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Abstract

Cytosolic extracts from a human lymphoblastoid B cell line, RPMI 8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca.sup.2+ --calmodulin dependent PDE (PDE1) and cAMP specific PDE4 subtypes. In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity. Using reverse transcription-polymerase chain reaction (RT-PCR), the mRNA encoding the 63 kDa form of PDE1 (PDE1B1) is expressed in RPMI 8392 cells, but not in normal, resting HPBL. This mRNA is, however, induced in HPBL following mitogenic stimulation by phytohemagglutinin (PHA). Also using RT-PCR, the full open reading frame for human PDE1B1 cDNA was cloned from RPMI 8392 cells and it encodes a protein of 536 amino acids with 96% identity to bovine, rat and mouse species. RT-PCR also identifies the presence of PDE1B1 in other human lymphoblastoid and leukemic cell lines. The PHA induced increase in PDE1B1, PDE4A and PDE4D mRNA is mimicked by incubation of HPBL with dibutyryl cAMP (dbcAMP) and 1-methyl-3-isobutylxanthine (IBMX). Inhibition of PDE1 or PDE4 activity by selective inhibitors induced RPMI 8392 cells, as well as the other cell lines, to undergo apoptosis. Culture of RPMI 8392 cells with an 18 nucleotide phosphorothioate antisense oligodeoxynucleotide, targeted against the translation initiation region of the RPMI 8392 mRNA, led to a specific reduction in the amount of PDE1B1 mRNA after 1 day, and its disappearance after 2 days, and induced apoptosis in these cells in a sequence specific manner. This suggests that PDEs, particularly PDE1B1 and some of the subtypes of PDE4, may be useful therapeutic targets for inducing the death of leukemic cells and for treatment of immoproliferative disorders and immune dysfunction.

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