Up-Regulation of Transferrin Receptor Gene Expression by Granulocyte Colony-Stimulating Factor in Human Myeloid Leukemia Cells1

[CANCER RESEARCH 50, 7955-7961, December 15, 1990] Up-Regulation of Transferrin Receptor Gene Expression by Granulocyte Colony-stimulating Factor in Human Myeloid Leukemia Cells1

Yoshihisa Morishita,2 Takae Kataoka, Masayuki Towatari, Takahiko Ito, Hideo Inoue, Michinori Ogura, Yasuo Morishima, and Hidehiko Saito First Department of Internal Medicine, Nagoya University School of Medicine [Y. M., T. K., M. T., T. I., Y. M., H. S.], Showa-ku, Nagoya 466; Department of Hematology and Chemotherapy, Aichi Cancer Center Hospital [M. O.], Chikusa-ku, Nagoya 464; and Kirin Brewery Co. [H. l.J, Shibuya-ku, Tokyo ISO; Aichi Blood Disease Research Foundation [H. S.], Moriyama-ku, Nagoya 463, Japan

ABSTRACT TfR is expressed in the monocytic differentiation to macro phage (13, 14), or in the reticulocytes which require iron for Granulocyte colony-stimulating factor (G-CSF) enhanced surface hemoglobin synthesis, and in the syncytial trophoblasts of transfcrrin receptor (TfR) expression in two human myeloid leukemia placentas which transport iron to the developing fetus (15). cell lines, NKM-1 and NOMO-1, which possess G-CSF receptors. Conversely, the total number of TfRs declines when cells are Radioligand-binding assay revealed that 10 ng/ml G-CSF significantly induced to differentiate and stop proliferating (16-18). increased TfR to 186 Â±20 and 276 Â±38% of control for NKM-1 cells and NOMO-1 cells, respectively, in a 24-h culture. Scali-hard analysis Despite the circumstantial evidence suggesting that myeloid showed the increase of transferrin(Tf)-binding sites but no change in the leukemia cells proliferate in the presence of various cytokines receptor affinity. The enhanced TfR expression was not mediated either (19-21), little is known of events that probably occur in the by the kinetic change of receptor cycling or by cellular iron content. process of proliferation. Among the cytokines that may support Immunoprecipitationwith anti-TfR antibody was used, and the increased the leukemia cell self-renewal, G-CSF is of particular interest, biosynthesis of the receptor was demonstratedin G-CSF-stimulated cells. because G-CSF has been proved to stimulate myeloid leukemia Northern blot analysis showed a 2- to 3-fold increase of TfR mRNA of cells and increase blast colony formation (22), while it has NKM-1 cells cultured in medium containing Tf and G-CSF, whereas the induced the terminal differentiation and suppressed self-re mRNA declined without G-CSF. The effect of G-CSF on the TfR mRNA newal capacity of murine WEHI-3B D+ cells (23) and human was observed within 2 h, which preceded the increase of surface TfR and HL-60 cells (24). In this context, the present study was designed the transition to the S phase of the cell cycle. G-CSF also potentiated TfR expression in freshly obtained myeloid leukemia cells. to evaluate the mechanism underlying the stimulative effect of The present study shows up-regulation of TfR expression by G-CSF G-CSF on human myeloid leukemia cells. We have demon in myeloid leukemia cells and provides evidence that the regulation is strated that G-CSF causes an induction of the Tf-binding ca mediated by controlling the steady-state level of the mRNA. pacity. This induction occurred only in the myeloid leukemia cells which were stimulated by G-CSF. The regulation of Tf receptor expression was not mediated by the redistribution of INTRODUCTION the receptor pool but by the elevation of steady-state levels of the Tf receptor mRNA. Eukaryocytic cells acquire iron through the endocytosis of diferric Tf3 bound to its high-affinity cell surface receptor (TfR) (1). Subsequently, receptor-ligand complexes are internalized MATERIALS AND METHODS and incorporated into an acidic compartment in which iron is released for storage as an iron-ferritin complex, and TfRs Cell Lines. Four myeloid cell lines, HL-60, NKM-1 (25), NOMO-1 (26), and NCO-2 (27), and a megakaryoblastic leukemia cell line, MEG- recycle back to the plasma membrane where apotransferrin is 01 (28), were used in this study. The HL-60 cell line was a gift of Dr. released from the cell (2). In vivo, a limited number of cells and B. Dupont (Memorial Sloan-Kettering Cancer Center, New York, NY) tissues express Tf receptors, but they are displayed by cultured and has been maintained in our laboratory for more than 3 years. The human hematopoietic cells (3) and human leukemia cells (4, 5). last four lines were established in our laboratory. NKM-1 cells were The interaction between Tf and its receptor plays a crucial role derived from a patient with acute myeloblastic leukemia (FAB classifi in the cellular metabolism related to growth since iron is cation M2), and NOMO-1 cells were from a patient with acute mono essential for many biological processes, particularly in control cytic leukemia (FAB classification M5a). Ph'-positive NCO-2 cells were ling cell proliferation (6, 7). A separate transferrin-independent established from a patient with myeloid crisis of chronic myelogenous leukemia. A binding analysis with '"I-labeled 'Tyr-'Tyr-G-CSF was pathway has also been proposed (8,9). It may be closely coupled to the other TfR signal for the cell activation that is independent used, and NKM-1 cells and NOMO-1 cells were found to have G-CSF receptors. The radiolabeling of G-CSF was greatly enhanced by the of the ability to deliver iron to the cell (10, 11). Normal resting presence of tyrosine residues at NH2 terminus and the specific activity peripheral lymphocytes cannot be shown to bind Tf, while was not altered by this engineering. NKM-1 cells expressed 60 binding mitogen-stimulated lymphocytes display a marked increase in sites/cell with a Adof 100 p\i and Scatchard analysis of the data yielded the binding sites (12). It has also been demonstrated that the a straight line, indicating a single class of binding sites (29). NOMO-1 cells also expressed 580 binding sites/cell with a KÂ¿of140 pM. Recep Received 6/11/90; accepted 9/17/90. The costs of publication of this article were defrayed in part by the payment tors for G-CSF were not found in HL-60 cells, NCO-2 cells, and MEG- of page charges. This article musi therefore be hereby marked advertisement in 01 cells, although the HL-60 cell line used in trlis study may be a accordance with 18 U.S.C. Section 1734 solely to indicate this fact. receptor-negative variant or may lose the receptors during continuous ' This work was supported in part by Grants-in-Aid for Scientific Research culture. The NKM-1 and NOMO-1 cells have been grown in RPM1 from the Ministry of Education, Science and Culture and a Grant-in-Aid from the Ministry of Health and Welfare in Japan. 1640 supplemented with 10% fetal calf serum without any growth 2To whom correspondence should be addressed. factor for more than 2 years. They retained the myeloid characteristics 3The abbreviations used are: Tf, transferrin; G-CSF, granulocyte colony- and the karyotypes of the patients' neoplastic cells. stimulating factor; TfR, transferrin receptor; FAB, French-American-British; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; BSA, bo Leukemia Cells. Leukemia cells were obtained from peripheral blood vine serum albumin; PI, propidium iodide; SDS, sodium dodecyl sulfate; AMI. of four patients (Y. N., R. S., M. S., and R. G.) with informed consent. acute myeloid leukemia; ALL, acute lymphoblastic leukemia. Cells were separated by Ficoll-Conray density gradient centrifugation. 7955

Leukemia cells made up >95% of all samples. 100 nmol/liter of a mixture of labeled and unlabeled Tf. The cells were Biological Reagents. Recombinant human G-CSF (KRN 8601) was washed with a cold medium and resuspended at 5 x 106/ml in RPMI produced at Kirin Brewery Co. (Tokyo, Japan) by transforming Esche- 1640 medium containing 0.1 mg/ml BSA and 500 n\i unlabeled Tf. richia coli with a cloned G-CSF complementary DNA. Human trans- They were then rewarmed to 37Â°Cand, at indicated times, a 100-^1 ferrin was purchased from Sigma Chemical Co. (St. Louis, MO) and aliquot was removed and spun over oil to determine residual cell- saturated with ferric chloride as previously described (30). As an anti- associated Tf. transferrin receptor antibody, OKT9 (Ortho Diagnostic System Inc., Cellular Ferritin Levels. Cells ( I x 10") in 1 ml distilled water were Raritan, NJ) was used. Monoclonal antibody 4E (31) recognizes HLA lysed by freezing and thawing three times on ethanol/dry ice. The cell- class I (mainly B locus) molecules. 9E8 (32), which recognizes the free supernatant was recovered by ultracentrifugation at 20,000 x g for murine leukemia virus antigen plS(E), was used as a negative control. 30 min at 4Â°Cand assayed for ferritin by radioimmunoassay using an Cell Cultures. Cells were cultivated in the microplates at a concen assay kit (Baxter Healthcare Corp., Cambridge, MA). tration of 3 x 10s ml in 100 >ilof either RPMI 1640 medium alone or Northern Blot Hybridization. Total cellular RNA was extracted by the medium containing various concentrations of iron-saturated trans the guanidine isocyanate method (38). Ten ^g RNA was electropho- ferrin. The viable cells were counted by the trypan blue exclusion resed in 1% agarose gel containing formaldehyde and transferred onto method or quantitated by a colorimetrie assay using a tetrazolium salt, nitrocellulose membranes (Nitroplus 2000; Micron Separations Inc., MTT, as described previously (33). Because the results with the MTT Westbord, MA). Hybridization was performed at 42Â°Cusing32P-labeled assay were consistently equivalent with the viable cell counts, data probes. The membrane was washed three times in 2x standard saline presented in this paper were obtained using the MTT assay. The plates citrate and 0.2% SDS for 30 min and in 0.2x standard saline citrate were read on a scanning mult iwell spectrophotometer (SLT 210, SLT- and 0.2% SDS for 60 min at 42Â°C.The probes used in this study were LAB Instruments, Salzburg, Austria) using a test wavelength of 545 a 1.7-kilobase Â£coRIfragment from the human transferrin receptor nm and a reference wavelength of 650 nm. The results were expressed complementary DNA clone, pTR43 (39) (provided by Dr. N. K. Spurr, as a fraction of absorbance (A x IO3 of samples â€”A x IO3 that of Imperial Cancer Research Fund, London, England) and a 1.4-kilobase background). HLA-B7 probe, pDPOOl (kindly provided by Dr. Lloyd J. Old, Me |3H|Thymidine Incorporation. Short-term proliferation was deter morial Sloan-Kettering Cancer Center, New York, NY). mined by measuring [3H]thymidine uptake (34) with cells seeded at 3 Immunoprecipitation of Metabolically Labeled Tf Receptors. Immu- x 10!/ml in 96-weIl microplates. After the indicated time of incubation noprecipitation was performed as described before (25) . Briefly, cells at 37'C, 0.2 nC\ of [3H]thymidine (6.7 Ci/mmol, NEN Research (1 x IO6)were pulse labeled with 200//Ci of [35S]methionine(Amersham Products, Boston, MA) was added and harvested 4 h later. Corp. Arlington Heights, IL) for 60 min and solubilized in 300 n\ of Cell Cycle Analysis. Cellular DNA of the cells that had been cultured Tris-buffered saline containing 1% Triton X-100, 1 mM phenylmeth- for various times in the presence of G-CSF was examined by staining ylsulfonyl fluoride, and 50 ng/ml aprotinin for 30 min at O'C. After with PI (35). The cells were then washed and resuspended in 0.1% centrifugation, a 100 //I aliquot of the supernatant was precleared with sodium citrate containing 0.2% Nonidet P-40, 50 ng/ml PI, and 250 Protein A Sepharose (CL-4B, Pharmacia) and then mixed with OKT9, ng/ml RNase (Boehringer-Mannheim, Mannheim, West Germany), 4E, or 9E8 and incubated overnight at 4Â°C.After Protein A Sepharose and incubated at O'C for 30 min and then at 37"C for 15 min. The cells was added and tumbled for l h at 4'C, the complexes were boiled at 90Â°Cfor 5 min in a sample buffer. SDS-polyacrylamide gel electropho- were examined by flow cytometry (EPICS PROFILE, Coulter Elec tronics, Inc., Hialeah, FL). The percentage of cells having S and G2 + resis was carried out using 7.5% polyacrylamide gel. The gel was fixed M content of DNA was estimated by a program developed by Ortho and dried for autoradiography after immersing in Enlightening (NEN Diagnostics. Research Products). Binding of Diferric |'-'fl|'l f to Cell Surface. Binding assay was per formed on cells grown in 16-mm wells in the presence or absence of 10 ng/ml G-CSF at a cell density of 5 x IO5/ml for 24 h. The cells were RESULTS washed and resuspended in ice-cold RPMI 1640 supplemented with 0.1 mg/ml BSA and 0.2% NaN3 (36). Various concentrations of [125I] Proliferative Responses to G-CSF. Table 1 shows [3H]thymi- Tf (0.68 /iCi/^g, NEN Research Products) were added to the 5 x 10s dine incorporation of NKM-1 and NOMO-1 at 24 h after cells and incubated for 2 h at O'C, since preliminary experiments initiating cultures. In the absence of Tf (RPMI medium alone), revealed that 2-h incubation was required to reach maximum binding no additive proliferation was observed in response to G-CSF, at O'C. Then, a 100 ,/1aliquot was layered over 200 jil of dibutylphthal- whereas the uptake was apparently increased by G-CSF in the ate oil:toluene, 4.5:1, in a 400-/d centrifuge tube. After centrifugation presence of extracellular Tf. Cell growth of HL-60, NCO-2, for 1 min at 8000 x g, the bottom of the tube containing cell pellets and MEG-01 cells was not influenced by adding exogenous G- was cut off and counted by a gamma counter. Nonspecific binding was estimated in cultures with a 1000-fold excess of milabeled diferric Tf. CSF. These results indicate that the increased cell growth of NKM-1 cells and NOMO-1 cells by G-CSF is highly dependent For the cells cultured in medium containing Tf or human serum, the cells were washed and incubated in a Tf-free medium for 30 min at on the concentration of extracellular Tf. Since Tf promoted the 37"C before the radioligand analysis in order to "wash out" Tf bound cell growth even in the absence of G-CSF, two cell types are to its receptor. not totally dependent on G-CSF for their proliferation. I ransferrin Uptake and Release. To measure cellular uptake of difer Effect of G-CSF on Tf Receptor Expression. The effect of G- ric Tf, the cells which had been preincubated for 24 h in RPMI 1640 CSF on Tf receptor surface expression was first demonstrated with or without 10 ng/ml G-CSF were incubated with 50 nM ['"I]Tf at by immunofluorescence staining with OKT9. Approximately 4*C for 60 min to saturate surface TfR. Then the cells were warmed to 60% of NKM-1 cells expressed TfR after a 24-h incubation 37'C in medium containing 300 UMcold Tf. At the indicated time, with G-CSF, whereas only about 30% of untreated cells ex radioactivity remaining on the cell surface was determined as follows. pressed the receptor. Because the immunofluorescence tech Intracellular Tf was estimated by incubating cells (100 n\) for 15 s in nique is not suitable for quantitative analysis, a radioligand- 100 Â¿ilof250 mvi acetic acid and 500 mM NaCl (pH 2.3), neutralizing binding assay was used to examine the effect of G-CSF on TfR with 50 n\ of l M sodium acetate, and pelleting through oil (37). The cell surface-bound Tf was determined by subtracting the acid-resistant surface expression. Results for NKM-1 cells and NOMO-1 Tf from the total cell-associated Tf. cells that had been cultivated for 24 h in RPMI 1640 medium To analyze the release of Tf, cells were allowed to achieve steady- with or without 10 ng/ml of G-CSF are shown in Fig. 1. state binding of Tf to the total cycling population of TfR by incubating Scatchard analysis demonstrated a single class of high-affinity- cells for 60 min at 37*C in RPMI 1640 containing 0.1 mg/ml BSA and binding sites in both cells. G-CSF enhanced the number of the 7956

Table 1 Proliferative responses to G-CSF in transferrin-supplemented medium Cells (2 X 10*) were cultured at 37"C in RPMI 1640 with various concentrations of diferric transferrin in the presence or absence of 10 ng/ml G-CSF. [3H]- Thymidine incorporation was measured after 24 h of culture. Transferrin (jig/ml) 0.5 20 NKM-1Without G-CSFWith Â±382Â°4,266 Â±2259,1 Â±3231 1,853Â±23,822 G-CSFNOMO-1Without Â±1191 17Â±65717,745 7,297 Â±598*20,770 Â±22,058

G-CSFWith 6,952 Â±1,02617,080 Â±4032 Â±72927,070 Â±27,344 G-CSF5,081 Â±2089,006 1,469 Â±657*11,822 Â±1,048'1 Â±1,3281,461*1,2231,033' " Mean Â±SD of triplicate cultures. *P < 0.01 control compared with G-CSF-treated cells. ' F < 0.05 control compared with G-CSF-treated cells.

Fig. 1. Transferrin binding to cell surface. Left, saturation curve of Tf binding to N KM - 1 cells. Shown is the amount of total bound Tf for control cells (O) and G-CSF-stimulated cells (â€¢).The nonspecific binding of Tf (â€¢) was determined as described in "Materials and Methods." Inset, Scatchard plot of the equilib rium data. Linear regression was used to lÃ¬mi best fit. Apparent KÂ¿- 1.4 x 10"' mol/liter and 1.3 x 10~' mol/liter for NKM-1 cells without G-CSF and NKM-1 cells with G-CSF, respectively. Maximum specific binding, 1.5 x 10' and 2.7 x 10* Tf molecules/cell, respec tively. Right, saturation curve of Tf binding to NOMO-1 cells. For control NOMO-1 cells without G-CSF and NOMO-1 cells with G- CSF, 1.9 x 10*and 5.6 x 10*specific binding sites/cell with Ka of 1.5 x 10'' and 1.7 x 10'Â» mol/liter, respectively. One of two represent ative experiments is presented. 10 20 30 40 50 60 10 20 30 40 50 60 Transferrin (nM) Transferrin (nM)

2001 NKM1 Table 2 Ferritin content Cells (5 X 10*/ml) were cultured in RPMI 1640 containing 0 or 5 ^g/ml of 150 diferric transferrin in the presence or absence of 10 ng/ml G-CSF for 24 or 48 h at 37'C. Cellular ferritin was determined as described in "Materials and Meth ods." Ferritin content before cultures was 6 Â±2 and 16 Â±3 ng/5 x 10*cells for 100 NKM-1 and NOMO-1 cells, respectively. Tf(-) Tf<+) 5Â° 24 h 48 h 24h 48h NKM-1Without \"4Â± 0.5 2 5 20 G-CSFWith 15Â± 115Â±256 Â±724 G-CSFNOMO 120 114 Â±5118 Tf concentration (ng/ml) 1Without G-CSFWith Â±236 Â±349 Â±4270 Â±13879 Â§400 NOM01 G-CSF4Â± Â±36Â± Â±512Â± Â±4630 Â±92 " Mean Â±SDof three separateexperiments. 300

200 CSF was apparent as early as 6 h and reached a maximum at about 24 h and gradually subsided thereafter. The increase of 100- surface-bound Tf was detected at as little as 0.1 ng/ml of G- CSF and elevated in a dose-responsive manner when assayed 24 h after G-CSF exposure (data not shown). [l25I]Tf-binding 0.5 2 5 20 analysis was also performed in HL-60 and NCO-2 cells which Tf concentration (ng/ml) had no receptors for G-CSF. The surface-bound [125I]Tfwas Fig. 2. Down-regulation of surface transferrin receptor in the presence of transferrin. Cells were subcultured in RPMI 1640 medium with various concen not significantly different between cells stimulated with 10 ng/ trations of diferric Tf in the presence (â€¢)orabsence (O) of 10 ng/ml G-CSF. The ml G-CSF for 24 h and cells without G-CSF; 2.6 versus 2.7 x specific binding capacity was measured with a fixed concentration of labeled Tf 10s Tf molecules/cell in HL-60 cells and 2.1 versus 2.0 x 10s (50 nM). Points, means Â±SD (bars) of three separate experiments. molecules/cell in NCO-2 cells. Regulation of Cell Surface Tf Receptor by Extracellular Tf. Tf-binding sites to 2- to 3-fold that of control but did not affect Because the TfR at the cell surface has been reported to be the receptor affinity. Note that, in this culture condition (RPMI regulated by iron availability, [125I]diferric Tf-binding analysis medium alone), neither NKM-1 nor NOMO-1 showed prolif- was performed on the cells grown for 24 h in either RPMI erative responses to G-CSF. The up-regulation of TfR by G- 1640 medium alone or the medium containing various concen- 7957

decreases when cellular iron increases (40). In order to deter 1CO mine whether the alterations of TfR expression by G-CSF in NKM-1 or NOMO-1 cells were due to changes in iron balance, intracellular ferritin levels were analyzed. No reduction was observed in G-CSF-treated cells in either RPMI medium alone or Tf-supplemented medium (Table 2). Moreover, the ferritin content of NOMO-1 cells was greatly increased after the stim ulation with G-CSF. These findings suggest that increased E JJ 10 expression of the surface TfR by G-CSF was not merely due to

5 10 decreased cellular iron. Time ( minutes ) Internalization and Release of Tf. One mechanism by which G-CSF may regulate TfR expression at the cell surface is to change the rates of endocytosis and exocytosis. The internalization was assessed by surface [125I]Tfdisappearance. As shown in Fig. 3 (top), the first-order rate constant of NKM-1 cells was calculated as 0.11 min"1. No differences were observed between control cells and G-CSF-treated cells. Likewise, the rate con stant was 0.28 min~' for NOMO-1 cells and, again, no effect was observed by G-CSF treatment. The regulation of TfR exocytosis by G-CSF was examined by monitoring [l25I]Tfrelease from the Tf-saturated cells. Treat ment of NKM-1 cells with G-CSF for 24 h caused no remark able changes in the rate, while G-CSF-treated NOMO-1 cells Time ( minutes ) Fig. 3. Top, surface ['"Ijtransferrin disappearance. Radioactivity remaining showed a delay of the release (Fig. 3, bottom). These findings on the cell surface was determined as described in "Materials and Methods" using suggest that the increased cell surface expression of TfR by G- the cells preincubated with (closed symbols) or without (open symbols) G-CSF. CSF is not explained by changes of intracellular recycling of The results are the means of observations in three separate experiments. Bottom, exocytosis of transferrin receptor. The rate of [125I]apotransferrin release from TfR. NKM-1 cells and NOMO-1 cells was measured as described in "Materials and Transferrin Receptor Gene Expression. The mRNA levels Methods." The results represent the means of determinations made in three separate experiments. The first-order rate constants were estimated as 0.11, 0.11, were measured by Northern blot analysis of total cellular RNA. 0.13, and 0.10 min'1 for control NKM-1 (O), G-CSF-stimulated NKM-1 (â€¢), As shown in Fig. 4, G-CSF caused a rapid induction of 4.9 control NOMO-1 (D), and G-CSF-stimulated NOMO-1 (â€¢),respectively. kilobases TfR mRNA within 2 h in the presence of 10 Mg/ml Tf, whereas the mRNA diminished in the same medium without trations of iron-saturated Tf. G-CSF increased the binding G-CSF. No significant changes were observed in the levels of capacity of [l25I]Tf in any given culture condition, although the HLA class I gene expression that were simultaneously exam magnitude of TfR appearance was inhibited by the presence of ined as a control. TfR mRNA peaked at 12-24 h after stimu external Tf during cultures (Fig. 2). Therefore, it is suggested lation of G-CSF, at which time approximately 2- to 3-fold the that the surface TfR expression was positively regulated by G- amount of the receptor transcript was present as compared with CSF and negatively regulated by extracellular Tf. the initial value. Note that in this culture condition the cells Intracellular Ferritin Levels. In proliferating cells, the surface entering the S phase of the cell cycle remained unchanged expression of TfR increases when cellular iron content falls and during 6 h after the exposure to G-CSF. The increased expres-

Control G-CSF hrs 0 3 24 2 3 6 24

TfR-Â» -28S

Fig. 4. Northern blot analysis of transferrin receptor mRNA in NKM-1 cells. Cells were incubated in RPMI medium containing 10 Â¿ig/ ml Tf in the presence or absence of 10 ng/ml G-CSF. At indicated times of culture, total cellular RNA was extracted. Percentage of S phase was determined by PI stain. -18S

-18S HLA-Â»-

S-phase(%) 19.5 18.4 19.8 18.7 18.9 28.3

â€¢control G-CSF( + ) creased by 180-260% of control 12 h after the stimulation of 200- G-CSF, although the binding capacity was much lower than leukemia cell lines. In contrast, G-CSF did not potentiate the TfR expression on ALL cells obtained from patient R. G. which were insensitive to the proliferative effect of G-CSF.

-TfR DISCUSSION

In the present study, we have demonstrated that G-CSF positively regulates the transferrin receptor expression in myeloid leukemia cells. The increased surface expression of the TfR was also demonstrated in the G-CSF-responsive myeloid leu kemia cells which were freshly isolated from patients with AML, whereas the TfR expression was not induced in HL-60 cells, NCO-2 cells, or ALL cells which did not respond to G- -HLA CSF. Therefore, the Tf-TfR pathway may represent a common 'N 45- requirement for G-CSF-induced proliferation of myeloid leu kemia cells. It has been reported that the TfR expression is closely coupled to events in hematopoietic cell growth such as the transition of resting cells (G0) to the DNA synthesis (S) phase of the cell 1 cycle (12). Increment in the TfR expression may be viewed as Fig. 5. Biosynthetic induction of transferrin receptor. Shown are immunopre- a consequence of cellular proliferation. However, the surface cipitates with 9E8 (lanes I and 4), 4E (lanes 2 and 5), or OKT9 (lanes 3 and 6). expression was up-regulated by G-CSF in the condition such as A nonspecific band of M, 47,000 (A') was seen in all lanes. RPMI 1640 medium alone in which the cell growth and the cell cycle distribution were identical during experimental pe Table 3 Transferrin-binding capacity of G-CSF-stimulated leukemia cells riods between G-CSF-untreated and -treated cells. Moreover, Leukemia cells isolated from peripheral blood were cultivated at 5 x 10'/ml in RPMI 1640 containing 5% human serum in the presence or absence of 10 ng/ in Tf-supplemented medium, the surface expression of TfR ml G-CSF. Cell surface Tf-binding capacity was determined at 12 h by incubating preceded the cell growth. Conceivably, the binding of G-CSF cells with a fixed concentration of [12!]Tf(5 nM) at O'C. Nonspecific binding was <10% of total activity bound. [3H]Thymidine was added 20 h after initiating and its receptor should transduce a signal which, in turn, drives cultures and harvested 4 h later. machinery to induce the TfR expression that is a prerequisite capacity to cell growth. (fmol/2 x 10' [3H]Thymidine uptake Recently, increased attention has been directed to the regu PatientY. (cpm)WithoutG-CSF cells) lation of the receptor for transferrin. The mechanisms control 977* N. r ling surface TfR expression may be divided into two categories: With 34.7 30,961 :: 1,179 R. S. AML (M2) Without 13.8 2,766 112 redistribution between the cell surface and an internal receptor With 26.0 7,352 257 pool and modulation of receptor synthesis and steady-state M.S. AML(M3) Without 18.4 2,969 173 mRNA levels. The receptor recycling is acutely regulated by With 48.8 10,000 993 R. G.TypeAML(Ml)ALLTf-bindingWithout 12.6 1,317 51 peptide growth factors (41). Manipulation of fibroblast with With15.3Â° 12.28,713 1,356t 38 epidermal growth factor or insulin-like growth factor I causes " Mean of duplicated cultures. a redistribution of TfR from the intracellular compartment to * Mean Â±SD of triplicated cultures. the cell surface. In phorbol diester-treated HL-60 cells, stimu lated internalization of TfR is triggered by the receptor phos- sion of the TfR gene was also demonstrated in NKM-1 cells phorylation, which results in a decline of the surface TfR (42). and NOMO-1 cells cultured in Tf-free media in the presence of On the other hand, a steady-state level of TfR mRNA is G-CSF (data not shown). modulated in T-cells with mitogen stimulation (43) and in HL- Biosynthesis of Transferrin Receptor. To study the effect of 60 cells treated with dibutyryl cyclic AMP (44) or various G-CSF on the TfR biosynthesis, NKM-1 cells that had been differentiation inducers (45). We selected culture conditions in preincubated for 18 h in RPMI 1640 with or without 10 ng/ml order to avoid cell density-dependent regulation or changes of G-CSF were labeled with [35S]methionine. Under reducing con cell viability. It is evident that G-CSF modulates the cell surface ditions, a protein of M, ~87,000 was detected in the control TfR through up-regulation of the TfR mRNA expression and lane 3, indicating a steady-state level of the TfR biosynthesis. the receptor protein biosynthesis, since no apparent differences G-CSF treatment greatly increased the TfR biosynthesis (Fig. of TfR endocytosis and exocytosis were observed after the 5). This increase was relatively specific for TfR protein, since exposure to G-CSF. A delay of exocytosis seen in G-CSF- the HLA class I molecule, as estimated by the immunoprecipi- treated NOMO-1 cells accounts for the intracellular accumu tation with 4E antibody, was not significantly different. lation of iron as a ferritin complex. G-CSF Effect in Fresh Leukemia Cells. The effect of G-CSF Iron has been shown to play a crucial role in the regulation on the TfR expression was also tested in leukemia cells freshly of the TfR gene expression in various cells that constitutively obtained from three patients with AML and a patient with express TfR. This is consistent with our observation that the ALL. In three AML cells, [3H]thymidine uptake increased by extracellular Tf down-regulated the cell surface expression of approximately 3-fold at 24 h in medium supplemented with 5% TfR and the mRNA level in the absence of G-CSF. In T-cell human serum (containing about 25 Mg/ml Tf) and 10 ng/ml G- proliferation, the mitogen stimulation programs T-lymphocytes CSF (Table 3). The surface [125I]Tf-binding capacity was in- into cell cycle progression. The TfR gene is then modulated via 7959

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1990 American Association for Cancer Research. TRANSFERRIN RECEPTOR EXPRESSION BY G-CSF a decline of the intracellular iron level which is amplified by E. W. Deferoxamine: a reversible S-phase inhibitor of human lymphocyte the interleukin 2-interleukin 2 receptor-signaling process (12, proliferation. Blood, 64: 748-753, 1984. Stein, B. S., and Sussman, H. H. Demonstration of two distinct transferrin 43). A similar regulation may also govern the TfR gene expres receptor recycling pathways and transferrin-independent receptor internali sion during cell growth in myeloid cells (46-48). Up-regulation zation in K562 cells. J. Biol. Chem., 26J: 10319-10331, 1986. 9. Bolt, D. H., Phillips, J. L., and Alcantra, O. Disparity between expression of in the monocyte-macrophage stimulated by 7-interferon is ac transferrin receptor ligand binding and non-ligand binding domains on companied by a decreased level of cellular ferritin and iron human lymphocytes. J. Cell. Physiol., 132: 331-336, 1987. content (9). In G-CSF-induced proliferation of myeloid cells, 10. Manger, B., Weiss, A., Hardy, K. J., and Stobo, J. D. A transferrin receptor antibody represents one signal for the induction of IL-2 production by a however, the increased TfR gene expression does not appear to human T cell line. J. Immunol., 136: 532-538, 1986. be due to an indirect effect of decreased iron availability but to 11. Esserman, L., Takahashi, S., Rojas, V., Warnke, R., and Levy, R. An epitope of the transferÃin receptor is expressed on the cell surface of high-grade but a direct action of G-CSF. This is substantiated by the following not low-grade human lymphomas. Blood, 74: 2718-2729, 1989. results: (a) ferritin levels were found to increase after G-CSF 12. Neckers, L. M., and Cossman, J. Transferrin receptor induction in miiogen- stimulation; and (b) TfR mRNA was enhanced by G-CSF even stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. Proc. Nati. Acad. Sci. USA, 80: in the presence of Tf in the culture medium, whereas TfR was 3494-3498, 1983. down-regulated without G-CSF. Similar observations have been 13. Taetle, R., and Honeysett, J. M. f-Interferon modulates human monocyte/ described by Tacile et al. (48) using HL-60 cells grown in macrophage transferrin receptor expression. Blood, 71: 1590-1595, 1988. 14. Hirata, T., Bitterman, P. B., Mornex, J.-F., and Crystal, R. G. Expression medium containing soluble iron. A transient increase in Tf of the transferrin gene during the process of mononuclear phagocyte matu receptors was observed during the first 24 h and it was not ration. J. Immunol., 136: 1339-1345, 1986. 15. Gatter, K. C., Brown, G., Trowbridge, I. S., Woolstone, F. E., and Mason, accompanied by reduced intracellular ferritin levels. Recently, D. Y. Transferrin receptors in human tissues: their distribution and possible iron-responsive elements have been identified (49, 50). Five clinical relevance. J. Clin. Pathol., 36: 539-545, 1983. iron-responsive elements in the 3'-untranslated region appear 16. Tei, I., Makino, Y., Sakagami, H., Hanamaru, I., and Konno, K. Decrease of transferrin receptor during mouse myeloid leukemia (Ml) cell differentia to play a major role in regulation of TfR mRNA expression by tion. Biochem. Biophys. Res. Commun., 107: 1419-1424, 1982. iron (51). The interaction of the 3'-untranslated region with a 17. Testa, U., Thomopoulos, P., Vinci, G., Titeux, M., Brettaieb, A., Vain- chenker, W., and Rochamt, H. Transferrin binding to K562 cell line. Exp. nuclear binding protein which is induced by iron deprivation Cell Res., 140: 251-260, 1982. may regulate the turnover of the transcript to protect the TfR 18. Yeh, C. J. G., Papamicheal, M., and Faul, K. W. P. Loss of transferrin mRNA from endonuclease cleavage. receptors following induced differentiation of HL-60 promyelocytic leukemia cells. Exp. Cell Res., 138: 429-433, 1982. Besides iron regulation of the TfR gene expression, a distinct 19. Griffin, J. D., Young, D., Herrman, F., Wiper, D., Wagner, K., and Sabbath, mechanism may act on the TfR gene transcription. Pelosi et al. D. K. Effects of recombinant GM-CSF on the proliferation of clonogenic (52) have suggested a distinction between the iron regulation cells in acute myeloblastic leukemia. Blood, 67:1448-1453, 1986. 20. Begley, C. G., Metcalf, D., and Nicola, N. A. Primary human myeloid and the increased gene expression that accompanies growth leukemia cells: comparative responsiveness to proliferative stimulation by arrest of fibroblasts by serum deprivation. Miskimis et al. (53) GM-CSF or G-CSF and membrane expression of CSF receptors. Leukemia (Baltimore), /: 1-8, 1987. have demonstrated a nuclear protein of M, 88,000 that binds 21. Miyauchi, J., Kelleher, C, Yang, Y.-C., Wong, G. C, Clark, S. C., Minden, to 5'-promoter regions as a result of serum stimulation of M. D., Minkin, S., and McCulloch, E. A. The effects of three recombinant quiescent Swiss/3T3 cells. Within the 5'-promotor region of growth factors, IL-3, GM-CSF, and G-CSF, on the blast cells of acute myeloblastic leukemia maintained in short term suspension culture. Blood, the TfR gene, an Eia enhancer homologue and a putative Spl 70:657-663, 1987. site have been identified (53, 54). These sequences may be 22. Vellenga, E., Young, D. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1990 American Association for Cancer Research. Up-Regulation of Transferrin Receptor Gene Expression by Granulocyte Colony-stimulating Factor in Human Myeloid Leukemia Cells

Yoshihisa Morishita, Takae Kataoka, Masayuki Towatari, et al.

Cancer Res 1990;50:7955-7961.

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