Objective: Expression of the cyclin-dependent kinase inhibitor p15(INK4B) frequently is altered in myeloid malignancies. We previously demonstrated that p15(INK4B) is expressed in normal myeloid cells. The aim of this study was to investigate whether p15(INK4B) expression is restricted to the granulomonocytic lineage and to evaluate its modulation during normal and leukemic myeloid differentiation.
Materials and methods: Normal CD34(+) cells were cultured in serum-free media to obtain granulomonocytic, erythroid, or megakaryocytic unilineage differentiation. NB4 promyelocytic cell line and fresh leukemic blasts from seven patients with acute promyelocytic leukemia were cultured with all-trans retinoic acid. At different times of culture, cell samples were collected to evaluate p15(INK4B) by semiquantitative reverse transcriptase polymerase chain reaction.
Results: p15(INK4B) mRNA was found during granulomonocytic and megakaryocytic, but not erythroid, differentiation. In the granulomonocytic lineage, p15(INK4B) was detectable when the majority of cells were at the promyelocytic stage and increased progressively in more mature elements. In the megakaryocytic lineage, p15(INK4B) was expressed in the early phase of differentiation, before megakaryoblasts had appeared, and was mantained throughout the time of culture. NB4 cell line and five of seven leukemic samples displayed undetectable or very low level of p15(INK4B) that rapidly increased during retinoic acid-induced differentiation. Two leukemic samples (both collected from two patients developing all-trans retinoic acid syndrome) showed high basal levels of p15(INK4B), which was not modified by retinoic acid treatment.
Conclusions: p15(INK4B) upregulation occurs specifically during normal granulomonocytic and megakaryocytic commitment. In acute promyelocytic leukemic blasts, p15(INK4B), which is detectable at a very low level, is promptly increased by retinoic acid. In contrast, two acute promyelocytic leukemia samples obtained from patients who developed all-trans retinoic acid syndrome showed high basal levels of p15(INK4B) that did not increase further during all-trans retinoic acid-induced differentiation.