Normal Cell Cycle Regulation Research Articles

Discovery of GJC1 as a prognostic biomarker in glioma cells: insights into its cell-cycle relationship and differential expression in non-neuronal cells.

Gliomas, originating from the most common non-neuronal cells in the brain (glial cells), are the most common brain tumors and are associated with high mortality and poor prognosis. Glioma cells exhibit a tendency to disrupt normal cell-cycle regulation, leading to abnormal proliferation and malignant growth. This study investigated the predictive potential of GJC1 in gliomas and explored its relationship with the cell cycle. Retrospective analysis of RNA-seq and single-cell sequencing data was conducted using the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases. The differential expression of GJC1 in gliomas with various pathological features and in different non-neuronal cell groups was analyzed. Functional data were examined using gene set variation analysis (GSVA). Furthermore, CellMiner was used to evaluate the relationship between GJC1 expression and predicted treatment response across these databases. GJC1 expression was enriched in high-grade gliomas and 1p/19q non-codeletion gliomas. GJC1 enrichment was observed in classical and mesenchymal subtypes within the TCGA glioma subtype group. In single-cell subgroup analysis, GJC1 expression was higher in glioma tissues compared to other non-neuronal cells. Additionally, the TCGA classical subtype of glioma cells exhibited more GJC1 expression than the other subgroups. GJC1 emerged as an independent prognostic factor for overall survival in glioma. GSVA unveiled potential mechanisms by which GJC1 may impact cell-cycle regulation in glioma. Finally, a significant correlation was observed between GJC1 expression and the sensitivity of multiple anti-cancer drugs. These findings confirmed GJC1 as a novel biomarker and provided insights into the differential gene expression in non-neuronal cells and the impact of the cell cycle on gliomas. Consequently, GJC1 may be used to predict glioma prognosis and has potential therapeutic value.

Abstract 4641: Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells

Abstract Among the myriad of molecular alterations associated with cancer, loss of the normal tumor suppressive functions of p53, the “guardian of the genome,” is the most consistent. Thus, the possibility of targeting oncogenic, mutant p53 proteins has long been a therapeutic goal. However, that aspiration has not been fully realized, leaving patients vulnerable to aggressive disease that is resistant to chemotherapy and to targeted agents such as PARP inhibitors and immunotherapy owing to the loss of wild type p53 functionality and the gain of new oncogenic functions when mutant p53 is expressed. An effective therapeutic strategy is urgently needed for p53 mutant cancers, and our team is developing novel analogues of curcumin with the potential to convert mutant p53 to a wild type conformation and restore its tumor suppressive functions. In this study, we investigated the effects of AKT-100, which was developed from the parent molecule and curcumin analogue HO-3867. Effects were compared to another drug believed to reactivate p53 functionality, APR-246, using CyQUANT IC50 cell proliferation assays and bulk and single cell RNA sequencing. Structural analyses are underway investigating the specific sites of interaction of AKT-100 with various mutant forms of p53. Our findings indicate that, first, AKT-100 exhibits impressive cell killing in multiple ovarian and serous endometrial cancer cells at concentrations ranging from 100-300 nM and is synergistic with the PARP inhibitor olaparib. Second, RNA sequencing demonstrates the reactivation of wild type p53 genes associated with normal cell cycle regulation (induction of CDKN1A encoding p21 and GADD45A), apoptosis (induction of PMAIP1 encoding Noxa and DR5 encoding Death Receptor 5), and the inhibition of DNA replication and multiple alternative mechanisms of DNA repair employed by malignant cells. Third, RNA sequencing indicates that AKT-100 acts through both p53-dependent and independent pathways to inhibit cancer cell proliferation. Gene expression is impacted in ways that portend a therapeutic effect as well as highlight potential resistance pathways (e.g. significant induction of PLK5 encoding polo like kinase 5) that can be further targeted to create synthetic lethal drug combinations with AKT-100 chosen specifically to target the various mutant forms of p53. Taken together, these results identify novel curcumin analogues in early development with the potential to improve therapy for p53 mutant cancers. Through this strategy it is also possible to identify and block resistance pathways that are activated by different forms of mutant p53 in response to AKT-100 and to create synthetic lethal precision drug combinations. Citation Format: Kimberly K. Leslie, Jamie L. Padilla, Geneva L. Williams, Hua-Ying Fan, Cristian G. Bologa, Eric R. Prossnitz, Jun-Yong Choe, Lane E. Smith, Alexander Goss, Robert J. Lake, Avinash D. Sahu. Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4641.

CDKN2A/p16 evaluation in cytology specimens.

CDKN2A/p16 evaluation in cytology specimens.

Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

ABSTRACT Cyclin-dependent kinase 4 (CDK4) is a master integrator that couples mitogenic/oncogenic signaling with the cell division cycle. It is deregulated in most cancers and inhibitors of CDK4 have become standard of care drugs for metastatic estrogen-receptor positive breast cancers and are being evaluated in a variety of other cancers. We previously characterized the T-loop phosphorylation at T172 of CDK4 as the highly regulated step that determines the activity of cyclin D-CDK4 complexes. Moreover we demonstrated that the highly variable detection of T172-phosphorylated CDK4 signals the presence or absence of the active CDK4 targeted by the CDK4/6 inhibitory drugs, which predicts the tumor cell sensitivity to these drugs including palbociclib. To date, the phosphorylation of CDK4 has been very poorly studied because only few biochemical techniques and reagents are available for it. In addition, the available ones including 2D-IEF separation of CDK4 modified forms are considered too tedious. The present report describes the generation, selection and characterization of the first monoclonal antibodies that specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide corresponding to the complete activation segment of CDK4. These monoclonal antibodies specifically recognize T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, as a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27. Abbreviations:2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch virus cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome entry site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence

Clinical and radiological features of orbito-temporal neurofibromatosis

Neurofibromatosis- 1 (NF- 1) is an autosomal dominant condition belonging to the well-known group of neurocutaneous syndromes. It affects 1 in 3500 persons. It is caused by a mutation in the neurofibromin gene localized to chromosome 17q11.2. which codes for a protein involved in the normal cell cycle regulation. Therefore NF-1 gene mutations predispose the individual to various benign and malignant tumors. The diagnosis of NF-1 remains largely clinical, given its characteristic lesions. Here we describe one such case with hallmark lesions of NF-1. A 12-year-old boy presented with a slow-growing and progressive right upper lid mass causing severe drooping, vision impairment and facial disfiguration. On detailed ophthalmic examination he was found to have severe ptosis, Lisch nodules and keratoconus. A systemic examination revealed café au lait spots and axillary freckling. Radiological imaging showed sphenoid wing dysplasia, encephalocele and optic pathway glioma. The patient was taken up for debulking surgery and a referral to the neurosurgery department was sent. He has been on follow up for last 12 months. This case first presented to an ophthalmology clinic and thus, as an ophthalmologist, keeping in mind the associated intraorbital and intracranial pathology can be life-saving. A multidisciplinary approach is needed in the management of such severe cases of NF-1.

NUSAP1 Promotes Cell Proliferation Via the DNA Damage Response Pathway in Diffuse Large B-Cell Lymphoma

Introduction: Nucleolar spindle-associated protein 1 (NUSAP1), a microtubule binding protein with molecular weight of 55KD, plays an important role to ensure the normal regulation of cell cycle in chromosome separation, spindle assembly and DNA repair. NUSAP1 has been shown to be highly expressed in a variety of tumors, involved in tumor occurrence, invasion, migration, and drug resistance. Moreover, it is associated with poor prognosis. Whereas, no research has been reported regarding the role of NUSAP1 in diffuse large B-cell lymphoma (DLBCL). Methods: Peripheral blood samples from de novo DLBCL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE83632 and GSE32918 were obtained from Gene Expression Omnibus. Kaplan-Meier survival curves with log-rank test of overall survival (OS) were analyzed. Immunohistochemistry staining (IHC) was performed to assess NUSAP1 expression in specimens. Expression levels of NUSAP1 mRNA and protein were detected by quantitative RT-PCR and western blotting. The DLBCL cells were transfected by lentiviral shRNA and vectors to stably silence and up-regulate NUSAP1. Effects of doxorubicin on cell viabilities were assessed by cell counting kit-8. Besides, apoptosis and cell cycle were respectively detected by annexin V-PE/7AAD and PI/RNase staining via flow cytometry. Invasion ability was analyzed by transwell assay. ShNUSAP1 cells and Scramble cells were subcutaneously injected to SCID-Beige mice to establish xenograft models. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of DLBCL patients was significantly increased than that of healthy donors (P<0.05) (Figure A, B). Besides, patients with high expression of NUSAP1 was related to shorter overall survival (HR=0.456, P<0.01), indicating poor prognosis (Figure C). Stronger positivity of NUSAP1 was markedly observed in DLBCL lymph nodes than reactive hyperplasia group, which was associated with Ann Arbor stage of DLBCL patients (Figure D). Compared with normal B cells, both the mRNA and protein level of NUSAP1 were up-regulated in DLBCL cell lines (Figure E, F). After transfected with lentivirus, the proliferation rate of NUSAP1 knockdown group was lower than that of the control group, while the overexpression group was faster than control (Figure G). Through flow cytometry, silencing of NUSAP1 led to increased apoptotic rates of LY1 and LY3 cells (Figure H). Along with the recovery of NUSAP1 expression level, apoptosis rates were improved again. With the addition of doxorubicin at 100nM, interference of NUSAP1 could increase the sensitivity of cells to doxorubicin (Figure I). NUSAP1 intervention induced obvious cell cycle arrest in G1 phase of LY1 and LY3 cell lines, with concomitant reduction of cell proportion in S phase (Figure J). Moreover, known as EMT biomarkers, the expression of ZEB1 and VIMENTIN were decreased with reduction of NUSAP1. Likewise, we also investigated NUSAP1 overexpression has promoted EMT-like process of LY1 and LY3 cells (Figure K). Surppression of NUSAP1 inhibited the action of DNA damage repair pathway (Figure L). Moreover, we also found that NUSAP1 knockdown induced striking reduction in invasion cells (Figure M). We found that tumor growth was significantly suppressed by inhibiting NUSAP1 in xenograft models (Figure N, O). IHC for NUSAP1 and Ki67 on tumor sections showed that tumors derived from shNUSAP1 cells exhibited significantly lower levels of Ki67 compared to control group (Figure P). Conclusions: This study first identified that the high expression of NUSAP1 in DLBCL patients is associated with poor prognosis through database analysis and in vitro experiments. Interference of NUSAP1 expression led to a slower DLBCL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest and promoted EMT-like process. Collectively, our study demonstrated that NUSAP1 plays a role in promoting tumor growth both in vivo and vitro through DNA damage response pathway, which providing a new direction for prognosis assessment and targeted therapy of DLBCL. Figure Disclosures No relevant conflicts of interest to declare.

Abstract 90: Inducing and targeting senescence in HPV-negative head and neck squamous cancer cells

Abstract Background: The loss of normal cell cycle regulation via disruption of CDKN2A in HPV-negative (-) head and neck squamous cell carcinoma (HNSCC) is universal. Targeting CDK4/6 with a specific inhibitor, such as palbociclib, is therefore a logical strategy in this disease. Previous work has demonstrated that CDK4/6 inhibition leads to cell cycle arrest and senescence in cancer cells, and specifically HPV(-) HNSCC. Recent studies have also shown that the BCL-xL inhibitor, ABT-263 (navitoclax), effectively targets senescent cells. This study aimed to evaluate the efficacy and mechanism of a combination approach utilizing palbociclib and ABT-263 in HNSCC. Methods: Three HPV (-) HNSCC cell lines (CAL27, HN31, PCI15B) were treated with palbociclib. Cresyl violet staining and annexin V apoptosis assays were used to assess cell viability in response to palbociclib, navitoclax, or the combination of the two drugs. Senescence following treatment with palbociclib was measured using β-galactosidase expression on western blot analysis, as well as via immunofluorescence staining assessed and quantified using confocal microscopy. Levels of BCL-xL were also measured using these methods. Results: Following treatment with palbociclib, all cell lines exhibited phenotypic evidence of senescence, and protein expression of β-galactosidase was significantly increased compared to untreated controls (mean fold change ± SD: CAL27 2.67±0.26; HN31 1.79±0.41; PCI15B 1.26±0.15). Protein expression of BCL-xL was concurrently increased under these conditions (mean fold change ± SD: CAL27 1.57±0.04; HN31 1.58±0.39; PCI15B 1.33±0.17). Immunofluorescent staining was also used to determine if cells exhibiting senescence specifically demonstrated higher BCL-xL expression. Co-expression of β-galactosidase and BCL-xL showed a strong positive correlation (CAL27 r = 0.94, p < 0.0001; HN31 r = 0.94, p < 0.0001; PCI15B r = 0.71, p < 0.0001). Treatment with the combination of palbociclib and navitoclax resulted in significantly decreased survival compared to each agent alone in all HNSCC cell lines tested (data summarized for the cell line panel: mean percent survival, navitoclax 76.40%; palbociclib 38.76%; combination 16.74%). Annexin V apoptosis assay also revealed that there was a significant increase in apoptosis observed in all cell lines tested comparing each agent to combination treatment (data summarized for the cell line panel: mean percent apoptotic cells, navitoclax 35.53%; palbociclib 29.95%; combination 60.98%). Apoptosis with combination of palbociclib and navitoclax approached or exceeded levels achieved with staurosporine treated controls. Conclusions: Palbociclib treatment leads to consistent induction of senescence in HPV (-) HNSCC. Adding a senolytic agent, such as the BCL-xL inhibitor ABT-263 (navitoclax) appears to be an effective strategy which holds promise in this disease. Citation Format: Nicholas Gadsden, Carlos Thomas, Daniel Li, Nitisha Shrivastava, Nicolas Schlecht, Michael Prystowsky, Jeffrey Segall, Thomas Ow. Inducing and targeting senescence in HPV-negative head and neck squamous cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 90.

Optogenetic dissection of mitotic spindle positioning in vivo.

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

The requirement of integrins for breast epithelial proliferation

Epithelial cells forming mammary gland ducts and alveoli require adhesion to the extracellular matrix for their function. Mammary epithelial cells need β1-integrins for normal cell cycle regulation. However, the role of β1-integrins in tumorigenesis has not been fully resolved. β1-integrin is necessary for tumour formation in transgenic mice expressing the Polyomavirus Middle T antigen, but it is dispensable in those overexpressing ErbB2. This suggests that some oncogenes can manage without β1-integrin to proliferate and form tumours, while others still require it. Here we have developed a model to test whether expression of an oncogene can surpass the need for β1-integrin to drive proliferation. We co-expressed the ErbB2 or Akt oncogenes with shRNA to target β1-integrin in mammary epithelial cells, and found that they show a differential dependence on β1-integrin for cell division. Moreover, we identified a key proliferative role of the Rac1-Pak axis downstream of β1-integrin signalling. Our data suggest that, in mammary epithelial cells, oncogenes with the ability to signal to Pak surpass the requirement of integrins for malignant transformation. This highlights the importance of using the correct combination therapy for breast cancer, depending on the oncogenes expressed in the tumour.

P16 Tumor Suppressor Gene Methylation in Diffuse Large B Cell Lymphoma: A Study of 88 Cases at Two Hospitals in the East Coast of Malaysia

Introduction:p16 gene plays an important role in the normal cell cycle regulation. Methylation of p16 has been reported to be one of the epigenetic events contributing to the pathogenesis of diffuse large B-cell lymphoma (DLBCL) which occurring at varying frequency. DLBCL is an aggressive and high-grade malignancy which accounts for approximately 30% of all non-Hodgkin lymphoma cases. However, little is known regarding the epigenetic alterations of p16 gene in DLBCL cases in Malaysia. Therefore, the objective of this study was to examine the status of p16 methylation in DLBCL.Methods:A total of 88 formalin-fixed paraffin-embedded DLBCL tissues retrieved from two hospitals located in the east coast of Malaysia, namely Hospital Tengku Ampuan Afzan (HTAA) Pahang and Hospital Universiti Sains Malaysia (HUSM) Kelantan, were chosen for this study. DNA specimens were isolated and subsequently subjected to bisulfite treatment prior to methylation specific-PCR. Two pairs of primers were used to amplify methylated and unmethylated regions of p16 gene. The PCR products were then separated using agarose gel electrophoresis and visualised under UV illumination. SPSS version 12.0 was utilised to perform all statistical analysis.Result:p16 methylation was detected in 65 of 88 (74%) samples. There was a significant association between p16 methylation status and patients aged >50 years old (p=0.04).Conclusion:Our study demonstrated that methylation of p16 tumor suppressor gene in our DLBCL cases is common and significantly increased among patients aged 50 years and above. Aging is known to be an important risk factor in the development of cancers and we speculate that this might be due to the increased transformation of malignant cells in aging cell population. However, this has yet to be confirmed with further research and correlate the findings with clinicopathological parameters.

Anticancer and Cytotoxic Potential of Turmeric (Curcuma longa), Neem (Azadirachta indica), Tulasi (Ocimum sanctum) and Ginger (Zingiber officinale) Extracts on HeLa cell line

Anticancer and Cytotoxic Potential of Turmeric (Curcuma longa), Neem (Azadirachta indica), Tulasi (Ocimum sanctum) and Ginger (Zingiber officinale) Extracts on HeLa cell line

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

Author SummaryFungal infections are a particularly challenging problem in medicine due to the small number of effective antifungal drugs available. Fluconazole, the most commonly prescribed antifungal, prevents cells from growing but does not kill them, giving the fungal population a window of opportunity to become drug resistant. Candida albicans is the most prevalent fungal pathogen, and many fluconazole-resistant strains of this microbe have been isolated in the clinic. Fluconazole-resistant isolates often contain an abnormal number of chromosomes (a state called aneuploidy), and the additional copies of drug resistance genes on those chromosomes enable the cells to circumvent the drug. How Candida cells acquire abnormal chromosome numbers is a very important medical question—is aneuploidy merely passively selected for, or is it actively induced by the drug treatment? In this study, we found that fluconazole and other related azole antifungals induce abnormal cell cycle progression in which mother and daughter cells fail to separate after chromosome segregation. Following a further growth cycle, these cells form an unusual cell type that we have termed “trimeras”—three-lobed cells with two nuclei. The aberrant chromosome segregation dynamics in trimeras produce progeny with double the normal number of chromosomes. Unequal chromosome segregation in these progeny leads to an increase in the prevalence of aneuploidy in the population. We postulate that the increase in aneuploidy greatly increases the odds of developing drug resistance.

A Novel Source of Cultured Podocytes

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex.

Curcumin binds tubulin, induces mitotic catastrophe, and impedes normal endothelial cell proliferation

Curcumin, a component of turmeric spice that imparts flavor and color to curry, is thought to possess anti-inflammatory and antioxidant properties in biological tissues. However, while such efficacies have been described in the context of carcinogenesis, the impact of curcumin on normal cell cycle regulation is poorly understood. Here, we provide evidence of curcumin toxicity in proliferating bovine aortic endothelial cells, at concentrations relevant to the diet and below those previously reported in cancer models. Upon confirming curcumin's ability to upregulate hemeoxygenase-1 in a dose-dependent fashion, we found the minimally efficacious curcumin concentration to also inhibit endothelial cell DNA synthesis. Moreover, curcumin concentrations below the minimum 2 μM threshold required to induce hemeoxygenase-1 bound tubulin protein in vitro and triggered hallmark evidence of mitotic catastrophe in vivo. Concentrations as low as 0.1 μM curcumin led to disproportionate DNA segregation, karyorrhexis, and micronucleation in proliferating endothelial cells. While suggesting a mechanism by which physiological curcumin concentrations inhibit cell cycle progression, these findings describe heretofore unappreciated curcumin toxicity with potential implications for endothelial growth, development, and tissue healing.

Multiparametric Cell-Based Assay for the Evaluation of Transcription Inhibition by High-Content Imaging

Loss of normal cell cycle regulation is a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and have been actively pursued as promising therapeutic targets. Likewise, members of the CDK family are functionally related to transcriptional modulation, a molecular pathway suitable for therapeutic intervention. We used a set of 2500 compounds in the U2OS cell line to evaluate its effect in the cell division process. Interestingly, out of this analysis, we identified a subpopulation of compounds that are able to inhibit RNA polymerase activity, thus interfering with gene transcription processes. After this finding, we developed, validated, and fully automated a multiparameter high-content imaging (HCI) assay to measure phosphorylation of the RNA polymerase II carboxyl terminal domain (pCTD). Simultaneously, we measured both the DNA content and cell proliferation index in the treated cells. The linear regression analysis comparing the IC50 for pCTD and the 4N EC50 for DNA content or IC50 for cell proliferation showed an excellent agreement (r2 = 0.84 and r2 = 0.94, respectively). Our results confirm that this method allows discriminating between cell cycle and transcription inhibition and confirms HCI as a powerful technology for the identification of compounds with an effective and selective pathway phenotype.

Checkpoint kinase 1 in DNA damage response and cell cycle regulation

Originally identified as a mediator of DNA damage response (DDR), checkpoint kinase 1 (Chk1) has a broader role in checkpoint activation in DDR and normal cell cycle regulation. Chk1 activation involves phosphorylation at conserved sites. However, recent work has identified a splice variant of Chk1, which may regulate Chk1 in both DDR and normal cell cycle via molecular interaction. Upon activation, Chk1 phosphorylates a variety of substrate proteins, resulting in the activation of DNA damage checkpoints, cell cycle arrest, DNA repair, and/or cell death. Chk1 and its related signaling may be an effective therapeutic target in diseases such as cancer.

Cyclin D1 gene amplification in multiple myeloma and its impact on the disease outcome and drug resistance

Background Cyclin D1 is involved in the normal regulation of the cell cycle and in neoplasia. Inhibition of cyclin D1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer. However, the prognostic value of overexpression of the cyclin D1 gene in multiple myeloma (MM) is still a point of debate. This study aims at evaluating the prognostic significance of cyclin D1 gene amplification in MM and whether it is correlated with MDR1 expression as an indicator of chemoresistance. Patients and methods Twenty-five patients with MM were studied retrospectively. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients by fluorescence in-situ hybridization. Immunohistochemical study of the bone marrow biopsies was carried out to detect multidrug resistance (MDR1) gene expression. The correlations between the cyclin D1 gene amplification on the one hand and overall survival and MDR1 expression on the other were studied and analyzed statistically. Results Cyclin D1 gene amplification was found in 20% of patients with myeloma and was associated with a higher percentage of plasma cell infiltration of the bone marrow and increase liability for multiple osteolytic lesions. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients. Cyclin D1 levels showed a statistically highly significant positive correlation with MDR1 levels (r: 0.802 and P Conclusion We suggest that there is an association between cyclin D1 gene amplification and higher disease severity, unfavorable prognosis, and increased expression of MDR1 in patients with MM.

Nuclear Factor of Activated T-Cells (NFAT1): Potential Novel Target in T-Cell Acute Lymphocytic Leukemia (T-ALL)

Abstract 4634 Background.T-cell transcription factor NFAT1 (NFATc2) has been reported to regulate cell cycle regulatory proteins including cyclins, cyclin-dependent kinases (CDKs), and p21. MicroRNA-184 (miR-184) has been noted to have close sequence homology to the 3' UTR of NFAT1 and to regulate NFAT1 mRNA translation in human adult and umbilical cord blood (UCB) CD3+CD4+ T-cells. In this study, we are characterizing the functional role of NFAT1 in T-ALL cells to determine whether its dysregulation may alter normal cell cycle regulation and thereby may contribute to T-ALL leukemogenesis. Materials/Methods.In this study we used MOLT-4 cell line to investigate the role of NFAT1 in T-ALL, since MOLT-4 represents early stage of human T-ALL (CD4+CD8+ double positive) compared with other T-cell lines including Jurkat. We developed two distinct approaches to explore NFAT1 signalling, either by manipulating the expression level of NFAT1 protein by blocking miR-184, or by overexpressing a constitutively active form of NFAT1 (caNFAT1) holding a HA-tag (Addgene plasmid 11792, A. Rao, PhD, Harvard University). caNFAT1 was introduced into gateway system expression vector pEF-DEST51. MOLT-4 cells were maintained in complete RPMI 1640 medium according to ATCC guidelines. Electroporation transfection was performed on Amaxa Nucleofector I using program C-05. 200 nM of anti-miR-184 miRNA inhibitor (Dharmacon) or 2 μ g of plasmid containing caNFAT1 was transfected into 2×10e6 MOLT-4 cells in each sample. Transfection efficiency was tested using pmaxGFP (Lonza). Proteins were extracted at various time points after transfection using RIPA buffer (PIERCE) with protease inhibitor cocktail and tested for the expression of NFAT1 (BD Biosciences), phosphorylated NFAT1 (Santa Cruz Biotechnology), HA (Roche), and beta-actin (Sigma-Aldrich). Cell cycle was measured at various time points by pelleting down 0.5–1 X10e6 cells, followed by ethanol fixation, and incubation in propidium iodide (PI) (10 μ g/ml) staining solution and flow cytometric measurement for PI incorporation. Cell apoptosis was measured by PE-conjugated annexin V staining (BD Biosciences) in transfected MOLT-4 cells including empty plasmid controls. Results/Discussion.The over-expression of caNFAT1 in MOLT-4 led to increased proportion of cells in G2 phase than in controls (10.8% vs. 7.3% on day5, and 11.8% vs. 8.1% on day6). In addition to these cell cycle changes in MOLT-4 treated with caNFAT1 plasmid, higher rates of apoptosis were noted on day 2 (28.3% vs. 15.5%), which notably leveled off from day 3 to day 6 potentially attributed to compensatory cellular responses. Previous work by this group has shown higher expression of miR-184 in MOLT-4 by 56 fold compared with normal human CD4/8 double positive T-cells. Here, miR-184 knockdown was also notable for a larger proportion of MOLT-4 cells in G2 cell cycle phase and higher apoptosis, albeit modest changes compared with that exerted by caNFAT1. Conclusion.In MOLT-4 cell line, this preliminary data suggests that increasing active non-phosphorylated NFAT1 is associated with enhanced proportion of cells in G2 phase as well as enhanced resultant apoptosis. Taken together, regulation of NFAT1 may be exploited as potential T-ALL targeted therapy. [Display omitted] Disclosures:No relevant conflicts of interest to declare.