The object of this proposal is to identify the control mechanisms responsible for retinoic acid induced differentiation and reversal of malignancy, a central unsolved problem in retinoid biology and biochemistry. Recently novel approaches have developed in treating patients with leukemia using agents that modify differentiation and growth properties of preleukemic cells; 13-cis- and all-trans-retinoic acids have been promising agents. Beneficial clinical effects of 13-cis-retinoic acid have been described for a subgroup of patients with acute promyelocytic leukemia. Retinoids have a practical importance in clinical medicine if toxicity and resistance problems can be overcome. Since 13-cis-retinoic acid has a relatively low toxicity in humans compared to all-trans-retinoic acid but is equipotent in differentiation of HL-60 cells, the mechanisms that control the biologic activity these retinoids should be defined.
The specific aims of this proposal are 1) to develop a stable 13cisretinoic acid resistant HL60 (differentiation defective, HL60D-) cell line from the original differentiation positive HL60D+ that is characterized by proliferative capacity, alteration in nitroblue tetrazolium reduction capacity, by karyotypic and two dimensional electrophoretic analysis and compare it to HL-60D+; 2) to create revertants from HL60D+ and HL60D- that have been mutagenized with ethyl methane sulfonate (EMS) that will not differentiate, HL60EMSD-, or differentiate, HL60EMSD+ in presence of 13cis-retinoic acid; 3) to identify the cellular localization of 13cisretinoic acid in HL- 60-, HL60D+, HL-60EMSD- and HL60EMSD+ after treatment with 13-cis-retinoic acid using polyclonal and monoclonal anti(13cisretinoic acidhemocyanin) antibody; 4) to characterize the proteins from HL60D+, D-, EMSD+, and EMSD- that interact with retinoic acid, by affinity chromatography and amino acid sequence analysis. The role of 13cis-retinoic acid in gene expression will be examined. Gene expression will be evaluated at 1) the chromosome level by karyotypic analysis, 2) the phenotypic level by developing a 13cisretinoic acid resistant cell line, 3) the genotypic level by mutagenizing HL60 with a selective transition (point mutation) type mutagenic agent, EMS and 4) at the protein level by monitoring expression of transglutaminase in a specific response for retinoic acidinduced differentiation. Gene expression will be assessed at the cellular level by correlating the subcellular localization of 13cisretinoic acid and the ability to differentiate in HL60 and in constructed HL60 variants.
| Salyers, K L; Cullum, M E; Zile, M H (1993) Glucuronidation of all-trans-retinoic acid in liposomal membranes. Biochim Biophys Acta 1152:328-34 |
