MECHANISM OF ACTION OF BIFUNCTIONAL ALKYLATING AGENTS

Project: Research project

Description

This study will examine in detail changes in DNA synthesis and cell cycle
progression following bifunctional alkylating agent treatment and correlate
the changes with cytotoxicity and DNA damage. Using a variety of cell
types, both mono- and bifunctional alkylating agents, and drugs which
modify the DNA damage and toxicity of the alkylating agents, we will be
able to determine whih functional abnormalities of DNA synthesis and cell
cycle result from the DNA damage of bifunctional alkylators and result in
cell death. Alterations in rate and initiation and elongation of DNA
synthesis will be measured by the pH step modification of the alkaline
elution method. Alterations will be measured at a series of time points
following drug treatment. Cell cycle changes will be measured utilizing
flow microfluorometry. Alterations in cell cycling and percentage of cells
in different cycle compartments will be measured at various times following
drug treatment. Abnormalities of DNA synthesis and cell cycle progression
will be correlated with known parameters of DNA damage (DNA interstrand
crosslinking) and cytotoxicity. The toxic importance of bifunctional
alkylations in the nitrosoureas will be measured by comparing mono- and
bifunctional nitrosoureas. Platinum complex mono-alkylations can be
compared to bifunctional alkylations by the addition of thiourea following
cis-platinum treatments. The use of trans-platinum will further
distinguish those functional aberrations which correlate with
cytotoxicity. Modifications of mustard toxicity (L-PAM) will be made with
thiourea. We will use methylxanthines (caffeine and theophylline) to
modify cytotoxicity and study changes in DNA synthesis. Polyamine
synthesis inhibitors modify drug effects with resultant changes in DNA
synthesis and cytotoxicity as well as DNA damage parameters. We will
develop and isolate cell lines with altered alkylating agent sensitivity
and contrast these with their parent line as to DNA synthesis and cell
cycle changes. Where cytotoxic or DNA damage data is lacking, this will be
measured utilizing the alkaline elution technique. Finally, if major
alterations in intracellular dNTP pools are seen, changes in pool sizes
will be measured by an in vitro DNA synthesis rate technique. This
information will allow identification of functional consequences of
bifunctional alkylating agent treatment and enlarge our understanding of
how DNA crosslinking affects DNA synthesis and cell cycling and hence cell
death.
StatusFinished
Effective start/end date7/1/836/30/86

Funding

  • National Institutes of Health

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Alkylating Agents
DNA Damage
DNA
Platinum
Alkylation
Cell Cycle
Pharmaceutical Preparations
Cytophotometry
Thiourea
Mustard Plant
Melphalan
Poisons
Theophylline
Caffeine
Cell Line

ASJC

  • Medicine(all)