Preliminary study on CASK/Id1 pathway in fibroblasts of human keloid / 中华整形外科杂志

<p><b>OBJECTIVE</b>To verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.</p><p><b>METHODS</b>Immunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin. RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts. The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.</p><p><b>RESULTS</b>CASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances. Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts. The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 +/- 0.024, which was lower than that in the normal control group (1.076 +/- 0.008, t = 11.159, P < 0.05). The expression of Id1 mRNA was 0.497 +/- 0.014, which was higher than that in the normal control group (0.307 +/- 0.017, t = 15.148, P < 0.05). The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 +/- 0.006, which was lower than that in the normal control group (0.168 +/- 0.012, t = 13.524, P < 0.05); the expression of Id1 protein was 0.812 +/- 0.035, which was higher than that in the normal control group (0.368 +/- 0.031, t = 16.356, P < 0.05). The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate, while CASK also could be detected in the Id1 precipitate. There was a natural binding of CASK and Id1 in keloid fibroblasts.</p><p><b>CONCLUSION</b>CASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.</p>

Role of histone deacetylase activity in the developing lateral line neuromast of zebrafish larvae

Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.

Characteristics of antisense non-coding RNA in the INK4 locus and its roles in disease / 中国医学科学杂志(英文版)

With the development of genome-wide sequencing technology, 195 types of functional long non-coding RNAs (lncRNAs) have so far been found, and their cellular roles are gradually being revealed. Now lncRNAs have become a hotspot in the life science. These small molecules exist in almost all higher eukaryotes, and have very important regulatory roles in these organisms. This review briefly summarizes recent progress in researches on antisense non-coding RNA in the INK4 locus.

role of pharmacokinetics and pharmacodynamics in early drug development with reference to the cyclin-dependent kinase [CdK] inhibitor - Roscovitine

Pharmacokinetics, pharmacodynamics and pharmacogenetics play an important role in drug discovery and contribute to treatment success. This is an essential issue in cancer treatment due to its high toxicity. During the last decade, cyclin-dependent kinase inhibitors were recognized as a new class of compounds that was introduced for the treatment of several diseases including cancer. Cyclin-dependent kinases [Cdks] play a key role in the regulation of cell cycle progression and ribonucleic acid transcription. Deregulation of Cdks has been associated with several malignancies, neurodegenerative disorders, viral and protozoa infections, glomerulonephritis and inflammatory diseases. [R]-roscovitine is a synthetic tri-substituted purine that inhibits selectively Cdk1, 2, 5, 7 and 9. Roscovitine has shown promising cytotoxicity in cell lines and tumor xenografts. In this paper, we present several aspects of pharmacokinetics [PK] and pharmacodynamics [PD] of roscovitine. We present also some of our investigations including bioanalysis, haematotoxicity, age dependent kinetics, PK and effects on Cdks in the brain. Unfavourable kinetic parameters in combination with poor distribution to the bone marrow compartment could explain the absence of myelosuppression in vivo despite the efficacy in vitro. Higher plasma and brain exposure and longer elimination half-life found in rat pups compared to adult rats may indicate plasma and brain exposure and longer elimination half-life found in rat pups compared to adult rats may indicate that roscovitine can be a potential candidate for the treatment of brain tumours in children. Cdk5 inhibition and Erk1/2 activation that was detected in brain in rat pups may suggest the use of roscovitine in neurodegenerative diseases. Early pharmacokinetic/ pharmacodynamic studies are important issues in drug discovery and may affect further development of promising drug candidates

Gene profiling after knocking-down the expression of NS gene in prostate cancer PC-3 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To screen the genes and possible signal transduction pathways involved in the mechanism of nucleostemin (NS) in the proliferation of prostate cancer.</p><p><b>METHODS</b>Oligonucleotide DNA microarray was used to screen the genome changes after knocking-down expression of NS in PC-3 cells and quantitative real-time PCR was used to further confirm the important differentially expressed genes.</p><p><b>RESULTS</b>219 differentially expressed genes were found and theses genes were involved in cell cycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation, etc. INK4 family genes (p15, p16, p18) were up-regulated and cyclin D1, HDAC1 were down-regulated, the main action points were CDK4/6-cyclin D and pRb-E2F1 complexes.</p><p><b>CONCLUSION</b>NS may promote the progression of prostate cancer by inhibiting the expression of p15, p16, and p18 in PC-3 cells. NS is an important G(1)/S checkpoint regulator and its regulatory activity has been certified at gene level.</p>

Expression of cell cycle control genes in myelodysplastic syndromes / 中华血液学杂志

<p><b>OBJECTIVE</b>To study aberrant expression of cell cycle control genes in patients with myelodysplastic syndromes (MDS).</p><p><b>METHODS</b>Reverse transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of cell cycle control genes (cyclin D2, cyclin D3, cyclin A1, cyclin E, CDK2, CDK4, CDK6, p21, p27, p57, Rb and E2F1) in bone marrow mononuclear cells (BMMNCs) from 29 normal control, 27 MDS and 19 de novo acute myeloid leukemia (AML).</p><p><b>RESULTS</b>The expression levels of cyclin D3 (0.65 +/- 0.17, P < 0.05) and cyclin A1 (0.48 +/- 0.04, P < 0.05) in MDS were higher than those in normal control and significantly lower than those in AML. The expression rates and levels of cyclin D2 (40.7% and 0.78 +/- 0.21) and cyclin E (51.9% and 0.52 +/- 0.10) in MDS were statistically higher than those in normal control and AML. The expression level of CDK2 in MDS (0.66 +/- 0.19, P < 0.01) was higher than that in normal control (0.42 +/- 0.04) and the expression rate of CDK6 in MDS (25.9%) higher than in normal control (3.4%, P < 0.05). There was no significant difference of the expression rates and levels of CDK4 in MDS, AML and normal control. The expression rates and levels of p21 (77.8% and 1.18 +/- 0.21) and p27 (48.1% and 1.14 +/- 0.40) in MDS were statistically higher than those in normal control and AML. The expression level of p57 in MDS (0.69 +/- 0.06) was higher than that (0.53 +/- 0.05, P < 0.01) in normal control but lower than in AML (0.96 +/- 0.16, P < 0.01). The expression rate (55.6%) and level (0.85 +/- 0.17) of Rb in MDS were significantly higher than those in normal control and AML. The expression rate (7.4%) and level (0.39 +/- 0.04) of E2F1 in MDS were comparable to those in normal control but lower than those in AML.</p><p><b>CONCLUSION</b>MDS clones have aberrant mechanism of cell cycle control: high expressions of cyclin family members, CDK2 and CDK6 may lead to high proliferation; high expression of p21 and p27 may cause the G1 phase arrest.</p>

Expression of Cyclin Dependent Kinase Inhibitors of KIP Family in Gastric Cancer / 대한소화기학회지

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.