Subsequent Cell Cycle Research Articles

Mode of action of ethyl tertiary-butyl ether hepatotumorigenicity in the rat: Evidence for a role of oxidative stress via activation of CAR, PXR and PPAR signaling pathways

To elucidate possible mode of action (MOA) and human relevance of hepatotumorigenicity in rats for ethyl tertiary-butyl ether (ETBE), male F344 rats were administered ETBE at doses of 0, 150 and 1000mg/kg body weight twice a day by gavage for 1 and 2weeks. For comparison, non-genotoxic carcinogen phenobarbital (PB) was applied at a dose of 500ppm in diet. Significant increase of P450 total content and hydroxyl radical levels by low, high doses of ETBE and PB treatments at weeks 1 and 2, and 8-OHdG formation at week 2, accompanied accumulation of CYP2B1/2B2, CYP3A1/3A2 and CYP2C6, and downregulation of DNA oxoguanine glycosylase 1, induction of apoptosis and cell cycle arrest in hepatocytes, respectively. Up-regulation of CYP2E1 and CYP1A1 at weeks 1 and 2, and peroxisome proliferation at week 2 were found in high dose ETBE group. Results of proteome analysis predicted activation of upstream regulators of gene expression altered by ETBE including constitutive androstane receptor (CAR), pregnane-X-receptor (PXR) and peroxisome proliferator-activated receptors (PPARs). These results indicate that the MOA of ETBE hepatotumorigenicity in rats may be related to induction of oxidative stress, 8-OHdG formation, subsequent cell cycle arrest, and apoptosis, suggesting regenerative cell proliferation after week 2, predominantly via activation of CAR and PXR nuclear receptors by a mechanism similar to that of PB, and differentially by activation of PPARs. The MOA for ETBE hepatotumorigenicity in rats is unlikely to be relevant to humans.

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.

Pharmacodynamic separation (PDS) of gemcitabine (Gem) and the epidermal growth factor receptor (EGFR) inhibitor erlotinib (E) in patients (Pts) with advanced pancreatic cancer: Phase II results.

e15073 Background: Continuously dosed E given concurrently with Gem results in only a modest survival improvement in pts with advanced pancreatic cancer. Preclinical data suggest that there is antagonism between chemotherapy and EGFR tyrosine kinase inhibitors (TKIs) when these are delivered concurrently due to cell cycle effects. Work by our group and others has shown that EGFR TKIs induce cytostasis due to G1 arrest, which reduces subsequent cell cycle phase-dependent activity of chemotherapy. We tested a PDS approach for E + Gem as a means of overcoming the hypothesized negative interaction of EGFR TKIs and chemotherapy. Methods: Pts with measurable, previously untreated locally advanced unresectable/metastatic pancreatic cancer with adequate organ function and PS 0-2 were eligible. Tumor tissue was collected for molecular studies. PDS Regimen: Gem 1000 mg/m2 IV days 1, 8, 15, and E 150 mg/day on days 2-5, 9-12, 16-26 of each 28-day cycle. Primary endpoint was PFS; secondary endpoints included response rate (RR) and safety. To have 80% power at 5% significance, 70 pts were planned to detect an increase of PFS from 3.75 to 5.25 months. The study was terminated due to funding considerations. Results: 30 pts enrolled with median age 67 years (range 46-84); PS: 0 (23%), 1 (47%), and 2 (30%); locally advanced (13%) & metastatic (87%). Median PFS was 2.07 months (95% CI; 1.87 – 5.50 months). Median OS was 5.67 months (95% CI; 2.83 – 11.87 months). Median cycles completed were 2 (range 0-8). Two pts withdrew consent in the first cycle without progression and were not evaluable for response. RR was 11% (3 PRs); DCR was 46%. Grade 3/4 adverse events occurred in 63% of pts; the most common were neutropenia (23%), fatigue (13%), anemia (10%), thrombocytopenia (10%), rash (10%), nausea (10%), and diarrhea (10%). Conclusions: Although Gem and E in a PDS dose-schedule was feasible & tolerable in pts with advanced pancreatic cancer, no signal for increased efficacy was seen (compared to historic controls) in this molecularly unselected cohort. Correlative studies to identify potential molecular biomarkers of benefit are ongoing and will be presented. Clinical trial information: NCT00810719.

Abstract 3427: The dinuclear platinum agent, BBR3610-DACH, exhibits a significant anti-proliferative effect: Regulation of G1/S and G2/M cell cycle arrest and apoptosis induction.

Abstract BBR3610-DACH, a long-chain, bifunctional dinuclear PTII complex with a similar DNA binding mode to the Phase II clinical drug, BBR3464 and its potent analogue, BBR3610, was shown previously to be resistant to metabolic decomposition by sulfur-containing nucleophiles. Initial observations utilizing the alkaline comet assay indicate that BBR3610-DACH forms interstrand crosslinks. Cell cycle analysis using flow cytometry showed that BBR3610-DACH inhibited cell proliferation and caused G1/S and G2/M cell cycle arrest in HCT116 colorectal cells in a time dependent manner accompanied with significant s phase depletion. Similar experiments with synchronized cells showed a distinct G1 phase arrest elicited by the drug following release from arrest. Immunoblotting revealed a stabilization of p53 between 6-24hr after treatment with 20μM (IC90) BBR3610-DACH, with a concomitant increased levels of the cyclin dependent kinase inhibitors, p21 and p27. Cell proliferation assay using MTT and subsequent cell cycle studies with HCT116 p53-/- and HCT116 p21-/- indicated that the cytotoxicity profile of BBR3610-DACH was p21 dependent and partially dependent on the p53 status. However, no change was observed in the protein levels of cyclin E, CDK2, and CDC25A which are G1 phase regulators. Decrease in the levels of cyclin A and cyclin B1 at 24hrs suggested a G2/M block indicative of a possible mitotic catastrophe. Next, we examined whether the DNA damage sensor protein kinases, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3 related (ATR) had a role in the cell cycle regulation after drug treatment. Studies using the ATM inhibitor, KU-60019 and caffeine (inhibits both ATM and ATR) revealed no apparent role of ATM but a possible dependence on ATR. Following the cell cycle effects, BBR3610-DACH induced cell death as seen from cleaved poly (ADP-ribose) polymerase-1 (PARP-1) levels as early as 6hrs after drug treatment. Interestingly, no cleaved caspase 3 was seen suggesting a caspase-independent PARP cleavage. Together these findings suggest that BBR3610-DACH is a major cell cycle inhibitory DNA binding platinum agent that could be further developed as a major chemotherapeutic. Citation Format: Vijay Menon, Erica Peterson, Sumitra Deb, Lawrence Povirk, Nicholas Farrell. The dinuclear platinum agent, BBR3610-DACH, exhibits a significant anti-proliferative effect: Regulation of G1/S and G2/M cell cycle arrest and apoptosis induction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3427. doi:10.1158/1538-7445.AM2013-3427

The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1-3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle.

The nucleolus stress response is coupled to an ATR-Chk1–mediated G2 arrest

We report experiments on the connection between nucleolar stress and cell cycle progression, using HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator. Nucleolar stress elicited by brief exposure of cells to a low concentration of actinomycin D that selectively inhibits rRNA synthesis had no effect on traverse of G1 or S, but stalled cells in very late interphase. Additional experiments revealed that a switch occurs during a specific temporal window during nucleolar stress and that the subsequent cell cycle arrest is not triggered simply by the stress-induced decline in the synthesis of rRNA or by a ribosome starvation phenomenon. Further experiments revealed that this nucleolus stress-induced cell cycle arrest involves the action of a G2 checkpoint mediated by the ataxia telangiectasia and Rad3-related protein (ATR)-checkpoint kinase 1 (Chk1) pathway. Based on analysis of the cell cycle stages at which this nucleolar stress effect is put into action, to become manifest later, our results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.

Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264.7 macrophages

The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.

Hypermorphic expression of centromeric retroelement-encoded small RNAs impairs CENP-A loading

The proper functioning of centromeres requires a complex cascade of epigenetic events involving chromatin and kinetochore assembly; however, the precise mechanism by which this cascade proceeds is unknown. The pivotal event during kinetochore formation is the "loading," or deposition, of CENP-A. This histone H3 variant is specific to centromeres and replaces conventional H3 in centromeric chromatin. Failure to load CENP-A into mammalian centromeres in late telophase/early G1 of the cell cycle leads to malsegregation and cell division defects in subsequent cell cycles. Mounting evidence supports the hypothesis that an RNA component is involved, although how RNAs participate in centromere formation in mammals has remained unknown. Using the marsupial model, the tammar wallaby, we show that centromeric retroelements produce small RNAs and that hypermorphic expression of these centromeric small RNAs results in disruption of CENP-A localization. We propose that tight regulation of the processing of this new class of small RNAs, crasiRNAs, is an integral component of the epigenetic framework necessary for centromere establishment.

Functional replacement of fission yeast γ-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3

Microtubule-organizing centers such as the γ-tubulin ring complex (γ-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast γ-tubulin small complex (γ-TuSC) of the γ-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast γ-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the γ-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human γ-TuSC proteins, we found that GCP3 assembles normally into the >2000 kDa fission yeast γ-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of γ-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast γ-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4-GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during γ-TuRC assembly. Our findings have broad importance for understanding the essential domains of γ-TuSC proteins in the γ-TuRC mechanism.

UV-induced G2 checkpoint depends on p38 MAPK and minimal activation of ATR-Chk1 pathway

In response to UV light, single-stranded DNA intermediates coated with replication protein A (RPA) are generated, which trigger the ATR-Chk1 checkpoint pathway. Recruitment and/or activation of several checkpoint proteins at the damaged sites is important for the subsequent cell cycle arrest. Surprisingly, upon UV irradiation, Rad9 and RPA only minimally accumulate at DNA lesions in G2 phase, suggesting that only a few single-stranded DNA intermediates are generated. Also, little phosphorylated Chk1 is observed in G2 phase after UV-irradiation, and UV light fails to elicit efficient accumulation of typical DNA damage response proteins at sites of damage in this phase. By contrast, p38 MAPK is phosphorylated in G2 phase cells after UV damage. Interestingly, despite the lack of an obvious activation of the ATR-Chk1 pathway, only the combined inhibition of the ATR- and p38-dependent pathways results in a complete abrogation of the UV-induced G2/M arrest. This suggests that UV light induces less hazardous lesions in G2 phase or that lesions created in this phase are less efficiently processed, resulting in a low activation of the ATR-Chk1 pathway. UV-induced G2 checkpoint activation in this situation therefore relies on signalling via the p38 MAPK and ATR-Chk1 signalling cascades.

Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.

Targeted Radiosensitization by the Chk1 Inhibitor SAR-020106

To explore the activity of a potent Chk1 inhibitor (SAR-020106) in combination with radiation. Colony and mechanistic invitro assays and a xenograft invivo model. SAR-020106 suppressed-radiation-induced G2/M arrest and reduced clonogenic survival only in p53-deficient tumor cells. SAR-020106 promoted mitotic entry following irradiation in all cell lines, but p53-deficient cells were likely to undergo apoptosis or become aneuploid, while p53 wild-type cells underwent a postmitotic G1 arrest followed by subsequent normal cell cycle re-entry. Following combined treatment with SAR-020106 and radiation, homologous-recombination-mediated DNA damage repair was inhibited in all cell lines. A significant increase in the number of pan-γH2AX-staining apoptotic cells was observed only in p53-deficient cell lines. Efficacy was confirmed invivo in a clinically relevant human head-and-neck cell carcinoma xenograft model. The Chk1 inhibitor SAR-020106 is a potent radiosensitizer in tumor cell lines defective in p53 signaling.

O6-Methylguanine DNA lesions induce an intra-S-phase arrest from which cells exit into apoptosis governed by early and late multi-pathway signaling network activation

The O(6)-methylguanine (O(6)MeG) DNA lesion is well known for its mutagenic, carcinogenic, and cytotoxic properties, and understanding how a cell processes such damage is of critical importance for improving current cancer therapy. Here we use human cells differing only in their O(6)MeG DNA methyltransferase (MGMT) or mismatch repair (MMR) status to explore the O(6)MeG/MMR-dependent molecular and cellular responses to treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We find that O(6)MeG triggers MMR-dependent cell cycle perturbations in both the first and second cell cycle post treatment. At lower levels of damage, we show that a transient arrest in the second S-phase precedes survival and progression into subsequent cell cycles. However, at higher levels of damage, arrest in the second S-phase coincides with a cessation of DNA replication followed by initiation of apoptotic cell death. Further, we show that entry into apoptotic cell death is specifically from S-phase of the second cell cycle. Finally, we demonstrate the key role of an O(6)MeG/MMR-dependent multi-pathway, multi-time-scale signaling network activation, led by early ATM, H2AX, CHK1, and p53 phosphorylation and followed by greatly amplified late phosphorylation of the early pathway nodes along with activation of the CHK2 kinase and the stress-activated JNK kinase.

The nuclear membrane as a lipid ‘sink’—linking cell cycle progression to lipid synthesis

The nuclear membrane as a lipid ‘sink’—linking cell cycle progression to lipid synthesis

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.

The chloroethylating anticancer drug ACNU induces FRA1 that is involved in drug resistance of glioma cells

FRA1 belongs, together with c-Fos and FosB, to the family of Fos proteins that form with members of the ATF and Jun family the transcription factor AP-1 (activator protein 1). Previously we showed that c-Fos protects mouse embryonic fibroblasts against the cytotoxic effects of ultraviolet (UV) light by induction of the endonuclease XPF, leading to enhanced nucleotide excision repair (NER) activity. Here, we analyzed the regulation of FRA1 in glioma cells treated with the anticancer drug nimustine (ACNU) and its role in ACNU-induced toxicity. We show that FRA1 is upregulated in glioblastoma cells following ACNU on mRNA and protein levels. Knockdown of FRA1 by either siRNA or shRNA clearly sensitized glioma cells towards ACNU-induced cell death. Despite decreased AP-1 binding activity upon FRA1 knockdown, this effect is independent on regulation of the AP-1 target genes fasL, ercc1 and xpf. In addition, FRA1 knockdown does not affect DNA repair capacity. However, lack of FRA1 attenuated the ACNU-induced phosphorylation of CHK1 and led to a reduced arrest of cells in G2/M and, thereby, presumably leads to enhanced cell death in the subsequent cell cycle.

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

Abstract 2784: Nutlin-3 induces cell cycle arrest in p53 wild-type ovarian carcinomas via upregulation of p21

Abstract Despite the fact that ovarian epithelial ovarian cancer is a molecularly and histologically heterogeneous disease, standard treatment is the same for all subtypes. Most high-grade serous ovarian carcinomas (OCs) are responsive to initial platinum/taxane chemotherapy, but low-grade serous OCs and clear cell OCs are relatively chemoresistant with limited treatment options upon recurrence, and most patients succumb to the disease. It is therefore of interest to exploit the molecular differences between high-grade serous OCs and other histological types in order to develop novel therapeutics. Over 90% of high-grade serous OCs harbor p53 mutations, whereas low-grade serous OCs are p53 wild-type but more frequently contain KRAS and BRAF mutations. Similarly, ovarian clear cell carcinomas are p53 wild-type but more frequently contain PI3K mutations. Nutlin-3 functions via inhibition of MDM2, thereby activating wild-type p53 and inducing apoptosis. We therefore sought to validate nutlin-3 as a potential novel therapeutic compound for p53 wild-type ovarian carcinomas. Two low-grade serous OC cell lines (HOC-7, MPSC-1), two clear cell OC cell lines (TOV21G, OVAS), and one high-grade serous OC p53-null cell line (SKOV3) were treated with varying concentrations of nutlin-3 (0-70 µm) for 72 hours, and cellular proliferation assays were performed. The three serous OC cell lines were then treated with nutlin-3 (5 µm), and quantitative reaction real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were employed to quantify MDM2, p53, and p21 expression at 24, 48, and 72 hours after treatment. The MPSC-1 cell line was then sequenced for p53 mutations in exons 5, 6, 7, and 8. Nutlin-3 inhibited cellular proliferation in a dose-dependent manner in the clear cell and low-grade serous cell lines, but not the p53-null high-grade serous cell line, as expected. Both PI3K-mutant clear cell ovarian cancer cell lines exhibited decreased growth in response to nutlin-3, but to a lesser extent than the low-grade serous cell lines. HOC-7, which bears a KRAS mutation, was more sensitive to inhibition than BRAF-mutated MPSC-1. Sequencing for p53 mutation in MPSC-1 surprisingly revealed a heterozygous p53 40 bp deletion in exon 5, which accounted for the reduced sensitivity of MPSC-1 to nutlin-3. On qRT-PCR and WB, levels of p53 were slightly decreased after nutlin-3 treatment and were absent in SKOV3. MDM2 and p21 were initially upregulated, but returned to near-baseline levels after 72 hours. Nutlin-3 is a novel potential therapeutic agent for low-grade serous ovarian carcinoma, and is dependent on an intact p53 pathway. It appears to function via upregulation of p21 with subsequent cell cycle arrest. Further studies characterizing response to nutlin-3 according to tumor mutation status are underway. 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 2784. doi:1538-7445.AM2012-2784

Control of MCAK degradation and removal from centromeres

Mitotic centromere associated kinesin (MCAK) is a kinesin related protein with the ability to stimulate microtubule depolymerization. It is found at spindle poles, where it may be involved in poleward microtubule flux, and at kinetochores and centromeres where it plays a role in correcting chromosome alignment errors. Its microtubule depolymerase activity and recruitment to centromeres is regulated by phosphorylation, but little is known about how MCAK is maintained at appropriate levels. We previously reported that MCAK accumulates during the cell cycle and is then degraded during mitosis. Using proteomic analysis, we have now identified a new phosphorylation site on MCAK that is responsible for its degradation. Mutation of the site to prevent phosphorylation prolonged the stability of the protein beyond the metaphase to anaphase transition and into the subsequent cell cycle whereas a phosphomimetic mutation accelerated degradation. Unexpectedly, the mutation that prevented phosphorylation also inhibited the removal of MCAK from centromeres causing it to remain attached throughout the cell cycle. Even low expression of phosphorylation-resistant MCAK delayed mitosis and interfered with cell division. Mitotic defects were also observed by overexpressing a green fluorescent protein-tagged version of wild-type MCAK that similarly escaped degradation and accumulated to toxic levels, but did not remain associated with kinetochores during interphase. The results demonstrate that degradation is an important mechanism for controlling the activity of MCAK.

Transcriptional control of cell cycle-dependent kinase 4 by Smad proteins—implications for Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by deregulation of neuronal cell cycle and differentiation control eventually resulting in cell death. During brain development, neuronal differentiation is regulated by Smad proteins, which are elements of the canonical transforming growth factor β (TGF-β) signaling pathway, linking receptor activation to gene expression. In the normal adult brain, Smad proteins are constitutively phosphorylated and predominantly localized in neuronal nuclei. Under neurodegenerative conditions such as AD, the subcellular localization of their phosphorylated forms is heavily disturbed, raising the question of whether a nuclear Smad deficiency in neurons might contribute to a loss of neuronal differentiation control and subsequent cell cycle re-entry. Here, we show by luciferase reporter assays, electromobility shift, and RNA interference (RNAi) technique a direct binding of Smad proteins to the CDK4 promoter inducing transcriptional inhibition of cell cycle-dependent kinase 4 (Cdk4). Mimicking the neuronal deficiency of Smad proteins observed in AD in cell culture by RNAi results in elevation of Cdk4 and retardation of neurite outgrowth. The results identify Smad proteins as direct transcriptional regulators of Cdk4 and add further evidence to a Smad-dependent deregulation of Cdk4 in AD, giving rise to neuronal dedifferentiation and cell death.