Cell Cycle Inhibitory Protein Research Articles

Modelling how plant cell-cycle progression leads to cell size regulation.

Populations of cells typically maintain a consistent size, despite cell division rarely being precisely symmetrical. Therefore, cells must possess a mechanism of "size control", whereby the cell volume at birth affects cell-cycle progression. While size control mechanisms have been elucidated in a number of other organisms, it is not yet clear how this mechanism functions in plants. Here, we present a mathematical model of the key interactions in the plant cell cycle. Model simulations reveal that the network of interactions exhibits limit-cycle solutions, with biological switches underpinning both the G1/S and G2/M cell-cycle transitions. Embedding this network model within growing cells, we test hypotheses as to how cell-cycle progression can depend on cell size. We investigate two different mechanisms at both the G1/S and G2/M transitions: (i) differential expression of cell-cycle activator and inhibitor proteins (with synthesis of inhibitor proteins being independent of cell size), and (ii) equal inheritance of inhibitor proteins after cell division. The model demonstrates that both these mechanisms can lead to larger daughter cells progressing through the cell cycle more rapidly, and can thus contribute to cell-size control. To test how these features enable size homeostasis over multiple generations, we then simulated these mechanisms in a cell-population model with multiple rounds of cell division. These simulations suggested that integration of size-control mechanisms at both G1/S and G2/M provides long-term cell-size homeostasis. We concluded that while both size independence and equal inheritance of inhibitor proteins can reduce variations in cell size across individual cell-cycle phases, combining size-control mechanisms at both G1/S and G2/M is essential to maintain size homeostasis over multiple generations. Thus, our study reveals how features of the cell-cycle network enable cell-cycle progression to depend on cell size, and provides a mechanistic understanding of how plant cell populations maintain consistent size over generations.

Rice Mitogen-Activated Protein Kinase regulates serotonin accumulation and interacts with cell cycle regulators under prolonged UV-B exposure

Stress conditions such as UV-B exposure activates MAPKs in Arabidopsis and rice. UV-B radiation is hazardous to plant as it causes photosystem disruption, DNA damage and ROS generation. Here we report its effect on biological pathways by studying the global changes in transcript profile in rice seedling exposed to UV-B radiation for 1 h and 16 h. Short UV-B exposure (1 h) led to moderate changes, while a drastic change in transcript landscape was observed after long term UV-B exposure (16 h) in rice seedlings. Prolonged UV-B exposure negatively impacts the expression of cell cycle regulating genes and several other metabolic pathways in developing seedlings. MAP kinase signaling cascade gets activated upon UV-B exposure similar to reports in Arabidopsis indicating conservation of its function in both dicot and monocot. Expression analysis in inducible overexpression transgenic lines of MPK3 and MPK6 shows higher transcript abundance of phytoalexin biosynthesis gene like Oryzalexin D synthase and Momilactone A synthase, along with serotonin biosynthesis genes. An accumulation of serotonin was observed upon UV-B exposure and its abundance positively correlates with the MPK3 and MPK6 transcript level in the respective over-expression lines. Interestingly, multiple cell cycle inhibitor proteins including WEE1 and SMR1 interact with MPK3 and MPK6 thus, implying a major role of this pathway in cell cycle regulation under stress condition. Overall overexpression of MPK3 and MPK6 found to be detrimental for rice as overexpression lines shows higher cell death and compromised tolerance to UV-B.

Cell Cycle Control by Optogenetically Regulated Cell Cycle Inhibitor Protein p21.

The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Human primary myoblasts derived from paraspinal muscle reflect donor age as an experimental model of sarcopenia

BackgroundLow back pain is a general phenomenon of aging, and surgery is an unavoidable choice to relieve severe back pain. The discarded surgical site during surgery is of high value for muscle and muscle-related research. This study investigated the age-dependent properties of patients' paraspinal muscles at the cellular level. MethodsTo define an association of paraspinal muscle degeneration with sarcopenia, we analyzed lumbar paraspinal muscle and myoblasts isolated from donors of various ages (25–77 years). Preoperative evaluations were performed by bioimpedance analysis using the InBody 720, magnetic resonance (MR) imaging of the lumbar spine, and lumbar extension strength using a lumbar extension dynamometer. In addition, the growth and differentiation capacity of myoblasts obtained from the donor was determined using proliferation assay and western blotting. ResultsThe cross-sectional area of the lumbar paraspinal muscle decreased with age and was also correlated with the appendicular skeletal muscle index (ASM/height2). Human primary myoblasts isolated from paraspinal muscle preserved their proliferative capacity in vitro, which tended to decrease with donor age. The age-dependent decline in myoblast proliferation was correlated with levels of cell cycle inhibitory proteins (p16INK4a, p21CIP1, and p27KIP1) associated with cellular senescence. Primary myoblasts isolated from younger donors differentiated into multinucleate myotubes earlier and at a higher rate than those from older donors in vitro. Age-dependent decline in myogenic potential of the isolated primary myoblasts was likely correlated with the inactivation of myogenic transcription factors such as MyoD, myogenin, and MEF2c. ConclusionsMyoblasts isolated from human paraspinal muscle preserve myogenic potential that correlates with donor age, providing an in vitro model of sarcopenia.

SEMA6C: a novel adhesion-independent FAK and YAP activator, required for cancer cell viability and growth

Transmembrane semaphorins are signaling molecules, controlling axonal wiring and embryo development, which are increasingly implicated in human diseases. Semaphorin 6C (Sema6C) is a poorly understood family member and its functional role is still unclear. Upon targeting Sema6C expression in a range of cancer cells, we observed dramatic growth suppression, decreased ERK phosphorylation, upregulation of cell cycle inhibitor proteins p21, p27 and p53, and the onset of cell senescence, associated with activation of autophagy. These data are consistent with a fundamental requirement for Sema6C to support viability and growth in cancer cells. Mechanistically, we unveiled a novel signaling pathway elicited by Sema6C, and dependent on its intracellular domain, mediated by tyrosine kinases c-Abl and Focal Adhesion Kinase (FAK). Sema6C was found in complex with c-Abl, and induced its phosphorylation, which in turn led to FAK activation, independent of cell–matrix adhesion. Sema6C-induced FAK activity was furthermore responsible for increased nuclear localization of YAP transcriptional regulator. Moreover, Sema6C conferred YAP signaling-dependent long-term cancer cell survival upon nutrient deprivation. In conclusion, our findings demonstrate that Sema6C elicits a cancer promoting-signaling pathway sustaining cell viability and self-renewal, independent of growth factors and nutrients availability.

Design, synthesis, and biological activities of 3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-ones

Novel (Z)-3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-one derivatives were designed and synthesized to find chemotherapeutic agents. Derivative 9 was selected based on its clonogenicity against cancer cells and synthetic yield for further biological experiments. It showed decreases in aurora kinase A, B, and C phosphorylation from western blot analysis. Derivative 9 upregulated the expression of G1 cell cycle inhibitory proteins including p21 and p27, and G1 progressive cyclin D1, and downregulated G1-to-S progressive cyclins, resulting in cell cycle arrest at the G1/S boundary. It stimulated the cleavage of caspase-9, -3, -7, and poly (ADP-ribose) polymerase, resulting in triggering apoptosis through a caspase-dependent pathway. In addition, derivative 9 inhibited in vivo tumor growth in a syngeneic tumor implantation mouse model. The findings of this study suggest that derivative 9 can be considered as a lead compound for chemotherapeutic agents.

Antrodia salmonea extract inhibits cell proliferation through regulating cell cycle arrest and apoptosis in prostate cancer cell lines.

Antrodia salmonea (AS) is a fungus, which belongs to a fungal family of Taiwanofungus salmoneus with the features of anti-oxidant, anti-inflammatory, and anticancer. Recent studies have shown that AS has anti-cancer functions in ovarian and breast cancer. However, the effects of AS on prostate cancer (PCa) proliferation remain unknown. Therefore, we investigated the role of AS in PCa proliferation through apoptosis, and cell cycle regulation in PCa cell lines. Our results showed that Antrodia salmonea extract (ASE) inhibited PCa cells growth with a dose-dependent manner. In addition, ASE decreased the anchorage-independent growth formation ability in PC3 cells. Moreover, ASE-induced cell growth inhibition in PCa cells (DU145, PC3) was correlated to decreased cell cycle-related proteins such as cyclin A/B and cyclin-dependent kinase CDK1/2/4, and increased cell cycle inhibitor proteins p21. Besides, ASE decreased the total protein level of epidermal growth factor receptor and its downstream signaling pathways Akt and Erk in both PCa cells. We found that apoptotic markers such as cleaved-PARP protein levels increased significantly in DU145 cells indicating ASE might induce apoptosis. In conclusion, our results suggest that ASE may have the ability to induce PCa cell death through regulating cell cycle arrest and apoptosis pathways.

Profiling Activins and Follistatin in Colorectal Cancer According to Clinical Stage, Tumour Sidedness and Smad4 Status.

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded (n = 90 patients) alongside fresh (n = 40 patients) specimen cohorts. Activin β-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours (n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The βA-subunit and activin-A increased, whilst βB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro, activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein.

P53, p21, and cyclin d1 protein expression patterns in patients with breast cancer.

Background and Aim:The mutation in the wild-type tumor suppressor gene p53 is the most common genetic change in human tumors. In addition, the normal function of p21, which is both antiproliferative and an inhibitor of the cell cycle, is disrupted in some types of cancer. Meanwhile, cyclin D1 is a member of the cyclin protein family that is involved in regulating cell cycle progression. This study aimed to assess the expressions of the cell cycle inhibitory proteins p21, cyclin D1, and tumor suppressor gene p53, as well as their influence on the expressed histopathological changes in breast cancer tissues.Materials and Methods:Overall, 40 breast tissue specimens were investigated in this study, 30 of which were cancerous, while 10 were healthy tissues. p53, p21, and cyclin D1 expression patterns were detected using an immunohistochemistry (IHC) system.Results:The IHC reactions for p53 were positively observed in 27/30 (90%) cancerous tissues, compared with 2/10 (20%) normal breast tissues. For p21, reactions were observed in 28/30 (93.33%) cancerous tissues and 3/10 (30%) control tissues. For cyclin D1, reactions were observed in 25/30 (83.33%) cancerous tissues and 1/10 (10%) control tissues. The differences between the breast cancer tissues and the control tissues were statistically highly significant (p<0.01).Conclusion:The high expression rates of p21, cyclin D1, and p53 in malignant breast cancer cells with little or no regulatory role might imply mutational events in these proteins operating in concert with a variety of other genetic mutations in these tissues, which may play a molecular role in the development and/or progression of breast carcinogenesis.

Cdkn1a transcript variant 2 is a marker of aging and cellular senescence.

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo, variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice.

Fatty acid‐binding Protein‐3 (FABP3) is a Novel Regulator of Endothelial Function

Introduction Peripheral Artery Disease (PAD) is characterized by plaque deposition in the arteries that supply blood to the lower extremities. Endothelial dysfunction plays a key role in the development of PAD. Recently, we demonstrated a direct correlation between elevated serum FABP3 (Fatty acid-binding protein-3) and PADs’ severity in patients. However, the role of FABP3 in the endothelium is not known. To understand the pathophysiological role of endothelial FABP3, we hypothesized that FABP3 is a novel regulator of endothelial function. Methods and Results: To test our hypothesis, we cultured human umbilical vein endothelial cells (HUVECs) and confirmed the basal expression of FABP3. HUVECs were then treated with different doses (0, 10, 20, 50 and 80 mg/ml) of oxidized low-density lipoprotein (oxLDL), which is an in vitro model to study atherosclerosis-associated endothelial dysfunction, for 24 hours and the expression level of FABP3 was measured. To our surprise, we observed a significant reduction in the expression level of FABP3 in oxLDL-treated cells in a dose-dependent manner. Next, we measured the expression level of FABP3 in HUVECs using 80 mg/ml of oxLDL and evaluated for FABP3 expression at 0, 6, 12 and 24 hours. We observed a significant reduction in FABP3 expression for 6, 12 and 24 hours of oxLDL treatment with maximum reduction at 24 hours of oxLDL treatment. We also measured the expression level of the fatty acid-binding proteins FABP4 and FABP5 following similar oxLDL treatment to HUVECs and observed a significant down-regulation for both proteins. Next, we transfected HUVECs with siFABP3 or scrambled control to silence FABP3 and confirmed silencing. Loss of FABP3 in HUVECs caused reduced proliferation, which was associated with increased expression of the cell cycle inhibitor protein p21. Conclusion: Our preliminary findings support our hypothesis and warrant investigation. Our data demonstrate that FABP3 is basally expressed in endothelial cells and regulates proliferation. We also, for the first time, report that oxLDL-treatment significantly reduced FABP3 expression in a dose- and time-dependent manner in endothelial cells in vitro. Studies are underway to elucidate the potential role of FABP3 in endothelial cells.

Abstract PO-014: 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors

Abstract Background: Peptide receptor radionuclide therapy (PRRT) is a novel radiotherapy approved for gastroenteropancreatic neuroendocrine tumors (GEP-NETs); however, PRRT has a relatively low treatment response rate of 20-30%. We hypothesize that addition of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a ribonucleotide reductase (RnR) inhibitor that inhibits the rate-limiting enzyme of DNA synthesis and repair, can improve radiotherapy outcomes. The purpose of this study is to determine the therapeutic rationale of RnR-targeted therapy and evaluate 3-AP as a radiosensitizer in treating GEP-NET in vitro and in vivo. Methods: We utilized two human GEP-NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) Cells were treated with 3-AP for 96h and cell proliferation assays were performed using a sulforhodamine B (SRB) kit. Next, cells were treated with 3-AP for 24, 48, 72, 96h and cell cycle analyses were performed with Beckman Coulter cell cycle kit and quantified by flow cytometry. Expression of cell cycle arrest markers (i.e., pCdk-1, cyclin D1) was assessed by immunoblotting. (ii) To determine radiosensitivity in vitro, cells were treated with 3-AP for 16h, followed by X-ray radiotherapy (XRT), and incubation in drug-free medium. Cell survival was assessed by colony formation assay and quantified by SRB kit at 14d post-XRT. Apoptosis was measured at 72 and 96h post-XRT, and expression of apoptotic proteins (i.e., cleaved PARP and cleaved CASPASE 3) was examined by immunoblotting. (iii) To determine radiosensitivity in vivo, we randomized 20 nude mice bearing QGP-1 xenografts into four groups: control; 3-AP (10mg/Kg); XRT (2 Gy); and combination (XRT immediately followed by 3-AP); each mouse received 5 consecutive treatments. Results: (i) A dose of 2500nM 3-AP arrested cells before entering G2/M phase, significantly reducing percentage of G2/M cells from 8.4% to 0.5% at 72h. Concurrently, we observed increased expression of checkpoint proteins (pRb, pCdk1, pCdk2, pCdc2) and decreased expression of cell cycle inhibitory proteins (cyclin D1, p21, p27 Kip). (ii) Exposure of QGP-1 and BON cells to 3-AP prior to XRT resulted in a synergistic decrease (i.e., 85% and 90%, respectively) in clonogenicity. Furthermore, the combination of 3-AP and XRT increased apoptosis 5-fold and cleaved PARP expression in BON cells. (iii) A significant decrease in tumor size was noted in mice with XRT alone compared to control (146mm2 vs. 280 mm2, respectively); combination treatment (3-AP + XRT) demonstrated a further 48% decrease in tumor size compared to XRT alone. There was no significant decrease in tumor size with 3-AP treatment alone. Conclusion: 3-AP treatment prior to XRT therapy resulted in significant increase in apoptosis in vitro and decrease in tumor size in vivo, suggesting synergistic cytotoxicity and radiosensitivity. Selective RNR inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs. Citation Format: Zeta Chow, Jeremy Johnson, Aman Chauhan, Tadahide Isumi, Lowell Anthony, Courtney M. Townsend, B. Mark Evers, Piotr Rychahou. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-014.

Herpetic Oncogenic Virus Co ? Infections Impinge Cell Cycle Regulatory Gene Expressions in Oral Cavity Malignancies

Oral squamous cell carcinoma (OSCC) is the most encountered type of oral malignant neoplasm. P27 is a nuclear cell cycle inhibitory protein, that can be transported to the cytoplasm to deactivate it's role, Similarly the mechanism is used by cancer cells to unlock cell cycle arrest. In reaction to damage to DNA caused, either by the cell cycle arresting or apoptosis, the tumor suppressor p73 is activated. Epstein-Barr virus (EBV) that infects human beings has oncogenic capacity in different kinds of human neoplasia. Human Cytomegalovirus (HCMV) is a prevalent opportunistic pathogen that causes severe disease in various immunosuppressed patient. The aim of this study is to investigate the impact of EBV and HCMV contagions togetherness on p27 and p73 gene products in Oral cavity carcinomas. This study included a total number of seventy three (73) formalin-fixed, paraffin-embedded tissue blocks from OSCC patients. The confirmatory re-examination for histopathological samples were donea Chromogenic In Situ Hybridization assay (CISH) for EBV-EBERs and HCMV-PP65 DNA localization and immunohistochemistry assay (IHC) for p73 and p27 gene expression were performed). The results illustrate that the presence of these two viruses in OSCC were at highest percentage in undifferentiated grade (74.47%%). On the other hand, co-presence of p27 and p73 was highest in undifferentiated group (70.27%).

The prognostic significance of p16 expression pattern in diffuse gliomas.

BackgroundCDKN2A is a tumor suppressor gene that encodes the cell cycle inhibitor protein p16. Homozygous deletion of the CDK-N2A gene has been associated with shortened survival in isocitrate dehydrogenase (IDH)–mutant gliomas. This study aimed to analyze the prognostic value of p16 and to evaluate whether p16 immunohistochemical staining could be used as a prognostic marker to replace CDKN2A genotyping in diffuse gliomas.Methodsp16 immunohistochemistry was performed on tissue microarrays of 326 diffuse gliomas with diagnoses that reflected IDH-mutations and 1p/19q codeletion status. The results were divided into three groups (negative, focal expression, overexpression) according to the presence and degree of p16 expression. Survival analysis was performed to assess the prognostic value of p16 expression.ResultsA loss of p16 expression predicted a significantly worse outcome in all glioma patients (n = 326, p < .001), in the IDH-mutant glioma patients (n = 103, p = .010), and in the IDH-mutant astrocytoma patients (n = 73, p = .032). However, loss of p16 expression did not predict the outcome in the IDH-wildtype glioma patients (n = 223, p = .121) or in the oligodendroglial tumor patients with the IDH-mutation and 1p/19q codeletion (n = 30, p = .457). Multivariate analysis showed the association was still significant in the IDH-mutant glioma patients (p = .008; hazard ratio [HR], 2.637; 95% confidence interval [CI], 1.295 to 5.372) and in the IDH-mutant astrocytoma patients (p = .001; HR, 3.586; 95% CI, 1.649 to 7.801). Interestingly, patients who presented with tumors with p16 overexpression also had shorter survival times than did patients with tumors with p16 focal expression in the whole glioma (p < .001) and in IDH-mutant glioma groups. (p = .046).ConclusionsThis study suggests that detection of p16 expression by immunohistochemistry can be used as a useful surrogate test to predict prognosis, especially in IDH-mutant astrocytoma patients.

Transmembrane and Immunoglobulin Domain Containing 1, a Putative Tumor Suppressor, Induces G2/M Cell Cycle Checkpoint Arrest in Colon Cancer Cells

Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target.

HPO iron chelator, CP655, causes the G1/S phase cell cycle block via p21 upregulation.

Iron is known not only for its importance in cellular and metabolic pathways but also for its role in causing cellular toxicities such as production of reactive oxygen species and growth of pathogens. The inability of the human body to physiologically excrete excess iron highlights the need to develop a cheap yet effective iron chelator. This study provides initial evidence of the therapeutic and prophylactic properties of 3‐hydroxypyridin‐4‐one (HPO) chelators in murine collagen‐induced arthritis. To determine whether these chelators would be effective on human cells, we tested a panel of different HPO chelators and identified 7‐diethylamino‐N‐((5‐hydroxy‐6‐methyl‐4‐oxo‐1,4‐dihydropyridin‐3‐yl)methyl)‐N‐methyl‐2‐oxo‐chromen‐3‐carboxamide (CP655) as the most effective compound targeting human CD4+ T cells. Treatment with CP655 causes significant inhibition of cell proliferation and production of inflammatory cytokines such as interferon‐γ and interleukin‐17. Microarray analysis revealed dysregulation in cell cycle‐related genes following CP655 treatment. This was validated by flow cytometry demonstrating a G1/S phase block caused by CP655. Finally, mechanistic experiments revealed that the chelator may be causing an upregulation of the cell cycle inhibitor protein CDKN1A (p21) as a possible mechanism of action. In conclusion, this study demonstrates that HPO chelators could prove to have therapeutic potential for diseases driven by excessive T cell proliferation.

Extrinsic modulation of integrin α6 and progenitor cell behavior in mesenchymal stem cells.

Mesenchymal stem cells (MSC) are heterogeneous cells of complex nature that show different potentials while different culture conditions can modify their functionalities through interactions with the microenviroment. Here, we found that bone marrow (BM) MSC from different donor sources and passages that expressed higher levels of α6 integrin subunit (ITGA6), showed higher clonogenicity, migration and differentiation potential. ITGA6 showed important roles improving these potentials and regulating proliferation through protein kinase B (AKT) pathway and cell cycle inhibitor proteins p53 and p21. Moreover, ITGA6 downregulation impaired migration. Cell confluence regulated ITGA6, increasing its expression in low density cultures and decreasing in high density cultures. Besides, ITGA6- cells expressed ITGA6 when seeded at low densities. We found higher ITGA6 expression on fibronectin substrates at lower confluency. Fibronectin increased proliferation, clonogenicity, activation of AKT, decreased cell cycle inhibitor proteins and augmented growth factors expression. Spheres-derived MSC showed higher ITGA6 expression and enhanced potentials for migration, clonogenicity and proliferation. In conclusion, though there is an intrinsic regulation of ITGA6 expression, associated to the progenitor potential of BM-MSC, this expression is regulated by culture conditions and is translated in changes in cell behavior and proliferation. This knowledge could be used to enhance the potential of BM-MSC for clinical application.

Baicalein suppresses the proliferation and invasiveness of colorectal cancer cells by inhibiting Snail‑induced epithelial‑mesenchymal transition.

Scutellaria baicalensis (S. baicalensis) is a plant that is widely used for medicinal purposes. Baicalein, one of the primary bioactive compounds found in S. baicalensis, is thought to possess antitumor activity, although the specific mechanisms remain unclear. Therefore, the present study aimed to evaluate the ability of baicalein to disrupt the proliferation and metastatic potential of colorectal cancer (CRC) cells; a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometric method was employed for the identification of baicalein in an S. baicalensis aqueous extract and in rat plasma. To investigate the effects of baicalein, Cell Counting Kit-8 (CCK-8), western blotting, wound-healing and Transwell assays were performed. The data indicated that baicalein was absorbed into the blood and was able to effectively disrupt the proliferation, migration and invasion abilities of CRC cells in a dose- and time-dependent manner. Baicalein treatment was also revealed to decrease the expression of epithelial-mesenchymal transition (EMT)-promoting factors including vimentin, Twist1, and Snail, but to upregulate the expression of E-cadherin in CRC cells. The expression levels of cell cycle inhibitory proteins p53 and p21 also increased following baicalein treatment. In addition, Snail-induced vimentin and Twist1 upregulation, as well as E-cadherin downregulation, were reversed following treatment with baicalein. In conclusion, the results of the present study indicate that baicalein may suppress EMT, at least in part, by decreasing Snail activity.

Albumin promotes proliferation of G1 arrested serum starved hepatocellular carcinoma cells

Albumin is the most abundant plasma protein and functions as a transport molecule that continuously interacts with various cell types. Because of these properties, albumin has been exploited by the pharmaceutical industry to improve drug delivery into target cells. The immediate effects of albumin on cells, however, require further understanding. The cell interacting properties and pharmaceutical applications of albumin incentivises continual research into the immediate effects of albumin on cells. The HepG2/C3A hepatocellular carcinoma cell line is used as a model for studying cancer pathology as well as liver biosynthesis and cellular responses to drugs. Here we investigated the direct effect of purified albumin on HepG2/C3A cell proliferation in the absence of serum, growth factors and other serum originating albumin bound molecules. We observed that the reduced cell counts in serum starved HepG2/C3A cultures were increased by the inclusion of albumin. Cell cycle analysis demonstrated that the percentage of cells in G1 phase during serum starvation was reduced from 86.4 ± 2.3% to 78.3 ± 3.2% by the inclusion of albumin whereas the percentage of cells in S phase was increased from 6.5 ± 1.5% to 14.3 ± 3.6%. A significant reduction in the cell cycle inhibitor protein, P21, accompanied the changes in the proportions of cell cycle phases upon treatment with albumin. We have also observed that the levels of dead cells determined by DNA fragmentation and membrane permeabilization caused by serum starvation (TUNEL: 16.6 ± 7.2%, ethidium bromide: 13.8 ± 4.8%) were not significantly altered by the inclusion of albumin (11.6 ± 10.2%, ethidium bromide: 16.9 ± 8.9%). Therefore, the increase in cell number was mainly caused by albumin promoting proliferation rather than protection against cell death. These primary findings demonstrate that albumin has immediate effects on HepG2/C3A hepatocellular carcinoma cells. These effects should be taken into consideration when studying the effects of albumin bound drugs or pathological ligands bound to albumin on HepG2/C3A cells.

Yes-associated protein regulates podocyte cell cycle re-entry and dedifferentiation in adriamycin-induced nephropathy

Podocytes are terminally differentiated cells with little proliferative capacity. The high expression levels of cell cycle inhibitory proteins, including p21, p27, and p57, play an important role in maintaining the low level of proliferation of mature podocytes. In the present study, we aimed to explore the role of yes-associated protein (YAP) signalling in adriamycin-induced podocyte re-entry into the cell cycle and dedifferentiation. Proliferating cell nuclear antigen (PCNA)-, cyclin-dependent kinase 4 (CDK4)-, and Cyclin D1-positive podocytes were found in mice with adriamycin-induced nephropathy. In vitro, adriamycin administration increased the percentage of cells in S phase and the upregulation of mesenchymal-related marker proteins. CDK4 and cyclin D1 were significantly up-regulated after incubation with adriamycin. Overexpression of YAP in podocytes promoted their entry into the cell cycle; up-regulated cyclin D1, desmin, and snail2 expression and down-regulated Wilms’ tumour 1 (WT1) and nephrin production. Recombinant murine FGF-basic induced podocytes to re-enter the cell cycle, inhibited WT1 and nephrin, and increased desmin and snail2 expression. Pretreating podocytes with verteporfin, an inhibitor of YAP/ TEA domain transcription factor (TEAD), decreased the adriamycin-induced overexpression of cyclin D1 and reduced the ratio of S-phase podocytes. This result was further verified by knocking down YAP expression using RNA interference. In conclusion, adriamycin induced podocytes to re-enter the cell cycle via upregulation of CDK4 and cyclin D1 expression, which was at least partly mediated by YAP signalling. Re-entry into the cell cycle induced the over-expression of mesenchymal markers in podocytes.