Direct Correlation among Telomere Length, Cellular Aging, and Rejuvenation Effects of Honey Child Powder

Purpose: Telomere length (TL) is an indicator of age;however, hormonal influences complicate individual aging. It remains unclear whether TL shortening is a direct factor in both individual and cellular aging. Therefore, we examined the direct relationship between TL and cellular senescence at the cellular level. Methods: Telomerase activity, TL, and gene expression were measured in cultured human lung-, fetal-, and skin-derived fibroblasts, human skin keratinocytes, and telomerase reverse transcriptase (TERT) gene-immortalized cells using detection kits, Cawthon’s method, and reverse transcription-quantitative polymerase chain reaction, respectively. Novel substances that elongate telomeres were screened to confirm cell rejuvenation effects. Results: Long-term cell culture of TIG-1-20 normal human fibroblasts resulted in TL shortening, decreased division rate, and senescence progression, whereas in OUMS-36T-2 cells, TL elongation via TERT gene transfer increased the division rate, reduced endoplasmic reticulum stress, and upregulated genes associated with young individuals, indicating that cellular rejuvenation occurs via TL elongation. In addition, a honey child powder (HCP) extract was found through screening, and the HCP extract strongly suppressed the menin gene, resulting in increased telomerase activity and extended cell lifespan. Upon addition of the HCP extract to skin fibroblasts, gene expression of moisturizing components, including collagen, hyaluronic acid, and elastin, increased, and exhibited a rejuvenating effect with an increase in elastin amount. Conclusions: TL elongation or shortening is involved in cell proliferation rate and cellular aging, and TL elongation rejuvenates cells. In addition, HCP extract has a rejuvenating effect on cells and is expected to be a rejuvenating compound.

The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance （ p 〈 0. 05, p 〈 0.01 ）. The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

EXPRESSION OF A MUTANT hTERT IN HUMAN BLADDER CARCINOMA CELL LINE T24 AND ITS CLINICAL SIGNIFICANCE

To construct a mutant pEGFP- hTERTexpression vector, to observe its steady expression intransfected human bladder carcinoma cell line T24 and its role in molecular regulatory mechanisms of telomerase, and to provide a new target gene for bladder cancer. Methods: PCR amplification was performed by using primers basedon the known gene sequence of hTERT. PCR productionwas cloned into plasmid pGEMT-T easy and the sequenceof mutant hTERT gene was analyzed. A recombinantmutant hTERT vector (pEGFP-hTERT) was constructed at the EcoR I and Sal I sites of the pEGFP-C1 vector. Aftertransfecting the fusion gene into bladder carcinoma cell line T24 by calcium phosphate-DNA coprecipitation, the steady expression of GFP-hTERT fusion protein was tested by fluorescent light microscopy. The proliferation changes ofbladder carcinoma cell line T24 were detected by lightmicroscopy and senescence correlated b-galactosidase staining. Results: Identification of pEGFP-hTERT byenzyme digestion showed that mutant hTERT fragment had been cloned into EcoR I and Sal I sites of the pEGFP-C1 vector. The steady expression of GFP-hTERT fusion protein was localized in the nucleus of transfected cells. Expression of senescence-associated b-galactosidase in transfected cells gradually increased with extended cultured time and cellgrowth was suppressed. Conclusion: The mutant-type hTERT gene suppresses the proliferation of bladder carcinoma cell line T24 by competitive effect on telomerase activity. This suggests that hTERT gene might be a suitable gene target for bladder cancer therapy.

Gain of human telomerase RNA gene is associated with progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ

Background The 3q26 chromosome region, where the human telomerase RNA gene （hTERC） is located, is a biomarker for cervical cancer and precancerous lesions. The aim of this study was to confirm the value of measuring hTERC gene gain in predicting the progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ （CIN-Ⅰ and -Ⅱ, respectively） to CIN-Ⅲ and cervical cancer. Methods Liquid-based cytological samples from 54 patients with CIN-Ⅰ or CIN-Ⅱ lesions were enrolled in this study. Follow-up was performed with colposcopy and biopsy within 24 months after the diagnosis of CIN-Ⅰ or CIN-Ⅱ. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization with a dual-color probe mix containing the hTERC gene probe （labeled red） and the control, the chromosome 3 centromere-specific probe （labeled green）.Results All patients whose lesions progressed from CIN-Ⅰ or CIN-Ⅱ to CIN-Ⅲ displayed a gain of the hTERC gene, whereas patients where the hTERC gene was not amplified did not subsequently progress to CIN-Ⅲ or cervical cancer. The signal ratio pattern per cell was recorded as N：N （green： red）. The numbers of cells with the signal ratio pattern of 4：4 or N：≥5 in patients whose lesions progressed to CIN-Ⅲ were significantly higher than those whose lesions did not progress. Significantly, none of the patients with a 4：4 signal ratio pattern regressed spontaneously.Conclusions In conclusion, measurement of hTERC gene gain in CIN-Ⅰ or CIN-Ⅱ patients using liquid-based cytological samples could be a useful biomarker to predict the progression of such cervical lesions. In addition, a 4：4 or N：≥5 signal ratio pattern may indicate the unlikeness of spontaneous regression of CIN-Ⅰ or CIN-Ⅱ lesions.

Telomerase activity and expression of the telomerase catalytic subunit gene in non small cell lung cancer: correlation with decreased apoptosis and clinical prognosis

To investigate the level of telomerase activation (TA) and telomerase catalytic subunit (hEST 2) gene mRNA in patients with non small cell lung cancer, and determine whether they are associated with tumor cell apoptosis, stage, and clinical outcome Methods Primary tumor specimens from 58 patients untreated with chemotherapy and 10 cases of histologically benign and adjacent lung tissue were analyzed TA and hEST 2 were measured by means of a modified telomerase repeat amplification protocol (TRAP) assay and in situ hybridization (ISH), respectively Terminal deoxynucleotidyl transferase (TdT) mediated biotin dUTP nick end labeling (TUNEL) was used to evaluate apoptotic cells Reverse transcription polymerase chain reaction (RT PCR) was used to detect bcl 2 mRNA expression Results TA and hEST 2 were detected in 45 (77 6%) and 43 (74 1%) of 58 tumor specimens, respectively, and not detected in specimens of adjacent and benign lung tissue One case expressed hEST 2 as a weak positive Statistically significant positive association was found between the level of TA and hEST 2( r =0 85, P =0 001) TA and hEST 2 were associated with tumor stage, but not associated with tumor grade, gender and patient age Positive rate of bcl 2 mRNA was 38 (65 5%) of 58 tumor specimens The mean apoptotic index in the bcl 2 positive group (9 5±1 3) was lower than that in the bcl 2 negative one (19 8±2 1, P <0.05), suggesting that apoptotic index may be inversely associated with bcl 2 expression ( r =-0 48, P =0 041) Bcl 2 expression in the TA and hEST 2 positive group (92 1% and 89 4%) was higher than that in the negative one (50 0% and 45 0%, P =0 043 and P =0 032, respectively) The apoptotic index was lower in the TA or hEST 2 positive group (8 2±1 4, 10 7±1 1) than in the negative one (20 5±1 6, 24 2±2 1, P <0 05) A statistically significant inverse association was found between TA or hEST 2 and apoptotic index ( r =-0 45, P =0 02 and r = -0 51, P =0 001, respectively) Positive correlation was also detected between TA or hEST 2 and bcl 2 expression ( r =0 86, P =0 01 and r =0 73, P =0 024, respectively) The level of hEST 2 mRNA and apoptotic index were associated with clinical outcome in a multivariate cox regression analysis Conclusions High TA and hEST 2 were frequently detected in primary non small cell lung cancer untreated with chemotherapy, having high bcl 2 expression and a low tumor cell apoptotic rate This suggests that both TA and hEST 2 are correlated with the deregulation of apoptosis hEST 2 and apoptotic index have prognostic significance in patients with non small cell lung cancer

Expression of telomerase hTERT in human non-small cell lung cancer and its correlation with c-myc gene

Objective To investigate the expression of human telomerase catalytic subunit,hTERT, in human non-small cell lung cancer (NSCLC) and its correlations to c-myc gene.Methods hTERT and c-myc mRNA expressions were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Statistical correlation analysis was made to estimate whether there was interrelation between them.Results Positive rate of hTERT expression in 51 surgically resected lung cancer specimens was 86.3%,significantly higher than that in adjacent non-neoplastic lung tissues and benign lesions,which were 14.3% and 27.3% respectively. No statistical significance was observed between the frequency of hTERT expression and histologic types,degree of differentiation,TNM stages,tumor size or lymph nodes metastases. Correlation analysis revealed that the expression of c-myc gene was significantly related to that of hTERT (correlation coefficient,r =0.633,P <0.001).Conclusions hTERT may be a useful tumor marker in diagnosing lung cancer. Significant correlation between the expression of hTERT and c-myc mRNA indicates that the activation and up-regulation of hTERT might be conferred by over-expression of c-myc gene.