A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress

Abstract

It has long been known that prolonged culture or serial transplantation leads to the loss of hematopoietic stem cells (HSCs); however, the mechanisms for this loss are not well understood. We hypothesized that expression of p16 Ink4a or p19 Arf or both may play a role in the loss of HSCs during conditions of enhanced proliferation, either in vitro or in vivo. Arf was not expressed in freshly isolated HSCs from adult mice but was induced in phenotypically primitive cells after 10 to 12 days in culture. When cultured bone marrow cells from either Arf ؊/؊ or Ink4a-Arf ؊/؊ mice were compared to wild-type cells in a competitive repopulation assay, no significant differences in HSC activity were seen. We then evaluated the role of p19 Arf IntroductionRapid turnover of mature hematopoietic cells requires production of tens of billions of cells every day during the life span of mammals. Hierarchical organization of the hematopoietic system helps to ensure the lifelong production of blood cells of different lineages. Hematopoietic stem cells (HSCs) have the ability to self-renew, differentiate into all hematopoietic lineages, and repopulate lethally irradiated hosts. Proliferative activity of HSCs is tightly regulated by incompletely understood mechanisms. HSCs are relatively quiescent and generally cytokine-resistant, with a long life span, whereas more committed progeny have a robust proliferation potential, are highly cytokine-responsive, and have a more limited life span. Although the low proliferation potential of HSCs may play a role in maintenance of HSCs over time, it is an obstacle in gene therapy applications and in clinical strategies to expand HSCs in vitro. 1 Several candidate genes have recently been implicated in the HSC self-renewal process. It has recently been demonstrated that HSC entry into the cell cycle is regulated by a cyclin-dependent kinase inhibitor (CKI), p21 Cip1 , whereas proliferation of progenitors is regulated by another cell-cycle inhibitor, p27 Kip1 . 2,3 Deletion of an early G 1 -phase CKI, p18(INK4C), results in increased self-renewal of the primitive hematopoietic cells and improved long-term engraftment. 4 Low levels of telomerase in HSCs have been implicated in the replicative senescence of HSCs 5 ; however, overexpression of TERT, a catalytic reverse transcriptase component of telomerase, does not lead to enhancement of HSC self-renewal. 6 Overexpression of the HOXB4 homeodomain transcription factor is able to induce expansion of HSCs both in vivo 7 and in vitro. 8 Overexpression of P-glycoprotein can also cause an HSC expansion, later followed by development of a myeloproliferative disorder. 9,10 HSC expansion was also noted in murine HSCs expressing the acute myeloid leukemia 1-eight-twenty-one (AML1-ETO) fusion protein, a product of a chromosomal translocation common in human AML. 11,12 Bmi1, a polycomb group repressor, is essential for the selfrenewal of neuronal and hematopoietic stem cells. Bmi1 is expressed in stem cells of neuronal and hematopoietic origin, and deletion...

Rb is dispensable for self-renewal and multilineage differentiation of adult hematopoietic stem cells

“…However, with the exception of p18 INK4c , the HSC phenotype of these mutants is relatively subtle, with a 2-fold increase in HSC frequency after the loss of p27 Kip1 (3). The loss of p21 Cip1 reveals a significant biological difference in HSC function only after quaternary transplantation (2), with the loss of both p16 INK4a and p19 ARF resulting in only a modest increase in serial transplant potential (5). Although p18 INK4c does result in an increased frequency of HSCs and increased serial transplantation potential, it also results in a lymphoproliferative disorder and predisposes the animals to leukemogenesis (4,29,30).…”

“…Deletion of p27 Kip1 resulted in an increased frequency of serially transplantable HSCs in addition to an expanded progenitor compartment (1,3). In contrast to loss of p18 INK4c , loss of both p16 INK4a and p19 INK4d had little effect on HSC function, with only a subtle increase in serial repopulation reported (5). Loss of p18 INK4c , however, resulted in enhanced repopulating capacity and frequency of HSCs (4).…”

“…Recently, a number of studies have reported intrinsic roles for negative regulators of the cell division cycle in determining the fate of HSCs (1)(2)(3)(4)(5). These studies have predominantly focused on inhibitors of G 1 -S phase cell cycle progression, in particular the CIP͞KIP and INK4 family of cyclin-dependent kinase inhibitors, and they have revealed that the loss of these genes can alter HSC self-renewal and differentiation.…”

Cell intrinsic and extrinsic mechanisms of stem cell aging depend on telomere status

“…(Molofsky et al, 2006;Krishnamurthy et al, 2006;Janzen et al, 2006;Stepanova and Sorrentino, 2005) No improvement in organ maintenance and lifespan in p16Ink4A, P19ARF compound mutant mice (Khoo et al, 2007) (Lynch and Lynch, 2005); Mismatch repair defect Colorectal cancer, extra-CRC cancers.…”

Hematopoietic Stem Cell Injury Induced by Ionizing Radiation

“…However, knockout of the Arf gene alone does not provide any advantage for HSC/HPC expansion and selfrenewal (113). In contrast, knockout p16 increases the lifespan of HSCs by promoting HSC self-renewal (74,144). Furthermore, mutation of the ATM gene also results in upregulation of p16 and Arf in HSCs (69,113).…”