Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.</p><p><b>METHODS</b>The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.</p><p><b>RESULTS</b>The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.</p><p><b>CONCLUSIONS</b>Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.</p>

Correlation between the Expression Levels of Transforming Growth Factor-beta (TGF-beta) Receptors and Responsiveness to TGF-beta1-Induced Growth Inhibitory Effects in Leukemic Cell Lines / 대한혈액학회지

BACKGROUND: Defects in TGF-beta receptors have been found in a variety of malignant cells, we therefore investigated whether these defects could be also demonstrated in leukemic cells. In addition, we analyzed the relation between TGF-beta receptor expression and responsiveness to TGF-beta-induced growth inhibitory effects. METHODS: Eleven human leukemic cell lines and two normal cell lines were recruited for the study. To evaluate the expression of TGF-beta receptor type I (RI) and type II (RII), Western blotting analysis was conducted utilizing two kinds of primary antibodies against both RI and RII. Band strength was quantitated with densitometry. Moreover, specific peptides against primary antibodies were employed for competitive inhibition assay. Responsiveness to TGF-beta1 was assessed by [3H] thymidine uptake. RESULTS: Bands of same molecular size were demonstrated by two different primary antibodies. Peptides against RI or RII antibodies successfully blocked the emergence of RI or RII message, respectively, verifying that former bands represented specific RI or RII. Relative RI levels in leukemic cells except HL-60 compared with CCL-64, normal lung epithelial cells, were in the range of 0.48~1.14. In contrast, relative RII levels, although variable between individual cells, were less than 0.25 in all the leukemic cells. Percents [3H]thymidine uptake of leukemic cells at 10 ng/mL of TGF-beta1 compared with untreated control were widely distributed in the range of 7.7~62.9%. Positive correlation between RII levels and TGF-beta responsiveness was observed (P=0.025). CONCLUSION: Defective RI expression seems to be rare, however, defective RII expression appears to be rather common in leukemic cells. Positive correlation between RII levels and TGF-beta responsiveness suggests role of defective RII expression in the acquisition of resistance to TGF-beta in leukemic cells.

Density and Affinity of IL-6 Receptors in Human Leukemic Cells / 中国肿瘤生物治疗杂志

Objective: To make a study of density and affinity of IL-6R in human leukemic cell lines, and discuss the affection of high affinity IL-6R to the targeted treatment of leukemia with IL-6-PE40 fusion protein. Methods: Radial binding assay with scatchard plot and FACS were used to analysis the density and affinity of IL-6R and protein expression of IL-6Rα and β subunits in totally 8 representative human leukemic cell lines. Results: Myelocytie, monocytic and erythrocytic leukemic cell lines U937, HL-60, KG1 and TF1 express high affinity IL-6R, whose average density per cell is 2 502,2 874, 2 319 and 9 329 respectively, however no 125I-IL-6 binding was detected on chronic myelocytic leukemic cell line K562 and lymphoblastic leukemic cell lines such as Raji, CEM and HUT28. These results correlate with those of FACS highly. Conclusion:These observations suggest that acute nonlymphoblastic leukemic cells may be more suitable for targeted treatment with IL-6-PFA0 fusion protein.

Growth Inhibition of Leukemic Cells by C-myb Antisense Oligos: Use of Simulational Target Site Search and Enhanced Delivery of Antisense Oligos by Cationic Liposomes / 대한혈액학회지

BACKGROUND: Aberrant expression of c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos was shown to inhibit growth of normal as well as leukemic cells. C-myb antisense oligo for inhibition of tumor cell growth was, however, not decisive enough to be an effective anti-cancer agent. Thus, we set out to devise a systematic approach to find effective target sites for c-myb antisense oligos and to compare cellular uptake of antisense oligos complexed with different liposomes. METHODS: A computer simulation program for RNA secondary structures was employed to choose 8 potential target sites free of secondary structures along the entire c-myb mRNA sequence. Linear phosphorothioate-capped antisense oligos complementary to the selected target sites were synthesized and delivered into HL-60 and K562 cancer cell lines as liposomes complexes. RESULTS: Three of the 8 target sites were found to be relatively effective for reducing c-myb message. The three oliogs, MIJ-4, -17 and -18 were able to reduce c-myb message by more than 70% and suppressed tumor cell growth by about 70%. When three different cationic liposomes were used to facilitate the cellular uptake of antisense c-myb oligos, distinct liposome formulations were found to be comparably effective for reduction of c-myb message and inhibition of tumor cell growth. CONCLUSION: These results show that simulation of RNA secondary structure can be used to search effective target sites for antisense oligos and oligo uptake can be significantly enhanced by liposomes. However, cellular uptake of antisense oligos by liposomes needs further improvement.

Development of Covalently Closed c-myb Antisense Oligonucleotides for Growth Inhibition of Leukemic Cells / 대한암학회지

PURPOSE: Aberrant expression of the c-myb gene is often detected in transformed leukemic cells. Inhibition of c-myb expression by antisense oligos could be an effective way to abort rapid growth of leukemic cells. Developing stable antisense oligos combined with enhanced delivery into cells would be of great use in developing an effective anti-cancer molecular agent. MATERIALS AND METHODS: Selection of target sites was carried out by employing computer simulation of mRNA secondary structures. Multiple antisense oligo sequences were adjoined and AS-oligos were then covalently closed to evade exonuclease activities. C-myb antisense oligos with a novel structure were complexed with cationic liposomes and used to treat HL-60 leukemic cells. RESULTS: We developed covalently closed antisense oligos which harbor four adjoined antisense sequences. The c-myb antisense oligos were found to be exceptionally stable and effective in specifically ablating c-myb mRNA. The antisense oligos were able to inhibit growth of leukemic cell line (HL-60) by about 80%. Antisense effect was more pronounced when the cells were treated twice with the antisense oligos at lower concentrations. CONCLUSION: The novel covalently closed antisense oligo (CMAS-oligos) was found to be effective and exceptionally stable, Growth of HL-60 was significantly inhibited, showing a rational way to develop an effective molecular anti-cancer agent.

Apoptotic mechanism of leukemic K562 cells induced by mangiferin / 中草药

Objective To study the apoptosis mechanism of K562 cell lines induced by mangiferin. Methods The mRNA expression levels of apoptosis-related genes including bcl-2, bax, survivin of K562 cells treated by mangiferin (25—200 ?mol/L) for 24, 48, 72, and 96 h were determined by RT-PCR; the BCR/ABL protein P210 level was detected by Western blotting. Results Mangiferin up-regulated bax gene of K562 cells significantly and down-regulated bcl-2 gene slightly, resulting in an enhancement of the ratio of bax/bcl-2. Mangiferin down-regulated the expression levels of P210 in K562 cells in a time-and concentration-dependent manner and so is the expression level of survivin mRNA in K562 cells. ConclusionThe mechanism of mangiferin-induced apoptosis in K562 leukemic cells might be involved in up-regulating the gene expression of bax and down-regulating the mRNA expression of BCR/ABL protein P210, bcl-2, and survivin.

A complete remission can be achieved despite persistence of abnormal bone marrow promyelocytes in acute promyelocytic leukemia: experience in 2 patients

Acute promyelocytic leukemia (APL) is a distinct subset of acute myeloid leukemia (AML) and is distinguished from other subsets of AML by its distinctive morphology, specific chromosomal abnormality, associated consumptive coagulopathy, and response to treatment. Interestingly, patients with APL frequently enter complete remission without undergoing a characteristic period of bone marrow hypoplasia. In two cases in this report, complete remission was achieved without bone marrow hypoplasia without further additional course of chemotherapy despite the appearance of persistent malignant cells in the bone marrow after first induction chemotherapy. During the period of treatment, severe coagulopathy occurred in both cases but resolved as the patients entered into remission. Remission in patients with APL may occur even when induction therapy fails to cause marrow hypoplasia or to eradicate replicative cells. To avoid unnecessary exposure to toxic therapy, caution should be exercised in assessing the adequacy of remission induction treatment.

EXPERIMENTAL TREATMENT OF L801 MYELOID LEUKEMIC MICE WITH LOW MOLECULAR WEIGHT TUMOR SUPPRESSOR OF NEW BORN CALF LIVER ORIGIN / 解放军医学杂志

With techniques of biochemical isolation and pruification, a semi-purified preparation of new born calf liver (BLS) with the peculiarity of low molecular weight has been found to have tumor surppressor activity inhibiting selectively the growth of murine myeloid leukemic cell (L801). But its effect is less marked against normal bone marrow granulocyte / macrophage progenitors in in vitro liquid or agar culture. Mice after inoculation of L801 cells died within 15 days due to development of leukemia. After consecutive injections of BLS, 25% - 53.8% of treated mice survived. Furthermore, it has been found that the survival rate was raised from 50% to 66% when Cyclophosphamide was first injected in the dose of 300?g / kg body weight once, followed by the consecutive injections of BLS. NO pathological changes have been observed in the mice which survived longer than 100 days after inoculation of L801 leukemic cells.