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Cell Death and Differentiation logoLink to Cell Death and Differentiation
. 2010 Jul 30;18(1):174–182. doi: 10.1038/cdd.2010.85

Very low oxygen concentration (0.1%) reveals two FDCP-Mix cell subpopulations that differ by their cell cycling, differentiation and p27KIP1 expression

A V Guitart 1,2,3,6, C Debeissat 1,2,3,6, F Hermitte 1,2,3,7, A Villacreces 1,2,3, Z Ivanovic 4, H Boeuf 1,2,3, V Praloran 1,2,3,5,*
PMCID: PMC3131863  PMID: 20671746

Abstract

Oxygen (O2) concentrations in bone marrow vary from 4% in capillaries to <0.1% in subendosteum, in which hematopoietic stem cells reside in specific niches. Culture at low O2 concentrations (3, 1 and 0.1%) influences hematopoietic stem and progenitor cells survival, proliferation and differentiation, depending on their level of differentiation. Culture of human CD34+ cells at low O2 concentrations (O2 ⩽3%) maintains stem cell engraftment potential better than at 20% O2 (NOD/Scid xenograft model). In contrast, progenitors disappear from cultures at/or <1% O2 concentrations. A very low O2 concentration (0.1%) induces CD34+ quiescence in G0. The exploration of molecules and mechanisms involved in hematopoietic stem and progenitor cells' quiescence and differentiation related to low O2 concentrations is unfeasible with primary CD34+ cells. Therefore, we performed it using murine hematopoietic nonleukemic factor-dependent cell Paterson (FDCP)-Mix progenitor cell line. The culture of the FDCP-Mix line at 0.1% O2 induced in parallel G0 quiescence and granulo-monocytic differentiation of most cells, whereas a minority of undifferentiated self-renewing cells remained in active cell cycle. Hypoxia also induced hypophosphorylation of pRb and increased the expression of p27KIP1, the two proteins that have a major role in the control of G0 and G1 to S-phase transition.

Keywords: hematopoiesis, hypoxia, cell cycle, quiescence, progenitors, p27

Physiological oxygen (O2) concentrations in tissues are much lower than in atmospheric air (20%), ranging from 14% in pulmonary alveoli to <1% in several organs.1 Numerous publications show the major role of variations of O2 concentration in the responses of normal cells, transformed cells and leukemic cells from primoculture or from continuous cell lines.2, 3, 4, 5, 6, 7, 8 Low O2 concentration induces apoptosis or cell survival, proliferation or quiescence, self-renewal or differentiation, depending on the degree of hypoxia, the cell type and the level of differentiation.

Response to hypoxia is largely dependent on hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor. At O2 concentrations <5%, the HIF-1α subunit is no longer degraded by the proteasome9, 10 and dimerizes with HIF-1β. The active complex then induces the expression of numerous genes including those of growth factors (erythropoietin (Epo) and vascular endothelial growth factor (VEGF)), transcription factors and cell cycle proteins.11, 12, 13, 14

In bone marrow, hematopoietic cells are permanently exposed to O2 concentrations ranging from 4% in capillaries to <0.1% in endosteal areas,15, 16, 17 in which the most primitive stem cells reside.18, 19 Hematopoietic stem cells (HSCs) are better preserved in cultures at low O2 (1–3%) than at atmospheric O2 concentration (20%).5, 20, 21 We previously showed that culture at 0.1% O2 prevents cord blood G0 CD34+ cells to enter in active cell cycle and induces those in active proliferation to re-enter in G0.22 The effects of low O2 concentration on the survival, proliferation and differentiation of normal and leukemic hematopoietic progenitors are more controversial. Indeed, in some articles a pro-differentiative effect6, 23, 24, 25 was pointed out, whereas in others it was suggested that oxygen levels <1% block cell proliferation and induce their rapid disappearance from cultures, probably by inducing apoptosis.4, 21, 26, 27

Culture at low O2 concentration (0.5%) causes cell cycle arrest in G1 of mouse embryonic fibroblasts and splenic B lymphocytes by inducing Rb hypophosphorylation and p27KIP1 upregulation.28, 29 All these results led us to explore the effects of low O2 concentration on the proliferation and differentiation of hematopoietic progenitors. Extensive protein studies using normal human or murine primoculture progenitor cells were quite unfeasible for two reasons: heterogeneity of the CD34+ cell population and difficulty of obtaining them in large numbers. As the murine cell line, factor-dependent cell Paterson (FDCP)-Mix, which is less heterogeneous, has major features of normal hematopoietic progenitors, we tested its response to hypoxia. Indeed, FDCP-Mix cells have a normal karyotype, are non-leukemic, strictly depend on IL-3 for their survival and proliferation, and differentiate toward different myeloid lineages when cultured with other cytokines.30 Varying the concentrations of O2 from 20 to 0.1% in cultures of FDCP-Mix cells had a time-dependent effect on their proliferation and cell cycle, with cells reaching quiescence (G0) after 72 h of culture at 0.1% O2. We then used these cells to analyze the self-renewal/proliferation/differentiation balance and the variations of p27KIP1 levels and pRb phosphorylation, the two proteins potentially involved in G0 cell cycle arrest.

Results

Very low O2 concentration inhibits FDCP-Mix cell growth

Low O2 concentration induced a time-dependent FDCP-Mix growth inhibition (Figure 1a). The number of living cells at 24 h was not significantly different in 20% O2 versus 0.1% O2 concentration cultures, nor was their doubling time (approximately 30 h in both conditions; Figure 1b). At later time points, the growth of FDCP-Mix cells cultured at 0.1% O2 was dramatically reduced, with a doubling time that reached 104 h after >48 h of culture. As the percentage of apoptotic cells in two conditions was similar (Figure 1c) after 24, 48 and 72 h of culture, we concluded that the growth arrest observed at 0.1% O2 was because of the inhibition of proliferation of FDCP-Mix cells after 24 h.

Figure 1.

Figure 1

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Influence of O2 concentration on the proliferation and apoptosis of FDCP-Mix cells. Cells were seeded at 1 × 105 cells/ml and cultured for 24, 48 and 72 h at 20, 3, 1 or 0.1% O2. (a) Living cell counts. (b) The cell doubling time was calculated as follows: t*ln2/ln (cf/ci) where t is time of culture in hours; ln is natural logarithm; and cf and ci are living cells concentrations at final and initial culture time points, respectively. (c) Percentage of apoptotic cells (annexin V-positive cells). Values are the mean±S.D. of five independent experiments (†P<0.05; Kruskal–Wallis test)

Very low O2 concentration (0.1%) induces FDCP-Mix cell arrest in G0

We then analyzed the FDCP-Mix cell cycle status after 24, 48 and 72 h of culture at 20 and 0.1% O2, using double labeling with propidium iodide (PI, a quantitative DNA fluorescent dye) and anti-Ki-67 antibodies (Ki-67 antigen is expressed in the nucleus of cycling cells and absent from G0 cells) to discriminate cells in G0 (DNA=2N; Ki-67−) from cells in G1 (DNA=2N; Ki-67+), S (2N < DNA <4N; Ki-67+) and G2/M (DNA=4N; Ki-67+). As shown in Figure 2a, the percentage of FDCP-Mix cells in G0 remained stable (13.6±2.3%) during 72 h of culture at 20% O2. In contrast, at 0.1% O2 the percentage of G0 cells significantly increased to 48.8±5.4% after 72 h of culture, whereas the percentage of cells in G1, S and G2/M decreased in parallel (Figure 2b–d). To determine whether this G0 phase accumulation was due to cells previously in G0 and G1 or because of cells entering in G0 after one or several divisions, FDCP-Mix cells labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE; a stable vital cellular fluorescent dye whose decay allows to analyze cell divisions) were cultured for 72 h at 20% O2 and at 0.1% O2. At this time point, division rates (CFSE labeling) and cell cycle status (Ki-67+ or Ki-67−) of cultured FSCP-Mix cells were co-analyzed by flow cytometry (FCM). As shown by the comparison of panels b and c in Figure 3, the division rate of FDCP-Mix cells was slower at 0.1% than at 20% O2. This is because of the association of two cell cycle events: (1) the extension of the cell cycle duration as concluded from the respective percentages of Ki-67+ cells in each division class and (2) the progressive accumulation of G0 cells after each division (revealed by comparing the percentages of Ki-67− cells in each division class at 20 and 0.1% O2).

Figure 2.

Figure 2

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Influence of O2 concentration on the cell cycle of FDCP-Mix cells. Cells were seeded at 1 × 105 cells/ml and cultured at 20% or 0.1% O2 for 24, 48 and 72 h. Double labeling with PI and Ki-67 antibody identifies cells (a) in G0 (DNA=2N; Ki-67−), (b) in G1 (DNA=2N; Ki-67+), (c) in S (2N< DNA <4N; Ki-67+) and (d) in G2/M (DNA=4N; Ki-67+). Values are the mean±S.D. of seven independent experiments (†P<0.05, *P<0.01; Friedman test)

Figure 3.

Figure 3

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Hypoxia (0.1% O2) reduces the number of cell divisions of FDCP-Mix cells. Dot plots of FDCP-Mix cells labeled with CFSE and anti Ki-67 antibody. Cells before culture (a) and after 72 h of culture at 20% (b) and 0.1% (c) O2. The Ki-67 antibody labeling discriminates quiescent cells (Ki-67−) from cycling cells (Ki-67+). Decreasing levels of CFSE fluorescence (from right to left) reflect the increasing number of cell divisions. One representative experiment out of three is shown

Very low O2 concentration (0.1%) induces the differentiation of a majority of FDCP-Mix cells

May-Grünwald/Giemsa staining of FDCP-Mix cytospins showed that after 72 h of culture at 0.1% O2, the majority of cells acquired a granulo-monocytic differentiation (Figure 4a) that was confirmed by FCM analysis of the expression of Gr-1 (granulocytic differentiation marker), Mac-1 (monocytic differentiation marker), as well as CD34 and CD117 (markers of primitive hematopoietic progenitors) cell surface markers (Figure 4b–d). Indeed, after 3 days of culture at 0.1% O2, the percentage of cells positive for Gr-1 and CD117, respectively, increased from 26 to 63% and decreased from 27 to 14% (Figure 4b and c). In parallel, as expected, we observed a decrease in the mean fluorescence intensity (MFI) of the CD34 primitive marker together with an increase in the MFI of the granulo-monocytic marker Mac-1 and Gr-1 (Figure 4d). These results confirmed that culture at 0.1% O2 induces the granulo-monocytic differentiation of the FDCP-Mix cell line.

Figure 4.

Figure 4

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Hypoxia (0.1% O2) increases granulo-monocytic differentiation of FDCP-mix cells. (a) After 72 h of culture at 20 and 0.1% O2, cells were harvested, cytospun and stained with May-Grünwald/Giemsa. (b) After 72 h of culture at 20 and 0.1% O2, cells were labeled with appropriate isotypic antibodies, anti-Gr-1, anti-Mac-1, anti-CD34 or anti-CD117-specific antibodies and analyzed by FCM. Histograms of one representative experiment are shown. (c) This table gives the mean±S.D. of the percentage of cells positive for the Gr-1, Mac-1, CD34 and CD117 markers after 3 days of culture at 20 and 0.1% O2. These percentages were calculated from three independent experiments. (d) Variation of the MFI of Gr-1-, Mac-1-, CD34- and CD117-positive cells after 72 h of culture at 20 or 0.1% O2. For each marker, the value represents the mean ratio±S.D. of the MFI measured at 0.1% O2/MFI measured at 20% O2 in three independent experiments

Very low O2 concentration (0.1%) also maintains a minor population of FDCP-Mix cells with culture-repopulating ability (CRA)

FDCP-Mix cells issued from 72 h of liquid culture (LC1) at 0.1% O2 were sequentially replated every 72 h at 1.105 cells/ml at 20% O2 to test their ability to repopulate later LCs (LC2–LC5), as determined by counting the total number of cells in LC2 to LC5 (CRAcell). The growth rate of cells incubated in LC1 at atmospheric O2 concentration remained stable throughout the experiment (Figure 5a, left part of the graph). The growth rate of cells issued from LC1 incubated at 0.1% O2 (Figure 5a, right part of the graph) in nonselective, normoxic LC2–LC5 (Figure 5a) remained stable during LC2 and LC3, increased during LC4 and reached the growth rate showed by cells incubated in normoxic LC1 during LC5. In the meantime, the percentage of cells in G0 decreased and the percentage of cells in G1, S and G2/M started to increase during LC2 to reach control levels at LC4 (data not shown). To confirm that this progressive repopulation of LC was because of the maintenance in hypoxia of a limited cell subpopulation, cells rescued from nonselective (normoxic) LC2–LC5 were cultured in semisolid medium to identify and count, after 11 days, the number of colonies issued from colony-forming cell (CFC). The percentage of these CRACFC cells at the end of 0.1% O2 LC1 was 14 times lower than after 20% LC1 and progressively returned to similar values in LC4 (Figure 5b).

Figure 5.

Figure 5

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Hypoxia (0.1%) maintains a minor population of FDCP-Mix cells with culture-repopulating ability (CRA). After 3 days of primary liquid culture (LC1) at 20 or 0.1% O2, cells were serially replated at 1 × 105 cells/ml at 20% O2 every 3 days (LC2–LC5). At the end of each LC (LC1– LC5), (a) the number of living cells (CRAcell) in liquid culture was counted and (b) their CFC content were revealed by the CFA assay that allowed to count the number of colonies after 11 days of culture at 20% in semisolid medium (CRACFC). Values are the mean±S.D. from four independent experiments. Statistical analysis of differences between similar LC at 20 and at 0.1% O2 was performed using Mann–Whitney test (†P<0.05, *P<0.01). Statistical analysis of differences between serial LCs in the 20 and 0.1% O2 group used the Friedman test (†P<0.05)

Relationships between G0 quiescence, Gr-1 expression and CRACFC cells

As the differentiation after 3 days of culture at 0.1% O2 was accompanied by the G0 entry of a large percentage of FDCP-Mix cells, we explored the relationship between these two phenomena. As a first step, we sorted Gr-1+ and Gr-1− cells after 3 days of LC1 at 20 or 0.1% O2. The microscopic observation on May-Grünwald/Giemsa cytospins (Figure 6a) confirmed that Gr-1+ cells had a typical morphology of terminally differentiated granulocytes including donut-shaped nuclei (arrows ◂), whereas Gr-1- cells kept round nuclei with more basophilic cytoplasm as observed in control culture conditions (Figure 4a). At both O2 concentrations, all CRAcell- and CRACFC-type cells were contained within the Gr-1− population (Figure 6b and c). Analysis of the cell cycle status of Gr-1+ and Gr-1− cells showed that CRA-type undifferentiated Gr-1 cells were mostly cycling, whereas differentiated Gr-1+ cells without CRA were in G0 (Figure 6d). In addition, a 24-h treatment of FDCP-Mix cells with 5FU (10 μg/ml) at the end of LC1, followed by medium renewal and replating in LC2, dramatically decreased CRAcell and CRACFC similarly in both O2 conditions (data not shown). Altogether, these data show that at very low 0.1% O2 concentration, most FDCP-Mix cells differentiate and enter in the quiescent G0 status, whereas a minority of undifferentiated cells, those that maintain the self-renewal of the cell line, remain in active cell cycle.

Figure 6.

Figure 6

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Gr-1+ and Gr-1− FDCP-Mix cells have different morphology, CRAcell and CRACFC capacities and cell cycle status. After 3 days of LC1 at 20 or 0.1% O2, cells were sorted according to their absence or presence of the Gr-1 antigen. (a) Gr-1− and Gr-1+ cells issued from 20 and 0.1% O2 LC1 were cytospun and stained with May-Grünwald/Giemsa and observed at × 600 magnification. Arrows (◂) indicate cells with donut-shaped nuclei characteristic of terminally differentiated murine granulocytes. (b) Gr-1− and Gr-1+ cells issued from 20 and 0.1% O2 LC1 were serially replated at 20% O2 every 3 days (LC2 and LC3) and living cells were counted. (c) Gr-1− and Gr-1+ cells issued from 20 and 0.1% O2 LC1 were plated in semisolid culture to measure their CFA. (d) Distribution of the percentage of G0 cells in the Gr-1− and Gr-1+ cell subpopulations issued from 20 and 0.1% O2 LC1. Values are the mean±S.D. of three independent experiments (*P<0.001; Mann–Whitney test)

FDCP-Mix cells in G0 overexpress hypophosphorylated pRb and p27KIP1 proteins

Because of their role in the regulation of cell cycling, we analyzed the total amount of p27KIP1 and pRb proteins and the phosphorylation status of pRb after 24, 48 and 72 h of culture at 20% O2 and 0.1%. O2. As expected, the total quantity of pRb protein remained stable and similar during 72 h culture at 20 and 0.1% O2 (Figure 7a). The S-phase transition is dependent on pRb phosphorylation on several specific sites. Therefore, we studied this protein phosphorylation status. Indeed, unphosphorylated pRb progressively increased whereas phosphorylated pRb disappeared during 72 h culture of FDCP-Mix cells at 0.1% O2 (data not shown). We then analyzed several pRb phosphorylation sites involved in the G1 to S-phase transition. The Ser 608, Ser 807/811 and Thr 821 sites (stably phosphorylated at 20% O2) became very faint at 0.1% O2 (Figure 7b). In the meantime, the p27KIP1 level in FDCP-Mix cells strongly increased during cultures at 0.1% O2, whereas it remained stable at 20% O2 (Figure 8a). Upregulation of the p27KIP1 protein by hypoxia in FDCP-Mix cells was partly related to transcriptional mechanisms as p27KIP1 mRNA level significantly increased during cultures at 0.1% O2 (2.76-, 3- and 2.4-fold at 24, 48 and 72 h, respectively; Figure 8b). To know whether the regulation of the p27KIP1 protein was linked to the G0 quiescence and differentiation of the FDCP-Mix cell line, we explored its expression in the Gr-1− and Gr-1+ populations by flow cytometry. Both the level of p27KIP1 fluorescence intensity (Figure 8c and d) and the percentages of cells expressing high levels of p27KIP1 were different in Gr-1− and Gr-1+ cells.

Figure 7.

Figure 7

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Time-dependent modifications of pRb during culture in hypoxia. After 24, 48 or 72 h of culture at 20% O2 or 0.1% O2, FDCP-Mix cell lysates were analyzed by western blot. (a) Time- and O2 concentration-dependent expression of pRb, and (b) time- and O2 concentration-dependent expression of phospho-Ser 608 pRb, phospho-Ser 807/811 pRb and phospho-Thr 821 pRb. Detection of β-actin was used as loading control

Figure 8.

Figure 8

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Increase in p27KIP1 during culture of FDCP-Mix cells at 0.1% O2 is related to their terminal granulocytic differentiation. (a) Time- and O2 concentration-dependent expression of p27KIP1 was analyzed by western blot. Detection of β-actin was used as loading control. (b) p27KIP1 mRNA level (arbitrary units) was quantified by RT-qPCR after 24, 48 and 72 h of culture at 20% O2 or at 0.1% O2. Results are the mean±S.D. of four independent experiments performed in duplicates (**P<0.005, *P<0.05; Mann–Whitney test). (c) Flow cytometric analysis of the p27KIP1 expression in Gr-1− and Gr-1+ populations. FDCP-Mix cells were double labeled (anti-Gr-1 and anti-p27KIP1 antibodies) after 3 days of culture at 20 and 0.1% O2. (d) Percentages of Gr-1− and Gr-1+ cells with high and low p27KIP1 expression after 3 days of culture at 20 and 0.1% O2. High and low p27KIP1 cells were defined as indicated on the flow cytometry histogram (left panel). The right panel graph shows the mean percentage±S.D. of four independent experiments

Discussion

In the past 10 years, an increasing number of publications were devoted to the physiological low bone marrow O2 concentrations (from <0.1% O2 in sub-endosteal bone marrow niches to 4–5% in vascular areas compared with 21% in atmospheric air) and to the exploration of their role in the regulation of hematopoiesis.1, 15, 16 We and others showed that varying O2 concentrations (from 20 to 0.1%) in cultures of hematopoietic stem and progenitor cells modified their quiescence versus proliferation and self-renewal versus differentiation balances.2, 4, 5, 6, 7, 19, 20, 21, 22, 23, 31, 32, 33 To investigate the molecules and mechanisms involved in these phenomena, we used the FDCP-Mix hematopoietic cell line that is considered to share numerous phenotypic and functional properties with bone marrow hematopoietic progenitors. Indeed, FDCP-Mix cells have a normal karyotype, are non-leukemic, strictly depend on IL-3 for their survival and proliferation, and differentiate towards different myeloid lineages when cultured with other cytokines.30 In addition, when cultured at 0.1% O2, cycling FDCP-Mix cell line progressively accumulated in G0 after one to few divisions.

This accumulation in G0 was accompanied by a major granulo-monocytic differentiation, as shown by sorting Gr-1+ (differentiated) and Gr-1− (undifferentiated) FDCP-Mix cells after 3 days of culture at 20 and 0.1% O2. In both O2 conditions, the Gr-1− FDCP-Mix subset had a high CRA and a high percentage of cycling cells, whereas the Gr-1+ differentiated fraction was mostly in G0 with a quite null CRA. This result suggests a link between cell cycle arrest and differentiation.

Some articles found a differentiating effect of low O2 concentrations on leukemic cells, either from primoculture or from cell lines6, 23, 24, 25 similar to the one observed with FDCP-Mix cells. In contrast, Giuntoli et al.8 showed that when the K562 cell line (issued from a blast-crisis chronic myeloid leukemia – an HSC malignancy – patient) was cultured at 0.1% O2 for 7 days, the number of cells dramatically decreased (by 1 log) and most cells underwent apoptosis, whereas a minor hypoxia-resistant subpopulation repopulated LC2 incubated at 20% O2, showing full CRA maintenance. Using the murine MEL cell line, they obtained similar results, and found, in addition, at 0.1% O2 a quiescent subset of CRA cells that resisted to 5FU treatment.4 However, in these two articles, cell differentiation was not studied. When the FDCP-Mix cell line was cultured under similar conditions, apoptosis did not increase significantly, and most cells underwent proliferation arrest and differentiated, whereas a small population of slowly cycling CRA cells (as killed by 24 h of 5FU) persisted after 3 and even 7 days of culture at 0.1% O2. These differences between FDCP-Mix (and the leukemic cell lines that differentiate in hypoxia) on one hand and K562, MEL and CD34+ cells on the other could be due (1) to the fact that FDCP-Mix and the leukemic cell lines that differentiate in hypoxia6, 23, 24, 25 derive from progenitors and not from HSC, (2) to their lower sensitivity to apoptosis than primary CD34+ cells in hypoxic cultures owing to their immortalized status (but this does not apply to the K562 and MEL cell lines) or (3) to an association of both phenomena.

Our previous results showing that primoculture progenitors disappear rapidly during cultures of murine bone marrow or human CD34+ cells at 1% O221, 27 whereas leukemic and FDCP-Mix cell lines survive and differentiate strengthen the hypothesis that the maturation of FDCP-Mix cells in hypoxia is linked to their acquired immortalized status. This allows their prolonged survival as differentiated cells in hypoxia, whereas primoculture progenitors rapidly die in these conditions before reaching maturation.

The permanent cycling of CRA type and the absence of apoptosis in cultures of FDCP-Mix cells at very low O2 concentration that contrasts with the apoptosis of a large percentage and the quiescence of a minority of primoculture CD34+ and MEL cells cannot be explained by the immortalized status of FDCP-Mix, as MEL is also a permanent (and leukemic) cell line. We suggest that this major functional difference in response to very low O2 concentrations is because of the ‘stem' or ‘progenitor' cell status of these different cell types. Together, the above-mentioned results with primary CD34+ cells, K562 cells, other leukemic cell lines and our present work imply that regulation by very low O2 concentrations (<1%) of cycling, differentiation and survival, depending on the differentiation level of cells (stem cells versus progenitors) whether normal or leukemic tissue, is in question.

These results led us to explore the molecules involved in the cell cycle modifications induced by hypoxia. The G0 and/or G1 (undefined as neither Ki-67 nor any other G0 marker was used) quiescence of fibroblasts and B lymphocytes induced by serum starvation or hypoxia is linked to p27KIP1 increase that blocks the cyclin/cyclin-dependent kinase (CDK) activity, thus leading to pRb hypophosphorylation.11, 28, 29, 34, 35, 36, 37 Conversely, phosphorylations of pRb on multiple Ser/Thr sites by cyclin/CDK during the G1 phase lead to the release of molecules required for the transition to S phase.38 In brief, the phosphorylation of Thr 821 releases histone deacetylase; the phosphorylation of Ser 807/811 releases c-abl and the phosphorylation of Ser 608 participates in the release of the master transcription factor E2F. These three molecules have a major role in the G1 to S transition. Hypoxia dramatically reduced the phosphorylation of these three pRb sites in FDCP-Mix cells, suggesting that dephosphorylated pRb participate to their quiescence. Hypoxia also induced a significant increase in the p27KIP1 protein and mRNA in FDCP-Mix cells. Finally, we showed that the quiescent differentiated Gr-1+ cells expressed higher p27KIP1 levels than the Gr-1 cycling cells, a fraction containing CRA cells. That is most probably responsible for the accumulation of unphosphorylated pRb. Our present data that extend personal preliminary results39 are similar to those very recently published by Eliasson et al.40 Altogether, they show that p27KIP1 is involved in the regulation of FDCP-Mix cell cycling in hypoxia.

The last important point of our work is that it revealed a minor stem cell-like subpopulation (that repopulates LCs after returning them to 20% O2) of the FDCP-Mix cell line that kept its self-renewal potential after 3–7 days of culture at 0.1% O2. Similar to both normal stem cells and cancer cells, the CRA-type FDCP-Mix cells are highly O2 independent, as they survive and proliferate in nearly anoxic conditions (0.1% O2). Further experiments will explore: (1) the metabolic peculiarities of the low-energy proliferation mechanism of Gr-1− FDCP-Mix cells, (2) the relationship of this mechanism to self-renewal divisions uncoupled to differentiation and (3) the roles of HIF-1, p27KIP1 and pRb in the regulation of balance between quiescence and differentiation of FDCP-Mix.

Materials and Methods

Maintenance of the FDCP-Mix cell line in culture

The FDCP-Mix cells (kindly provided by G Mouchiroud; Lyon, France) were routinely replated at 5 × 104 cells/ml every 3 days in Iscove's modified Dulbecco's medium (IMDM, Invitrogen, Carlsbad, CA, USA) complemented with penicillin (50 units/ml), streptomycin (50 μg/ml), 20% horse serum (HS; Invitrogen), and 10% WEHI conditioned medium (CM) as a source of IL-3. All cultures were carried out at 37°C in a humidified 5% CO2 incubator (Binder, Tuttlingen, Germany). For experiments, cells in exponential growth at day 3 of culture were centrifuged, washed and resuspended in complete medium containing WEHI CM as required.

Cell proliferation, viability and apoptosis in ambient and low O2 concentration

FDCP-Mix cells were seeded at 1 × 105 cells/ml and cultured for 24, 48 and 72 h in the same culture medium, either at 20% O2 in a humidified 5% CO2 incubator or at 0.1% O2 concentration in an O2/CO2 incubator (Xvivo, BioSpherix, Redfield, NY, USA). After culture, viable cells were counted on Malassez Cell with Trypan Blue exclusion. Apoptosis was measured using the Annexin-V-FITC Kit (PN IM3546; Beckman Coulter, Fullerton, CA, USA) following the manufacturer's protocol. Samples were analyzed by FCM with Becton Dickinson FACSCanto II and FACSDiva software (San Jose, CA, USA).

Analysis of cell cycle and divisions by FCM

Cell cycle was analyzed at 24, 48 and 72 h of culture at 20% O2 and at 0.1% O2. To distinguish G0, G1, S and G2/M cell cycle phases, cells were double labeled with PI (quantitative DNA labeling) and Ki-67 antibody (absent only in G0 phase cells). In brief, cells washed in PBS were incubated for 30 min at room temperature (RT) in a PFA/saponin solution (0.4% PFA, 10 mM HEPES, 0.02% saponin and H2O). Cells were washed two times in ‘washing buffer' (5% FBS, 0.05% sodium azide, 55 mg/l EDTA and 0.02% saponin) and incubated for 30 min at RT with an anti-Ki-67 antibody or isotype (Alexa Fluor 647 conjugated anti-Ki-67 no. 558615, and Alexa Fluor 647 conjugated isotype no. 557732; BD Pharmingen, San Diego, CA, USA). Washed cells were resuspended in PBS supplemented with PI (20 μg/ml) to quantitatively label DNA. Double-labeled cells were then analyzed by FCM using a Becton Dickinson FACSCanto II and FACSDiva software. To know whether G0 cells were issued from cells previously in G0 and G1 phases or from cells in S, G2 and M phases that divided before entering in G0, cells were labeled with CFSE (a stable vital cellular fluorescent dye whose decay allows to analyze cell divisions; no. C34554, Invitrogen) according to the manufacturer's instructions. In brief, 1 × 106 FDCP-Mix cells were incubated in the dark for 15 min at 37°C in 1 ml of pre-warmed PBS + 1% BSA and 10 μM CFSE. Cells were then washed in FDCP-Mix culture medium and incubated for 30 min at 37°C in the dark. After a last wash, cells were resuspended in complete culture medium (3 × 105 cells/ml) and incubated (37°C, 20% O2, 5% CO2) for 24 h to eliminate dead cells and use cell populations that are able to proliferate. Cell aliquots were then cultured either at 20 or 0.1% O2 for 24, 48 or 72 h before being labeled with anti Ki-67 and PI as described and analyzed by FCM.

Cytospin and May-Grünwald/Giemsa staining

1.5 × 105 cells resuspended in 50 μl of HS were cytospun at 300 r.p.m. for 20 min using a cytocentrifuge (Shandon CytoSpin III; GMI, Ramsey, MN, USA), and dried and stained with May-Grünwald before microscopical examination at × 100 and × 600 magnification (AX70, Olympus, Tokyo, Japan).

Analysis of cell surface markers and cell sorting

Cells cultured at 20% O2 and at 0.1% O2 were washed with washing buffer, incubated for 30 min with fluorescein isothiocyanate- or phycoerythrin-conjugated isoptype, anti Mac-1, Gr-1, CD34 and CD117 antibodies (respectively, no. 22225033, no. 22159113, no. 22155243, no. 22150344 and no. 22151173; Immunotools, Friesoythe, Germany). Washed cells were then analyzed by FCM using a Becton Dickinson FACSCanto II and FACSDiva software or sorted according to their Gr-1 expression using a Becton Dickinson FACSAria.

Analysis of p27KIP1 expression in Gr-1− and Gr-1+ cells

Cells were double labeled first with anti-Gr-1 (as described in the previous paragraph) and then with anti-p27KIP1 antibody according mostly to the procedure described for cell cycle analysis. In brief, cells were incubated for 30 min with biotinylated anti-p27KIP1 antibody (diluted 1 : 300, no. MS-256-B; Thermo Scientific, Rockford, IL, USA) instead of anti-Ki-67. Cells were then washed twice with washing buffer before a final 20-min incubation with streptavidin-PE (diluted 1 : 1000, no. IM0557; Beckman Coulter).

CRA assay

After 72 h of LC at 20% or 0.1% O2 (LC1), cells were centrifuged for 5 min at 300 g at RT and transferred at 1 × 105 cells/ml in fresh medium into secondary cultures always incubated at 20% O2, irrespective of whether LC1 had been incubated at 20% or 0.1% O2 (nonselective LC2). Cells were then further replated every 3 days and always incubated at 20% O2 (LC3–LC5). At the end of each LC, cell aliquots were harvested for counting after Trypan Blue exclusion (CRAcell) and colony-forming ability (CFA) assay (CRACFC).

The CRA assay estimates the maintenance of the stem cell subset contained within a cell population that has undergone a treatment (e.g., incubation in hypoxia) in a selective LC1 by means of cell transfer into nonselective (e.g., normoxic) LC2 and a further incubation therein. LC2 repopulation is then measured by counting the total number of cells (CRAcell) or the number of clonogenic (CFC) cells (CRACFC) using CFA assay (see below). In normal hematopoietic cell populations, the standard CRA assay has been shown to detect short-term repopulating hematopoietic stem cells.41

CFA assay

1 × 103 FDCP-Mix cells were seeded in 250 μl of methyl-cellulose (Methocult, no. H4230; Stemcell Technologies, Vancouver, Canada) with 10 ng/ml of mIL3 (no. 213–13; Peprotech, Rocky Hill, NJ, USA) in duplicate. Culture dishes were incubated for 11 days at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 before counting the number of colonies (aggregates >50 cells).

Western blot analysis

Cells cultured at 20% O2 and at 0.1% O2 were washed in PBS and lysed in loading buffer (2% SDS, 10% glycerol, 50 mM Tris pH 7.8). Protein concentration was determined using the BCA Pierce assay (Thermo Scientific). Then, 40 μg of whole protein cell extract was resolved in SDS-PAGE and transferred onto a PVDF Membrane (no. RPN303F; GE Healthcare, Uppsala, Sweden) to probe for pRb and p27 proteins, respectively. The membrane was saturated with nonfat milk (5%) in TBS-T (10 mM Tris pH 7.8, 150 mM NaCl and Tween 0.1%) and then incubated overnight at 4°C with specific antibodies to phospho-Ser 608 pRb, phospho-Ser 807/811 pRb, p27KIP1 (respectively, no. 2181, no. 9308 and no. 2552; Cell Signaling Technology, Beverly, MA, USA), pRb (no 554136, BD Pharmingen) or phospho-Thr 821 pRb (no. 44–582, Invitrogen) diluted as indicated by the manufacturer. After three washes in TBS-T, membranes were incubated for 1 h with anti-rabbit or anti-mouse horseradish peroxydase-conjugated antibodies. The bands were visualized using the ECL system (no. WBKLS; Millipore, Billerica, MA, USA). The membrane was washed again, saturated, stained with an anti-β-actin antibody (no. A5316; Sigma, St Louis, MO, USA) and finally revealed as described above.

Reverse transcription and quantitative PCR

Cells cultured at 20% O2 and at 0.1% O2 were washed in PBS and treated with 250 μl Trizol (no. 15596–026; Invitrogen). Total RNA extraction, DNAse treatment (no. M6101; Promega, Madison, WI, USA) and reverse transcription with M-MLV Reverse Transcriptase (no. M1705, Promega) were realized according to the manufacturer's instructions. Samples were run three times in duplicates using the brilliant SYBR Green QPCR (no. 600548; Stratagene, La Jolla, CA, USA) in the MX4000 apparatus (Stratagene) according to the manufacturer's instruction. The real-time PCR reactions were carried out in the presence of mouse p27KIP1 gene-specific oligonucleotide primers (forward primer: 5′-AGGCGGTGCCTTTAATTGGG-3′, reverse primer: 5′-TTACGTCTGGCGTCGAAGGC-3′) or 36B4 gene-specific oligonucleotide primers (forward primer: 5′-GCTTCATTGTGGGAGCAGAC-3′ and reverse primer: 5′-CATGGTGTTCTTGCCCATCAG-3′) as follows: 10 min activation at 95°C, followed by 40 cycles at 95°C for 30 s, 58°C for 1 min and 72°C for 1 min.

A relative quantification of p27KIP1 transcript expression was obtained by standardizing to 36B4 levels that was shown to be stable in hypoxia.42 The final value was calculated by taking an average of relative transcript copy numbers amplified from the three sets of PCR reaction performed in duplicates.

Statistics

All data are presented as mean±S.D. of 3–7 independents experiments. The Friedman or Kruskal–Wallis tests were, respectively, used to compare >2 experimental conditions paired or not. For statistical analysis of differences between two not paired experimental conditions, Mann–Whitney test was used.

Acknowledgments

This work was funded by grants from la ligue française contre le cancer (comité sud-ouest) and association Laurette Fugain. AG, CD and FH received a doctoral fellowship from the French ministère de l'Education Nationale, de la Recherche et de la Technologie. The language correction of this paper by Mrs Elisabeth Volkmann is highly appreciated.

Glossary

O2

oxygen

HIF-1

hypoxia-inducible factor-1

Epo

erythropoietin

VEGF

vascular endothelial growth factor

HSC

hematopoietic stem cell

PI

propidium iodide

CFSE

carboxyfluorescein diacetate succinimidyl diester

MFI

mean fluorescence intensity

FDCP

factor-dependent cell Paterson

FCM

flow cytometry

CRA

culture-repopulating ability

CFC

colony-forming cell

LC

liquid culture

KIP

kinase inhibitor protein

CDK

cyclin-dependent kinase

IMDM

Iscove's modified Dulbecco's medium

CM

conditioned medium

HS

horse serum

RT

room temperature

CFA

colony-forming ability

The authors declare no conflict of interest.

Footnotes

Edited by RA Knight

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