A) Western blot (WB), immunoprecipitation (IP), and supernatants (SN) of the immunoprecipitation reaction with anti-IL-1IRAcP is shown in the left panel (Fig 4A).

<p>Westernblot of whole lysates of KMH2 is shown in the left lane (WB), the immunoprecipitated IL-1IRAcP is shown in the middle lane (IP), and the supernatant of this IP-reaction (SN), is shown in the right lane. The blot was detected also with the same antibody, anti-IL-1IRAcP, as a control reaction for the IP-reaction. The band in the supernatant indicates, that there was too much protein to get all immunoprecipitated, so that the amount of IL-1IRAcP, which was not bound by the antibody, occurs in the supernatant. IL-1IRAcP positive band is shown at the expected size of 66kDa (black arrow). Y-axis, size markers. <b>B)</b> After stripping the blot, detection was carried out with a specific anti-IL-1R2 antibody. Reactivity of the antibody was shown in the WB-, IP- and SN-lanes of KMH2, showing that the cell line expresses high amounts of membrane bound IL-1R2 (WB), which in part is able to interact with IL-1IRAcP (IP).</p

IL-1R2 concentrations in supernatants and plasma.

<p><b>A)</b> IL-1R2 concentration (pg/ml) in supernatants of HL derived cell lines as measured by ELISA (for details see Materials and Methods). <b>B)</b> IL-1R2 concentration in plasma of patients with Hodgkin’s disease (CR, complete remission; DP, active disease present) in comparison to control persons (CO) as measured by ELISA (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138747#sec002" target="_blank">Patients and Methods</a>). Each circle represents 1 individual person. Statistical comparison (Mann-Whitney test) revealed significant differences when values were compared as follows: DP vs. CR (p = .037), DP vs. CO (p = .018), CR vs. CO (p = .34).</p

IL-1beta expression in HRS cells as observed by ISH after 7, 14, and 21 days of autoradiography.

<p>MCHL, mixed cellularity (n = 6). NSHL, nodular sclerosis (n = 11). Each circle represents 1 individual patient. Y-axis shows the relative expression level of positive cells, which comprises the number of grains per cell and the expression intensity throughout the slide, with mean values and standard deviation. There was a statistically significant difference between the histological subtypes after 21 days (p = .042, Mann-Whitney test).</p

IL-1beta, IL-1R1, IL-1R2 expression in lymph node sections of patients with Hodgkin’s disease as observed with <i>in situ</i> Hybridization (ISH) (Fig 1A–1F).

<p>Positive cells are characterized by accumulation of black stains (examples illustrated with arrows). <b>A)</b> IL-1beta expression in few stromal and endothelial cells throughout the lymph node of MC type HL. <b>B)</b> IL-1beta expression in the stromal cells of areas with tissue remodeling and sclerosis in NSHL. <b>C, D)</b> IL-1R1 expression in HRS cells and few positive lymphocytes in MCHL <b>(C)</b>, and NSHL <b>(D)</b>. <b>E, F)</b> IL-1R2 positive HRS cells in a case of MCHL (E) and NSHL <b>(F)</b>.</p

Cyclin A1 expression in human and murine leukemic blasts.

<p>A. and B. Cyclin A1 was analyzed in mRNA microarray expression data from the purified fraction of human mononuclear bone marrow cells after Ficoll density centrifugation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref019" target="_blank">19</a>]. A. The expression of cyclin A1 was significantly increased in AML blasts compared to normal bone marrow (NBM) (p<0.001, t-test). Shown here are log arbitrary units. B. The expression of cyclin A1 was significantly decreased in AML blasts with complex karyotype and increased in AML M3 blasts compared with normal karyotype and (*p<0.001, t-test). C. Cyclin A1 expression was significantly induced in bone marrow cells from human AML patients with FAB subtype M3 (p = 0.015, t-test) and with FAB subtype M5/5a/5b (p = 0.05, two-tailed t-test) compared to normal bone marrow cells (NBM). Cyclin A1 expression was determined by qRT-PCR and normalized to GAPDH expression level. D. In bone marrow cells of PML-RARα-knockin mice, cyclin A1 expression was significantly upregulated upon a full-blown leukemic phenotype compared to non-leukemic PML-RARα-knockin mice (p = 0.04, t-test).</p

Cyclin A1 expression does not significantly influence PML-RARα-driven leukemogenesis.

<p>A. Kaplan-Meier survival curves of heterozygous PML-RARα-knockin mice with (SCL-cyclinA1-tg; n = 12) or without (control; n = 14) ectopic human cyclin A1 expression in the bone marrow. Although there was a trend towards an accelerated disease in the presence of ectopic cyclin A1 expression, latency and penetrance did not differ significantly between the two genotypes (p = 0.282, log-rank test). Also, the phenotype of acute myeloid leukemia was not changed by the presence of cyclin A1 (B). Shown here are examples of FACS analysis of bone marrow (upper panels) and spleen cells (lower panels) of diseased mice. Murine PML-RARα-driven leukemic blasts are characterized by the surface expression of CD34 and GR-1 which does not occur in non-leukemic mice. The number of CD34⁺/GR-1⁺ cells in diseased mice did not alter significantly upon cyclin A1 expression. SCL, Stem Cell Leukemia enhancer driving tTA expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref023" target="_blank">23</a>]; FI, fluorescent intensity; P/Rα-KI, PML-RARα-knockin mice. C. Kaplan-Meier survival curves of PML-RARα-knockin mice with wild type (PML-RARα-KI/cyclin A1^+/+; n = 30) or cyclin A1-knockout (PML-RARα-KI/cyclin A1^-/-; n = 35). Cyclin A1^-/- mice without PML-RARα-expression did not develop a lethal phenotype (control/cyclinA1^-/-; n = 3). Absence of murine cyclin A1 did not affect PML-RARα-driven leukemia. D. The PML-RARα-leukemic phenotype was not altered by the absence or presence of murine cyclin A1. May-Grünwald staining of blood smears showed the same distribution of leukemic blasts and high numbers of differentiated myeloid cells (upper panels). FACS analysis revealed comparable numbers of myeloid cells in the blood (CD11b⁺/GR-1⁺, lower panels).</p

Development of a cyclin A1-transgenic mouse model.

<p>A. Schematic overview about the constructs used to develop transgenic mouse lines. The driver mouse line SCL-tTA expresses the tetracycline-dependent transactivator protein (tTA) in hematopoietic stem cells under the control of the stem cell leukemia-factor enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129147#pone.0129147.ref024" target="_blank">24</a>]. In the novel cyclin A1-bi-luciferase responder mouse line, the cDNA of cyclin A1 and luciferase as reporter gene were expressed in absence of tetracycline in parallel and inducibly under control of the bidirectional tTA-responsive promoter element Pbi-1. B. Bars indicate expression levels of human cyclin A1 expression as detected by qRT-PCR in bone marrow cells that were transduced with a retroviral tTA-containing construct and cultured with or without tetracycline in methylcellulose for 10 days (n = 2 for each sample). C. Three months old mice carrying either cyclin A1 alone (control) or together with the driver construct SCL-tTA (SCL-tTAxcyclinA1-tg; n = 3 for each genotype) were induced for seven weeks and investigated for luciferase activity and cyclin A1 mRNA in the bone marrow and spleen. High luciferase activity was only detectable in induced SCL-tTAxcyclinA1-tg bone marrow and spleen cells. Numbers indicate mean luciferase levels.</p

Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

<div><p>Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of <i>Inca1</i> led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, <i>Inca1</i>-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in <i>Inca1</i>-deficient cells <i>in vivo</i>. As a consequence, leukemia induction and leukemia maintenance were severely impaired in <i>Inca1^−/−</i> bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of <i>Inca1^−/−</i> in MLL—AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.</p></div

Cytotoxic stress exhausts the stem cell pool in <i>Inca1</i>-deficient bone marrow.

<p><b>A.</b> The Kaplan-Meier plot illustrates the survival of <i>Inca1^+/+</i> (n = 7) and <i>Inca1^−/−</i> (n = 13) mice after weekly administration of the myeloablative agent 5-fluorouracil (5-FU). 5-FU treatment targeted predominantly proliferating bone marrow cells and led to a significantly shortened lifespan of <i>Inca1^−/−</i> mice compared to their wild type littermates (p = 0.02, log-rank). <b>B.</b> Cell cycle analysis of total bone marrow from mice treated twice with 5-FU using BrdU/PI staining and subsequent FACS analysis. <i>Inca1^−/−</i> bone marrow cells of mice that had been exposed twice to 5-FU showed more cells in S-phase than 5-FU treated wild type mice. Shown here is a representative example of three independent experiments. <b>C.</b> Histological bone sections from 5-FU treated mice showed significant depletion of hematopoietic cells in the bone marrow of <i>Inca1^−/−</i> sternum (right-hand side) with replacement by fat cells compared to wild type control mice. In contrast, hematopoietic cell numbers were only mildly decreased in wild type mice (left panel). Lower panels show higher magnifications of the areas marked in the upper panels. <b>D.</b> The total number of nucleated bone marrow cells was significantly decreased in <i>Inca1^−/−</i> mice after two cycles of 5-FU treatment (mean ± SD, p = 0.002, t-test). All mice were 16 weeks old at the time of analysis. <b>E.</b> Bone marrow cellularity in non-challenged mice did not differ between <i>Inca1^−/−</i> and <i>Inca1^+/+</i> genotypes (mice aged 70 to 425 days; mean ± SD, p = 0.83, t-test).</p

Inca1 is required for AML1-ETO9a-driven leukemia initiation and maintenance <i>in vivo</i>.

<p><b>A.</b> Survival curve of recipient mice which were transplanted in two independent experiments with bone marrow cells of <i>Inca1^+/+</i> and <i>Inca1^−/−</i> mice that were retrovirally transduced with AML1-ETO9a. Left-hand side: Transplantation with 100,000 AML1-ETO9a⁺ cells (both genotypes: n = 15); right-hand side: Transplantation with 250,000 AML1-ETO9a⁺ cells (<i>Inca1^+/+</i>: n = 14, <i>Inca1^−/−</i>: n = 10). Only one <i>Inca1^−/−</i> bone marrow transplanted with 250,000 AML1-ETO9a⁺ cells led to a lethal leukemic phenotype, while about half of the <i>Inca1^+/+</i> AML1-ETO9a transplanted mice died within 300 days. <b>B.</b> Bone marrow smears (upper panel) and spleen sections (lower panel) of <i>Inca1^+/+</i> and <i>Inca1^−/−</i> AML1-ETO9a transplanted mice that died of a leukemic phenotype. Arrows in the upper panels hint at leukemic blasts, indicating acute myeloid leukemia. HE stained sections revealed morphological disruption of the splenic structure and accumulation of myeloid cells in both genotypes (lower panels). <b>C.</b> Leukemic phenotypes of recipients that received AML1-ETO9a-transduced <i>Inca1^+/+</i> and <i>Inca1^−/−</i> bone marrow cells are characterized by an infiltration of bone marrow and spleen with GFP⁺c-kit⁺ leukemic blasts in both genotypes. <b>D.</b> Survival curve of secondary recipient mice which were transplanted with bone marrow cells of leukemic mice derived from the primary transplantation shown in Fig. 5A. All mice transplanted with primary leukemic <i>Inca1^+/+</i>; AML1-ETO9a bone marrow cells died within 60 days, whereas secondary recipients of <i>Inca1^−/−</i>; AML1-ETO9a cells died with an increased latency of 150 days (<i>Inca1^+/+</i>: n = 18, <i>Inca1^−/−</i>: n = 5; p = 0.001).</p