Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells

Abstract

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.

FIG. 1.

Expression of PES1 and UBF1 in 32D-derived cells. 32D, 32D IGF-IR, 32D IGF-IR/IRS-1, and 32D IRS-1 cells were infected with retroviruses expressing either PES1 or UBF1 as described in Materials and Methods. Mixed populations were collected, lysates were made, and Western blots were developed with an antibody to the FLAG epitope. Parental 32D cells were used as negative controls for the FLAG antibody. The positions of ectopic PES1 (A) and UBF1 (B) are indicated by arrows.

FIG. 2.

Growth curves for 32D-derived cell lines. The growth of the cell lines in either IGF-I (50 ng/ml) or IL-3 was determined. Open bars indicate parental cells, either 32D IGF-IR cells (upper panels) or 32D IGF-IR/IRS-1 cells (lower panels). The number of cells was counted at 24, 48, and 96 h after shifting to IGF-I or replating in IL-3. The numbers on the ordinate represent the percent increase over the number of plated cells (e.g., 300 indicates a threefold increase). Note the different ordinate scales for 32D IGF-IR and 32D IGF-IR/IRS-1 cells. We omitted an analysis of parental 32D cells, which grow very well in IL-3 and die quickly after a shift to IGF-I. Error bars indicate standard deviations.

FIG. 3.

Cell cycle analysis of 32D-derived cells. 32D IGF-IR and 32D IGF-IR/IRS-1 cells and the same cell lines expressing exogenous PES1 or UBF1 were grown in IGF-1 (24 h after a shift from IL-3). (A) FACS analysis of six cell lines for cell cycle distribution. Under these conditions, all cell lines grew exponentially. In PES1-expressing cells, there was a marked increase in the fraction of cells in the G₂ peak and a decrease in the fraction of cells with an S-phase amount of DNA (see the text). These experiments were repeated four times up to 6 days after a shift from IL-3 to IGF-I, with similar results (the differences in IL-3 were more modest). (B) Percentage of cells labeled by a 1-h exposure to BrdU. Error bars indicate standard deviations; values at right indicate means and standard deviations. Statistically significant differences are marked with an asterisk (comparison between each parental cell line and its PES1- and UBF1-expressing counterparts by a t test). All cell lines expressing PES1 showed a decrease in the percentage of BrdU-labeled cells.

FIG. 4.

Cell size of 32D-derived cells expressing UBF1 or PES1. All cell lines were grown in IL-3. (A) Representative data from FACS analysis for cell size (angle scattering) only for 32D cells, either parental or expressing either UBF1 or PES1. (B) Similar analysis for other 32D-derived cells. Cell size was expressed in arbitrary units; error bars indicate standard deviations. The asterisks indicate significant differences (P < 0.05) between the parental cell lines and the cell lines expressing ectopic UBF1 or PES1, as determined by a t test. (C) Actual means and standard deviations for cell size. Overexpression of UBF1 and PES1 increased cell size for all three cell lines, although the increase was more dramatic for parental 32D cells (see the text). These experiments were done with cells in IL-3, but the same results were obtained with cells in IGF-I, at either 2 or 6 days after a shift from IL-3 to IGF-I.

FIG. 5.

Differentiation of 32D-derived cells into granulocytes. The cells examined were 32D IGF-IR cells, 32D IGF-IR cells expressing PES1, 32D IGF-IR cells expressing UBF1, and 32D IGF-IR/IRS-1 cells. The cells were stained with Giemsa stain 6 days after a shift from IL-3 to IGF-I. All images were taken at the same magnification, ×40. The arrows in panels A, B, and C indicate differentiating cells (bilobed, irregular nuclei and some well-differentiated granulocytes). The 32D IGF-IR/IRS-1 cells in panel D have round nuclei, and one of them (vertical arrow) is actually in mitosis. Note the difference in size between parental 32D IGF-IR cells (B) and the same cells expressing PES1 (C), UBF1 (A), or IRS-1 (D).

FIG. 6.

Expression of MPO and 24p3 mRNAs and of ID2 protein in 32D-derived cells. (Upper panel) Northern blots of mRNAs for two markers of differentiation, 24p3 and MPO. Times, cell lines, and treatments are indicated above the lanes. Only IRS-1 inhibited the appearance of these markers in 32D IGF-IR cells. The bottom section gives the mRNA amount in each lane. (Lower panels) Western blots showing the expression of the ID2 protein, which was markedly increased in 32D IGF-IR cells, in which differentiation was inhibited by IRS-1 (2, 47). Only IRS-1 was capable of inducing ID2 expression. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

FIG. 7.

PES1 localizes to the nuclei and nucleoli of cells. (A) Confocal microscopy of cells stained with propidium iodide (PI) (red) and counterstained with an anti-FLAG antibody (green). The merged image shows the localization of PES1 (FLAG tagged) within the nuclei and nucleoli of infected cells. (B and C) Interactions among IRS-1, PES1, and UBF1. (B) Lysates from various cell lines were immunoprecipitated (IP) with an anti-FLAG antibody, and the Western blots (WB) were successively developed with antibodies to either IRS-1 or FLAG. FLAG-tagged PES1 was detectable in both cells lines expressing it, but IRS-1 was visible only in 32D IGF-IR/IRS-1 cells. (C) Lysates from cells expressing either FLAG-tagged UBF1 (first two lanes) or FLAG-tagged PES1 (last two lanes) were immunoprecipitated with an anti-FLAG antibody, and the Western blot was developed with an antibody to UBF1.

FIG. 8.

Expression of endogenous PES1 in 32D-derived cells. (A) 32D IGF-IR and 32D IGF-IR/IRS-1 cells were shifted from IL-3 to IGF-I, and the levels of endogenous PES1 expression in whole-cell lysates were measured. The time after the shift is indicated above the lanes in hours. PES1 expression persisted in transformed 32D IGF-IR/IRS-1 cells but not in differentiating 32D IGF-IR cells. (B) Expression of exogenous UBF1 and PES1 in differentiated 32D-derived cells. The cell lines examined were 32D IGF-IR cells overexpressing either PES1 or UBF1 3 days after a shift to IGF-1. Lanes IL-3 contained the same cells at zero time. Western blotting was done with antibodies to PES1 or UBF1. An anti-Grb2 antibody was used to monitor protein amounts.

FIG. 9.

PES1 and UBF1 induce the transformation of normal MEFs. The cells examined were parental R12 and R508 cells, which are 3T3-like MEFs incapable of forming colonies in soft agar (see the text). Both cell lines were infected with retroviruses expressing either PES1 or UBF1, and mixed populations were collected. (A) Parental and retrovirus-infected cells were tested for their ability to form colonies in soft agar. The ordinate shows the number of colonies after 3 weeks. The colonies counted measured at least 125 μm. Error bars indicate standard deviations. (B) Western blotting of lysates from infected cells expressing FLAG-tagged proteins.