In this study the effects of red orpiment on NB4 and HL-60 cells were tried to determine. Semi-quantitative RT-PCR to determine the PML mRNA expression, immuno-fluorcscence study, together with the fluorescence stain and morphological observations were used. The results showed that: (1) red orpiment induces apoptosis morphologically in NB4 and HL-60 cells, the morphology of typical apoptosis can be seen in NB4 and HL-60 cells after 12 hours of treatment with red orpiment. Through the Wright's stain, we can see the extensive membrane blebbing, condensation of heterochromatin, and cell fragmentation, apoptotic body appearing. Many dead cells can be found on the second day. (2) in NB4 cells, red orpiment is shown to induce the PML-RARalpha chimera disappearance and to reorganize then to degradation of PML nuclear bodies which also seen in HL-60 cells, indirect immunofluorescence staining of PML with a specific monoclonal antibody was performed in control and treated cells. In NB4 cells, the control was diffusely microspeckled pattern of immunoreactivity. Upon red orpiment treatment, the microspeckled pattern disappeared, PML protein reversed into normal location. and the the size and the brightness of the particles were increased obviously. The normal nuclear distribution of PML protein was seen in untreated HL-60 cells. After treatment with red orpiment, in the nuclei of HL-60 cells, the size and the brightness of the particles were also increased. After two days of treatment with red orpiment, the immunofluorescent particles in cells almost disappeared. (3) the expression of PML mRNA is not changed in red orpiment-treated cells, RT-PCR to determine the PML mRNA expression in NB4 and HL-60 cells treated with red orpiment, the expression results are similar to the controls, that to say, the PML mRNA lever is unaffected. It was concluded that, red orpiment induced PML to play the effects of induce apoptosis in leukemia cells at the translational level and inhibited the proliferation of leukemia cells.

OBJECTIVETo investigate if CYP3A5 is involved in drug resistances mechanisms of acute leukemia.

METHODSBy using RT-PCR, immunohistochemistry and MTT assay, CYP3A5 mRNA and protein were detected in leukemia cell lines and acute leukemia patients, meanwhile transcriptional regulation of CYP3A5 induced by daunorubicin was observed. A pcDNA3-CYP3A5 reconstituted plasmid and its stably transfected cell line HL-60/CYP3A5 were both established.

RESULTSCYP3A5 mRNA was detected in K562 and U937 cells, whose IC(50) values of daunorubicin were 2.1-fold higher than those of NB4 and HL-60 cells. Bone marrow CYP3A5 positive blast cell percentage at the time of diagnosis in primary drug resistance group (17.2%) was significantly higher than that of continuous complete remission (CCR) group (0.4%) and secondary drug resistance group (5.4%). In their first complete remission of the early relapsed group, the positive rate had been 23.9% as compared with that of CCR group (1.3%). Daunorubicin increased CYP3A5 mRNA level in K562/A02 and activated its transcription in HL-60/ADR. HL-60/CYP3A5 cell was significantly resistant to daunorubicin and vincristine than HL-60 cells did (3.0 and 4.0 times, respectively).

CONCLUSIONCYP3A5 expressed in leukemia cells may cause in situ metabolization of many kinds of anticancer drugs, thus led to drug resistance.

Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Daunorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Leukemia ; drug therapy ; enzymology ; RNA, Messenger ; analysis ; Tumor Cells, Cultured