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.