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. 2008 Nov 15;22(22):3115-20.
doi: 10.1101/gad.1715808.

Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16Ink4a and Arf

Robert A J Signer  1 , Encarnacion Montecino-Rodriguez, Owen N Witte, Kenneth Dorshkind
Affiliations

Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16Ink4a and Arf

Robert A J Signer et al. Genes Dev. .

Abstract

Lymphoid progenitors exhibit severe growth defects during aging while myelopoiesis is relatively unperturbed. These effects are due in part to the preferential expression of p16(Ink4a) and Arf in aged lymphoid progenitors. Their increased expression contributes to reduced growth and survival of lymphoid progenitors and makes them refractory to malignant transformation. Down-regulation of p16(Ink4a) and Arf in aged lymphoid progenitors reverted the senescent phenotype and restored susceptibility to transformation. These data provide a molecular explanation for the preferential effects of aging on lymphopoiesis, suggest that inhibiting p16(Ink4a) and Arf expression can rejuvenate B lymphopoiesis, and link aging and cancer resistance.

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Figures

Figure 1.
Figure 1.
p16Ink4a and Arf are preferentially expressed in aging B lineage progenitors. (A–F) Representative immunostaining profiles used to purify HSCs and CLPs (A,B); common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), and megakaryocyte erythroid progenitors (MEPs) (C,E); and pre-pro-B cells, pro-B cells, and pre-B cells (D,F) from mice. The frequency of each population is indicated in parentheses. The frequencies of lymphoid progenitors are decreased and the frequencies of HSC and myeloid progenitors are increased in the BM of old mice. Expression of p16Ink4a (G) and Arf (H) relative to β-Actin in the indicated young and old purified populations was assessed by qPCR. P values for each population are based on comparison of old versus young cells. (*) P < 0.005; (**) P < 0.1; (#) expression below level of detection. Each population was purified in two independent experiments from BM cell suspensions pooled from four to six young or old C57BL/6 mice, respectively. Reactions for each population were run a total of eight times.
Figure 2.
Figure 2.
Ectopic expression of p16Ink4a and Arf mimic the effects of aging in young B lineage cells. (A) Experimental system used for transduction and growth of pro-/pre-B cells. (B) Number of cells harvested from cultures 5 d following transduction of purified Lin−Ly6C−IgM−CD93+CD45R+CD19+ pro-/pre-B cells with retroviruses encoding either GFP, p16Ink4a or Arf. P values are based on comparisons between each population and the young GFP transduced cells. (*) P < 0.05. The data are based on transduced cells only. (C,D) Ectopic expression of p16Ink4a (C, top left panel) or Arf (C, top right panel) results in an increase in the frequency of apoptotic AnnexinV+ cells in young pro-/pre-B cells when compared with young and old GFP transduced pro-/pre-B cells (D). Plots shown are gated on live GFP+ cells.
Figure 3.
Figure 3.
Bmi-1, Id1, and Ets1 exhibit differential, age-related patterns of expression in hematopoietic progenitors. Changes in expression of Bmi-1 (A), Id1 (B), and Ets1 (C) relative to β-Actin in the indicated populations with age. Values are reported as the mean ratio of expression in old versus young cells. P values are based on comparison of expression levels in old versus young cells within each population (*P < 0.05; #P < 0.0006). Samples were the same as used in Figure 1 and Supplemental Figure S1.
Figure 4.
Figure 4.
Restoring expression of Bmi-1 rescues proliferative defects in aged B lineage cells. (A) Number of cells harvested from cultures 15 d following transduction of purified Lin−Ly6C−IgM−CD93+ CD45R+CD19+ pro-/pre-B cells with retroviruses encoding either GFP or Bmi-1. Ectopic expression of Bmi-1 in old pro/pre-B cells restores their growth potential compared with their GFP transduced counterparts. P values are based on comparisons between each population and the young GFP transduced cells. (*) P < 0.01; (**)P < 0.0002. (B) Expression of Bmi-1, p16Ink4a, and Arf relative to β-Actin in young and old pro-/pre-B cells 13 d following transduction with retroviruses encoding either Bmi-1 or GFP. Values are normalized to expression of each gene in the GFP transduced old pro-/pre-B cells. P values are based on comparison of the expression in each population relative to the expression in old GFP+ cells. (*) P < 0.04; (#) P < 0.0008. (C) The frequency of proliferating Ki67+ cells is elevated in cultures established with old Bmi-1 transduced pro-/pre-B cells as compared with old GFP transduced pro-/pre-B cells. (D) Ectopic expression of Bmi-1 (right panel) decreases the frequency of AnnexinV+ cells in old pro-/pre-B cells to levels comparable with their GFP transduced young counterparts (left panel). (E) Bmi-1 transduced young (top) and old (bottom) pro-/pre-B cells can differentiate into IgM+ cells in vitro.
Figure 5.
Figure 5.
Ectopic expression of Bmi-1 makes old B lineage cells susceptible to leukemic transformation by BCR-ABL. (A) Pro-/pre-B cells from young and old mice were simultaneously cotransduced with BCR-ABL and Bmi-1 or with BCR-ABL alone and were transplanted into Rag1−/− mice. Mice were sacrificed when they showed symptoms of disease. BCR-ABL transduced old cells did not generate leukemia while 100% of recipients repopulated with BCR-ABL and Bmi-1 cotransduced cells developed disease by day 41 post-transplantation. (B–E) Representative phenotypic profiles of spleen cells from Rag1−/− mice transplanted with young or old pro-/pre-B cells transduced with Bmi-1 and/or BCR-ABL. (B) Representative Rag1−/− recipient of BCR-ABL transduced old pro-/pre-B cells showing no YFP+ leukemic cells. (C) Representative Rag1−/− recipient of BCR-ABL transduced young pro-/pre-B cells showing abundant YFP+ leukemic cells with a CD45R+CD19+IgM−CD43+ pro-B-cell phenotype. Representative Rag1−/− recipients of young (D) and old (E) pro-/pre-B cells cotransduced with BCR-ABL and Bmi-1 showing abundant GFP+YFP+ leukemic cells with a CD45R+CD19+IgM−CD43+ pro-B-cell phenotype. (F–I) Photomicrographs of spleen cell cytospins from (F) a Rag1−/− recipient of BCR-ABL transduced old pro-/pre-B cells showing no lymphoid blasts present, and Rag1−/− recipients of BCR-ABL transduced young (G), Bmi-1 and BCR-ABL cotransduced young (H) and old (I) pro-/pre-B cells showing abundant lymphoid blasts. Images in F–I were viewed at 500× magnification with a Leitz Laborlux D microscope using a FLUOTAR 50×/0.70 NA phase-contrast objective.
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