The objectives of this project are to identify those abnormalities in RNA which are mediators of L-PAM cytotoxicity. The working hypothesis is that while alterations in cell cycling may relate to cytotoxicity, these changes are not necessarily in the S phase. Therefore, the cycle abnormalities are most likely translated into cytotoxicity via alterations in RNAs. Using a model with a human lymphoma cell line and L-phenylalanine mustard (L-PAM), specific populations of RNAs will be separated by cell cycle compartment using an elutriator rotor technique to avoid complications of other biochemical manipulations to synchronize cells. Following compartmental separation, nuclei and cytoplasmic contents will be separated by detergent lysis and sucrose density sedimentation. If the cells contain high levels of RNAses an alternate method of separation utilizing guanidinium isothiocyanate and cesium chloride will be used. This latter method will not allow the separation of nuclear from cytoplasmic material. The resultant RNA will be separated on oligo(T) cellulose columns into poly(A)+ and poly(A)- RNAs. r-RNA will be quantified and analyzed by polyacrylamide gel electrophoresis. Quantitation of r-RNA will be performed by densitometry of the stained gels. Synthesis will be analyzed by the incorporation of tritiated uridine and scintillation counting of solubilized gel slices. m-RNA synthesis will be analyzed by radiolabeled precursor incorporation and TCA precipitation of the poly(A)+ material. Analysis of type and amount of messages present will be made using a reticulocyte lysate in vitro translation system and two-dimensional polyacrylamide gel electrophoresis of the protein products. For both m-RNA and r-RNA, changes in quantity over time will be correlated with rates of synthesis and if necessary uridine pool sizes and rates of RNA degradation will be measured if the results are discordant. Following identification of RNA populations showing major alterations following drug treatment, other alkylating agents and drug modifiers will be used to vary DNA damage, cell cycle, and cytotoxic parameters and to ascertain if the targeted RNA populations also change. In the performance of this project products of translation will be analyzed for the appearance of new proteins which might be DNA damage induced proteins similar to heat shock proteins seen in other systems.
| Widstrom, R L; Ducore, J M (1991) A DNA crosslinking drug alters synthesis of several low molecular weight proteins in human lymphoma cells. Biochem Biophys Res Commun 176:717-21 |
