Increased expression of the INK4a/ARF locus in polycythemia vera.
The retinoblastoma (Rb), cyclin-dependent kinase (CDK), and CDK inhibitor genes regulate cell generation, and deregulation can produce increased cell growth and tumorigenesis. Polycythemia vera (PV) is a clonal myeloproliferative disease where the mechanism producing increased hematopoiesis is still unknown. To investigate possible defects in cell-cycle regulation in PV, the expression of Rb and CDK inhibitor gene messenger RNAs (mRNAs) in highly purified human erythroid colony-forming cells (ECFCs) was screened using an RNase protection assay (RPA) and 11 gene probes. It was found that RNA representing exon 2 of p16(INK4a) and p14(ARF) was enhanced by 2.8- to 15.9-fold in 11 patients with PV. No increase of exon 2 mRNA was evident in the T cells of patients with PV, or in the ECFCs and T cells from patients with secondary polycythemia. p27 also had elevated mRNA expression in PV ECFCs, but to a lesser degree. Because the INK4a/ARF locus encodes 2 tumor suppressors, p16(INK4a) and p14(ARF) with the same exon 2 sequence, the increased mRNA 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 mRNAs from the first exons of both genes were increased in erythroid and granulocyte-macrophage cells and Western blot analysis showed that the INK4a protein (p16(INK4a)) was increased in PV ECFCs. Sequencing revealed no mutations of INK4a or ARF in 10 patients with PV. p16(INK4a) is an important negative cell-cycle regulator, but in contrast with a wide range of malignancies where inactivation of the INK4a gene is one of the most common carcinogenetic events, in PV p16( INK4a) expression was dramatically increased without a significant change in ECFC cell cycle compared with normal ECFCs. It is quite likely that p16(INK4a) and p14(ARF) are not the pathogenetic cause of PV, but instead represent a cellular response to an abnormality of a downstream regulator of proliferation such as cyclin D, CDK4/CDK6, Rb, or E2F. Further work to delineate the function of these genes in PV is in progress. (Blood. 2001;97:3424-3432)
Dai
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Department of Veterans Affairs Medical Service, Vanderbilt University, Nashville, TN 37232, USA.
Krantz
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D000368:Aged; D002453:Cell Cycle; D002455:Cell Division; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004252:DNA Mutational Analysis; D015672:Erythroid Precursor Cells / Q000378:metabolism / Q000473:pathology; D005091:Exons; D005260:Female; D015870:Gene Expression; D006098:Granulocytes / Q000737:chemistry; D006412:Hematopoietic Stem Cells / Q000737:chemistry; D006801:Humans; D008264:Macrophages / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D011087:Polycythemia Vera / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000737:chemistry / Q000378:metabolism; D025803:Tumor Suppressor Protein p14ARF
D019941:Cyclin-Dependent Kinase Inhibitor p16; D011506:Proteins; D012333:RNA, Messenger; D025803:Tumor Suppressor Protein p14ARF; D018844:Cyclin-Dependent Kinases