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The study was supposed to investigate the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide (ASPSODN) targeting hTERT mRNA on gene of interest in K562 cells and influence of ASPSODN on telomerase activity and apoptosis of K562 cells. Human leukemia cell line K562 was transfected by liposome with ASPSODN and SPSODN (sense phosphorothioate oligodeoxynucleotide) at different concentrations (0.2, 0.6, 1.0 micromol/L). At the same time, blank control, liposome control and SPSODN groups were designed for comparison. The transfected cells were collected and detected at 24 and 48 hours; the expression of target gene hTERT mRNA and telomerase activity were detected by RT-PCR and TRAP-ELISA respectively, and cell apoptosis was assayed by flow cytometry. The results showed that after K562 cells were transfected for 24 hours, the expression of hTERT mRNA had no difference between liposome control (0.80+/-0.24), 0.2 micromol/L ASPSODN (0.69+/-0.12), 0.2 micromol/L SPSODN (0.72+/-0.25) and blank control (0.85+/-0.28), but the expression of hTERT mRNA in 0.6 micromol/L ASPSODN group (0.42+/-0.16) remarkably decreased as compared with liposome control group, 0.6 micromol/L SPSODN (0.69 +/- 0.26) had no obvious effect on the expression of hTERT mRNA, the expression of hTERT mRNA in 1.0 micromol/L ASPSODN and SPSODN groups both decreased; mortality of K562 cells transfected by liposome with 1.0 micromol/L ASPSODN and SPSODN remarkably increased. After 24 hours, telomerase relative activity of K562 cells showed no significant difference between blank control (88.9%) and liposome control (77.7%). The telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 60.6%, 52%, 58.2% respectively. There was significant difference as compared with blank control; 0.6 micromol/L ASPSODN showed significant difference (p=0.037), as compared with liposome control group. The telomerase relative activities in K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN were 76.1%, 72.2%, 65.7% respectively, but the telomerase relative activities of K562 cells in 0.2, 0.6 micromol/L SPSODN groups was not inhibited obviously. When K562 cells were treated for 48 hours, telomerase relative activity of K562 cells in each ASPSODN groups restored. It showed that telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L ASPSODN were 84.1%, 82.3%, 79.6% respectively, while telomerase relative activities of K562 cells treated with 0.2, 0.6, 1.0 micromol/L SPSODN for 48 hours were 74.8%, 74.5%, 67.9% respectively. Telomerase activity of K562 cells could not be inhibited by 0.2 and 0.6 micromol/L SPSODN. After culturing for 48 hours, the cell apoptosis rates of K562 in 0.6 micromol/L ASPSODN, 0.6 micromol/L SPSODN, liposome control and blank control groups were (4.82+/-0.39)%, (1.83+/-0.34)%, 1.84+/-1.04)%, (1.07+/-0.74)% respectively. There was difference between ASPSODN and SPSODN groups (p<0.05), but the significant difference was found in ASPSODN group as compared with liposome control and blank control (p<0.01). It is concluded that the ASPSODN targeting hTERT can specifically inhibit the expression of hTERT mRNA in K562 cells and significantly suppress the telomerase activity of K562 cells at 0.6 micromol/L, which inhibitory time is short. The ASPSODN at high concentration (1.0 micromol/L) shows definite cytotoxicity. 0.6 micromol/L of ASPSODN significantly induces cell apoptosis, while no such effect was seen in SPSODN group.

This study was aimed to investigate the effect of plasmid-mediated RNAi targeting hTERT on blocking hTERT gene and inhibiting telomerase activity in leukemia cell line K562. For inhibiting hTERT mRNA, three siRNA strands were chemosynthesized and transfected into K562 cells, two effective and specific siRNA strands were chosen. Then plasmid pSUPER-U6-Kan rhTERT-1, pSUPER-U6-Kan rhTERT-2 targeting hTERT mRNA were constructed and transfected into K562 cells by liposome. The expression of hTERT mRNA, telomerase activity and cell apoptosis were detected at 48 hours and 72 hours. The results showed that three chemosynthesized strands began to significantly inhibit target gene expression at 48 hours, but the inhibiting rates were different. The inhibiting effect disappeared after 72 hours. After plasmid pSUPER-U6-Kan rhTERT -1 (P-1 group) and pSUPER-U6-Kan rhTERT -2 (P-2 group) were transfected into K562 cells, the expressions of hTERT mRNA both decreased in the two groups. The expression of hTERT mRNA in P-1 group was 0.39+/-0.13 at 48 hours, 0.57+/-0.32 at 72 hours. The expression of hTERT mRNA in P-2 group was 0.55+/-0.20 at 48 hours, 0.88+/-0.23 at 72 hours. Telomerase activity in P-1 group was 0.42+/-0.07 at 48 hours, 0.31+/-0.08 at 72 hours; the telomerase activity in group P-2 was 0.49+/-0.27 at 48 hours, 0.39+/-0.03 at 72 hours while telomerase activities in both groups were significantly lower than that in negative control group (0.88+/-0.30, 0.88+/-0.32). At 48 hours, the apoptosis rates in P-1 group (18.39+/-3.08%) and P-2 group (15.5+/-3.59%) were significantly higher than that in negative control group (7.64+/-3.73%). At 72 hours the apoptosis rate in P-1 group (13.2+/-1.18%) and in P-2 group (12.86+/-3.09%) had no significant difference as compared with negative control group (8.07+/-0.19%). It is concluded that RNAi targeting hTERT inhibits the expression of hTERT mRNA, and therefore inhibits telomerase activity. The inhibiting effect is closely correlated with target site. The si-hTERT-1 effect is better than si-hTERT-2, while si-hTERT-3 almost has no effect at all. The effective time of plasmid-induced RNAi is obviously longer than that of chemosynthesized siRNA. The former is longer than 72 hours, while the latter is only 48 hours. After the telomerase activity is inhibited, cell apoptosis increases a little than control group at 48 hours. At 72 hours cell apoptosis has no difference with control group. It is supposed that some cells can be induced to differentiation when telomerase activity has been down regulated.

This study was aimed to investigate the modulating effects on telomere length and telomerase activity in K562 cells treated by arsenic trioxide, ginseng saponin, beta-elemene alone or in combination with cyclophosphamide (CTX) and to explore the possible mechanism and new therapy for acute leukemia. Human erythroleukemic cell line K562 was co-cultured with the above-mentioned drugs. Cells were collected after 24, 48 and 72 hours for further detection. Telomere length and telomerase activity were detected by Southern-blot and PCR-ELISA respectively. The effects of these drugs were observed at different concentrations and exposure time. The results showed that (1) ginseng saponin, arsenic trioxide, beta-elemene, or CTX could completely inhibit the telomerase activity of K562 cells at proper concentrations and exposure time. The inhibiting effects were enhanced when the three former drugs were used with CTX. Telomerase activity decreased proportionally with the concentrations and length of time. (2) viability of K562 cells was decreased after being co-cultured with arsenic trioxide, ginseng saponin, beta-elemene and CTX. The level of inhibition depends on the concentration and exposure time. (3) telomere length of K562 cells was 5.36 +/- 0.18 kb. After being co-cultured with those drugs for 72 hours, telomere length was 5.90 kb -6.50 kb, significantly longer than that of control (5.18 - 5.35 kb). It is concluded that arsenic trioxide, ginseng saponin, and beta-elemene can inhibit the growth and telomerase activity of K562 cells. The inhibiting effects were enhanced when they were used in combination with CTX. The depression of telomerase activity may be one of the mechanisms of anti-tumor effect. Less dosage and shorter course can be expected when arsenic trioxide, ginseng saponin, and beta-elemene are used in combination with CTX. When telomerase activity was depressed, the telomere length prolonged a little, indicating K562 cell line may extend telomeres by some alternative way other than telomerase activation.

The aim was to explore the modulating and inhibiting effects of arsenic trioxide, ginseng saponin and beta-elemene on telomere length and telomerase activity in K562 cell line, and to study their anti-tumor mechanism and seek new method of therapy for acute leukemia. Human erythroleukemia cell line K562 was co-cultured with arsenic trioxide, ginseng saponin, beta-elemene separately, cells were collected after 24, 48 and 72 hours for further detecting. Telomere length and telomerase activity were detected by the methods of Southern-blot and PCR-ELISA respectively. The effects of these drugs on telomere length and telomerase activity were observed at different concentrations and length of time. The results showed that (1) telomerase activity of K562 cells decreased after co-cultured with arsenic trioxide, ginseng saponin and beta-elemene. The inhibiting effects depended on drug concentrations and length of time. When co-cultured at proper concentration and period of time, telomerase activity could be inhibited; (2) viability of K562 cells decreased after co-cultured with arsenic trioxide, ginseng saponin and beta-elemene, the inhibiting effect depends on drug concentrations and length of time; (3) after co-cultured with arsenic trioxide, ginseng saponin, and beta-elemene for 72 hours, telomere length of K562 cell line prolonged a little. It is concluded that (1) arsenic trioxide, ginseng saponin and beta-elemene can inhibit telomerase activity in K562 cell line, the suppression of telomerase activity may be one of the mechanisms of anti-tumor effect; (2) arsenic trioxide, ginseng saponin and beta-elemene can inhibit the growth of K562 cell line, the inhibiting effect depends on concentration and time; (3) when telomerase activity was suppressed, the telomere length prolonged a little, indicating that in K562 cell line may exist another mechanism to regulate telomere length, except telomerase activation.

To explore the change of telomerase activity in acute leukemia (AL) cells and its relationship with cell cycle, PCR-ELISA was used to detect telomerase activity of bone marrow cells from 148 AL patients, including 92 cases with acute non-lymphocytic leukemia (ANLL) and 56 cases with acute lymphocytic leukemia (ALL). Thirty-six patients without bone marrow disorders were detected as normal control. The cell cycle of 16 patients and 4 controls was detected with flow cytometry. The results showed that the positive rate of telomerase was 71.6% (106/148) in the cells from AL patients, which was higher than that in the control group 5.6% (2/36). It was 88.9% (32/36) in the relapse group and 81.3% (61/75) in the untreated group. Both rates were higher than that in the CR group (35.1%, 13/37). There was no significant difference in the ALL and ANLL groups. The cell number in various phases of cell cycle had no significant difference between telomerase positive and negative groups. It was concluded that the activation of telomerase was very common in acute leukemia cells. Telomerase positive rate was closely associated with the different stages and progress of acute leukemia, and it might be a molecular marker for increased proliferation of leukemic cells during the process of the disease. Activation of telomeras had no correlation with cell number in different phases of cell cycle, while telomerase activity is modulated by other biological factors in addition to cell cycle.