PABA/NO is a
diazeniumdiolate selectively activated by
glutathione S-transferase P (GSTP) to release
nitric oxide (NO) and is a potent inducer of
protein S-glutathionylation, a redox-sensitive post-translational modification of
cysteine residues. Using a procedure that incrementally increased exposure of cells to
PABA/NO, an acquired
drug resistant human promyelocytic
leukemia HL60 cell line (HL60(
PABA)) that exhibited 1.9-fold resistance to the
drug (IC(50) 15 μM vs ~8 μM for wild-type) was created. HL60(
PABA) cells had a decreased growth rate attributable to altered cellular differentiation, as measured by increased expression of CD11b; decreased expression of CD14; decreased nuclear to cytoplasmic ratios and a condensation of nuclear
chromatin. This was accompanied by alterations in both plasma and mitochondrial membrane potentials. Both GSTP expression and
nitric oxide release were reduced two-fold, while increased expression levels of genes involved in the unfolded protein response (UPR) were evident in HL60(
PABA) cells. Wild type cells treated with
PABA/NO had increased levels of
protein S-glutathionylation and JNK activation, while JNK was constitutively active in HL60(
PABA) cells and these cells had reduced levels of S-glutathionylation. By removing
PABA/NO from the growth medium, HL60(
PABA) cells reverted to sensitivity within 21 days suggesting that resistance was not genetically stable. Mechanistically,
PABA/NO resistance is mediated through reduced levels of GSTP resulting in reduced NO release and its subsequent alterations in cellular response to nitrosative stress.