Inn-eased expression of the INK4a/ARF locus in polycythemia vera

The retinoblastoma (Rb), cyclin-dependent kinase (CDK), and CDK inhibitor g enes regulate cell generation, and deregulation can produce increased cell growth and tumorigenesis. Polycythemia vera (PV) is a clonal myeloprolifera tive disease where the mechanism producing increased hematopoiesis is still unknown. To investigate possible defects in cell-cycle regulation in PV, t he expression of Rb and CDK inhibitor gene messenger RNAs (mRNAs) in highly purified human erythroid colony-forming cells (ECFCs) was screened using a n RNase protection assay (RPA) and 11 gene probes. It was found that RNA re presenting exon 2 of p16(INK4a) and p14(ARF) was enhanced by 2.8- to 15.9-f old in 11 patients with PV. No increase of exon 2 mRNA was evident in the T cells of patients with PY or in the ECFCs and T cells from patients with s econdary polycythemia, p27 also had elevated mRNA expression in PV ECFCs, b ut to a lesser degree. Because the INK4a/ARF locus encodes 2 tumor suppress ors, p16(INK4a) and p14(ARF) With the same exon 2 sequence, the increased m RNA fragment could represent either one. To clarify this, mRNA representing the unique first exons of INK4a and ARF were analyzed by semiquantitative reverse transcription-polymerase chain reaction. This demonstrated that mRN As from the first exons of both genes were increased in erythroid and granu locyte-macrophage cells and Western blot analysis showed that the INK4a pro tein (p16(INK4a)) was increased in PV ECFCs. Sequencing revealed no mutatio ns of INK4a or ARF in 10 patients with PV, p16(INK4a) is,, important negati ve cell-cycle regulator, but in contrast with a wide range of malignancies where inactivation of the INK4a gene is one of the most common carcinogenet ic events, in PV p16(INK4a) expression was dramatically increased without a significant change in ECFC cell cycle compared with normal ECFCs. It is qu ite likely that p16(INK4) and p14(ARF) are not the pathogenetic cause of PV , but instead represent a cellular response to an abnormality of a downstre am regulator of proliferation such as cyclin D, CDK4/CDK6, Rb, or E2F, Furt her work to delineate the function of these genes in PV is in progress. (Bl ood, 2001; 97:3424-3432) (C) 2001 by The American Society of Hematology.