P27 Kip1 Protein Research Articles

The PI3K/Akt/FOXO3a pathway regulates regeneration following spinal cord injury in adult rats through TNF-α and p27kip1 expression.

The aim of the present study was to elucidate the expression and role of the phosphatidylinositol 3‑kinase (PI3K)/Akt/forkhead boxO3 (FOXO3a) pathway in the regeneration of the spinal cord following spinal cord injury (SCI), and its regulatory effect on tumor necrosis factor (TNF)-α and cyclin-dependent kinase inhibitor 1B (p27kip1) expression. Firstly, in a Sprague-Dawley rat model of SCI, western blot analysis revealed that the protein levels of PI3K, phosphorylated Akt and FOXO3a were markedly inhibited compared with those in the sham control group. Invitro experiments were also conducted, in which primary dissociated cultures of rat dorsal spinal cord cells were induced with lipopolysaccharide (LPS;4µg/ml). The downregulation of PI3K using LY294002 markedly suppressed cell viability, reduced the protein levels of FOXO3a and p27kip1, and increased TNF-α protein production in the LPS-induced spinal cord cells. In addition, when the LPS-induced spinal cord cells were infected with FOXO3a adenoviral vectors, the overexpression of FOXO3 markedly promoted cell proliferation, activated p27kip1 protein levels and inhibited TNF-α protein production in the spinal cord cells. These results suggest that the PI3K/Akt/FOXO3a pathway regulates regeneration following SCI in adult rats via its modulatory effects on TNF-α and p27kip1 expression.

Expression of cyclin D1 correlates with p27KIP1 and regulates the degree of oral dysplasia and squamous cell carcinoma differentiation

The aim of this study was to identify an association or link between cyclin D1 and p27KIP1 protein expression and dysplastic changes or progression. Oral mucosal biopsies with a diagnosis of non-neoplastic tissue (gingivitis) (n = 10), mild to moderate oral epithelial dysplasia (n = 12), and oral squamous cell carcinoma (n = 11) were evaluated by using immunohistochemistry. Scanning software was used to determine cyclin D1 and p27KIP1 intensity of expression, location, and pattern. A significant increase in expression of cyclin D1 and a decrease in expression of p27KIP1 proteins were identified in oral epithelial dysplasia and less differentiated oral squamous cell carcinoma (OSCC). There was a more diffuse distribution of cyclin D1 protein expression extending from the basal cell layer into the prickle cell layers in epithelial dysplasia and extending within all epithelial layers in OSCC. Cases of oral epithelial dysplasia had moderate infrequent expression of p27KIP1. There were no p27KIP1-positive cells in OSCC. The percentage of cells with both nuclear and cytoplasmic cyclin D1 staining was higher in OSCC specimens than control groups and oral epithelial dysplasia. The expression of both cyclin D1 and p27KIP1 correlated with the grade of oral epithelial dysplasia and degree of OSCC differentiation. The results obtained will be verified through a basic follow-up of the cases to determine the prognosis/progression of oral dysplasia.

Cold-inducible RNA-binding protein (CIRP) induces translation of the cell-cycle inhibitor p27Kip1.

The CDK inhibitor p27Kip1 plays a central role in controlling cell proliferation and cell-cycle exit. p27Kip1 protein levels oscillate during cell-cycle progression and are regulated by mitogen or anti-proliferative signaling. The abundance of the protein is frequently determined by post-transcriptional mechanisms including ubiquitin-mediated proteolysis and translational control. Here, we report that the cold-inducible RNA-binding protein (CIRP) selectively binds to the 5′ untranslated region of the p27Kip1 mRNA. CIRP is induced, modified and relocalized in response to various stress stimuli and can regulate cell survival and cell proliferation particularly during stress. Binding of CIRP to the 5′UTR of the p27Kip1 mRNA significantly enhanced reporter translation. In cells exposed to mild hypothermia, the induction of CIRP correlated with increased translation of a p27Kip1 5′UTR reporter and with the accumulation of p27Kip1 protein. shRNA-mediated CIRP knockdown could prevent the induction of translation. We found that p27Kip1 is central for the decreased proliferation at lower temperature, since p27Kip1 KO mouse embryonic fibroblasts (MEFs) hardly increased their doubling time in hypothermic conditions, whereas wild-type MEFs significantly delayed proliferation in response to cold stress. This suggests that the CIRP-dependent p27Kip1 upregulation during mild hypothermia contributes to the cold shock-induced inhibition of cell proliferation.

Phospholipase Cβ1 regulates proliferation of neuronal cells.

Cells have developed lineage-specific mechanisms to control proliferation and drive morphologic changes upon differentiation. A hallmark of differentiation is the assembly of signaling molecules that transduce extracellular signals, such as the production of the G protein-regulated enzyme phospholipase Cβ (PLCβ), which generates calcium signals from sensory stimuli. We found that in most cancerous cell lines there is positive correlation between PLCβ1 levels and cell proliferation. In cells of neuronal lineage, however, reducing PLCβ1 levels increases the rate of proliferation. Using a combination of biochemical and biophysical methods, we find that, in the G1 phase, a cytosolic population of PLCβ1 associates with cyclin-dependent kinase 16 (CDK16), a neuron-specific enzyme that is activated by cyclin Y to inactivate the antioncogenic protein p27Kip1. Binding of PLCβ1 directly inhibits CDK16 activity and in turn reduces the ability of cells to enter the S phase. Activation of Gαq by carbachol causes movement of PLCβ from the cytosol to the plasma membrane, reducing its association with CDK16. Similarly, the overexpression of activated Gαq moves PLCβ1 to the membrane, reverses G1 arrest, and promotes proliferation, thereby connecting external stimuli with cell proliferation. Our results present a model in which the transient high expression of PLCβ1 that occurs at the onset of differentiation arrests cells in the G1 phase through its association with CDK16 and allows CDK16 to transition to its postmitotic function of neurite outgrowth and trafficking of synaptic vesicles. The novel role of PLCβ1 in neuronal cell proliferation offers a unique interaction that can be manipulated to guide cells into a neuronal phenotype or to develop therapies for neuroblastomas.-Garwain, O., Valla, K., Scarlata, S. Phospholipase Cβ1 regulates proliferation of neuronal cells.

Morin inhibits PDGF-induced proliferation, migration, and invasion of vascular smooth muscle cells via modulating p27KIP1, AKT, and MMP-9 activities.

Hyper-proliferation and migration of vascular smooth muscle cells (VSMCs) are closely associated with atherosclerosis. Recently, the flavonol morin has been reported to exhibit potent anti-oxidant and anti-inflammatory activities. Therefore, we investigated molecular mechanisms of morin in VSMCs stimulated by PDGF. Morin effectively inhibited PDGF-stimulated proliferation of VSMCs through a G1 cell-cycle arrest, leading to down-regulation of CDK2, CDK4, cyclin D1, and cyclin E proteins. Interestingly, PDGF markedly down-regulated p27KIP1 protein expression; however, morin treatment restored the p27KIP1expression to the basal level. Morin did not affect phosphorylation of MAPKs (ERK, p38, and JNK); however, phosphorylation of AKT was dramatically suppressed by morin in PDGF-stimulated VSMCs. Using the PI3K inhibitor, LY294002, we revealed that AKT is a key regulator in the inhibitory mechanism of morin against PDGF-induced proliferation of VSMCs. Morin disturbed migratory and invasive potential of VSMCs via suppression of matrix metalloproteinase-9 (MMP-9) activity. Using electrophoretic mobility shift assays, we verified that NF-κB, AP-1, and Sp-1 transcription factors are implicated in the mode of action of morin, which suppresses the MMP-9 activity in PDGF-induced VSMCs. Based on the results, we believe that morin may be a potential therapeutic agent for atherosclerosis without negative side effect.

Expression of p27Kip1, A Cell Cycle Repressor Protein with Dual Roles for Both Cancer Prevention and Promotion, Is Regulated Primarily at the Level of Unusual p27Kip1 mRNA—A Short Concept Proposal

The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0–20 μM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

Upregulation of miR-221 and -222 in response to increased extracellular signal-regulated kinases 1/2 activity exacerbates neointimal hyperplasia in diabetes mellitus

Background and aimsDiabetes is associated with accelerated arterial intimal thickening that contributes to the increased cardiovascular disease seen in this population. In healthy arteries, intimal thickening is inhibited by elevated levels of the cyclin-dependent kinase inhibitor, p27Kip1, and intimal thickening is promoted by activation of the mammalian Target of Rapamycin to promote degradation of p27Kip1 protein. Recently, we reported that two microRNAs, miR-221 and -222, which promote intimal thickening via down-regulation of mRNA encoding p27Kip1, are elevated in the arteries of diabetic patients. To determine if these miRNAs are critical to the increased intimal thickening under diabetic conditions, we examined the regulation of p27Kip1in a mouse model of diabetes. MethodsComparisons of p27Kip1 signaling in NONcNZO10 mice fed a diabetogenic versus control diet were performed using immunochemistry and real-time PCR. ResultsVascular smooth muscle cells and arteries of diabetic mice exhibited decreased levels of p27Kip1 that derived from destabilization of p27Kip1 mRNA in an extracellular signal response kinase-1/2 (ERK-1/2) dependent manner. The activity of ERK-1/2 is increased in the arteries of diabetic mice and promotes an increase in miR-221 and -222. Inhibition of miR-221 and -222 restores normal levels of p27Kip1 mRNA and protein in the arteries of diabetic mice and reduces intimal thickening following wire injury. ConclusionsThese data suggest diabetes is accompanied by increases in arterial miR-221 and -222 expression that promotes intimal thickening. Inhibition of the increased miR-221 and -222 may be efficacious in the prevention of the cardiovascular complications of diabetes.

The relationship between cervical precancerous lesion galectin-3 and p27 protein expression and clinical prognosis.

The relationship between galectin-3 and p27kip1 protein expression levels in cervical precancerous lesions and clinical prognosis were studied. A total of 74 patients with cervical intraepithelial neoplasia [(CIN), 20 cases classified as stage I, 24 cases as stage II and 30 cases as stage III] were enrolled in this study. Tissue galectin-3, p27kip1, vascular endothelial growth factor (VEGF)-2 and cyclin D protein levels were detected via immunohistochemical staining and reverse transcription polymerase chain reaction (PCR). Follow-up median duration was 13.5 months and recurrence rate was determined. Galectin-3, VEGF-2, and cyclin D expression was elevated in patients with higher stage CIN, whereas p27kip1 showed the opposite trend (p<0.05). During follow-up, there were 3 cases (15.0%) of recurrence in the CIN-I group, 5 cases (20.8%) in the CIN-II group and 9 cases (30.0%) in CIN-III the group. No significant difference in recurrence rate was noted among the groups (p>0.05). The upregulation of galectin-3 and downregulation of p27kip1 in CIN tissues may be related to tumor progression. This phenomenon will require further verification.

ChIP-Seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts.

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

Human mesenchymal factors induce rat hippocampal- and human neural stem cell dependent oligodendrogenesis.

The generation of new oligodendrocytes is essential for adult brain repair in diseases such as multiple sclerosis. We previously identified the multifunctional p57kip2 protein as a negative regulator of myelinating glial cell differentiation and as an intrinsic switch of glial fate decision in adult neural stem cells (aNSCs). In oligodendroglial precursor cells (OPCs), p57kip2 protein nuclear exclusion was recently found to be rate limiting for differentiation to proceed. Furthermore, stimulation with mesenchymal stem cell (MSC)-derived factors enhanced oligodendrogenesis by yet unknown mechanisms. To elucidate this instructive interaction, we investigated to what degree MSC secreted factors are species dependent, whether hippocampal aNSCs respond equally well to such stimuli, whether apart from oligodendroglial differentiation also tissue integration and axonal wrapping can be promoted and whether the oligodendrogenic effect involved subcellular translocation of p57kip2. We found that CC1 positive oligodendrocytes within the hilus express nuclear p57kip2 protein and that MSC dependent stimulation of cultured hippocampal aNSCs was not accompanied by nuclear p57kip2 exclusion as observed for parenchymal OPCs after spontaneous differentiation. Stimulation with human MSC factors was observed to equally promote rat stem cell oligodendrogenesis, axonal wrapping and tissue integration. As forced nuclear shuttling of p57kip2 led to decreased CNPase- but elevated GFAP expression levels, this indicates heterogenic oligodendroglial mechanisms occurring between OPCs and aNSCs. We also show for the first time that dominant pro-oligodendroglial factors derived from human fetal MSCs can instruct human induced pluripotent stem cell-derived NSCs to differentiate into O4 positive oligodendrocytes.

ARHI is a novel epigenetic silenced tumor suppressor in sporadic pheochromocytoma.

Pheochromocytoma (PCC) is related to germline mutations in 12 susceptibility genes. Although comparative genomic hybridization array has revealed some putative tumor suppressor genes on the short arm of chromosome 1 that are likely to be involved in PCC tumorigenesis, the molecules involved, except for those encoded by known susceptibility genes, have not been found in the generation of sporadic tumors. In the present work, we first identified that the unmethylated allele of Aplasia Ras homolog member I (ARHI) was deleted in most PCC tumors which retained a hypermethylated copy, while its mRNA level was significantly correlated with the unmethylated copy. De-methylation experiments confirmed that expression of ARHI was also regulated by the methylation level of the remaining allele. Furthermore, ARHI overexpression inhibited cell proliferation, with cell cycle arrest and induction of apoptosis, in ARHI-negative primary human PCC cells, whereas knockdown of ARHI demonstrated the opposite effect in ARHI-positive primary human PCC cells. Finally, we demonstrated that ARHI has the ability to suppress pAKT and pErK1/2, to promote the expression of p21Waf1/Cip1 and p27Kip1, and also to increase p27Kip1 protein stability. In summary, ARHI was silenced or downregulated in PCC tissues harboring only one hypermethylated allele. ARHI contributes to tumor suppression through inhibition of PI3K/AKT and MAKP/ERK pathways, to upregulate cell cycle inhibitors such as p27Kip1. We therefore reasoned that ARHI is a novel epigenetic silenced tumor suppressor gene on chromosome 1p that is involved in sporadic PCC tumorigenesis.

Silencing heme oxygenase-1 increases the sensitivity of ABC-DLBCL cells to histone deacetylase inhibitor in vitro and in vivo

Heme oxygenase-1 (HO-1) can promote tumor growth and reinforce the resistance of diffuse large B-cell lymphoma (DLBCL) cells to chemotherapeutic drug vincristine. We herein found that HO-1 protein expression was higher in high-risk DLBCL patients than in low-risk ones. Silencing HO-1 gene expression resisted vorinostat-induced apoptosis and arrested cell cycle in the G0/G1 phase of LY-10 cells. Western blot, co-immunoprecipitation and chromatin immunoprecipitation assays confirmed that the possible mechanisms may be increased cleaved caspase-3 protein expression, decreased phospho-histone deacetylase 3 protein expression, and activated histone acetylation of P27Kip1 promoter. Moreover, silencing HO-1 gene expression enhanced vorinostat-induced tumor cell apoptosis, prolonged survival time and promoted P27Kip1 protein expression in a xenograft mouse model.In conclusion, HO-1 is a potential therapeutic target of DLBCL. The findings provide a valuable preclinical evidence for sensitizing DLBCL patients with poor prognosis to histone deacetylase inhibitors.

Characterization of a novel p110β-specific inhibitor BL140 that overcomes MDV3100-resistance in castration-resistant prostate cancer cells.

Our previous studies demonstrated that the class IA PI3K/p110β is critical in castration-resistant progression of prostate cancer (CRPC) and that targeting prostate cancer with nanomicelle-loaded p110β-specific inhibitor TGX221 blocked xenograft tumor growth in nude mice, confirming the feasibility of p110β-targeted therapy for CRPCs. To improve TGX221's aqueous solubility, in this study, we characterized four recently synthesized TGX221 analogs. TGX221 analog efficacy were examined in multiple prostate cancer cell lines with the SRB cell growth assay, Western blot assay for AKT phosphorylation and cell cycle protein levels. Target engagement with PI3K isoforms was evaluated with cellular thermal shift assay. PI3K activity was determined with the Kinase-Glo Plus luminescent kinase assay. Cell cycle distribution was evaluated with flow cytometry after propidium iodide staining. As expected, replacing either one of two major functional groups in TGX221 by more hydrophilic groups dramatically improved the aqueous solubility (about 40-fold) compared to TGX221. In the CETSA assay, all the analogs dramatically shifted the melting curve of p110β protein while none of them largely affected the melting curves of p110α, p110γ, or Akt proteins, indicating target-specific engagement of these analogs with p110β protein. However, functional evaluation showed that only one of the analogs BL140 ubiquitously inhibited AKT phosphorylation in all CRPC cell lines tested with diverse genetic abnormalities including AR, PTEN, and p53 status. BL140 was superior than GSK2636771 (IC50 5.74 vs 20.49 nM), the only p110β-selective inhibitor currently in clinical trials, as revealed in an in vitro Kinase-Glo assay. Furthermore, BL140 exhibited a stronger inhibitory effect than GSK2636771 on multiple CRPC cell lines including a MDV3100-resistant C4-2B cell subline, indicating BL140 elimination of MDV3100 resistance. Mechanistic studies revealed that BL140 blocked G1 phase cell cycle entry by reducing cyclin D1 but increasing p27kip1 protein levels. These studies suggested that BL140 is a promising p110β-specific inhibitor with multiple superb properties than GSK2636771 worthy for further clinical development.

Aberrant Level of Skp2 and p27KIP1 in Intraductal Proliferative Lesions is Associated with Tumorigenesis

ABSTRACTBreast cancer is one of the leading causes of cancer-related death in women worldwide. Here we aimed to examine the expression status of S-phase kinase-associated protein 2 (Skp2) and p27KIP1, and assess the significance of Skp2 plus p27KIP1 expression in patients with intraductal proliferative lesions, including ductal carcinoma in situ (DCIS) and atypical ductal hyperplasia (ADH). Skp2 and p27KIP1 mRNA levels in DCIS, ADH, flat epithelial atypia, and usual ductal hyperplasia (UDH) were evaluated by quantitative real-time reverse transcription polymerase chain reaction and protein expression was evaluated immunohistochemically in 60 fresh tissues and 120 paraffin-embedded tissues from patients with the four subtypes above. We found that the protein and mRNA level of Skp2 were significantly increased in DCIS and ADH as compared with that in UDH. In contrast, p27KIP1 protein and mRNA levels were reduced. Based on the above findings, abnormal levels of Skp2 and p27KIP1 have probably been involved in the pathogenesis of ADH and DCIS. Thus, Skp2 and p27KIP1 may serve as important diagnosis markers.

Expression and clinical significance of Ser10 phosphorylated p27(kip1) protein in human breast carcinoma

Objective: To explore the expression and clinicopathological significance of Ser10 phosphorylated p27(kip1)(P-p27Ser10) protein in human breast carcinoma tissue. Methods: Tissue specimens were obtained from 111 cases with breast carcinoma from January 2004 to December 2005. And the expression of P-p27Ser10 protein for each specimen was detected by immunohistochemical (EnVision) analysis. Six representative paired samples of cancerous and paired adjacent normal tissues were collected and detected by Western blot. The relationships between the expression levels of P-p27Ser10 protein and its clinicopathological characteristics in primary breast carcinoma were retrospectively analyzed.The association of P-p27Ser10 and Jab1 protein was detected by co-immunoprecipitation method. Results: A high-level expression of P-p27Ser10 was present in cancerous tissues but not in paired adjacent normal tissues. The positive expression rate of P-p27Ser10 protein was as high as 88.3% (98/111) and 7.2% (8/111) in cancerous and paired adjacent normal tissues respectively (P<0.05). P-p27Ser10 expression was correlated with tumor size, lymph node metastasis and TNM stage (all P<0.05). P-p27Ser10 expression was significantly correlated with Ki-67 (r=0.582, P<0.05), and inversely correlated with p27 (r=-0.426, P<0.05). Co-immunoprecipitation revealed that the association of P-p27Ser10 and Jab1 protein was increased in breast carcinoma. Conclusion: Over-expression of P-p27Ser10 protein might play an important role in the pathogenesis of breast carcinoma. P-p27Ser10 protein therefore represents a novel diagnostic marker and therapeutic target in patients with breast carcinoma.

Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

AbstractThe kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D‐DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α‐helical structure, upon binding to cyclin‐dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real‐time observation of 13C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time‐dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D‐DNP NMR experiment.

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy.

The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13 C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.

Characterisation of cell cycle arrest and terminal differentiation in a maximally proliferative human epithelial tissue: Lessons from the human hair follicle matrix

Human hair follicle (HF) growth and hair shaft formation require terminal differentiation-associated cell cycle arrest of highly proliferative matrix keratinocytes. However, the regulation of this complex event remains unknown. CIP/KIP family member proteins (p21CIP1, p27KIP1 and p57KIP2) regulate cell cycle progression/arrest, endoreplication, differentiation and apoptosis. Since they have not yet been adequately characterized in the human HF, we asked whether and where CIP/KIP proteins localise in the human hair matrix and pre-cortex in relation to cell cycle activity and HF-specific epithelial cell differentiation that is marked by keratin 85 (K85) protein expression. K85 expression coincided with loss or reduction in cell cycle activity markers, including in situ DNA synthesis (EdU incorporation), Ki-67, phospho-histone H3 and cyclins A and B1, affirming a post-mitotic state of pre-cortical HF keratinocytes. Expression of CIP/KIP proteins was found abundantly within the proliferative hair matrix, concomitant with a role in cell cycle checkpoint control. p21CIP1, p27KIP1 and cyclin E persisted within post-mitotic keratinocytes of the pre-cortex, whereas p57KIP2 protein decreased but became nuclear. These data imply a supportive role for CIP/KIP proteins in maintaining proliferative arrest, differentiation and anti-apoptotic pathways, promoting continuous hair bulb growth and hair shaft formation in anagen VI. Moreover, post-mitotic hair matrix regions contained cells with enlarged nuclei, and DNA in situ hybridisation showed cells that were >2N in the pre-cortex. This suggests that CIP/KIP proteins might counterbalance cyclin E to control further rounds of DNA replication in a cell population that has a propensity to become tetraploid. These data shed new light on the in situ-biography of human hair matrix keratinocytes on their path of active cell cycling, arrest and terminal differentiation, and showcase the human HF as an excellent, clinically relevant model system for cell cycle physiology research of human epithelial cells within their natural tissue habitat.

Integrated Cox’s model for predicting survival time of glioblastoma multiforme

Glioblastoma multiforme is the most common primary brain tumor and is highly lethal. This study aims to figure out signatures for predicting the survival time of patients with glioblastoma multiforme. Clinical information, messenger RNA expression, microRNA expression, and single-nucleotide polymorphism array data of patients with glioblastoma multiforme were retrieved from The Cancer Genome Atlas. Patients were separated into two groups by using 1 year as a cutoff, and a logistic regression model was used to figure out any variables that can predict whether the patient was able to live longer than 1 year. Furthermore, Cox's model was used to find out features that were correlated with the survival time. Finally, a Cox model integrated the significant clinical variables, messenger RNA expression, microRNA expression, and single-nucleotide polymorphism was built. Although the classification method failed, signatures of clinical features, messenger RNA expression levels, and microRNA expression levels were figured out by using Cox's model. However, no single-nucleotide polymorphisms related to prognosis were found. The selected clinical features were age at initial diagnosis, Karnofsky score, and race, all of which had been suggested to correlate with survival time. Both of the two significant microRNAs, microRNA-221 and microRNA-222, were targeted to p27Kip1 protein, which implied the important role of p27Kip1 on the prognosis of glioblastoma multiforme patients. Our results suggested that survival modeling was more suitable than classification to figure out prognostic biomarkers for patients with glioblastoma multiforme. An integrated model containing clinical features, messenger RNA levels, and microRNA expression levels was built, which has the potential to be used in clinics and thus to improve the survival status of glioblastoma multiforme patients.