M6A modification regulates tumor suppressor DIRAS1 expression in cervical cancer cells.

DIRAS family GTPase 1 (DIRAS1) has been reported as a potential tumor suppressor in other human cancer. However, its expression pattern and role in cervical cancer remain unknown. Knockdown of DIRAS1 significantly promoted the proliferation, growth, migration, and invasion of C33A and SiHa cells cultured in vitro. Overexpression of DIRAS1 significantly inhibited the viability and motility of C33A and SiHa cells. Compared with normal cervical tissues, DIRAS1 mRNA levels were significantly lower in cervical cancer tissues. DIRAS1 protein expression was also significantly reduced in cervical cancer tissues compared with para-cancerous tissues. In addition, DIRAS1 expression level in tumor tissues was significantly negatively correlated with the pathological grades of cervical cancer patients. DNA methylation inhibitor (5-Azacytidine) and histone deacetylation inhibitor (SAHA) resulted in a significant increase in DIRAS1 mRNA levels in C33A and SiHa cells, but did not affect DIRAS1 protein levels. FTO inhibitor (FB23-2) significantly down-regulated intracellular DIRAS1 mRNA levels, but significantly up-regulated DIRAS1 protein levels. Moreover, the down-regulation of METTL3 and METTL14 expression significantly inhibited DIRAS1 protein expression, whereas the down-regulation of FTO and ALKBH5 expression significantly increased DIRAS1 protein expression. In conclusion, DIRAS1 exerts a significant anti-oncogenic function and its expression is significantly downregulated in cervical cancer cells. The m6A modification may be a key mechanism to regulate DIRAS1 mRNA stability and protein translation efficiency in cervical cancer.

The novel gene LRP15 is regulated by DNA methylation and confers increased efficiency of DNA repair of ultraviolet-induced DNA damage.

LRP15 is a novel gene cloned from lymphocytic cells, and its function is still unknown. Bioinformatic data showed that LRP15 might be regulated by DNA methylation and had an important role in DNA repair. In this study, we investigate whether the expression of LRP15 is regulated by DNA methylation, and whether overexpression of LRP15 increases efficiency of DNA repair of UV-induced DNA damage in HeLa cells. The results showed (1) the promoter of LRP15 was hypermethylated in HeLa cells, resulting a silence of its expression. Gene expression was restored by a demethylating agent, 5-aza-2'-deoxycytidine, but not by a histone deacetylase inhibitor, trichostatin A; (2) overexpression of LRP15 inhibited HeLa cell proliferation, and the numbers of cells in the G2/M phase of the cell cycle in cells transfected with LRP15 increased about 10% compared with controls; (3) cyclin B1 level was much lower in cells overexpressing LRP15 than in control cells; and (4) after exposure to UV radiation, the LRP15-positive cells showed shorter comet tails compared with the LRP15-negative cells. From these results we conclude that the expression of LRP15 is controlled by methylation in its promoter in HeLa cells, and LRP15 confers resistance to UV damage and accelerates the DNA repair rate.

[Effect of 2-methoxyestradiol on differentiation of primary myeloma cells].

In order to investigate the differentiation-inducing effect of 2-methoxyestradiol (2ME2), an estrogen derivative, on CD138+ (Syndecan-1) primary myeloma cells from 7 patients with myeloma, primary myeloma cells from 7 patients were enriched by using CD138 immunomagenetic beads. The enriched CD138+ (Syndecan-1) myeloma cells treated with 0.5 micromol/L 2ME2 for 36 h and 72 hours were used to observe the differentiation changes with expression of CD49e and to quantitatively detect the light-chain secretion in the supernatants. The results indicated that 2ME2 caused morphological and immunophenotypic changes with light-chain secretion in the supernatant, and the typical features of differentiation of CD138+ (Syndecan-1) primary myeloma cells. CD138+ (Syndecan-1) myeloma cells became more and more mature in morphology, with decreased ratio of nucleus to plasma, disappearance of nucleoli, and pyknosis of chromatin. The result of detecting the expression of CD49e of CD138+ (Syndecan-1) myeloma cells showed the obvious up-regulation of CD49e expression after exposure to 2ME2 for 72 hours. Quantitative assay for light chain secretion in the supernatant of bone marrow CD138+ cells from patients showed remarkable increment at 72 hours. It is concluded that lower concentrations of 2-methoxyestradiol can induce differentiation of bone marrow CD138+ cells from multiple myeloma patients.

Preliminary investigation of 2-methoxyestradiol inducing differentiation of myeloma cell line CZ-1.

This study was aimed to investigate whether 2-methoxyestradiol (2ME2) could exert effect of inducing differentiation on myeloma cells. A myeloma cell line CZ-1 secreting lambda light chain protein was used as an object of study. The CZ-1 cell morphology was observed by Wright's staining, the CD49e expression on cell surface after treatment with 2ME2 was detected by flow cytometry, the concentration of lambda light chain protein in the supernatant was assayed by immuno-scattering turbidity method. The results showed that treatments with 0.1-0.5 micromol/L 2ME2 for 72 hours resulted in some mature morphological changes of CZ-1 cells, such as the ratio of karyoplasms going down, nucleolus reducing or disappearing, chromatin getting rougher and more compacted; the CD49e positive CZ-1 cells increased by 2ME2 with concentrations of 0.1 micromol/L to 0.5 micromol/L in a concentration-dependent manner. The statistical difference from the control group was significant; the concentration of lambda light chain protein increased from control group 29.3 +/- 2.77 microg/ml to 35.97 +/- 2.6 microg/ml (P < 0.05) after exposure to 0.1 micromol/L 2ME2 for 72 hours, and the treatment of 0.5 micromol/L 2ME2 up-regulated lambda light chain protein to 79.67 +/- 1.88 microg/ml (P < 0.01) continuously. It is concluded that 2ME2 at low-concentration can induce differentiation of the CZ-1 cells to mature, which provides a new, and safe strategy for myeloma therapy.

[Effect of 2-methoxyestradiol on cell differentiation of myeloma cell line CZ-1].

OBJECTIVE: To investigate the differentiation induction effect of 2-methoxyestradiol (2ME2), an estrogen derivative on myeloma cell line CZ-1. METHODS: The changes of CZ-1 cells in morphology, expression of surface CD49e and quantity of light chain secretion in the supernatant were observed when treated with 0.1 approximately 0.5 micromol/L 2ME2 for 48 h. RESULTS: 2ME2 could induce differentiation of CZ-1 cells. The cells appeared decreased in size of nucleus, increased in cytoplasma, decreased in the ratio of nucleus to plasma, decreased in number or disappearance of nucleolus, and thickness and pyknosis of chromatin. The expression of CD49e was increased from (12.20 +/- 1.57)% to (24.80 +/- 1.26)% (P < 0.05). Light chain secretion in the supernatant was increased from (35.97 +/- 2.60) microg/ml to (79.67 +/- 1.88) microg/ml (P < 0.05). CONCLUSION: Low concentrations of 2ME2 could induce differentiation of myeloma cell line CZ-1.

[Investigation of the effect of 2-methoxyestradiol and arsenic trioxide on the apoptosis-associated gene expression profile of myeloma cells].

OBJECTIVE: To explore the effect of 2-methoxyestradiol (2ME2) and arsenic trioxide (As2O3) on the apoptosis related gene expression in multiple myeloma cell line CZ-1. METHODS: Total RNA was isolated from CZ-1 cells which had been treated with 2ME2 and As2O3 at an apoptosis-inducing concentration and reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP, and then hybridized it with a microarray containing up to 96 key genes involved in apoptosis. The gene expression profile of the 2ME2 and As2O3 treated CZ-1 cells were analyzed with GEArray Analyzer software. The microarray results were confirmed by RT-PCR. RESULTS: As2O3 treatment caused the alteration in the expression of 52 genes (54.2% of total genes on microarray). Among them, 42 (80.8%) were upregulated and 10 (19.2%) were downregulated. The upregulated genes were mainly involved in caspases family, P53 and ATM pathway, death effector domain family, TNF receptor family and CIDE family. 2ME2 treatment resulted in the alteration of 42 genes (43.8% of the total genes on microarray). Of them, 32 (76.2%) were downregulated and 10 (23.8%) were upregulated. The downregulated genes mainly belonged to bcl-2 family, inhibitor of apoptosis protein family (IAP), TRAF family, TNF ligand family, and CARD family. CONCLUSION: 2ME2 and As2O3 induce the CZ-1 cells apoptosis by different pathways. As2O3 mainly induces upregulation of proapoptotic genes, and 2ME2 downregulation of anti-apoptotic genes expression.

[Effect of 2-methoxyestradiol on proliferation and apoptosis of myeloma cell lines].

The objective was to explore the in vitro effects of growth inhibition and apoptosis induction of 2-methoxyestradiol (2ME2), an estrogen derivative, on seven myeloma cell lines NCI-H929, HS-sultan, KM3, SKO-007, CZ-1, U266 and LP-1and to observe its synergistic effects in combination with some other drugs, such as dexamethasone, As(2)O(3), thalidomide and zoledronic acid. Seven myeloma cell lines NCI-H929, HS-sultan, KM3, SKO-007, CZ-1, U266 and LP-1 were cultured at different concentrations with or without dexamethasone, As(2)O(3), thalidomide and zoledronic acid. Cell viability was assessed by trypan blue assay, plasma cell labeling index (PCLI) was detected by BrdU assay, terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay were used to determine apoptosis cells in situ. Synergistic effects of 2ME2 in combination with other drugs were judged by King's formula. The results showed that after treatment with 1, 4, 8, 12, 16 micromol/L 2ME2 at 12, 24, 36 and 48 hours respectively, 2ME2 caused a dose- and time-dependent inhibition of the cell viability. The concentration of 50% growth inhibition (IC(50)) was between (20.8 +/- 0.27) and (34.1 +/- 0.57) micromol/L. After treatment with 12 micromol/L 2ME2 within 24 hours, 2ME2 led to a progressive decline in the fraction of S-phase cells by BrdU assay, plasma cell labeling index (PCLI) declined from (30.14 +/- 4.28)% to (14.71 +/- 6.27)% (P < 0.05). After treatment with 1, 4, 8, 12, 16 micromol/L 2ME2 at 12, 24, 36 and 48 hours respectively, 2ME2 can induce a dose- and time-dependent apoptosis of myeloma cell lines. The percentage of apoptosis was between 9% - 33% (P < 0.05). Q value of synergistic effects was between 1.13 to 1.43. It is concluded that 2ME2 can inhibit proliferation and induce apoptosis of myeloma cell lines and has synergistic effects with dexamethasone, As(2)O(3), thalidomide and zoledronic acid.

[Molecular mechanism of myeloma cell line differentiation induced by 2-methoxyestradiol].

OBJECTIVE: To study the molecular mechanism of differentiation induction by 2-methoxyestradiol (2ME2) of myeloma cell lines. METHODS: Differentiation induction effect on myeloma cell lines LP-1, CZ-1 and NCI-H929 which were incubated with 2ME2 and XBP-1, Blimp-1, pax-5 phosphorothioate antisense oligodeoxynucleotide (ASODN) was evaluated by cell morphology, CD49e expression, quantitation of light chain secretion, and the level of pax-5 and XBP-1 mRNA expression. RESULTS: 2ME2 caused morphological, immunophenotypic and the supernatant light chain secretion changes typical of differentiation in all the three myeloma cell lines. 2ME2 up-regulated the XBP-1 mRNA expression. XBP-1 and Blimp-1 ASODNs partially inhibited the differentiation of LP-1, CZ-1, NCI-H929 cells induced by 2ME2; whereas pax-5 ASODN did the contrary. After incubated with pax-5 ASODN for 72 hours, LP-1, CZ-1, NCI-H929 cells exhibited characteristic morphologic feature of differentiation. The expression of CD49e was increased statistically (P < 0.05). Light chain secretion in the supernatant was also increased statistically (P < 0.05). After incubation with Blimp-1 ASODN, the level of XBP-1 mRNA was declined, while the level of pax-5 mRNA increased. CONCLUSION: 2ME2 could induce cell differentiation and up-regulate XBP-1 mRNA expression in myeloma cell lines. Blimp-1 could help 2ME2 with inducing differentiation of myeloma cells through downregulating pax-5 mRNA and upregulating XBP-1 mRNA.