Abstract
Telomere shortens and telomerase activation was found both in malignant hematological cell line cells and leukemia samples. However, the mechanism of telomerase activation is unclear. The regulation of telomerase activity could be explained by many aspects, telomere binding protein is one of the new one. Pinx1 is an inhibitor of telomerase activity. Overexpression of Pinx1 inhibits telomerase activity, shortens telomeres, and induces crisis, whereas depletion of endogenous Pinx1 increases telomerase activity and elongates telomeres. However, how does Pinx1 act on telomerase in malignant tumor cells is not clear. In this study, we detected the expression of telomerase inhibitor Pinx1 in acute leukemia cells and during the differentiation of acute promyelocytic leukemia cells to realize its effect on telomerase activity and the probable mechanism inside. 30 acute leukemia cases were all enrolled at their first diagnose, 17 samples were of acute non-lymphocytic leukemia (ANLL) while 13 of acute lymphoblastic leukemia (ALL). Realtime quantitative PCR with fluorescence probe hybridization was used to measure expression of Pinx1 and hTERT mRNA in acute leukemia cells and during differentiation of NB4 cells induced by ATRA. NB4 cells at the initial concentration of 1×105/ml were treated with 1uM ATRA. Cells were collected at 5 time points as 0h, 12h, 24h, 48h and 72h. Pinx1 mRNA expression in acute leukemia samples(0.00312, 5.42×10−4~0.024)is significantly higher than that in normal bone marrow mononuclear cells(7.89×10−4, 0~0.00863)(P<0.01=. Both its expression in ANLL cells(0.00296, 0.00103~0.0182)( P<0.05=and ALL cells(0.00327, 5.42×10−4~0.024) (P<0.05= were higher than that in normal bone marrow mononuclear cells. The expression of Pinx1 mRNA had significant positive correlation with hTERT mRNA expression (r=0.296, P<0.05=. Pinx1 mRNA expression decreased during NB4 cell differentiation, its expression was positive correlated with hTERT mRNA expression (r=0.900, P<0.05=. Why telomerase was activated in malignant tumor cells is unclear yet. It was considered to be activated after telomere length shortened to an extent. So telomere binding proteins may play an important role in telomerase activation. Pinx1 was regarded as a negative regulator of telomerase. It was reported that Pinx1 expression was significantly reduced in many human tumor samples including liver, prostate, colon and lung carcinoma. But some studies showed Pinx1 expression has no significant change in human gastrointestinal tract carcinoma and in human hepatocellular carcinoma. We found Pinx1 expression increased in acute leukemia cells and decreased during NB4 cell differentiation, positive correlated with hTERT mRNA expression As Pinx1 is the only telomere binding protein that can bind to hTERT directly, we hypothesized that the variation of Pinx1 mRNA expression during differentiation acted as a negative feedback to hTERT for stabilization of telomerase activity. When hTERT levels increased, more Pinx1 would be bound with hTERT resulting in reduced free Pinx1 protein in cells. This in turn caused compensate increase in Pinx1 transcription. When hTERT decreased, amount of free Pinx1 protein would increase and result in decrease in Pinx1 mRNA expression. So Pinx1’s variation may be a subsequent reaction induced by that of hTERT during differentiation, though the exact mechanism needs further studies.
Peer Review #2 of "The β-carboline alkaloid harmine inhibits telomerase activity of MCF-7 cells by down-regulating hTERT mRNA expression accompanied by an accelerated senescent phenotype (v0.1)"
