Expression of leukocyte alkaline phosphatase gene in normal and leukemic cells: regulation of the transcript by granulocyte colony- stimulating factor

Abstract

The levels of leukocyte alkaline phosphatase (LAP) messenger RNA (mRNA) are evaluated in B and T lymphocytes, monocytes, and polymorphonuclear cells (PMNs), and this transcript is found to be present only in PMNs. Precursors of the myelomonocytic pathway, represented by leukemic cells isolated from several cases of chronic myelogenous leukemia (CML) in its stable and blastic phase and acute myelogenous leukemia (AML), are devoid of LAP transcript. These data support the notion that LAP is a marker of the granulocyte terminal differentiation. Despite the absence of LAP mRNA in both the myeloid and the lymphoid precursors, nuclear run-on experiments show constitutive transcription of the LAP gene in leukemic cells obtained from AML, CML, as well as acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (B-CLL). In CML and in chronic myelo-monocytic leukemia (CMML) PMNs, granulocyte colony- stimulating factor (G-CSF) specifically accumulates LAP mRNA without showing a substantial increase in the rate of transcription of the LAP gene. Once increased by G-CSF, LAP mRNA is very stable, showing a half- life of more than 4 hours in the presence of actinomycin-D. G-CSF is suggested to play a pivotal role in the modulation of LAP transcript in PMNs.

Leucocyte alkaline phosphatase identifies terminally differentiated normal neutrophils and its lack in chronic myelogenous leukaemia is not dependent on p210 tyrosine kinase activity

“…In addition, although the function of LAP is not yet known, the presence of mature granulocytes negative for the expression of both the surface and the cytoplasmic form of the protein, suggests that in normal myeloid differentiation LAP does not play a necessary role in the release of mature granulocytes from the bone morrow into the blood stream. We and others previously showed that G-CSF regulates LAP mRNA expression in normal and CML granulocytes (Rambaldi et al, 1989(Rambaldi et al, , 1990Sato et al, 1991). In this study we have provided evidence that pharmacological doses of G-CSF increase in vitro the cell surface expression of LAP protein in normal granulocytes.…”

“…Short-term incubation (2-6 h) was not associated with modification in the fluorescence profile of 1B12.1 MoAb as compared to control samples (data not shown). Not surprisingly, this G-CSF-mediated up-regulation of LAP surface expression occurred in parallel with a concomitant modulation of apoptosis (Rambaldi et al, 1990;Sato et al, 1991;De Renzo et al, 1990;Colotta et al, 1992;Homburg et al, 1995). In fact, as determined by the annexin V assay, we found that only 20% of normal granulocytes cultured for 24 h with G-CSF showed an apoptotic phenotype as compared to 40% of granulocytes cultured in medium alone (data not shown).…”

“…Indeed, the chronic phase of CML is typically associated with a marked reduction of LAP enzymatic activity as a consequence of LAP mRNA deficiency (Rambaldi et al, 1989). These defects can be corrected in vitro after incubation of CML granulocytes with G-CSF (Yuo et al, 1987;De Renzo et al, 1990;Rambaldi et al, 1990). Moreover, increase of LAP enzymatic activity was observed in CML patients with clinical conditions characterized by increased circulating levels of G-CSF such as infections and paraneoplastic syndromes (Peeillie et al, 1967;Masuda et al, 1995).…”

“…RNA preparation and Northern blot analysis. Northern blot analysis was performed as previously described (Rambaldi et al, 1990). Briefly, granulocytes were lysed in 4 mol/l guanidium isothyocyanate and total cellular RNA was recovered by ultra centrifugation through a caesium chloride gradient.…”

“…In order to define more precisely the kinetics of LAP surface expression on neutrophil granulocytes, we took advantage of previously characterized experimental models whereby granulocyte colony-stimulating factor (G-CSF) regulated LAP messenger RNA (mRNA) expression of granulocytes in normal donors (Rambaldi et al, 1990;Sato et al, 1991) and patients with chronic myelogenous leukaemia (CML) (Rambaldi et al, 1989). During the chronic phase of CML, LAP enzymatic activity is substantially decreased or absent because of a deficiency in LAP mRNA (Rambaldi et al, 1989).…”