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(Circulation Research. 1996;79:757-764.)
© 1996 American Heart Association, Inc.

Articles

The Fumagillin Analogue TNP-470 Inhibits DNA Synthesis of Vascular Smooth Muscle Cells Stimulated by Platelet-Derived Growth Factor and Insulin-like Growth Factor-I

Possible Involvement of Cyclin-Dependent Kinase 2

Hidenori Koyama, Yoshiki Nishizawa, Masayuki Hosoi, Shinya Fukumoto, Kyoko Kogawa, Atsushi Shioi, Hirotoshi Morii

the Second Department of Internal Medicine, Osaka (Japan) City University Medical School.

Correspondence to Yoshiki Nishizawa, MD, Second Department of Internal Medicine, Osaka City University Medical School, 1-5-7, Asahi-machi, Abeno-ku, Osaka 545, Japan.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The effect of an angiogenesis inhibitor, TNP-470, on DNA synthesis and its underlying signaling cascades stimulated by platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I were examined in bovine vascular smooth muscle cells (SMCs). PDGF-BB (10 ng/mL)– and IGF-I (100 ng/mL)–stimulated increase in DNA synthesis was completely abolished by simultaneous treatment with TNP-470 (1.0 ng/mL). TNP-470 had no effects on PDGF receptor autophosphorylation or early signal transduction, such as activation of mitogen-activated protein kinase and immediate early gene expression. PDGF-BB induced an increase in mRNA levels of cyclin D1, cyclin-dependent kinase (cdk) 4, and cdk2, as well as the activity of cdk2, which preceded the G1/S boundary, as estimated by the kinetics of DNA synthesis. The PDGF-BB–induced activation of cdk2 was inhibited by TNP-470, which was correlated with decreased cdk2 mRNA levels. In contrast, TNP-470 had no or less marked effect on cyclin D1 and cdk4 mRNA levels induced by PDGF-BB. TNP-470 also inhibited a much smaller increase in cdk2 mRNA levels and activation stimulated by IGF-I. In conclusion, TNP-470 potently inhibits DNA synthesis of SMCs, and this inhibition is associated with decreased levels of cdk2 mRNA and activity.

Key Words: TNP-470 • bovine vascular smooth muscle cells • platelet-derived growth factor • insulin-like growth factor-I • cyclin-dependent kinase 2

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Migration and proliferation of SMCs from the media into the intima leads to accumulation of SMCs during the development of atherosclerosis.¹ Proliferation of SMCs can be stimulated by a number of growth factors present in the vascular wall and atherosclerotic lesions, including PDGF and IGF-I.^{2 3 4 5} PDGF is one of the major mitogens for SMCs^{6 7} and a potent chemoattractant for them.⁸ PDGF has been shown to be involved in the accumulation of SMCs after angioplasty of rat carotid artery in vivo.^{9 10} IGF-I is also a potent chemoattractant for SMCs¹¹ and stimulates DNA synthesis with less potency than PDGF in rat,¹² bovine,¹³ porcine,¹⁴ and human strains.¹⁵

In the G0 phase of the cell cycle, growth and cell division are arrested, but several growth factors are able to initiate a complex series of events causing cells to traverse G1, replicate DNA during the S phase, and ultimately divide. On entering the cell cycle, certain gene products are stimulated, such as MAP kinase,¹⁶ ornithine decarboxylase, c-myc,¹⁷ and cell-cycle kinase p34^cdc2,¹⁶ while others are downregulated, such as smooth muscle -actin¹⁸ and a diverged homeobox gene, Gax.¹⁹ In contrast, transforming growth factor-ß1 can inhibit SMC replication, leading to G1 arrest, which is associated with a decrease in p34^cdc2 kinase activity.²⁰ However, only limited information is available regarding the regulation of G1 cyclins and cdks and their control of cell-cycle progression in SMCs.²¹

TNP-470, a synthetic analog of fumagillin, can inhibit the angiogenic response in vivo and in vitro and possibly suppress the growth of solid tumors.²² It has been shown that TNP-470 selectively inhibits the proliferation of endothelial cells, since as much as 10⁶ times higher concentrations were needed to inhibit proliferation of a variety of cancer cell lines.²³ However, the effects of TNP-470 on the proliferation of normal cells have not been examined extensively.

In the present study, we examined the effects of TNP-470 on SMC DNA synthesis. We found that TNP-470 potently inhibits DNA synthesis in bovine SMCs stimulated by PDGF-BB and IGF-I at concentrations comparable to those used for endothelial cells. The suppressive effect of TNP-470 on PDGF-BB–stimulated cell growth was associated with a decrease in activity and mRNA levels of cdk2 without reducing the mRNA levels of cyclin D1. TNP-470 also inhibits SMC proliferation induced by IGF-I, although the effects of IGF-I on cdk2 mRNA levels and activity were much smaller than those of PDGF.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Medial SMCs were obtained from bovine aorta by the explant method as originally described.²⁴ Cells were grown in DMEM supplemented with 10% FCS and were transferred when they reached 80% subconfluence to serum-free DMEM containing 0.1% BSA (fraction V, essentially fatty acid–free; Sigma Chemical Company), 2.5 µg/mL holotransferrin (Sigma), and 35 µg/mL ascorbic acid for 72 hours to make the cells quiescent. Thereafter, the following test materials were added: TNP-470 (a generous gift of Dr K. Sudo, Takeda Pharmaceutical Co, Osaka, Japan), recombinant human PDGF-BB (Genzyme), and recombinant human IGF-I (Malinckrodt). Cells were used in passages 5 to 10 and were characterized by expression of smooth muscle -actin, as determined by immunoblot and immunocytochemical analysis. Similar results were obtained with two different strains of SMCs.

Measurements of DNA Synthesis and Proliferation
The DNA synthesis of SMCs was evaluated by [³H]thymidine incorporation assays. Cells were grown in 48-well plates and, when they reached subconfluence, were changed to serum-deprived DMEM for 72 hours. After 72 hours, test materials were added, and the cells were incubated for the indicated periods of time and labeled with 2 µCi/mL [³H]thymidine (New England Nuclear) for the last 2 hours in the presence of growth factors and reagents. [³H]Thymidine incorporation into DNA was measured as TCA-insoluble radioactivity as described previously.²⁵ Cell proliferation was measured by determining cell number. Cells were suspended by digestion with 0.01% trypsin-0.1 mmol/L EDTA, fixed in Holley's fixative (3.7% formaldehyde, 86 mmol/L NaCl, and 106 mmol/L Na₂SO₄), and counted in a cell counter (Coulter).

Evaluation of Protein Tyrosine Phosphorylation, MAP Kinase, and cdk2
Cells in 100-mm dishes were quickly rinsed with PBS, harvested in lysis buffer-I (20 mmol/L Tris-HCl, pH 8.0, 1% Triton X-100, 10% glycerol, 137 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EDTA, 50 mmol/L NaF, 2 mmol/L Na₃VO₄, 1 mmol/L PMSF, 20 µmol/L leupeptin, and 10 µg/mL of aprotinin) for evaluation of protein tyrosine phosphorylation and MAP kinase assay or lysis buffer-II (50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 2.5 mmol/L EGTA, 0.1% Tween 20, 10% glycerol, 0.1 mmol/L PMSF, 20 µmol/L leupeptin, 10 µg/mL of aprotinin, 10 mmol/L ß-glycerophosphate, 1 mmol/L NaF, and 0.1 mmol/L Na₃VO₄) for cdk2 assay. For assay of cdk2, samples were sonicated twice for 10 seconds each on ice and clarified by centrifugation at 14 000g for 10 minutes. For Western blot analysis of tyrosine-phosphorylated proteins, the supernatant was separated on 8.0% nonreducing SDS-PAGE as described by Laemmli.²⁶ The proteins were transferred to polyvinylidene fluoride membranes (Immobilon, Millipore Corp) and immunoblotted with antiphosphotyrosine antibody (clone PY20, ICN Biomedicals Inc), using a horseradish peroxidase–conjugated rabbit anti-mouse IgG as the second antibody, and developed by the enhanced chemiluminescence method (Amersham International). MAP kinase and cdk2 activities were determined by immunoprecipitation followed by in vitro kinase assay. Cell extracts precleared with protein A–Sepharose (Pharmacia) were incubated with anti-rat MAP kinases R2 (UBI) or anti-human cdk2 (UBI), followed by precipitation with protein A–Sepharose. After the immunoprecipitates were washed three times with lysis buffer and twice with kinase buffer (in mmol/L: HEPES 50, pH 7.5, MgCl₂ 10, EGTA 2.5, ß-glycerophosphate 10, NaF 1, and Na₃VO₄ 0.1), they were incubated at 30°C for 10 minutes (MAP kinase) or 30 minutes (cdk2) in 30 µL of kinase buffer containing 20 µmol/L [-³²P]ATP, 1 mmol/L dithiothreitol, and substrates (30 µg of myelin basic protein [Sigma] for MAP kinase; 1 µg of histone H1 [Boehringer Mannheim] for cdk2). The reactions were stopped by the addition of 2x Laemmli sample buffer, and samples were analyzed by nonreducing SDS-PAGE followed by autoradiography or cutting substrate bands from the gels to measure the radioactivities by scintillation counting. Protein concentration was determined by a method described by Bradford.²⁷

Northern Blot Analysis and cDNA Probes
For Northern blot analysis, 20 µg of total cellular RNA, prepared by the guanidine isothiocyanate/phenol/chloroform extraction method,²⁸ was separated by formaldehyde/1.0% agarose gel electrophoresis and transferred to a nylon membrane (Hybond N, Amersham). cDNAs for human c-fos and c-myc (obtained from the Japanese Cancer Research Resources Bank); mouse cyclins D1, D2, and D3; and mouse cdk2 and cdk4 (cDNAs for cyclin D1, cdk2, and cdk4 were kindly provided by Dr H. Matsushime, University of Tokyo, Japan) were used as probes for hybridization. The probes were labeled by the random-primed method, and hybridization was performed as described.²⁹ Laser densitometry (Pharmacia LKB 2222 UltroScan XL) was used to quantify the relative signal intensities of the bands obtained.

Statistical Analysis
Statistical analysis was done by using Student's t test or ANOVA combined with multiple comparison (Scheffe's type). These analyses were performed with the assistance of a computer program (SuperANOVA, Ver 1.1, Abacus Concepts).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA Synthesis Is Stimulated in SMCs by PDGF-BB and IGF-I and Inhibited by TNP-470
Subconfluent quiescent cultures of SMCs were stimulated with increasing concentrations of PDGF-BB and IGF-I (Fig 1A). At 22 hours, the incorporation of [³H]thymidine into cellular DNA was determined. Both PDGF-BB and IGF-I increased DNA synthesis in a dose-dependent manner, with their maximal increases observed at 10 and 100 ng/mL, respectively (Fig 1A). To determine the kinetics of DNA synthesis, the time course of thymidine incorporation was examined after treatment with the growth factors. The kinetics of thymidine incorporation after stimulation with PDGF-BB and IGF-I were very similar (Fig 1B); the G1/S boundary was estimated to be around 12 hours, maximal DNA synthesis was 21 hours, and return to basal levels was observed by 48 hours (Fig 1B). On the basis of these experiments, 10 ng/mL PDGF-BB and 100 ng/mL IGF-I were used, and thymidine incorporation was determined at 21 hours in subsequent experiments to evaluate the effect of the antiangiogenic compound TNP-470. Fig 1C and 1D shows that TNP-470 inhibited PDGF-BB– and IGF-I–stimulated DNA synthesis of SMCs. Complete inhibition was achieved at 1.0 ng/mL TNP-470 (IC₅₀, 0.3 ng/mL) for both PDGF-BB and IGF-I stimulation. TNP-470 also marginally but significantly suppressed basal DNA synthesis (Fig 1D). DNA synthesis was also inhibited by 48 hours in TNP-470 (10 ng/mL)–treated cells stimulated with both growth factors (data not shown). The decrease in DNA synthesis in cells treated with TNP-470 was associated with suppression of cell number increase (Fig 1E). Both PDGF-BB and IGF-I failed to increase cell number in the presence of TNP-470 (Fig 1E). In contrast to PDGF-BB and IGF-I, SMC DNA synthesis stimulated by 10% FCS was only partially inhibited by TNP-470, at concentrations as much as 100 ng/mL; however, IC₅₀ of TNP-470 action appeared to be comparable to PDGF- and IGF-I–treated conditions (Fig 1F). Cell viability, as determined by trypan blue dye exclusion, was always >95% after the addition of TNP-470 at concentrations up to 100 ng/mL. No changes in cell shape were observed in cells treated with TNP-470. From these experiments, 10 ng/mL TNP-470 was used for further experiments to evaluate the molecular mechanism of TNP-470 action.

View larger version (38K): [in this window] [in a new window]   Figure 1. DNA synthesis in bovine SMCs. A, Both PDGF-BB and IGF-I stimulate increase in a dose-dependent DNA synthesis in bovine SMCs. Subconfluent cells were incubated in serum-free DMEM supplemented with 0.1% BSA, 2.5 µg/mL holotransferrin, and 35 µg/mL ascorbic acid for 72 hours before addition of growth factors. Thereafter, cells were incubated for 20 hours in the presence of indicated concentrations of PDGF-BB or IGF-I followed by labeling with 2 µCi/mL [3H]thymidine for 2 hours. DNA synthesis was measured as TCA-insoluble radioactivity. , PDGF-stimulated and , IGF-I–stimulated DNA synthesis. Values are presented as mean±SD of quadruplicate samples. *P<.05 vs 0 ng/mL, multiple comparison (Scheffe's type). B, The kinetics of DNA synthesis induced by PDGF-BB and IGF-I are comparable in bovine SMCs. Quiescent bovine SMCs were treated with 10 ng/mL PDGF-BB or 100 ng/mL IGF-I for the indicated time. DNA synthesis was determined as described above. , PDGF-stimulated and , IGF-I–stimulated DNA synthesis. Values are presented as mean±SD of quadruplicate samples. C and D, TNP-470 dose dependently inhibits basal as well as PDGF- and IGF-I–stimulated DNA synthesis. Quiescent bovine SMCs were treated for 21 hours with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I in the presence or absence of indicated concentrations of TNP-470. DNA synthesis was determined as described above. Values represented on graphs are mean±SD. *P<.05 vs TNP-470 (-), multiple comparison (Scheffe's type). E, TNP-470 inhibits cell number increase induced by PDGF-BB and IGF-I. Quiescent SMCs were treated with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I alone or in combination with 10 ng/mL TNP-470. Cell numbers were counted after 72 hours. Values are mean±SD of triplicate samples. *P<.05 vs control; **P<.05 vs TNP-470 (-). F, TNP-470 inhibition of FCS-stimulated DNA synthesis is less marked than that observed with PDGF or IGF-I. Quiescent bovine SMCs were treated for 21 hours with 10% FCS alone or in combination with the indicated concentrations of TNP-470. DNA synthesis was determined as described above. Values are mean±SD of quadruplicate samples. *P<.05 vs TNP-470 (-), multiple comparison (Scheffe's type). Each experiment was repeated at least twice, with identical results.

Protein Tyrosine Phosphorylation
To examine the possibility that TNP-470 inhibits early signaling events stimulated by PDGF-BB and IGF-I, cellular protein tyrosine phosphorylation and MAP kinase activation were studied. For these experiments, TNP-470 was added 1 hour before the cells were treated with the growth factors. Pretreatment periods of up to 12 hours with TNP-470 did not alter the results observed. Shown in Fig 2 is the Western blot analysis of phosphotyrosine-containing proteins. Stimulation of SMCs with PDGF-BB for 10 minutes led to increases in the amount of tyrosine-phosphorylated proteins, including a 180-kD PDGF receptor. TNP-470 treatment resulted in a small but reproducible decrease in PDGF-BB–stimulated protein tyrosine phosphorylation. In contrast, IGF-I–stimulated protein tyrosine phosphorylation was barely detected in this system, and TNP-470 had no apparent modulatory effects on IGF-I–induced phosphorylation.

View larger version (108K): [in this window] [in a new window]   Figure 2. Protein tyrosine phosphorylation in bovine SMCs. Subconfluent bovine SMCs were incubated in serum-free DMEM containing 0.1% BSA, 2.5 µg/mL holotransferrin, and 35 µg/mL ascorbic acid for 3 days to make the cells quiescent. After the cells were pretreated with or without 10 ng/mL TNP-470 for 1 hour, they were stimulated for 10 minutes with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I in the presence (+) or absence (-) of 10 ng/mL TNP-470 at 37°C. Cell lysates were fractionated in 8.0% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antiphosphotyrosine antibody.

MAP Kinase Activation
We next examined the effect of TNP-470 on growth factor–stimulated MAP kinase activation. PDGF-BB caused an increase in MAP kinase activity as early as 2.5 minutes, with its peak at 10 minutes (Fig 3A). TNP-470 did not suppress either basal or PDGF-BB–stimulated MAP kinase activity at either 5 or 10 minutes (Fig 3B). In contrast to PDGF-BB, IGF-I failed to enhance MAP kinase activity by 30 minutes (Fig 3A), and TNP-470 did not show any major modulatory effect on IGF-I action (Fig 3B).

View larger version (90K): [in this window] [in a new window]   Figure 3. MAP kinase activation in bovine SMCs. A, Quiescent cells were stimulated with 10 ng/mL PDGF-BB or 100 ng/mL IGF-I for the indicated times. B, MAP kinase activation induced by PDGF-BB is not inhibited by TNP-470. Quiescent cells were pretreated with (+) or without (-) 10 ng/mL TNP-470 for 1 hour and then treated with vehicle, 10 ng/mL PDGF-BB, or 100 ng/mL IGF-I for 5 and 10 minutes. Cell lysates were incubated with anti-MAP kinase antibody, followed by precipitation with protein A–Sepharose. Immune complexes were assayed for kinase activity, with myelin basic protein as a substrate. Lower panels of A and B show the summary of results obtained from three separate experiments. Values represented on graphs are mean±SD. A, , PDGF-BB; , IGF-I. *P<.05 vs 0 minute, multiple comparison (Scheffe's type). B, Open bars show results at 5 minutes; solid bars, 10 minutes.

Immediate Early Gene Expression
We next examined the effect of TNP-470 on immediate early gene expression induced by PDGF-BB and IGF-I. Preliminary experiments revealed that both PDGF-BB and IGF-I increased the levels of c-fos mRNA, with the maximum response observed at 30 to 60 minutes (data not shown). c-myc mRNA levels were also increased by both PDGF-BB and IGF-I, with a maximal effect at 60 minutes (data not shown). On the basis of these observations, the effect of TNP-470, added 1 hour before the addition of growth factors, on immediate early gene expression was examined at 60 minutes after treatment with growth factors. As shown in Fig 4, TNP-470 did not affect c-fos or c-myc mRNA levels induced by either PDGF-BB or IGF-I.

View larger version (43K): [in this window] [in a new window]   Figure 4. Immediate early gene expression in bovine SMCs. After quiescent SMCs were pretreated for 1 hour with (+) or without (-) TNP-470, the cells were stimulated for 60 minutes with vehicle, 10 ng/mL PDGF, or 100 ng/mL IGF-I, and the cells were harvested for RNA isolation. Total RNA (20 µg per lane) was separated on 1.0% agarose gel, blotted onto a nylon membrane, and hybridized with c-fos and c-myc cDNAs. The right panel shows the summary of the results of densitometric analysis for c-fos and c-myc mRNA levels corrected for rRNA, obtained from three individual experiments.

cdk2 Activity
The lack of effect of TNP-470 on early signaling events involved in G0/G1 transition suggested the possibility that TNP-470 might arrest cells in G1. We therefore focused on cdk2 activity, since cdk2 is a primary candidate for regulation of G1, possibly by interacting with its regulatory partners, cyclins D, E, and A.³⁰ As shown in Fig 5A, after the addition of PDGF-BB, cdk2 activity associated with anti-cdk2 immunoprecipitates was increased as early as 8 hours and reached a maximum at 20 hours (Fig 5A). This time course of cdk2 activation preceded that of S-phase activity determined by [³H]thymidine incorporation. Unexpectedly, the effect of IGF-I on cdk2 activity was much less than that of PDGF-BB, despite the fact that the maximal effect of IGF-I was almost 70% to 80% as much as that of PDGF-BB in inducing DNA synthesis. As shown in Fig 5B, when TNP-470 was simultaneously added with PDGF-BB, PDGF-stimulated cdk2 activity was almost completely abrogated at 20 hours, while marginal inhibition was observed at 12 hours (Fig 5B). The smaller increase in anti-cdk2–immunoprecipitated kinase activity induced by IGF-I at 20 hours was also significantly suppressed by TNP-470 (Fig 5B).

View larger version (78K): [in this window] [in a new window]   Figure 5. cdk2 activity in bovine SMCs. Subconfluent bovine SMCs were incubated in serum-free DMEM containing 0.1% BSA, 2.5 µg/mL holotransferrin, and 35 µg/mL ascorbic acid for 3 days. These quiescent cells were incubated for the indicated times with 10 ng/mL PDGF or 100 ng/mL IGF-I in the presence (+) or absence (-) of 10 ng/mL TNP-470. Cell lysates were incubated with anti-cdk2 antibody, followed by precipitation with protein A–Sepharose. Immune complexes were assayed for cdk2 activity, using histone H1 as a substrate. A, Time course analysis of cdk2 activity was performed after the stimulation with PDGF-BB or IGF-I. The lower panel summarizes three separate experiments. , PDGF-BB; , IGF-I. *P<.05 vs 0 hour, multiple comparison (Scheffe's type). B, TNP-470 inhibits cdk2 activity to varying extents, depending on the time of analysis. The lower panel shows the summary of results of three experiments. Open bars show results at 12 hours; solid bars, 20 hours. Values represented on graphs are mean±SD. *P<.05 vs TNP-470 (-), Student's t test.

cdk2 Gene Expression
To further understand the molecular mechanism involved in TNP-470 suppression of cdk2 activity, Northern blot analysis of cdk2 was performed. As shown in Fig 6A, PDGF-BB increased the mRNA levels of cdk2 and cdk4 as early as 6 hours, with the maximal effect observed at 12 to 16 hours. Increases in mRNA levels for cyclin D1, which appears to be a regulatory subunit of cdk2 and cdk4, preceded the increased levels of cdk2 and cdk4 mRNA, with maximal increases observed at 6 hours and maintained for up to 20 hours. Consistent with the smaller effect of IGF-I on cdk2 activity, IGF-I had only a small effect on mRNA levels of cdk2 (Fig 6B). Although IGF-I increased the levels of cyclin D1 mRNA by 100%, increases in cdk4 mRNA levels were about 150%. Cyclin D2 and D3 mRNA were not detected in any of these experiments. On the basis of these results, we examined the effect of TNP-470 on mRNA levels at 12 hours. As shown in Fig 6C, simultaneous addition of TNP-470 completely suppressed PDGF-induced increases in cdk2 mRNA levels, with a partial decrease in cdk4 mRNA levels. In contrast, PDGF-stimulated cyclin D1 mRNA levels were further increased by the simultaneous treatment with TNP-470. IGF-I induced smaller increases in cdk2 mRNA levels that also appeared to be suppressed by TNP-470. IGF-I stimulated modest increases in cdk4 mRNA levels that were only partially (about 30%) suppressed by TNP-470.

View larger version (143K): [in this window] [in a new window]   Figure 6. Cyclin D1, cdk2, and cdk4 mRNA levels in bovine SMCs. Quiescent SMCs were stimulated for the indicated hours with 10 ng/mL PDGF (A) or 100 ng/mL IGF-I (B), and the cells were harvested for RNA isolation. Total RNA (20 µg per lane) was separated on 1.0% agarose gel, blotted onto a nylon membrane, and hybridized with the indicated cDNA probes. The graphs in A and B show the results of densitometric analysis of mRNA levels for time points, corrected for rRNA, summarized from three separate experiments. , cyclin D1; , cdk2; and , cdk4 mRNA levels. Values are mean±SD. Cyclin D1, P<.05 vs 0 hours; cdk2, P<.05 vs 0 hours; and cdk4, P<.05 vs 0 hours, multiple comparison (Scheffe's type). C, Effects of TNP-470 on cyclin D1, cdk2, and cdk4 mRNA levels. The graph on the right shows the summary of the densitometric analysis of mRNA levels for each condition, corrected for rRNA, obtained from three separate experiments. Values are mean±SD. P<.05 vs TNP-470 (-), Student's t test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present studies were undertaken to determine the effect of the antiangiogenic substance TNP-470 on PDGF-BB– and IGF-I–stimulated DNA synthesis of SMCs. We demonstrate that TNP-470, at concentration levels comparable to those used on endothelial cells, inhibited SMC DNA synthesis stimulated by both PDGF-BB and IGF-I. The inhibitory effect of TNP-470 on DNA synthesis was associated with suppression of increases in cdk2 mRNA levels and cdk2 activation.

TNP-470 Potently Inhibits DNA Synthesis in SMCs Stimulated by PDGF-BB and IGF-I
Previous reports showed that the growth-inhibitory effect of TNP-470 appears to be specific for normal endothelial cells compared with cancer cell lines. Abe et al³¹ reported that WI-38 human fibroblasts were less sensitive to TNP-470. It was also reported that TNP-470 had no or less suppressive effect on the growth of leukemic cells or several other cancer cells in vitro.^{22 23} The effect of TNP-470 on diploid cells other than endothelial cells has not been examined, with the exception of human embryonic lung fibroblasts, which appear to be as sensitive to TNP-470 inhibition as endothelial cells.²² The possible pathophysiological role of SMC proliferation in atherogenesis led us to examine the effect of TNP-470 on the proliferation of normal vascular SMCs in vitro. TNP-470 inhibits SMC DNA synthesis, with its IC₅₀ at 0.3 ng/mL. As reported previously,^{22 23 31 32 33} TNP-470 inhibited DNA synthesis of bovine and human endothelial cells, with their IC₅₀s at 0.03 ng/mL and 0.3 ng/mL, respectively, but not human leukemic cells (HL-60 cells), with IC₅₀ up to 10 ng/mL (data not shown). These results suggest that SMCs appear as sensitive to TNP-470 as endothelial cells. Consequently, TNP-470 as low as 1.0 ng/mL completely inhibited DNA synthesis of SMCs stimulated both by PDGF-BB and IGF-I.

Mechanism of Action of TNP-470
The mechanism of action by which TNP-470 inhibits cell proliferation is unclear. Antoine et al³² reported that TNP-470 prevents quiescent bovine endothelial cells from entering the G1 phase of the cell cycle. In contrast, Abe et al,³¹ using human endothelial cells, showed that this inhibitor may act in late G1, where cyclin E and cyclin A, but not cyclin D, mRNA levels were inhibited. They also observed that TNP-470 inhibits cdk2 activation and retinoblastoma phosphorylation. Recently, Hori et al,³³ using human endothelial cells, have shown that TNP-470 inhibits the increases in cyclin D1 mRNA levels. In each report, different combinations of several growth factors, including FCS, were used to stimulate endothelial cells to enter the cell cycle and thus may be responsible for the discrepancies within different reports. We observed that the inhibitory effect of TNP-470 on SMC DNA synthesis was much less marked when the cells were stimulated with FCS than with PDGF-BB and IGF-I. This observation suggests that the mechanism of TNP-470 action may differ among different growth factors. Bovine aortic SMCs provide a system in which the mechanism of inhibition of DNA synthesis stimulated by a single growth factor can be examined in serum-free defined media. Furthermore, the time course of DNA synthesis was monophasic and returned to basal levels after 48 hours, suggesting that cells are relatively synchronized by serum deprivation and that a large proportion of the cells accumulated to pass through S phase to G2 interphase in response to PDGF or IGF-I.

TNP-470 completely abrogated DNA synthesis in bovine SMCs 21 hours after treatment with PDGF-BB or IGF-I. In TNP-470–treated cells, both growth factors failed to induce DNA synthesis for as long as 48 hours, suggesting that the cells were arrested at G0 or G1 and were not delayed in the cell cycle. Furthermore, TNP-470 did not influence early signaling events induced by PDGF-BB or IGF-I, such as PDGF receptor autophosphorylation, tyrosine protein phosphorylation, MAP kinase activation, and immediate early gene expression, suggesting that TNP-470 arrests the SMC cycle at G1. These observations agree with recent observations in endothelial cells.³¹ The possible arrest of SMCs at G1 by TNP-470 led us to examine a possible G1/S regulating molecule, cdk2.³⁰ cdk2 activity in bovine SMCs was dramatically stimulated by PDGF-BB as early as 8 hours, and the maximal activation was observed at 20 hours. This time course of cdk2 activity preceded the G1/S boundary, as estimated by the time course of thymidine incorporation. TNP-470 almost completely abrogated cdk2 activity at 20 hours, whereas only partial inhibition was observed at 12 hours. TNP-470 inhibition of kinase activity was associated with decreased mRNA levels for cdk2. mRNA levels for cdk2 were suppressed to basal levels by TNP-470 12 hours after treatment with PDGF-BB. The inhibitory effect of TNP-470 on PDGF-stimulated increases in cdk2 mRNA levels was relatively specific to cdk2, since cyclin D1 mRNA levels were slightly increased at the time point examined and cdk4 mRNA levels were suppressed to a lesser extent. Since the cyclin E-cdk2 complex is regarded as a checkpoint of G1/S transition, and cyclin A-cdk2 is thought to play a role in sustaining S phase, our data raise the possibility that the decrease in cdk2 activity after treatment with TNP-470 may be involved in its inhibition of DNA synthesis.

PDGF-BB– and IGF-I–Stimulated DNA Synthesis Might Be Mediated by Distinct Signaling Systems
Our data further suggest that PDGF-BB– and IGF-I–stimulated DNA synthesis may be mediated by distinct signaling systems. Although IGF-I stimulated maximal DNA synthesis at a level equivalent to 70% to 80% of that observed with PDGF-BB, much less increase in the mRNA levels of cyclin D1 (100%) and cdk2 (30%) was observed with IGF-I than with PDGF stimulation. PDGF-BB caused a 500% increase in cyclin D1 and a 200% increase in cdk2 mRNA levels. IGF-I was reported to induce cyclin D1 mRNA levels in MG63 human osteosarcoma cells;³⁴ however, in mouse and human fibroblasts, IGF-I was found to have no effect.^{35 36} These discrepancies may be explained by the differences in responsiveness of cells to IGF-I. For MG63 cells, IGF-I is a potent mitogen, whereas for fibroblasts, IGF-I is a relatively poor stimulant of DNA synthesis. Thus, in bovine SMCs there is a clear discrepancy of IGF-I actions between induction of DNA synthesis and cyclin D1 expression. cdk4, a possible regulator of G1 transition, is induced by IGF-I to almost a similar extent as by PDGF. cdk4 expression in controlling the cell cycle has been implicated in the case of the action of transforming growth factor-ß in other cell systems.³⁷ Studies are currently under way to determine whether IGF-I can influence cdk4 protein expression and kinase activity.

After cells were treated with IGF-I, we failed to detect significant tyrosine phosphorylation of cellular proteins, including IGF-I receptor. This finding may be the result of downregulation of IGF-I receptor, since depriving cells of serum for 72 hours was reported to cause marked downregulation of IGF-I receptor in SMCs.³⁸ IGF-I also failed to stimulate MAP kinase activity, in contrast to PDGF-BB. IGF-I could induce c-myc and c-fos, suggesting that induction of immediate early gene expression by IGF-I may be mediated by a signaling system distinct from the MAP kinase cascade. Depending on the cell type, IGF-I has been reported to activate MAP kinase in some cells,^{39 40} as has insulin.^{41 42} However, since IGF-I failed to induce MAP kinase in human SMCs,¹¹ the signaling cascade induced by IGF-I is undoubtedly cell-type specific. Therefore, it will be important to identify early signaling events induced by IGF-I in SMCs.

In summary, TNP-470 potently inhibits DNA synthesis of bovine SMCs stimulated by PDGF-BB or IGF-I and is associated with an inhibition of cdk2 mRNA levels and activity.

	Selected Abbreviations and Acronyms

 

cdk = cyclin-dependent kinase IGF-I = insulin-like growth factor-I MAP = mitogen-activated protein PDGF = platelet-derived growth factor SMC = smooth muscle cell

	Acknowledgments

 
We thank Dr Hitoshi Matsushime (University of Tokyo, Japan) for providing us with cDNAs for murine cyclin D1, cdk2, and cdk4 and Dr K Sudo, Takeda Pharmaceutical Co, Osaka, Japan, for providing TNP-470. We are grateful to Dr Karin E. Bornfeldt, Dr Elaine W. Raines, and Dr Russell Ross (University of Washington, Seattle) for helpful suggestions and critical reading of the manuscript.

Received June 13, 1996; accepted July 24, 1996.

	References

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