The TKTL1 gene influences total transketolase activity and cell proliferation in human colon cancer LoVo cells.

A plasmid carrying DNA to be transcribed into a small interfering RNA against transketolase-like-1 mRNA was constructed and transfected into a human colon cancer cell line. The mRNA expression of transketolase gene family in the human colon cell line was determined by real-time polymerase chain reaction. The effect of anti-transketolase-like-1 small interfering RNA on cell proliferation and cell cycle in the human colon cancer cell line cells was detected by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide. The transketolase-like-1 gene was significantly downregulated in human colon cancer cell line cells transfected with small interfering RNA transketolase-like-1 constructs compared with the cells transfected with control vector and the cells without transfection. In addition, the anti-transketolase-like-1 small interfering RNA construct significantly decreased the level of transketolase in the transfected human colon cancer cell line cells, arrested them in G0/G1 phase and substantially inhibited cell proliferation. No significant difference was found in the other two genes (transketolase and transketolase-like-2 genes) between the transfected human colon cancer cell line cells and the controls (P>0.05). Our data demonstrated that the transketolase-like-1 gene plays an important role in total transketolase activity and in the cell proliferation of human colon cancer. Transketolase-like-1 may serve as a target for novel anticancer therapies.

[Effect of glucose-6-phosphate dehydrogenase on intracellular gsh level in Raji cells during oxidative stress].

AIM: To explore a role of G6PD in replenishment of intracellular GSH during oxidative stress. METHODS: In vitro Raji cell was cultured, intracellular GSH levels and G6PD, GR, GPX activities were determined at different time points after PMS treatment when G6PD activity was inhibited or not by DHEA. RESULTS: Intracellular GR, GPX, G6PD activities elevated significantly combined with GSH level decreased dramatically before 30 minutes, replenished gradually after 30 minutes and restore normal levels about 6 h after PMS treatment when G6PD was not inhibited. No change in GR and significant increase in GPX activity were shown following depleted GSH after PMS treatment when G6PD was inhibited by DHEA. CONCLUSION: G6PD contributes to replenish intracellular GSH and is a critical factor regulating GSH levels during oxidative stress.

Detection of three common G6PD gene mutations in Chinese individuals by probe melting curves.

OBJECTIVE: In order to develop and validate an assay for rapid detection of three common G6PD gene mutations in Chinese individuals. METHODS: In this report we design two sets of primers and fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon; after annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplication process. At the completion of PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each mutation was obtained. By using the fluorescence method and PCR/RE, a total of 57 samples obtained from two groups of G6PD-deficient individuals were studied. RESULT: A rapid method for detection of three common G6PD gene mutations in Chinese individuals by probe melting curves was developed. This method shows 100% accordance with the traditional method. CONCLUSION: This fluorescent melting curve analysis is a simple, rapid, and effective method for clinical diagnosis and screening of G6PD deficiency.