Cell cycle inhibitors are important regulators in normal tissue regeneration and disruption of the regulators are involved in cancer development. Our recent study showed that the absence of the CDK inhibitor p18INK4C (p18) enhances self-renewal of normal hematopoietic stem cell (HSC) in vivo, whereas previous studies by others showed an increased incidence of leukemogenesis in older p18-null mice. Here, we have examined potential leukemogenesis during experimentally induced regeneration of HSC in the absence of p18 in order to gauge the relation between these two processes. Reconstituted mice with p18-deficient HSCs under the condition of repetitive proliferative stress (serial transplantation) were followed for >3 years. T cell leukemia from the p18−/− origin was recapitulated 24 months after secondary transplantation. However, no myeloid leukemia was found in the recipients. The T cell leukemia–initiating cells (mainly in a CD3lo cell subset) did not share the same immunophenotype with normal HSCs and, in fact, the function of HSCs was significantly compromised with decreased abundance in the leukemic mice. Furthermore, we found that the p15 or p16 gene promoters were frequently methylated in the leukemic cells but not in HSCs. Our present study argues against the possibility of overgrowth of p18-null HSCs leading to a leukemic phenotype. The data also support the notion that p18 has an independent role in T cell maintenance such that CD3+CD8+ cells, unlike HSCs, are more accessible to leukemogenic transformation after the loss of p18. (Cancer Res 2006; 66(1): 343-51)

The hematopoietic stem cell (HSC) has defined therapeutic roles in clinical transplantation, but it might also directly or indirectly contribute to the development of leukemia due to its ability to self-renew and differentiate into multiple lineages over a lifetime (1, 2). The unique feature of HSC self-renewal must be physiologically balanced with cell differentiation or apoptosis. Imbalance of these processes may cause leukemogenesis, during which the leukemia-initiating cells (LICs) or leukemia stem cells (LSCs) must acquire a competitive self-renewal potential coupled with decreased cell death or a disrupted differentiation program to yield a leukemic phenotype (3). Therefore, it is vital that we gain a greater understanding of the relationship among these critical processes that underlie HSC kinetics in leukemogenesis as well as in normal hematopoietic regeneration. One of the fundamental mechanisms that coordinate these critical processes is cell cycle regulation.

In mammalian cells, cell cycle progression is largely controlled at the G1 phase (4). The G1 phase is regulated by the sequential activation and inactivation of cyclin-dependent kinases (CDKs; refs. 5, 6). Two families of low molecular weight cyclin-dependent kinase inhibitors (CKIs), Cip/Kip and INK4, have been identified as capable of interacting with CDKs to suppress progression through G1. The Cip/Kip family, which includes p21Cip1/Waf1, p27kip1, and p57Kip2 (p21, p27, and p57 hereafter), may interact with a broad range of cyclin-CDK complexes; whereas the INK4 family, which includes p16INK4A, p15INK4B, p18INK4C, and p19INK4D (p16, p15, p18, and p19 hereafter), specifically inhibit CDK4 and CDK6 kinases at early G1 phase. Increasing lines of evidence suggest that CKIs may represent an interface between the cell cycle and upstream stem cell regulatory pathways (3). For instance, Bmi-1, a polycomb protein, critically regulates self-renewal of different adult stem cell populations through inhibition of p16 expression and its alternative reading frame (ARF; refs. 79). With knockout mouse models, previous work from several laboratories showed that p21 is crucial for the maintenance of stem cell quiescence in both hematopoietic and central nervous systems (1014), whereas p27 more specifically inhibits the proliferation of early progenitor cells (10, 15, 16).

Based on studies of the hematopoietic system, p18 seems to be an interesting and unique molecule because its absence results in the most significant enhancement of hematopoietic engraftment in mouse transplant models compared with the absence of other CKIs (p21, p27, and p16; refs. 10, 15, 17, 18). Loss of p18 increases in vivo self-renewing divisions of HSCs over a prolonged period of time and is able to compensate for the exhausting effect of irradiated hosts on transplanted HSCs (19), which implies that targeting of p18 may be used for therapeutic manipulations of human HSCs. However, p18-deficient mice exhibit predisposition to the development of both spontaneous and carcinogen-induced tumors in multiple organs (2023). In hematopoietic and lymphoid systems, a small percentage (12%) of p18-null mice begin to develop T cell leukemia/lymphomas after reaching 1 year in age (21).

Given the enhanced regeneration of HSC and the risks of T cell malignancy in the absence of p18, we sought to further define the relation between HSC self-renewal and potential leukemia development, and to explore the other molecular disruptions that, in addition to p18 deletion, lead to a leukemic phenotype. These issues are important if targeting of p18 is proposed to improve HSC expansion, especially because there is at least partially overlapping immunophenotype of the LSCs with HSCs in certain cases of acute lymphoblastic leukemia (ALL; refs. 2427). In this study, we have extensively employed a serial competitive bone marrow transplantation (cBMT) model and analyzed the incidence of leukemogenesis of different subpopulations of bone marrow cells in transplanted mice. We were able to recapitulate the development of T lymphoid leukemia, but not other types of leukemia, from the p18-null origin during multiple rounds of cBMT. T cell LICs (T-LICs) or T cell LSCs (T-LSCs) do not share the same immunophenotype with the regenerated HSCs in the absence of p18. In addition to the loss of p18, deregulation of other cell cycle regulators, such as p15 and p16 in T cells, seems to be involved in the leukemic transformation.

Mice. The p18+/− mice in a C57BL/6;129sv genetic background were originally created by Dr. David Franklin et al. in Dr. Yue Xiong's laboratory (18), kindly provided by Dr. David Franklin (Department of Biological Science, Purdue University, West Lafayette, IN), and maintained in the certified animal facility at University of Pittsburgh Cancer Institute. The p18−/− or +/+ mice were generated from p18+/− breeding pairs. The mice were genotyped by a PCR method as described in our previous article (17). Littermates or age-matched mice (2-3 months old) were used in each experiment. Wild-type C57BL/6;129sv recipient mice for transplantation experiments were purchased from the Jackson Laboratory (Bar Harbor, ME). All procedures involved in the animal work were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

cBMT in conjunction with serial transfer. The procedure was detailed in our previous articles (17, 19). Briefly, an equal number (2 × 106) of wild-type bone marrow nucleated cells (BMNCs) as competitor cells from young mice (8-12 weeks old) were cotransplanted with male test cells into lethally irradiated female recipients for each cBMT, thereby serving as the same standard control for varied types of test input cells in each cBMT. Blood was collected at varied time points after each cBMT and the relative contribution to hematopoiesis from each genotype (−/− versus +/+) was quantified by a semiquantitative PCR assay (17). The engraftment level and leukemic occurrence after each cBMT are summarized in Table 1.

Table 1.

Development of T cell leukemia in p18−/− competitively reconstituted mice

Serial cBMT*No. of mice transplantedMonths after each cBMTAccumulative age of p18−/− cellsMean percentage of p18−/− engraftmentNo. of leukemic mice observed
First cBMT
Group 1 10 12 15 97.5
Group 2 10 8-14 14-20 94.3-99.1
Group 3 10 12 16 90.1
Second cBMT§
Group 1 10 14 30 88.5 (n = 7)
Group 2 10 12 30 N/A 10
Third cBMT 36 54.8 (n = 8)
Fourth cBMT 2-6 38-42 N/A
Serial cBMT*No. of mice transplantedMonths after each cBMTAccumulative age of p18−/− cellsMean percentage of p18−/− engraftmentNo. of leukemic mice observed
First cBMT
Group 1 10 12 15 97.5
Group 2 10 8-14 14-20 94.3-99.1
Group 3 10 12 16 90.1
Second cBMT§
Group 1 10 14 30 88.5 (n = 7)
Group 2 10 12 30 N/A 10
Third cBMT 36 54.8 (n = 8)
Fourth cBMT 2-6 38-42 N/A
*

Test and wild-type competitor cells were cotransplanted at a 1:1 ratio (2 × 106 cells/each) for each cBMT as detailed in Materials and Methods and our previous published work (17). Donor BMNCs for each subsequent transplantation were pooled from three nonleukemic animals, 6 to 14 months after the last round of cBMT.

Age of the original p18−/− donors plus months after cBMT accumulatively.

Engraftment levels were evaluated by the p18 genotyping PCR as described previously (17) only for the nonleukemic mice.

§

Groups 1 and 2 of the second cBMT were transplanted with the donor cells from the group 2 of the "first cBMT", 10 and 12 months, respectively, after transplantation.

Donor cells were obtained from group 1 of the second cBMT, 14 months after transplantation.

Donor cells were obtained from the nonleukemic mice 6 months after the third cBMT.

Transplantation of leukemic cells. Different doses of BMNCs, or sorted subpopulations from leukemic or nonleukemic mice alone or with normal wild-type female BMNCs (competitor cells), were transplanted into female wild-type recipient mice irradiated with 9.5 or 10 Gy. All the leukemic cells were of male p18−/− origin.

Multicolor flow cytometry analysis and cell sorting. For HSC analysis, the BMNCs were stained with a mixture of biotinylated antibodies against mouse CD3, CD4, CD8, B220, Gr-1, Mac-1, and TER-119 (Caltag, Burlingame, CA), then costained with streptavidin-PE-Cy7, Sca-1-PE, c-Kit-APC, and CD34-FITC (BD PharMingen, San Diego, CA). MoFlo High-Speed Cell Sorter (DakoCytomation, Fort Collins, CO) and Summit software (version 3.1, DakoCytomation) were used for data acquisition, analysis, or cell sorting. For lineage analysis of the peripheral blood cells, blood was stained with CD3, B220, MAC-1, and Gr-1 antibodies. The red cells were lysed with fluorescence-activated cell sorting lysing solution (BD Biosciences, San Jose, CA) and analyzed on CyAn LX (DakoCytomation) or Beckman Coulter XL cytometer.

Single HSC culture. Single CD34LKS cells were sorted and deposited into 96-well plates (one cell per well). Each well contained 100 μL of Iscove's modified Dulbecco's medium supplemented with 50 ng/mL of Flt3 ligand (Flt3-L), 50 ng/mL of stem cell factor, 10 ng/mL thrombopoietin, and 10 ng/mL interleukin3 (IL-3; Peprotech, Rocky Hill, NJ). 10 days after seeding, each colony was examined under a microscope and lysed for PCR.

Methylation-specific PCR. Methylation-specific PCR (MSP) was done as previously described (28). Briefly, DNA was isolated from the hematopoietic or leukemic subsets using the Wizard Genomic DNA isolation kit (Promega, Madison, WI). The DNA was denatured in 0.2 mol/L NaOH for 10 minutes and treated with sodium bisulfite (Sigma, Saint Louis, MO) in the presence of hydroxyquinone (Sigma) for 16 hours at 50°C. Modified DNA was collected using the Wizard DNA Purification Kit (Promega) and redissolved in nuclease-free water. PCR reactions were carried out with the primers below for 35 cycles (30 seconds at 95°C, 30 seconds at the indicated annealing temperature, and 1 minute at 72°C). The primers used are as follows:

• p15 methylated forward-TAGTGACGCGGGTTTGGTTATCGTC,

• p15 methylated reverse-GCCTCCCGAAACGATTCAAAACGT,

• p15 unmethylated forward-GTAGTAGTGATGTGGGTTTGGTTATTGTTG,

• p15 unmethylated reverse-CACCTCCCAAAACAATTCAAAACATT,

• p16 methylated forward-CGATTGGGCGGGTATTGAATTTTCGC,

• p16 methylated reverse-CACGTCATACACACGACCCTAAACCG,

• p16 unmethylated forward-GTGATTGGGTGGGTATTGAATTTTTGTG,

• p16 unmethylated reverse-CACACATCATACACACAACCCTAAACCA.

The annealing temperatures for the p15 and p16 primers were 60°C and 66°C, respectively. p15 MSP on sorted cell populations was carried out as described (29). After sorting, the cells were embedded in agarose beads and lysed overnight with 0.5 mol/L EDTA (pH 8.0)/2 mg/mL proteinase K at 50°C. The cells were then treated with sodium bisulfite in the presence of hydroxyquinone for 4 hours at 50°C. The DNA was recovered as above and a nested PCR approach was employed. The DNA was first amplified with the two nested primers (forward-GTTTAGAGATTAGGTTGTAGTAAT and reverse-CCCAAATAATACCCAATTACAAC) for 35 cycles as above with an annealing temperature of 55°C. One microliter of this reaction was then used for p15 MSP as described above for 20 cycles. The PCR reactions were run on a 3.0% agarose gel, stained with ethidium bromide, and visualized under UV light.

Western blot analysis. Total cell extract from spleen tissue was prepared according to standard protocols and resolved on 12% SDS-polyacrylamide gels. After transferring to nitrocellulose, the membranes were blocked overnight and incubated with anti-cyclin D3 (sc-182, Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution, anti-cdk4 (sc-260, Santa Cruz Biotechnology) 1:100 dilution, or anti-actin (sc-8432, Santa Cruz Biotechnology) 1:500 for 1 hour. The membranes were washed and incubated with the species-appropriate secondary antibody conjugated with horseradish peroxidase at 1:1,000 for 1 hour. The membranes were again washed and bands visualized with the Western lighting chemiluminescence kit (Perkin-Elmer Life Sciences, Boston, MA) on X-ray film (Kodak, Rochester, NY) for 1 to 10 minutes.

Flow cytometry-based measurement of telomere length (flow fluorescence in situ hybridization). Telomeres in leukemic cells were detected with the PNA Kit for Flow Cytometry (DAKO A/S, Glostrup, Denmark), according to the manufacturer's protocol. Briefly, 2 × 106 cells from normal wild-type or leukemic bone marrow cells were mixed with an equal number of control cells (1301 T cell leukemia line) and divided into four 1.5 mL tubes. The cellular DNA was denatured for 10 minutes at 82°C and hybridized with the telomere PNA probe (FITC) at room temperature overnight. After washing, the cells were stained with the DNA dye, propidium iodide. The cells were then analyzed on a Beckman Coulter XL cytometer. Photomultiplier tube linearity of the flow cytometer was checked with eight-peak Rainbow Beads (Spherotech, Inc., Libertyville, IL). The relative telomere length (RTL) of test versus control cells was calculated as described in Fig. 5A.

Spontaneous T cell leukemogenesis is recapitulated in p18−/− reconstituted mice. According to previous reports by others, there is a low frequency of spontaneous T cell lymphomas/leukemias in aged p18−/− mice and the frequency increases following exposure to carcinogens (21, 22). In order to recapitulate the potential leukemogenesis and dissect it from the enhanced HSC regeneration in the p18−/− reconstituted model, we extensively followed up the serial cBMT model (17, 19). HSCs deficient in p18 sustained their competitiveness to freshly isolated wild-type HSCs from nontransplanted young mice (8-12 weeks old) after multiple cycles of bone marrow transplantation over 30 months. Furthermore, p18 absence significantly decelerated the exhaustion of hematopoietic repopulation caused by p21 deficiency (19). Notably, the incidence of leukemia increased after the aging of hematopoietic cells in our transplantation model, whereas no leukemia developed in the primarily transplanted recipients (n = 30) in 14 months. Leukemia was observed in one group after the second cBMT (30 months of cellular age accumulatively; Table 1). The leukemic mice had enlarged lymph nodes and spleens with increased WBC counts (>1 × 108 cells/mL). Morphology of the leukemic cells in blood and bone marrow seemed to have lymphocytic features (data not shown). The passage number of serial transfer did not seem to closely correlate with the incidence of leukemia development, perhaps due to the limited size of animal cohorts and uneven distribution of leukemic cell clones in the recipients. But multiple rounds of cBMT did result in a higher penetrance of the leukemia as evidenced by low leukemic incidence (1 of 9) after the third cBMT but a 100% (9 of 9) occurrence after the fourth cBMT. Flow cytometry analysis revealed that the main cell population in the blood of leukemic mice expressed CD3 expression at a lower fluorescent intensity (CD3lo), as compared with CD3 in normal wild-type mice (Fig. 1A). Further analysis indicated that the majority of the CD3lo cells were CD8-positive in the primarily developed leukemic samples (Fig. 1B). T cell leukemic cells were of p18−/− origin in all of the examined leukemic mice (n = 20). Interestingly, whereas p18−/− phenotype dominance favored myeloid lineage in nonleukemic transplanted mice undergoing serial transplantation (19), we did not observe myeloid leukemia development in all serial recipients (n = 68).

Figure 1.

Phenotype of leukemia developed in the p18−/− reconstituted mice. A, flow cytometry analysis showing the dominance of T cells (CD3+), not B cells (B220+) or myeloid cells (Mac-1+ or Gr-1+). B, further flow cytometry analysis indicating substantial coexpression of CD8, not CD4, within the CD3+ leukemic cells. Data shown are from representative leukemic mice in group 2 of the second cBMT (Table 1).

Figure 1.

Phenotype of leukemia developed in the p18−/− reconstituted mice. A, flow cytometry analysis showing the dominance of T cells (CD3+), not B cells (B220+) or myeloid cells (Mac-1+ or Gr-1+). B, further flow cytometry analysis indicating substantial coexpression of CD8, not CD4, within the CD3+ leukemic cells. Data shown are from representative leukemic mice in group 2 of the second cBMT (Table 1).

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To determine whether leukemic cells were transplantable, we transplanted varied numbers of the primary leukemic bone marrow cells along with 2 × 106 normal p18+/+ competitor cells (n = 10 mice/group/dose) into new recipients. All the new recipient mice developed T cell leukemia within 35 days after transplantation, including the mice that received the lowest dose of 1 × 104 leukemic cells per mouse. The morphology of the leukemia cells was very similar with that of primarily developed leukemia cells (data not shown).

T-LICs predominantly reside in the T cell compartment but not HSC pools. Increasing evidence has suggested that LICs may originate from HSCs in some leukemias (2). In our previous report, we have shown that HSCs deficient in p18 sustained their competitiveness to freshly isolated wild-type HSCs from nontransplanted young mice and retained their multilineage differentiation potential after serial bone marrow transplantation over a period of 30 months (19). These facts raise the possibility that enhanced regeneration of HSCs, or perhaps overgrown HSCs, might directly or indirectly contribute to T cell leukemia in the absence of p18.

To formally address whether the leukemic transformation initiates in the HSC compartment, we first transplanted BMNCs, the lineage positive (Lin+) cell populations (dominated by CD3loCD8+ as shown in Fig. 1) or the enriched HSC/HPC populations (Linc-Kit+Sca-1+, LKS) from the leukemic bone marrow into lethally irradiated wild-type mice (n = 10 mice/group). All the mice transplanted with Lin+ cells developed leukemia much faster than mice transplanted with the same number of LKS cells from the same leukemic donors (Fig. 2A and B). Transplantations of leukemic Lin+ or BMNCs were able to rapidly induce leukemia within 50 days (Fig. 2B). In contrast, mice receiving LKS cells that were fractionated from the same leukemic mice developed leukemia at a much slower pace (3 months after transplantation and ∼80% of mice in this group had T cell leukemia within 7 months after transplantation). This finding suggested that T-LICs are not predominantly contained in LKS population. The much delayed onset of leukemic phenotype in the p18−/− LKS transplanted group might be due to the indirect contribution of HSCs/early progenitors to the leukemogenesis or the contamination of T-LICs contained in the CD3+ population. This issue was further defined with a more homogeneous population for T-LIC or HSC in our subsequent studies below. Notably, the overall shorter latency of the leukemia in the secondary leukemic transplants, as compared with that in the original transplants, suggests that additional genetic or epigenetic abnormalities contributing to the leukemogenesis may have occurred during the serial transplantation or aging process.

Figure 2.

T-LICs predominantly contained in CD3lo but not CD34LKS population. A, WBC counts in the transplanted mice 30 days after transplantation. A fractioned marrow cell subset (3 × 105 Lin+ or 1 × 103 LKS cells) or unfractioned marrow cells (1 × 106) from the leukemic mice were sorted and cotransplanted with 1 × 106 normal wild-type BMNCs into a 10-Gy–irradiated female p18+/+ mouse (n = 10/group). Abbreviations: N-LKS, LKS cells from nonleukemic p18−/− marrow; L-LKS, LKS cells from leukemic marrow; L-Lin+, Lin+ cells from leukemic marrow; L-BM, BMNCs from leukemic mice. All the leukemic samples were of p18−/− origin as verified by the genotyping PCR (17). There is a significant difference (P < 0.01, Student's t test) between L-Lin+ or L-BM and N-LKS or L-LKS. There is no significant difference (P > 0.05, Student's t test) between LKS groups or between L-Lin+ and L-BM. B, survival curves of the transplanted mice described in (A). Data were plotted with Prism 3.0 Software (Graphpad, San Diego, CA) and expressed as Kaplan-Meier survival curves. There is a significant difference between N-LKS and L-LKS or between L-Lin+ or L-BM and N-LKS or L-LKS (P < 0.01, log-rank nonparametric test). C, survival curve of recipient mice transplanted with CD3lo cells in comparison with CD34LKS cells from the same leukemic mice or normal p18+/+ mice. CD3lo cells at varied doses isolated from the leukemic mice or 600 CD34LKS cells from the leukemic (L-CD34LKS) or normal (N-CD34LKS) mice along with 2 × 105 female competitor cells were injected into 9.5 Gy–irradiated female recipients (n = 5/group). All the leukemic cells were of p18−/− origin. Data were plotted with Prism 3.0 Software and expressed as Kaplan-Meier survival curves. Each group injected with a specific cell dose is pointed to its corresponding line in the graph. There is a significant difference between groups (P < 0.05, log-rank nonparametric test) except between CD3lo-103 and CD3lo-105 or between CD3lo-103 and CD3lo-104. D, functional loss of hematopoietic activity of CD34LKS cells from the leukemic mice in vivo. Blood was collected from the recipients 2 months after transplantation with CD34LKS cells and donor contribution to irradiated host was assessed by the specific PCR detection for mouse Y chromosome (10). p18−/− cells were originally from male donors. Each recipient in groups “1 to 5” received 600 CD34LKS cells from normal mice and each recipient in groups “1′ to 4′” received 600 CD34LKS cells from the leukemic marrow. “Sry” indicates the band for the specific mouse Y-chromosome DNA and “Myo” indicates the band for a housekeeping gene, myogenin. The leukemic bone marrow samples were obtained from group 2 of the second and fourth cBMT (Table 1).

Figure 2.

T-LICs predominantly contained in CD3lo but not CD34LKS population. A, WBC counts in the transplanted mice 30 days after transplantation. A fractioned marrow cell subset (3 × 105 Lin+ or 1 × 103 LKS cells) or unfractioned marrow cells (1 × 106) from the leukemic mice were sorted and cotransplanted with 1 × 106 normal wild-type BMNCs into a 10-Gy–irradiated female p18+/+ mouse (n = 10/group). Abbreviations: N-LKS, LKS cells from nonleukemic p18−/− marrow; L-LKS, LKS cells from leukemic marrow; L-Lin+, Lin+ cells from leukemic marrow; L-BM, BMNCs from leukemic mice. All the leukemic samples were of p18−/− origin as verified by the genotyping PCR (17). There is a significant difference (P < 0.01, Student's t test) between L-Lin+ or L-BM and N-LKS or L-LKS. There is no significant difference (P > 0.05, Student's t test) between LKS groups or between L-Lin+ and L-BM. B, survival curves of the transplanted mice described in (A). Data were plotted with Prism 3.0 Software (Graphpad, San Diego, CA) and expressed as Kaplan-Meier survival curves. There is a significant difference between N-LKS and L-LKS or between L-Lin+ or L-BM and N-LKS or L-LKS (P < 0.01, log-rank nonparametric test). C, survival curve of recipient mice transplanted with CD3lo cells in comparison with CD34LKS cells from the same leukemic mice or normal p18+/+ mice. CD3lo cells at varied doses isolated from the leukemic mice or 600 CD34LKS cells from the leukemic (L-CD34LKS) or normal (N-CD34LKS) mice along with 2 × 105 female competitor cells were injected into 9.5 Gy–irradiated female recipients (n = 5/group). All the leukemic cells were of p18−/− origin. Data were plotted with Prism 3.0 Software and expressed as Kaplan-Meier survival curves. Each group injected with a specific cell dose is pointed to its corresponding line in the graph. There is a significant difference between groups (P < 0.05, log-rank nonparametric test) except between CD3lo-103 and CD3lo-105 or between CD3lo-103 and CD3lo-104. D, functional loss of hematopoietic activity of CD34LKS cells from the leukemic mice in vivo. Blood was collected from the recipients 2 months after transplantation with CD34LKS cells and donor contribution to irradiated host was assessed by the specific PCR detection for mouse Y chromosome (10). p18−/− cells were originally from male donors. Each recipient in groups “1 to 5” received 600 CD34LKS cells from normal mice and each recipient in groups “1′ to 4′” received 600 CD34LKS cells from the leukemic marrow. “Sry” indicates the band for the specific mouse Y-chromosome DNA and “Myo” indicates the band for a housekeeping gene, myogenin. The leukemic bone marrow samples were obtained from group 2 of the second and fourth cBMT (Table 1).

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To further measure the frequency of T-LIC in the leukemogenic cell population, we then sorted the CD3lo cells from leukemic mice and transplanted different doses of the cells along with normal competitor cells into wild-type recipients after a lethal dose of 9.5 Gy irradiation. All the mice transplanted with CD3lo leukemic cells, including those transplanted with as low as 100 cells, developed T cell leukemia and died within 3 months after transplantation (Fig. 2C), thereby indicting a high potency of CD3lo leukemic cells in reinitiating leukemogenesis in vivo.

Functional and phenotypic exhaustion of HSCs in leukemic mice. In contrast to the rapid induction of the CD3lo cells in T cell leukemia, the mice that received 600 CD34LKS cells (one of the primitive HSC immunophenotypes; ref. 30) showed no apparent signs of leukemia during 8 months posttransplantation, although some recipients died of bone marrow failure or complications from irradiation (Fig. 2C). Two months after transplantation, a PCR assay for detecting donor Sry sequence in blood cells was used to quantify the level of engraftment (10). Although all the mice that received wild-type CD34LKS cells showed engraftment of donor cells as expected, the mice that received leukemic transplants had barely detectable amounts of donor cells in the blood (Fig. 2D). Furthermore, we isolated CD34LKS cells from the bone marrow to determine whether these cells from leukemic mice are still responsive to the cytokines that have been commonly used to stimulate HSC growth in vitro (30, 31). Single CD34LKS cells were sorted into 96-well plates with one cell per well and cultured in liquid medium supplemented with stem cell factor, thrombopoietin, Flt3-L, and IL-3 for 10 days. Although CD34LKS cells from nonleukemic donor mice (third cBMT) responded to the cytokine stimulation by forming multilineage colonies in the culture, CD34LKS cells from leukemic mice failed to form colonies (Table 2). Finally, we used an independent method to measure the abundance of phenotypic HSCs in leukemic marrow compared with that in nonleukemic marrow. The HSC phenotype was significantly diminished in all leukemic marrow compared with nonleukemic marrow, as assessed by either Linc-Kit+ or SP fraction (32) in Sca-1+-enriched populations (Fig. 3). These data show that p18-null HSCs from leukemic mice were not only unable to transfer the leukemic phenotype but were also unable to exert their normal hematopoietic functions at this stage thereby contrasting the overwhelming effect of p18-null HSCs against the exhausting effect of the irradiated hosts in which leukemia had not developed (19).

Table 2.

Response of single HSCs (CD34LKS) to cytokine stimulation in vitro

Bone marrow after the third cBMTTotal no. of single cells culturedPercentage of responseTotal no. of colonies genotypedPercentage of p18-null in total
Nonleukemic 360 67.8 93 76.3
Leukemic 180 20.6 37
Bone marrow after the third cBMTTotal no. of single cells culturedPercentage of responseTotal no. of colonies genotypedPercentage of p18-null in total
Nonleukemic 360 67.8 93 76.3
Leukemic 180 20.6 37
Figure 3.

Phenotypic exhaustion of HSCs in the leukemic marrow. BMNCs were enriched with Sca-1-conjugated beads (Miltenyi Biotec) and then stained for Sca-1, c-Kit and Lineage antibodies or Hoeschest33343 to quantify the abundance of the c-Kit+ fraction (indicated by the percentage in each figure) in the LinSca-1+ population (left) or in the side population (SP; indicated by the percentage in each figure; right) with multicolor flow cytometry analysis. Data shown are from one of three experiments with similar results.

Figure 3.

Phenotypic exhaustion of HSCs in the leukemic marrow. BMNCs were enriched with Sca-1-conjugated beads (Miltenyi Biotec) and then stained for Sca-1, c-Kit and Lineage antibodies or Hoeschest33343 to quantify the abundance of the c-Kit+ fraction (indicated by the percentage in each figure) in the LinSca-1+ population (left) or in the side population (SP; indicated by the percentage in each figure; right) with multicolor flow cytometry analysis. Data shown are from one of three experiments with similar results.

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Deregulation of other cell cycle regulators in the leukemic cell subset. Molecular disruption in cancer development is a multistep process (33). The fact that T cell leukemia developed in aged bone marrow cells in our reconstitution model suggests that down-regulation of p18 alone is not sufficient for triggering the full leukemic transformation, and other genetic or epigenetic changes may be induced during either bone marrow reconstitution or the aging process, or both. Because hypermethylation of p15 or p16 promoter is frequently found in leukemias (3), we did methylation-specific PCR to assess the methylation status of CpG islands at the promoter sites of p15 and p16. Interestingly, 80% (8 of 10) of the leukemic samples showed methylation at the p15 promoter, whereas 20% (2 of 10) of the samples showed methylation at both p15 and p16 promoters (n = 10), and 20% of the samples showed no methylation in either gene promoter (Fig. 4A). In the leukemic marrow, where p15 promoter was methylated in the bulk leukemic bone marrow cell populations, we isolated different cell subsets including LKS, CD3+CD4+ and CD3+CD8+, and determined the methylation status of p15 gene promoter from each cell subset. The methylation of p15 promoter was only observed in CD3+CD8+ cells, not in CD3+CD4+ or LKS cells (Fig. 4B), which suggests the specific association of additional molecular abnormalities with CD3+CD8+ cells that contain the T-LICs. To investigate the consequence of these additional disruptions of cell cycle regulators, we did Western blot analysis for the expression of the target molecule of INK proteins, CDK4 and its functional partner, cyclin D3. The leukemic cells showed an elevated level of cyclin D3 as well as CDK4 as compared with the nonleukemic p18+/+ or p18−/− controls (Fig. 4C). Moreover, telomere length increased significantly in the leukemic cells (n = 6, P < 0.001), as assessed by a flow cytometry-based fluorescence in situ hybridization assay (Fig. 5B). Notably, neither CDK4 activity (Fig. 4C) nor telom4ere length (data not shown) were increased in p18−/− nonleukemic cells that had undergone the same passage of serial transfer as the leukemic cells. These results illustrate one snapshot of the subsequent molecular events that, in addition to the loss of p18, likely contribute to the leukemic transformation.

Figure 4.

Deregulation of additional cell cycle regulators in the leukemic cells. A, methylation of p15 or p16 promoters in leukemic cells. Methylation of CpG islands at the p15 and p16 promoters was assessed by the methylation-specific PCR. Numerals indicate individual leukemic mice in group 2 of the second cBMT (Table 1). “U” and “M” indicate the PCR result with unmethylated and methylated primers, respectively (details in Materials and Methods). B, a representative gel showing the methylation of p15 promoter in CD3+CD8+ cells, not in LKS or CD3+CD4+ cells. C, Western blot analysis for CDK4 and cyclin D3 in comparison with β-actin. All the samples were BMNCs. “WT” and “p18−/−” indicate normal wild-type and p18−/− transplant, respectively. Numbers under “leukemic” indicate three different leukemic mice.

Figure 4.

Deregulation of additional cell cycle regulators in the leukemic cells. A, methylation of p15 or p16 promoters in leukemic cells. Methylation of CpG islands at the p15 and p16 promoters was assessed by the methylation-specific PCR. Numerals indicate individual leukemic mice in group 2 of the second cBMT (Table 1). “U” and “M” indicate the PCR result with unmethylated and methylated primers, respectively (details in Materials and Methods). B, a representative gel showing the methylation of p15 promoter in CD3+CD8+ cells, not in LKS or CD3+CD4+ cells. C, Western blot analysis for CDK4 and cyclin D3 in comparison with β-actin. All the samples were BMNCs. “WT” and “p18−/−” indicate normal wild-type and p18−/− transplant, respectively. Numbers under “leukemic” indicate three different leukemic mice.

Close modal
Figure 5.
Increased telomere length in the leukemic cells. Telomere length was analyzed by flow cytometry as detailed in Materials and Methods. A, establishment of the assay for measuring the relative telomere length in test cells (R1 and R3) as compared with the immortalized leukemia cell line 1301 (R2 and R4). Left, without PNA probe hybridization; right, with PNA probe hybridization. Telomere labeling was compared only in the cells at G1/0 phase determined by propidium iodide staining (circled). The RTL (%) was calculated with the following formula:
$\frac{\mathrm{(mean\ FITC\ intensity\ of\ R3{-}mean\ FITC\ intensity\ of\ R1)\ {\times}\ DNA\ index\ of\ test\ cells\ {\times}100}}{\mathrm{(mean\ FITC\ intensity\ of\ R4\ {-}\ mean\ FITC\ intensity\ of\ R2)\ {\times}\ DNA\ index\ of\ control\ cells}}.$
B, the actual RTL of p18−/− leukemic cells (black column) compared with p18−/− nonleukemic cells (gray column). The difference is statistically significant between these two groups (n = 5, P < 0.001; Student's t test). These cells were BMNCs from the mice that had undergone three serial rounds of cBMT. There was no difference between p18−/− and p18+/+ transplanted cells in the nonleukemic mice (data not shown).
Figure 5.
Increased telomere length in the leukemic cells. Telomere length was analyzed by flow cytometry as detailed in Materials and Methods. A, establishment of the assay for measuring the relative telomere length in test cells (R1 and R3) as compared with the immortalized leukemia cell line 1301 (R2 and R4). Left, without PNA probe hybridization; right, with PNA probe hybridization. Telomere labeling was compared only in the cells at G1/0 phase determined by propidium iodide staining (circled). The RTL (%) was calculated with the following formula:
$\frac{\mathrm{(mean\ FITC\ intensity\ of\ R3{-}mean\ FITC\ intensity\ of\ R1)\ {\times}\ DNA\ index\ of\ test\ cells\ {\times}100}}{\mathrm{(mean\ FITC\ intensity\ of\ R4\ {-}\ mean\ FITC\ intensity\ of\ R2)\ {\times}\ DNA\ index\ of\ control\ cells}}.$
B, the actual RTL of p18−/− leukemic cells (black column) compared with p18−/− nonleukemic cells (gray column). The difference is statistically significant between these two groups (n = 5, P < 0.001; Student's t test). These cells were BMNCs from the mice that had undergone three serial rounds of cBMT. There was no difference between p18−/− and p18+/+ transplanted cells in the nonleukemic mice (data not shown).
Close modal

Given the increase of HSC self-renewal and T cell malignancy in p18−/− mice, as previously shown by different laboratories (17, 19, 21), our present study further shows that there is no direct link between enhanced HSC regeneration and potential leukemogenesis in the absence of p18. Additional molecular disruption in addition to the absence of p18 seems to be required for the development of T cell leukemia. Unlike chronic myeloid leukemia and most types of acute myeloid leukemia (AML; ref. 34), T cell leukemia has not been generally considered to be closely related to HSC deregulation. However, the relation between T cell leukemia and HSCs remains obscure because it has been shown that prethymic T cell progenitors exist in the LKS population of mouse bone marrow (35). Moreover, several studies by others showed an overlapped phenotype (CD34+CD38) of LIC with that of HSC in some cases of human ALL (2427). Therefore, our current study also addresses a critical issue in possible scenarios in which targeting of p18 is used for therapeutic manipulation (including expansion and/or gene therapy) of HSC.

The existence of multiple CKIs with overlapping biochemical activity suggests that each CKI may function in a cell type–specific or differentiation-specific fashion. In the case of HSC regulation, although p18 restricts self-renewing divisions of HSC, p16, and ARF alone have a limited role in the regulation of HSC proliferation under homeostatic conditions (18). On the other hand, p15 and p16 are frequently inactivated in human cancers, whereas p18 is not. For example, based on a survey of 4,700 primary cases of leukemia or lymphoma and 320 leukemia-lymphoma cell lines, p15 and p16 are frequently deleted or silenced by hypermethylation in their promoters; in contrast, very few cases had p18 or p19 deletion or hypermethylation (36). These genetic or epigenetic data in human cancers are in agreement with the occurrence of tumor development in mice deficient in these CKIs. Mice lacking p16 have thymic hyperplasia with enhanced mitogenic responsiveness in T cells. Like ARF deficiency, p16 deficiency is significantly associated with an increased incidence of spontaneous and carcinogen-induced cancers (37). Loss of the p18 gene in mice results in organomegaly with higher cellularity, and to a lesser degree, an increase in the incidence of spontaneous pituitary tumor with advanced age, or multiple tumor types in the presence of carcinogens or in synergy with loss of another CKI such as p27 (2023, 38, 39). In contrast to p15 or p16, p18 has been proposed to function as a haploinsufficient tumor suppressor (22). However, the molecular links of p18 loss and epigenetic deregulation of p15/p16 expression, as shown in our current study, are currently unknown. In addition, the fact that 20% of the leukemia samples had no abnormality of p15 or p16 promoter suggests that other molecular disruptions, yet to be defined, are also involved in the leukemogenesis.

Given the different tumor susceptibilities and the distinct effects of these CKIs, we propose that increased HSC self-renewal by down-regulation of p18 is not sufficient to cause leukemia, and that the disruption of p15 or p16 may set the stage for forming T-LIC to out-compete normal hematopoiesis and lymphopoiesis (3). We speculate that normal and malignant stem cells might thus employ or favorably employ different CKIs during their divisions, at least in some tissues. This similar paradigm has also been suggested by studies on different molecules. For example, ectopic expression of HoxB4 strikingly enhances self-regeneration of normal HSC, whereas ectopic expression of HoxA9 induces AML (40). In addition, aberrant signaling in the nuclear factor κB pathway may be specifically associated with AML LSC, but not with normal HSC (41). Therefore, further defining the unique molecular interplay in LSC versus normal HSC is of great importance and our current study focusing on CKIs may provide a unique angle or model to address this important question in other kinds of leukemia or cancer as well.

Since the common lymphoid progenitor (IL-7R+Thy-1Linc-KitloSca-1lo) population from adult mouse bone marrow was discovered by Kondo et al. (42), other pathways for T-lineage development also have been reported. Studies from Jacobsen's group showed that Flt3+CD34+LKS cells are primarily responsible for rapid lymphoid reconstitution, which represents the initial commitment from the long-term repopulating HSCs (43). In addition, after the thymic early T cell progenitor was shown to develop via a common lymphoid progenitor–independent pathway (44), Spangrude's group found a T cell progenitor population (L-selectin+Thy-1.1LKhiS) in close relation to HSC in mouse bone marrow, from which early T cell progenitors likely originate (35). Our phenotypic analysis of the leukemogenic cells shows that neither HSCs nor these bone marrow–derived T cell progenitors are the direct target of leukemogenesis in the absence of p18. Rather, the transformation of T cell leukemia ultimately occurs in the mature T cell population, based on the expression of CD3 and CD8 in the T-LICs (Fig. 1B). However, our study cannot completely rule out the possibility of pre-LSC–like cells in the LKS population given the delayed leukemic development in LKS-transplanted mice (Fig. 2B). If this is the case, however, the primitive HSC phenotype (CD34LKS), which has been shown to be solely responsible for the long-term hematopoietic reconstitution in lethally irradiated recipients (30), does not seem to be directly involved based on the results that CD34LKS cells from the leukemic marrow were not able to transfer the leukemic phenotype, and moreover, the normal hematopoietic function of CD34LKS cells was apparently lost in leukemic mice (Figs. 2D and 3). The phenotypic and functional exhaustion of p18-null HSCs in leukemic mice is intriguing because we have observed strikingly improved engraftment of the p18-null cells in the serially transplanted mice in which leukemia was not developed (Table 1; ref. 19). This might be attributable to some epigenetic factors, yet to be defined, uniquely produced by the leukemic cells or microenvironment. Therefore, further investigation for such an interesting phenomenon is warranted.

A previous study by Kovalev et al. (21) showed that whereas loss of p18 resulted in a hyperproliferative response of T cells to CD3 stimulation in a bulk splenic cell culture, the major G1 cell cycle regulatory proteins were not altered in p18-null T cells. Furthermore, the activated p18+/+ T cells expressed a constant level of p18 protein. In our current study, we observed an unchanged level of cyclin D3 in both p18+/+ and p18−/− hematopoietic cells, but an elevated level was associated with elongated telomere in the leukemic cells in which p15 or p16 was frequently methylated (Figs. 4C and 5B). These data suggest that p18 absence alone does not simply cause a general enhancement of cell cycle progression in the T cell compartment and that in fact, there might be a cell cycle–independent mechanism modulating the fate of proliferating T cells, as we proposed for HSC repopulation in the absence of p18 (17). On the other hand, the susceptibility of the T cell subset (CD3+CD8+) versus HSCs to leukemogenesis in the absence of p18 seems to differ significantly during aging, suggesting that different signaling pathways control cellular senescence as opposed to self-renewal. Therefore, it will be of considerable interest to determine which T cell subset can be regulated by p18 as an analogue to the self-renewable HSC population under homeostasis, and how p18 may be engaged with other molecular players in the development of T cell leukemia but not at the HSC level after oncogenic insults.

Note: Y. Yuan and H. Yu contributed equally to this work.

Grant support: NIH grants (DK02761 and HL70561) and the Innovative Award from PNC Foundation (2002). T. Cheng was a recipient of the Scholar Award from American Society of Hematology (2003).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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