SUPPLEMENTAL RESEARCH DATA
Aging and Cancer Resistance in Lymphoid Progenitors Are Linked Processes Conferred by
p16Ink4a and Arf
Robert A.J. Signer1, Encarnacion Montecino-Rodriguez1, Owen N. Witte2, & Kenneth Dorshkind1
1 Departments of Pathology and Laboratory Medicine and 2Microbiology, Immunology, and Molecular Genetics, 2Molecular and Medical Pharmacology and 2The Howard Hughes Medical Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Corresponding author: Kenneth Dorshkind
Department of Pathology and Laboratory Medicine
DavidGeffenSchool of Medicine
University of California, Los Angeles
10833 Le Conte Avenue
Los Angeles, CA90095
USA
Tel.: 1-310-206-9535
Fax: 1-310-206-9391
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Running Title: Lymphoid aging is mediated by p16Ink4a and Arf
Key Words: aging, hematopoiesis, cancer, p16Ink4a, Arf, Bmi-1,
Supplemental Methods
Cell Sorting
Hematopoietic populations were purifed by flow cytometry based on the following phenotypes: hematopoietic stem cells (HSC; Lin–CD117HiSca-1HiCD150+), common lymphoid progenitors (CLP; LinCD117LoSca-1LoCD127+); common myeloid progenitors (CMP; Lin-Sca-1-CD127-CD117HiCD34+CD16/32Lo), granulocyte macrophage progenitors (GMP; Lin-Sca-1-CD127-CD117HiCD34+CD16/32Hi), megakaryocyte erythroid progenitors (MEP; Lin-Sca-1-CD127-CD117HiCD34+CD16/32Lo); pre-proB cells (Lin-Ly-6C-IgM-CD93+CD45R+CD19-), proB cells (Lin-Ly-6C-IgM-CD93+CD45R+CD19+CD43+) and preB cells (Lin-Ly-6C-IgM-CD93+CD45R+CD19+CD43-).
Cell Cycle Analysis
Proliferating cells were detected by Ki-67 staining as previously described (Min et al. 2006). Apoptotic cells were detected with the Annexin V-PE Apoptosis Detection Kit (BD Biosciences). Cells were acquired on a FACScan, FACScanto, or LSRII and analyzed with Cell Quest, FACSDiva, or Flowjo software.
Generation of Retroviral Stocks
High-titer helper-free retroviruses were prepared as previously described (Signer et al. 2007b). The retroviral vector pMSCV (Hawley et al. 1994) which contains a 5’ long-terminal repeat–driven Bmi-1, p16Ink4a, Arf, or BCR-ABL followed by an internal ribosome entry site (IRES) and an enhanced green or yellow fluorescent protein (EGFP, YFP), or control vectors containing a 5’ long-terminal repeat–driven IRES EGFP or YFP were used. Retroviral vectors containing shRNAs for p16Ink4a and Arf were a kind gift from Dr. Scott Lowe.
Pro/preB cells were transduced in complete media with 4 µg/mL polybrene (Sigma). Complete media is defined as RPMI 1640 (Gibco) supplemented with 10% fetal calf serum (HyClone), 1 mM L-glutamine (Gibco), 100 U/mL streptomycin (Gibco), 100 µg/mL penicillin (Gibco), 50 µM β-mercaptoethanol (Sigma), 50 µg/mL gentamicin (Sigma), 20 ng/mL SCF (Biosource), 20 ng/mL IL-3 (Biosource), 10 ng/mL Flt-3L (R&D Systems), 30 ng/mL IL-7 (Biosource).
B Cell Differentiation Potential
Differentiation potential was tested by seeding cells directly onto confluent S17 stromal cells in RPMI 1640 supplemented with 10% fetal calf serum, 1 mM L-glutamine,100 U/mL streptomycin, 100 µg/mL penicillin, 50 µM β-mercaptoethanol for 3-5 days, at which time cells in suspension were tested for B lineage cell antigen expression (Whitlock and Witte 1982).
Models of Leukemogenesis
Purified pro/preB cells from young and old mice were transduced with retroviruses encoding Bmi-1-IRES-EGFP or BCR-ABL-IRES-YFP as described above, except that each virus was added 4-6 times over a 60 hour period. Following 7 days in culture, transduced cells expressing GFP and/or YFP were sorted and reseeded in the modified progenitor cell-culture system for an additional 7 to 10 days. Following expansion in vitro, 3x105 cells were injected intravenously into Rag1-/-mice that were preconditioned with 250 R from a 137Cs irradiator (120 R/min; Mark I-68A; JL Shepperd and Associates, San Fernando, CA) 24 hours earlier. Mice were sacrificed when they showed signs of disease characterized by poor grooming, slowed movement, and wasting. If no symptoms were observed, mice were sacrificed at day 85 post-transplantation. BM and spleen cells from the transplanted mice were prepared and tested for the presence of GFP+YFP+, YFP+, or GFP+ B cell tumors. BM and spleen cells were also deposited onto slides with a cytocentrifuge and stained with Wright-Giemsa. Pictures were taken using an Olympus DP11 camera and processed using Adobe Photoshop 7.0 image-acquisition software.
Supplemental References
Hawley, R.G., Lieu, F.H., Fong, A.Z., and Hawley, T.S. 1994. Versatile retroviral vectors for potential use in gene therapy. Gene Ther1: 136-138.
Whitlock, C.A. and Witte, O.N. 1982. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc Natl Acad Sci U S A79: 3608-3612.
Supplemental Figure Legends
Figure S1. Age-related changes in expression of p16Ink4a and Arf. Expression of p16Ink4a (A) and Arf (B) in total BM, HSCs, mature B cells (IgM+), and myeloid cells (CD11b+Gr-1+) relative to -Actin. P values are based on comparison of old versus young cells for each population (*, P<0.005; #, P<0.1; **, P<0.03). Each population was purified in two independent experiments from BM cell suspensions pooled from 4-6 young and 4-6 old C57BL/6 mice. Reactions for each population were run a total of eight times.
Figure S2. Retroviral transduction of young pro/preB cells with p16Ink4a and Arf results in rapid and severe decrease of growth in B lineage cells that correlates with expression levels of these genes exceeding those found in old pro/preB cells. (A) Number of cells harvested from cultures 48 hours following transduction of purified Lin–Ly6C-IgM–CD45R+CD19+ pro/preB cells with retroviruses encoding either GFP, p16Ink4a or Arf. P values are based on the comparison of each transduced population versus the young GFP transduced cells (*, P<0.05; **, P<0.0007). Representative qPCR amplification plots for GAPDH (B), p16Ink4a (C), and Arf (D) from the indicated populations 48 hours after retroviral transduction: GFP transduced young and old pro/preB cells (Young GFP and Old GFP), p16Ink4a transduced young pro/preB cells (Young p16Ink4a), and Arf transduced young pro/preB cells (Young Arf). Transduced cells in each population were purified based on GFP expression. The total amount of RNA used for each reaction was maintained constant for each individual population, but varied between populations. The over-expression of Arf (D) is evident when comparing expression levels relative to GAPDH which is expressed at much higher levels in Young and Old GFP populations than in the Young Arf population (B).
Figure S3. Knockdown of p16Ink4a expression rescues proliferative defects in aged B lineage cells. (A) Number of cells harvested from cultures 13 days following transduction of purified Lin–Ly6C-IgM-CD45R+CD19+ pro/preB cells with retroviruses encoding either GFP or an shRNA to p16Ink4a (shP16). Expression of shP16 in old pro/pre-B cells restores their growth potential when compared to their GFP transduced counterparts. An additional control group of pro/preB cells using an shRNA targeted to the luciferase gene was used, and there was no difference in growth relative to the GFP transduced control (not shown). (B) Expression of p16Ink4a and Arf relative to -Actin in young and old pro/preB cells 13 days following transduction with retroviruses encoding either GFP or shP16. Values are normalized to expression in the GFP transduced old pro/preB cells. P values are based on comparison ofthe expression in each population relative to the expression in old GFP transduced cells (*, P<0.05).(C) Expression of p16Ink4a relative to -Actin in untransduced 3T3-L1 cells and 3T3-L1 cells 13 days following transduction with a retrovirus encoding shP16. Values are normalized to expression in the untransduced 3T3-L1 cell line. (D) Western blot for p16Ink4a and -Actin in untransduced 3T3-L1 cells and 3T3-L1 cells 13 days following transduction with a retrovirus encoding shP16 showing effective knockdown of p16Ink4a protein and correlation to mRNA levels detected by qPCR.
Figure S4. Representative phenotypic profiles of BM cells from Rag1-/- mice transplanted with young or old pro/preB cells transduced with Bmi-1 and/or BCR-ABL. (A) Representative Rag1-/- recipient of BCR-ABL transduced old pro/preB cells showing no YFP+ leukemic cells. (B) Representative Rag1-/- recipient of BCR-ABL transduced young pro/preB cells showing abundant YFP+ leukemic cells with a CD45R+CD19+IgM-CD43+ proB cell phenotype.Representative Rag1-/- recipients of young (C) and old (D) pro/preB cells co-transduced with BCR-ABL and Bmi-1 showing abundant GFP+YFP+ leukemic cells with a CD45R+CD19+IgM-CD43+ proB cell phenotype. The mice are the same as those shown in Figure 5B-E.