RNA CHANGES BY L-PAM IN HUMAN CELLS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date8/1/868/31/90

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.