Subsequent Cell Cycle Progression Research Articles

RBTT-05. A SINGLE-ARM, OPEN-LABEL, PHASE II STUDY EVALUATING THE EFFICACY AND SAFETY OF ABEMACICLIB IN PATIENTS WITH RECURRENT OLIGODENDROGLIOMA: TRIALS IN PROGRESS

Abstract BACKGROUND Oligodendroglioma is a rare primary brain tumor characterized by mutation in the IDH1/IDH2 genes and codeletion of chromosomes 1p and 19q. Although median overall survival extends beyond 10 years for patients with oligodendroglioma who receive adjuvant radiation and alkylating chemotherapy, the disease uniformly relapses and has no other effective treatments. The CIC gene, which encodes a high-mobility group box transcriptional repressor, is mutated in about 70% of oligodendrogliomas and is associated with worse prognosis and overexpression of cyclin D1 (CCND1). Cyclin D1 assembles with the cyclin-dependent kinases CDK4 and CDK6 to cause phosphorylation of Rb protein and subsequent cell cycle progression from the G1 to S phase. Therapeutic inhibition of CDK4/6 activity, which leads to Rb dephosphorylation and cell cycle arrest, has revolutionized the care of breast cancer. The current phase 2 study will investigate the efficacy and safety of abemaciclib, a selective and potent small molecule inhibitor of CDK4 and CDK6 with significant brain-brain barrier penetration, in patients with 1p/19q codeleted oligodendroglioma that has recurred after standard radiation and alkylating chemotherapy. METHODS This protocol (NCT03969706) has a single-arm, open-label, single-stage phase 2 design. Subjects will be treated with abemaciclib 200mg tablet twice daily on 28-day cycles until disease progression or unacceptable toxicity. Tumor response will be assessed according to modified Response Assessment in Neuro-Oncology (mRANO) criteria. The primary objective of the study is to determine the efficacy of abemaciclib for recurrent oligodendroglioma. The primary endpoint is the progression-free survival status of the subject at 6 months after enrollment (PFS-6). The alternative hypothesis is that the proportion of subjects without an event at 6 months will be ³85%. We will test this hypothesis against a null hypothesis of PFS-6 = 50%. With enrollment of 10 planned subjects, the power to detect this difference is 82% (one-sided alpha = 0.05).

Anti-tumor effect of CDK inhibitors on CDKN2A-defective squamous cell lung cancer cells.

Squamous cell lung cancer (SqCLC) is a distinct histologic subtype of non-small cell lung cancer (NSCLC). Although the discovery of driver mutations and their targeted drugs has remarkably improved the treatment outcomes for lung adenocarcinoma, currently no such molecular target is clinically available for SqCLC. The CDKN2A locus at 9p21 encodes two alternatively spliced proteins, p16INK4a (p16) and p14ARF (p14), which function as cell cycle inhibitors. The Cancer Genome Atlas (TCGA) project revealed that CDKN2A is inactivated in 72% of SqCLC cases. In addition, it was found that CDKN2A mutations are significantly more common in SqCLC than in adenocarcinoma. Down-regulation of p16 and p14 by CDKN2A gene inactivation leads to activation of cyclin-dependent kinases (CDKs), thereby permitting constitutive phosphorylation of Rb and subsequent cell cycle progression. Here, we hypothesized that CDK inhibition may serve as an attractive strategy for the treatment of CDKN2A-defective SqCLC. We investigated whether the CDK inhibitors flavopiridol and dinaciclib may exhibit antitumor activity in CDKN2A-defective SqCLC cells compared to control cells. The cytotoxic effect of the CDK inhibitors was evaluated using cell viability assays, and the induction of apoptosis was assessed using TUNEL assays and Western blot analyses. Finally, anti-tumor effects of the CDK inhibitors on xenografted cells were investigated in vivo. We found that flavopiridol and dinaciclib induced cytotoxicity by enhancing apoptosis in CDKN2A-defective SqCLC cells, and that epithelial to mesenchymal transition (EMT) decreased and autophagy increased during this process. In addition, we found that autophagy had a cytoprotective role. Our data suggest a potential role of CDK inhibitors in managing CDKN2A-defective SqCLC.

15-Lipoxygenase/15-hydroxyeicosanoid and activator protein 1 contribute to hypoxia-induced pulmonary artery smooth muscle cells phenotype alteration

We have previously shown that 15-lipoxygenase (15-LOX) and its metabolite 15-hydroxyeicosanoid (15-HETE) play a critical role on hypoxia-triggered pulmonary artery smooth muscle cell (PASMC) phenotype alteration through multifactorial pathways, like extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Here, we hypothesize that activator protein 1 (AP-1) was also involved in the PASMC phenotype alteration. Hypoxia elevated AP-1 expression in pulmonary arterials and in cultured PASMCs with a time-dependent manner. Both the gene disruption and pharmacological inactivation of 15-lipoxygenase (15-LOX) significantly attenuated the hypoxia-elevated AP-1 expression. Silencing of AP-1 with small interference RNA abrogated the hypoxia- and 15-HETE-increased cell viability, proliferating cell nuclear antigen expression, and Ki67 and α-tubulin staining. Moreover, AP-1 knockdown prevented hypoxia- and 15-HETE-promoted cyclin D1 expression and subsequent cell cycle progression into G2/M+S phase. Interestingly, AP-1 knockdown also inhibited the expression of 15-LOX, indicating a feedback regulation of 15-LOX/15-HETE signaling by AP-1. These findings shed light on the involvement of AP-1 in the PASMCs phenotype alteration via the hypoxia/15-LOX/15-HETE signaling.

Phase II trial of palbociclib (PD-0332991) in patients with metastatic urothelial cancer (UC) after failure of first-line chemotherapy.

500 Background: UC is a common malignancy with poor outcomes in patients with metastatic disease. The majority of urothelial cancers harbor alterations in key retinoblastoma (Rb) pathway genes with CDKN2A alterations in approximately 35% of tumors, leading to loss of Rb tumor suppressor function with subsequent cell cycle progression and unchecked cell proliferation. Palbociclib is an oral, selective inhibitor of CDK4/6 that prevents Rb phosphorylation to promote cell cycle arrest. Methods: In this phase II trial, biomarker-selected (p16 loss and intact Rb by tumor immunohistochemistry [IHC]) patients with metastatic platinum-refractory UC received palbociclib 125mg po daily for 21 days of a 28-day cycle. The primary endpoint was progression-free survival at 4 months (PFS4) using a Simon’s two-stage design. Prespecified tumor analysis with next generation sequencing (NGS) including Rb pathway alterations was conducted. Results: 12 patients (67% male, median age 68 years) were enrolled. Post-platinum prognostic factors included hemoglobin < 10 g/dL, 17%; liver metastases, 0%; median time from prior therapy, 5.0 months; and Eastern Cooperative Oncology Group performance status < = 1, 92%. Overall, two of 12 patients (17%) achieved PFS4 with insufficient activity to advance to stage 2. 58% of patients had grade ≥3 hematologic toxicity with 25% grade 3 anemia and 17% grade 3 thrombocytopenia. Using NGS, the most frequently observed somatic mutations were ARID1A, MLL2, PIK3CA, and TP53 (55% for each). No patients had CDKN2A alterations, although the majority (82%) of patients had a cell cycle pathway alteration (CDKN1A 27%, CDKN2B 27%, E2F3 18%). There was no correlation between genomic alterations and clinical outcome. Conclusions: Palbociclib demonstrated limited activity in patients with platinum-refractory metastatic UC selected for p16 loss and intact Rb by IHC. Further development of palbociclib should only be considered with improved integral biomarker selection such as NGS or in rational combination with other therapies. Clinical trial information: NCT02334527.

Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance.

The Saccharomyces cerevisiae Forkhead Box (Fox) orthologs, Forkheads (Fkh) 1 and 2, are conserved transcription factors required for stress response, cell cycle progression and longevity. These yeast proteins play a key role in mitotic progression through activation of the ubiquitin E3 ligase Anaphase Promoting Complex (APC) via transcriptional control. Here, we used genetic and molecular analyses to demonstrate that the APC E3 activity is necessary for mitotic Fkh1 protein degradation and subsequent cell cycle progression. We report that Fkh1 protein degradation occurs specifically during mitosis, requires APCCdc20 and proteasome activity, and that a stable Fkh1 mutant reduces normal chronological lifespan, increases genomic instability, and increases sensitivity to stress. Our data supports a model whereby cell cycle progression through mitosis and G1 requires the targeted degradation of Fkh1 by the APC. This is significant to many fields as these results impact our understanding of the mechanisms underpinning the control of aging and cancer.

Trans-translation ensures timely initiation of DNA replication and DnaA synthesis in Escherichia coli.

The trans-translation pathway, mediated by the transfer messenger RNA (tmRNA; encoded by the ssrA gene) and the SmpB protein (tmRNA-binding protein expressed in Salmonella enterica), which is conserved in bacteria, is required for various cellular processes. A previous study has shown that trans-translation is required to ensure timely (non-delayed) dnaA transcription and consequent initiation of DNA replication in Caulobacter crescentus. In this study, we observed that initiation of chromosome replication was delayed in Escherichia coli lacking the smpB and/or ssrA genes (DssrA, DsmpB, or DsmpBDssrA mutants). We observed that the growth rate of the mutant cells was much slower than that of its wild-type counterpart. However, the delayed initiation of replication and slower growth in the DssrA or DsmpB mutants were reversed by ectopic expression of tmRNA or SmpB. A synchronized DsmpBDssrA cell culture containing the dnaC2 mutant allele showed delayed protein (total and DnaA) accumulation per cell; DnaA accumulation was also delayed in the DsmpB. These results indicated that absence of trans-translation leads to a delay in initiation of DNA replication, synthesis of total protein (including DnaA), and a decrease in E. coli growth rate. In summary, we propose that the trans-translation pathway is required to ensure timely initiation of replication, protein synthesis, and subsequent cell cycle progression.

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

Control of hepatocyte proliferation and survival by Fgf receptors is essential for liver regeneration in mice

ObjectiveFibroblast growth factors (Fgfs) are key orchestrators of development, and a role of Fgfs in tissue repair is emerging. Here we studied the consequences of inducible loss of Fgf receptor...

P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy.

Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24-72 h) in response to 70% PH were impaired in P2Y2-/- mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2-/- remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2-/- mice were treated with ATP or ATPγS for 5-120 min and 12-24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.

Regulation and function of pyruvate kinase M2 in cancer

Altered metabolism is fundamental to the growth and survival of cancer cells. Pyruvate kinase M2 (PKM2), a key enzyme in cancer metabolism, has been demonstrated to play a central role not only in metabolic reprogramming but also in direct regulation of gene expression and subsequent cell cycle progression. This review outlines the current understanding of PKM2 protein kinase activity and regulatory mechanisms underlying PKM2 expression, enzymatic activity, and nuclear localization, thus highlighting PKM2 as a potential therapeutic target.

Regulation of a Novel Androgen Receptor Target Gene, the Cyclin B1 Gene, through Androgen-Dependent E2F Family Member Switching

The malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor (AR) in the surrounding stroma. However, the function and mechanisms of AR signaling in prostate cancer (PCa) stroma remain elusive. Here we report, by using proteomics pathway array analysis (PPAA), that androgen and its receptor inhibit the proliferation of prostate stromal cells through transcriptional suppression of cyclin B1, and we confirmed our findings at mRNA and protein levels using AR-negative or -positive primary prostate stromal cells. Furthermore, AR showed a negative correlation with cyclin B1 expression in stroma of human PCa samples in vivo. Mechanistically, we identify cyclin B1 as a bona fide AR target gene in prostate stromal cells. The negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to the cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR-negative stromal cells or AR-positive stromal cells when androgens are depleted. Upon stimulation with androgen in AR-positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to the recruitment of the silencing mediator for retinoid and thyroid hormone receptor (SMRT)/histone deacetylase 3 (HDAC3) corepressor complex and repression of cyclin B1 at the chromatin level. The switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as the cytoplasmic-to-nuclear translocation of E2F4. Thus, we identified a novel mechanism for E2F factors in the regulation of cell cycle gene expression and cell cycle progression under the control of AR signaling.

Abstract SY09-02: Molecular mechanisms in prostate cancer in African Americans

Abstract African American men have long been known to have the highest rates of prostate cancer (PCa) in the world. In addition, age adjusted mortality rates from PCa patients are two times worse among African Americans (AA) than Caucasians (CA). The reasons for the disparity in PCa risk, incidence, clinical progression, and disease-specific death are still controversial. In addition to socioeconomic and environmental factors, such as diet, access to care, and screenings, a biologically more aggressive cancer is proposed as one important explanation for the racial disparity observed in AA men. Androgens and the androgen receptor pathway are well established key regulators of aggressive PCa behavior. Using a novel proteomic pathway analysis comparing the distinct signal pathway and network proteins different between AA and CA PCa, we identified a number of markers including stem/progenitor cell and EMT related proteins that are overexpressed in PCa among AA. Our ongoing study indicates a novel link among Notch, WNT and androgen receptor (AR) pathways. In contrast to increase in epithelial AR, our data revealed a decrease in the expression of the stromal AR in AA PCa compared to CA PCa. Androgen and its receptor inhibit proliferation of prostate stromal cells through transcriptional suppression of cyclin B1. Mechanistically, the negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR negative stromal cells or AR positive stromal cells under androgen depleted condition. Upon stimulation with androgen in AR positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to recruitment of the SMRT/HDAC3 co-repressor complex and repression of cyclin B1. Furthermore, the switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as cytoplasm to nucleus translocation of E2F4. In summary, our study revealed an association between distinct epithelial and stromal AR expression and PCa racial disparity as well as the molecular mechanism involved in regulating AR expression in epithelial and stromal prostate cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY09-02. doi:1538-7445.AM2012-SY09-02

Distinct roles of the mTOR components Rictor and Raptor in MO7e megakaryocytic cells

During megakaryopoiesis, hematopoietic progenitor cells in the bone marrow proliferate and ultimately differentiate in mature megakaryocytes (MK). We and others have recently described a role for the mammalian target of Rapamycin (mTOR) in proliferation and differentiation of MK cells. Two non-redundant complexes of mTOR have been described; mTORC1 containing rapamycin-associated TOR protein (Raptor) and mTORC2 containing Rapamycin-insensitive companion of mTOR (Rictor). The individual roles of these complexes in MK development have so far not been elucidated, and were investigated in this study. We have used an siRNA approach to selectively knock down either Rictor or Raptor expression in MO7e megakaryoblastic cells. Using flow cytometry, nuclear ploidity, and cell cycling as assessed by BrdU incorporation were investigated. Electron microscopy and cotransductions with GFP-LC3 were used to quantify autophagy. Activation of intracellular signal transduction pathways was studied by Western blot analysis. We observed a reduced cell cycling upon Rictor siRNA transduction, resulting in decreased numbers of polypoid cells. Knocking down Raptor expression resulted in a reduced expansion and a reduced cell size. In addition, increased autophagy was observed in Raptor siRNA-transduced cells, in correspondence with an attenuation of activation of the p70S6K/S6, and 4E-BP pathways. The current study shows that the mTORC1 and mTORC2 complexes have distinct, non-redundant functions in MO7e MK cell proliferation, and development. The mTOR/Rictor complex affects megakaryopoiesis by regulating nuclear division and subsequent cell cycle progression, whereas Raptor signaling protects MK cells from autophagic cell death, enabling normal megakaryopoiesis to take place.

Ca2+ signals coordinate zygotic polarization and cell cycle progression in the brown alga Fucus serratus.

Zygotes of the fucoid brown algae provide excellent models for addressing fundamental questions about zygotic symmetry breaking. Although the acquisition of polarity is tightly coordinated with the timing and orientation of the first asymmetric division--with zygotes having to pass through a G1/S-phase checkpoint before the polarization axis can be fixed--the mechanisms behind the interdependence of polarization and cell cycle progression remain unclear. In this study, we combine in vivo Ca2+ imaging, single cell monitoring of S-phase progression and multivariate analysis of high-throughput intracellular Ca2+ buffer loading to demonstrate that Ca2+ signals coordinate polarization and cell cycle progression in the Fucus serratus zygote. Consistent with earlier studies on this organism, and in contrast to animal models, we observe no fast Ca2+ wave following fertilization. Rather, we show distinct slow localized Ca2+ elevations associated with both fertilization and S-phase progression, and we show that both S-phase and zygotic polarization are dependent on pre-S-phase Ca2+ increases. Surprisingly, this Ca2+ requirement cannot be explained by co-dependence on a single G1/S-phase checkpoint, as S phase and zygotic polarization are differentially sensitive to pre-S-phase Ca2+ elevations and can be uncoupled. Furthermore, subsequent cell cycle progression through M phase is independent of localized actin polymerization and zygotic polarization. This absence of a morphogenesis checkpoint, together with the observed Ca2+-dependences of S phase and polarization, show that the regulation of zygotic division in the brown algae differs from that in other eukaryotic model systems, such as yeast and Drosophila.

A cell-type-specific transcriptional network required for estrogen regulation of cyclin D1 and cell cycle progression in breast cancer

Estrogen stimulates the proliferation of the most common type of human breast cancer that expresses estrogen receptor alpha (ERalpha) through the activation of the cyclin D1 (CCND1) oncogene. However, our knowledge of ERalpha transcriptional mechanisms remains limited. Hence, it is still elusive why ERalpha ectopically expressed in ER-negative breast cancer cells (BCC) is functional on ectopic reporter constructs but lacks activity on many endogenous target genes, including CCND1. Here, we show that estradiol (E2) stimulation of CCND1 expression in BCC depends on a novel cell-type-specific enhancer downstream from the CCND1 coding region, which is the primary ERalpha recruitment site in estrogen-responsive cells. The pioneer factor FoxA1 is specifically required for the active chromatin state of this enhancer and as such is crucial for both CCND1 expression and subsequent cell cycle progression. Interestingly, even in BCC, CCND1 levels and proliferation are tightly controlled by E2 through the establishment of a negative feedforward loop involving the induction of NFIC, a putative tumor suppressor capable of directly repressing CCND1 transcription. Taken together, our results reveal an estrogen-regulated combinatorial network including cell-specific cis- and trans-regulators of CCND1 expression where ERalpha collaborates with other transcription factors associated with the ER-positive breast cancer phenotype, including FoxA1 and NFIC.

Calmodulin regulates the post-anaphase reposition of centrioles during cytokinesis

A transient postanaphase repositioning of the centriole is found to control the completion of cytokinesis. Using a green fluorescent protein-calmodulin fusion protein as a living cell probe, we have previously found that calmodulin is associated with the initiation and progression of cytokinesis. In this study, we further studied the effect of calmodulin on the repositioning of the centriole and subsequent cell cycle progression. When activity of calmodulin is inhibited, the regression of the centriole from the intercellular bridge to the cell center is blocked, and thus the completion of cell division is repressed and two daughter cells are linked by longer cell bridge in perturbed cells. W7 treatment during cytokinesis also results in unfinished cytokinesis and stopped G1 phase. These results suggest that calmodulin activity is required for centriole repositioning and can affect the completion of cytokinesis and cell cycle progression.

B Cell Receptor (BCR) Ligation Enhances Cell Survival in Progressive Chronic Lymphocytic Leukemia (CLL).

Recent gene profiling studies demonstrated that signalisation through the BCR plays a central role during CLL disease progression. However, incidence of BCR engagement on cell survival remains unclear as both increased and decreased apoptosis have been reported after BCR ligation by anti-mu antibodies. We investigated the effect of anti-mu exposure on fresh cells from 32 untreated CLL cases. Cell viability was assessed both by MTT assay and Annexin V/ IP labelling. Culture conditions were set after time and dose experiments. A 25% increase of viability, obtained by MTT assay after 72h of culture, was considered as significant. When a coated rabbit anti-human IgH mu specific antibody was used, it significantly prevented apoptosis in 20/32 cases (named responders) and never increased it. On the same CLL cases, soluble goat F(ab')2 anti-human IgH mu specific antibody increased apoptosis in all but one case. However, coating of this latter antibody to the culture plate cancelled the pro-apoptotic effect and restored cell survival promoting effect in 13/14 cases tested. Unspecific effects were ruled out using control antibodies. Cytometry experiments and confocal microscopy demonstrated that uncoated antibody was rapidly internalized and degraded. In responders cases, enhanced survival was supported by increased expression of early cell cycle protein cyclin D2 and cdk4. There was no subsequent cell cycle progression as shown by IP staining and absence of pRB phosphorylation most likely related to the lack of p27 down regulation. There was a strong correlation between the extent of apoptosis prevention after anti-mu exposure and disease progression. Indeed, the 12 over 32 cases who were found to have no IgVH somatic hypermutations, happened to be all responders. Conversely among the 20 mutated cases, 12/20 were non responders and had a clinically stable disease. As for the 8 responders with somatic mutations, four had detectable levels of ZAP 70 protein and 2 others had a clinically progressive disease. In conclusion, coated anti-mu antibody is likely to mimic in vivo conditions of stimulation, suggesting that BCR ligation could play a major role in the pathophysiology of the progressive disease, giving a survival advantage to these CLL cells

Egr-1 Null Mice Exhibit Impaired Hepatic Regeneration.

The liver regenerates itself in response to a wide variety of injuries. The rodent partial hepatectomy model has been a useful tool with which to investigate the signals that regulate this regenerative response. These signals include activation of an immediate-early gene expression program during early liver regeneration which directs growth factor-dependent hepatocellular proliferation and leads to restoration of normal hepatic mass. The early growth response 1 transcription factor (EGR-1), whose expression is known to be regulated in a variety of models of cellular growth and differentiation, has been shown to be induced as part of the immediate early gene expression response during liver regeneration. In the studies reported here the functional significance of EGR-1 expression during liver regeneration was examined by characterizing the hepatic regenerative response to partial hepatectomy in EGR-1 null mice. The results of these studies showed that liver regeneration in EGR-1 null mice is impaired. Although the early signaling events leading up to the first wave of hepatocellular DNA synthesis occurred normally following partial hepatectomy in EGR-1 null mice, subsequent signaling events and cell cycle progression after the first round of DNA synthesis were deranged. This derangement was characterized by increased activation of the p38 mitogen activated protein kinase and inhibition of hepatocellular metaphase-to-anaphase mitotic progression. Together these observations suggest that Egr-1 is an important regulator of hepatocellular mitotic progression through the spindle-assembly checkpoint. In support of this, microarray-based gene expression analysis showed that induction of expression of the cell division cycle 20 gene (CDC20), a key regulator of the mitotic anaphase promoting complex, is significantly reduced in EGR-1 null mice. Taken together these data define a novel functional role for EGR-1 in regulating hepatocellular mitotic progression during liver regeneration.

Delayed Hepatocellular Mitotic Progression and Impaired Liver Regeneration in Early Growth Response-1-deficient Mice

The early growth response-1 transcription factor (Egr-1) is induced as part of the immediate-early gene expression response during early liver regeneration. In the studies reported here the functional significance of EGR-1 expression during liver regeneration was examined by characterizing the hepatic regenerative response to partial hepatectomy in Egr-1 null mice. The results of these studies showed that liver regeneration in Egr-1 null mice is impaired. Although activation of interleukin-6-STAT3 signaling, regulation of expression of hepatic C/ebpalpha, C/ebpbeta, cyclin D, and cyclin E and progression through the first wave of hepatocellular DNA synthesis occurred appropriately following partial hepatectomy in Egr-1 null mice, subsequent signaling events and cell cycle progression after the first round of DNA synthesis were deranged. This derangement was characterized by increased activation of the p38 mitogen-activated protein kinase and inhibition of hepatocellular metaphase-to-anaphase mitotic progression. Together these observations suggest that EGR-1 is an important regulator of hepatocellular mitotic progression. In support of this, microarray-based gene expression analysis showed that induction of expression of the cell division cycle 20 gene (Cdc20), a key regulator of the mitotic anaphase-promoting complex, is significantly reduced in Egr-1 null mice. Taken together these data define a novel functional role for EGR-1 in regulating hepatocellular mitotic progression through the spindle assembly checkpoint during liver regeneration.

A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression.

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1-cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.