Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Cytosine arabinoside- induced apoptosis in human myeloblastic leukemia cells: role of NF-ƒÛB/Rel activity

MC Turco 1, MF Romano 1, A Lamberti 1, R Bisogni 1, A Petrella 1, T Ribecco 1, S Venuta 2

1 Dipartimento di Biochimica e Biotecnologie Mediche, Federico II University, Napoli;, 2 Dipartimento di Medicina Sperimentale e Clinica, University Catanzaro, Cantanzaro, Italy,

The activity of NF-ƒÛB/Rel nuclear factors is known to inhibit apoptosis in various cell types. The apoptosis- controlling activity of NF-ƒÛB/Rel factors suggests anti- tumor strategies based on inhibition of NF-ƒÛB/Rel. Indeed, NF-ƒÛB/Rel inhibitors can be expected to increase tumour cell sensitivity to apoptosis, leading to enhanced effects of chemotherapeutic compounds. We investigated whether the subtraction of NF-ƒÛB/Rel activity influenced the response of 11 AML (M1, M2 and M4) patients' cells to AraC. To block NF-ƒÛB/Rel in primary AML cells, we used a phosphorothioate double- stranded decoy oligodeoxynucleotide (ODN) carrying the NF-ƒÛB/Rel- consensus sequence (Romano et al., Blood 1998; Blood 1999). As verified by electrophoretic mobility shift assay (EMSA) of cells' nuclear extracts, AML cell incubation with this ODN, but not its mutated (scrambled) form used as a control, resulted in abating the NF-ƒÛB/Rel nuclear levels in these cells. We incubated the leukemic cells with AraC (32 or 1 ƒÝM), either in the absence or presence of the decoy or the scrambled ODN, and analyzed cell apoptosis. The spontaneous cell apoptosis detectable in the absence of AraC (<25%) was not modulated by the oligonucleotide presence in cell cultures. On the other hand, in 10 of the 11 samples tested, the decoy ƒÛB, but not the scrambled, ODN significantly (p<0.01 in a Student's t test) enhanced cell apoptotic response to AraC. Such effect was particularly remarkable at low AraC doses (1ƒÝM). These findings indicate that NF-ƒÛB/Rel activity influence response to AraC in human primary myeloblastic cells, and suggest that the inhibition of NF-ƒÛB/Rel factors can improve the effect of chemotherapy in AML. This piece of evidence contributes to support the possible use of NF-ƒÛB/Rel- specific inhibitors, such as ODN, for improving antineoplastic programs and contrasting chemoresistance ( Romano et al., Leuk. Lymphoma 2000; Gene Therapy 2000). Relating NF-ƒÛB/Rel activities to individual target genes can represent a first step in a progress, toward a deeper understanding of life-death balance and envisioning of apoptosis- modulating therapies. Although some anti- apoptotic genes regulated by NF-ƒÛB/Rel have been identified, the whole survival pathway governed by these factors remains to be elucidated. In Jurkat leukemic cells transfected with either an IƒÛBƒÑ- expressing or a control void vector (Lamberti et al., Apoptosis 1999), we identified by a differential display strategy a novel gene, named ƒÛB-R (for NF-ƒÛB- Repressed), whose expression is under the negative control of NF-ƒÛB/Rel activity. Based on sequence analysis and expression studies of the full- length cDNA, this apparently encodes a 20 kD protein, whose involvement in the apoptotic process is currently investigated.

KEY WORDS: apoptosis, leukemia, NF-ƒÛB/Re.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on chemotherapy.