-100 세포의 증식이 처리 시간 의존적으로 억제되었으며, 이는 핵의 형태변화 및 DNA 단편화 현상을 수반한 apoptosis 유발과 연관성이 있었다. 또한 DNA flow cytometry 분석결과 cAMP는 세포주기 G1기 특이적 arrest를 유발하였다. cAMP 처리에 의하여 retinoblastoma 단백질(pRB)의 인산화가 억제되었으며, 전사조절인자 E2F-1과의 결합이 증대되었다. cAMP는 cyclin-dependent kinase (Cdk) 2 및 cyclin E 단백질의 발현변화에는 영향을 미치지 않았으나, 그들의 kinase 활성은 처리시간 의존적으로 매우 감소되었다. 또한 cAMP 처리에 의하여 Cdk inhibitor인 <TEX>$p21^{WAF1/CIP1$</TEX>의 발현이 증가되었으며, 증가된 p21 단백질은 Cdk2와 강한 결합을 형성하고 있었다. 이상의 결과에서 cAMP의 암세포 성장억제 효과에 pRB 및 p21이 매우 중요한 역할을 함을 알 수 있었다. To develop a new approach to the treatment of neuroblastoma cells we evaluated the effect of cAMP on the Ewing's sarcoma cell line CHP-100. We observed that the proliferation-inhibitory effect of cAMP analogs was due to cell cycle arrest and induction of apoptosis, which was confirmed by observing the morphological changes and DNA fragmentation. DNA flow cytometric analysis revealed that cAMP arrested the cell cycle progression at the G1 phase, which effects were associated with inhibition of phosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB and the transcription factor E2F-1. cAMP also suppressed the cyclin-dependent kinase (Cdk) 2 and cyclin E-associated kinase activity without changes of their expressions. Furthermore, cAMP induced the levels of Cdk inhibitor <TEX>$p21^{WAF1/CIP1$</TEX> expression and p21 proteins induced by cAMP were associated with Cdk2. Overall, our results identify a combined mechanism involving the inhibition of pRB phosphorylation and induction of p21 as targets for cAMP, and this may explain some of its anti-cancer effects.

Activation of c-Myc Contributes to Bovine Papillomavirus Type 1 E7-induced Cell Proliferation

Inactivation of the tumor suppressor pRB by the human papillomavirus (HPV) oncoprotein E7 is a mechanism by which HPV promotes cell growth. The bovine papillomavirus type 1 (BPV-1) E7 does not bind pRB efficiently yet is required for full transformation of murine cells by BPV-1. In the present study, we investigated the mechanism of BPV-1 E7-induced cell proliferation. Our studies indicate that expression of BPV-1 E7 induces DNA synthesis and stimulates cells to enter S phase in quiescent cells. The induction of cell proliferation by BPV-1 E7 can occur in the retinoblastoma gene (Rb)-null cells, suggesting an Rb-independent mechanism. Consistent with this observation, BPV-1 E7 does not efficiently activate the transcription of the E2F family of transcription factors (E2F)-responsive promoters. Notably, c-Myc is able to induce cells to enter S phase in quiescent cells through an Rb/E2F-independent pathway. Significantly, c-Myc levels are increased in BPV-1 E7-expressing cells. Moreover, expression of a dominant negative c-Myc mutant inhibited BPV-1 E7-induced DNA synthesis. Consistent with the notion that c-Myc could down-regulate p27 and activate Cdk2, p27 level is decreased while both cyclin A and cyclin E-associated kinase activities are up-regulated in BPV-1 E7-expressing cells. These studies indicate an important role for c-Myc in BPV-1 E7-induced cell proliferation.

Quercetin elevates p27(Kip1) and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1.

Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27(Kip1). Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27(Kip1) with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27(Kip1) was due not only to increased protein half-life, but also to increased mRNA transcription.

HPV E7 expression in skeletal muscle cells distinguishes initiation of the postmitotic state from its maintenance.

The E7 oncogene is an essential tool used by papillomaviruses to interfere with the cell cycle and cellular differentiation. We investigated the effects of E7 expression on both cellular functions in skeletal muscle cells, a terminally differentiating system. When expressed in myoblasts, E7 impaired differentiation only partially, but allowed continuation of DNA synthesis during and after differentiation. Surprisingly, E7 expression in terminally differentiated myotubes could not reactivate DNA synthesis even though the oncogene bound the retinoblastoma protein, reduced its levels, and increased E2F transcriptional activity. Despite the high cyclin E protein levels induced by E7, the myotubes remained devoid of cyclin E-associated kinase activity. Enforcement of such activity in the presence of E7 brought myotubes into S phase. These results show that E7, unlike other DNA tumor-virus oncogenes, cannot reactivate the cell cycle in postmitotic myotubes. In contrast, E7 allows significant differentiation to occur in the presence of persisting DNA synthesis. These observations distinguish E7 from other functionally related oncogenes and bear significance for the understanding of the natural life cycle of human papillomaviruses. The fact that E7 alone inhibits the initiation but not the maintenance of the postmitotic state indicates that the mechanisms underlying these two functions are at least partially distinct.

Identification of distinct and common gene expression changes after oxidative stress and gamma and ultraviolet radiation.

The human genome is exposed to many different kinds of DNA-damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (gamma) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G(1) checkpoint induction and slow down in early S-phase progression, as reflected by a comparable reduction in cyclin E-associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 microM of tert-butyl hydroperoxide (t-butyl-OOH)), or UV radiation (UVC) (7.5 J/m(2)) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to gamma radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E-associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after gamma radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, gamma radiation had its major influence on cell-cycle progression in S-phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage.

Suppression of human prostate cancer cell growth by beta-lapachone via down-regulation of pRB phosphorylation and induction of Cdk inhibitor p21(WAF1/CIP1).

The product of a tree (Tabebuia avellanedae) from South America, beta-lapachone, is known to exhibit various pharmacological properties, the mechanisms of which are poorly understood. The aim of the present study was to further elucidate the possible mechanisms by which beta-lapachone exerts its anti-proliferative action in cultured human prostate cancer cells. We observed that the proliferation-inhibitory effect of beta-lapachone was due to the induction of apoptosis, which was confirmed by observing the morphological changes and cleavage of the poly(ADP-ribose) polymerase protein. A DNA flow cytometric analysis also revealed that beta-lapachone arrested the cell cycle progression at the G1 phase. The effects were associated with the down-regulation of the phosphorylation of the retinoblastoma protein (pRB) as well as the enhanced binding of pRB and the transcription factor E2F-1. Also, beta-lapachone suppressed the cyclin-dependent kinases (Cdks) and cyclin E-associated kinase activity without changing their expressions. Furthermore, this compound induced the levels of the Cdk inhibitor p21(WAF1/CIP1) expression in a p53-independent manner, and the p21 proteins that were induced by beta-lapachone were associated with Cdk2. beta-lapachone also activated the reporter construct of a p21 promoter. Overall, our results demonstrate a combined mechanism that involves the inhibition of pRB phosphorylation and induction of p21 as targets for beta-lapachone. This may explain some of its anticancer effects.

TGF-β Induced G 1 Cell Cycle Arrest Requires the Activity of the Proteasome Pathway

Transforming growth factor-β (TGF-β) induces a potent G 1/S-phase cell cycle arrest of epithelial cells by inhibiting the activities of cyclin D- and cyclin E-associated kinase complexes. Downregulation of the kinase activities is mediated by induction of cyclin dependent kinase (CDK) inhibitor p15 Ink4b which blocks CDK4 and CDK6 kinases and leads to binding of p27 Kip1 to CDK2-cyclin E complex. Levels of several of these factors are controlled by the ubiquitin–proteasome pathway. We demonstrate here that proteasomal inhibitors release the cells from TGF-β imposed G 1-phase arrest and instigate the entry of the cells into S-phase. Proteasomal inhibitors are shown to specifically increase the activity of the cyclin D–kinase complex by increasing the levels of p27 Kip1 and cyclin D and by maintaining CDK4/6 protein levels leading to phosphorylation of the retinoblastoma protein without increasing cyclin E-associated kinase activity. The results indicate caution in the potential therapeutic use of the proteasome inhibitors due to unscheduled initiation of DNA replication in the presence of a physiological growth inhibitor.

Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells

Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.

Active RB Elicits Late G1/S Inhibition

The retinoblastoma tumor suppressor protein (RB) is activated/dephosphorylated to mediate cell cycle inhibition in response to antimitogenic signals. To elucidate the mode of RB action at this critical transition, we utilized cell lines that can be induced to express a constitutively active allele of RB (PSM-RB). As expected, induction of PSM-RB, but not wild-type protein (WT), inhibited progression into S phase. It has been well documented that active RB inhibits E2F reporter activity, and this observation was confirmed upon induction of PSM-RB. Additionally, active RB inhibited E2F-2-mediated stimulation of cyclin E. By contrast, PSM-RB did not affect the mRNA or protein levels of endogenous cyclin E when mediating cell cycle inhibition. Similarly, there was no observable effect on cyclin E protein levels when p16ink4a was utilized to activate endogenous RB. CDK2/cyclin E complex formation was not disrupted and cyclin E-associated kinase activity was retained in the presence of PSM-RB. Additionally, centrosome duplication, a CDK2/cyclin E-dependent event, was not altered in the presence of active RB. Together, these data indicate that active RB does not block the G1/S transition through inhibition of cyclin E expression or activity. In contrast, PSM-RB leads to a dramatic reduction in cyclin A protein levels by coordinate transcriptional repression and degradation. This attenuation of cyclin A protein correlates with cell cycle inhibition. These studies indicate that RB inhibits cell cycle progression by targeting CDK2/cyclin A-dependent events at the G1/S transition to inhibit cell cycle progression.

Lack of intestinal bile results in delayed liver regeneration of normal rat liver after hepatectomy accompanied by impaired cyclin E–associated kinase activity

Background. The importance of bile in liver regeneration after hepatectomy is unknown, although we have recently shown that preoperative internal biliary drainage is superior to external biliary drainage for liver regeneration in obstructive jaundiced rats. This study examined the hypothesis that the presence or absence of bile in the intestinal tract modulates cyclins and cyclin-dependent kinases after hepatectomy in rats. Methods. In male Wistar rats, bile was drained externally (ED group) or into the duodenum (ID group) for 7 days before 70% hepatectomy. Relative liver weight, DNA synthesis rate, and proliferating cell nuclear antigen labeling index were determined at the time of hepatectomy (day 0) and on days 1, 3, and 7 after hepatectomy. Posthepatectomy expressions of cyclin D1 and E and of cyclin D1- and E-associated kinases were serially analyzed. Hepatic function tests were performed. Results. No significant difference in liver function was found between the 2 groups at hepatectomy except for the lower albumin level in the ED group. The relative liver weight was lower in the ED group than in the ID group on day 3 after hepatectomy (ED, 2.58% ± 0.06%; ID, 2.84% ± 0.08%; P <.05). Both the DNA synthesis rate and proliferating cell nuclear antigen labeling index in the ED group (77 ± 36 disintegrations per minute/μg DNA and 8.3% ± 1.9%, respectively) were lower than those in the ID group (262 ± 50 disintegrations per minute/μg DNA and 21.6% ± 5.6%, respectively) on day 1 after hepatectomy (P <.05, respectively). Cyclin D1-associated kinase activity and cyclin D1 expression were not significantly different between the 2 groups. Cyclin E-associated kinase activity was lower in the ED group than in the ID group at 18 hours after hepatectomy (ED, 84% ± 17%; ID, 146% ± 28% of the value at 0 hour in the ID group; P <.05), although expressions of cyclin E and p27 binding to cyclin E were not significantly different between the 2 groups. Conclusions. These results suggest that the absence of bile in the intestine delays liver regeneration associated with cyclin E-associated kinase inactivation after hepatectomy. (Surgery 2002;131:564-73.)

Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells.

Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E-associated kinase activity transiently within 9-12 h; total cyclin-dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27(KIP1)) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27(KIP1) expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27(KIP1). The activity of cyclin E/Cdk2 complexes from zearalenone-treated lysates was inhibited in vitro by recombinant p27(KIP1), and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27(KIP1) by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone-treated cells was depleted by anti-p27(KIP1) and anti-Cdc2 interacting protein (p21(CIP1)) antibodies. Overexpression of the Cdk4/6-specific Cdk inhibitor of Cdk4 p16(INK4A) was associated with increased association of p27(KIP1) with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2-leu-leu-leu-H aldehyde (MG-132) was relatively ineffective in inhibiting the initial, sequestration-dependent activation of cyclin E/Cdk2 yet was as effective as p16(INK4A) in inhibiting activation of cyclin A/Cdk2 later in G(1). Downregulation of p27(KIP1) proceeded in p16(INK4A)-expressing cells after zearalenone treatment, and G(1) arrest afforded by p16(INK4A) expression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF-7 cells elicited expression of F-box protein S phase kinase-associated protein 2 (p45(SKP2)), a substrate-specific component of the ubiquitin-ligase complex that targets p27(KIP1) for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27(KIP1) play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF-7 cells.

The Cell Cycle Regulatory Effects of High Dose 5-fluorouracil on Breast Cancer Cell Line

Background: Chemotherapy with 5-fluorouracil (5-FU) has been one of the mainstay in breast cancer treatment. The effects of high dose 5-FU on cell cycle regulation were studied in breast caner cells. Methods: A breast cancer cell line MCF-7 was used. Protein expressions of G1/S cyclins, p21, cdk2, E2F1 and retinoblastoma were tested by western blot analysis. Immunoprecipitation and immune complex kinase assay were done for the assessment of E2F1/RB interacton and the activity of cdk2 respectively. Results: p21 expression was barely detectable in control cells. With addition of 5-FU level of p21 were induced and cyclin D3 level was decreased as cell growth decreases. In accordance with increased expression of p21, cyclin E-associated cdk2 kinase activity was reduced. Retinoblastoma protein (RB) became dephosphorylated and E2F-1 binding activity with RB was increased. Conclusion: In this situation of high concentration of 5-FU breast cancer cells tend to be G1/S cell cycle arrested. Overexpression of p21 and dephosphorylation of RB may mediate the effectss of 5-FU by inhibiting E2F-1 activity, which contributes to G1/S cell cycle arrest. These results could be an indicating landmark for further study of high dose chemotherapy with 5-FU. (Immune Network 2002;2(1):60-64)