EFFECTS OF PML AND PML/RMRα ANTISENCE OLIGO-NUCLEOTIDE ON PROMYELOCYTIC LEUKEMIA CELL NB4

Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and PML-RAR(/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RAR( mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RAR(/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RAR( fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RAR( mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RAR( antisence oligonucleotides can specially block the expression of PML-RAR( at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.

EFFECTS OF ANTI-PML/RARα OR ANTI-PML ANTISENCE ON THE GROWTH, DIFFERENTIATION AND APOPTOSISOF NB4 CELL LINES

Objectire To investigate the ellects of anti - PML/RARx or anti - PML antisence on the growth,dtherentiation and apoptosis of NB4 cell lines. Methods Wright’s stain for cell morphology, flow cytometry andDNA gel electronphoresis for cell apoptosis, methylcellulose assays for leukemic colony forming unit andtrypan - blue exclusion for cell counts. Results Both the start cordon region of the PML or PML - RARx mRNA(STAS) and the fusion point region of the long type PML - RARx mRNA (FUAS) could inhibit cell growth. Cellsbecame partially differentiated at 5d of treatment, and FUAS - treated cells showed more significant differentiationthan STAS- treated cells. Morphology of typical apoptosis could be seen at 7, 9d incubation with antisenceoligodeoxynucleotides (AS). In contrast, no cell growth inhibition, no morphology changes were seen in Sen or Rantreated cells compared with untreated cells. The number of acute myelocytic leukemia colony forming unit(AML - CFU) markedly decreased in STAS and FUAS treated cells. Cell DNA content analyzed by flow cytometryshowed the typical profile of apoptotic cells, in which pre - G1 peak appear before G1 peak at 7,9d of treatment withSTAS or FUAS. Conclusion Anti - PML/RARx or anti- PML antisence inhibit the cell growth, inducedifferentiation and differentiated cell apoptosis of NB4 cells.