All trans retinoic acid (atra) fails to upregulate retevoic acid receptor beta (rar β) in a non m3 acute myelogenous leukemia cell line

Pharmacologie doses of ATRA are able to overcome the differentiation block observed in acute promyelocytic leukemia (APL). When added to standard chemotherapy this combination improves survival in APL; a similar benefit has been difficult to demonstrate in the other sub-types of AML (non M3 AML). A possible mechanism is that the differentiation block is more complex in non M3 AML. We hypothesize that retinoid responsive genes are silenced by mechanisms such as methylation of CpG islands in promoter regions. To test this hypothesis two ATRA responsive cell lines were compared: NB4 which represents APL and OCI AML2 a non M3 AML cell line. Response to ATRA was assessed by growth in colony forming assays, morphologic changes, and changes in specific gene expression as measured by RT-PCR. Both the NB4 and AML2 cell lines demonstrated inhibition of growth to increasing ATRA concentrations with almost complete growth inhibition at levels of liiM. This was associated with decreased bcl-2 expression in both cell lines. Only the NB4 cell line displayed morphologic changes in response to ATRA 1 jiM. This suggests that genes important for differentiation arc not retinoid responsive in AML2 cell line. One candidate gene is RAR βthat is normally upregulated in response to ATRA. RAR βexpression increased in response to ATFLA 1 |iM in the NB4 cell line alone. STAT1 expression is responsive to RAR βand it was upregulated in response to ATRA only in the NB4 cell line. If retinoid responsive genes are transcriptionally silent secondary to CpG methylation then the addition of 5-aza-2'deoxycytidine (5-Aza-CdR), an inhibitor of DNA methyltransferase, to cell culture should restore upregulation of RAR βin response to ATRA in AML2. 5-Aza-CdR was added to AML2 cell culture for 48 hours prior to the addition of ATRA lM and doses of 5-AzaCdR 200-400 ng/ml re-established RAR βexpression in response to ATRA 1 jilV. To determine the methylation status of the RARβ promoter in both cell lines genomic DNA was subjected to methylation specific PCR. Both the NB4 and AML2 cell line demonst'ated that the RAR βpromoter is not methylated. In conclusion, the AML2 cell line was unable to differentiate in response to pharmacolgic levels of ATRA. This was associated wi :h an inability of the AML2 cell line to upregulate RARβ. The RARβ promoter wa, not methylated in the AML2 cell line but inhibition of CpG methylation by 5-aza restored RARβ responsiveness. These results suggest that methylation of CpG islands has a role in preventing normal retinoid signalling in the AML2 cell line.