This study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and selfrenewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16 INK4a expression in the p16 INK4a -deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16 INK4a and related INK4 proteins, cyclindependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34 ؉ bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells from p16 INK4a؊/؊ mice and wild-type mice. The results from p16 INK4a؊/؊ marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT of p16 INK4a IntroductionDeletion of the tumor-suppressor gene p16 INK4a has been implicated in tumorigenesis in general 1 and in leukemogenesis in particular. [2][3][4][5] It has been found in 25% to 60% of cases of acute leukemia and 20% to 50% of cases of lymphoma. 6 Although deletion of the p16 INK4a gene is uncommon in acute myeloid leukemia, messenger RNA expression is frequently undetectable, possibly as a result of DNA hypermethylation or mutations in the p16 INK4a promotor region. 7 These observations raise the possibility that p16 INK4a expression plays an important role in the regulation of normal hematopoiesis. There is ample evidence that restoration of p16 INK4a into p16 INK4a -deficient leukemic cell lines reduces their proliferation rate in liquid culture and some evidence that it suppresses their ability to form colonies in semisolid culture, 8,9 but the effects of p16 INK4a expression on primary normal hematopoietic cell proliferation have not been examined. In particular, it is not known whether p16 INK4a has any effects on the kinetics of progenitor cell renewal and differentiation. This is important because transformation of leukemic target cells must be associated with an increase in self-renewal probability above the steady-state value of 0.5 before the leukemic clone can expand, become established, and eventually predominate over normal hematopoiesis. [10][11][12] The p16 INK4a protein is the prototypic member of the INK4 13 This pathway (the pRB pathway) regulates the transition from G 1 to S phase of the cell cycle. The formation of complexes between cyclin D and CDK4 is inhibited by p16 INK4a through binding to CDK4/6. The cyclin D-CDK4/6 complex phosphorylates pRB, resulting in the release of transcription factors so that the genes necessary for cell cycle progression can be transcribed. 14 The pRB family of pocket proteins consists of pRB itself and the structurally related proteins p107 and p130. They are negative ...

The reference value ranges established in this study reflect significant differences between genders. It is possible that these reference ranges may be generalizable to adults living in Thailand. The findings of this study emphasize the importance of establishing specific hematologic reference values for specific populations.