Genes Cyclin Research Articles

Abstract 254: Apolipoprotein B Mediates Hepatic Proliferation And Cell Survival Signalling: A Crispr/cas-9 Gene Editing ApoB-knockout Study

Introduction: Apolipoprotein B (ApoB) is a key structure protein in very-low-density lipoprotein (VLDL). Ample research has been focusing on its crucial role in lipoprotein metabolism and the onset of cardiovascular disease (CVD). However, in vivo research on the broader pathophysiological function of ApoB has been hindered by the embryonic lethality of ApoB knockout (ApoB-KO) animal models. Thus, the objective of this study is to establish ApoB-KO cell models and demonstrate ApoB as a key determinant in governing hepatocyte proliferation and apoptosis. Approaches and Results: the Crispr/Cas-9 gene editing technology was applied to genetically disrupt ApoB expression in both human and mouse hepatocytes. Total RNAs of the parental and ApoB-KO cells were subjected to RNA sequencing profiling for whole transcriptome. Subsequent bioinformatics analysis revealed that ApoB-KO significantly altered expression of more than 2000 genes in hepatocytes. More importantly, expression of genes involved in DNA replication, cell cycle, cell proliferation and apoptotic signaling were the genes mostly affected, which were either up- or down-regulated by more than 5-folds. These included the histone associated genes H2ac10-12, cyclin Ccna2, Ccnb1 and Cdc20, cell proliferation gene Fosb and apoptosis gene P53. A BrDU cell proliferation assay detecting the actively proliferating cells further demonstrated that depletion of ApoB greatly inhabited cell proliferation, indicated by the markedly reduced cell numbers and the prolonged cell doubling time in ApoB-KO cells compared to their parental cells. Mechanistic study revealed that cAMP signaling, and the insulin associated PI3K kinase pathway that medicated gene transcription, cell growth and proliferation were profoundly impaired upon ApoB depletion. Reconstitution of ApoB in the KO cells by expressing a truncated ApoB protein, ApoB72, was able to promote cell growth rate and improve cell survival to a level comparable to the parental cells. Conclusion: This is the first study that characterizes a novel function of ApoB as a key determinant in hepatic cell proliferation and survival pathways. This novel finding greatly advances our knowledge of ApoB beyond the boundary of VLDL metabolism and CVD.

Neutrophil extracellular traps promote bronchopulmonary dysplasia-like injury in neonatal mice via the WNT/β-catenin pathway.

Bronchopulmonary dysplasia (BPD) is one of the most common and severe chronic diseases in preterm infants. Premature infants are susceptible to BPD due to immature lungs and adverse perinatal episodes of infection, hyperoxia, and mechanical ventilation. Neutrophils are the first line of host defence, and the release of neutrophil extracellular traps (NETs) is an important strategy to immobilize and kill invading microorganisms. This study examined whether NETs were associated with BPD in preterm infants and contributed to hyperoxia-induced lung injury in neonatal mice via the WNT/β-catenin pathway. In this study, we found that preterm infants with BPD had higher levels of NETs in their tracheal aspirates than those without BPD. Neonatal mice treated with NETs after birth exhibited BPD-like changes in their lungs. Furthermore, the levels of Aquaporin 5 (AQP5) and surfactant-associated protein C (SPC), which represent alveolar differentiation and development, were significantly lower than those in the controls. The WNT/β-catenin pathway is one of the most well-known signalling pathways involved in lung growth. We found that the expression of the target genes c-MYC, cyclin D, and vascular endothelial growth factor (VEGF) and the important proteins WNT3a and β-catenin significantly decreased. Moreover, heparin, which is a NET inhibitor, attenuated changes in gene and protein expression, thereby attenuating BPD-like changes. This finding indicates that NETs are associated with BPD and can induce BPD-like changes in neonatal mice via the WNT/β-catenin pathway.

Transglutaminase 2 regulates terminal erythroid differentiation via cross-linking activity.

Transglutaminase 2 (TGM2) is a versatile enzyme that modulates cell survival and differentiation. However, its role in terminal erythroid differentiation is poorly understood. In this study, we investigated the function of TGM2 in primary fetal liver erythroid differentiation. We predicted TGM2 as an upstream regulator via ingenuity pathway analysis (IPA), and found that its expression was increased at both RNA and protein level during terminal erythroid differentiation. TGM2 cross-linking activity inhibitors GK921 and Z-DON suppressed erythroid maturation and enucleation, while its GTPase inhibitor LDN27219 had no such effect. Z-DON treatment arrested differentiation at basophilic erythroblast stage, and interfered with cell cycle progression. RT-PCR demonstrated decreased GATA-1 and KLF1, and disarranged cyclin, CDKI and E2F family genes expression after Z-DON treatment. In conclusion, TGM2 regulates terminal erythroid differentiation through its cross-linking enzyme activity.

Populus D-type cyclin gene PsnCYCD1;1 accelerates cell division and participates in secondary growth of vascular bundles.

Plant growth and development rely heavily on cyclins, which comprise an important class of cell division regulators. D-type cyclins (CYCDs) are responsible for the rate-limiting step of G1 cells. In the plant kingdom, despite the importance of CYCDs in herbaceous plants, there is little knowledge of these proteins in perennial woody plants. Here, the gene of a nucleus-localized cyclin, PsnCYCD1;1, was cloned from Populus simonii × P. nigra. PsnCYCD1;1 was highly expressed in tissues with active cell division, especially the leaf buds, and could be induced by sucrose and phytohormones. Moreover, overexpression of PsnCYCD1;1 in poplar could stimulate cell division, resulting in the generation of small cells and causing severe morphological changes in the vascular bundles, resulting in 'S'-shaped tortuous stems and curled leaves. Furthermore, transcriptomic analysis revealed that endogenous genes related to cell division and vascular cambium development were significantly up-regulated in the transgenic plants. In addition, using yeast two-hybrid and bimolecular fluorescence complementation assays PsnCDKA1, PsnICK3, and PsnICK5 were identified as proteins interacting with PsnCYCD1;1. Our study demonstrates that PsnCYCD1;1 accelerates plant cell division and participates in secondary growth of vascular bundles in poplar.

Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gβγ and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, β-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

Comprehensive analysis and expression profiles of the AP2/ERF gene family during spring bud break in tea plant (Camellia sinensis)

BackgroundAP2/ERF transcription factors (AP2/ERFs) are important regulators of plant physiological and biochemical metabolism. Evidence suggests that AP2/ERFs may be involved in the regulation of bud break in woody perennials. Green tea is economically vital in China, and its production value is significantly affected by the time of spring bud break of tea plant. However, the relationship between AP2/ERFs in tea plant and spring bud break remains largely unknown.ResultsA total of 178 AP2/ERF genes (CsAP2/ERFs) were identified in the genome of tea plant. Based on the phylogenetic analysis, these genes could be classified into five subfamilies. The analysis of gene duplication events demonstrated that whole genome duplication (WGD) or segmental duplication was the primary way of CsAP2/ERFs amplification. According to the result of the Ka/Ks value calculation, purification selection dominated the evolution of CsAP2/ERFs. Furthermore, gene composition and structure analyses of CsAP2/ERFs indicated that different subfamilies contained a variety of gene structures and conserved motifs, potentially resulting in functional differences among five subfamilies. The promoters of CsAP2/ERFs also contained various signal-sensing elements, such as abscisic acid responsive elements, light responsive elements and low temperature responsive elements. The evidence presented here offers a theoretical foundation for the diverse functions of CsAP2/ERFs. Additionally, the expressions of CsAP2/ERFs during spring bud break of tea plant were analyzed by RNA-seq and grouped into clusters A-F according to their expression patterns. The gene expression changes in clusters A and B were more synchronized with the spring bud break of tea plant. Moreover, several potential correlation genes, such as D-type cyclin genes, were screened out through weighted correlation network analysis (WGCNA). Temperature and light treatment experiments individually identified nine candidate CsAP2/ERFs that may be related to the spring bud break of tea plant.ConclusionsThis study provides new evidence for role of the CsAP2/ERFs in the spring bud break of tea plant, establishes a theoretical foundation for analyzing the molecular mechanism of the spring bud break of tea plant, and contributes to the improvement of tea cultivars.

Abstract 5469: Characterization of CCNE1 amplifications and associated genomic features in ovarian and uterine cancers

Abstract Amplification of the Cyclin E1 gene (CCNE1) is a recurrent genetic alteration in ovarian and uterine cancers. Therapeutic approaches to target cancers harboring CCNE1 amplification are being developed (e.g. PKMYT1, WEE1, and CDK2 inhibitors). In this study, we report the genomic landscape and architecture of CCNE1 amplifications using a combination of whole-exome (WES) and whole-genome sequencing (WGS) approaches. WES data from 361 ovarian and 580 uterine cancers was retrieved from TCGA. WGS data from 113 ovarian and 51 uterine cancers was retrieved from PCAWG. For both WES and WGS, amplification was defined as CN minus ploidy (CN-ploidy) >= 4. CCNE1 amplification was detected in up to 21.2% of ovarian and 13.7% of uterine cancers from both methods. A review of the co-alteration landscape using WES revealed that TP53 alterations frequently co-occurred with CCNE1 amplifications in both ovarian and uterine cancers (ovarian: 95.8% vs 87.6%, OR = 3.24, p=0.1; uterine: 89.6% vs 36.0%, OR = 15.2, p =2.14e-13) and BRCA1 alterations were mutually exclusive with CCNE1 amplifications in ovarian cancer (2.1% vs 17.9%, OR=0.2, p = 3.1e-3). In addition, alterations in ARID1A (0% vs 40.5%, OR = 0, p = 1.3e-10) and PTEN (4.2% vs 60.1%, OR = 0.03, p =2.7e-15) were mutually exclusive with CCNE1 amplifications in uterine cancer. The number of CCNE1 copies ranged widely, with median CN-ploidy values of 9.3 (range: 4.0 -35.0) in ovarian and 7.7 (range: 4.0 -40.3) in uterine cancers from the WES cohort, and 7.4 (range: 4.1 -34.3) in ovarian and 11.0 (range: 4.6 - 81.9) in uterine cancers from WGS. We also observed a broad range of CCNE1 amplicon sizes, from highly focal to chromosome arm-level amplifications. WGS data showed frequent loss of heterozygosity (LOH) of chromosome 19 arms and an enrichment of fold-back inversions proximal to the CCNE1 amplicon implicating breakage-fusion-bridge in formation of CCNE1 amplicons. Whole genome duplication (WGD) was detected significantly more frequently in ovarian and uterine cancers with CCNE1 amplification than the wild-type group (ovarian: 90.9% vs 48.3%, p = 6.4e-10; uterine: 78.7% vs 21.9%, p = 7.1e-15). As compared to CCNE1 wild-type cancers, CCNE1-amplified cancers were found to display an enrichment for certain structural variant signatures: RS1, dominated by 100Kb-1Mb duplications (median = 30, p=0.08); RS2, dominated by translocations (median = 16, p=0.001); and RS7, dominated by 100Kb-1Mb deletions (median=23, p=0.0002). In conclusion, CCNE1-amplified ovarian and uterine cancers are enriched for TP53 alterations and display a high degree of genome instability manifested as WGD and characteristic structural variants. Fold-back inversions and LOH were frequently observed at the CCNE1 locus in tumors with CCNE1 amplification, suggesting a potential breakage-fusion-bridge mechanism in the genesis of this amplicon. Citation Format: Sunantha Sethuraman, Dominik Glodzik, Pier Selenica, Adrienne Johnson, Jorge S. Reis-Filho, Artur Veloso, Ian M. Silverman. Characterization of CCNE1 amplifications and associated genomic features in ovarian and uterine cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5469.

Abstract 1281: Oncoprotein YB-1 modulation of Lyn-MAPK-NF-kB pathway, a potential therapeutic strategy combined with sorafenib for advanced stage HCC

Abstract The Y-Box Binding protein 1 (YB-1) is a 324 amino acid-long member of the Cold Shock Domain (CSD) protein superfamily. YB-1 interacts with micro RNAs and non-coding RNAs in the cytoplasm, acting as a regulator of mRNA translation. YB-1 translocation into the nucleus is circadian clock-dependent and leads to the upregulation of cyclin gene expression, among others. Another mechanism of nuclear translocation of YB-1 is mediated by phosphorylation of the Ser 102 residue in the CSD. The YB-1 oncoprotein acts as a transcription factor when bound to DNA, modulating the transcription of genes that regulate biological processes such as Multidrug Resistance Mutation 1 (MDR1). YB-1 is linked to poor prognosis in breast, prostate, and liver cancer. Despite advances in biomedicine, the incidence and mortality of hepatocellular carcinoma (HCC) remain high. Sorafenib, a tyrosine kinase Inhibitor (TKI), is the first-line treatment for advanced HCC. Sorafenib also inhibits downstream activation of NF-kB by inhibition of Lyn phosphorylation (Y397). However, it was observed in many patients that its sorafenib’s effectiveness is hampered by drug resistance, but the mechanism is still unclear. We have found that SK-HEP-1 HCC cells transfected with a YB-X-1 overexpression plasmid subsequently developed into a stable YB-1 overexpression cell line (SK-HEP-1+YB-1) displayed increased invasion, migration, proliferation, and colony formation property as compared to the vector control (SKHEP1+Vec). The SK-HEP-1+YB-1 cell line also showed increased IC50 for sorafenib and elevated phosphorylation of Lyn (Y397) and further activated NF-kB (S165/S176) phosphorylation. This indicates YB-1 may cause increased Sorafenib resistance in HCC by activating tyrosine kinases (TK), such as lyn, and simultaneous activation of NF-kB. The combination of YB-1 inhibition and sorafenib might provide a novel therapeutic strategy and improve the survival of patients with advanced-stage HCC. Citation Format: Ana G. Ayala Pazzi, Omar Karkoutly, Kristopher Ezell, Samantha Lopez, Kyle D. Doxtater, Elias George, Vijian Dhevan, Manish K. Tripathi. Oncoprotein YB-1 modulation of Lyn-MAPK-NF-kB pathway, a potential therapeutic strategy combined with sorafenib for advanced stage HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1281.

Dps-dependent in vivo mutation enhances long-term host adaptation in Vibrio cholerae.

As one of the most successful pathogenic organisms, Vibrio cholerae (V. cholerae) has evolved sophisticated regulatory mechanisms to overcome host stress. During long-term colonization by V. cholerae in adult mice, many spontaneous nonmotile mutants (approximately 10% at the fifth day post-infection) were identified. These mutations occurred primarily in conserved regions of the flagellar regulator genes flrA, flrC, and rpoN, as shown by Sanger and next-generation sequencing, and significantly increased fitness during colonization in adult mice. Intriguingly, instead of key genes in DNA repair systems (mutS, nfo, xthA, uvrA) or ROS and RNS scavenging systems (katG, prxA, hmpA), which are generally thought to be associated with bacterial mutagenesis, we found that deletion of the cyclin gene dps significantly increased the mutation rate (up to 53% at the fifth day post-infection) in V. cholerae. We further determined that the dpsD65A and dpsF46E point mutants showed a similar mutagenesis profile as the Δdps mutant during long-term colonization in mice, which strongly indicated that the antioxidative function of Dps directly contributes to the development of V. cholerae nonmotile mutants. Methionine metabolism pathway may be one of the mechanism for ΔflrA, ΔflrC and ΔrpoN mutant increased colonization in adult mice. Our results revealed a new phenotype in which V. cholerae fitness increases in the host gut via spontaneous production nonmotile mutants regulated by cyclin Dps, which may represent a novel adaptation strategy for directed evolution of pathogens in the host.

Abstract OT3-23-01: VELA: A first-in-human phase 1/2 study of BLU-222, a potent, selective cyclin-dependent kinase (CDK) 2 inhibitor in patients with cyclin E1 gene (CCNE1)-amplified or CDK4/6 inhibitor-resistant advanced solid tumors

Abstract Background The regulation of cell growth and proliferation is dependent on cyclins and CDKs. The formation of the cyclin D-CDK4/6 complex increases the expression of cyclin E1 and E2. Cyclin E1 and E2 bind to and activate CDK2; this results in a cyclin E/CDK2 complex that assists with downstream expression of DNA synthesis machinery. The use of CDK4/6 inhibitors such as palbociclib or ribociclib is an effective treatment in patients with hormone receptor-positive (HR+), human epithelial growth factor receptor-2 negative (HER2-) breast cancer; however, resistance to treatment eventually occurs. Aberrant cyclin E/CDK2 activity has been identified as a potential resistance mechanism by which tumors can evade CDK4/6 inhibitors. BLU-222 is an oral, investigational, potent, and selective CDK2 inhibitor. In preclinical studies, BLU-222 treatment in combination with ribociclib led to durable tumor regression in both CDK4/6-resistant and sensitive models of HR+HER2- breast cancer. Trial design VELA (NCT05252416) is an international, open-label, first-in-human phase 1/2 study evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of BLU-222 in adult patients with CCNE1-amplified tumors or with HR+HER2- breast cancer with disease progression on CDK4/6 inhibitors. In phase 1 and 2, patients aged ≥18 years with an Eastern Cooperative Oncology Group performance status 0–2 are eligible. In phase 2, all patients must have ≥1 measurable target lesion per Response Evaluation Criteria in Solid Tumors version 1.1. Primary endpoints include assessing the safety of BLU-222 as a single agent or BLU-222 in combination with either carboplatin or ribociclib and/or fulvestrant (phase 1 and 2), identifying the maximum tolerated dose and/or recommended phase 2 dose (phase 1), and determining the objective response rate (phase 2). In the phase 1 dose-escalation part, patients with any advanced solid tumor with progression on standard of care (SOC) will receive BLU-222; patients with gastric or endometrial cancer (EC) with progression on ≥2 prior therapies (including ≥1 platinum-based therapy) or with CCNE1-amplified platinum-resistant/refractory ovarian cancer (OC) will receive BLU-222 and carboplatin; patients with HR+HER- breast cancer with progression on CDK4/6 inhibitors will receive BLU-222, ribociclib, and fulvestrant. In the phase 2 dose-expansion part, patients with CCNE1-amplified tumors including EC (progression on ≥2 prior therapies), platinum-resistant/refractory OC, or other advanced solid tumors (progression after SOC) will receive BLU-222 monotherapy; patients with CCNE1-amplified platinum-resistant/refractory OC will receive BLU-222 and carboplatin; and patients with CDK4/6 inhibitor-resistant HR+HER2- breast cancer will receive BLU-222 and fulvestrant with/without ribociclib. Pharmacokinetic parameters will be calculated using standard non-compartmental methods from the plasma concentration–time data. Tissue biopsies will be collected during cycle 1 to assess the phosphorylation of retinoblastoma 1 (Rb1) protein which will be used as a pharmacodynamic marker to assess target inhibition. Dose escalation is ongoing and approximately 50 sites are anticipated to enroll patients across North America, Europe, and the Asia/Pacific region. Citation Format: Manish R Patel, Dejan Juric, Brian S Henick, Kathleen N Moore, Doreen Do, Joshua Chapman, Hui Zhang, Maria Roche, Kate J Newberry, Mikael Rinne, Timothy A Yap. VELA: A first-in-human phase 1/2 study of BLU-222, a potent, selective cyclin-dependent kinase (CDK) 2 inhibitor in patients with cyclin E1 gene (CCNE1)-amplified or CDK4/6 inhibitor-resistant advanced solid tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-23-01.

Abstract P3-10-01: Deciphering the heterogeneity of cell cycle in breast cancer and relevance to clinical application of CDK inhibitors

Abstract BACKGROUND: Deregulation of cellular proliferation represents a defining hallmark of cancer. The mechanisms driving aberrant cell cycle progression across different breast cancer subtypes is highly diverse and likely contributes to therapeutic resistance in a variety of contexts. The significance of cell cycle heterogeneity is most clear in the context of metastatic HR+/HER2- breast cancer, wherein acquired and intrinsic resistance to CDK4/6 inhibition represents a significant clinical challenge. However, aberrant cell cycle regulatory networks likely further confound the use of targeted therapies in the context of TNBC. METHODS AND RESULTS: We employed unbiased analyses of large number of breast cancer models to decipher the genetic requirements for different CDK and Cyclins which drive proliferation and the significance to response to CDK4/6 inhibitors. Breast cancer cells harbor heterogeneity in the requirement for a given G1/S regulatory CDK or Cyclin. These dependencies are conditioned by the expression of the respective CDK or Cyclin, as well as other gene expression or genetic features present within the tumor that could represent putative biomarkers. In general, conventional dependency for CDK4 and Cyclin D1 is strongest in HR+/HER2- breast cancer models, whereas TNBC exhibits dependency on a variety of different CDK or Cyclin genes. Resistance to CDK4/6 inhibitors in HR+/HER2- breast cancer models, while occurring through a number of different primary mechanisms, functionally deregulates the phosphorylation of RB. However, genetically enforcing RB activation is sufficient to block proliferation, underscoring the opportunity to ameliorate resistance to CDK inhibitory strategies. CRISPR screens in HR+/HER2- and TNBC models are identifying new mediators of sensitivity and resistance to CDK4/6 inhibitors. Inherently, elevated expression of Cyclin E and p16INK4A are associated with resistance to multiple therapeutic approaches, including CDK4/6 inhibitors, but denote a requirement for CDK2. The cell cycle conditions disparately dependent on CDK4/D1 vs. CDK2/E can be visualized by multispectral imaging and used to direct CDK inhibitor based strategies. CONCLUSIONS: Together these studies illustrate the complex nature of breast cancer cell cycles which can confound therapy. However, understanding of this heterogeneity can create new opportunities for precision approaches targeting CDK/Cyclin-dependence therapeutically. Citation Format: Erik S. Knudsen, Vishnu M. Kumarasamy, Jianxin Wang, Agnieszka Witkiewicz. Deciphering the heterogeneity of cell cycle in breast cancer and relevance to clinical application of CDK inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-10-01.

Adipose Tissue-Mesenchymal Stem Cells Caused to Change the Methylation Status of hTERT Gene Promoter CpG Islands of Molt-4 Leukemia Cells as Cell-based Therapy.

DNA methylation was considered as prognostic information in some hematological malignancies. Previous studies have reported the in vitro and in vivo biology role of mesenchymal stem cells (MSCs) on leukemic cells. The aim of this study was to investigate the effect of MSCs on the promoter methylation status of hTERT as a catalytic subunit of telomerase enzyme. In the experimental study, the Molt-4 leukemic cells were co-cultured with MSCs for 7 days. At the end of the co-culture period, the Molt-4 cells were collected, DNA and protein were extracted. Then methylation specific-PCR and western blotting were done for evaluating the hTERT gene promoter methylation status and cyclin D1 and hTERT protein expression, respectively. In the following, the flow cytometry was done for cell cycle distribution assay. It was found that MSCs resulted in a significant decrease in the cyclin D1 and hTERT protein expression levels. Also, MSCs caused changes in the methylation status of the CpG islands in the hTERT gene promoter region. The following results showed that MSCs caused a significant increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as decrease in the cell proliferation of Molt-4 cells. It is concluded that co-culture of MSCs with Molt-4 cells could be involved in changing the methylation status of hTERT gene promoter, cell cycle and hTERT protein expression; it could be potentially beneficial for further investigations regarding the cell transplantation and cell-based therapy.

Hgc1 Independence of Biofilm Hyphae in Candida albicans.

Biofilm and hypha formation are central to virulence of the fungal pathogen Candida albicans. The G1 cyclin gene HGC1 is required for hypha formation under diverse in vitro and in vivo growth conditions. Hgc1 is required for disseminated infection and is a linchpin in the argument that hyphal morphogenesis itself is required for pathogenicity. We report here that HGC1 is dispensable for hypha formation during biofilm formation both in vitro, under strong inducing conditions, and in vivo, in a mouse oropharyngeal candidiasis model. These findings are validated with two or more C. albicans isolates. Systematic screening of overexpressed cyclin genes indicates that CCN1 and CLN3 can compensate partially for Hgc1 function during biofilm growth. This conclusion is also supported by the severity of the hgc1Δ/Δ ccn1Δ/Δ double mutant biofilm defect. Our results suggest that hypha formation in biofilm is accomplished by combined action of multiple cyclins, not solely by Hgc1. IMPORTANCE The HGC1 gene encodes a cyclin that is required for virulence of the fungal pathogen Candida albicans. It is required to produce the elongated hyphal filaments of free-living planktonic cells that are associated with virulence. Here, we show that HGC1 is not required to produce hyphae in the alternative growth form of a biofilm community. We observe Hgc1-independent hyphae in two infection-relevant situations, biofilm growth in vitro and biofilm-like oropharyngeal infection. Our analysis suggests that hypha formation in the biofilm state reflects combined action of multiple cyclins.

Search for polymorphic variants of candidate genes contributing to individual radiosensitivity

Background.Cytogenetic damage (СD) in lymphocytes induced by low doses (up to 0.1 Sv) of ionizing radiation (IR) is the main cytogenetic sign of individual radiosensitivity of the human body. In addition to DNA repair and cell death, which affect the formation of СD and its elimination, IR effects on the cell can be manifested through changes in proliferation of cells with unrepaired DNA damage. The system of cyclins and cyclin-dependent kinases (CDK), which provide coordination of mitotic events during passage of a cell through the cell cycle, plays a crucial role in regulation of cell proliferation.Aim.To evaluate the relationship of single-nucleotide polymorphisms (SNPs) of cell cycle genes with an increased frequency of СD in workers of a nuclear power plant affected by chronic occupational radiation exposure in the dose range of 100–500 mSv.Materials and methods.The object of the study was blood of 55 conditionally healthy workers of Siberian Chemical Plant (SCP) who were affected by chronic occupational radiation exposure (gamma radiation) in the dose range of 100–500 mSv. A standard cytogenetic analysis of blood lymphocytes was performed for all examined individuals. Genomic DNA was isolated from the blood of the workers using the QIAamp DNA Blood Mini Kit (QIAGEN, Germany). DNA was genotyped using 257 SNPs of cyclin genes and neighboring intergenic regions using DNA microarrays from the high-density CytoScan HD Array (Affymetrix, USA).Results.Taking into account the Bonferroni correction, only statistically significant associations of SNPs with the frequency of dicentric chromosomes were found; all other types of chromosomal aberrations did not show statistical significance. The rs803054CCNI2was associated with an increased frequency of dicentric chromosomes arising under the influence of chronic occupational radiation exposure.Conclusion.The discovered SNP (rs803054), whose recessive genotype is associated with an increased frequency of dicentric chromosomes in workers of SCP exposed to radiation at doses of 100–500 mSv over a long time, can be considered as a potential marker of individual radiosensitivity. To confirm the identified associations, further validation studies are needed on an expanded sample of people affected by chronic occupational radiation exposure.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector β-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/β-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/β-catenin signaling in OVX mice.

Ethylcoprostanol modulates colorectal cancer cell proliferation and mitigates cytotoxicity of cholesterol metabolites in non-tumor colon cells.

Sterols can be metabolized by gut microbiota. The cholesterol metabolites have been proposed as promoters of colorectal cancer (CRC), while the effect of plant sterol metabolites is unknown. This study aimed to evaluate the cytotoxicity of metabolites from cholesterol (coprostanol, cholestanol, coprostanone and cholestenone) and β-sitosterol (ethylcoprostanol) on human colon tumor (Caco-2) and non-tumor (CCD-18Co) cells at physiological concentrations (9-300 μM) and exposure time (24 h). Ethylcoprostanol reduced the tumor cell proliferation (MTT), showing in flow cytometry assays induction of apoptosis via production of reactive oxygen species (ROS) and ceramide. Transcriptomic analysis (qPCR) showed activation of the intrinsic apoptosis pathway (BAX/BCL2 ratio and CASP9 increased), accompanied by downregulation of the p21 gene. Cholesterol metabolites, mainly the most hydrophobic, induced apoptosis and G0/G1 phase arrest in non-tumor cells through overproduction of ROS. Both the intrinsic and extrinsic (CASP8 increased) apoptosis pathways occurred. In turn, a reduction in the expression of the cyclin E1 gene confirmed the cell cycle arrest. In addition, ethylcoprostanol protected non-tumor cells from the most cytotoxic cholesterol metabolite (cholestenone). In conclusion, ethylcoprostanol is a promising candidate as a therapeutic adjuvant in CRC, while cholesterol metabolites could act as CRC promoters through their cytotoxicity.

Vanillin prevents the growth of endometriotic lesions through anti-inflammatory and antioxidant pathways in a mouse model.

Endometriosis is an estrogen-dependent chronic inflammatory gynecological disease defined by the presence of endometrial glands and mesenchyme outside the uterine cavity, named ectopic endometrium. Recent studies showed that endometriosis is associated with hormone imbalance, inflammation and oxidative stress. As the main component of vanilla bean extract, vanillin is widely used as a flavoring agent in the food, pharmaceutical, and cosmetic industries. It is known for its anti-inflammatory, antibacterial, and antitumor properties, but its therapeutic efficacy in endometriosis has not been studied. In this study, we evaluated the roles of vanillin in this disease using an induced endometriotic mouse model. The results showed that vanillin significantly inhibited the growth of endometrial lesions. Compared with the control group, the weight and volume of lesions were reduced considerably in the vanillin-treated group, showing its fantastic ability to inhibit cell proliferation and promote apoptosis. In addition, in the treatment group, mRNA expression of the pro-inflammatory cytokines Tnfa, Infg, Il1b, and Il6 was reduced, the number of macrophages and neutrophils was decreased, and the NF-κB signaling pathway was inhibited, indicating that vanillin suppressed the inflammatory response in the ectopic endometrium. Besides, we found that the intensity of tissue reactive oxygen species (ROS) was significantly lower, and mitochondrial complex IV expression was reduced in the vanillin-treated group. Meanwhile, treatment of the immortalized human endometriotic epithelial cell line (11Z) with vanillin resulted in the downregulation of cyclin genes that drive the cell proliferation process, inhibited cell proliferation, promoted apoptosis, and downregulated the expression of LPS-induced inflammatory cytokines. Most importantly, our data showed that the vanillin treatment had only minimal effects on the eutopic endometrium with respect to the pregnancy process, indicating its safety to be used in treating endometriosis in adults. In conclusion, our data suggest that vanillin has potential therapeutic properties for endometriosis as a regulatory molecule of cell proliferation, apoptosis, inflammation, and oxidative stress.

NOTCH1 mutation in chronic lymphocytic leukaemia is associated with an enhanced cell cycle G1/S transition and specific cyclin overexpression: Preclinical ground for targeted inhibition.

Studies prior to next-generation sequencing (NGS) showed that the frequent indolent course of chronic lymphocytic leukaemia (CLL) is related to most cells remaining quiescent in the G0 -G1 cell cycle phase, due to the expression of dysregulated cyclin genes. Of note, the activating nature of the NOTCH1 mutation in T lymphoblastic leukaemia also drives the dysregulation of cell cycle genes. Our goal was to comprehensively revisit the cell cycle in NOTCH1-mutated CLL (NOTCH1MUT ) to test for potential therapeutic targets. Among 378 NGS-annotated CLL cases, NOTCH1MUT cells displayed a unique transcriptome profile of G0 -G1 cell cycle components, with an overexpression of early-phase effectors, reaching a 38-, 27- and ninefold change increase for the complex elements CCND3, CDK4 and CDK6, respectively. This NOTCH1MUT cells' profile was related to more cells traversing through the cell cycle. In-vitro targeted inhibition of NOTCH1 gamma-secretase and CDK4/6 reversed the distribution of cells through the cycle phases and enhanced the killing of NOTCH1MUT CLL cells, suggesting new therapeutic approaches.

Patterns of the Expression of Cyclin Genes in Bortezomib-Sensitive and Resistant Cells of Multiple Myeloma

Patterns of the Expression of Cyclin Genes in Bortezomib-Sensitive and Resistant Cells of Multiple Myeloma

PDZD2 Is Essential for Steady-State Hematopoiesis and Its 37-Kda Secreted Product, sPDZD2, Functions As a Soluble Tumor Suppressor in AML

PDZ domain-containing protein 2 (PDZD2) is expressed in normal hematopoietic stem and progenitor cells (HSPC), specifically HSC, MPP and erythroid progenitors, however, it is almost universally lost through hypermethylation in myeloid malignancies. PDZD2 encodes for a 300kDa protein containing 6 PDZ domains. The full-length protein is cleaved in the cytoplasm in a caspase 3-dependent manner, and the C-terminal fragment containing PDZ domains 5 and 6 is secreted to the tissue microenvironment as a 37kDa protein known as secreted PDZD2 (sPDZD2). Given that sPDZD2 has been reported to function as a tumor suppressor in solid tumors and its almost universal loss in AML, we hypothesized that PDZD2 is required for normal hematopoiesis and that sPDZD2 functions as a soluble tumor suppressor in the hematopoietic system. To determine if sPDZD2 is secreted to the bone marrow (BM) microenvironment we analyzed sPDZD2 levels in BM plasma isolated from primary AMLs and healthy donors using an ELISA-based detection method. We confirmed sPDZD2 secretion by normal CD34+ HSPC and detected significantly lower levels in AMLs (169 ± 17.53 vs 46.12 ± 6.70 ng/ml; p<0.0001). In addition, AML cell lines secreted significantly lower levels of sPDZD2 into the culture medium than HSPCs (28.71 ± 1.76 vs 4.62 ± 1.19 ng/mL; p<0.0001). Notably, treatment of a panel of AML cell lines (n=9) and primary human AML cells (n=4) with recombinant sPDZD2 (r-sPDZD2; dose: 100nM-300nM/day) led to growth inhibition (CellTiter-Glo, p<0.05 for all), differentiation (confirmed by upregulation of CD15/CD11b by flow cytometry) and cell cycle arrest in G0/G1. qRT-PCR analysis of cell cycle regulators in MV4-11 cells treated with r-sPDZD2 confirmed upregulation of CDKN2A (4.01-fold) and E2F4 (4.66-fold) and marked downregulation of multiple cyclin genes, including cyclins D and E, compatible with the cell cycle arrest observed by flow cytometry. To identify signaling pathways downstream of sPDZD2, we treated MV4-11 cells with 100nM r-sPDZD2 and performed phospho-kinase protein array followed by immunoblot for validation. In vitro treatment with r-sPDZD2 led to inhibition of ERK1/2 (T202/Y204, T185/Y187) and CREB (S133) phosphorylation, as well as decreased ß-Catenin expression and increased GSK3ß (S9), RSK1/2 (S221/S227) and p53 (S46) phosphorylation. In order to determine whether PDZD2 plays a role in normal hematopoiesis we performed in vitro differentiation in CRISPR-edited human HSPCs. We used a Cy3-labeled recombinant Cas9 protein and specific single guide RNAs (gRNAs) targeted against PDZD2. Cy3-positive cells were then used for in vitro myeloid or erythroid differentiation, and cell surface markers were evaluated by flow-cytometry analysis over a period of 11 days. While PDZD2-edited cells (PDZD2 KO) displayed a minor defect on myeloid differentiation due to insufficient upregulation of CD15, erythropoiesis appears to be more compromised, with a significant failure to adequately upregulate both CD235a (39 vs 15%) and CD71 (58 vs 26%). To determine the role of Pdzd2 in vivo, we took advantage of a Pdzd2Gt/Gt gene-trap knock-out mouse model and performed peripheral blood (PB) and BM analysis. PB counts at 8-weeks of age showed anemia, with a significant decrease in both red blood cell counts (9.04 ± 0.26 vs 7.58 ± 0.47 x106/µL) and hemoglobin levels (14.88 ± 0.46 vs 12.30 ± 0.81 g/dL) (p<0.05 for both), an observation compatible with the impaired erythropoiesis seen in vitro after PDZD2 KO in human HSPCs. In addition, we observed a trend to decreased MEPs and short-term HSCs, in the BM of 12-week-old mice (n=3), though the small size of the cohort prevented robust statistical analysis. In summary, our findings are the first to shed light on a previously unrecognized role for PDZD2 in hematopoiesis. Loss of PDZD2 in HSPC resulted in impaired erythroid differentiation both in vitro and in vivo. Moreover, our findings validate a soluble tumor suppressor role for sPDZD2 in AML, similar to that seen in solid tumors. Importantly, we further demonstrate a potential role for recombinant sPDZD2 as a novel therapeutic approach for AML.