P16 and P21 are involved in the pathogenesis of endometrial thinning: A cross-sectional study.

P16 plays a role in the negative regulation of cell proliferation, regulating cell apoptosis to control the growth of tumor cells. P21 is a nuclear protein that suppresses DNA synthesis and inhibits cell division. This study aimed to examine the expression and roles of P16 and P21 in endometrial thinning. Thirty cases of endometrial biopsy diagnosed as endometrial thinning were assessed by p16 and p21 immunohistochemistry from March 2014 to August 2020 in Huazhong University of Science and Technology Union Shenzhen Hospital. Another thirty cases of normal endometrium in the same period were assessed as controls. The specimens underwent histological analysis, and P16 and P21 were assessed by immunohistochemistry. There were no statistically significant differences in age, menstrual cycle, BMI, sex hormone levels, gravidity and parity between the two groups (all P > .05). In the endometrial thinning group, P16 was expressed in the endometrial adenoid nucleus, cytolymph and interstitial cell nucleus. In the normal group, P16 was mainly expressed in the endometrial adenoid nucleus, with some P16 signals detected in the endometrial interstitial nucleus. P21 expression was mainly detected in the endometrial adenoid nucleus. P16 and P21 amounts in endometrial thinning cases were significantly lower than those of the normal endometrial group. There was no correlation between p16 and p21 amounts. This study revealed aberrant expression of P16 and P21 in the endometrium might be due to a compensatory effect of the thin endometrium to increase cell proliferation and suppress cell apoptosis. However, the pathological roles of P16 and P21 in endometrial thinning and the contribution of cell senescence deserve further investigation.

Gene expression profile in functioning and non-functioning nodules of autonomous multinodular goiter from an area of iodine deficiency: unexpected common characteristics between the two entities.

PURPOSE: Toxic multinodular goiter is a heterogeneous disease associated with hyperthyroidism frequently detected in areas with deficient iodine intake, and functioning and non-functioning nodules, characterized by increased proliferation but opposite functional activity, may coexist in the same gland. To understand the distinct molecular pathology of each entity present in the same gland, the gene expression profile was evaluated by using the Affymetrix technology. METHODS: Total RNA was extracted from nodular and healthy tissues of two patients and double-strand cDNA was synthesized. Biotinylated cRNA was obtained and, after chemical fragmentation, was hybridized on U133A and B arrays. Each array was stained and the acquired images were analyzed to obtain the expression levels of the transcripts. Both functioning and non-functioning nodules were compared versus healthy tissue of the corresponding patient. RESULTS: About 16% of genes were modulated in functioning nodules, while in non-functioning nodules only 9% of genes were modulated with respect to the healthy tissue. In functioning nodules of both patients and up-regulation of cyclin D1 and cyclin-dependent kinase inhibitor 1 was observed, suggesting the presence of a possible feedback control of proliferation. Complement components C1s, C7 and C3 were down-regulated in both types of nodules, suggesting a silencing of the innate immune response. Cellular fibronectin precursor was up-regulated in both functioning nodules suggesting a possible increase of endothelial cells. Finally, Frizzled-1 was down-regulated only in functioning nodules, suggesting a role of Wnt signaling pathway in the proliferation and differentiation of these tumors. None of the thyroid-specific gene was deregulated in microarray analysis. CONCLUSION: In conclusion, the main finding from our data is a similar modulation for both kinds of nodules in genes possibly implicated in thyroid growth.

Low myocardial transcript variant alt-a of cyclin dependent kinase inhibitor p21 expression differentiates hypothermia from cardiac/respiratory causes of death.

Gene expressions in the myocardium have been shown to vary between different causes of death, which can be utilized in the recognition of varied processes. Our previous work with a limited number of cases showed a high messenger ribonucleic acid expression of the transcript variant alt-a of cyclin dependent kinase inhibitor p21 (p21 alt-a) in chronic cardiac ischemia deaths and a low expression in hypothermia deaths and acute myocardial ischemia deaths. In present work, p21 alt-a expression in the myocardium of human cadavers was calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene. In this collection of 143 samples, the p21 alt-a expression was significantly lower in hypothermia than in chronic cardiac ischemic heart disease with (P < .001) or without (P < .001) acute myocardial infarction and in other cardiac and respiratory disease deaths (P < .000). Chronic ischemic heart disease in hypothermia cases did not increase the expression. The p21 alt-a expression did not correlate with postmortem interval, quality of RNA or with the age of the deceased. The p21 alt-a referenced to GAPDH expression in cadaver myocardium has apparent potential as a marker distinguishing between hypothermia and cardiac/respiratory diseases as causes of death.

The Prognostic Value of G1 Cyclins, p21 and Rb Protein in Patients With Colon Cancer.

BACKGROUND/AIM: Cyclins D1 and E play different roles in the cell cycle. Cyclin E promotes chromosome instability, whereas cyclin D1 regulates apoptosis of cells. This study evaluated the prognostic significance of G1 cyclins, p21 and pRb in tumor proliferation. PATIENTS AND METHODS: A total of 102 patients with colon cancer were operated on and staged according to TNM. Follow-up was 2 to 68 months (mean 38.3±16.7 months). Expression of cyclin E and D1 were evaluated using immunohistochemistry. RESULTS: Levels of cyclin E expression were correlated with cyclin D1 expression (p=0.038), p21 expression (p=0.047), and pRb expression (p=0.004). The 5-year survival rate along with prognosis of patients with advanced stage (III, IV) colon cancer and cyclin D1 positive tumors, were significantly worse (p=0.009). Statistically significant association was observed between tumor proliferative capacity Ki-67, cyclin D1 (p=0.009), pRb (p=0.031) and p21 (p=0.050). CONCLUSION: Cyclin D1 is highly expressed in advanced stage colon cancer patients, implying a potential prognostic value.

IL-1Ra protects hematopoietic cells from chemotoxicity through p53-induced quiescence.

The protection of constantly proliferating gut epithelia and hematopoietic tissues from cytotoxicity could improve conventional chemotherapy efficacy and widen its therapeutic window. Previously, we reported that, in mouse models, pretreatment of recombinant human IL-1 receptor antagonist (rhIL-1Ra) protected both types of vulnerable tissues from chemotherapeutics. Here, we showed that rhIL-1Ra treatment up-regulated the protein levels of phosphorylated p38, p53, and p21 and induced transient hematopoietic stem/progenitor cell (HS/PC) quiescence. Knockout of IL-1 receptor I (IL-1RI), p53, or p21 alleles and pharmacological inactivation of p38 mapped the rhIL-1Ra pathway in the induction of HS/PC quiescence. Therefore, rhIL-1Ra administration before but not after chemotherapy alleviated 5-fluorouracil-induced neutropenia. In addition, in vivo and in vitro cell proliferation assays revealed that the rhIL-1Ra treatment did not affect cancer cell proliferation or chemosensitivity. Lastly, we propose an IL-1/IL-1Ra pathway (IL-1RI → p38 → p53 → p21), which regulates HS/PC quiescence. The rhIL-1Ra may provide a new route for p53-based cyclotherapy, which spares normal cells but kills cancer cells during chemotherapy.-Ye, H., Qian, L., Zhu, S., Deng, S., Wang, X., Zhu, J., Chan, G. L., Yu, Y., Han, W. IL-1Ra protects hematopoietic cells from chemotoxicity through p53-induced quiescence.

Reduction of plasma angiopoietin-like 2 after cardiac surgery is related to tissue inflammation and senescence status of patients.

OBJECTIVES: A strong relationship between high circulating angiopoietin-like 2 (ANGPTL2) levels, a proinflammatory adipokine, and cardiovascular diseases has been reported. Our objective was to determine whether plasma ANGPTL2 and high-sensitivity C-reactive protein (hs-CRP) levels change postoperatively in patients who underwent heart valve surgery and/or coronary artery bypass grafting. We hypothesized that a corrective cardiac surgery would decrease ANGPTL2 levels. METHODS: In 47 prospectively recruited patients who underwent coronary artery bypass grafting (n = 16), valve replacement (n = 16), or both (n = 15), we measured plasma ANGPTL2 and hs-CRP levels preoperatively, at 24 hours, at 3 to 5 days (hospital discharge), and at 30 to 90 days (follow-up) after surgery. Mediastinal adipose tissue and distal fragments of the left internal mammary artery (IMA) were harvested during surgery and mRNA expression of inflammatory and senescence markers was assessed using real-time quantitative polymerase chain reaction. RESULTS: ANGPTL2 and hs-CRP levels were elevated 24 hours after surgery and then returned to baseline levels. We noted, however, a dichotomy among patients: compared with baseline, plasma ANGPTL2 levels either significantly decreased (n = 21/47) or increased (n = 26/47) after surgery. In contrast, hs-CRP levels were identical between groups (P = .997). Patients in the increased group were older (P = .002) with a higher systolic blood pressure (P = .038) at baseline. Moreover, changes in ANGPTL2 levels (ΔANGPTL2 = final minus initial levels) positively correlated with mRNA expression of tumor necrosis factor α and interleukin 8 in mediastinal adipose tissue and IMA (P < .05) and with the senescence-associated marker cyclin-dependent kinase inhibitor 1 in IMA (P = .009). CONCLUSIONS: In younger patients with lower levels of tissue inflammation and arterial senescence load, ANGPTL2, but not hs-CRP levels decreased after cardiac surgery, suggesting that circulating ANGPTL2 reflects tissue inflammation and senescence.

Immunohistochemical analysis of c-erbB-2, Bcl-2, p53, p21WAF1/Cip1, p63 and Ki-67 expression in hydatidiform moles.

Hydatidiform moles (HM) are characterized by an abnormal proliferating trophoblast with a potential for a malignant transformation. Similar to other human tumors, trophoblastic pathogenesis is likely a multistep process involving several molecular and genetic alterations. The study was performed to investigate the expression patterns of c-erbB-2 and Bcl-2 oncoproteins, p53, p21WAF1/CIP1 and p63 tumor suppressor proteins and Ki-67 cell proliferation marker in HM. We conducted a retrospective study of 220 gestational products, including 39 hydropic abortions (HA), 41 partial HM (PHM) and 140 complete HM (CHM). The expression of c-erbB-2, Bcl-2, p53, p21WAF1/CIP1, p63 and Ki-67 was investigated by immunohistochemistry on archival tissues. c-erbB-2 expression was observed in three PHM and 10 CHM. Bcl-2 immunostaining was significantly higher in PHM (61%) and CHM (70.7%) compared with HA (7.7%, p = 0.001 and p < 0.0001, respectively). p53 expression was stronger in CHM (73.6%) compared with PHM (24.4%, p < 0.0001) and HA (12.8%, p < 0.0001). p21WAF1/CIP1 staining was observed as well in molar and non-molar gestations (p > 0.05). p63 immunoexpression was significantly described in CHM (85.7%) and PHM (78%) compared with HA (10.2%, p < 0.0001 and p = 0.0001, respectively). Ki-67 was significantly expressed in CHM (72.1%) compared with HA (46.2%, p = 0.005). Altered expression of Bcl-2, p53, p63 and Ki-67 reflects the HM pathological development. Immunohistochemical analysis is beneficial to recognize the HM molecular and pathogenic mechanisms. Furthermore, it could serve as a useful adjunct to conventional methods for refining HM diagnosis.

[Significance of HPV and cell cycle related proteins in basaloid squamous cell carcinoma of the larynx].

Objective: To study the significance of HPV and cell cycle related proteins in basaloid squamous cell carcinoma (BSCC) of the larynx. Methods: Twenty-nine cases of laryngeal BSCC from Beijing Tongren Hospital from January 2005 to December 2011 were reviewed. HPV typing by polymerase chain reaction-reverse dot blot (PCR-RDB) and p53, Ki-67, p16, p21 and cyclin D1 expression by immunohistochemistry were performed. The relationship between these indicators, various pathologic parameters (TNM, tumor size, tumor site and lymph node metastasis) and HPV status was analyzed. Results: There were 27 male and 2 female patients. The median age was 62 years. Lymph node metastasis and supraglottic tumor location were slightly higher than that of "usual" SCC, but not statistically significant (P>0.05). HPV DNA was detected in 27.6% (8/29) of the laryngeal BSCC, and all were HPV16. The expression of HPV was not related to age, alcohol consumption, tumor stage and tumor size. p53 was expressed in 31.0%(9/29) of laryngeal BSCC, and these cases were more likely supraglottic and had lymph node metastases (P<0.05). p16 staining was seen in 24.1% (7/29) of laryngeal BSCC, and these cases showed slightly higher rate of lymph node metastasis compared to p16 negative cases. The expression rates of p21 and cyclinD1 were 27.6% (8/29) and 69.0%(20/29), respectively, which were not related to age, tumor size, stage, lymph node metastasis, smoking and drinking. There were only 3 p16+ /HPV+ cases, which showed higher p21 and Ki-67 index compared to the HPV negative group (P<0.05). Conclusion: Some laryngeal BSCC expresses HPV DNA, possibly indicating an association with HPV; but p16 expression is not a reliable indicator for HPV infection.

Acral Spitz Nevi: A Clinicopathologic Study of 50 Cases With Immunohistochemical Analysis of P16 and P21 Expression.

Spitz nevi on acral sites are rare and poorly documented. The combination of Spitzoid cytomorphology and atypical architectural features of the junctional component may lead to an erroneous diagnosis of melanoma. To study the clinicopthologic and immunohistochemical features, 50 Spitz nevi localized on the distal extremities were retrieved from departmental files. Clinical data and follow-up were obtained and the histologic features were analyzed. P16 and P21 immunohistochemical staining of the dermal component was compared with that of 10 acral lentiginous melanomas and 10 acral nevi. Acral Spitz nevi affected young adults (median: 24.5 y; range: 4 to 61 y) with a female predominence and a predilection for the feet. They were pigmented measuring 1 to 15 mm (median: 4 mm). Follow-up, available for 45 patients (median: 48 mo; range: 4 to 228 mo), revealed no local recurrence, metastasis or mortality. Histologically, acral Spitz nevi were composed of large epithelioid and/or spindled melanocytes. They were well circumscribed with occasional asymmetry. The junctional component was broad with a lentiginous and nested growth and shoulder formation. Marked pagetoid spread and transepidermal elimination of junctional nests were common features. Focal active regression of the dermal component was frequently noted but there was no nuclear pleomorphism or dermal mitotic activity. Acral Spitz nevi were characterized by strong and diffuse P16 and P21 expression, which differs from acral nevi and acral lentiginous melanoma. Acral Spitz nevi are a distinctive subgroup of Spitz nevi with benign behavior. Awareness of the subtle histologic differentiating features and the distinctive P16/P21 expression pattern allows separation from melanoma.

Hyperoxia-induced lung injury increases CDKN1A levels in a newborn rat model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common, chronic lung disease of infants. Presently, high oxygen exposure and mechanical ventilation considerably influence the development of BPD. To clarify the pathological mechanisms of this disease, we developed a hyperoxia-induced BPD rat model and investigated the role of CDKN1A in the pathogenesis of BPD. Newborn rats were randomly assigned to the hyperoxia (85% O2) and control (normoxia, 21% O2) groups. Lung tissues were collected on days 1, 3, 7, 14, or 21 after the start of hyperoxia or normoxia exposure. The expression of CDKN1A was detected by immunohistochemistry, western blot, and real-time PCR. Starting from day 3, CDKN1A mRNA expression was higher in the hyperoxia group. From day 7, the radial alveolar count was significantly different between the two groups, and on day 14, the hyperoxia group had high CDKN1A protein expression compared to the control group. These results suggest that increased CDKN1A expression may be involved in the pathogenesis of BPD through alveolarization and lung retardation.

Interaction between mTOR pathway inhibition and autophagy induction attenuates adriamycin-induced vascular smooth muscle cell senescence through decreased expressions of p53/p21/p16.

Cellular senescence is related to aging and extremely stable proliferative arrest with active metabolism. Senescent cells can activate mammalian target of rapamycin (mTOR) pathway, which plays a crucial role in the regulation of cell metabolism, cellular growth, and autophagy in senescence-associated cardiovascular diseases. Therefore, we examined whether mTOR pathway could induce cellular senescence by inhibition of autophagy in vascular smooth muscle cells (VSMCs). We found that adriamycin-induced VSMC senescence is accompanied by increased activity of mTOR, a major controller of cell growth and a negative regulator of autophagy. VSMC senescence induced by activation of mTOR pathway led to reduced levels of signal-associated autophagy proteins, and inhibition of mTOR pathway resulted in a drastic decrease in the number of senescence-associated ß-galactosidase (SA-ß-gal)-stained cells and increased levels of signal-associated autophagy proteins. Autophagic inhibition potentiated adriamycin-induced mTOR pathway activation as well as increase in the number of SA-ß-gal-stained VSMCs. Results of further experiments showed that mTOR pathway inhibition regulates adriamycin-induced expression of senescence markers (p53/p21/p16), which plays an important role in different aspects of cellular aging. Taken together, these results support the idea that intervention to modulate the interaction between mTOR pathway and autophagy could be a potential strategy for longevity.

Are synchronous and metachronous bilateral breast cancers different? An immunohistochemical analysis focused on cell cycle regulation.

INTRODUCTION: The biology and pathomechanisms of bilateral breast cancers is not fully understood. We compared the morphological and immunohistochemical characteristics of primary tumors in patients with synchronous (sBBC) and metachronous bilateral breast cancers (mBBC), with special focus on cell cycle regulation and its correlation with markers determining intrinsic phenotype. METHODS: Immunohistochemical expression of p16Ink4A, p21(WAF1/CIP1), p27Kip1, p53, cyclin A, cyclin B, cyclin D1, cyclin D3 and cyclin E was assessed in tissue microarrays containing primary breast tumor cores from 113 mBBC and 61 sBBC. Expression of these markers was correlated with tumor grade and expression of estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2) and Ki-67. RESULTS: In univariate analysis, mBBC demonstrated higher expression of p16Ink4A (both cytoplasmic: p=0.002 and nuclear: p=0.014), cyclin A (p=0.024) and B (cytoplasmic; p=0.015). In multivariate analysis mBBC were associated with lower expression of p21: p=0.038 and higher cytoplasmic expression of cyclin B: p=0.019. Lower ER expression for all BBCs and mBBC, respectively, was associated with stronger p16 expression (cytoplasmic: both p<0.000001 and nuclear: p<0.000001, p=0.00002), p53: p<0.000001, p=0.000001, cyclin A: p=0.00002, p=0.00045, cyclin B (cytoplasmic: p=0.00037, 0.00015 and nuclear: both p=0.0004) and cyclin E: p=000003, p<0.000001, and weaker expression of p27: p=0.00003, p=0.0001 and cyclin D1: both p<0.000001; for sBBC some of these correlations were absent. Higher p27 score correlated with lower HER2 expression in sBBC: p=0.018, whereas higher HER2 expression was associated with higher p53: 0.024 and cyclin E: p=0.048 expression in all BBC and higher nuclear expression of cyclin B in sBBC: p=0.027. Higher Ki-67 expression was correlated with higher expression of p16 (cytoplasmic: p=0.000015, p=0.086, p=0.0002 and nuclear: p=0.000009, p=0.016, p=0.00003) in all subsets [all BBC, sBBC (non-significant for cytoplasmic score), mBBC, respectively], p21 (all BBC: p=0.05) and sBBC: p=0.017), p53 (all BBC: p=0.0003 and mBBC: p=0.0002), cyclin A: all p<0.000001, cyclin B (cytoplasmic: p<0.000001, p=0.004, p<0.000001, respectively and nuclear: p=0.0002, p=0.047, p=0.0026, respectively), cyclin D3 (all BBC: p=0.005 and mBBC: p=0.02) and cyclin E (all BBC: p<0.000001 and mBBC: p=0.000002), and lower expression of cyclin D1 (all BBC: p=0.046 and mBBC: p=0.035) and p27 (sBBC: p=0.048). CONCLUSION: Compared to sBBC, mBBC are characterized by lower expression of p21 and higher cytoplasmic expression of cyclin B, suggesting its more aggressive behavior. Correlations between cell-cycle regulation proteins and markers of breast cancer phenotype parallel those reported for unilateral breast cancer.

Tumoral FOXP3 expression is associated with favorable clinicopathological variables and good prognosis in gastric adenocarcinoma: the tumor suppressor function of tumoral FOXP3 is related with the P21 expression in gastric adenocarcinoma.

The function and contribution of tumoral FOXP3 in gastric cancer development remain poorly understood. Thus, we studied the expression of tumoral FOXP3 and its relationship with the well-known tumor suppressor proteins P21 and P53 in gastric adenocarcinoma. The tissue microarray was constructed from 182 cases of gastric adenocarcinoma. The immunohistochemistry was performed on 4-µm tissue sections from each tissue microarray block. We found that positive tumoral FOXP3 expression was significantly correlated with a lower T category, a lower N category, a lower recurrence rate, and less lymphatic invasion. Furthermore, the survival analysis revealed that the tumoral FOXP3-positive group had significantly increased overall survival and disease-free survival rates compared with the tumoral FOXP3-negative group. Additionally, P21 expression showed a significant positive correlation with tumoral FOXP3 expression in gastric adenocarcinoma cells. Taken together, these findings demonstrate that tumoral FOXP3 expression is associated with favorable clinicopathological variables and good prognosis in gastric adenocarcinoma through increased expression of the tumor suppression protein P21.

Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

Protein expression patterns of cell cycle regulators in operable breast cancer.

BACKGROUND-AIM: To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes. METHODS: Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis. RESULTS: Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively. CONCLUSIONS: It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer.

[Correlation between Mic60 haploid insufficiency and cardiac aging in mouse].

Objective: To investigate the role of Mic60 in cardiac aging. Methods: Wild-type and Mic60(+ /-) male mice at age of 4-6 months (young group, n=6) and 18-20 months (aged group, n=9) were used. H&E and Masson staining of frozen and paraffin sections were subjected to morphologic evaluation of the cardiac tissue samples. SA-ß-Gal staining was utilized to detect the activity of senescence-associated ß-galactosidase. Western blot was performed to detect the expression of Mic60 and p21 in cardiac tissues. Results: Expression of Mic60 in mouse cardiac tissue increased in an age-dependent manner. Haploid insufficiency of Mic60 resulted in an increased left ventricular wall thickness [(1.32±0.09) mm vs.(1.12±0.09) mm, P<0.05], cardiomyocyte hypertrophy[(474.9±27.6) µm(2) vs.(358.8±48.7) µm(2), P<0.05] and interstitial fibrosis [ (38.24±7.58) ×10(3)µm(2) vs.(25.81±4.12)×10(3)µm(2,) P<0.05], increased activity of SA-ß-Gal (2.26±0.24 vs.0.25±0.05, P<0.01) and higher expression of p21 (P<0.01) in aged mouse cardiac tissue, but not in young mice. Conclusion: Haploid insufficiency of Mic60 leads to cardiac hypertrophy, interstitial fibrosis, increased activity of SA-ß-Gal and higher expression of p21 in aged cardiac tissue in mice, suggesting that Mic60 may prevent cardiac aging.

Urinary DcR2 is a novel biomarker for tubulointerstitial injury in patients with diabetic nephropathy.

Tubulointerstitial injury (TII) plays a crucial role in the progression of diabetic nephropathy (DN), but lack of specific and sensitive biomarkers for monitoring TII in DN management. This study is to investigate whether urinary decoy receptor 2 (uDcR2) could serve as a novel noninvasive biomarker for assessing TII in DN. We recruited 311 type 2 diabetics and 139 DN patients who were diagnosed by renal biopsy. uDcR2 levels were measured by ELISA, and renal DcR2 expression was detected immunohistochemically. Associations between uDcR2 and renal DcR2 and renal functional parameters were evaluated. Receiver operating characteristics (ROC) curve analyzed area under the curve (AUC) of uDcR2 for assessing TII. Double staining was undertaken for renal DcR2 with proximal and distal tubular markers; senescent markers p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal); and fibrotic markers collagen I and IV. We found DcR2 was primarily expressed in renal proximal tubules; uDcR2 levels were elevated per albuminuria stratum and correlated with renal functional parameters in diabetics and were associated with percentage of tubular DcR2 and TII score in DN. The uDcR2 had an AUC of 0.909 for assessing TII in DN by ROC analysis. Almost all tubular DcR2 was coexpressed with p16 and p21, and nearly more than one-half of tubular DcR2 was positive for SA-ß-gal, primarily in collagen I- and IV-positive regions of DN. Our results indicate uDcR2 could potentially serve as a novel biomarker for TII and may reflect senescence of renal proximal tubular cells in DN pathogenesis.

[Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

[Role of high mobility group protein B1 in IL-1α-induced endothelial cell senescence].

OBJECTIVE: To explore the effect of interleukin-1α (IL-1α) on the senescence of human umbilical vein endothelial cells (HUVECs) and the function of high mobility group protein 1 (HMGB1).
 Methods: HUVECs were randomly divided into a control group, a IL-1α group (10 ng/mL IL-1α), a HMGB1 group (100 ng/mL HMGB1), and a HMGB1+IL-1α group (100 ng/mL of HMGB1 plus 10 ng/mL of IL-1α). Senescence associated ß-galactosidase (SA ß-gal) staining was used to assess the number of senescent cells, Western blot were performed to detect the protein levels of silent information regulator 1(SIRT1), and quantitative real-time PCR (qRT-PCR) was used to detect the mRNA levels of p53, p21 and p16.
 Results: Compared with the control group, the number of SA ß-gal positive cells were significantly increased in the IL-1α group (P<0.05), while the expression of SIRT1 protein significantly decreased (P<0.01). Compared with the IL-1α group, the expression of SA ß-gal positive cells in the HMGB1+IL-1α group was decreased and the mRNA levels of p21 and p53 were down-regulated (all P<0.05), however, there was no statistical significance in the mRNA expression of p16 (P>0.05).
 Conclusion: IL-1α can induce the senescence of HUVECs, and HMGB1 may inhibit IL-1α-induced endothelial cell senescence via p53-p21 pathway.

Decreased DACH1 expression in glomerulopathy is associated with disease progression and severity.

Cell fate determination factor dachshund1 (DACH1) is a chromosome-associated protein that regulates cellular differentiation throughout development. Recent genome-wide association studies have show that missense mutation in DACH1 leads to hereditary renal hypodysplasia. Renal DACH1 expression can be used to estimate glomerular filtration rate (eGFR). We firstly characterized the function of DACH1 in normal and diseased renal tissue using immunohistochemistry to assess DACH1 in human renal biopsy specimens from 40 immunoglobulin A nephropathy (IgAN) patients, 20 idiopathic membranous nephropathy (IMN) patients, and 15 minimal change disease (MCD) patients. We found that DACH1 expression was decreased in the nephropathy group relative to healthy controls. DACH1 staining in the glomerulus correlated positively with eGFR (r = 0.41, p < 0.001) but negatively with serum creatinine (r = -0.37, p < 0.01). In vitro, DACH1 overexpression in human podocytes or HK2 cells decreased expression of cyclin D1, but increased expression of p21 and p53, which suggested that DACH1 overexpression in human podocytes or HK2 cells increased the G1/S phase or G2/M cell arrest. Together, These findings indicate that DACH1 expression is decreased in glomerulopathy imply a potential role for DACH1 in the this development of human chornic glomerulopathy. These data suggest that DACH1 is a potential a marker of disease progression and severity for glomerular diseases.