Endogenous Cdk2 Research Articles

Noncanonical CDK4 signaling rescues diabetes in a mouse model by promoting β cell differentiation.

Expanding β cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain β cell number. β cell failure in the IRS2-deletion mouse type 2 diabetes model is, in part, due to loss of CDK4 regulator cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of β cell mass in IRS2-deficient mice. Surprisingly, not only β cell mass but also β cell dedifferentiation was effectively rescued, despite no improvement in whole body insulin sensitivity. Ex vivo studies in primary islet cells revealed a mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical β cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation and reduced FOXO1 abundance. Taken together, these results demonstrate a differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that β cell mass can be expanded without compromising function.

Expression, purification and characterisation of a human anti-CDK4 single-chain variable fragment antibody

BackgroundCyclin-dependent kinase 4 (CDK4) when hyperactivated drives development and maintenance of most tumour types, thus prompting its use as an essential cancer treatment target and a diagnostic tool. Target-binding molecules, such as single-chain variable fragment (scFv) antibodies, hold tremendous potential for use in a wide range of cancer diagnostic and therapeutic applications.ResultsA human anti-CDK4 scFv antibody (AK2) derived from a human phage display library was expressed in soluble form in Escherichia coli and shown to be secreted into the culture supernatant. Next, soluble AK2 within culture supernatant was successfully purified using affinity chromatography then was shown, using enzyme-linked immunosorbent assays, to bind to recombinant human CDK4 with high affinity and specificity. Further analyses of AK2 interactions with intracellular components demonstrated that AK2 recognised and interacted specifically with endogenous CDK4 and thus could be useful for detection of CDK4 within tumour cells.ConclusionsA novel anti-CDK4 scFv antibody that can recognise and interact specifically with recombinant human CDK4 and endogenous CDK4 in tumour cells was expressed and purified successfully. These results suggest that the anti-CDK4 scFv antibody may serve as a new and promising tool for achieving CDK4-targeted diagnosis, prognosis and treatment of numerous types of cancers.

Stochastic Endogenous Replication Stress Causes ATR-Triggered Fluctuations in CDK2 Activity that Dynamically Adjust Global DNA Synthesis Rates

SUMMARYFaithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that reenter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events.

Abstract 151: Inhibition of AKT-1 for the treatment of human non-small cell lung cancer (NSCLC) in-vitro

Abstract AKT is a serine-threonine kinase implicated in tumorigenesis as a central regulator of cellular growth, proliferation, survival, metabolism, and migration. Activated AKT is overexpressed in 50-70% of NSCLC tumors and has exhibited an association with poor prognosis as well as chemotherapeutic resistance to platinum-based therapy. Accordingly, AKT inhibitors such as MK-2206 are currently undergoing clinical investigation for the treatment of human NSCLC however, these agents broadly target all three (1-3) AKT isoforms. Recent evidence suggests opposing roles of the AKT isoforms in tumorigenesis where loss of AKT-1 inhibits while the loss of AKT-2 enhances lung tumor development in transgenic mouse models. Based on these findings, we hypothesized that preferential inhibition of AKT-1 would warrant a more effective therapeutic strategy for NSCLC compared to the current clinical approach of broad AKT inhibition. WST-1 cell viability assays have revealed that a selective AKT-1 inhibitor A-674563 is a more potent regulator of survival in 6 NSCLC cell lines compared to a pan-AKT inhibitor MK-2206. Furthermore, off-target CDK2 inhibition likely contributes to the observed benefits of the AKT-1 inhibitor as the reduction in cell viability largely parallels the effects of a CDK2 inhibitor PHA-848125. In addition, the cell lines with higher endogenous CDK2 and p-CDK2 expression are more sensitive to the AKT-1 inhibitor relative to the pan-AKT inhibitor MK-2206. Basal protein and RNA levels in each of the cell lines have also shown that high AKT-3 expression may confer resistance to the pan-AKT inhibitor MK-2206. Thus, AKT-3 expression has the potential to serve as a predictive marker for patient response to AKT-1 versus broad AKT-inhibition. Cell cycle analysis demonstrated that the AKT-1 inhibitor decreases the proportion of cells in the Go/G1 phase and increases the proportion of cells in the S-phase, indicating a possible S-phase cell cycle arrest. These differences are also more significant in the cell lines with augmented sensitivity to the AKT-1 inhibitor. Therefore, altered cell cycle progression could be the major driver of the therapeutic benefits of the AKT-1 inhibitor. Overall, our findings suggest that AKT-1 inhibition is significantly more effective at reducing NSCLC cell viability in-vitro compared to pan-AKT inhibition. Furthermore, cell lines with higher CDK2 and AKT-3 expression have marginally increased sensitivity to the AKT-1 inhibitor A-674563 compared to the pan-AKT inhibitor MK-2206. Future research will focus on understanding the mechanism of action of the AKT-1 inhibitor through combined western blot, AKT antibody array, flow cytometry, and confocal microscopy data. Additionally, we will investigate the toxicity of these inhibitors on normal human small airway epithelial cells (HSAECs) to ensure preferential activity against malignant over somatic cells. Citation Format: Paige M. Chorner, Roger A. Moorehead. Inhibition of AKT-1 for the treatment of human non-small cell lung cancer (NSCLC) in-vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 151. doi:10.1158/1538-7445.AM2017-151

Abstract IA20: Exploiting CDK2-driven replication stress to repurpose cancer chemotherapy

Abstract Cyclin-dependent kinase 2 (CDK2) coordinates diverse process, including cell cycle entry and progression, DNA replication, and DNA damage and replication stress responses. Cancers frequently contain mutations in genes that regulate CDK2, which is also activated by oncogenic signaling. Because cancers have abnormally high CDK2 activity, most therapeutic strategies seek to inhibit CDK2. Our studies of CDK2 function during replication stress suggest an alternate approach: to harness high CDK2 activity as a therapeutic modality to cause irreparable DNA damage. In order to study the functions of CDK2 inhibitory phosphorylation without perturbing other cell cycle proteins, we used gene targeting in human cells to mutate the endogenous CDK2 T14 and Y15 Wee1 phosphorylation sites. CDK2AF cells exhibited abnormal G1 progression, abnormal DNA replication dynamics, and genome instability, indicating essential regulatory roles for CDK2 inhibitory phosphorylation in these processes. Most strikingly, cells that cannot inhibit CDK2 during stalled S-phase rapidly accumulate massive and irreparable DNA damage and double stranded DNA breaks (termed CDK2-dependent replication stress failure). Importantly, even transient S-phase arrest produces irreversible DNA damage and permanent cell cycle exit, and genetic and pharmacologic studies revealed that that persistent CDK2 activity is both necessary and sufficient to drive replication stress failure. CDK2 inhibitory phosphorylation is thus essential for S-phase checkpoint function. Because cancers do not contain CDK2AF mutations, we used MK1775, a pharmacologic Wee1 inhibitor, to prevent CDK2 inhibitory phosphorylation during replication stress. Pharmacologic Wee1 inhibition of cells treated with S-phase toxins (e.g. hydroxyurea-HU, aphidicolin) recapitulated the CDK2AF replication stress failure phenotype, and brief (5hr) treatment of HU-phase arrested cells caused catastrophic DNA damage and permanent cell cycle exit, and this was seen in multiple cell types. Wee1 inhibition thus converts HU from an agent that reversibly inhibits S-phase to one that acts irreversibly through DNA damage. Moreover, HU/MK-1775 combination therapy exhibited synergy at HU and MK-1775 concentrations that were ineffective as single agents. Because CDK2-driven replication stress failure requires CDK2 activity to be maintained in S-phase arrested cells, rapidly dividing cells are extremely sensitive to this approach compared with slowly cycling cells, such as stem cells and many normal tissues. Importantly, we have identified common oncogenic mutations that greatly sensitize cancer cells to CDK2-driven replication stress failure, thus providing an even greater therapeutic index that favors killing cancer cells while sparing normal tissues, and identifying tumor genotypes appropriate for clinical trials of induced replication stress failure. These concepts are now being employed in mouse leukemia and solid tumor models. Citation Format: Hannah Richards, Hui Zhao, Bridget T. Hughes, Bruce E. Clurman. Exploiting CDK2-driven replication stress to repurpose cancer chemotherapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA20.

Brk/Protein tyrosine kinase 6 phosphorylates p27KIP1, regulating the activity of cyclin D-cyclin-dependent kinase 4.

Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity.

Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Cell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.

Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation

Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.

Protection of cisplatin cytotoxicity by an inactive cyclin-dependent kinase

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition

Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr(161) by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr(14) and Tyr(15) phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G(2)/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr(15). In addition, Tyr(15)-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G(2) and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr(161) phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G(2)/M is tightly coupled and regulated by Cdc25 phosphatases.

Abstract C68: Using a chemical genetic approach to define the role of CDK2 in normal and transformed cell lines

Abstract Cyclin dependent kinases (CDKs) drive progression through the eukaryotic cell cycle, making them an attractive target for cancer therapeutics. In particular, CDK2 is thought to regulate mammalian S-phase entry and progression, and frequently shows increased activity in a variety of tumors. Surprisingly, recent studies using conventional genetic approaches suggest that CDK2 is dispensable for cellular proliferation, both during normal development and in malignant cell lines. Here we use an alternative chemical genetic approach to determine whether acute ablation of CDK2 activity has the same affect as genetic deletion. We have replaced the endogenous CDK2 allele in mouse embryonic fibroblasts (MEFs) with a mutant analogue sensitive CDK2 allele, allowing us to selectively inactivate CDK2 using bulky ATP analogs. In contrast to genetically deleting CDK2, the acute inhibition of CDK2 resulted in severe defects in proliferation of non-transformed MEFs, as well as MEFs that were transformed using various oncogenes. These findings reveal a requirement for CDK2 in mammalian cell proliferation and suggest its potential as a therapeutic target in tumor cells. Citation Information: Cancer Res 2009;69(23 Suppl):C68.

Genetic substitution of Cdk1 by Cdk2 leads to embryonic lethality and loss of meiotic function of Cdk2

It was believed that Cdk2-cyclin E complexes are essential to drive cells through the G1-S phase transition. However, it was discovered recently that the mitotic kinase Cdk1 (Cdc2a) compensates for the loss of Cdk2. In the present study, we tested whether Cdk2 can compensate for the loss of Cdk1. We generated a knockin mouse in which the Cdk2 cDNA was knocked into the Cdk1 locus (Cdk1Cdk2KI). Substitution of both copies of Cdk1 by Cdk2 led to early embryonic lethality, even though Cdk2 was expressed from the Cdk1 locus. In addition, we generated Cdk2-/- Cdk1+/Cdk2KI mice in which one copy of Cdk2 and one copy of Cdk1 were expressed from the Cdk1 locus and the Cdk2 gene was deleted from the endogenous Cdk2 locus. We found that both male and female Cdk2-/- Cdk1+/Cdk2KI mice were sterile, similar to Cdk2-/- mice, even though they expressed the Cdk2 protein from the Cdk1 locus in testes. The translocational and cell cycle properties of knockin Cdk2 in Cdk2-/- Cdk1+/Cdk2KI cells were comparable to those of endogenous Cdk2, but we detected premature transcriptional activation of Cdk1 during liver regeneration in the absence of Cdk2. This study provides evidence of the molecular differences between Cdk2 and Cdk1 and highlights that the timing of transcriptional activation and the genetic locus play important roles in determining the function of Cdk proteins in vivo.

Inhibition of Cyclin-dependent Kinase-2 Induces Apoptosis in Human Diffuse Large B-cell Lymphomas

Cyclin-dependent kinase (cdk) inhibitors have the potential to induce growth arrest and apoptosis in cancer cells. The genes encoding cdks involved in G1-S progression are often amplified in B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Here, we evaluated the in vitro cytotoxic activity of the cdk2 inhibitor CVT-313 against several human DLBCL cells. Treatment of DLBCL cells with CVT-313 resulted in apoptosis. CVT-313 treatment reduced cdk2-mediated phosphorylation of the retinoblastoma gene product (Rb) on T821, but did not affect cyclin D-cdk4/6-mediated Rb phosphorylation on S807/811. Depletion of endogenous cdk2 by short interfering (si)RNA also resulted in apoptosis in human LY3, LY8 and LY18 DLBCL cells. Importantly, inhibition of cdk2 with CVT-313 or knockdown of endogenous cdk2 with siRNA resulted in down-regulation of the anti-apoptotic factor Myeloid cell leukemia-1 (Mcl-1), suggesting that decreased levels of cellular Mcl-1 contribute to apoptosis. In support of this, siRNA-mediated knockdown of Mcl-1 was sufficient to induce apoptosis in LY3 and LY18 DLBCL. Further, cdk2 inhibition led to decreased Mcl-1 mRNA levels, which was proceeded by reduced phosphorylation of serine 2 on the carboxyl terminal domain (CTD) of RNA polymerase II. Taken together, these data suggest that cdk2 activity is necessary for the survival of human DLBCL.

Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression

Cell-cycle inhibitors of the Cip/Kip and INK4 families are involved in cellular senescence and tumor suppression. Some of these proteins, p21(Cip1), p16(INK4a) and p15(INK4b), are coexpressed in response to antiproliferative signals such as cellular senescence resulting in cell-cycle arrest. To understand the roles of these inhibitors and their synergistic effect, we have characterized the growth properties and senescent behavior of primary cells deficient in p21(Cip1) and expressing an endogenous Cdk4(R24C) (cyclin-dependent kinase) mutant (Cdk4(R24C) knock-in cells) insensitive to INK4 proteins. Inactivation of both p21(Cip1) and INK4 pathways strongly cooperate in suppressing cellular senescence in vitro. These double mutant cells behavior as immortal cultures and display high sensitivity to cellular transformation by oncogenes. Moreover, mice double mutant in the INK4 and p21(Cip1) pathways (Cdk4(R24C); p21(Cip1)-null mice) display an increased incidence of specific sarcomas, suggesting a significant cooperation between these two families of cell-cycle inhibitors in senescence responses and tumor suppression in vivo.

Antisense to CDK4 inhibits the growth of human colon cancer cells HT29

Antisense CDK4 was transfected into human colorectal cancer cells HT29 to examine the effect of it on the growth and proliferation of HT29 cells. Antisense CDK4 transfected the HT29 cells with lipofectamine. The transfected effects were verified by the Northern blot, Western blot, morphological method, flow cell measure, etc. The transformant cells showed the expression of exogenous antisense CDK4 mRNA, and downmodulation of endogenous CDK4 mRNA expression and inhibition of its protein synthesis. The transformant cells exhibited the partial reversion of the malignant phenotype and behavior, and that the cell growth and the ability of colony formation in soft agar were inhibited. The arrest of G1 phase was also revealed. the apoptosis rate is higher in HT29-asCDK4. The antisense CDK4 gene can inhibited the cell growth and proliferation, and induce the cell apoptosis. It might provide a new way to gene therapy of the colorectal carcinoma.

Evidence for a cancer-specific switch at the CDK4 promoter with loss of control by both USF and c-Myc.

USF and c-Myc are basic helix-loop-helix transcription factors with similar DNA-binding specificities, but antagonistic effects on cellular transformation. In order to determine how these opposite functions correlate with the transcriptional activities of the two factors on particular downstream targets, we investigated the roles of USF and c-Myc in expression of CDK4, a known direct target of c-Myc. Overexpression of either c-Myc or USF2, but not USF1, stimulated the expression of CDK4 promoter-driven reporter genes in the non-tumorigenic mammary epithelial MCF-10A cells. Dominant-negative mutants specific to either Myc or USF family proteins inhibited reporter gene activity as well as endogenous CDK4 expression, demonstrating involvement of both USF and Myc in CDK4 transcriptional control. In contrast, in two different breast cancer cell lines where USF is transcriptionally inactive and c-Myc is overexpressed, CDK4 promoter activity was no longer responsive to either transcription factor. Accordingly, chromatin immunoprecipitation revealed significantly lower levels of both USF and c-Myc bound to the endogenous CDK4 promoter in breast cancer cells than in MCF-10A cells, with a concomitant decrease in associated histone H3 acetylation. These results suggest that a major switch in the transcriptional control of CDK4 occurs during breast carcinogenesis, with likely alteration of cell cycle regulation.

Activation of Cyclin-dependent Kinase 2 by Full Length and Low Molecular Weight Forms of Cyclin E in Breast Cancer Cells

Cyclin E, a positive regulator of the cell cycle, controls the transition of cells from G(1) to S phase. Deregulation of the G(1)-S checkpoint contributes to uncontrolled cell division, a hallmark of cancer. We have reported previously that cyclin E is overexpressed in breast cancer and such overexpression is usually accompanied by the appearance of low molecular weight isoforms of cyclin E protein, which are not present in normal cells. Furthermore, we have shown that the expression of cyclin E low molecular weight isoforms can be used as a reliable prognostic marker for breast cancer to predict patient outcome. In this study we examined the role of cyclin E in directly activating cyclin-dependent kinase (CDK) 2. For this purpose, a series of N-terminal deleted forms of cyclin E corresponding to the low molecular weight forms detected only in cancer cells were translated in vitro and mixed with cell extracts. These tumor-specific N-terminal deleted forms of cyclin E are able to activate CDK2. Addition of cyclin E into both normal and tumor cell extracts was shown to increase the levels of CDK2 activity, along with an increase in the amount of phosphorylated CDK2. The increase in CDK2 activity was because of cyclin E binding to endogenous CDK2 in complex with endogenous cyclin E, cyclin A, or unbound CDK2. The increase in CDK2 phosphorylation was through a pathway involving cyclin-activating kinase, but addition of cyclin E to an extract containing unphosphorylated CDK2 can still lead to increase in CDK2 activity. Our data suggest that the ability of high levels of full-length and low molecular weight forms of cyclin E to activate CDK2 may be one mechanism that leads to the constitutive activation of cyclin E.CDK2 complexes leading to G(1)/S deregulation and tumor progression.

The cyclin D3-CDK4-p27 kip1 holoenzyme in thyroid epithelial cells: activation by TSH, inhibition by TGFβ, 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 p27 kip1 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 β (TGFβ) 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 TGFβ.

Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number.

Lack of Cdk4 expression in mice leads to insulin-deficient diabetes and female infertility owing to a reduced number of pancreatic beta cells and prolactin-producing pituitary lactotrophs, respectively. Cdk4 null mice display also reduced body and organ size. Here, we show that Cdk4 is essential for the postnatal proliferation of pancreatic beta cells but not for embryonic neogenesis from ductal epithelial cells. Re-expression of endogenous Cdk4 in beta cells and in the pituitary gland of Cdk4 null mice restores cell proliferation and results in fertile and normoglycemic animals, thus, demonstrating that the proliferation defects in these cellular populations are cell autonomous because of the lack of Cdk4 expression. However, these mice remain small in size, indicating that this phenotype is not because of pancreatic- or pituitary-mediated endocrine defects. This phenotype is a consequence of reduced cell numbers rather than reduced cell size. Thus, mammalian Cdk4 is not only involved in controlling proliferation of specific cell types but may play a wider role in establishing homeostatic cell numbers.