Cell Cycle Progression Markers Research Articles

Cell-Membrane-Coated and Cell-Penetrating Peptide-Conjugated Trimagnetic Nanoparticles for Targeted Magnetic Hyperthermia of Prostate Cancer Cells.

Prostate malignancy represents the second leading cause of cancer-specific death among the male population worldwide. Herein, enhanced intracellular magnetic fluid hyperthermia is applied in vitro to treat prostate cancer (PCa) cells with minimum invasiveness and toxicity and highly specific targeting. We designed and optimized novel shape-anisotropic magnetic core-shell-shell nanoparticles (i.e., trimagnetic nanoparticles - TMNPs) with significant magnetothermal conversion following an exchange coupling effect to an external alternating magnetic field (AMF). The functional properties of the best candidate in terms of heating efficiency (i.e., Fe3O4@Mn0.5Zn0.5Fe2O4@CoFe2O4) were exploited following surface decoration with PCa cell membranes (CM) and/or LN1 cell-penetrating peptide (CPP). We demonstrated that the combination of biomimetic dual CM-CPP targeting and AMF responsiveness significantly induces caspase 9-mediated apoptosis of PCa cells. Furthermore, a downregulation of the cell cycle progression markers and a decrease of the migration rate in surviving cells were observed in response to the TMNP-assisted magnetic hyperthermia, suggesting a reduction in cancer cell aggressiveness.

Do Aging and Parity Affect VEGF-A/VEGFR Content and Signaling in the Ovary?-A Mouse Model Study.

In this study, the effects of aging and parity on VEGF-A/VEGFR protein content and signaling in the mice ovaries were determined. The research group consisted of nulliparous (virgins, V) and multiparous (M) mice during late-reproductive (L, 9-12 months) and post-reproductive (P, 15-18 months) stages. Whilst ovarian VEGFR1 and VEGFR2 remained unchanged in all the experimental groups (LM, LV, PM, PV), protein content of VEGF-A and phosphorylated VEGFR2 significantly decreased only in PM ovaries. VEGF-A/VEGFR2-dependent activation of ERK1/2, p38, as well as protein content of cyclin D1, cyclin E1, and Cdc25A were then assessed. In ovaries of LV and LM, all of these downstream effectors were maintained at a comparable low/undetectable level. Conversely, the decrease recorded in PM ovaries did not occur in the PV group, in which the significant increase of kinases and cyclins, as well phosphorylation levels mirrored the trend of the pro-angiogenic markers. Altogether, the present results demonstrated that, in mice, ovarian VEGF-A/VEGFR2 protein content and downstream signaling can be modulated in an age- and parity-dependent manner. Moreover, the lowest levels of pro-angiogenic and cell cycle progression markers detected in PM mouse ovaries sustains the hypothesis that parity could exert a protective role by downregulating the protein content of key mediators of pathological angiogenesis.

Natural Steroidal Lactone Induces G1/S Phase Cell Cycle Arrest and Intrinsic Apoptotic Pathway by Up-Regulating Tumor Suppressive miRNA in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with minimal treatment options. In the present work, Withaferin A (WA), a natural steroidal lactone found in Withania somnifera (Solanaceae), was studied to deduce the miRNA expression modulation mediated anticancer mode of action in TNBC cells. Small RNA next generation sequencing (NGS) of WA (2 µM) and vehicle (0.1% DMSO)-treated MDA-MB-231 cells revealed a total of 413 differentially expressed miRNAs (DEMs) and demonstrated that WA potentially up-regulates the miR-181c-5p, miR-15a-5p, miR-500b-5p, miR-191-3p, and miR-34a-5p and down-regulates miR-1275, miR-326, miR-1908-5p, and miR-3940-3p among total DEMs. The NGS and qRT-PCR expression analysis revealed a significantly higher expression of miR-181c-5p among the top 10 DEMs. Predicted target genes of the DEMs showed enrichment in cancer-associated gene ontology terms and KEGG signaling pathways. Transient up-expression of mir-181c-5p showed a time-dependent decrease in MDA-MB-231 and MDA-MB-453 cell viability. Co-treatment of miR-181c-5p mimic and WA (at varying concentration) down-regulated cell cycle progression markers (CDK4 and Cyclin D1) at mRNA and protein levels. The treatment induced apoptosis in MDA-MB-231 cells by modulating the expression/activity of Bax, Bcl2, Caspase 3, Caspase 8, Caspase 3/7, and PARP at mRNA and protein levels. Confocal microscopy and Annexin PI assays revealed apoptotic induction in miRNA- and steroidal-lactone-treated MDA-MB-231 cells. Results indicate that the Withaferin A and miRNA mimic co-treatment strategy may be utilized as a newer therapeutic strategy to treat triple-negative breast cancer.

First Evidence of the Expression and Localization of Prothymosin α in Human Testis and Its Involvement in Testicular Cancers.

Prothymosin α (PTMA) is a phylogenetically conserved polypeptide in male gonads of Vertebrates. In Mammals, it is a ubiquitous protein, and, possessing a random-coil structure, it interacts with many other partners, in both cytoplasmic and nuclear compartments. PTMA has been widely studied during cell progression in different types of cancer because of its anti-apoptotic and proliferative properties. Here, we provided the first evidence of PTMA expression and localization in human testis and in two testicular cancers (TC): classic seminoma (CS) and Leydig cell tumor (LCT). Data showed that its protein level, together with that of proliferating cell nuclear antigen (PCNA), a cell cycle progression marker, increased in both CS and LCT samples, as compared to non-pathological (NP) tissue. Moreover, in the two-cancer tissue, a decreased apoptotic rate and an increased autophagic flux was also evidenced. Results confirmed the anti-apoptotic action of PTMA, also suggesting that it can act as a switcher from apoptosis to autophagy, to favor the survival of testicular cancer cells when they develop in adverse environments. Finally, the combined data, even if they need to be further validated, add new insight into the role of PTMA in human normal and pathological testicular tissue.

HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory Tcells.

SummaryHIV-1 replicates in CD4+ T cells, leading to AIDS. Determining how HIV-1 shapes its niche to create a permissive environment is central to informing efforts to limit pathogenesis, disturb reservoirs, and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate T cells in vitro to make them permissive to infection. This dramatically alters T cell biology and virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient, productive infection of resting memory T cells without prior activation. Strikingly, we find that HIV-1 infection primes resting T cells to gain characteristics of tissue-resident memory T cells (TRM), including upregulating key surface markers and the transcription factor Blimp-1 and inducing a transcriptional program overlapping the core TRM transcriptional signature. This reprogramming is driven by Vpr and requires Vpr packaging into virions and manipulation of STAT5. Thus, HIV-1 reprograms resting T cells, with implications for viral replication and persistence.

The c-Myc/TBX3 Axis Promotes Cellular Transformation of Sarcoma-Initiating Cells.

Sarcomas are highly aggressive cancers of mesenchymal origin whose clinical management is highly complex. This is partly due to a lack of understanding of the molecular mechanisms underpinning the transformation of mesenchymal stromal/stem cells (MSCs) which are presumed to be the sarcoma-initiating cells. c-Myc is amplified/overexpressed in a range of sarcomas where it has an established oncogenic role and there is evidence that it contributes to the malignant transformation of MSCs. T-box transcription factor 3 (TBX3) is upregulated by c-Myc in a host of sarcoma subtypes where it promotes proliferation, tumor formation, migration, and invasion. This study investigated whether TBX3 is a c-Myc target in human MSCs (hMSCs) and whether overexpressing TBX3 in hMSCs can phenocopy c-Myc overexpression to promote malignant transformation. Using siRNA, qRT-PCR, luciferase reporter and chromatin-immunoprecipitation assays, we show that c-Myc binds and directly activates TBX3 transcription in hMSCs at a conserved E-box motif. When hMSCs were engineered to stably overexpress TBX3 using lentiviral gene transfer and the resulting cells characterised in 2D and 3D, the overexpression of TBX3 was shown to promote self-renewal, bypass senescence, and enhance proliferation which corresponded with increased levels of cell cycle progression markers (cyclin A, cyclin B1, CDK2) and downregulation of the p14ARF/MDM2/p53 tumor suppressor pathway. Furthermore, TBX3 promoted the migratory and invasive ability of hMSCs which associated with increased levels of markers of migration (Vimentin, SLUG, SNAIL, TWIST1) and invasion (MMP2, MMP9). Transcriptomic analysis revealed that genes upregulated upon TBX3 overexpression overlapped with c-myc targets, were involved in cell cycle progression, and were associated with sarcomagenesis. Together, the data described indicate that the c-Myc/TBX3 oncogenic molecular pathway may be a key mechanism that transforms hMSCs into sarcomas.

Long Non-Coding RNA CCAT2 Activates RAB14 and Acts as an Oncogene in Colorectal Cancer.

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound−healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3β signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3β signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

Abstract 1403: Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss

Abstract Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC is an aggressive subtype of breast cancer that lacks the expression of estrogen and progesterone receptors and HER2 protein. This lack of functioning receptors results in limited treatment options, usually resulting in the use of taxanes, platinums, and anthracyclines. Although TNBC often starts as being sensitive to traditional chemotherapies, tumor relapse and/or resistance often develops. Paclitaxel (PTX) is a commonly used taxane for TNBC, well known to induce mitotic arrest through stabilization of microtubules (MT) leading to an interference with the treadmilling function of MT through the activation of the spindle assembly checkpoint. We have previously shown that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor significantly increases resistance to PTX in APC knockdown (APCKD) cells generated in two human TNBC cell lines, MDA-MB-157 and MDA-MB-231. Given this striking PTX resistance, we were interested in seeing if resistance in APCKD can be seen using other non-taxane MT targeting agents. To accomplish this, we have used an epothilone (Epothilone B) and a vinca alkaloid (Vinblastine), that function by stabilizing and destabilizing the MTs, respectively. Our previous investigations demonstrated that APC loss results in increased CDK1 and CDK6, so these have been used as markers of cell cycle progression after drug treatment. In addition, changes in apoptosis were measured through the use of Annexin V and PI staining, and cleaved caspase 3 expression. Examined together, a better understanding of how APC loss influences chemoresistance in TNBC can lead to more efficient treatments for TNBC patients resistant to taxanes. Given the critical interaction between APC and MTs, this may be an avenue for targeted therapies to overcome taxane resistance. Citation Format: Pamela D. Arenas, Sara M. Maloney, Jenifer R. Prosperi. Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1403.

HSPA4 Knockdown Retarded Progression and Development of Colorectal Cancer.

PurposeColorectal cancer (CRC) is a common malignancy associated with high morbidity and mortality. Heat shock 70 kDa protein 4 (HSPA4) has been shown to exert regulatory roles during tumor progression in different cancer types. Here, we investigated the expression and cellular functions of HSPA4 in CRC.Materials and MethodsExpression of HSPA4 in CRC tissues and paracancerous tissues was analyzed by RT-qPCR and immunohistochemistry IHC staining. The functional roles of HSPA4 were explored using shRNA-mediated knockdown in HCT116 and RKO CRC cell lines, both in vitro and in tumor xenograft studies.ResultsHSPA4 expression was significantly increased at the RNA and protein levels in CRC tissues compared with noncancerous tissues. Moreover, HSPA4 expression was positively associated with tumor stage and its high expression of HSPA4 indicated poor patient prognosis. In vitro studies established that HSPA4 knockdown inhibited proliferation and migration, causing arrest in the G2-phase of the cell cycle along with increased levels of apoptosis. This phenotype was recapitulated in vivo where HSPA4 knockdown suppressed xenograft growth. Mechanistic investigations showed silencing of HSPA4 reduced activation of the PI3K, Akt signaling axis while also downregulating the cell cycle progression markers, CCND1 and CDK6. Similarly, there was altered expression of apoptosis-related proteins consistent with the increase in apoptosis.ConclusionOur findings demonstrate clinical significance for HSPA4 in CRC, further showing that HSPA4 contributes to CRC tumorigenesis through effects on proliferation, migration and survival. Thus, HSPA4 represents a novel prognostic indicator as well as a promising therapeutic target in CRC.

Cell Cycle Regulatory Protein Expression in Multinucleated Giant Cells of Giant Cell Tumor of Bone: do They Proliferate?

Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive (p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15INK4b, p16INK4a, p27KIP1, p53 through its effector p21WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB.

Effects of RAC1 on Proliferation of Hen Ovarian Prehierarchical Follicle Granulosa Cells.

Simple SummaryThe growth and development of ovary follicles is an intricate, highly organized process involving many local intra-ovarian factors. Ras-related C3 botulinum toxin substrate1 (RAC1) is speculated to be associated with prehierarchical follicle development of hen ovaries. The current study initially revealed RAC1 mRNA to be expressed in varied-size follicles and stroma and its expression levels in the prehierarchical follicles of 1.0–3.9 mm, 6.0–6.9 mm and 7.0–8.0 mm in diameter were remarkably higher than the other groups. Moreover, RAC1 protein was mainly expressed in the oocytes and granulosa cells (GC), as well as in stromal tissues of the follicles. To understand the exact roles of the RAC1 gene in regulation of follicular GC proliferation and differentiation, siRNA interference and overexpression of the RAC1 gene were conducted. Our experiments demonstrated that the RAC1 gene can significantly promote the expression of mRNA and proteins of FSHR, CCND2, CYP11A1, PCNA and StAR genes in GC and directly elevate the proliferation of GC in vitro. These results indicated RAC1 played a crucial role in regulation of GC proliferation and differentiation and steroidogenesis during the development of prehierarchical follicles. This study provided a base for elucidating the molecular mechanisms underlying the biological effect of RAC1 on the hen ovary follicle growth and development.RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.

Abstract 1910: Resistance to anti-HER2 therapy in HER2+ breast cancer is mediated by genomic alterations that switch pathway dependence from PI3K/AKT to RAS/MAPK

Abstract Although HER2 overexpression activates both PI3K-AKT and RAS-MAPK signaling, HER2+ breast tumors are primarily driven by PI3K-AKT. Thus, tumor resistance to HER2-targeted agents has been thought to emerge via downstream reactivation of PI3K signaling. We performed genomic profiling on 272 HER2-amplified breast tumors to identify genetic alterations associated with de novo or acquired resistance to anti-HER2 therapies. Surprisingly, we identified enrichment of mutations in RAS-MAPK pathway members, including NF1, ERBB2, and KRAS, in patients who had received prior HER2-targeted therapies, raising the possibility that activation of this pathway contributes to therapy resistance. To determine the impact of RAS-MAPK activation on response to anti-HER2 therapy in HER2+ breast cancer, we generated isogenic HER2+ breast cancer cell line and patient derived xenograft models harboring MAPK pathway alterations enriched in patients. Depletion of NF1 expression in HER2+ breast cancer cell lines using shRNA or CRISPR/Cas9 indeed promoted resistance to the HER2 inhibitors (HER2i) lapatinib and neratinib in vitro, as measured by increased proliferation and markers of cell cycle progression compared to parental lines. Unexpectedly, resistant models exhibited diminished dependence on AKT (MK2206 IC50 increase from 200 nM to 4500 nM) and were sensitized to MEK/ERK inhibition (trametinib IC50 decrease from 740 nM to 13 nM and SCH772984 from 2900 nM to 250 nM). To establish the basis for this change in pathway dependence, we examined the cell cycle profile of the cell line models upon drug treatment. Both cell cycle analysis by DNA content and immunoblot analysis revealed that NF1 null cells arrested in G1 upon MEK inhibition (but not upon AKT inhibition). Given the known regulation of CDK4/6 and CDK2 kinases by the RAS-MAPK pathway, we used both shRNA knockdown and small molecular inhibitors to define the G1 CDK responsible and found CDK2 to be required for resistance to HER2i and MEK sensitivity. Further, in vitro kinase assays using a recombinant RB substrate showed CDK2 activity to be MEK dependent in resistant cells and not in parental. We observed similar phenomena in isogenic HER2+ models in which we introduced mutant BRAF, KRAS, and ERBB2, and found each of these mutations also promoted HER2i resistance and MAPK/CDK2 dependence. Finally, using lentiviral CRISPR/Cas9, we knocked out NF1 in a HER2+ patient derived xenograft model and again found that this alteration promoted profound MEK dependence in vivo. Overall, these data suggest that MAPK-activating mutations constitute an important potential mode of acquired resistance to HER2-targeted therapies and demonstrate the potential utility of MEK/ERK targeted therapies for a subset of anti-HER2 therapy resistant HER2+ breast cancers. Citation Format: Alison E. Smith, Sarat Chandarlapaty. Resistance to anti-HER2 therapy in HER2+ breast cancer is mediated by genomic alterations that switch pathway dependence from PI3K/AKT to RAS/MAPK [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 1910.

Abstract 372: Misoprostol Attenuates Hypoxia-induced Cardiomyocyte Proliferation Through Bnip3 and Perinuclear Calcium Signaling

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3 Δ Exon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNC’s, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, promoting nuclear calcium accumulation leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited a hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, without fibrosis which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells.

While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53+/+ cells. Using dCas9, we determined this arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells compared to TP53−/− cells and DD-Cas9, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing recovery in primary and TP53+/+ cell lines.

Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

2066-P: Dyrk1a Antisense Oligonucleotides Conjugated to GLP-1R Agonist Promote Pancreatic Beta-Cell Proliferation

Loss of functional beta cell mass is a major contributor to the pathogenesis of both type 1 and type 2 diabetes. Enhancing proliferation of existing beta cells in the pancreatic islet is one of the promising approaches for restoring functional beta cell mass. Increasing evidence indicates inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a) stimulates beta cell proliferation. However, to date DYRK1a small molecule inhibitors have not been therapeutically viable due to their ability to increase proliferation in peripheral tissues. Previously we demonstrated that generation 2.5 antisense oligonucleotides (ASO) conjugated to a glucagon like peptide 1 receptor (GLP-1R) agonist can specifically target the pancreatic beta cell. When compared to control ASO treated isolated islets, islets administered a GLP-1/Dyrk1a ASO conjugate reduces DYRK1a mRNA and protein expression and significantly increases Ki67 mRNA, a cell proliferation marker, in a dose-response manner in vitro. Furthermore, combination treatment of a GLP-1/Dyrk1a ASO conjugate and the small molecule TGF-β inhibitor Ly364947, can synergistically increase Ki67 mRNA expression, cell cycle progression markers cyclin D1, cyclin E2, Cdk1, Cdk4 mRNAs and beta cell markers Pdx1 and Glut2 mRNAs when compared to a DYRK1a conjugated ASO or Ly364947 alone. Administration of a Dyrk1a conjugated ASO to wild type and db/db mice reduced Dyrk1a mRNA and increased Ki67 mRNA expression in islets when compared to a control ASO. Cell cycle markers cyclin D2, cyclin E2 and Cdk1 mRNAs are also significantly upregulated. In conclusion, a GLP-1/Dyrk1a ASO conjugate can specifically inhibit beta cell Dyrk1a mRNA expression and promote beta cell proliferation, suggesting that this may be an important approach for the treatment of type 1 and type 2 diabetes. Disclosure W. Fu: Employee; Self; Ionis Pharmaceuticals, Inc. R. Lee: Employee; Self; Ionis Pharmaceuticals, Inc. E.C. Pirie: Employee; Self; Ionis Pharmaceuticals, Inc. T. Prakash: None. S. Murray: Employee; Self; Ionis Pharmaceuticals. A. Powers: Employee; Self; Ionis Pharmaceuticals, Inc. Stock/Shareholder; Self; Ionis Pharmaceuticals, Inc. A.E. Mullick: None. R.M. Crooke: Employee; Self; Ionis Pharmaceuticals, Inc. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.

Oleanolic Acid Ameliorates Benign Prostatic Hyperplasia by Regulating PCNA-Dependent Cell Cycle Progression In Vivo and In Vitro.

Oleanolic acid (OA) is a natural, biologically active pentacyclic triterpenoid found in Cornus officinalis. Although C. officinalis and OA have antiproliferative actions, the effects and mechanisms of OA in benign prostatic hyperplasia (BPH) are unclear. We examined the effect of OA in an animal model of testosterone-induced BPH. Male rats were injected with testosterone propionate with or without OA. The inhibitory effect of OA on BPH-1 cells was determined in vitro. Rats with BPH exhibited outstanding BPH symptoms, including prostatic enlargement, upregulated dihydrotestosterone and 5α-reductase 2 levels, and histological changes. Compared with the BPH group, the OA group showed fewer pathological alterations and regular androgen events. OA inhibited prostate cell proliferation by downregulating the expression of proliferating cell nuclear antigen (PCNA) and cell cycle markers in BPH-induced animals. This indicated that OA has superior therapeutic effect in the BPH animal model than finasteride. In vitro studies demonstrated upregulation of PCNA and cell cycle proteins, whereas OA clearly reduced this upregulation. Thus, OA may inhibit the development of BPH by targeting cell cycle progression markers. These suggest that OA is a potential agent for BPH treatment.

Pharmacological Stimulation of Nurr1 Promotes Cell Cycle Progression in Adult Hippocampal Neural Stem Cells.

Nuclear receptor related-1 (Nurr1) protein performs a crucial role in hippocampal neural stem cell (hNSC) development as well as cognitive functions. We previously demonstrated that the pharmacological stimulation of Nurr1 by amodiaquine (AQ) promotes spatial memory by enhancing adult hippocampal neurogenesis. However, the role of Nurr1 in the cell cycle regulation of the adult hippocampus has not been investigated. This study aimed to examine changes in the cell cycle-related molecules involved in adult hippocampal neurogenesis induced by Nurr1 pharmacological stimulation. Fluorescence-activated cell sorting (FACS) analysis showed that AQ improved the progression of cell cycle from G0/G1 to S phase in a dose-dependent manner, and MEK1 or PI3K inhibitors attenuated this progression. In addition, AQ treatment increased the expression of cell proliferation markers MCM5 and PCNA, and transcription factor E2F1. Furthermore, pharmacological stimulation of Nurr1 by AQ increased the expression levels of positive cell cycle regulators such as cyclin A and cyclin-dependent kinases (CDK) 2. In contrast, levels of CDK inhibitors p27KIP1 and p57KIP2 were reduced upon treatment with AQ. Similar to the in vitro results, RT-qPCR analysis of AQ-administered mice brains revealed an increase in the levels of markers of cell cycle progression, PCNA, MCM5, and Cdc25a. Finally, AQ administration resulted in decreased p27KIP1 and increased CDK2 levels in the dentate gyrus of the mouse hippocampus, as quantified immunohistochemically. Our results demonstrate that the pharmacological stimulation of Nurr1 in adult hNSCs by AQ promotes the cell cycle by modulating cell cycle-related molecules.

Isolation of Cardiomyocytes Undergoing Mitosis With Complete Cytokinesis.

Adult human cardiomyocytes do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result, polyploidization and multinucleation occur. To get a deeper understanding of the mechanisms surrounding division of cardiomyocytes, there is a crucial need for a technique to isolate cardiomyocytes that complete cell division/cytokinesis. Markers of cell cycle progression based on DNA content cannot distinguish between mitotic cardiomyocytes that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MBs) targeting specific mRNAs, we aimed to identify truly proliferative cardiomyocytes derived from human induced pluripotent stem cells. Fluorescence-activated cell sorting combined with MBs was performed to sort cardiomyocyte populations enriched for mitotic cells. Expressions of cell cycle specific genes were confirmed by means of reverse transcription-quantitative polymerase chain reaction and single-cell RNA sequencing (scRNA-seq) combined with gene signatures of cell cycle progression. We characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive cardiomyocyte subpopulation exhibited patterns consistent with the processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs enabled the enrichment of cytokinetic events (CDC20highSPG20high). Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20lowSPG20high while polyploid cardiomyocytes that replicated DNA but failed to complete karyokinesis were found to be CDC20lowSPG20low. This study demonstrates a novel alternative to existing DNA content-based approaches for sorting cardiomyocytes with true mitotic potential that can be used to study the unique dynamics of cardiomyocyte nuclei during mitosis. Our technique for sorting live cardiomyocytes undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.

A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules.

Legume plants benefit from a nitrogen-fixing symbiosis in association with rhizobia hosted in specialized root nodules. Formation of root nodules is initiated by de novo organogenesis and coordinated infection of these developing lateral root organs by rhizobia. Both bacterial infection and nodule organogenesis involve cell cycle activation and regulation by auxin and cytokinin is tightly integrated in the process. To characterize the hormone dynamics and cell division patterns with cellular resolution during nodulation, sensitive and specific sensors suited for imaging of multicellular tissues are required. Here we report a modular toolkit, optimized in the model legume Lotus japonicus, for use in legume roots and root nodules. This toolkit includes synthetic transcriptional reporters for auxin and cytokinin, auxin accumulation sensors and cell cycle progression markers optimized for fluorescent and bright field microscopy. The developed vectors allow for efficient one-step assembly of multiple units using the GoldenGate cloning system. Applied together with a fluorescence-compatible clearing approach, these reporters improve imaging depth and facilitate fluorescence examination in legume roots. We additionally evaluate the utility of the dynamic gravitropic root response in altering the timing and location of auxin accumulation and nodule emergence. We show that alteration of auxin distribution in roots allows for preferential nodule emergence at the outer side of the bend corresponding to a region of high auxin signaling capacity. The presented tools and procedures open new possibilities for comparative mutant studies and for developing a more comprehensive understanding of legume-rhizobia interactions.