Induction of Expressions of c-fos and c-myc Protooncogenes by Basic Calcium Phosphate Crystal: Effect of β-Interferon Herman S. Cheung, Peter G. Mitchell and W. Jackson Pledger Cancer Res 1989;49:134-138. Published online January 1, 1989.
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[CANCER RESEARCH 49, 134-138. January 1, 1989]
Induction of Expressions of c-fos and c-myc Protooncogenes by Basic Calcium Phosphate Crystal: Effect of ß-Interferon1 Herman S. Cheung, Peter G. Mitchell,2 and W. Jackson Pledger Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 [H. S. C.], and Department of Cell Biology, Vanderbill University, School of Medicine, Nashville, Tennessee 37232 [P. G, M., W. J. P.]
ABSTRACT
factors in platelet-poor plasma (10). BCP crystals can substitute for PDGF as a "competence" factor (11).
Basic calcium phosphate (BCP) crystals control the traverse of cells from the Go-Gi to S-phase of the cell cycle and initiate proliferation byrendering fibroblasts competent to respond to insulin-like growth factors in plasma. The present study examines whether BCP crystals induce transcription of the protooncogenes c-fos and c-myc and the effect of iiinterferon (IFN-/3) on protooncogene transcription as well as BCP crystalinduced DNA synthesis. Stimulation of density-arrested BALB/C-3T3 cells with either BCP crystal or platelet-derived growth factor (PDGF) results in maximal accumulation of c-fos mRNA at 30 min after stimu lation. Induction of c-myc transcription by BCP crystal or PDGF occurs within l h and is maximal at around 3 h after stimulation. Simultaneous addition of IFN-0 with either BCP crystals or PDGF had little effect on c-fos induction but delayed both c-myc message accumulation and entry into S phase. The delay in c-myc message induction after IFN-/3 treatment cannot account for the observed delay in the onset of DNA synthesis, since IFN-/3 can be added at up to 6 h after stimulation with either PDGF or BCP crystals, and a similar delay in the onset of DNA synthesis is still observed.
PDGF directly activates the expression of several genes, and at least 5 PDGF-inducible genes have been isolated (12). More over, c-fos and c-myc appear to belong to the competence gene family in 3T3 cells (13-15). The induction of c-fos by PDGF occurs within minutes, and the amount of c-fos mRNA is maximal at 30 min after stimulation (15, 16). Induction of cmyc transcription by PDGF occurs within an hour and is maximal at 3 h after stimulation. Unlike c-fos, stimulated cells maintain elevated transcription of c-myc for at least 5 h (13, 14). The addition of IFN-/8 concomitant with serum or a combi nation of growth factors to density-arrested 3T3 cells delays or completely blocks the G0-Gi- to S-phase transition (17-19). Furthermore, Einat and coworkers have shown that IFN-/3 inhibited the PDGF-initiated increase of transcription of c-fos and c-myc. They suggest that such inhibition of the expression of genes belonging to the competence gene family may account for the block in the G0-Gi- to S-phase transition (20). Since BCP crystal is a competence growth factor (11), we were interested in whether BCP crystal could stimulate tran scription of c-fos and c-myc and the effect of IFN-0 on such transcriptions as well as on the BCP crystal-induced mito genesis. In the present study, we show that BCP crystals induce the expression of both protooncogenes in BALB/C-3T3 cells with a similar time course as PDGF. Since endocytosis and dissolution of the BCP crystals require several hours. This argues against these processes being directly involved in the initiation of cell activation, although these processes may be essential for the progression phase of ( >,.Concomitant addition of IFN-0 with either BCP crystals or PDGF delays but does not block transcription of c-myc and possibly c-fos. Addition ally, such treatment delays the onset of DNA synthesis.
INTRODUCTION Synovial hyperplasia is a feature of chronic synovitis associ ated with BCP3 crystals which include hydroxyapatite, octacalcium phosphate, and tricalcium phosphate (1, 2) or calcium pyrophosphate dihydrate (3, 4). Each of these crystals, as well as calcium unite, and Ca2Cl2-methylene-diphosphonate have been shown to stimulate mitosis of cultured human skin fibro blasts or canine synovial fibroblasts in a concentration-depend ent fashion (5, 6). Both the onset and the peak of thymidine incorporation after crystal addition to quiescent cells were delayed by 2 to 3 h as compared to the effect of 10% serum. Conditioned media from these cultures were not mitogenic nor were control particles such as sodium urate, diamond crystals, or latex beads (5). Synthetic BCP crystals labeled uniformly with 45Ca were solubilized after endocytosis by synovial cells, fibroblasts, and peripheral blood monocytes (7, 8). Such solubilization was inhibited by lysosomotropic agents, such as chlo roquine and ammonium chloride, in concentrations that signif icantly blocked the mitogenic effect of crystals but not of serum, suggesting that mitogenesis and intracellular dissolution are related phenomena (6). Serum contains two types of growth factors that function sequentially to stimulate mitosis in density-arrested BALB/c3T3 cells and possibly other anchorage-dependent cell types as well (9). PDGF is the serum component that renders quiescent BALB/C-3T3 cells "competent" to respond to "progression" Received 7/6/88; revised 9/29/88; accepted 10/4/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported in part by USPHS Grants AR38421 (H. S. C.), CA42713 (W. J. P.), and CA42636 (W. J. P.) and by Research Career Develop ment Award AM 1065 (H. S. C.). 2 Present address: Division of Rheumatology, Department of Medicine, Med ical College of Wisconsin, 8700 W. Wisconsin Ave., Milwaukee, WI 53226. 3The abbreviations used are: BCP, basic calcium phosphate: PDGF, plateletderived growth factor, IFN-tf, (i-interferon; DMEM, Dulbecco-Vogt-modified Eagle's medium; PBS, phosphate-buffered saline.
MATERIALS AND METHODS Cell Culture. Stock cultures of BALB/C-3T3 cells (clone A31) were grown in DMEM supplemented with 10% (v/v) calf serum, 4 BIMLglutamine, penicillin at 50 units/ml, and streptomycin at 50 ¿ig/mlin humidified 5% CO2/95% air at 37°C.For all experimental procedures, cells were grown to confluence in serum-containing medium and used 2 to 3 days after growth cessation. DNA Synthesis Assay. Cells in 96-cell plates were incubated with [3H]thymidine (5 ¿iCi/ml),rinsed with PBS, treated twice for 10 min with methanol, and then rinsed with water. The bottom of each well was then punched out, and thymidine incorporation was measured by scintillation counting. Preparation of RNA. RNA was prepared by scraping the cells from 100-mm plates and washing in PBS, followed by disruption of cells in guanidinium isothiocyanate. The RNA was then precipitated in 4 M lithium chloride as described by Cathala et al. (21). RNA Blot Analysis. RNA (5 Mg/lane) was electrophoresed on form aldehyde gels and transferred to nitrocellulose filters as described previously (22). The filters were hybridized with inserts purified from plasmid-carrying sequences of the desired gene. The inserts were labeled to a specific activity greater than 10s cpm/^g using the random primer method (23). 134
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CRYSTAL-INDUCED
C-/DJAND c-myc EXPRESSION
•g1g1
Plasmiti. The fos insert used as a probe in this work was a 1.0kilobase Pstl \-fos fragment from the pfos-\ plasmid (24). The myc probe was a 4.8-kilobase Xbal-BamH\ fragment from the pSVc-myc-1 plasmid which carries the mouse myc sequence (25). Preparation of BCP Crystals. BCP crystals were prepared as described (11). They were crushed and sieved to yield 10- to 20-^m aggregates, sterilized by heating at 200°Cfor 90 min, weighed, and suspended in
80'1 60*_f
DMEM. The suspensions were sonicated before use. Material. Highly purified PDGF was prepared as described (26). The pfos-l and pSVc-myc-l plasmids were kindly provided by I. Verma (24) and R. A. Weinberg (25), respectively. [3H]Thymidine and [a-32P]dCTP
20-¿F^IFN(U/n.1)CÕKTDTKÌ'1K_i_'r5,10.Ì10'
were from Amersham Corporation (Arlington Heights, IL). Probes were randomly primed with kits from Boehringer Mannheim (Indian Fig. 2. Relationship between IFN-/3 concentration and inhibition of [3H]apolis, IN). TPA was obtained from Sigma (St. Louis, MO). Mouse ß- thymidine incorporation into DNA of BALB/C-3T3 cells. Cells were grown and interferon (specific activity, 4.2 x IO6 IRU/ml; Catalogue No. 20181) maintained in 96-multiwell culture plates until confluence. They were then fed was purchased from Lee Biomedicai Research Laboratory (San Diego, CA). All cell culture supplies are products of Gibco Laboratory (Grand Island, NY).
with DMEM and 5% platelet-poor plasma containing either BCP crystals (SO Mg/ml) or PDGF (50 ng/ml) in the presence or absence of various concentrations of IFN-/3. [3H]Thymidine incorporation was estimated 24 h after stimulation as described in "Materials and Methods." Columns, mean; him. SEM (n = 4). D. BCP crystals (50 Mg/ml); D, PGDF (50 ng/ml).
RESULTS 14-J
Exposure of quiescent BALB/C-3T3 cells to medium contain ing various concentrations of BCP crystals and 5% plateletpoor plasma caused a dose-dependent increase in DNA synthe sis as determined by [3H]thymidine incorporation in cells har vested after 24 h of incubation (Fig. 1). The maximum stimu lation of DNA synthesis plateaued between 50 and 100 fig/m\ of BCP crystals and was approximately equal to 70% of that achieved with 50 ng/ml of PGDF. The quantity of BCP crystals required for stimulation of DNA synthesis in the present cell system is similar to that required to stimulate other fibroblasts (5, 6). The same concentration of BCP crystals (50 Mg/ml) was used in all subsequent experiments. To define the conditions under which mouse IFN-/3 inhibited the proliferation of BALB/C-3T3 cells, we first examined the dosage of IFN-0 required to significantly suppress the BCP crystal-induced [3H]thymidine incorporation by these cells in 24 h. Concomitant treatment of BALB/C-3T3 cells with BCP crystals (50 ng/m\) and IFN-/3 at 1000 units/ml, 5000 units/ ml, and 10,000 units/ml produced reductions of 20%, 57%, and 61 % of [3H]thymidine incorporation, respectively (Fig. 2).
>*loll'".I)-N XXJLN IFN-L._u 10-1 8' ? 6'i
4.Un2 2-i.E[&ZIJLeoi(5(«ral
Oh
2K
4h
6h
8)1
10h
TIMEOF IFNADDITION Fig. 3. [3H]Thymidine incorporation into DNA of BALB/C-3T3 cells as a function of delayed addition of IFN-0 (5000 units/ml) after stimulation with BCP crystals (50 *tg/ml). Columns, mean; bars, SEM (n = 4). PDGF IFN+ PDGF BCP Cr„tol,
Almost identical inhibition data were obtained against PGDF (50 ng/ml). As a result, the concentration of IFN-/3 used in all subsequent experiments was 5000 units/ml. The suppression of S-phase entry, as a function of the time of IFN-/3 addition, was measured to determine the time in the 6050!»0I"i?
5 1^-:5
10
15
20
25
30
35
HOURSAFTERADDITION
Fig. 4. S-phase entry of control and IFN-/3-treated BALB/C-3T3 cells. Time course after simultaneous addition of BCP crystals (50 ng/ml) or PDGF (50 ng) to quiescent cells. Points, mean; bars, SEM (n = 4).
0 BCP(|ig/>*1) 0PDGF
20
50
10050
cell cycle at which the IFN-mediated block occurred. IFN-/3 could be added as late as 6 h after the cells were stimulated with BCP crystals. Thereafter the ability of IFN-/3 to suppress entry into S-phase significantly decreased. By the tenth hour after stimulation, addition of IFN-0 had no inhibitory effect on BCP crystal-stimulated [3H]thymidine incorporation by cells as
(ng/.l),1A1
Fig. 1. Effects of different concentrations of BCP crystals on [3H]thymidine incorporation in BALB/C-3T3 cells. Cells were grown and maintained in 96multiwell culture plates until confluence. They were exposed to DMEM and 5% platelet-poor plasma containing various concentrations of BCP crystals or PGDF (50 ng/ml) for 24 h. Plates were fixed, and individual wells were counted as described in "Materials and Methods." Columns, mean; ears, SEM (n = 4).
measured at 24 h (Fig. 3). To determine whether IFN-/3 delays or blocks BCP crystals or PDGF-stimulated entry into S phase, [3H]thymidine incor poration by cells was examined at times up to 48 h after stimulation in the presence or absence of IFN-/3 (Fig. 4). Ex135
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CRYSTAL-INDUCED
trapolation of the data revealed a similar time of entry into S phase, although as observed previously, cells stimulated with BCP crystals lagged by 1 to 2 h when compared to PDGF stimulation (5). Although the stimulation of DNA synthesis by BCP crystals was only 40% of that of PDGF in this experiment, the usual maximum stimulation by BCP crystals is approxi mately 60% that of PDGF. IFN-0 clearly delayed but did not block the BCP crystal- and PDGF-stimulated entry into S phase by approximately 4 to 5 h. Almost identical data were obtained when a higher dose of IFN-/3 (10,000 units/ml) was used (data not shown). The effect of IFN-jtf on the induction of expression of c-fos by BCP crystals and PDGF is summarized in Fig. 5, a and ¿>, respectively. Stimulation of density-arrested BALB/C-3T3 cells with either BCP crystals or PDGF resulted in maximal accu mulation of c-fos mRNA at 30 min after stimulation. In the presence of IFN-/8, the level of c-fos mRNA at 30 min appeared to be similar and remained detectable at l h after stimulation. Induction of c-myc transcription by BCP crystals (Fig. 6a) and PDGF (Fig. 6¿>) occurred within l h and was maximal at around 3 h after stimulation. Unlike c-fos, stimulated cells maintained elevated transcription of c-myc for at least 5 h. The presence of IFN-ßappeared to delay the expression of the cmyc message by at least an hour.
c-fos AND c-myc EXPRESSION Myc Hrs O
Hrs O i
BCP 1 2
i
1
3
25,
5
!,
12
BCP+Int 1235
35
Fig. 6. Northern blot of c-myc mRNA of control and IFN-/3-treated cells after stimulation with BCP crystals (50 (ig/ml) (A) and PDGF (50 ng/ml) (B).
DISCUSSION Barnes and Colowick (27) were the first to describe stimula tion of [3H]thymidine incorporation into mouse 3T3 cells in culture by precipitates of calcium phosphate. A similar growthstimulatory effect on 3T3 cells was also observed with precipi tates of calcium pyrophosphate (28, 29). Our previous studies showed that mitogenic properties can be demonstrated for other calcium-containing crystals (for re view see Refs. 30 and 31), and that BCP crystals can substitute for PDGF as a competence growth factor in vitro (11). MoreFOB
BCP
BCP + Int i 1 3
FM PF Mrs 0
i
PF+ Int 1 3
over, BCP crystals and PDGF exert a number of similar bio logical effects on cultured cells (32), such as stimulation of prostaglandin E2 production via the phospholipase Aj/cyclooxygenase pathway (33), activation of phospholipase C and inositol phospholipid hydrolysis (34), and induction of col IngerÃ-ase and neutral proteases synthesis (35-37). Our present study demonstrated that stimulation of densityarrested BALB/C-3T3 cells with either BCP crystals or PDGF resulted in a transient increase in the level of c-fos with maximal c-fos mRNA levels reached at 30 min after stimulation, followed by a rapid decline to the basal level (Fig. 5, a and h). Induction of c-myc transcription by BCP crystals and PDGF occurred within an hour and was maximal at around 3 h after stimulation. Stimulated cells maintained an elevated level of transcription of c-myc for at least 5 h (Fig. 6, a and b). This is the first demonstration that BCP crystals activate the expression of both protooncogenes in cultured cells. Previous work has shown that intracellular crystal dissolution and BCP crystal-induced mitogenesis are related events (6, 38). Our earlier hypothesis was that BCP crystals were solubili/ed in the acidic environment of secondary lysosomes after endocytosis, as free Ca2+ appeared in the extracellular medium within 2 h. Ca2+ in transit from crystal dissolution either diffused or was actively pumped from the lysosome, raising the intracellular Ca2+ concentration. As a consequence, many Ca2+activated processes, such as microtubule depolymerization, pro tein phosphorylation, and protease activation, occurred as part of a program leading eventually to DNA synthesis (6, 31). However, even though approximately 60% of added BCP crys tals are intimately associated with cells within 30 min (8), intracellular dissolution probably does not proceed fast enough to account for the rapid induction of c-fos and possibly c-myc. It is likely that dissolution and elevation of intracellular ("a2*
Fig. 5. Northern blot of c-fos mRNA of control and IFN-0-treated cells after stimulation with BCP crystals (50 Mg/ml (A) and PGDF (50 ng/ml) (B).
as the result of dissolution are required for later cellular events occurring in the progression phase of d. 136
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Our present report demonstrates that simultaneous addition of IFN-/3 with either PDGF or BCP crystals delays the activa tion of c-myc (Fig. 6, a and b) and the entry into S-phase ([3H] -thymidine incorporation) (Fig. 4). This is in agreement with the observation of Tommaga and Lengyel (39) that there was no reduction in the amounts of the PDGF messages c-myc, JE, and KC when BALB/C-3T3 cells were exposed to IFN-/3 for 48 h before PDGF treatment. Under such conditions, cell repli cation was inhibited. Similar to an earlier report (19), it is necessary to add IFN within 8 h after stimulation with growth factors in order to see this delay (Fig. 2). The quantitative differences between ours and Lin et al. (19) may stem from the different IFN preparation and/or the technique used to measure DNA synthesis. Addi tionally, we also confirmed the requirement of at least 1000 units/ml of IFN-0 in order to see any significant reduction in ['H]thymidine incorporation in BALB/C-3T3 cells (Fig. 3). In contrast to our data, Einat et al. (20) reported that as little as 2 units/ml of IFN-0 were needed to reduce [3H]thymidine incorporation into DNA of their BALB/C-3T3 cells. Further more, they showed that addition of mouse IFN-/5 (100 units/ ml) together with PGDF to the same cells inhibited the induc tion of c-myc, ornithine decarboxylase, and /3-actin. We do not have any explanation for these discrepancies other than these may be due to differences in the BALB/C-3T3 cells and/or culturing condition. Substantial reductions in the amount of cmyc transcription were also observed after IFN-/? treatment of lymphoblastoid cells (18, 40). Our present study and others (19, 41) have shown that IFN.)'can be added as late as 8 h to block or delay DNA synthesis
7. 8. 9.
10.
11. 12. 13. 14.
15. 16. 17. 18.
19.
after the cells have been stimulated with PDGF. Since induction of c-myc expression by PGDF (13,14) and BCP crystals occurs within an hour and is maximal at 3 h after stimulation, this would argue against the inhibitory effect of IFN-/3 on DNA synthesis being mediated through a block or a delay in c-myc activation. We would like to suggest that the antagonistic effects of IFN-0 on cell replication are mainly exerted at a later time in the Gìphase of the cell cycle after the induction of the c-fos and c-myc messages. Since we have only examined the effects of IFN-/3 on mRNA induction, the other possibility is that the antagonistic effect of IFN-/3 may be mediated through a posttranscriptional mechanism. In summary, we have demonstrated that BCP crystals induce the expression of the c-fos and c-myc protooncogenes in a time course similar to that of PDGF. IFN-/3 delayed but did not block the induction of DNA synthesis and c-myc expression. Our results suggest that the antagonistic actions of IFN-/3 on the cell cycle traverse do not appear to be mediated through an inhibition or a delay in induction of c-fos and c-myc expression.
20. 21. 22. 23. 24. 25. 26.
27. 28. 29.
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30.
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c-fos AND c-myc EXPRESSION
39. Tommaga, S., and Lengyel, P. /3-Interferon alters the pattern of proteins secreted from quiescent and platelet-derived growth factor-treated BALB/c3T3 ce|ls j Biol Cnem., 260: 1975-1978, 1985. 40 Jonak «ÕÕU r j h and rprlnrrinn mvc mRNA 4°'Ã-^rti u Knieht a , Frf Ir Selertivp P N TT T¿ nf cÕKA »iriwìin D,a"dl c„e'ls by human ^-"«erferon- Pr°c-Nati. Acad. Sci. USA, Sì:1747l750' 19844I- Kimchi, A., Shure, H., Lapido«,Y., Rapoport, S., Panel, A., and Revel, M. Antimitogenic effects of interferon and (2'-5')-oligoadenylate in synchronized 3T3 cells. FEBS Lett., 134: 212-214, 1981.
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