regulation of deoxyribonucleic acid synthesis in ......this report investigates the regulation of...

0013.7227/95/$03.00/0 Endocrinology Copyright 0 1995 by The Endocrine Society

Vol. 136, No. 1 Printed in U.S.A.

Regulation of Deoxyribonucleic Acid Synthesis in Proliferating and Differentiating Trophoblast Cells: Involvement of Transferrin, Transforming Growth Factor-P, and Tyrosine Kinases*

GARY P. HAMLIN AND MICHAEL J. SOARES

Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160

ABSTRACT This report investigates the regulation of DNA synthesis in tro-

phoblast stem cells and differentiating trophoblast cells. Experiments in this study were performed on the Rcho-1 trophoblast cell line, which was established from a transplantable rat choriocarcinoma. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate along the trophoblast giant cell pathway. DNA synthesis in quiescent trophoblast stem cells (maintained in serum-free medium) was stimulated by fetal bovine serum and donor horse serum or transferrin to a level approximately 30- and lo-fold above the basal level, respectively. Transferrin and horse serum were ineffective at maintaining trophoblast cell proliferation. In contrast, serum-starved differentiating trophoblast cells synthesize DNA at maximal levels and could not be further stimulated by the addition of exogenous

factors. Fetal bovine serum-stimulated proliferation was effectively inhibited by transforming growth factor-pl. Experiments with the tyrosine kinase inhibitor genistein implicate tyrosine kinase involvement in the regulation of DNA synthesis and proliferation in trophoblast stem cells and DNA synthesis in differentiating trophoblast cells. Proliferating and differentiating trophoblast cells differ in their levels of tyrosine kinase activities and express unique tyrosine-phosphorylated proteins.

In summary, DNA synthesis and proliferation in trophoblast stem cells are under extrinsic control, whereas DNA synthesis in differentiating trophoblast cells is under intrinsic control. Both mechanisms require tyrosine kinase activity, but the nature of the tyrosine kinase pathways in each process appears to be distinct. (Endocrinol- ogy 136: 322-331, 1995)

R ODENT trophoblast cells synthesize DNA during two different cellular programs: proliferation and en-

doreduplication (l-6). Proliferation results in the expansion of trophoblast precursor cells that can differentiate along the trophoblast multilineage pathway (l-4). Endoreduplication is characterized by the continuation of DNA synthesis in the absence of cell division, resulting in the formation of trophoblast giant cells (5, 6). Trophoblast giant cells represent one of the differentiated trophoblast cell lineages and possess a number of specialized functions, including the biosynthesis of steroid and peptide hormones (4-7). Steroid and peptide hormone production follow a stage-specific pattern and oc- cur concomitantly with endoreduplication (7).

The Rcho-1 trophoblast cell line provides a means of dis- secting the trophoblast giant cell differentiation pathway (4, 8, 9). Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate @,9). Proliferating trophoblast cells can be maintained by culturing Rcho-1 trophoblast cells in the presence of fetal bovine serum (FBWsupplemented culture medium and preventing the cells from becoming confluent, whereas differentiation can be induced by substituting horse serum (HS) supplementation for FBS and increasing the density of the cultures (9). Differentiating Rcho-1 trophoblast cells undergo endoreduplication and a stage-specific pattern of expression of peptide and steroid hormones mimicking

Received June 28, 1994. Address all correspondence and requests for reprints to: Dr. Michael

J. Soares, Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160.

* This work was supported by a grant from the NICHHD (HD-20676).

the behavior of trophoblast giant cells developing in situ (8-10). These initial observations suggested significant differences in the control of proliferating and differentiating trophoblast cells.

In the present report, we use the Rcho-1 trophoblast cell line to investigate the regulation of DNA synthesis in proliferating and differentiating trophoblast cells. Evidence is presented to support the notion of differential regulation of proliferating and differentiating trophoblast cells and for the involvement of transferrin, transforming growth factor-p (TGFP), and tyrosine kinase signaling pathways in the regulation of trophoblast cells.

Materials and Methods

Reagents

FBS and HS were purchased from JRH Bioscience (Lenexa, KS). [3H]Thymidine was obtained from ICN Biomedicals (Irvine, CA). The growth factors and serum supplements used in the experiments included human TGFSl, human insulin-like growth factor-I, and mouse nerve growth factor (Austral Biologicals, San Ramon, CA); human insulin-like growth factor-II and mouse epidennal growth factor (Sigma Chemical Co., St. Louis, MO), and colony stimulating factor-I (National Institute for Biological Standards and Control BiologGal Response Mod- ifiers Program, NCI-FCRDC, Frederick, MD); c-Kit ligand, granulocyte- macrophage colony-stimulating factor, and granulocyte colony-stimulating factor (R & D Systems, Minneapolis, MN); mouse leukemic inhibitorv factor (Gibco-BRL. Gaithersbure. MD): rat euidermal growth factor (iioproducts for Science, Indian&olis,.IN); bovine fibyoblast growth factor and MITO+ serum extender (Collaborative Research, Bedford, MA); human platelet-derived growth factor-AA (Upstate Biotechnology, Lake Placid, NY); human iron-saturated transferrin

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TROPHOBLAST GROWTH AND DIFFERENTIATION 323

(Boehringer Mannheim, Indianapolis, IN); ovine PRL (National Hor- mone and Pituitary Program, Baltimore, MD); and human hepatocyte growth factor and keratinocyte growth factor (generous gifts from Dr. Jefferv S. Rubin, NCI, Bethesda, MD). Genistein, lavendustin-A, and the tyrosine kinase assay kit containing the RR-Src peptide were obtained from Gibco-BRL. Antiohosohotvrosine antibodies couoled to horseradish peroxidase were p&chased from Transduction Laboratories (RC20; Lexington, KY) and Upstate Biotechnology (4GlO). Reagents for the detection of immune complexes by enhanced chemiluminescence were obtained from Amersham Corp. (Arlington Heights, IL). Reagents for sodium dodecyl sulfate-polyacrylamide gel ilectrophore& (SDS- PAGE) were purchased from Bio-Rad Laboratories (Richmond, CA). Unless otherwise stated, all other reagents were purchased from Sigma.

Maintenance of the Rcho-1 cell line

The Rcho-1 cell line was routinely maintained under subconfluent conditions with either RPMI-1640 or NCTC-135 culture medium supplemented with lo-20% FBS, as previously reported (8, 9). Differenti- ation was induced by growing the cells to confluence in FBS-supplemented culture medium and then replacing the serum supplementation with HS (l-10%). High cell density and the absence of sufficient growth stimulatory factors ?removal of FBS) facilitate trophoblast giant cell formation (8, 9). Rcho-1 trophoblast cells that are in the proliferative stage of culture will be refe-med to as stem cells, and cells’ induced to differentiate will be referred to as differentiating cells.

phenylmethylsulfonylfluoride, 50 kallikrein inhibitor units aprotinin] and centrifuged at 800 X g for 10 min to remove nuclei and cellular debris. High speed centrifugation of the supernatants at 150,000 X g for 40 min yielded cytosolic and membrane fractions. The membrane fraction was solubilized 150 mM Tris-HCl (pH 7.5),20 mM Mg acetate, 5 mM NaF. 1 mM EDTA. 1 mM DTT. 30 U..M Na,VO,. and 0.5% Nonidet P-401 on ice for 1 h. The solubilized membrane Fraction was collected by ultracentrifugation (at 150,000 X g for 40 min), and the protein concentrations of the membrane and cytosolic fractions were estimated by the Bradford assay (13). Equal amounts of protein were assessed for tvrosine kinase activity, as pre;iously described (12). Briefly, 10 pg protein were added to duplicate tubes either with or without the RR-Src peptide substrate (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) in a kinase buffer mix (60 mM HEPES, 20 mM MgCI,, 40 ~.CLM EDTA, 0.2 mM DTT, 50 pg/ml BSA, 0.3% Nonidet P-40, 14cp~ Na,VO,, and 120 PM ATP). The reaction was started bv the addition of 13’PlATP and stouued after a lo-min incubation at room temperature by the addition ojcold 10% trichloroacetic acid. The synthetic peptide was removed from the reaction mix by absorption to phosphocellulose disks. The disks were then placed in liquid scintillation cocktail, and the amount of 32P in- corporated into the peptide was measured.

Western blotting for phosphotyrosine-containing proteins

DNA synthesis

Rcho-1 cells were plated at a density of 13,333 cells/cm’ in 20% FBS-NCTC (day 0) in 24-well plates. Cultures were maintained in 20% FBS-NCTC until day 3 of culture, when the medium was changed to 10% HS-NCTC. Cultures were then maintained in 10% HS-NCTC. Medium was changed every other day during the culture period, except during the serum starvation and restimulation periods (see below).

Assessment of DNA synthesis in trophoblast stem cell cultures was performed by washing day 1 cultures three times with serum-free RPM1 to remove serum factors. Assessment of DNA synthesis in differentiating cultures was performed by washing day 8 cultures three times with serum-free RPMI. Both types of cultures (stem and differentiated) were then incubated for 24 h in serum-free RPM1 before the addition of various treatments in RPM1 for an additional 24-h incubation. DNA synthesis was estimated by pulsin (1 @i/ml) for 6 h. Estimation of 1 B

treated cultures with [3H]thymidine Hlthymidine incorporation by liquid

scintillation counting was performed as previously described (9, 11).

Tyrosine-phosphorylated proteins were analyzed in trophoblast stem cell cultures and differentiating trophoblast cultures by Western blotting with antiphosphotyrosine antibodies. Whole cell lysates were prepared in RIPA buffer [lo mM Tris (pH 7.2), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM NasVO,, 50 mM NaF, and 10 wg/ml aprotininl, and equal amounts of protein (40 pg/lane) were separated by SDS-PAGE on 10% gels, as previously described (141, and transferred to nitrocellulose, as previously described (15). Nitrocellulose filters were blocked with 3% BSA in TBS (10 mM Tris, pH 7.5, and 100 mM NaCl) for 1 h at 37 C before incubation with RC-20-horseradish peroxidase-cou- pled primary antibody. Immune complexes were then detected using the enhanced chemiluminescence system.

Statistical analysis

The data were analyzed by analysis of variance. The source of vari- ation from significant F ratios was determined with the Newman-Keuls multiple comparison test (16).

Assessment of Rcho-1 stem cell proliferation Results

Cultures were established by plating 500 or 5000 cells in 20% FBS- NCTC/well in a 24-well plate (day 0). Treatments were added at the time of plating and replaced every 2 days during the culture period. Visual examination of treated cultures 24 h after plating was conducted to ensure consistent plating efficiency. Cultures were maintained for 4-8 days (depending upon seeding density), followed by assessment of proliferation, as previously described (9, 11). Briefly, cultures were rinsed with PBS (10 mM sodium phosphate, pH 7.2, and 150 mM NaCl) and stained with a crystal violet solution (300 pi/well; 5% formalin, 50% ethanol, 150 mM NaCI, and 0.5% crystal violet) for 10 min. Monolayers were extensively washed in tap water to remove excess stain before elution of the dye with ethylene glycol. Cell density was quantified by measuring the absorbance of the eluate at 600 nm. In this assay, cell number is positively correlated with absorbance of the cellular eluates (9).

Tyrosine kinase assay

Tyrosine kinase activity in fractionated tmlphpblast cell cultures was measured by estimating the incorporation of I’ mto a synthetic peptide substrate. Approximately lo7 trophoblast stem cells or differentiating trophoblast cells were fractionated, as previously described (12). Briefly, cells were sonicated in extraction buffer 110 mM Tris-HCl (pH 7.4), 1 mM MgCI,, 1 mM EDTA, 1 mM dithiothreitol (D’IT), 0.25 M sucrose, 1 mM

Zdentification of extracellular modulators regulating trophoblast cell DNA synthesis and proliferation

In the initial series of experiments, we compared the responses of stem and differentiating trophoblast cells to various extracellular modulators. The results presented below demonstrate that DNA synthesis is differentially regulated in proliferating and differentiating trophoblast cells.

Serum supplementation and DNA synthesis in stem and di’er- entiating trophoblast cells. To obtain an initial understanding of the nature of the regulation of proliferating and differentiating trophoblast cells, we examined the effects of serum supplementation (FBS, HS, or serum free) on DNA synthesis by stem and differentiating Rcho-1 trophoblast cells. DNA synthesis in trophoblast stem cells was dependent on and significantly stimulated by the presence of serum (Fig. 1). FBS was a more potent stimulator of DNA synthesis than HS (Fig. 1). In contrast, DNA synthesis in differentiating trophoblast cells was maximal under serum-free conditions (Fig. 1). Sup- plementation with FBS was without effect, whereas HS ap-



324 TROPHOBLAST GROWTH AND DIFFERENTIATION Endo l 1995 Vol 136. No 1

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STEM CELLS DIFFERENTIATING CELLS

FIG. 1. Serum stimulation of DNA synthesis in trophoblast stem cells and differentiating trophoblast cells. Rcho-1 cultures were established by plating 25,000 cells/well in a 24-well plate (day 0). Cultures were placed in serum-free medium on day 1 of culture (trophoblast stem cells) or day 8 of culture (differentiating trophoblast cells) for 24 h before the addition of various treatments and incubated for an additional 24 h. Treated cultures were then incubated with 1 @X/ml 13Hlthymidine for 6 h to assess DNA synthesis. SF, Cultures maintained in serum-free medium during the treatment period; FBS, cultures treated with 20% FBS; HS, cultures treated with 10% HS. The left half of the figure represents responses measured in trophoblast stem cells, and the right halfof the figure represents responses measured in differentiating trophoblast cells. Bars indicate the mean for each treatment, and vertical lines indicate the SEM. *, Significantly different from proliferating cells maintained in SF medium (P < 0.05); **, significantly different from differentiating cells maintained in SF medium (P < 0.05).

peared to moderately inhibit DNA synthesis in differentiating trophoblast cells (Fig. 1).

Transferrin and DNA synthesis in stem and differentiating trophobzast ceZIs. Transferrin is known to be a significant growth regulatory component present in serum (17). In the next series of experiments, we examined the effects of transferrin on DNA synthesis and proliferation of Rcho-1 trophoblast stem cells and on DNA synthesis in differentiating Rcho-1 trophoblast cells. Transferrin significantly stimulated DNA synthesis in serum-deprived Rcho-1 trophoblast stem cells (Fig. 2), but was not sufficient to promote their proliferation (data not shown). The EDso for transferrin-stimulated DNA synthesis was approximately 0.25 pg/ml (Fig. 2). DNA synthesis in differentiating Rcho-1 trophoblast cells was not significantly affected by transferrin supplementation (Fig. 2).

Growth factors, DNA synthesis, and proliferation of trophoblast cells. Our initial experiments indicated the dependence of trophoblast stem cell DNA synthesis and proliferation on serum. Transferrin was identified as a stimulatory component, but was not sufficient to promote proliferation or maximal DNA synthesis (Fig. 2). In the following experiments, we investigated the actions of a number of different peptide growth factors on the proliferation and DNA synthesis of Rcho-1 trophoblast stem cells. Many of the factors tested in these experiments have been reported to be produced in the

placenta or have been speculated to influence the behavior of trophoblast cells (18, 19). The factors (see Materials and

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FIG. 2. Transferrin stimulation of DNA synthesis in trophoblast stem cell cultures. Rcho-1 cultures were established by plating 25,000 cells/well in a 24-well plate (day 0). Cultures were placed in serum- free medium on day 1 of culture (trophoblast stem cells) or day 8 of culture (differentiating trophoblast cells) for 24 h before the addition of various treatments and incubated for an additional 24 h. Treated cultures were then incubated with 1 &i/ml L3Hlthymidine for 6 h to assess DNA synthesis. Bars indicate the mean for each treatment, and vertical lines indicate the SEM. Top panel, Responses of trophoblast stem cells and differentiating trophoblast cells to treatment with 10 pg/ml transferrin. SF, Cultures maintained in serum-free medium during the treatment period; FBS, cultures treated with 20% FBS; Tr, cultures treated with iron-saturated transferrin. *, Significantly different from proliferating cells maintained in SF medium (P < 0.05); **, significantly different from differentiating cells maintained in SF medium (P < 0.05). Bottom panel, Responses of trophoblast stem cells to treatment with increasing concentrations of transferrin are presented. Cultures were treated with serum-free medium (C) or serum- free medium plus increasing concentrations of transferrin (0.1-10.0 &ml). *, Significantly different from serum-free control (P < 0.05).



TROPHOBLAST GROWTH AN’D DIFFERENTIATION 325

Methods) were tested at a variety of doses to evaluate their actions on trophoblast stem cell proliferation or DNA synthesis. Additionally, combinations of growth factors were assessed. The only growth modulator shown to have a consistent influence on the Rcho-1 trophoblast cells was TGF/31.

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TGFPl had a modest, but significant, inhibitory effect on serum-stimulated Rcho-1 trophoblast stem cell DNA synthesis (Fig. 3; P < 0.05) and a pronounced significant inhibitory effect on the proliferation of FBS-stimulated Rcho-1 trophoblast stem cells (Fig. 3). Significant inhibition of serum-stimulated proliferation was seen with doses of TGFPl at and above 0.01 rig/ml (P < 0.05). The ED,, for TGFPl inhibition of Rcho-1 trophoblast stem cell proliferation was between 0.01-0.05 rig/ml (Fig. 3). TGFPl did not significantly influence DNA synthesis by differentiating Rcho-1 trophoblast cells maintained under serum-free or FBS-supplemented conditions (Fig. 3).

Identification of intracellular pathways controlling DNA synthesis in stem and differentiating trophoblast cells

The following series of experiments was designed to investigate signal transduction mechanisms involved in regulating DNA synthesis in proliferating and differentiating trophoblast cells. Preliminary experiments indicated that ac- tivators of protein kinase-C (phorbol12-myristate 12-acetate) and protein kinase-A (8-bromo-CAMP) athways had minimal effects on Rcho-1 trophoblast cell [ cp Hlthymidine incorporation or proliferation (data not shown). The results presented below implicate the involvement of tyrosine kinase pathways in the regulation of DNA synthesis of both proliferating and differentiating trophoblast cells.

Genistein and DNA synthesis in stem and differentiating tropho- bZust cells. Rcho-1 trophoblast cells were treated with the tyrosine kinase inhibitor, genistein, to assess the role of tyrosine kinases in regulating DNA synthesis during proliferation and differentiation. FBS or transferrin stimulated DNA synthesis in trophoblast stem cells, and DNA synthesis in differentiating trophoblast cells was significantly inhibited by genistein at concentrations of 10 PM and above (Fig. 4; P < 0.05). The ED,, for the inhibition of

FIG. 3. The effect of TGFPl on trophoblast cell DNA synthesis and proliferation. Top panel, Rcho-1 cultures were established by plating 25,000 cells/well in a 24-well plate (day 0). Cultures were placed in serum-free medium on day 1 of culture (trophoblast stem cells) or day 8 of culture (differentiating trophoblast cells) for 24 h before the addition of 10 @ml TGFpl for 24 h. Treated cultures were then incubated with 1 @Z/ml [3H]thymidine for 6 h to assess DNA synthesis. SF, Cultures maintained in serum-free medium during the treatment period; FBS, cultures treated with 20% FBS; FBS + TGFPl, cultures treated with 10 rig/ml TGFpl in the presence of 20% FBS. Bars indicate the mean for each treatment, and vertical lines indicate the SEM *, Significantly different from FBS (P < 0.05). Middle and bottom panels, Rcho-1 cells were plated at 500 cells/well in a 24-well plate in 20% FBS-NCTC (C) or 20% FBS-NCTC plus increasing concentrations of TGFpl (0.0001-1.0 rig/ml). Culture medium, including treatments, was replaced at a-day intervals. On day 8, cultures were stained with crystal violet to estimate cell density. Crystal violet specifically taken up by the cells was eluted with ethylene glycol and quantitated by measuring the absorbance of the eluate at 600 nm. The middle panel is a photomicrograph of repre- sentative cultures treated with increasing amounts of TGFpl in the presence of 20% FBS-NCTC for 8 days. The bottom panel is a plot of the absorbance at 600 nm of the crystal violet eluates for each treatment. Cultures treated with 0.01 rig/ml TGFPl or more were significantly growth inhibited (P < 0.05) compared to control cultures. The bar in the bottom panel indicates the mean value for the control culture (20% FBS-NCTC), and the vertical lines indicate the SEM.



326 TROPHOBLAST GROWTH AND DIFFERENTIATION Endo. 1995 Vol 136. No 1

DNA synthesis by genistein in both proliferating and dif- The inhibitory effects of genistein were reversible after re- ferentiating cells was approximately 20 PM (Fig. 4). moval of the drug (data not shown). The related tyrosine Genistein was also an effective inhibitor of FBS-stimulated kinase inhibitor, lavendustin-A, was ineffective at influ- trophoblast stem cell proliferation (Fig. 5). The potency of encing the behavior of trophoblast stem or differentiating genistein in these assays was similar to its inhibitory ac- cells at concentrations equivalent to or higher than those tions on DNA synthesis by Rcho-1 trophoblast stem cells. used for genistein (data not shown).

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FIG. 4. Inhibition of trophoblast cell DNA synthesis by the tyrosine kinase inhibitor, genistein. Rcho-1 cells were plated at 25,000 cells/well in a 24-well plate in 20% FBS-NCTC (day 0). Cultures were then washed and incubated in serum-free medium on day 1 (trophoblast stem cells) or day 8 (differentiating trophoblast cells) of culture for 24 h before the addition of various treatments for an additional 24 h. Treated cultures were labeled with 1 @X/ml [3H]thymidine for 6 h. Bars indicate the mean for each treatment, and vertical lines represent the SEM. A, Trophoblast stem cell and differentiating trophoblast cell responses to genistein. The treatments included: SF, cultures maintained in serum-free medium during the 24-h treatment period; FBS, cultures treated with 20% FBS; FBS + Gen, cultures treated with 20% FBS in the presence of 20 pM genistein; Tr, cultures treated with 10 pg/ml transferrin; Tr + Gen, cultures treated with 10 pg/ml transferrin and 20 pM genistein; SF + Gen, cultures maintained in serum-free medium and treated with 20 pM genistein. * and **, Significantly different (P < 0.05) from trophoblast stem cell cultures treated with FBS or Tr, respectively; ***, significantly different from differentiating trophoblast cultures treated with SF (P < 0.05). B, Trophoblast stem cell responses to increasing concentrations of genistein. Trophoblast stem cells were treated with 10 pg/ml transferrin (C), 10 pg/ml transferrin plus a maximal dose of dimethylsulfoxide vehicle (01, 10 pg/ml transferrin plus increasing concentrations of genistein (2.5-40.0 PM), or serum-free medium (SF). *, Significantly different from control cultures treated with 10 pg/ml transferrin and 10 pg/ml transferrin plus dimethylsulfoxide vehicle (P < 0.05). C, Trophoblast stem cell responses to increasing concentrations of genistein. Trophoblast stem cells were treated with 20% FBS (Cl, 20% FBS plus a maximal dose of dimethylsulfoxide vehicle (0),20% FBS plus increasing concentrations of genistein (2.5-80 PM), or serum-free medium (SF). *, Significantly different from cultures treated with 20% FBS and 20% FBS plus dimethylsulfoxide vehicle (P < 0.05). D, Differentiating trophoblast cell responses to increasing concentrations of genistein. Differentiating trophoblast cultures were maintained in serum-free medium (C), serum-free medium plus maximal dimethylsulfoxide vehicle concentration (O), or serum-free medium plus increasing concentrations of genistein (5-80 PM). *, Significantly different from cultures maintained in serum-free medium or serum-free medium plus dimethylsulfoxide vehicle (P < 0.05).




2.5 5 10 20 40 80

Genistein (JJM)

C 0 2.5 5 10 20 40 80

Genistein ( p M)

FIG. 5. Inhibition of trophoblast stem cell proliferation: dose response to the tyrosine kinase inhibitor genistein. Trophoblast stem cells were plated at 500 cells/well (in 24-well plates) in 20% FBS- NCTC (C), 20% FBS-NCTC plus maximal DMSO vehicle concentration (O), or 20% FBS-NCTC plus increasing concentrations of genistein (2.5-80 PM). Culture medium including treatments was replaced at 2-day intervals. On day 8, cultures were stained with crystal violet to estimate cell density. The top panel is a photomicrograph of the trophoblast stem cells after 8 days of culture stained with crystal violet. Cell density was estimated by elution of crystal violet with ethylene glycol and measurement of absorbance at 600 nm (bottom panel). Bars indicate the mean for each treatment, and vertical lines represent the SEM. Cultures treated with 5 ~.LM genistein or more were significantly growth inhibited compared to the DMSO vehicle control (P < 0.05).

Tyvosine kinase activities in proliferating and differentiating trophoblast cells. A peptide containing a consensus tyrosine phosphorylation site from the tyrosine kinase c-SYC was used to examine tyrosine kinase activities in stem and differentiating trophoblast cells. Tyrosine kinase activities were detectable in both proliferating and differentiating trophoblast cells (Fig. 6). FBS-treated trophoblast stem cells possessed significantly higher tyrosine kinase activities than serum- deprived trophoblast stem cells (Fig. 6). Transferrin-treated trophoblast stem cells also showed significantly higher tyrosine kinase activities than serum-deprived trophoblast stem cells (Fig. 6). FBS-responsive tyrosine kinase activities were present in both membrane and cytosolic fractions, whereas transferrin-responsive tyrosine kinase activities were restricted to the membrane fraction (Fig. 6). Tyrosine kinase activities were detectable in serum-deprived differentiating cultures and were significantly increased after FBS treatment (Fig. 6). However, the magnitude of the tyrosine kinase activity increase after FBS treatment of differentiating

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FIG. 6. Comparison of tyrosine kinase activity in trophoblast stem cells and differentiating trophoblast cells. Trophoblast cells were plated at 1 X 10s tells/75-cm2 flask in 20% FBS-NCTC (day 0). Tro- phoblast stem cell (day 1) and differentiating trophoblast cell cultures (day 8) were washed and incubated in serum-free medium for 24 h before the addition of various treatments for 24 h. Cultures were then harvested and fractionated into membrane (MEM) and cytosolic (CYT) fractions. Bars indicate the mean for each treatment, and vertical lines represent the SEM. Top panel, Tyrosine kinase activities measured in membrane and cytosolic fractions of trophoblast stem cell cultures treated with serum-free medium, 10 wg/ml transferrin, or 20% FBS. *, Significantly different from the activity measured in the membrane fraction of stem cells treated with serum-free medium (P < 0.05); **, significantly different from the activity measured in the membrane fraction of stem cell treated with serum-free medium or transferrin (P < 0.05); ***, significantly different from the activity measured in the cytosolic fraction of stem cells treated with serum- free medium or transferrin. Bottom panel, Tyrosine kinase activities measured in membrane and cytosolic fractions of differentiating trophoblast cell cultures treated with serum-free medium or 20% FBS. *, Significantly different from the activity measured in the membrane fraction of differentiating trophoblast cells treated with serum-free medium (P < 0.05).

trophoblast cells was modest compared to the response of trophoblast stem cells (Fig. 6).

Identification of putative substrates for tyrosine kinases present in proliferating and differentiating trophoblast cells. As both proliferating and differentiating trophoblast cells were responsive to a tyrosine kinase inhibitor and possessed tyrosine kinase activities, we next analyzed the spectrum of phosphotyrosine-containing proteins in each population of cells.



328 TROPHOBLAST GROWTH AND DIFFERENTIATION Endo . 1995 Vol 136. No 1

The distribution of phosphotyrosine-containing proteins present in differentially treated trophoblast stem cells was examined by Western blot analysis. After a 24-h exposure to serum-free medium, trophoblast stem cells were exposed to one of four treatments for an additional 24 h before they were harvested and analyzed for their distribution of phosphotyrosine-containing proteins. The treatments included 1) serum-free medium, 2) serum-free medium plus transferrin (10 pg/ml), 3) 10% FBS, and 4) 10% FBS plus TGFPl (10

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rig/ml). Unique patterns of phosphotyrosine-containing proteins were evident for each treatment. Treatment with transferrin resulted in a decrease in the intensity of phosphotyrosine-containing proteins migrating at approximately 37 and 40 kilodaltons (kDa) and others between 50-60 kDa (compare lanes A and B of Fig. 7, top panel). Treatment with FBS resulted in a further decrease in the phosphotyrosine content of the 37-, 40-, and 50- to 60-kDa species and an increase in the phosphotyrosine content of species migrating at approximately 38, 39, and 62 kDa (compare lanes A and C of Fig. 7, top panel). TGFP-treated cells showed a pattern very similar to that of the FBS-treated cells except for the appearance of an additional phosphotyrosine-containing protein migrating at approximately 93 kDa (compare lanes C and D of Fig. 7, top panel).

Western blot analyses of proliferating and differentiating trophoblast cell lysates with antiphosphotyrosine antibodies indicated different distributions of phosphotyrosine-containing proteins in each preparation (Fig. 7, bottom panel). Phosphoproteins of approximately 32,34, and 49 kDa were in greater abundance in proliferating trophoblast cells, whereas phosphoproteins of approximately 26,41,55,57,75, and 93 kDa were in greater abundance in differentiating trophoblast cells. The unique 93-kDa phosphotyrosine-containing species present in the TGFP-treated stem cells (see Fig. 7, top pane0 showed a similar mobility to the 93-kDa phosphotyrosine-containing band present in differentiated trophoblast cells (see Fig. 7, bottom paneI).

Discussion

In this report, the Rcho-1 trophoblast cell line was used to examine the regulation of DNA synthesis in proliferating and differentiating trophoblast cells. The results indicate the uti- lization of different regulatory mechanisms for the control of DNA synthesis in proliferating VS. differentiating trophoblast cells and the dependence of each process on tyrosine kinase signaling pathways (see Fig. 8 for an overview).

FIG. 7. Western blot analysis of tyrosine-phosphorylated proteins in trophoblast stem cells and differentiating trophoblast cells. Tropho- blast cells were lysed in RIPA buffer, and whole cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Nitrocel- lulose filters were probed with antiphosphotyrosine antibodies. Mo- lecular mass standards (X10e3) are shown, Top panel, Analysis of phoshotyrosine-containing proteins present in trophoblast stem cells acutely exposed to conditions that influence DNA synthesis and proliferation. Trophoblast stem cells were incubated in serum-free medium for 24 h, followed by an additional 24-h incubation in one of four treatments. The treatments included: A) serum-free medium (SF), B) transferrin (10 pg/ml), C) 10% FBS, or D) 10% FBS plus TGFpl (10 rig/ml). Arrozuos indicate the locations of some of the phosphotyrosine- containing species differentially affected by the treatments. Bottom panel, Analysis of phosphotyrosine-containing proteins in proliferating and differentiating trophoblast cells. The left panel depicts unique tyrosine-phosphorylated proteins in trophoblast stem cells (day 2) and differentiating trophoblast cells (day 9). The locations of proliferation- or differentiation-enhanced phosphotyrosine-containing protein species are identified by arrows. The rightpanel is a representation of the negative control experiment in which antibody was preabsorbed with excess phosphotyrosine (1 mM).




PROLIFERATION DIFFERENTIATION

EXTRINSIC REGULATION INTRINSIC REGULATION OF DNA SYNTHESIS OF DNA SYNTHESIS

Transferrin TGF-B

Tyrosine Kinases

unique P-Tyr proteins 32,34,49 kDa

Tvrosine Kinasts

unique P-Tyr proteins 26,41,55,57,75,93 kDa

FIG. 8. Control of DNA synthesis in proliferating and differentiating trophoblast cells. This is a schematic diagram that depicts our current knowledge of the regulation of DNA synthesis in trophoblast stem cells and differentiating trophoblast cells. DNA synthesis in trophoblast stem cells is regulated by exogenous factors predominantly found in fetal serum. Transferrin is a positive regulator of DNA synthesis in trophoblast stem cell cultures, and TGFpl is a negative regulator of trophoblast stem cell proliferation. DNA synthesis in differentiating trophoblast cells appears to be under the control of an intrinsic mechanism(s). Differentiating trophoblast cells may secrete factors required to maintain DNA synthesis, or the maintenance of DNA synthesis may be an intrinsic part of the trophoblast giant cell differentiation program. Tyrosine kinases play an important role in the regulation of trophoblast cell proliferation and DNA synthesis. Unique tyrosine kinase activities and tyrosine phosphorylation patterns in proliferating and differentiating trophoblast cells suggest that tyrosine kinases may also be involved in these processes. P-Tyr, Tyrosine-phosphorylated proteins enriched in whole cell lysates from trophoblast stem cells or differentiating trophoblast cells.

Extracellular modulation of trophoblast cell DNA synthesis and proliferation

Proliferating and differentiating trophoblast cells respond differently to extracellular modulators. Trophoblast stem cells use extrinsic mechanisms (exogenous factors: serum, transferrin, and TGFPI) to regulate proliferation and DNA synthesis, whereas DNA synthesis in differentiating trophoblast cells is much less responsive to extracellular modulators and appears to be under the control of intrinsic mechanisms.

Extrinsic modulation of trophoblast stem cells. Rcho-1 trophoblast stem cell proliferation is maximally stimulated by factors present in FBS. The addition of other growth- related supplements has some of the actions of FBS, but none is fully capable of maintaining trophoblast stem cell proliferation. Transferrin is a positive regulator of DNA synthesis in trophoblast stem cells, but is not capable of completely replacing the growth-promoting properties of FBS. Other cells of the trophoblast lineage have also been shown to respond positively to transferrin (20).

The identification of trophoblast cell growth modulatory factors has been elusive. In the present study, we were unable to identify factors that promote trophoblast stem cell proliferation. The placenta is a rich source of a variety of different growth factors and receptors for growth factors (19, 211, many of which have been proposed to regulate trophoblast cell proliferation. However, analysis of the literature does not

provide convincing evidence for the identification of any growth factor that promotes trophoblast cell proliferation. There are a number of technical problems associated with studying the proliferation of trophoblast cells. First of all, trophoblast cell proliferation in vitro is an aberration. Upon removal from their in situ location, trophoblast cells spon- taneously differentiate (2, 4, 22). The factors necessary to prevent spontaneous differentiation have not been eluci- dated. Secondly, homogeneous cultures of primary trophoblast cells are difficult to obtain from any species. Some reports of in vitro growth promotion of placental cells are potentially confounded by nontrophoblast elements of the placenta (19,22). Finally, as DNA synthesis can be associated with trophoblast cell differentiation, the only reliable mea- sure of trophoblast cell proliferation is an increase in trophoblast cell number. These apparent difficulties have made identification of factors that promote trophoblast cell proliferation an arduous task.

Nonetheless, we were successful in identifying a factor that negatively modulates trophoblast cell proliferation. TGFPl acts as an inhibitor of FBS-stimulated Rcho-1 trophoblast stem cell proliferation. These actions appear to be phys- iologically relevant given the localization of TGFPs in the uterine decidua (23,241. Furthermore, the actions of TGFPl are at sites distinct from modulation of trophoblast cell DNA synthesis and are, thus, compatible with endoreduplication and the presence of trophoblast giant cells at the placental- uterine interface. Inhibition of trophoblast cell proliferation may be a necessary first step in the differentiation pathway. The observations are also consistent with the proposed involvement of TGFPs in regulating trophoblast cell invasion in the human (23, 25).

In these experiments, we used Rcho-1 trophoblast cells. Rcho-1 cells have undergone an unidentified transformation event(s) enabling them to proliferate in vitro (8,9,26,27). The nature of the aberration(s) in Rcho-1 trophoblast cells may account for their responses or lack of response to various growth modulators examined in this report. However, most importantly, Rcho-1 trophoblast stem cells are responsive to exogenous stimuli (FE%, transferrin, and TGFPl) and retain the ability to exit the proliferation program and progress along the trophoblast giant cell differentiation program (8,9).

Intrinsic modulation of differentiating trophoblast cells. In contrast to the extrinsic control of trophoblast stem cell DNA synthesis, differentiating trophoblast cells maximally synthesize DNA in the absence of serum and could not be stimulated above this intrinsic level. Modulators of DNA synthesis in proliferating trophoblast cells (serum, transferrin, and TGFPl) had minimal, if any, detectable effects on DNA synthesis in differentiating trophoblast cells. Thus, the nature of the control of DNA synthesis during the endocycle of giant cells may be quite different from that during the cell cycle of diploid cells. Giant cells may secrete factors required for progression of the endocycle. These factors may be the same as those required for progression through the cell cycle of trophoblast stem cells. Preliminary experiments examining the effects of conditioned medium from differentiated trophoblast cells on proliferation or DNA synthesis of trophoblast stem cells did not support this hypothesis (Hamlin, G.



330 TROPHOBLAST GROWTH AND DIFFERENTIATION Endo l 1995 Vol 136 l No 1

P., and M. J. Soares, unpublished results). Alternatively, giant cells may secrete unique regulatory molecules or may use unique intracellular mechanisms for controlling DNA synthesis that bypass extracellular activation.

Tyrosine kinase pathways and trophoblast cells

Our difficulty in elucidating extracellular modulators of trophoblast cell proliferation and differentiation led to an analysis of intracellular pathways used by trophoblast stem cells and differentiating trophoblast cells. We report here the involvement of tyrosine kinase-activated pathways in the control of trophoblast stem cell DNA synthesis and proliferation and in the control of DNA synthesis in differentiating trophoblast cells.

Treatment of stem and differentiating trophoblast cells with the tyrosine kinase inhibitor, genistein, disrupted the abilities of the cells to proliferate and/or synthesize DNA. The related tyrosine kinase inhibitor, lavendustin-A, was ineffective at influencing the behavior of trophoblast stem and differentiating cells even at high doses. In other systems, lavendustin-A and genistein have been reported to possess different effects (28, 29). Genistein is a well documented broad spectrum inhibitor of both receptor (30-32) and nonreceptor (30,33,34) tyrosine kinase pathways. Both receptor and nonreceptor tyrosine kinases have been implicated in the control of the growth and differentiation of a number of different cell lineages (3537). DNA synthesis in trophoblast stem cells and differentiating trophoblast cells may involve nonreceptor tyrosine kinase signaling pathways. This pos- tulate is consistent with the absence of stimulation of DNA synthesis in trophoblast stem cells treated with a number of ligands known to activate receptor tyrosine kinases (present study) and the differential activation of three members of the src family of nonreceptor tyrosine kinases (src, yes, and Zyn) during the transition from trophoblast cell proliferation to differentiation (Hamlin, G. P., and M. J. Soares, unpublished results).

Although tyrosine kinase activities were detectable in both proliferating and differentiating trophoblast cells, there were significant differences in their levels. Tyrosine kinase activities in PBS- or transferrin-treated trophoblast stem cells were considerably higher than those in differentiating trophoblast cells. This observation may be related to the types of tyrosine kinases present in proliferating VS. differentiating trophoblast cells. Different tyrosine kinases may possess different substrate specificities. Consequently, tyrosine kinases present in each cell population may differ in their abilities to use the synthetic peptide (RR-Src) containing the c-src consensus tyrosine phosphorylation site used in our analysis of tyrosine kinase activity. Such a hypothesis is further supported by the unique distributions of phosphotyrosine-con- taming proteins in proliferating VS. differentiating trophoblast cells. Trophoblast stem cells stimulated to proliferate or differentiate were characterized by both the loss and gain of specific phosphotyrosine-containing proteins. Most interest- ingly, TGFP treatment, which appears to be capable of ini- tiating the exit of trophoblast stem cells from the proliferation program, was associated with the appearance of a 93-kDa phosphotyrosine-containing protein also present in differ-

entiated trophoblast cells. Determination of the identity of the 93-kDa and other unique phosphotyrosine-containing proteins and the kinases responsible for their phosphorylation should provide information regarding up- and down- stream regulatory events involved in trophoblast cell proliferation, DNA synthesis, and differentiation.

In conclusion, regulatory mechanisms controlling DNA synthesis in trophoblast cells differ depending on their differentiation state. Proliferating trophoblast cells are under extrinsic control, whereas differentiating trophoblast cells are under intrinsic control. Tyrosine kinase-mediated signal transduction pathways are involved in the regulation of trophoblast cell proliferation, DNA synthesis, and differentiation.

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