Aging is an independent risk factor for chronic diseases in the elderly, and understanding aging mechanisms is one of the keys to achieve early prevention and effective intervention for the diseases. Aging process is dynamic and systemic, making it difficult for mechanistic study. With recent advances in aging biomarkers and development of live-imaging technologies, more and more reporter mouse models have been generated, which can live monitor the aging process, and help investigate aging mechanisms at systemic level and develop intervention strategies. This review summarizes recent advances in live-imaging aging reporter mouse models based on widely used aging biomarkers (p16^Ink4a, p21^Waf1/Cip1, p53 and Glb1), and discusses their applications in aging research.
Humans ; Animals ; Mice ; Aged ; Aging ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cyclin-Dependent Kinase Inhibitor p21/metabolism* ; Biomarkers ; Tumor Suppressor Protein p53

BACKGROUNDBoth p16(INK4) and p21(Waf1) are tumor suppressors with similar biological functions in the regulation of cellular senescence. Previous reports showed that p16(INK4) could be activated by p21(Waf1) through transcriptional factor Sp1 in HeLa cells. This study was undertaken to determine the effects of p16(INK4) on the expression and functions of p21(Waf1).

METHODSHuman diploid fibroblast 2BS cells were stably transfected with sense (2BS/p16(INK4)), antisense p16(INK4) (2BS/asp16(INK4)) or empty vector (2BS/neo). Then they were assayed by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence activated cell sorting (FACS) and Western blot.

RESULTS2BS/p16(INK4) cells exhibited cell cycle arrest in both G1 and G2/M phases. Endogenous p21(Waf1) protein levels increased twofold in the 2BS/p16(INK4) cells, but not decreased in the 2BS/asp16(INK4) cells. p21(Waf1) mRNA levels were not affected in neither 2BS/p16(INK4) nor 2BS/asp16(INK4) cells.

CONCLUSIONp16(INK4) may play an important role in the regulation of cellular senescence by modulating the p21(Waf1) protein level via the posttranscriptional mechanism.

Cell Cycle ; Cells, Cultured ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; physiology ; Cyclin-Dependent Kinase Inhibitor p21 ; physiology ; Fibroblasts ; metabolism ; Humans ; Transcription, Genetic

OBJECTIVE: To investigate the expression and clinicopathological significance of the cell cycle regulators cyclin E, cyclin D1, p21, p16 in laryngeal carcinogenesis tissus. METHOD: The expression of cell cycle regulators were detected by flow cytometry method in 23 cases of polyps of vocal cord, 69 cases of laryngeal precancerous change and 33 cases of laryngeal squamous cell carcinoma (LSCC), which tissue was paraffin embedded, sliced, dewaxed, and prepared into the cell suspension, then fluorescently labeled by cyclin E, cyclin D1, p21 and p16. RESULT: In polyps of vocal cord, laryngeal precancerous change and LSCC, The positive expression rate of cyclin E and cyclin D1 were respectively 13.04%, 20.29D, 42.420 and 26.09%, 43.48% and 93.94%. The positive expression rate of p16 and p21 were respectively 61.90%, 40.98%, 14.28% and 47.62%, 23.81%, 26.23%. Those showed the positive expression rate of cyclin D1, cyclin E gradually decreased from vocal cord polyps, laryngeal precancerous change to LSCC, (P < 0.05, P < 0.01), while the positive expression rate of p21 and p16 gradually decreased (P < 0.01). CONCLUSION The abnormal expression of cell cycle regulatory factors is the molecular events of laryngeal carcinoma. High expression of positive regulatory factors cyclin D1 and cyclin E, and low expression of negative regulatory factors p16 and p21, which showed the imbalance of multiple positive and negative regulatory factors related with cell cycle play an important role in the occurrence of laryngeal cancer.
Adult ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Female ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Oncogene Proteins ; metabolism

BACKGROUND/AIMS: The cyclin-dependent kinase inhibitors (CDKI) including p21, p27, and p57 of the kinase inhibitor protein (KIP) family are negative regulators of cell cycle progression and potentially act as tumor suppressor. Tumor behavior and growth are influenced by the extent of tumor cell proliferation. The aim of this study was to evaluate the expression of KIP family CDKI in gastric cancer tissue, and to examine the relationship between these expression and various clinicopathological parameters including tumor cell proliferation. METHODS: We conducted an immunohistochemical analysis of p21, p27, and p57 expression in 109 gastric cancer tissues. Tumor cell proliferation was assessed by immunohistochemistry with antibody against Ki-67. RESULTS: Negative expression of p21, p27, and p57 was demonstrated in 45.9%, 65.1%, and 57.8% of cancer tissues, respectively. Negative expression of p21 correlated with larger tumor size, poor differentiation, depth of invasion, lymph node metastasis and advanced TNM stage (p=0.048, 0.041, 0.001, 0.005, and 0.001 respectively). Negative expression of p21 correlated with poor survival (p=0.037). Tumors with negative p21 expression had higher Ki-67 expression than those with positive p21 expression (p=0.024). No significant correlation could be observed between status of p27 and p57 expression and various clinicopathological parameters including survival and tumor cell proliferation. CONCLUSIONS: These results suggest that negative expression of p21 may play an important role in carcinogenesis by stimulating tumor cell proliferation, and may help in predicting the prognosis of gastric cancer.
Adult ; Aged ; Cell Division ; Cyclin-Dependent Kinase Inhibitor Proteins/*metabolism ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cyclin-Dependent Kinase Inhibitor p57/metabolism ; English Abstract ; Female ; Humans ; Male ; Middle Aged ; Stomach Neoplasms/*metabolism/mortality/pathology ; Survival Rate

BACKGROUND/AIMS: Eukaryotic cell cycle is regulated by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDKs) and their partner cyclins, or by interaction with CDK inhibitors. Thioacetamide (TA) is a weak hepatocarcinogen causing several types of liver damage in a dose dependent manner and ultimately producing malignant transformation. We investigated alterations of expression of cell cycle regulators in the rat liver, involved in G1 entry and progression during TA administration. METHODS: We studied expression patterns of cyclin D1, CDK4, CDK6, p21(CIP1) and p16(INK4a) during daily intraperitoneal injection of low dose TA (50 mg/kg) till 7 day. We used western blot and immunohistochemistry for detection. RESULTS: Expression of cyclin D1, CDK4, CDK6 and p21(CIP1) increased from 6 hour and peaked at 2, 3 day, then decreased next 2 days, and re-increased at 6 day. Cytoplasmo-nuclear translocation of cyclin D1 and p21(CIP1) was evident within 1 day and prominent at 2 and 7 day. Expression of p16(INK4a) increased immediately after TA treatment and remarkably increased from 3 day and progressed till 7 day, showing cytoplasmic location, suggestive of inactive form. Most of in situ immunoreactions occurred at the centrilobular hepatocytes. Concomitant nuclear translocation of p21(CIP1) and cyclin D1, different with p16(INK4a) suggests that p21(CIP1) might be a transporter for nuclear translocation rather than cell cycle inhibitor. CONCLUSIONS: Daily administration of low dose TA makes cell cycle open and G1 progress, possibly due to cyclin D1, CDK4 and CDK 6, their transporter p21(CIP1), and inactive p16(INK4a), which occur at quiescent hepatocytes, not stem cells.
Animals ; Cell Cycle Proteins/*metabolism ; Cyclin D1/metabolism ; Cyclin-Dependent Kinase 4/metabolism ; Cyclin-Dependent Kinase 6/metabolism ; Cyclin-Dependent Kinase Inhibitor p16/metabolism ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; G1 Phase ; Immunohistochemistry ; Liver/*drug effects/enzymology/metabolism ; Liver Diseases/chemically induced/metabolism/pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Thioacetamide/*toxicity

This study is to examine the effects of NNIspm-mediated cellular senescence of HepG2 cells and elucidate its potential molecular mechanism. Cellular senescence was detected with senescence-associated beta-galactosidase staining. Cell cycle distribution, intracellular fluorescence intensity and accumulation of intracellular reactive oxygen species (ROS) were detected by high content screening (HCS). Protein expression was detected by Western blotting. Polyamines content was analyzed by high performance liquid chromatography (HPLC). The results demonstrated that NNIspm significantly induced HepG2 cells senescence. This effect was due to the decrease of intracellular polyamines, the arrest at G0/G1 phase and an increase of ROS level. The molecular senescence marker p21 increased significantly after NNIspm treatment. In contrast, the protein expressions of Cyclin E and CDK2 were obvious down-regulation. The results indicated that cellular senescence induced by NNIspm was one of its antitumor mechanisms.
Antineoplastic Agents ; metabolism ; pharmacology ; Cellular Senescence ; drug effects ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; G1 Phase ; Hep G2 Cells ; Humans ; Oncogene Proteins ; metabolism ; Polyamines ; metabolism ; pharmacology ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the relationship between the sensitivity to chemotherapeutic drugs and the expression of cyclin D1 and P21WAF1 in gastric carcinoma.

METHODSThe sensitivity of 80 samples of gastric cancer cells to HCPT, CDDP, 5-FU, ADM and MMC was evaluated with MTT assay. The protein expression of cyclin D1 and P21WAF1 was detected by immunohistochemistry.

RESULTSThe sensitivity of gastric carcinoma cells to chemotherapeutic drugs was different. Inhibitory rates of tumor cells exposed to 5-FU, MMC and DDP were significantly higher than those exposed to ADM and HCPT (P <0.05). Positive expression rates of cyclin D1 and P21WAF1 in gastric cancer were 70.0% and 47.5% respectively. Cyclin D1 positive cells showed more sensitive to 5-FU and HCPT than cyclin D1 negative cells. P21WAF1 negative cells showed more sensitive to MMC, 5-FU and DDP than P21WAF1 positive cells.

CONCLUSIONExpression of cyclin D1 and P21WAF1 is related with drug-resistance of tumor. Examination of cyclinD1 and P21WAF1 would have reference value in selection of chemotherapeutic drugs.

Adult ; Aged ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Drug Screening Assays, Antitumor ; Female ; Humans ; Male ; Middle Aged ; Stomach Neoplasms ; drug therapy ; metabolism

The purpose of this study was to elucidate the apoptosis, apoptotic pathway of HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Apoptosis of HL-60 cells was detected by flow cytometry, the expression of P21, P27 and P53 proteins in HL-60 cells treated with MG132 was assayed by Western blot. The HL-60 cells were treated with 1 µmol/L MG132 for 48 h, and irradiated by 75 Gy of (60)Co γ-ray, but their antigenicity was preserved. The effect of irradiated HL-60 cells treated with MG132 on proliferation of peripheral blood mononuclear cells (PBMNC) was measured by CCK-8 method. The results showed that the apoptotic rate of MG132-treated HL-60 cells increased in dose-and time-dependent manner. No significant changes in MG132-induced apoptosis were observed after inhibiting caspase-8 and caspase-9 pathway. The expression of P21 and P27 protein increased after treatment of HL-60 cells with MG132. CCK-8 test showed that HL-60 cells induced with low-dose of MG132 displayed the enhancing effect on proliferation of PBMNC. It is concluded that high dose of MG132 can induce the apoptosis of HL-60 cells, and has direct killing effect on HL-60 cells, but this inducing apoptotic effect on HL-60 cells can not be realized through caspase-8 and caspase-9 pathway. The P21 and P27 protein may be involved in MG132 induced HL-60 cell apoptosis. Low dose of MG132 promotes the proliferation of PBMNC in healthy individuals and enhance the immunity of organism.
Apoptosis ; drug effects ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; HL-60 Cells ; Humans ; Leupeptins ; pharmacology ; Proteasome Inhibitors ; pharmacology

This study was aimed to investigate the effect of arsenic trioxide (As2O3) on proliferation and cell cycle of human Burkitt lymphoma cells and its related molecular mechanism, so as to provide experimental evidence for treatment of Burkitt lymphoma with As2O3. Human Burkitt lymphoma cell line Namalwa was used as the model, the effect of As2O3 on cell proliferation, cell cycle and apoptosis, as well as the expression of cell cycle modulation related genes, including mRNA and protein level, were detected by MTT method, flow cytometry, real-time quantitative PCR (RQ-PCR) and Western blot, respectively. The results showed that the As2O3 inhibited significantly the growth and proliferation of Namalwa cells in concentration-and time-dependent manner. The As2O3 arrested obviously cell cycle of Namalwa cells in G1 phase, and showed significant concentration-effect relationship. The As2O3 induced the apoptosis of Namalwa cells in concentration-and time-dependent manner, downregulated the expression of the important driving genes of cell cycle including Cyclin E and CDK2 in mRNA and protein level, upregulated the expression of the important inhibiting gene of cell cycle-P21 in mRNA and protein level in concentration-dependent manner. It is concluded that As2O3 inhibits significantly the growth and proliferation of Namalwa cells, and the effect was closely relates with its inducing the apoptosis and blocking the cell cycle of Namalwa. The action of blocking cell cycle is closely associated with its downregulating the expression of driving genes of cell cycle-Cyclin E and CDK2, upregulating the expression of the inhibiting gene of cell cycle-P21.
Apoptosis ; Arsenicals ; pharmacology ; Burkitt Lymphoma ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Oncogene Proteins ; metabolism ; Oxides ; pharmacology

OBJECTIVETo investigate the effect of Shenkang Injection (SI) on hypertrophy and expressions of p21 and p27 in glomerular mesangial cells of rats cultured in high glucose fluid.

METHODSCultured rat glomerular mesangial cells were divided into 6 groups: the normal control group, mannitol-treated group, high glucose-treated group, high, middle and low dose SI treated groups. The synthesis of cell protein and DNA were determined with incorporation of 3H-TdR and 3H-Leu. The changes of p21 mRNA and expressions of p21 and p27 protein were determined by reverse transcriptase polymerase chain reaction and Western blot.

RESULTSHigh glucose caused incorporation of 3H-TdR reduced, incorporation of 3H-Leu increased. SI could decrease incorporation of 3H-Leu, increase incorporation of 3H-TdR. Compared with the high glucose-treated group, SI could inhibit the overexpression of p2l mRNA and protein,p27 protein induced by high glucose.

CONCLUSIONSI could inhibit hypertrophy of glomerular mesangial cells cultured in high glucose partly through inhibiting overexpressions of p2l mRNA, p21 and p27 protein.

Animals ; Cell Enlargement ; Cells, Cultured ; Culture Media ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; pharmacology ; Mesangial Cells ; cytology ; drug effects ; metabolism ; Rats