Loss-of-function variants in CSDE1 have been strongly linked to neuropsychiatric disorders, yet the precise role of CSDE1 in neurogenesis remains elusive. In this study, we demonstrate that knockout of Csde1 during cortical development in mice results in impaired neural progenitor proliferation, leading to abnormal cortical lamination and embryonic lethality. Transcriptomic analysis revealed that Csde1 upregulates the transcription of genes involved in the cell cycle network. Applying a dual thymidine-labelling approach, we further revealed prolonged cell cycle durations of neuronal progenitors in Csde1-knockout mice, with a notable extension of the G1 phase. Intersection with CLIP-seq data demonstrated that Csde1 binds to the 3' untranslated region (UTR) of mRNA transcripts encoding cell cycle genes. Particularly, we uncovered that Csde1 directly binds to the 3' UTR of mRNA transcripts encoding Cdk6, a pivotal gene in regulating the transition from the G1 to S phases of the cell cycle, thereby maintaining its stability. Collectively, this study elucidates Csde1 as a novel regulator of Cdk6, sheds new light on its critical roles in orchestrating brain development, and underscores how mutations in Csde1 may contribute to the pathogenesis of neuropsychiatric disorders.
Animals ; Neurogenesis/genetics* ; Cell Cycle/genetics* ; Mice, Knockout ; Mice ; Neural Stem Cells/metabolism* ; DNA-Binding Proteins/metabolism* ; Cyclin-Dependent Kinase 6/genetics* ; Cell Proliferation ; 3' Untranslated Regions ; Cerebral Cortex/embryology* ; RNA-Binding Proteins ; Mice, Inbred C57BL

Ovarian cancer is the most lethal malignancy affecting the female reproductive system. Pharmacological inhibitors targeting CDK4/6 have demonstrated promising efficacy across various cancer types. However, their clinical benefits in ovarian cancer patients fall short of expectations, with only a subset of patients experiencing these advantageous effects. This study aims to provide further clinical and biological evidence for antineoplastic effects of a CDK4/6 inhibitor (TQB4616) in ovarian cancer and explore underlying mechanisms involved. Patient-derived ovarian cancer organoid models were established to evaluate the effectiveness of TQB3616. Potential key genes related to TQB3616 sensitivity were identified through RNA-seq analysis, and TRIM4 was selected as a candidate gene for further investigation. Subsequently, co-immunoprecipitation and GST pull-down assays confirmed that TRIM4 binds to hnRNPDL and promotes its ubiquitination through RING and B-box domains. RIP assay demonstrated that hnRNPDL binded to CDKN2C isoform 2 and suppressed its expression by alternative splicing. Finally, in vivo studies confirmed that the addition of siTRIM4 significantly improved the effectiveness of TQB3616. Overall, our findings suggest that TRIM4 modulates ubiquitin-mediated degradation of hnRNPDL and weakens sensitivity to CDK4/6 inhibitors in ovarian cancer treatment. TRIM4 may serve as a valuable biomarker for predicting sensitivity to CDK4/6 inhibitors in ovarian cancer.
Humans ; Female ; Ovarian Neoplasms/pathology* ; Animals ; Tripartite Motif Proteins/genetics* ; Mice ; Cyclin-Dependent Kinase 4/antagonists & inhibitors* ; Cell Line, Tumor ; Cyclin-Dependent Kinase 6/antagonists & inhibitors* ; Protein Kinase Inhibitors/pharmacology* ; Ubiquitin/metabolism* ; Xenograft Model Antitumor Assays ; Ubiquitination ; Antineoplastic Agents/pharmacology*

Objective To prepare and identify rabbit anti-cyclin dependent kinase 6 (CDK6) antibody. Methods The recombinant pET21a (+)/CDK6 was successfully constructed, then the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells and was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression, which was detected by SDS-PAGE and Western blot analysis. The expressed protein was purified by nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose and then analyzed by SDS-PAGE. Japanese white rabbits were immunized with purified CDK6 protein for many times every two weeks. The blood was collected at 0, 2, 4 and 6 weeks after immunization, and serum was separated from blood. The titer was detected by indirect ELISA. Western blot analysis, immunofluorescence assay and immunohistochemistry were employed to determine the specificity. Results High purity CDK6 protein and high specificity of rabbit anti-CDK6 antibody were successfully prepared. The titer of CDK6 rabbit serum antibody reached 1:30 000 after immunization, which could specifically recognize the CDK6 protein expressed in cervical cancer cell line and cervical cancer tissues. Conclusion The high titer and specificity of rabbit anti-CDK6 antibody is successfully prepared.
Animals ; Female ; Humans ; Rabbits ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cyclin-Dependent Kinase 6 ; Enzyme-Linked Immunosorbent Assay ; Uterine Cervical Neoplasms

Cyclin-dependent kinases 4/6 (CDK4/6) inhibitors are anti-tumor agents for the treatment of hormone receptor-positive breast cancer. Palbociclib, abemaciclib and dalpiciclib have been approved for the treatment of breast cancer in China. Common adverse effects of CDK4/6 inhibitors include bone marrow suppression, gastrointestinal toxicities, liver dysfunction, and skin or subcutaneous tissue adverse reactions (AEs). The Breast Cancer Expert Group of Chinese Society of Clinical Oncology (CSCO) summarized the incidence, clinical manifestations, and grading of the AEs. This expert consensus reports measures of AE management on the basis of experience of clinical practice and the latest advances worldwide, aiming to guide clinical practice by the way of managing AE and help to choose the best treatment regimen.
Female ; Humans ; Aminopyridines/adverse effects* ; Antineoplastic Agents/adverse effects* ; Breast Neoplasms/drug therapy* ; Consensus ; Cyclin-Dependent Kinase 4/antagonists & inhibitors* ; Protein Kinase Inhibitors/adverse effects* ; Cyclin-Dependent Kinase 6/antagonists & inhibitors*

The development and progression of most cancers have been well recognized as the result of highly activated cell cycle. Cyclin dependent kinase 4/6 plays important roles not only in mitosis, but also in multiple biological processes that contribute to cancer development, such as aging, apoptosis and histone modification. Three FDA approved CDK4/6 inhibitors, Palbociclib, Ribociclib and Abemaciclib, have been used as targeted cancer therapeutic agents to benefit patients with endocrine therapy-resistant breast cancer and other types of cancer, prolonging their survival. However, the clinical application of these inhibitors also leads to acquired drug resistance and other problems. This paper reviews the regulatory roles of CDK4/6, the application of CDK4/6 inhibitors in cancer and the challenge of drug resistance.
Breast Neoplasms ; Cyclin-Dependent Kinase 4/therapeutic use* ; Cyclin-Dependent Kinase 6/therapeutic use* ; Female ; Humans ; Molecular Targeted Therapy ; Protein Kinase Inhibitors/therapeutic use* ; Signal Transduction

The introduction of cyclin-dependent kinase (CDK) 4/6 inhibitors has revolutionized the clinical management paradigm of hormone receptor (HR) positive/human epidermal growth factor receptor (HER) 2 negative breast cancer. As of today, CDK 4/6 inhibitors including Palbociclib, Ribociclib, and Abemaciclib have been widely approved by regulatory agencies. Randomized clinical trials demonstrated that CDK 4/6 inhibitors in combination with an aromatase inhibitor (AI) or fulvestrant in the first-, second- or later-line setting for HR positive/HER2 negative locally advanced or metastatic breast cancer led to substantial reduction in the risk of disease progression or death. Adverse effects of treatment were manageable and as or better than expected in terms of patient satisfaction. Considering CDK4/6 inhibitors in combination with endocrine therapy being a novel approach in China clinical practice, the panel developed the consensus comprehensively describing the pharmacology properties, monitoring strategy during treatment and adverse events management, to facilitate greater understanding in Chinese oncologists of a whole new therapeutic class of drug, promote accuracy of clinical decision and help reach the ultimate goal of improving survival and quality of life of the target patient population.
Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Breast Neoplasms/drug therapy* ; China ; Consensus ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase 6 ; Humans ; Protein Kinase Inhibitors/therapeutic use* ; Quality of Life ; Receptor, ErbB-2

BACKGROUND: We aimed to determine the effect of fibronectin (FN)-immobilized microgrooved titanium (Ti) on human gingival fibroblast proliferation, gene expression and protein expression. METHODS: Photolithography was used to fabricate the microgrooved Ti, and amine funtionalization (silanization) was used for FN immobilization on titanium surfaces. Cell proliferation, gene expression and protein expression were analyzed, followed by multiple regression analysis for determining the influential factors on cell proliferation. RESULTS: FN-immobilized microgrooved Ti significantly enhanced the fibroblast proliferation in various timelines of culture, among which a burst of fivefold increase is induced at 96 h of culture compared to that on the control smooth Ti. We suggest a presence of the synergistic promotion effect of microgrooves and FN immobilization on fibroblast proliferation. Through a series of analyses on the expression of various genes and proteins involved in cell adhesion and proliferation, cyclin-dependent kinase 6, cyclin D1, integrin α5, oncogene c-Src, osteonectin, paxillin and talin-2 were determined as influential factors on promoting fibroblast proliferation induced by FN-immobilized microgrooved Ti. CONCLUSION: FN-immobilized microgrooved Ti can act as an effective surface for enhancing fibroblast proliferation, and can be used for promoting soft tissue response on the connective tissue attachment zone of biomaterial surfaces.
Cell Adhesion ; Cell Proliferation* ; Connective Tissue ; Cyclin D1 ; Cyclin-Dependent Kinase 6 ; Fibroblasts* ; Fibronectins ; Gene Expression ; Humans* ; Immobilization ; Oncogenes ; Osteonectin ; Paxillin ; Titanium*

BACKGROUND: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. METHODS: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. RESULTS: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. CONCLUSIONS PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Mice ; Piperazines ; pharmacology ; Pyridines ; pharmacology

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.
Animals ; Cell Proliferation ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cytokines ; genetics ; metabolism ; DNA Damage ; Fibroblasts ; drug effects ; Interleukin-6 ; secretion ; Mice ; Mitomycin ; pharmacology ; NIH 3T3 Cells ; Phenotype

OBJECTIVETo investigate the expression of p16INK4a protein in breast cancer and analyze its clinical significance.

METHODSA total of 132 surgical specimens of primary breast cancer obtained between 2014 and 2015 were examined for expressions of ER, PR, CK5/6, Her-2 and p16INK4a proteins using immunohistochemistry.

RESULTSThe breast cancer samples were classified into 5 molecular subtypes, namely Luminal A (58 cases), Luminal B (32 cases), Her-2-positive (21 cases), basal-like (12 cases) and normal-like (9 cases) types. p16INK4a expression was negative in 7/132 (5.30%) cases, weakly positive in 15/132 (11.36%) cases, positive in 40/132 (30.30%) cases, and strongly positive in 70/132 (53.03%) cases. When categorizing negative and weakly positive cases into negative group and the positive and strongly positive cases into positive group, the total negative and positive expression rates of p16INK4a were 16.67% (22/132) and 83.33% (110/132) in the carcinoma tissues. Statistical analysis showed the expression intensity of p16INK4a differed significantly between the age groups (P<0.05) but was not significantly correlated with ER, PR, Her-2, molecular subtypes or metastasis of the tumors.

CONCLUSIONThe compensatory high expression of p16INK4a is the main mechanism of cell cycle deregulation in invasive breast cancer and can be an important specific molecular marker for invasive breast cancer.

Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; classification ; diagnosis ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Female ; Humans ; Keratin-5 ; metabolism ; Keratin-6 ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism