Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis.

Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

AL amyloidosis manifesting as a vertebral amyloidoma secondary to an unrecognized plasmacytoma expressing cyclin D1 case report.

INTRODUCTION: Immunoglobin-related (AL) amyloidosis is the production of amyloidogenic immunoglobulin light chains from clonal plasma cells or, rarely, B-cell lymphomas with plasmacytic differentiation. Amyloid deposition causes progressive end organ destruction with profound morbidity. PRESENTATION OF CASE: We present a rare case of a lambda light chain AL amyloidoma localized to a thoracic vertebra of an 87-year-old woman who had a remote history of an unspecified non-Hodgkin B-cell lymphoma (NHL). Our patient presented with upper extremity neuropathy and was found by MRI to have a malignant-appearing lesion throughout the T1 vertebra. Initial biopsy showed amyloid deposition and staging evaluation found localized disease. Prior to planned surgery and radiation the following year, she had worsening neuropathy including multiple falls. Repeat MRI confirmed lesion progression with concern for cord compression. Urgent surgical resection was performed. Histology showed numerous plasma cells with abundant amyloid deposition that was found by amyloid typing to be lambda light chain. An incidental B-cell rich lymphoid aggregate was also seen in a bone marrow fragment that required additional immunohistochemical evaluation, showing the aggregate to be benign while revealing the plasma cells to be positive for cyclin D1. She received localized radiation and has been asymptomatic. DISCUSSION: Amyloidosis and plasma cell neoplasms require appropriate staging evaluation. The cyclin D1-positive plasma cells raises the possibility of the t(11;14)/IGH::CCND1 translocation that portends better prognosis and therapeutic response with venetoclax. CONCLUSION: Amyloidomas are uncommon and may present in nearly any site, requiring a high index of clinical suspicion for proper diagnosis.

[Bioinformatic analysis of CCND2 expression in papillary thyroid carcinoma and its impact on immune infiltration].

OBJECTIVE: To investigate cyclin D2 (CCND2) expression in papillary thyroid carcinoma (PTC) and its association with the clinicopathological features. METHODS: The public databases TCGA, TIMER 2.0 and UALCAN were used to explore CCND2 expression level in PTC and adjacent tissues, and its diagnostic value for PTC was analyzed using ROC curves. GO enrichment analysis of CCND2-related differentially expressed genes (DEGs) in PTC was performed, and tumor immune infiltration of CCND2 in thyroid cancer was analyzed using TIMER database and CIBERSORT data source. RT-qPCR and Western blot were used to detect CCND2 expression in normal human thyroid cell line Nthy-ori-3-1 and human PTC cell lines TPC-1 and BCPAP. CCND2 expression was also detected in clinical specimens of PTC and adjacent tissues by immunohistochemistry, and its correlation with clinicopathological features of the patients were analyzed. RESULTS: Informatic analysis revealed significantly higher CCND2 mRNA expression in thyroid cancer than in the adjacent tissues (P < 0.001) in close correlation with tumor stage, gender, age, pathological subtype, and lymph node involvement (P < 0.05). ROC curve analysis showed that at the cutoff value of 4.983, the diagnostic sensitivity, specificity, and accuracy of CCND2 expression for PTC was 83.6%, 94.9%, and 78.5%, respectively. CCND2 expression was positively correlated with B cells, CD4+ T cells, and macrophages (P < 0.001) and negatively with CD8+ T cells (P < 0.01), and also correlated with memory B-cell infiltration, CD4+ T-cell memory activation, M2 macrophages, resting mast cells, and mast cell activation (P < 0.05). RT-qPCR, Western blot and immunohistochemistry showed significantly higher CCND2 expression in the PTC cells than in Nthy-ori-3-1 cells (P < 0.01) and also in clinical PTC tissues than in the adjacent tissues (P < 0.05) in correlation with tumor size, lymph node metastasis and TNM stage (P < 0.05). CONCLUSION: CCND2 overexpression is closely correlated with tumor progression and immune cell infiltration in PTC patients..

KMT2A and chronic inflammation as potential drivers of sporadic parathyroid adenoma.

BACKGROUND: Sporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, yet the mechanisms involved in its pathogenesis remain incompletely understood. METHODS: Surgically removed PA samples, along with normal parathyroid gland (PG) tissues that were incidentally dissected during total thyroidectomy, were analysed using single-cell RNA-sequencing with the 10× Genomics Chromium Droplet platform and Cell Ranger software. Gene set variation analysis was conducted to characterise hallmark pathway gene signatures, and single-cell regulatory network inference and clustering were utilised to analyse transcription factor regulons. Immunohistochemistry and immunofluorescence were performed to validate cellular components of PA tissues. siRNA knockdown and gene overexpression, alongside quantitative polymerase chain reaction, Western blotting and cell proliferation assays, were conducted for functional investigations. RESULTS: There was a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells. This is associated with high expression of histone-lysine N-methyltransferase 2A (KMT2A). High KMT2A levels potentially contribute to promoting PAC proliferation through upregulation of the proto-oncogene CCND2, which is mediated by the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3). PA tissues are heavily infiltrated with myeloid cells, while fibroblasts, endothelial cells and macrophages in PA tissues are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues. CONCLUSIONS: We revealed the previously underappreciated involvement of the KMT2A‒STAT3/GATA3‒CCND2 axis and chronic inflammation in the pathogenesis of PA. These findings underscore the therapeutic promise of KMT2A inhibition and anti-inflammatory strategies, highlighting the need for future investigations to translate these molecular insights into practical applications. HIGHLIGHTS: Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis.

Differential effects of montelukast and zafirlukast on MDA­MB­231 triple­negative breast cancer cells: Cell cycle regulation, apoptosis, autophagy, DNA damage and endoplasmic reticulum stress.

Montelukast and zafirlukast, cysteinyl leukotriene receptor antagonists (LTRAs), trigger apoptosis and inhibit cell proliferation of triple­negative breast cancer MDA­MB­231 cells. By contrast, only zafirlukast induces G0/G1 cell cycle arrest. The present study compared the effects of these drugs on proteins regulating cell proliferation, apoptosis, autophagy, and endoplasmic reticulum (ER) and oxidative stress using reverse transcription­quantitative PCR, western blotting and flow cytometry. The expression of proliferating markers, Ki­67 and proliferating cell nuclear antigen, was decreased by both drugs. Zafirlukast, but not montelukast, decreased the expression of cyclin D1 and CDK4, disrupting progression from G1 to S phase. Zafirlukast also increased the expression of p27, a cell cycle inhibitor. Both drugs decreased the expression of anti­apoptotic protein Bcl­2 and ERK1/2 phosphorylation, and increased levels of the autophagy marker LC3­II and DNA damage markers, including cleaved PARP­1, phosphorylated (p)­ATM and p­histone H2AX. The number of caspase 3/7­positive cells was greater in montelukast­treated cells compared with zafirlukast­treated cells. Montelukast induced higher levels of the ER stress marker CHOP compared with zafirlukast. Montelukast activated PERK, activating transcription factor 6 (ATF6) and inositol­requiring enzyme type 1 (IRE1) pathways, while zafirlukast only stimulated ATF6 and IRE1 pathways. GSK2606414, a PERK inhibitor, decreased apoptosis mediated by montelukast, but did not affect zafirlukast­induced cell death. The knockdown of CHOP by small interfering RNA reduced apoptosis triggered by montelukast and zafirlukast. In conclusion, the effects on cell cycle regulator proteins may contribute to cell cycle arrest caused by zafirlukast. The greater apoptotic effects of montelukast may be caused by the higher levels of activated caspase enzymes and the activation of three pathways of ER stress: PERK, ATF6, and IRE1.

Multiple-heated cooking oil promotes early hepatic and renal senescence in adult male rats: the potential regenerative capacity of oleuropein.

For economic purposes, cooking oil is repeatedly heated in food preparation, which imposes serious health threats. This study investigated the detrimental effects of multiple-heated cooking oil (MHO) on hepatic and renal tissues with particular focusing on cellular senescence (CS), and the potential regenerative capacity of oleuropein (OLE). Adult male rats were fed MHO-enriched diet for 8 weeks and OLE (50 mg/kg, PO) was administered daily for the last four weeks. Liver and kidney functions and oxidative stress markers were measured. Cell cycle markers p53, p21, cyclin D, and proliferating cell nuclear antigen (PCNA) were evaluated in hepatic and renal tissues. Tumor necrosis factor-α (TNF-α) and Bax were assessed by immunohistochemistry. General histology and collagen deposition were also examined. MHO disturbed hepatic and renal structures and functions. MHO-fed rats showed increased oxidative stress, TNF-α, Bax, and fibrosis in liver and kidney tissues. MHO also enhanced the renal and hepatic expression of p53, p21, cyclin D and PCNA. On the contrary, OLE mitigated MHO-induced oxidative stress, inflammatory burden, apoptotic and fibrotic changes. OLE also suppressed CS and preserved kidney and liver functions. Collectively, OLE displays marked regenerative capacity against MHO-induced hepatic and renal CS, via its potent antioxidant and anti-inflammatory effects.

Radiomic Prediction of CCND1 Expression Levels and Prognosis in Low-grade Glioma Based on Magnetic Resonance Imaging.

OJECTIVES: Low-grade glioma (LGG) is associated with increased mortality owing to recrudescence and the tendency for malignant transformation. Therefore, it is imperative to discover novel prognostic biomarkers as existing traditional prognostic biomarkers of glioma, including clinicopathological features and imaging examinations, are unable to meet the clinical demand for precision medicine. Accordingly, we aimed to evaluate the prognostic value of cyclin D1 (CCND1) expression levels and construct radiomic models to predict these levels in patients with LGG MATERIALS AND METHODS: A total of 412 LGG cases from The Cancer Genome Atlas (TCGA) were used for gene-based prognostic analysis. Using magnetic resonance imaging (MRI) images stored in The Cancer Imaging Archive with genomic data from TCGA, 149 cases were selected for radiomics feature extraction and model construction. After feature extraction, the radiomic signature was constructed using logistic regression (LR) and support vector machine (SVM) analyses. RESULTS: CCND1 was identified as a prognosis-related gene with differential expression in tumor and normal samples and plays a role in regulating both the cell cycle and immune response. Landmark analysis revealed that high-expression levels of CCND1 were beneficial for survival (P < 0.05) in advanced LGG. Four optimal radiomics features were selected to construct radiomics models. The performance of LR and SVM achieved areas under the curve of 0.703 and 0.705, as well as 0.724 and 0.726 in the training and validation sets, respectively. CONCLUSION: Elevated levels of CCND1 expression could impact the prognosis of patients with LGG. MRI-based radiomics, especially the AUC values, can serve as a novel tool for predicting CCND1 expression and understanding the correlation between elevated CCND1 expression and prognosis. AVAILABILITY OF DATA AND MATERIALS: The datasets analyzed during the current study are available in the TCGA, TCIA, UCSC XENA and GTEx repository, https://portal.gdc.cancer.gov/, https://www.cancerimagingarchive.net/, https://xenabrowser.net/datapages/, https://www.gtexportal.org/home/.

AMPK regulates immature boar Sertoli cell proliferation through affecting CDK4/Cyclin D3 pathway and mitochondrial function.

Sertoli cell (SC) proliferation plays an important role in sperm production and quality; however, the regulatory mechanism of SC proliferation is not well understood. This study investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in the regulation of immature boar SC activity. Cell counting kit-8, Seahorse XFe96, mitochondrial respiratory enzyme-related assay kits, and transmission electron microscopy were used to detect SC proliferative viability, oxygen consumption rate (OCR), mitochondrial respiratory enzyme activity, and the ultrastructure of primary cultured SCs in vitro from the testes of 21-day-old boars. A dual luciferase reporter assay was performed to determine the miRNA-mRNA target interaction. Western blotting was used to analyze cell proliferation-related protein expression of p38, p21, proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinase 4 (CDK4), Cyclin D3, and phosphorylated retinoblastoma protein (Rb). Each experiment had a completely randomized design, with three replicates in each experiment. The results showed that the AMPK inhibitor (Compound C, 20 µM-24 h) increased cell proliferation viability, ATP production, and maximal respiration of SCs by 0.64-, 0.12-, and 0.08-fold (p < 0.05), respectively; increased the SC protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.13-, 0.09-, 0.88-, and 0.12-fold (p < 0.05), respectively; and decreased the SC protein expression of p38 and p21 by 0.36- and 0.27-fold (p < 0.05), respectively. The AMPK agonist AICAR (2 mM-6 h) significantly inhibited SC ultrastructure, OCR, mitochondrial respiratory enzyme activity, and cell proliferation-related protein levels. AMPK was validated to be a target gene of miR-1285 based on the result in which the miR-1285 mimic inhibited the luciferase activity of wild-type AMPK by 0.54-fold (p < 0.001). MiR-1285 mimic promoted the OCR of SCs, with 0.45-, 0.15-, 0.21-, and 0.30-fold (p < 0.01) increases in ATP production, basal and maximal respiration, and spare capacity, respectively. MiR-1285 mimic increased the mitochondrial respiratory enzyme activity of SCs, with 0.63-, 0.70-, and 0.97-fold (p < 0.01) increases in NADH-Q oxidoreductase, cytochrome c oxidase, and ATP synthase, respectively. Moreover, the miR-1285 mimic increased the protein expression of PCNA, CDK4, Cyclin D3, and p-Rb by 0.24-, 0.30-, 0.22-, and 0.13-fold (p < 0.05), respectively, and reduced the protein expression of p38 and p21 by 0.58- and 0.66-fold (p < 0.001). MiR-1285 inhibitor showed opposite effects on the above indicators and induced numerous autophagosomes and large lipid droplets in SCs. A high dose of estradiol (10 µM-6 h, showed a promotion of AMPK activation in a previous study) significantly inhibited SC ultrastructure, mitochondrial function, and proliferation-related pathways, while these adverse effects were weakened by Compound C treatment or miR-1285 mimic transfection. Our findings suggest that the activation and inhibition of AMPK induced by specific drugs or synthesized targeted miRNA fragments could regulate immature boar SC proliferative activity by influencing the CDK4/Cyclin D3 pathway and mitochondrial function; this helps to provide a basis for the prevention and treatment of male sterility in clinical practice.

Atox1-cyclin D1 loop activity is critical for survival of tumor cells with inactivated TP53.

The search for relevant molecular targets is one of the main tasks of modern tumor chemotherapy. To successfully achieve this, it is necessary to have the most complete understanding of the functioning of a transcriptional apparatus of the cell, particularly related to proliferation. The p53 protein plays an important role in regulating processes such as apoptosis, repair, and cell division, and the loss of its functionality often accompanies various types of tumors and contributes to the development of chemoresistance. Additionally, the proliferative activity of tumor cells is closely related to the metabolism of transition metals. For example, the metallochaperone Atox1 - a copper transporter protein - acts as a transcription activator for cyclin D1, promoting progression through the G1/S phase of the cell cycle. On the other hand, p53 suppresses cyclin D1 at the transcriptional level, thereby these proteins have divergent effects on cell cycle progression. However, the contribution of the interaction between these proteins to cell survival is poorly understood. This work demonstrates that not only exists a positive feedback loop between Atox1 and cyclin D1 but also that the activity of this loop depends on the status of the TP53 gene. Upon inactivation of TP53 in A549 and HepG2 cell lines, the expression of ATOX1 and CCND1 genes is enhanced, and their suppression in these cells leads to pronounced apoptosis. This fundamental observation may be useful in selecting more precise interventions for combined therapy of p53-negative tumors.

Novel insights into gut health: Cilostazol strengthens gut integrity by adjusting TLR-2/NF-κB/IL-23 and CD44/AKT/GSK-3ß/cyclin-D1 trajectories in methotrexate-induced mucositis model.

Methotrexate (MTX)-induced gastrointestinal mucositis is a common adverse effect characterized by redox imbalance and overproduction of inflammatory mediators that perturb intestinal integrity. Currently, there is no definitive treatment for this condition and its prevention is still far beyond comprehension. Because of its pleiotropic pharmacological actions, we aimed to explore the potential mechanisms through which cilostazol (CILO) can protect against MTX-induced intestinal mucositis. Wistar rats were allocated into 4 groups, control, CILO (100 mg/kg, p.o for 14 days), MTX (7.5 mg/kg for 4 successive days), and CILO + MTX. The improving effect of CILO on the morphological structure was confirmed by an upturn in the histopathological and transition electron microscope examinations evidenced by the increased jejunal villus height/width and the crypt depth besides the maintenance of tight junctions. These findings were verified biochemically; on the molecular level, CILO reduced the MTX-induced lipid peroxidation, cleaved caspase-3, p53, and the inflammatory parameters (TLR-2, NF-κB, IL-23, TNF-α, IL-1ß), while increasing the anti-inflammatory marker IL-10 and the antioxidant enzyme SOD. Moreover, CILO decreased the injurious axis AKT/GSK-3ß/cyclin-D1, and CD44+, but increased the immunoexpression of the cell proliferating marker PCNA. CILO also upheld the intestinal barrier by enhancing the tight junction molecules (ZO-1, claudin-4) and the E-cadherin/ß-catenin complex while abating the mesenchymal marker vimentin. In conclusion, CILO protected gut integrity by reducing the epithelial-mesenchymal transition process, the MTX-induced oxidative, apoptotic, and inflammatory mediators, and turning off the CD44/AKT/GSK-3ß/cyclin D1 trajectory and intensifying the expression of PCNA.

Cyclin D1 expression in penile cancer.

The main goal of the present study was to analyze the expression profile of cyclin D1 in patients with PC, and to determine possible correlations with clinical and histopathological features. A survey was conducted with 100 patients diagnosed with PC, who were treated at two reference hospitals in São Luís, Maranhão, Brazil, between 2013 and 2017. A review of clinical, epidemiological, and histopathological data was performed, Human Papillomavírus (HPV) DNA was detected using polymerase chain reaction (PCR) and cyclin D1 expression analysis was performed using immunohistochemical techniques. The data revealed that the absence of cyclin D1 expression was significantly associated with HPV-positive histological subtypes (p = 0.001), while its expression was associated with high-grade tumors (p = 0.014), histological subtype (p = 0.001), presence of sarcomatoid transformation (p = 0.04), and perineural invasion (p = 0.023). Patients with cyclin D1 expression exhibited lower disease-free survival compared to the cyclin D1-negative group, although the difference was not statistically significant. The results suggest that cyclin D1 may be a potential biomarker for PC, especially for poorer prognosis.

Abemaciclib-induced epithelial-mesenchymal transition mediated by cyclin-dependent kinase 4/6 independent of cell cycle arrest pathway.

Abemaciclib (ABM), a cyclin-dependent kinase 4/6 inhibitor, shows pharmacological effects in cell cycle arrest. Epithelial-mesenchymal transition is an important cellular event associated with pathophysiological states such as organ fibrosis and cancer progression. In the present study, we evaluated the contribution of factors associated with cell cycle arrest to ABM-induced epithelial-mesenchymal transition. Treatment with 0.6 µM ABM induced both cell cycle arrest and epithelial-mesenchymal transition-related phenotypic changes. Interestingly, the knockdown of cyclin-dependent kinase 4/6, pharmacological targets of ABM or cyclin D1, which forms complexes with cyclin-dependent kinase 4/6, resulted in cell cycle arrest at the G1-phase and induction of epithelial-mesenchymal transition, indicating that downregulation of cyclin-dependent kinase 4/6-cyclin D1 complexes would mimic ABM. In contrast, knockdown of the Rb protein, which is phosphorylated by cyclin-dependent kinase 4/6, had no effect on the expression level of α-smooth muscle actin, an epithelial-mesenchymal transition marker. Furthermore, ABM-induced epithelial-mesenchymal transition was not affected by Rb knockdown, suggesting that Rb is not involved in the transition process. Our study is the first to suggest that cyclin-dependent kinase 4/6-cyclin D1 complexes, as pharmacological targets of ABM, may contribute to ABM-induced epithelial-mesenchymal transition, followed by clinical disorders such as organ fibrosis and cancer progression. This study suggests that blocking epithelial-mesenchymal transition might be a promising way to prevent negative side effects caused by a medication (ABM) without affecting its ability to treat the disease.

LKB1 Loss Correlates with STING Loss and, in Cooperation with ß-Catenin Membranous Loss, Indicates Poor Prognosis in Patients with Operable Non-Small Cell Lung Cancer.

To investigate the incidence and prognostically significant correlations and cooperations of LKB1 loss of expression in non-small cell lung cancer (NSCLC), surgical specimens from 188 metastatic and 60 non-metastatic operable stage I-IIIA NSCLC patients were analyzed to evaluate their expression of LKB1 and pAMPK proteins in relation to various processes. The investigated factors included antitumor immunity response regulators STING and PD-L1; pro-angiogenic, EMT and cell cycle targets, as well as metastasis-related (VEGFC, PDGFRα, PDGFRß, p53, p16, Cyclin D1, ZEB1, CD24) targets; and cell adhesion (ß-catenin) molecules. The protein expression levels were evaluated via immunohistochemistry; the RNA levels of LKB1 and NEDD9 were evaluated via PCR, while KRAS exon 2 and BRAFV600E mutations were evaluated by Sanger sequencing. Overall, loss of LKB1 protein expression was observed in 21% (51/248) patients and correlated significantly with histotype (p < 0.001), KRAS mutations (p < 0.001), KC status (concomitant KRAS mutation and p16 downregulation) (p < 0.001), STING loss (p < 0.001), and high CD24 expression (p < 0.001). STING loss also correlated significantly with loss of LKB1 expression in the metastatic setting both overall (p = 0.014) and in lung adenocarcinomas (LUACs) (p = 0.005). Additionally, LKB1 loss correlated significantly with a lack of or low ß-catenin membranous expression exclusively in LUACs, both independently of the metastatic status (p = 0.019) and in the metastatic setting (p = 0.007). Patients with tumors yielding LKB1 loss and concomitant nonexistent or low ß-catenin membrane expression experienced significantly inferior median overall survival of 20.50 vs. 52.99 months; p < 0.001 as well as significantly greater risk of death (HR: 3.32, 95% c.i.: 1.71-6.43; p <0.001). Our findings underscore the impact of the synergy of LKB1 with STING and ß-catenin in NSCLC, in prognosis.

Identification of CD5/SOX11 double-negative pleomorphic mantle cell lymphoma.

Cyclin D1 protein-positive diffuse large B cell lymphoma (DLBCL) has an immunophenotype of CD5(-) cyclin D1(+) SOX11(-), and most cases lack a CCND1 rearrangement and have a gene expression profile of DLBCL. Rarely, cyclin D1 protein-positive DLBCL harbors a CCND1 rearrangement, and some genetic copy number features typical of mantle cell lymphoma (MCL) have been detected. Since gene expression studies have not been performed, whether such CCND1-rearranged cases represent cyclin D1 protein-positive DLBCL or CD5/SOX11 double-negative pleomorphic MCL remains unclear. To date, no cases of CD5/SOX11 double-negative MCL have been reported. In this study, we collected eight cases initially diagnosed as cyclin D1 protein-positive DLBCL, including four with a CCND1 rearrangement and four without. Immunohistochemically, all four CCND1-rearranged cases had >50% of tumor cells positive for cyclin D1 protein, whereas only one (25%) non-rearranged case had >50% positive tumor cells. Analysis of genome-wide copy number, mutational, and gene expression profiles revealed that CCND1-rearranged cases were similar to MCL, whereas CCND1-non-rearranged cases resembled DLBCL. Despite the SOX11 negativity by immunohistochemistry, CCND1-rearranged cases had a notable trend (P = 0.064) of higher SOX11 mRNA levels compared to non-rearranged cases. Here, we show for the first time that CCND1 rearrangement could be useful for identifying CD5/SOX11 double-negative pleomorphic MCL in cases diagnosed as cyclin D1 protein-positive DLBCL. Cases with >50% cyclin D1 protein-positive tumor cells immunohistochemically and higher SOX11 mRNA levels are more likely to have a CCND1 rearrangement, and fluorescence in situ hybridization can be used to detect the rearrangement.

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Prenatal treatment with preimplantation factor improves early postnatal neurogenesis and cognitive impairments in a mouse model of Down syndrome.

Down syndrome (DS) is a genetic disease characterized by a supernumerary chromosome 21. Intellectual deficiency (ID) is one of the most prominent features of DS. Central nervous system defects lead to learning disabilities, motor and language delays, and memory impairments. At present, a prenatal treatment for the ID in DS is lacking. Subcutaneous administration of synthetic preimplantation factor (sPIF, a peptide with a range of biological functions) in a model of severe brain damage has shown neuroprotective and anti-inflammatory properties by directly targeting neurons and microglia. Here, we evaluated the effect of PIF administration during gestation and until weaning on Dp(16)1Yey mice (a mouse model of DS). Possible effects at the juvenile stage were assessed using behavioral tests and molecular and histological analyses of the brain. To test the influence of perinatal sPIF treatment at the adult stage, hippocampus-dependent memory was evaluated on postnatal day 90. Dp(16)1Yey pups showed significant behavioral impairment, with impaired neurogenesis, microglial cell activation and a low microglial cell count, and the deregulated expression of genes linked to neuroinflammation and cell cycle regulation. Treatment with sPIF restored early postnatal hippocampal neurogenesis, with beneficial effects on astrocytes, microglia, inflammation, and cell cycle markers. Moreover, treatment with sPIF restored the level of DYRK1A, a protein that is involved in cognitive impairments in DS. In line with the beneficial effects on neurogenesis, perinatal treatment with sPIF was associated with an improvement in working memory in adult Dp(16)1Yey mice. Perinatal treatment with sPIF might be an option for mitigating cognitive impairments in people with DS.

Furanocoumarins promote proteasomal degradation of viral HBx protein and down-regulate cccDNA transcription and replication of hepatitis B virus.

Nucleot(s)ide analogues, the current antiviral treatments against chronic hepatitis B (CHB) infection, are non-curative due to their inability to eliminate covalently closed circular DNA (cccDNA) from the infected hepatocytes. Preclinical studies have shown that coumarin derivatives can effectively reduce the HBV DNA replication. We evaluated the antiviral efficacy of thirty new coumarin derivatives in cell culture models for studying HBV. Furanocoumarins Fc-20 and Fc-31 suppressed the levels of pre-genomic RNA as well as cccDNA, and reduced the secretion of virions, HBsAg and HBeAg. The antiviral efficacies of Fc-20 and Fc31 improved further when used in combination with the hepatitis B antiviral drug Entecavir. There was a marked reduction in the intracellular HBx level in the presence of these furanocoumarins due to proteasomal degradation resulting in the down-regulation of HBx-dependent viral genes. Importantly, both Fc-20 and Fc-31 were non-cytotoxic to cells even at high concentrations. Further, our molecular docking studies confirmed a moderate to high affinity interaction between furanocoumarins and viral HBx via residues Ala3, Arg26 and Lys140. These data suggest that furanocoumarins could be developed as a new therapeutic for CHB infection.

p16 overexpression identifies oncogenic high-risk HPV infection in non-oropharyngeal squamous cell carcinoma of the head and neck.

BACKGROUND: Human papillomavirus (HPV) is an increasing risk factor for cancer. HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable outcome. Blockstaining for p16 is a surrogate marker for HPV+ OPSCC. In oral and laryngeal squamous cell carcinoma (OSCC/LSCC), the relevance of p16 immunohistochemistry, alone or in combination with other cell cycle-related proteins, to identify HPV-driven non-OPSCC is less well understood. METHODS: We stained for p16, pRb, cyclin D1, and p53 in 327 HNSCC. In 310 OPSCC, HPV-status was assessed by HPV DNA PCR. In 119 non-OPSCC, RNA in situ hybridization was additionally performed. HPV-status was correlated with staining patterns, p53 and clinical data. RESULTS: The OPSCC showed blockstaining for p16 in 36%, 8% were equivocal. Of these, HPV-testing was performed in 57%, and 53% were positive for HPV DNA. HPV-association correlated with absence of pRb and cyclin D1 and favorable outcome. In non-OPSCC, 18% showed p16-blockstaining, and 13% showed E6/E7 RNA. Six of seven HPV+ OSCC and 8/8 LSCC lost pRb and cyclin D1. Compared to HPV-negative counterparts, patients with HPV+ cancers had lower rates of alcohol consumption and keratinizing morphology. HPV-positive OSCC had a longer overall survival (p < 0.05). HPV subtype 16 was the most common. CONCLUSIONS: We conclude that HPV-positive non-OPSCC are associated with p16 overexpression and low levels of pRb and cyclin D1. High expression of pRb and cyclin D1 indicates HPV-negativity.

Chemotherapeutic potential of betanin/capecitabine combination targeting colon cancer: experimental and bioinformatic studies exploring NFκB and cyclin D1 interplay.

Introduction: Betanin (C24H26N2O13) is safe to use as food additives approved by the FDA with anti-inflammatory and anticancer effects in many types of cancer cell lines. The current experiment was designed to test the chemotherapeutic effect of the combination of betanin with the standard chemotherapeutic agent, capecitabine, against chemically induced colon cancer in mice. Methods: Bioinformatic approach was designed to get information about the possible mechanisms through which the drugs may control cancer development. Five groups of mice were assigned as, (i) saline, (ii) colon cancer, (iii) betanin, (iv) capecitabine and (v) betanin/capecitabine. Drugs were given orally for a period of six weeks. Colon tissues were separated and used for biological assays and histopathology. Results: In addition, the mRNA expression of TNF-α (4.58-fold), NFκB (5.33-fold), IL-1ß (4.99-fold), cyclin D1 (4.07-fold), and IL-6 (3.55-fold) and protein levels showed several folds increases versus the saline group. Tumor histopathology scores in the colon cancer group (including cryptic distortion and hyperplasia) and immunostaining for NFκB (2.94-fold) were high while periodic-acid Schiff staining demonstrated poor mucin content (33% of the saline group). These pathologic manifestations were reduced remarkably in betanin/capecitabine group. Conclusion: Collectively, our findings demonstrated the usefulness of betanin/capecitabine combination in targeting colon cancer and highlighted that betanin is a promising adjuvant therapy to capecitabine in treating colon cancer patients.

Identification of a new bisindolinone arresting IGROV1 cells proliferation.

In an initial screening, a series of novel Knoevenagel adducts were submitted to the National Cancer Institute for evaluation of antitumor activity in human cell lines. In particular, compound 5f showed remarkable selectivity against IGROV1, an ovarian cancer cell line, without affecting healthy human fibroblast cells. Analyses of cytotoxicity, cell proliferation, cell migration, epigenetic changes, gene expression, and DNA damage were performed to obtain detailed information about its antitumor properties. Our results show that 5f causes proliferation arrest, decrease in motility, histone hyperacetylation, downregulation of cyclin D1 and α5 subunit of integrin ß1 gene transcription. In addition, 5f treatment reduces transcript and protein levels of cyclin D1, which increases sensitivity to ionizing radiation and results in DNA damage comparable to cyclin D1 gene silencing.