Antisense sequences of the nbl gene induce apoptosis in the human promyelocytic leukemia cell line HL-60

Abstract

Apoptosis is induced by the transcriptional inhibitor actinomycin D (Act D) in various cell types, particularly many leukemic cell lines such as HL-60. A common feature of these cell lines is their high constitutive expression level of the nbl gene, which was originally isolated by virtue of its abundance in a Namalwa Burkitt lymphoma cDNA library. In contrast, cell lines which constitutively express nbl at low levels appear not to undergo typical apoptotic death in response to Act D. Apoptotic induction by Act D in cells which normally express nbl at high levels was found in this study to be closely associated with a decline in nbl mRNA levels, raising the possibility that apoptosis could be induced by lowering nbl expression levels in such cells. Transient expression of nbl antisense sequences in HL-60 cells decreased cell viability, and induced typical apoptotic morphology such as cell shrinkage, chromatin condensation and nuclear fragmentation. Incubation with nbl antisense oligomers also induced similar features in HL-60 cells and in another high nbl-expressing cell line, Jurkat, but had little effect in HepG2 cells which constitutively express nbl at low levels. These findings suggest that lowering constitutively high levels of nbl expression can induce apoptosis.

Altered Gene Expression in Liver from a Murine Model of Hyperhomocysteinemia

“…The gene encoding RPS3a was down-regulated, and it has been shown that apoptosis is induced in certain cell lines by lowering constitutively high levels of RPS3a expression using antisense RPS3a gene sequences (44). The gene encoding methylthioadenosine phosphorylase was up-regulated.…”

Down-regulated RPS3a/nbl Expression during Retinoid-induced Differentiation of HL-60 Cells. A Close Association with Diminished Susceptibility to Actinomycin D-stimulated Apoptosis.

“…In some experiments, aliquots of 1×10 6 cells/mL were taken after the indicated length of incubation with or without the differentiating agents, and resuspended in fresh media for 6 hours with or without 1µg/mL Act D (Sigma, St Louis, MO, USA) (Naora et al, 1996) or 1µg/ mL A23187 (Boehringer Mannheim GmbH, Mannheim, Germany) . Phosphorothioate oligomers used in the present experiments were the same as those used previously (Naora et al, 1998a). The RPS3a/nbl antisense oligomer contained the sequence complementary to the ATG initiation codon and the next four codons: 5'-CTTGCCAACCGCCAT-3'.…”

“…Briefly, viable cells were identified as those cells that excluded trypan blue dye. Apoptotic cells were identified as those cells which were refractile and showed membrane convolution but still excluded trypan blue dye, whereas those cells whose membrane integrity was compromised and failed to exclude trypan blue dye were considered non-viable (Wyllie et al, 1980;Naora et al, 1996Naora et al, , 1998a. In each experiment, a minimum of 400 cells were counted.…”

“…Enhanced RPS3a/nbl expression, either constitutive or transient, seems to be a prerequisite for "priming" a cell for apoptosis, while abrupt suppression of such enhanced expression leads to execution of apoptosis (Naora et al, 1998a(Naora et al, , 1998bNaora and Naora, 1999). Of particular interest is that many cancer cells, such as parental HL-60 cells, constitutively express RPS3a/nbl at high levels, whereas normal cells maintain low levels of RPS3a/nbl expression (Naora et al, , 1996.…”

“…While the molecular mechanisms underlying differences in apoptosis-susceptibility of normal and cancer cells are as yet unclear, our previous studies indicate an important role of the expression level of RPS3a/nbl, which apparently encodes a ribosomal protein (Naora et al, , 1996(Naora et al, , 1998a(Naora et al, 1998bNaora and Naora, 1999). Enhanced RPS3a/nbl expression, either constitutive or transient, seems to be a prerequisite for "priming" a cell for apoptosis, while abrupt suppression of such enhanced expression leads to execution of apoptosis (Naora et al, 1998a(Naora et al, , 1998bNaora and Naora, 1999).…”